Investigation of the active ingredients and pharmacological mechanisms of Porana sinensis Hemsl. Against rheumatoid arthritis using network pharmacology and experimental validation.

BACKGROUND: Porana sinensis Hemsl. has been widely used as a substitute for Erycibes Caulis to treat rheumatoid arthritis (RA) in traditional Chinese medicine (TCM). However, little is known about the active ingredients and pharmacological mechanisms that mediate the action of P. sinensis against RA.
METHODS: The compounds contained in P. sinensis were analyzed by Q Exactive Focus mass spectrometer. The active constituents and pharmacological mechanism of P. sinensis against RA were clarified using a network pharmacology-based investigation. LPS-induced RAW 264.7 cells was used to verify anti-inflammatory effects of the active compounds screened by network pharmacology. Collagen-induced arthritis model was used to further investigate the mechanism of P. sinensis against RA.
RESULTS: The potential components and targets of P. sinensis against RA were analyzed using network pharmacology, and five compounds, twenty-five targets, and eight pathways were identified. Experimental validation suggested that P. sinensis extract and five compounds (esculetin, umbelliferone, trans-N-feruloyltyramine, caffeic acid and scopolin) could inhibit the release of inflammatory mediators (NO, TNF-α, IL-1β and IL-6) in LPS-induced RAW 264.7 cell. P. sinensis extract attenuated the severity, pathological changes, and release of cytokines (IL-6 and HIF-1α) during RA progression by regulating the PI3K/AKT and HIF-1 pathways.
CONCLUSION: The study provides a basis for the application of P. sinensis against RA. Our findings may provide suggestions for developing P. sinensis into a substitute for Erycibes Caulis.

1. Introduction

With the increasing demand for traditional Chinese medicine (TCM), some natural medicinal resources are on the verge of extinction. Developing substitutes may alleviate the pressure on these endangered Chinese medicine resources [1,2]. Erycibes Caulis is widely used in treating rheumatoid arthritis (RA) in TCM [3-5]. The official species of Erycibes Caulis recorded in China Pharmacopoeia 2020 edition are Erycibe obtusifolia Benth. and Erycibe schmidtii Craib. During the course of reduction of natural sources of Erycibes Caulis, Porana sinensis Hemsl. has become the primary substitute on the market [3,6]. P. sinensis, which belongs to the family Convolvulaceae, is mainly found in limestone mountainous regions and is widely distributed in China North-Central, China South-Central, China Southeast and Vietnam [7].

Our previous studies indicated that the chemical composition of P. sinensis was similar to that of Erycibes Caulis, and they all contained large amounts of coumarins and quinic acid derivatives [8]. The 40% ethanolic extract of P. sinensis was almost non-toxic (oral administration, 5 g/kg) and exhibited anti-inflammatory and anti-nociceptive effects [3]. Xue et al. reported that 80% methanol extract of P. sinensis reduced the production of NO on LPS-stimulated RAW 264.7 cells, and dicaffeoylquinic acids were the active compounds [7]. Although considerable evidence exists to support P. sinensis used as a substitute for Erycibes Caulis, much work is still needed to demonstrate this. For example, Erycibes Caulis is widely used in treating RA in TCM; however, there has been no study of the efficacy and mechanism of P. sinensis for the treatment of RA.

RA is a chronic autoimmune disease, which mainly acts on synovium, cartilage and bone, resulting in the decline of physical function and quality of life [9]. At present, nonsteroidal anti-inflammatory drugs (NSAIDs) and disease-modifying anti-rheumatic drugs (DMARDs) are commonly used in the treatment of RA. Although these drugs are typically effective, they are also not satisfactory because of their low efficacy and side effects [9]. It is of great significance to develop anti-RA TCM with multi-target effect and clear pharmacological effect.

With the rapid development of bioinformatics, network pharmacology has become a hot area of pharmacology research. Because network pharmacology delivers systematic understanding of multi-component and multi-target actions, it helps clarify the effect of TCM on various diseases [10-12]. Thus, we investigated the active constituents and pharmacological mechanism of P. sinensis against RA by integrating network pharmacology with experimental validation.

In this work, the chemical profile of P. sinensis was analyzed by Q Exactive Focus mass spectrometer (MS), and 21 compounds were identified. Subsequently, these compounds were used as the basis for network pharmacological analysis. Protein-protein interaction (PPI) network for RA was established to identify potential drug targets. KEGG pathway analysis was then performed to elucidate the signaling pathway regulated by P. sinensis. The results of cell experiment suggested that P. sinensis extract and five compounds (esculetin, umbelliferone, trans-N-feruloyltyramine, caffeic acid and scopolin) could inhibit the release of inflammatory mediators (NO, TNF-α, IL-1β and IL-6) in LPS-induced RAW 264.7 cell. Animal experiments showed that P. sinensis treated RA by modulating PI3K-Akt and HIF-1 pathways, regulating cytokine release (IL-6 and HIF-1α). The experimental results are consistent with those of network pharmacology. This study systematically explains the effective substances and mechanisms of P. sinensis against RA. Our findings provide feasible suggestions for developing P. sinensis into a substitute for Erycibes Caulis. We also expect that the research on P. sinensis will accelerate the rational development and utilization of plants of the genus Porana.

2. Materials and methods

2.1. Materials and instrumentation

Caulis of P. sinensis was purchased from Kunming, China, in December 2018 (lot number: 20181205). The material was identified by Dr. Zhiyong Chen and deposited at the Shaanxi Academy of Traditional Chinese Medicine. Scopolin (lot number: 16040805), scopoletin (161208), chlorogenic acid (1701904), cryptochlorogenic acid (17061401), neochlorogenic acid (17062003), 3,5-dicaffeoylquinic acid (19061201), 3,4-dicaffeoylquinic acid (17121201), 4,5-dicaffeoylquinic acid (18070401), umbelliferone (18010202), esculetin (18092803) and caffeic acid (17122804) were bought from Qiming Bioengineering Institute (Shanghai, China), trans-N-feruloyltyramine (W01D9Z76497) was bought from Yuanye Bioengineering Institute (Shanghai, China), and the purities of the reference compounds were all above 98%. Bovine type II collagen (20021, Chondrex, USA), Incomplete Freund’s adjuvant (F5506-10ML, Sigma−Aldrich, USA). IL-1β, IL-6 and TNF-α ELISA kits were obtained from Cloud-clone Corporation (Wuhan, China). HIF-1α ELISA kits was supplied by Jiancheng Bioengineering Institute (Nanjing, China). The RAW 264.7 murine macrophage were obtained from Youersheng Bioengineering Institute (Wuhan, China). The following antibodies were used: Actin antibody (Mouse, Servicebio, Wuhan, China), PI3K antibody (Mouse, Bioss, Beijing, China), AKT antibody (Rabbit, Servicebio, Wuhan, China), p-AKT antibody (Rabbit, Affinity, USA), and HIF-1α antibody (Rabbit, Abcam, UK).

Q Exactive Focus MS (Thermo Finnigan, San Jose, USA) was applied to identify the compounds. Samples were separated on Accucure aQ C18 columns (2.1 mm × 150 mm, 2.6 μm) purchased from Thermo Fisher Scientific, USA. Network Pharmacology database and analysis platform: TTD (http://db.idrblab.net/ttd/); UniProt (http://www.uniprot.org/); OMIM (https://www.omim.org/); DrugBank (https://www.drugbank.ca/); GeneCards (https://www.genecards.org/); SwissTargetPrediction (http://www.swisstargetprediction.ch/); STRING (https://string-db.org/); DAVID (https://david.ncifcrf.gov/tools.jsp); R language (Version 4.0.2, https://www.r-project.org/).

2.2. Identification of compounds by LC-MS

2.2.1. Standard solutions and sample preparation

All reference compounds (1 μg/mL) were prepared in 60% methanol. Approximately 0.5 g P. sinensis powder (40 mesh) was extracted with 50 mL 80% methanol for 30 min by ultrasound extraction. The solution was filtered and diluted with an equal volume of 40% methanol. The sample was then filtered through 0.22-μm pore membrane.

2.2.2. Analytical conditions

Column temperature: 35°C; flow rate: 0.3 mL/min; injection volume: 2 μL. A linear gradient elution of 0.1% formic acid aqueous (A) and methanol (B) was used. The elution program was optimized as follows: 5–25% B within 12 min, 25–38% B with over 12–20 min, 38–60% B with the range of 20–35 min. The MS parameters were optimized: spray voltage: ±3500 V; atomization temperature: 350°C; capillary temperature: 320°C; sheath gas pressure: 45 arb; aux gas pressure, 15 arb; S-lens RF, 60 V; scan mode: full MS (resolution 70000) and MS/MS (17500).

2.2.3. LC-MS data analysis

The elemental compositions were calculated according to the high-precision precursor ions. All compounds reported in P. sinensis and Porana species plants were summarized to find the most reasonable molecular formula by searching literature sources. The fragmentation patterns of these compounds were used to differentiate compounds with the same formula.

2.3. Network pharmacology research of P. sinensis against RA

2.3.1. Targets prediction and screening

The compounds identified by UPLC-MS were used as the basis for network pharmacological analysis. This study predicted drug targets in databases such as TCMSP and GeneCards. Target prediction (TCMSP) was performed using the WES (Weighted Ensemble Similarity) model [13,14], which showed good performance with a consistency (82.83%), sensitivity (81.33%), and specificity (93.62%). We also predicted the constituent Target via the Swiss Target Prediction network server based on 2D and 3D similarity measures of known ligands [15]. The predicted targets were collated and imported into the UniProt database. Then, the mapping analysis of the normalized targets and the RA-related targets information obtained from databases (GeneCards, OMIM, TTD, and DrugBank) were conducted to screen out the potential anti-RA targets of P. sinensis. Finally, a component-target-disease (C-T-D) network was visualized using Cytoscape 3.6.0 plotted as an interaction network. The degree centrality (DC), betweenness centrality (BC), and closeness centrality (CC) were analyzed for each node in the C-T-D network using Cytoscape 3.6.0 software. The nodes with a DC, BC, and CC larger than the median were identified as the potential targets.

2.3.2. PPI network

The PPI network was generated by importing potential drug targets into string database. The PPI network for P. sinensis was then constructed using Cytoscape 3.6.0. Simultaneously, the CytoHubba plug [16] in the software was utilized to screen the hub genes, and the "Degree" algorithm was selected.

2.3.3. KEGG analysis and network construction

KEGG analysis for P. sinensis against RA was conducted using DAVID 6.8 database. The top 20 pathways with P < 0.05 were selected, and the R language was used for plotting. The component-target-pathway (C-T-P) network was generated using Cytoscape 3.6.0. A network analyzer was utilized for computing the topological parameters of the network. Generally, the highest-ranked target plays an essential role in anti-RA.

2.4. Experimental validation

2.4.1. Preparation of P. sinensis extract

The powder of 2 kg dry samples was extracted with 20 L 40% ethanol for 2 h by reflux extraction two times. The filtered solution was concentrated using a rotary evaporator at 50°C. The yield of P. sinensis (Pse) was 13.0%. Eight active compounds in Pse were determined by HPLC according to our previous work [17], and the contents are 13.4268 mg/g (neochlorogenic acid), 12.6935 mg/g (scopolin), 48.5457 mg/g (chlorogenic acid), 8.2953 mg/g (cryptochlorogenic acid), 20.9330 mg/g (scopoletin), 28.6063 mg/g (3,4-dicaffeoylquinic acid), 13.5660 mg/g (3,5-dicaffeoylquinic acid) and 18.3498 mg/g (4,5-dicaffeoylquinic acid), respectively.

2.4.2. Effects on LPS-induced RAW 264.7 cell

2.4.2.1. Cell culture and viability assay. RAW 264.7 cell line was cultured in DMEM medium supplemented with 10% fetal bovine serum, and were maintained at 37°C in a water-saturated 5% CO2 incubator. Cells in the mid-log phase were used for further experiments. Lipopolysaccharide (LPS, 1 μg/mL) was applied onto RAW 264.7 cell to trigger the inflammatory responses for 24 h. Methotrexate (MTX) was used as anti-inflammatory positive control.

Cell viability was measured by MTT assay (Sigma). The RAW 264.7 cells were cultured in 96-well plate at a density of 104 cells/well. Different concentrations of Pse and compounds were added 2 h before LPS treatment. After 24 h, 20 μL MTT (5 mg/mL) was added in each wells, and the cells continued to be incubated for 4 h. Then the formazan crystals were dissolved in DMSO and measured at 490 nm. Relative cell viability was calculated by comparing with that of the control group.

2.4.2.2. Determination of pro-inflammatory cytokines. The RAW 264.7 cells was prepared with the same procedure described above, then the cells were treated with samples (MTX, Pse, umbelliferone, caffeic acid: 120 μg/mL; scopolin, esculetin, trans-N-feruloyltyramine: 5 μg/mL). The supernatant was taken 24 h after administration and detected according to the instructions of NO, TNF-α, IL-1β and IL-6 kits.

2.4.3. Effects on collagen-induced arthritis model

2.4.3.1. Animals. The study protocol of animal experiments was reviewed and approved by the Experimental Animal Ethical Committee at the Shaanxi Academy of Traditional Chinese Medicine (license numbers AF/SL-01/01.2 and AF/SC-05/01.2). All rats received human care followed the National Institutes of Health Guide for the Care and Use of Laboratory Animals (NIH Publications No. 8023). Male SD rats (SPF grade, 180–220 g) were brought from the Experimental Animal Facilities of Xi’an Jiaotong University (SYXK2020-005). The animals were kept with a 12/12 h light/dark cycle at a temperature of 22 ± 1°C and 65 ± 5% humidity.

2.4.3.2. Induction of collagen-induced arthritis (CIA). The 48 rats were randomly divided into six groups: the normal, model, methotrexate (1 mg/kg), high-dose (Pse, 0.6 g/kg), middle-dose (Pse, 0.3 g/kg) and low-dose (Pse, 0.15 g/kg) groups. The selection of administered doses is based on our previous studies. At these doses, the extract shows good anti-inflammatory and analgesic effects [3]. The CIA model is one of the standard RA models, which shares several pathological features with RA, such as synovial inflammatory cell infiltration, synovial hyperplasia and bone erosion [9]. The CIA model was established as previously described [18]. Type II collagen was prepared with 0.1 mol/L acetic acid at 2 mg/mL. Then, the solution was emulsified with an equal volume of incomplete Freund’s adjuvant. For immunization, 0.2 mL of collagen was injected at the base of the tail of each rat. The rats were given a booster injection (0.2 mL of collagen) on day 7. On day 13, Pse (0.6, 0.3, 0.15 g/kg) was intragastric administrated once daily for 17 days, while methotrexate was given every 3 days.

During the experiment, body weights were measured every 7 days. The left hind paw’s ankle circumference and the arthritis index (AI) were recorded every 4 days from day 13. The AI criteria [19]: 0, no swelling; 1, swelling or redness of digit; 2, slight swelling of the ankle; 3, gross swelling of the paw; and 4, severe arthritis of the entire paw. Hind paws were used to calculate AI. Relative organ weights were assayed as well.

2.4.3.3. Biochemical assays. On day 29, all rats were anesthetized with pentobarbital sodium, and the abdominal aorta was punctured for blood collection. The blood was centrifuged to separate the serum for ELISA testing of HIF-1α and IL-6 using assay kits.

2.4.3.4. Histopathological examination. The right hind ankle joints were harvested after serum collection, fixed in 10% formalin, and decalcified in ethylenediaminetetraacetic acid. The joints were then embedded in paraffin, stained with hematoxylin and eosin (H&E), and observed under light microscope.

2.4.3.5. Quantitative PCR analysis. The mRNA was extracted using the TRIzol reagent from the synovium of the joint. For complementary DNA (cDNA) synthesis, reverse transcriptase and 2 μg RNA were used. PCR amplification was conducted using gene-specific PCR primers provided by Wuhan Servicebio Technology (Wuhan, China). Primers used in this study are listed in . The amplification was performed in a 15 μL reaction volume containing 2 μL cDNA, 1.5 μL 2.5 μmol/L primers, 7.5 μL 2 × qPCR mix, and 4 μL ddH2O. Each reaction was carried out for 40 cycles of denaturation at 95°C for 15 s and annealing at 60°C for 60 s. GAPDH was used as a control for normalization.

2.4.3.6. Western blot analysis. The experiment was performed following previous studies’ protocol with slight modification [9]. The protein extraction of the synovium of joints was performed with RIPA lysis buffer. The lysate was then centrifuged at 4°C, 12000 rpm for 10 min. Subsequently, the supernatant was collected and boiled for 15 min, subjected to SDS-PAGE, and transferred to PVDF membrane. After incubating with respective primary antibodies, the membrane was extensive washed with TBST, and treated with horseradish peroxidase-conjugated secondary antibodies. In the experiment, β-actin (GB12001, Wuhan Servicebio Technology) was regarded as the internal control. Protein visualization was conducted on an imaging system (Clinx, Shanghai, China).

2.5. Statistical analysis

All data were expressed as mean ± SD for each group. Comparisons between multiple groups were made with one-way ANOVA, and a value of p < 0.05 was considered statistically significant.

3. Results

3.1. LC-MS analysis of P. sinensis

The total ion chromatograms are shown in . A total of 21 compounds and isomers from P. sinensis were identified, including seven coumarins (5, 8, 10, 11, 12, 13, 15), seven chlorogenic acids (2, 4, 6, 9, 16, 17, 20), one tropane alkaloid (1), two amides (19, 21), one flavonoid (18), one lignan (14) and two other compounds (3, 7). Twelve compounds were unambiguously identified as neochlorogenic acid (2), chlorogenic acid (4), scopolin (5), cryptochlorogenic acid (6), caffeic acid (7), umbelliferone (8), scopoletin (10), esculetin (15), 3,4-dicaffeoylquinic acid (16), 3,5-dicaffeoylquinic acid (17), 4,5-dicaffeoylquinic acid (20), and trans-N-feruloyltyramine (21) by comparisons with reference compounds. Data for all compounds are listed in .

Total ion chromatograms of P. sinensis in positive ion (A) and negative ion (B) modes.

3.2. Network pharmacology analysis of P. sinensis

3.2.1. C-T-D network construction and core target screening

Through Swiss Target Prediction and DrugBank reverse Prediction, 466 potential targets for 21 chemical components were obtained. The 466 potential targets were mapped to RA targets, and 293 common potential targets against RA were obtained. Using these targets as network nodes, Cytoscape 3.6.0 software was used to analyze network topology parameters. The results showed that the median DC was 41, the average BC was 0.0026, and the average CC was 0.3639 for the compounds; the median DC was 2, the average BC was 0.0013, and the average CC was 0.4897 for the 293 common targets. There were ten compounds and 112 common targets with DC, BC, and CC values higher than the median, shown in , Tables and .

3.2.2. Construction of the PPI network

Searching for proteins playing important roles in the PPI network, we uploaded the 112 anti-RA targets to the STRING database to observe these protein interaction relationships. Then, we imported the obtained TSV file into Cytoscape 3.6.0 to build the PPI network. Using the Cytohubba plugin, the greater the node’s degree value, the redder its color, the more important it is in the network (). The hub genes include CAPDH (Degree = 92), AKT1 (Degree = 91), GASP3 (Degree = 81), EGFR (Degree = 78), SRC (Degree = 76), HSP90AA1 (Degree = 75), MAPK1 (Degree = 72), TNF (Degree = 67), ESR1 (Degree = 67), STAT3 (Degree = 66).

3.2.3. KEGG analysis and C-T-P construction

The 112 targets were mapped to 122 pathways using DAVID. Unrelated pathways, such as "Pathways in cancer", "Hepatitis B" and "Bladder cancer" were excluded. The top 20 KEGG pathways were obtained based on P-value (), shown in . A total of 64 targets were directly connected to the top 20 pathways. Most of these pathways are involved in inflammation, such as PI3K-Akt, HIF-1, ErbB, Rap1, TNF, VEGF, Ras signaling pathways.

The C-T-P network for the P. sinensis-mediated treatment of RA is shown in . The network comprises 94 nodes (10 compounds, 64 targets, and 20 pathways) and 400 edges. The green triangle represents the compounds, the yellow diamond denotes the potential targets, and the purple V graphics represents the pathways. The topological parameters of C-T-P were calculated using the Network Analyzer (). The results show that the median DC was 16, the median BC was 0.0351, and the median CC was 0.4115 for the compounds. There were 5 compounds with DC, BC and CC values higher than the median: esculetin (DC = 30, BC = 0.1328, CC = 0.4898), umbelliferone (DC = 20, BC = 0.0481, CC = 0.4348), trans-N-feruloyltyramine (DC = 21, BC = 0.0917, CC = 0.4471), caffeic acid (DC = 16, BC = 0.0351, CC = 0.4115) and scopolin (DC = 16, BC = 0.0547, CC = 0.4152). The median values of DC, BC, and CC were 7, 0.0093 and 0.4155 for targets nodes, respectively. There were 25 targets that exhibited high topological values. For the pathway nodes, the median values of DC, BC and CC were 12, 0.0224 and 0.4043. There were 8 pathways that exhibited high topological values.

3.3. Experimental validation for P. sinensis against RA

3.3.1. Effects on LPS-induced RAW 264.7 cell

Prior to validation of the effects of Pse and five active compounds on LPS-induced RAW 264.7 cell, we first analyzed their cellular toxicities using the MTT cell viability assay. The results showed that RAW 264.7 cell viability was 83.6% after induction with LPS (1 μg/mL) alone, so we chose this concentration for subsequent experiments. Based on the anti-inflammatory activity and cytotoxicity, we selected the dose concentration of the samples (). As shown in , Pse and the five compounds screened by network pharmacology all showed good anti-inflammatory activities, and the inhibition effect of Pse on NO and IL-6 was better than umbelliferone and caffeic acid at the same concentration. Inhibitory effects of trans-N-feruloyltyramine on IL-1β and IL-6 were not obvious.

Inhibitory effect of P. sinensis extract (Pse), methotrexate (MTX) and five compounds (esculetin, umbelliferone, trans-N-feruloyltyramine, caffeic acid and scopolin) on the release of inflammatory mediators (NO, TNF-α, IL-1β and IL-6) in LPS-induced RAW 264.7 cell.

*P < 0.05 and **P < 0.01 compared with model group; #P < 0.05 and ##P < 0.01 compared with normal group.

3.3.2. Effects on collagen-induced arthritis model

3.3.2.1. Effects of P. sinensis on ankle circumference and AI. Red swelling in the paws of model rats was first observed on day 11 following CIA-induced modeling. As shown in , edema of the left hind paw and AI of rats were significantly different between normal and model groups on day 13, which represents the successful model. The arthritis severity in the model group reached a peak on Day 21. Pse (0.6 g/kg) can suppress the paw swelling and AI on day 29.

3.3.2.2. Effects of P. sinensis on body weight and relative organ weight. As shown in , body weight in normal group rats increased more than CIA rats (p < 0.01), while Pse treated rats (0.6 g/kg, 0.3 g/kg) did not achieve a significant difference from model group rats (p > 0.05). The weight gain of rats in the methotrexate treated group was less than that in the model group (p < 0.05). As shown in , the spleen’s relative organ weight in methotrexate treated rats was significantly higher than rats of other groups (p < 0.01).

3.3.2.3. Effect of P. sinensis on serum cytokine levels. As shown in , collagen-induced the significant production of HIF-1α and IL-6 compared with the normal group. Pse (0.6 g/kg) inhibited the collagen-induced production of HIF-1α and IL-6.

3.3.2.4. Effect of P. sinensis on histopathological changes. The histopathological changes of ankle joints in each group are shown in . In the model group, inflammatory infiltration, synovial hyperplasia, and cartilage degradation were observed in the ankle joint. Methotrexate and Pse significantly attenuated the synovial hyperplasia and cartilage degradation.

Histopathological examination of ankle joints (×100).

(A) normal group, (B) model group, (C) methotrexate group, (D) P. sinensis extract group (0.6 g/kg), (E) P. sinensis extract group (0.3 g/kg), (F) P. sinensis extract group (0.15 g/kg).

3.3.2.5. Effects of P. sinensis on the mRNA expression. As shown in , compared with the normal group, the mRNA levels of HIF-1α, PI3K, and AKT in the model group were significantly increased (P < 0.05). The high-dose group of Pse has an effect of decreasing the mRNA levels of HIF-1α, PI3K, and AKT (P < 0.05).

3.3.2.6. Effects of P. sinensis on the protein expression levels. As shown in , the expression levels of PI3K, AKT, p-AKT, and HIF-1α proteins were significantly increased in the synovium of CIA model rats, compared to the normal group (p < 0.01). After the administration of Pse (0.6 g/kg), their expression levels were significantly reduced (p < 0.05).

Effects of P. sinensis extract (Pse) on the expression levels of PI3K, AKT, p-AKT, and HIF-1α proteins.

The specific bands of proteins (A), the expression levels of proteins (B~E).

4. Discussion

The similarity of chemical compositions is an essential indicator for judging the rationality of substitutes. In this study, a total of 21 compounds from P. sinensis were identified by UPLC-MS, including seven coumarins, seven chlorogenic acids, one tropane alkaloid, two amides, one flavonoid, one lignan, and two other compounds. By comparing with other studies [4,8,23,24], we discovered the chemical composition of P. sinensis is similar to that of E. obtusifolia and E. schmidtii. It should be noted that there are few reports on the systematic isolation of P. sinensis; only nine compounds were reported from this plant [20]. Therefore, systematic studies on the phytochemistry of P. sinensis need to be carried out. Future phytochemical studies of P. sinensis should focus on separating and identifying distinct chemical components to clarify the chemical similarities and differences between P. sinensis and Erycibes Caulis.

The potential components and targets of P. sinensis against RA were analyzed using a network pharmacology approach, and five compounds, twenty-five targets, and eight pathways were finally obtained. Esculetin was shown to possess an anti-inflammatory effect by suppressing the HIF-1α signaling pathways [25]. Caffeic acid attenuated hepatocellular carcinoma cells’ angiogenesis by reducing JNK-1-mediated HIF-1α stabilization [26]. Umbelliferone was useful for treating arthritis by suppressing the MAPK/NF-κB pathway [27]. In this study, KEGG analysis and C-T-P network showed that P. sinensis regulated the PI3K/Akt, HIF-1, Estrogen, Rap1 signaling pathways and so on. Recent studies demonstrated that PI3K/AKT pathway inhibits apoptosis in chondrocytes, and modulation of the pathway has been proposed as a potential therapy against RA [28,29]. HIF-1α can increase the production of inflammatory cytokines, and promote angiogenesis in RA patients [30,31]. The maintenance of Rap1 signaling in T cells can reduce the incidence rate and severity of CIA [32].

Macrophages play an important role in the pathogenesis of RA, and are the main source of inflammatory cytokines (such as NO, TNF-α, IL-1β, and IL-6) [33]. Therefore, we chose LPS-induced RAW 264.7 cells to verify the results of network pharmacology in vitro. A large number of literatures have reported the levels of TNF-α, IL-1β and IL-6 were significantly increased in serum of patients with RA [34]. At the same time, TNF is an important target of P. sinensis for the treatment of RA in our network pharmacology research. Therefore, these indexes were selected to verify the anti-inflammatory activity of Pse and five active compounds. It was reported that caffeic acid and caffeoylquinic acids could be active compounds of the anti-inflammatory potential of P. sinensis [7]. In this study, coumarins (esculetin, umbelliferone and scopolin) and amides (trans-N-feruloyltyramine) from P. sinensis showed good anti-inflammatory activities, especially amides, which should be attached great attentions to quality control of P. sinensis in the future.

Adjuvant-induced arthritis (AIA) and CIA models are classic animal models for RA research. However, AIA is different from RA in that it lacks a chronic pathological process [35]. At present, the CIA is recognized as the best RA model. We verified the computational prediction mechanisms of P. sinensis against RA based on the CIA rat model. The PI3K/AKT pathway is closely related to RA by deregulating activated immune cells’ proliferation and synovial fibroblasts [28]. Zou et al [36] reported that HIF-1 levels in the peripheral serum of CIA rats positively correlated with AI, and the CIA rat model regulated the expression of HIF-1α proteins via PI3K pathway. Combined with network pharmacology and literature reports, the PI3K/AKT and HIF-1 pathway was selected for confirmation. The results suggested that Pse attenuates the severity, pathological changes, and the release of cytokines (IL-6 and HIF-1α) during RA progression in a dose-dependent manner. Western blot analysis demonstrated that Pse significantly reduced protein levels of PI3K, p-AKT, and HIF-1α in the inflamed joints of CIA rats. The experimental results are consistent with those of network pharmacology. As a positive control, methotrexate significantly inhibited the arthritic response of rats; however, the relative organ weight of the spleen was significantly higher than in rats of other groups, and the weight gain of rats was less than that of the model group, which may be side effects of methotrexate. By contrast, P. sinensis treated RA without these side effects. This suggests P. sinensis may be a better choice than methotrexate when treating RA.

In recent years, with the decrease of natural resources of Erycibes Caulis, P. sinensis has been used as the primary substitute. The study on the anti-rheumatic effect of P. sinensis provided evidence for its use as a substitute. The genus Porana is widely distributed globally; however, there are few medicinal uses. We expect this study on P. sinensis will accelerate the rational development and utilization of genus Porana plants.

5. Conclusion

We identified 21 compounds containing in P. sinensis by UPLC-MS and offered evidence that P. sinensis reverses the pathological events during RA progression by regulating the PI3K/AKT and HIF-1 signaling pathways. P. sinensis extract and five compounds (esculetin, umbelliferone, trans-N-feruloyltyramine, caffeic acid and scopolin) could inhibit the release of inflammatory mediators (NO, TNF-α, IL-1β and IL-6) in LPS-induced RAW 264.7 cell. These findings provide the experimental basis for the application of P. sinensis against RA. In addition, we expect that our findings may help develop P. sinensis into a substitute for Erycibes Caulis, thereby setting an example for the study of substitutes for TCM.

The C-T-D network for P. sinensis in treatment of rheumatoid arthritis.

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The protein-protein interaction (PPI) network.

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The KEGG pathway for P. sinensis against rheumatoid arthritis.

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Effects of cytotoxicity on RAW264.7 cells of P. sinensis extract (Pse) and its effective constituents (**P < 0.01).

(PDF)

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Effects of P. sinensis extract (Pse) on ankle circumference (A), arthritis index (B) and body weight (C) of rats. Values shown are mean ± SD (n = 8); *P < 0.05 and **P < 0.01 compared with model group; #P < 0.05 and ##P < 0.01 compared with normal group.

(PDF)

Click here for additional data file.

Effects of P. sinensis extract (Pse) on HIF-1α and IL-6 levels in collagen-induced arthritis rats.

(PDF)

Click here for additional data file.

Effects of P. sinensis extract (Pse) on mRNA levels of HIF-1α (A), PI3K (B) and AKT (C).

(PDF)

Click here for additional data file.

Summary of gene-specific real-time PCR primer sequences.

(DOC)

Click here for additional data file.

The information of top 20 significant KEGG enrichment analysis.

(DOC)

Click here for additional data file.

(ZIP)

Click here for additional data file.

21 Sep 2021

2. The level of the extract showed in Fig S5 are extremely high, and a toxicity assay in untreated animals should be shown.

3. Please provide the information related to the cytotoxicity, including the graphs.

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Reviewer #1: Yes

Reviewer #2: Yes

**********

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Reviewer #1: Yes

Reviewer #2: I Don't Know

**********

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Reviewer #2: Yes

**********

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5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: The manuscript entitle " Investigation of the active ingredients and pharmacological mechanisms of Porana sinensis Hemsl. against rheumatoid arthritis using network pharmacology and experimental validation " reviewed. The study design is well, but some points should be revised by authors:

- Authors should add more points about P. sinensis properties in introduction.

- Authors should add some points about RA in introduction.

- Authors should mention to “ Investigation of the mechanism of action of Porana sinensis Hemsl. against gout arthritis using network pharmacology and experimental validation” in references.

- Authors should add references for Methods in Method & Material part.

- Authors should explain more about groups of study in M&M.

- CIA as a model should be explained with refernces in M&M.

- Authors should add references for model, AI and duration of treatment in M&M.

- Authors should explain how selected the doses of treatment in the M&M or add references.

Reviewer #2: The manuscript entitled “Investigation of the active ingredients and pharmacological mechanisms of Porana sinensis Hemsl. against rheumatoid arthritis using network pharmacology and experimental validation” (PONE-D-21-26996) basically deals on if P. sinesis may act as a substitute of Erycibes Caulis.

I have only one suggestion for the authors: you could add the accuracy or probability that the compounds, that were not unambiguously identified, are the ones mentioned.

**********

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Reviewer #1: No

Reviewer #2: No

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21 Oct 2021

Manuscript Reference Number: PONE-D-21-26996R1

Manuscript Title: Investigation of the active ingredients and pharmacological mechanisms of Porana sinensis Hemsl. against rheumatoid arthritis using network pharmacology and experimental validation

Journal: PLOS ONE

Response to the editors’ comments:

1. The figures provided are very blurry.

Response: Thanks for your suggestion. The figures have been revised and uploaded to PACE digital diagnostic tool to ensure that figures meet PLOS ONE's requirements.

2. The level of the extract showed in Fig S5 are extremely high, and a toxicity assay in untreated animals should be shown.

Response: Thanks for your suggestion. The toxicity of 40% ethanolic extract of Porana sinensis has been evaluated in our previous work [1]. A single dose of the extract (5.0 g/kg) was administered (ig) to 10 mice (five males and five females). Behaviors such as hyperactivity, sedation, increased or decreased respiration, loss of righting reflex and food and water intake were observed over a period of 14 days. On day 14, all animals were sacrificed and subjected to necropsies. The results showed that all animals gained weight, appeared normal and the necropsy revealed no visible lesions in any animals. Thus, the oral LD50 values, for 40% ethanolic extract of P. sinensis, for female and male mice must be greater than 5.0 g/kg.

3. Please provide the information related to the cytotoxicity, including the graphs.

Response: Thanks for your suggestion. Cytotoxicity data have been provided in S4 Fig in Supporting Information.

4. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming.

Response: We ensure that our manuscript meets PLOS ONE's style requirements.

5. We note that the grant information you provided in the ‘Funding Information’ and ‘Financial Disclosure’ sections do not match. When you resubmit, please ensure that you provide the correct grant numbers for the awards you received for your study in the ‘Funding Information’ section. We note that you have provided funding information that is not currently declared in your Funding Statement. However, funding information should not appear in the Acknowledgments section or other areas of your manuscript. We will only publish funding information present in the Funding Statement section of the online submission form. Please remove any funding-related text from the manuscript and let us know how you would like to update your Funding Statement. Please include your amended statements within your cover letter; we will change the online submission form on your behalf.

Response: We have removed funding-related text from the manuscript, and provided funding information in the Funding Statement section.

6. PLOS ONE now requires that authors provide the original uncropped and unadjusted images underlying all blot or gel results reported in a submission’s figures or Supporting Information files. This policy and the journal’s other requirements for blot/gel reporting and figure preparation are described in detail at https://journals.plos.org/plosone/s/figures#loc-blot-and-gel-reporting- requirements and https://journals.plos.org/plosone/s/figures#loc-preparing -figures-from-image-files. When you submit your revised manuscript, please ensure that your figures adhere fully to these guidelines and provide the original underlying images for all blot or gel data reported in your submission. See the following link for instructions on providing the original image data: https://journals.plos.org/plosone/s/figures#loc-original-images-for-blots-and-gels. In your cover letter, please note whether your blot/gel image data are in Supporting Information or posted at a public data repository, provide the repository URL if relevant, and provide specific details as to which raw blot/gel images, if any, are not available.

Response: We have prepared our figures adhere to these guidelines and provided the original underlying images for all blot data in Supporting Information files (S1_raw_images.pdf).

Response to the reviewers’ comments:

Reviewer #1: The manuscript entitle "Investigation of the active ingredients and pharmacological mechanisms of Porana sinensis Hemsl. against rheumatoid arthritis using network pharmacology and experimental validation" reviewed. The study design is well, but some points should be revised by authors.

1. Authors should add more points about P. sinensis properties in introduction.

Response: Thanks for your suggestion. “P. sinensis, which belongs to the family Convolvulaceae, is mainly found in limestone mountainous regions and is widely distributed in China North-Central, China South-Central, China Southeast and Vietnam.”

2. Authors should add some points about RA in introduction.

Response: Thanks for your suggestion. “RA is a chronic autoimmune disease, which mainly acts on synovium, cartilage and bone, resulting in the decline of physical function and quality of life [9]. At present, nonsteroidal anti-inflammatory drugs (NSAIDs) and disease-modifying anti-rheumatic drugs (DMARDs) are commonly used in the treatment of RA. Although these drugs are typically effective, they are also not satisfactory because of their low efficacy and side effects [9]. It is of great significance to develop anti-RA TCM with multi-target effect and clear pharmacological effect.”

3. Authors should mention to “Investigation of the mechanism of action of Porana sinensis Hemsl. against gout arthritis using network pharmacology and experimental validation” in references.

Response: Thanks for your suggestion. This literature has been cited.

4. Authors should add references for Methods in Method & Material part.

Response: Thanks for your suggestion. References have been added.

5. Authors should explain more about groups of study in M&M.

Response: Thanks for your suggestion. This part has been rewritten. “The 48 rats were randomly divided into six groups: the normal, model, methotrexate (1 mg/kg), high-dose (Pse, 0.6 g/kg), middle-dose (Pse, 0.3 g/kg) and low-dose (Pse, 0.15 g/kg) groups. The selection of administered doses is based on our previous studies. At these doses, the extract shows good anti-inflammatory and analgesic effects [3].”

6. CIA as a model should be explained with references in M&M.

Response: Thanks for your suggestion. “The CIA model is one of the standard RA models, which shares several pathological features with RA, such as synovial inflammatory cell infiltration, synovial hyperplasia and bone erosion [9]. The CIA model was established as previously described [18].”

7. Authors should add references for model, AI and duration of treatment in M&M.

Response: Thanks for your suggestion. References have been added.

8. Authors should explain how selected the doses of treatment in the M&M or add references.

Response: Thanks for your suggestion. “The selection of administered doses is based on our previous studies. At these doses, the extract shows good anti-inflammatory and analgesic effects [3].”

Reviewer #2: The manuscript entitled “Investigation of the active ingredients and pharmacological mechanisms of Porana sinensis Hemsl. against rheumatoid arthritis using network pharmacology and experimental validation” (PONE-D-21-26996) basically deals on if P. sinensis may act as a substitute of Erycibes Caulis.

1. I have only one suggestion for the authors: you could add the accuracy or probability that the compounds, that were not unambiguously identified, are the ones mentioned.

Response: Thanks for your suggestion. Based on the high-accuracy precursor ions and product ions obtained from Q Exactive Focus MS, the elemental compositions were calculated. By searching literature sources, all components reported in the literature on P. sinensis and plants of the same family were summarized in a Microsoft Office Excel table to search the most rational molecular formula. The fragmentation patterns of these compounds were used to differentiate compounds with the same formula. References for compounds identification have been added in Table 1.

References:

1. Chen Z, Liao L, Zhang Z, Wu L, Wang Z. Comparison of active constituents, acute toxicity, anti-nociceptive and anti-inflammatory activities of Porana sinensis Hemsl., Erycibe obtusifolia Benth. and Erycibe schmidtii Craib. Journal of ethnopharmacology, 150(2): 501-506.

Submitted filename: Response to Reviewers.doc

Click here for additional data file.

13 Dec 2021

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.

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If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols.

We look forward to receiving your revised manuscript.

Kind regards,

Horacio Bach

Academic Editor

PLOS ONE

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #2: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #2: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #2: N/A

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #2: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #2: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #2: (No Response)

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #2: No

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

16 Dec 2021

1. According to the new policies of PLoS One, authors should provide uncropped gels and blots. We have asked to provide this information, but now the raw data do not show the whole gels and blots. Please provide them.

Response: Thanks for your suggestion. We have provided uncropped gels and blots in S1_raw_images (pdf) in supporting information.

2. The cytotoxicity asked has not been fulfilled. Instead, the cytotoxicity of individual compounds has been included and not the extracts as mentioned.

Response: Thanks for your suggestion. The cytotoxicity of P. sinensis extract (Pse) has been provided in S4 Fig in supporting information.

Submitted filename: Response to Reviewers.doc

Click here for additional data file.

13 Jan 2022

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.

A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.

An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols.

We look forward to receiving your revised manuscript.

Kind regards,

Horacio Bach

Academic Editor

PLOS ONE

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

16 Feb 2022

1. We have sent two request to be addressed. The cropped gels are still an issue. You have provided the immunoblots already cropped from the original gel. The request is to show the original gel that was used to crop.

Response: Thanks for your suggestion. We have provided the original gel in S1_raw_images (pdf) in supporting information.

Submitted filename: Response to Reviewers.doc

Click here for additional data file.

17 Feb 2022

Investigation of the active ingredients and pharmacological mechanisms of Porana sinensis Hemsl. against rheumatoid arthritis using network pharmacology and experimental validation

PONE-D-21-26996R3

Dear Dr. Chen,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

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Kind regards,

Horacio Bach

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

22 Feb 2022

PONE-D-21-26996R3

Investigation of the active ingredients and pharmacological mechanisms of Porana sinensis Hemsl. against rheumatoid arthritis using network pharmacology and experimental validation

Dear Dr. Chen:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

If we can help with anything else, please email us at plosone@plos.org.

Thank you for submitting your work to PLOS ONE and supporting open access.

Kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Prof. Horacio Bach

Academic Editor

PLOS ONE

Table 1

Identification of chemical constituents from P. sinensis by UPLC-MS.

No.	tR (min)	Observed m/z	Calculated m/z	Mode	Error (ppm)	Molecular formula	Fragment ions (m/z)	Identification	References
1	0.97	144.1015	144.1019	[M+H]+	-2.77	C7H13NO2	144.1015, 126.0910, 108.0806, 98.0962, 84.0806, 68.0696	baogongteng C or erycibelline	[17]
2*	7.00	353.0862	353.0868	[M-H]-	-1.70	C16H18O9	353.0859, 191.0546, 179.0334, 173.0443, 161.0227, 135.0435	neochlorogenic acid	[7,8]
3	7.28	153.0177	153.0182	[M-H]-	-3.27	C7H6O4	153.0178, 141.9110, 123.0074, 109.0278, 61.9858	3,4-dihydroxybenzoic acid	[7,8]
4*	10.86	353.0862	353.0868	[M-H]-	-1.70	C16H18O9	191.0560, 179.0347, 161.0242	chlorogenic acid	[7,8]
5*	11.04	355.1014	355.1024	[M+H]+	-2.82	C16H18O9	193.0493, 178.0259, 165.0543, 133.0282	scopolin	[7,8]
6*	11.98	353.0862	353.0868	[M-H]-	-1.70	C16H18O9	191.0545, 179.0347, 173.0452, 135.0434	cryptochlorogenic acid	[7,8]
7*	12.07	179.0334	179.0339	[M-H]-	-2.79	C9H8O4	179.0334, 135.0435	caffeic acid	[7]
8*	12.21	163.0383	163.0388	[M+H]+	-3.07	C9H6O3	163.0385, 145.0281, 135.0437, 117.0332, 107.0490, 89.0384	umbelliferone	[20]
9	17.13	367.1017	367.1024	[M-H]-	-1.91	C17H20O9	191.0545, 173.0438, 135.0356, 111.0435, 93.0329, 87.0071	methyl chlorogenate	[21]
10*	17.44	193.0492	193.0495	[M+H]+	-1.55	C10H8O4	193.0491, 178.0255, 165.0539, 149.0591, 137.0595, 133.0281	scopoletin	[3]
11	19.85	695.1804	695.1818	[M-H]-	-2.01	C31H36O18	359.0966, 335.0766, 197.0441, 173.0437, 153.0538, 135.0438	eryciboside F	[21]
12	21.60	665.1702	665.1712	[M-H]-	-1.50	C30H34O17	198.0475, 191.0334, 176.0099, 153.0540, 121.0278	eryciboside A or B or C	[21]
13	21.75	635.1597	635.1607	[M-H]-	-1.57	C29H32O16	191.0334, 167.0334, 121.0433	eryciboside D or E	[21]
14	22.19	595.2012	595.2021	[M-H]-	-1.51	C28H36O14	447.4385, 433.1486, 418.1223, 403.1426, 373.1277, 358.1036, 239.7386, 181.0488, 139.0023	aketrilignoside B	[21]
15*	22.92	179.0333	179.0338	[M+H]+	-2.79	C9H6O4	179.0333, 151.0386, 133.0280, 123.0436	esculetin	[22]
16*	23.46	515.1171	515.1184	[M-H]-	-2.52	C25H24O12	353.0859, 335.0760, 191.0546, 179.0334, 173.0439, 161.0228, 135.0435, 93.0329	3,4-dicaffeoylquinic acid	[7,8]
17*	24.38	515.1171	515.1184	[M-H]-	-2.52	C25H24O12	353.0862, 191.0545, 179.0333, 173.0437, 135.0434	3,5-dicaffeoylquinic acid	[7,8]
18	24.67	287.0548	287.0550	[M-H]-	-0.697	C15H12O6	243.0648, 199.0746, 177.0539, 163.0380, 137.0223, 119.0485, 93.0330, 61.9869	eriodictyol	[21]
19	25.87	282.1123	282.1125	[M-H]-	-0.709	C17H17O3N	282.1122, 197.9011, 162.0543, 1485.0278, 136.0750, 119.0485	trans-N-(p-coumaroyl)tyramine	[20]
20*	27.23	515.1171	515.1184	[M-H]-	-2.52	C25H24O12	353.0858, 191.0545, 179.0333, 173.0438, 135.0434, 93.0327	4,5-dicaffeoylquinic acid	[7,8]
21*	29.85	312.1246	312.1238	[M-H]-	2.56	C18H19O4N	312.1238, 297.1001, 190.0508, 178.0506, 148.0528, 135.0451	trans-N-feruloyltyramine	[20]

* Compared with reference compounds.

Table 2

The topological parameter analysis of 10 compounds in P. sinensis against rheumatoid arthritis.

No.	compounds	molecular formula	Betweenness centrality (BC)	Closeness centrality (CC)	Degree centrality (DC)
1	Scopolin	C16H18O9	0.00554748	0.3665	45
2	Scopoletin	C10H8O4	0.01196908	0.3842	86
3	Neochlorogenicacid	C16H18O9	0.00316416	0.3642	40
4	4,5-Dicaffeoylquinicacid	C25H24O12	0.00264869	0.3639	44
5	3,5-Dicaffeoylquinicacid	C25H24O12	0.00366490	0.3639	42
6	3,4-Dicaffeoylquinicacid	C25H24O12	0.00264869	0.3639	44
7	Esculetin	C9H6O3	0.02573882	0.4049	116
8	Caffeic acid	C9H8O4	0.01098960	0.3787	64
9	Umbelliferone	C9H6O3	0.01219321	0.3861	81
10	trans-N-feruloyltyramine	C18H19O4N	0.02687441	0.4122	102

Table 3

The topological parameter analysis of 112 targets with rheumatoid arthritis for P. sinensis.

NO.	UniProt	Gene names	Betweenness centrality (BC)	Closeness centrality (CC)	Degree centrality (DC)
1	Q14790	CASP8	0.00441081	0.49481865	6
2	P08246	ELANE	0.00673174	0.49739583	8
3	P08183	ABCB1	0.00741662	0.49739583	8
4	P42574	CASP3	0.00719441	0.49739583	8
5	P17252	PRKCA	0.00719441	0.49739583	8
6	Q05655	PRKCD	0.00541423	0.4961039	7
7	P08253	MMP2	0.01899941	0.5	10
8	P05067	APP	0.00673172	0.49739583	8
9	P15121	AKR1B1	0.02883723	0.50395778	13
10	P21964	COMT	0.00114453	0.49226804	4
11	P56817	BACE1	0.01344752	0.49869452	9
12	P04626	ERBB2	0.00064866	0.49100257	3
13	P09874	PARP1	0.00114453	0.49226804	4
14	P03372	ESR1	0.00200793	0.49354005	5
15	P27338	MAOB	0.00224914	0.49354005	5
16	P05177	CYP1A2	0.00144712	0.49226804	4
17	P22303	ACHE	0.00078702	0.49100257	3
18	P35968	KDR	0.00570423	0.4961039	7
19	P12931	SRC	0.00247146	0.49354005	5
20	P00390	GSR	0.00114453	0.49226804	4
21	P49841	GSK3B	0.00114453	0.49226804	4
22	P09769	FGR	0.00114453	0.49226804	4
23	P0DMV8	HSPA1A	0.00114453	0.49226804	4
24	P53350	PLK1	0.00114453	0.49226804	4
25	Q05397	PTK2	0.00064866	0.49100257	3
26	P09917	ALOX5	0.00154686	0.49226804	4
27	Q92731	ESR2	0.00200793	0.49354005	5
28	P42262	GRIA2	0.00032983	0.48974359	2
29	P07550	ADRB2	0.00091322	0.49100257	3
30	P00533	EGFR	0.00455135	0.4961039	7
31	P28482	MAPK1	0.00130213	0.49100257	3
32	Q07820	MCL1	0.00067511	0.48974359	2
33	P04406	GAPDH	0.00067511	0.48974359	2
34	P01375	TNF	0.00116211	0.49100257	3
35	P11142	HSPA8	0.00067511	0.48974359	2
36	P20248	CCNA2	0.00234162	0.49354005	5
37	P78396	CCNA1	0.00234162	0.49354005	5
38	P24941	CDK2	0.00334262	0.49481865	6
39	P14635	CCNB1	0.00116211	0.49100257	3
40	P06493	CDK1	0.00116211	0.49100257	3
41	P11802	CDK4	0.00334262	0.49481865	6
42	P24385	CCND1	0.00234162	0.49354005	5
43	P11021	HSPA5	0.00067511	0.48974359	2
44	P60568	IL2	0.00067511	0.48974359	2
45	P19367	HK1	0.00067511	0.48974359	2
46	P52789	HK2	0.00067511	0.48974359	2
47	P30542	ADORA1	0.01223823	0.49481865	6
48	P0DMS8	ADORA3	0.0151816	0.49739583	8
49	P10275	AR	0.00067511	0.48974359	2
50	P35869	AHR	0.00111812	0.49100257	3
51	P35354	PTGS2	0.00546993	0.4961039	7
52	P18031	PTPN1	0.00140827	0.4961039	7
53	Q02750	MAP2K1	0.00184084	0.49100257	3
54	P29323	EPHB2	0.00028174	0.48974359	2
55	P04818	TYMS	0.00028174	0.48974359	2
56	O14672	ADAM10	0.00028174	0.48974359	2
57	P55072	VCP	0.00028174	0.48974359	2
58	P08238	HSP90AB1	0.00028174	0.48974359	2
59	P14625	HSP90B1	0.00028174	0.48974359	2
60	P50281	MMP14	0.00028174	0.48974359	2
61	P15144	ANPEP	0.00028174	0.48974359	2
62	AGTR1	AGTR1	0.00028174	0.48974359	2
63	P11387	TOP1	0.00023523	0.49100257	3
64	Q13547	HDAC1	0.00028174	0.48974359	2
65	P35462	DRD3	0.00028174	0.48974359	2
66	P14416	DRD2	0.00028174	0.48974359	2
67	O60760	HPGDS	0.00028174	0.48974359	2
68	P23443	RPS6KB1	0.00028174	0.48974359	2
69	Q05193	DNM1	0.00028174	0.48974359	2
70	P42345	MTOR	0.00028174	0.48974359	2
71	O14757	CHEK1	0.00028174	0.48974359	2
72	Q92769	HDAC2	0.00034993	0.48974359	2
73	P19838	NFKB1	0.00034993	0.48974359	2
74	P40763	STAT3	0.00045826	0.48974359	2
75	Q16236	NFE2L2	0.00045826	0.48974359	2
76	O15054	KDM6B	0.00045826	0.48974359	2
77	P42336	PIK3CA	0.00045826	0.48974359	2
78	P08684	CYP3A4	0.00045826	0.48974359	2
79	P42338	PIK3CB	0.00045826	0.48974359	2
80	P06239	LCK	0.00045826	0.48974359	2
81	O00206	TLR4	0.00045826	0.48974359	2
82	P14780	MMP9	0.00329356	0.49226804	4
83	P37840	SNCA	0.00066095	0.49100257	3
84	Q14289	PTK2B	0.00066095	0.49100257	3
85	P09619	PDGFRB	0.00066095	0.49100257	3
86	P08069	IGF1R	0.00023292	0.48974359	2
87	P07948	LYN	0.00066095	0.49100257	3
88	P31749	AKT1	0.00066095	0.49100257	3
89	P11388	TOP2A	0.00023292	0.48974359	2
90	P05771	PRKCB	0.00023292	0.48974359	2
91	P17612	PRKACA	0.00023292	0.48974359	2
92	O75469	NR1I2	0.00023292	0.48974359	2
93	P15559	NQO1	0.00023292	0.48974359	2
94	P23458	JAK1	0.00023292	0.48974359	2
95	P07900	HSP90AA1	0.00128134	0.49226804	4
96	Q99527	GPER1	0.00023292	0.48974359	2
97	P26358	DNMT1	0.00023292	0.48974359	2
98	Q16678	CYP1B1	0.00023292	0.48974359	2
99	Q00534	CDK6	0.00023292	0.48974359	2
101	P60709	ACTB	0.00023292	0.48974359	2
102	P08254	MMP3	0.00068484	0.49100257	3
103	P06400	RB1	0.00023292	0.48974359	2
104	P38936	CDKN1A	0.00023292	0.48974359	2
105	P10415	BCL2	0.00023292	0.48974359	2
106	P08473	MME	0.00074482	0.48974359	2
107	P29317	EPHA2	0.00127883	0.49100257	3
108	P00519	ABL1	0.00127883	0.49100257	3
109	P05556	ITGB1	0.00074482	0.48974359	2
110	P16109	SELP	0.00141277	0.49100257	3
111	P16581	SELE	0.00141277	0.49100257	3
112	P02766	TTR	0.00141277	0.49100257	3

Table 4

The topological parameter analysis of C-T-P for P. sinensis in treatment of rheumatoid arthritis.

Number	Node	Betweenness centrality (BC)	Closeness centrality (CC)	Degree centrality (DC)
1	trans-N-feruloyltyramine	0.0917	0.4471	21
2	umbelliferone	0.0481	0.4348	20
3	Caffeic acid	0.0351	0.4115	13
4	Esculetin	0.1328	0.4898	30
5	Scopolin	0.0547	0.4152	16
6	Ras signaling pathway	0.0225	0.4189	15
7	Chemokine signaling pathway	0.0244	0.4043	14
8	Rap1 signaling pathway	0.0380	0.4189	16
9	TNF signaling pathway	0.0460	0.4189	14
10	ErbB signaling pathway	0.0275	0.4227	16
11	Estrogen signaling pathway	0.0531	0.4227	17
12	PI3K-Akt signaling pathway	0.1271	0.4745	29
13	HIF-1 signaling pathway	0.0593	0.4387	19
14	MAP2K1	0.0379	0.4947	16
15	RPS6KB1	0.0057	0.3974	6
16	MTOR	0.0057	0.3974	6
17	NFKB1	0.0211	0.4471	11
18	PIK3CA	0.0331	0.4947	16
19	PIK3CB	0.0331	0.4947	16
20	PDGFRB	0.0044	0.4009	6
21	IGF1R	0.0064	0.4079	7
22	AKT1	0.0462	0.5054	18
23	PRKCB	0.0060	0.4009	8
24	PRKACA	0.0043	0.3875	6
25	HSP90AA1	0.0095	0.4306	6
26	CDKN1A	0.0071	0.4079	6
27	CASP3	0.0195	0.4115	8
28	PRKCA	0.0353	0.4346	12
29	KDR	0.0156	0.4152	9
30	SRC	0.0153	0.4189	10
31	GSK3B	0.0068	0.4079	7
32	EGFR	0.0445	0.4895	15
33	MAPK1	0.0558	0.5167	19
34	TNF	0.0185	0.4043	10
35	CDK2	0.0152	0.4471	8
36	CDK4	0.0172	0.4515	8
37	CCND1	0.0127	0.4266	8
38	PTGS2	0.0229	0.4387	9

Table 5

Relative organ weights in rats.

Data represent the mean ± SD.

Relative organ weight (g/100 g)	Normal	Model	Methotrexate (1 mg/kg)	Pse (0.6 g/kg)	Pse (0.3 g/kg)	Pse (0.15 g/kg)
Kidney	0.63±0.03	0.71±0.04##	0.71±0.06	0.75±0.03	0.72±0.05	0.69±0.06
Heart	0.32±0.04	0.33±0.03	0.35±0.06	0.34±0.03	0.33±0.03	0.34±0.05
Liver	3.50±0.25	3.07±0.15##	2.83±0.45	3.01±0.18	3.42±0.42*	3.17±0.43
Spleen	0.18±0.04	0.20±0.02	0.35±0.15**	0.20±0.03	0.20±0.03	0.19±0.02
Lung	0.53±0.07	0.67±0.12##	0.76±0.07	0.63±0.10	0.60±0.08	0.57±0.10

*P < 0.05 and

**P < 0.01 compared with model group

#P < 0.05 and

##P < 0.01 compared with normal group.