Cistanche promotes the adipogenesis of 3T3-L1 preadipocytes.

Cistanche deserticola Ma (cistanche) is a traditional herb with a wide range of therapeutic properties. However, no evidence of cistanche's effect on adipogenesis has been found. The effect of cistanche that promotes the adipogenesis of 3T3-L1 preadipocytes was proved by using MTT spectrophotometry, Nile Red staining, Oil Red O staining and transcriptome sequencing technology. The mRNA level of key transcription factors for adipogenesis such as PPAR, AP2 and LPL were examined by RT-PCR. The results showed that the intracellular lipid content in cistanche treated cells were notably increased when compared with the non-treated cells. Between the differentiation and cistanche treated groups, the expression of adipogenesis related genes such as grow hormone releasing hormone (Ghrp), BCL2/adenovirus E1B interacting protein 3 (Bnip3) and Gastric inhibitory polypeptide receptor (Gipr) were significantly increased. Our findings also verified that cistanche promoted adipogenesis, which was accompanied by up-regulated level of Bnip3 and PPAR. This study could uncover new signaling pathways involved in adipogenesis regulation.

Introduction

Adipocytes are a key component of adipose tissue, and their proliferation and differentiation are linked to the tissue’s function. Adipocyte metabolism is abnormal in a range of disorders, including obesity, nutritional insufficiency and diabetes [1]. The study of adipocytes in vitro has become a popular issue in recent years due to the rising prevalence of lipid metabolism disorders. The 3T3-L1 preadipocytes are one of the most often used cells to create adipocytes models, and under the right conditions, they can be differentiated into adipocyte cells [2]. Knowing the key factors and signaling pathways in the process of adipogenesis could lead to a more effective strategy for treating lipid metabolism problems. As a result, it is vital to investigate the critical components and signaling pathways involved 3T3-L1 preadipocyte adipogenesis.

Cistanche is a traditional Chinese medication that has been used to cure disease and maintain health for thousands of years [3]. Cistanche has been shown to reduce the level of malondialdehyde (MDA), which is formed during the lipid peroxidation process [4, 5]. And lipid peroxidation is the process of oxidation in lipids and finally results in cell damage [6]. Furthermore, a range of natural compounds in cistanche have been shown to affect lipid metabolism by regulating triglyceride (TG) and fatty acid levels [5, 7, 8]. Moreover, cistanche has been shown to stimulate the differentiation of bone mesenchymal stem cells (BMSCs) into osteoblasts [9]. Cistanche promoted the development of murine marrow-derived dendritic cells (BMDCs) in a considerable way [10]. Therefore, we postulated that cistanche could have the potential capacity to promote the differentiation of 3T3-L1 preadipocytes.

RNA sequencing (RNA-Seq) has become an effective method for large scale transcriptomic research. RNA-Seq was able to extract nearly all complementary sequences transcripts (cDNA) from cell RNA [11]. DEGs were analyzed using GO enrichment to analyze the biological processes, cellular components and molecular function in DEGs. The DEGs enrichment signaling pathway was investigated using the Kyoto Encyclopedia of Genes and Genomes (KEGG) Pathway [12]. Thus, specific components implicated in adipogenesis of cistanche treated 3T3-L1 cells could be obtained in our study using RNA-Seq technology [13, 14].

Materials and methods

Chemicals and reagents

Shandong Institute of Traditional Chinese Medicine was where Cistanche was obtained. Solebol Technology Co. LTD provided acid cellulose and acid pectinase. Nile red and Oil red O were purchased from Sigma (St. Louis, MO, USA). Dulbecco’s Modified Eagle’s Medium-Hight glucose (DMEM-H) and fetal bovine serum (FBS) were obtained from Gibco, lnc (Grand Island, NY). The following items were acquired from Sigma: isobutylmethylxanthine (IBMX), dexamethasone, insulin and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltertrazolium bromide(MTT)(St. Louis, Mo).

Preparation of cistanche

All plant experiments complied with the IUCN Policy Statement on Research Involving Species at Risk of Extinction and the Convention on the Trade in Endangered Species of Wild Fauna and Flora. The cistanche was divided into three different treatments: Boiled, Enzymed and Boiled with Enzymed. (1) Boiled ultramicro mill was used to crush cistanche into powder with a mesh greater 1500 mesh. The cistanche powder was dissolved in distilled water at a ratio of 1:9. After uniform stirring, the solution of cistanche was extracted by boiling at 100°C for 30 minutes. The supernatant was saved and suspended in a tenfold ethanol solution before being heat refluxed three times for 30minutes at 100°C. The sample was centrifuged at 10000 r/min and the supernatant was lyophilized for preservation. (2) Enzymed the cistanche was ultrafinely pulverized and dissolved in sterile water before being enzymed at 50°C for 2 hours with acid pectinase and acid cellulase (acid fiber and sour fruit amount accounted for 1 ‰ of solid). The sample was centrifuged at 10000 r/min and the supernatant was lyophilized for preservation. (3) Boiled with Enzymed after pulverized cistanche solution boiling at 100°C for 30 minutes, the boiled cistanche extraction was then done at 50°C for 2 hours by adding acid pectinase and acid cellulose. The supernatant was then saved and suspended in a tenfold ethanol solution, and the heat was refluxed three times for 30 minutes at 100°C. The sample was centrifuged at 10000 r/min and the supernatant was lyophilized for preservation.

3T3-L1 preadipocytes culture and differentiation

3T3-L1 preadipocytes (presented by professor Yanqing Li from Qilu hospital of China) were cultured in Dulbecco’s Modified Eagle’s Medium-Hight glucose (DMEM-H, Gibco, 12800–017) supplemented with 10% fetal bovine serum (FBS; v/v) (Hyclone, SV30087.02). Cells were kept at 37°C in a humidified atmosphere of 5% CO2 under standard conditions.

The 3T3-L1 preadipocytes were divided into 5 groups: the normal control group (Nor), the differentiation group (Differ), the boiled group (Boiled), the enzymed group (Enzymed) and the boiled with enzymed group (Boiled with Enzymed). The Boiled group, Enzymed group and Boiled with Enzymed group were respectively added with 0.5μg/ml of cistanche extraction into the induction medium [15].

3T3-L1 cells were seeded at a density of 1×105 cells/ml in a 6-well culture plate. Two days after cells reached confluence, Cell differentiation was then induced with 10% FBS DMEM contain MDI (0.5μM isobutylmethylxanthine IBMX, 5μM dexamethasone and 0.5 μg/ml insulin) with cistanche for 2 days. Next, the medium was replaced with 10% FBS DMEM containing 5 μg/ml insulin with cistanche for 2 days. Next, the differentiation medium was replaced with 10% FBS DMEM contains cistanche for another 2 days. On day 7 of the experiment, Oil red O and Nile Red staining was performed, and the triglyceride, lipoprotein and lipid of intracellular could be stained into jacinth and red by ORO and Nile Red staining.

Cell viability assay

3-[4,5-dimethylthiazol-2-yl -2,5-diphenyltertrazolium bromide (MTT) assay was used to assess cell viability in response to cistanche. Briefly, 3T3-L1 preadipocytes were seeded in 96-well plates at a density of 5 × 104 cells/100 μl and treated with 0.05, 0.5 and 1μg/ml concentration of different cistanche extraction. After 24h and 48h, the culture media was added 20 μl MTT reagent to each well for another 4 h at 37°C. The absorbance was measured using a SpectraMax ABS microplate spectrophotometer (Molecular Devices, USA).

Oil red O staining

3T3-L1 preadipocytes were washed with PBS and fixed in 10% formaldehyde in PBS for 1 hour. Then cells were washed with 60% isopropanol and stained with working solution oil red for 10 minutes. The stained cells were washed four times with double distilled water and photographed under LSM 510 inverted microscopy. In order to determination the lipid content in 3T3-L1 adipocytes, cells were dissolved in isopropanol and the absorbance values of eluates were measured using a SpectraMax ABS microplate spectrophotometer (Molecular Devices, USA).

Nile red staining

3T3-L1 preadipocytes were fixed with 4% paraformaldehyde for 20 minutes, and then the cells were stained with NR for 10 minutes. The stained cells were washed three times with phosphate-buffered saline (PBS). The images of stained lipid droplets were taken using fluorescence microscope BZ-ZX700 (Keyence, Osaka, Japan). To quantify the lipid content, cells were dried and isopropanol was added, Absorbance values of eluates were measured using a SpectraMax ABS microplate spectrophotometer (Molecular Devices, USA) at 520 nm wavelength [16].

Quantitative real-time RT-PCR (RT-qPCR)

Total RNA was extracted from 3T3-L1 preadipocytes by Trizol (Invitrogen, USA), and RNA was reverse transcribed into cDNA using an ABScript II RT Mix (ABclonal, Wuhan, China). The cDNAs were subjected to qRT-PCR using primer pairs 5′-AGATCATTTACACAATGCTGGC-3′ and 5′-TAAAGTCACCAAAAGGCTTTCG-3′ for PPAR, 5′-CATCCGGTCAGAGAGTACTTTT-3′ and 5′- TAGGGTTATGATGCTCTTCACC-3′ for AP2, 5′-CCTGATGACGCTGATTTTGTAG-3′ and 5′-CAATGAAGAGATGAATGGAGCG-3′ for LPL, 5′-CCACTAACGAACCAAGTCAGAC-3′ and 5′-CATCTCTGCTGCTCTCTCAT-3′ for Bnip3, 5′-TGTGTCCGTCGTGGATCTGA-3′ and 5′-TTGCTGTTGAAGTCGCAGGAG-3′ for GAPDH. (Sangon Biotech Co., Shanghai, China). The mRNA expressions levels were examined on Rotor-Gene Q instrument (QIAGEN, Shanghai, China) using a SYBR Green Fast qPCR Mix (ABclonal, Wuhan, China) to evaluate the amount of double stranded DNA. The purity and concentration of isolated RNA was determined using NanoDrop 1000 (NanoDrop Technologies, Wilmington, DE, USA). The primer amplification efficiencies were measured in cDNA dilutions from 10 to 105. And the amplification was linear over the range of 10 to 105. The standard curves efficiencies of the primers ranged from 97% to 102%. The thermal cycling included 1 cycle at 95°C for 30 s, 40 cycles at 58°C for 30 s, at 72°C for 30 s, and the final cycle at 72°C for 5min. LPL, AP2, PPAR and Bnip3 gene expression levels were calculated using the normalized relative quantification method followed by the ΔΔCT method.

Transcriptome profiling by RNA-Seq

Total RNA was isolated from 3T3-L1 preadipocytes using the TRIzol reagent (Invitrogen, Beijing, China) according to the manufacturer protocols. Equal amounts of total RNA samples were collected from 5 groups: the Nor group, Differ group, Boiled group, Enzymed group and Boiled with Enzymed group. The RNA samples were extracted from each group and different parallel set was obtained. Transcriptome sequence (RNA-Seq) was performed by HuaDa Gene Company.

Statistical analysis

Data were analyzed using Excel and Graph Pad Prism and presented as mean ± SEM. Differences between Nor, Differ, Boiled, Enzymed or Boiled with Enzymed groups were analyzed by one-way analysis of variance (ANOVA) with use of SPSS v11.5 (SPSS Inc., Chicago, IL). P values less than 0.05 were considered as significant.

Results

Effect of cistanche extraction on 3T3-L1 preadipocytes cell viability

To explore the cytotoxic effects of cistanche extraction on 3T3-L1 preadipocytes, cell viability was measured by using the MTT assay (Fig 1A). Our results showed that cistanche at 0.5μg/ml had no cytotoxic effect on 3T3-L1 preadipocytes after 24h and 48h treatments (P<0.05, P<0.01, P<0.001). Therefore, the concentration of cistanche at 0.5μg/ml was used in the Boiled, Enzymed and Boiled with Enzymed groups in the next research. The 3T3-L1 preadipocytes could be differentiated in induction medium containing FBS, IBMX, dexamethasone and insulin. The results indicated that the induction medium in differ group could increase the viability of 3T3-L1 preadipocytes by 7 day. Meanwhile, the cell viability in the Boiled group also showed a significant increase compared to that in the Nor group. However, the cell viability exhibited no significant differences between differ and cistanche-treated groups (Fig 1B) (P<0.05, P<0.01).

Fig 1

The cell viability and cell proliferation in 3T3-L1cells after treated with cistanche.

(A) 3T3-L1 preadipocyte cells were treated with boil, enzyme and boil with enzyme of cistanche for 24h and 48h. (B) 3T3-L1 preadipocyte cells were pretreated in differentiation medium with cistanche for 7days. (Data are expressed as means ± S.E. *p< 0.05, **p< 0.01, ***p< 0.001. Experiments were performed in three repetitions).

Cistanche increased lipid accumulation in 3T3-L1 preadipocytes cells

The ORO and NR stainings were used to detect the intracellular lipid droplets in 3T3-L1 preadipocytes treated with cistanche extraction to investigate the influence of cistanche on adipogenesis. The intracellular lipid deposition was significantly increased in the Differ, Boiled and Boiled with Enzymed groups, according to the results of ORO staining (Fig 2A). In comparison to the Differ group, the levels of lipid accumulation were significantly increased in Boiled and Boiled with Enzymed group (Fig 2A–2f) (P<0.01, P<0.001). According to the results of NR staining, the lipid droplets are demonstrated in red color. The stained lipid droplets were dissolved in 100% isopropanol and the absorbance at 520nm was measured to determine their quantification. The accumulation of intracellular lipids was significantly increased in the group of Boiled and Boiled with Enzymed (Fig 2B). These results suggested that the cistanche extraction could promote the accumulation of intracellular lipids, and the cistanche of Boiled group and Boiled with Enzymed group exhibited better effects (P<0.01, P<0.001).

Fig 2

Representative images of ORO-stained and Nile Red-stained 3T3-L1 cells differentiated.

(A) ORO staining of differentiated 3T3-L1 adipocytes. (a) Normal group. (b) 3T3-L1 cells were treated with differentiation medium. (c) 3T3-L1 cells were treated with cistanche of Boiled. (d) 3T3-L1 cells were treated with cistanche of Enzymed. (e) 3T3-L1 cells were treated with cistanche of Boiled with Enzymed. (f) Lipid accumulation was quantified by measuring the absorbance at 520 nm. (Data are expressed as means ± S.E.) The lipid drops was stained in red and nucleus was stained in blue, and the more red dots in the figure, the more lipid accumulation (B) NR staining of differentiated 3T3-L1 adipocytes. The lipid droplets are demonstrated in red color (a) Normal group. (b) 3T3-L1 cells were treated with differentiation medium. (c) 3T3-L1 cells were treated with cistanche of Boiled. (d) 3T3-L1 cells were treated with cistanche of Enzymed. (e) 3T3-L1 cells were treated with cistanche of Boiled with Enzymed. (f) Lipid accumulation was quantified by measuring the absorbance at 520 nm. (Data are expressed as means ± S.E. **p< 0.01, ***p< 0.001. Experiments were performed in three repetitions).

RNA-seq analysis

RNA-seq analysis was performed to explore the mechanism of cistanche promotion the accumulation of intracellular lipids in 3T3-L1 preadipocyte cells. We calculated the pairwise Pearson correlation coefficients of FPKM values between samples. And the heat map was produced [17]. The results showed that these samples were highly correlated and did not have larger difference in the same group. The experimental groups were separated from control group [18] (Fig 3).

Fig 3

The Pearson correlation coefficients of all gene expressions.

Pearson correlation coefficients were used to describe correlations between control and experimental group. The Pearson correlation coefficients of all gene expressions between each two samples were compared. The higher the correlation coefficient was, the more similar the gene expression level was. And the correlation coefficient values above 0.995 are presented as red and yellow, the correlation coefficient values above below 0.995 are indicated in blue. These samples were highly correlated and did not have larger difference in the same group, and cistanche experimental group were separated from control group.

As shown in the Venn diagram (Fig 4), 188, 231, 251, 220 and 233 genes were respectively detected in Nor, Differ, Boiled, Enzymed and Boiled with Enzymed groups (Fig 4A). A total of 14251 co-expressed genes were found between the Differ and experimental groups (Fig 4B). 368, 11 and 44 differential genes were respectively detected between Differ versus Boiled group, Differ versus Enzymed group, Differ versus Boiled with Enzymed group (Fig 4C). Among these detected differential gens, 9 genes were differentially expressed among Differ and experimental groups.

Fig 4

Venn diagram shows the overlap of control and experimental group.

(A) 188, 231, 251, 220 and 233 genes were respectively detected in Nor, Differ, Boiled, Enzymed and Boiled with Enzymed group. (B) 14251 co-expressed genes were found between the Differ and experimental groups. (C) 368, 11 and 44 differential genes were respectively detected between Differ versus Boiled group, Differ versus Enzymed group, Differ versus Boiled with Enzymed group.

These differentially expressed genes were shown in the volcano plot, which the red colors represented the up-regulated genes, and the green colors represented the down-regulated genes (Fig 5). Comparison of 368 diff-expressed genes revealed 312 genes up-regulated and 56 genes down-regulated by setting the significance and fold-change (P<0.05 and log2 fold change>|1|) (Fig 5A). Comparison of 11 diff-expressed genes revealed 10 genes up-regulated and 1 genes down-regulated by setting the significance and fold-change (P<0.05 and log2 fold change >|1|) (Fig 5B). Comparison of 44 diff-expressed genes revealed 40 genes up-regulated and 4 genes down-regulated by setting the significance and fold-change (P<0.05 and log2 fold change >|1|) (Fig 5C).

Fig 5

Volcano plot shows the up or down regulated genes between control and experimental group.

(a) 312 genes up-regulated and 56 genes down-regulated between Boiled versus Differ. (b) 10 genes up-regulated and 1 gene down-regulated between Enzymed versus Differ. (c) 40 genes up-regulated and 4 genes down-regulated between Boiled with Enzymed versus Differ. (P<0.05 and log2 fold change>|1|).

Enrichment analysis of the Gene Ontology (GO) terms of the DEGs functional

In order to analysis the biological relationship of DEGs in adipocytes of cistanche treated cells. We performed GO enrichment analysis of DEGs. The GO enrichment histogram directly showed the quantity distribution of different expressed genes in biological processes, cellular components and molecular function (Fig 6). Within the biological processes category, most DEGs were associated with cellular process and metabolic process. In the cellular component category, the majority of DEGs were related with cell part and organelle part. In the molecular function category, most DEGs were associated with catalytic activity and transcription regulator activity.

Fig 6

Go analysis of DEGs.

The figure is consisting of molecular functions, cellular components and biological process. The significance level of enrichment was set at corrected P-value. (P<0.05 log2 fold change>|1|) (A) Differ versus Boiled group. (B) Differ versus Enzymed group. (C) Differ versus Boiled with Enzymed group.

Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis of DEGs

The top 20 significant pathways of DEGs were selected and represented in KEGG enrichment scatter diagram (Fig 7). DEGs were mainly enriched in metabolic pathways, the Biosynthesis of secondary metabolites pathway and the Galactose metabolism pathway [19]. On the other hand, the down regulated genes were mainly enriched in the tumor necrosis factor (TNF) signaling pathway [20], forkhead box O (FoxO) signaling pathway [21], and other fundamental biochemical process, such as Glycolysis pathway [22], Biosynthesis of amino acids signaling pathway and Glucagon signaling pathway [23].

Fig 7

KEGG pathway analysis of DEGs between Differ, Boiled, Enzymed, Boiled with Enzymed group.

The figure shows top 20 pathways in a total number of enrichment of DEGs in signaling pathway. Size and color of bubble represent amount of DEGs enriched in pathway and enrichment significance. (A) Differ versus Boiled group. (B) Differ versus Enzymed group. (C) Differ versus Boiled with Enzymed group. (P<0.05 and log2 fold change>|1|).

Analysis of key transcription factors for adipogenesis

To validate changes in gene expression patterns, we examined the effect of cistanche on mRNA expression of important transcription factors such as AP2, LPL and PPAR, which are considered as adipogenic markers. The data showed that the levels of AP2, LPL and PPAR were significantly increased in Differ group. Compared with those in Differ group, the level of PPAR was significantly up-regulated in Boiled group. And in the Boiled with Enzymed group, levels of PPAR, AP2 and LPL were significantly higher than that in the Differ group (Fig 8) (P<0.01). These experimental results suggested that the mRNA levels of AP2, LPL and PPAR were increased in ciatanche of Boiled with Enzymed group.

Fig 8

Analysis of key transcription factors.

The mRNA levels of three key transcription factors AP2(A), LPL(B), and PPAR(C) Bnip3(D) in 3T3-L1 adipocytes were detected. Data are expressed as means ± S.E. *p< 0.05, **p< 0.01 and ***p< 0.001. Nor: Normal group. Differ: 3T3-L1 cells were treated with differentiation medium. Boiled: 3T3-L1 cells were treated with cistanche of Boiled. Enzymed: 3T3-L1 cells were treated with cistanche of Enzymed. Boiled with Enzyme: 3T3-L1 cells were treated with cistanche of Boiled with enzymed.

Identify and verify the key genes in the regulation of adipogenesis

In order to identify the candidate key genes that potentially regulates adipogenesis of 3T3-L1 cells with cistanche [24], we compared the DEGs expression changes between control and experimental group. In comparison the Nor versus Differ group and Differ versus experimental group, the mRNA level of Bnip3 gene was all significantly increased (S1 Fig). And the mRNA level of Bnip3 also up-regulated in Boiled group, which is in line with the results of RNA-sequences (Fig 8D) (P<0.05). The results suggested that the accumulation of lipid in cistanche treated 3T3-L1 cell might be related to Bnip3.

Discussion

Cistanche is a very valuable herb in Chinese traditional medicine [25]. Until now, a large number of biologically active substances in cistanche have been proved [6]. The polysaccharides of cistanche could modulate the lipid metabolism by regulating triglyceride (TG) and fatty acid [4]. And the echinacoside of PhGs could affect the leptin biosynthesis pathway by regulating the gene of SOCS-3 [5]. Therefore, we indicated that cistance has an effect on adipogenesis, and it was selected to explore the accumulation of lipid in 3T3-L1 preadipocyte. Previous studies has showed that cistanche has a wide range of possible effects on lipid peroxidation and lipid accumulation, but its regulatory effects and mechanism on 3T3-L1 adipocytes are unknown. We expounded the mechanism of cistanche promoted the adipogenesis from the droplet formation and genetic level, which may help bridge this gap. Moreover, our present study also demonstrated that the levels of key transcription factors and DEGs were significantly increased in cistanche treated group, which help us to understanding the various adipocyte specific genes in the process of adipogenesis. This research has advanced our understanding of adipogenesis mechanisms while also demonstrating a potential target for adipogenesis regulation.

The process of adipogenesis is accompanied by altered expression of various transcription factors and adipocyte specific genes. Acid-binding protein (AP2), and Lipoprotein lipase (LPL) and Peroxisome proliferator activated receptor gamma (PPAR) are the key transcription factors in the process of fat synthesis. To further characterize the effect of cistanche on adipogenesis, we examined the expression of these key transcription factors using quantitative real-time PCR. We found that the expression of AP2, LPL and PPAR were up-regulated in differ group, which is consistent with the previous results. It was showed that the mRNA levels of these key transcription factors were also increased in cistanche treated cells, and the cistanche of Boiled with Enzymed group exhibited better (P<0.05, P<0.01), which to verify the results of ORO and NR stainings. It is might be that cistanche could be decomposed thoroughly by Boiled and enzyme, and the active ingredients of cistanche could be fully promoted in the group of Boiled with Enzymed. Therefore, the selection of extraction method is very important in future investigation of cistanche, and different extraction method may play different functional effects.

Meanwhile, the RNA-seq technology was used to explore the DEGs and gene expression patterns in control and cistanche treated 3T3-L1 cells. In additionally, our results showed that the mRNA levels of Bnip3 were up-regulated in cistanche treated group. A relatively studies have reported that Bnip3 encodes the mitophagy signaling pathway, which is associated with the lipid metabolism in the liver [26]. Additionally, Bnip3 is essential for mitochondrial bioenergetics during adipocyte remodeling, and previous study has addressed that Bnip3 expression positively relates with adipose storage capacity [27]. Moreover, the latest studies have indicated that Bnip3 is a key effector of PPAR mediated adipose expression, and Bnip3-/- mice lead to systemic metabolic dysfunction [28]. Our findings suggest that cistanche promotes adipogenesis in 3T3-L1 cells, which is accompanied by up-regulated mRNA expression of Bnip3 and PPAR signaling pathway. Moreover, the Biosynthesis of secondary metabolites pathway and the Galactose metabolism pathway were primarily enriched in the top 20 significant DEGs pathways. These signaling pathways are closely related to the accumulation of lipids.

Conclusion

In conclusion, we discovered that cistanche could promote the adipogenesis of 3T3-L1 preadipocytes. The genomic changes were analyzed using transcriptome sequencing technology. The transcriptome analysis revealed that the Differ and experimental groups had 14251 co-expressed genes. Between the Differ versus Boiled group, Differ versus Enzymed group, Differ versus Boiled with Enzymed group Differ vs Boiled group, 368, 11 and 44 differential genes were detected, respectively. And DEGs were mainly enriched in adipogenesis-related pathways such the insulin signaling pathway and AMP-activated protein kinase (AMPK) signaling pathway. Moreover, in the cistanche-treated group, the mRNA levels of key transcription factors for adipogenesis such as PPAR, AP2, LPL and DEGs Bnip3 were up-regulated. These findings provide new insight into the mechanism by which cistanche promote the adipogenesis of 3T3-L1 preadipocyte and identify Bnip3 as a new molecular target for adipogenesis.

The heatmap of important DEGs associated with adipogenesis.

DEGs were presented in heatmap. Red indicates high expression genes, while blue indicates low expression genes. Color changing from red to blue indicate that log 10 (FPKM+1) gradually changes from big to small. Nor: Normal group. Differ: 3T3-L1 cells were treated with differentiation medium. Boiled: 3T3-L1 cells were treated with cistanche of Boiled. Enzymed: 3T3-L1 cells were treated with cistanche of Enzymed. Boiled with Enzyme: 3T3-L1 cells were treated with cistanche of Boiled with enzymed.

(TIF)

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The 3T3-L1 cell under optical microscope.

The figure of 3T3-L1 cells in pre-staining. (A) Normal group. (B) 3T3-L1 cells were treated with differentiation medium. (C) 3T3-L1 cells were treated with cistanche of Boiled. (D) 3T3-L1 cells were treated with cistanche of Enzymed. (E) 3T3-L1 cells were treated with cistanche of Boiled with Enzymed.

(TIF)

Click here for additional data file.

(TIF)

Click here for additional data file.

14 Oct 2021

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Reviewer #1: The authors have shown the effect of Cistanche on adipogenesis. Although the work is novel, the experimental details, the representation of results and the discussion don’t meet the publication criteria as of now. However, the authors can consider revising and improve the manuscript.

Major comments

1.The English needs to be improved throughout the text. The figure numbers need to be uniform. The figure legends should be detailed and explanatory. Example Figure 2A should mention the details of all the stains used. Figure 3 showing RNA sequencing result should be detailed.

2. Figure a in both Figure 2 A and 2B representing the normal group shows very less cell numbers as compared to the treated group. As the cell number is different in each group, the quantification of lipid accumulation in terms of absorbance will not confirm the increase in lipid content. Either fixed cell number or amount of protein needs to be considered during final calculation.

Further, figure legend or material section doesn’t show the time of incubation in different treatments.

3. Both RNA sequencing results along with the Enrichment analysis needs to be explained properly in the result section.

5. As the authors mentioned about the expression of Ghrp, Bnip3 and Gipr genes were significantly increased, representation of selected genes with a heatmap can emphasize the difference.

4. Authors should rewrite the discussion while properly discussing the outcomes and the significance of the outcomes. The pathways which significantly changed with the treatment of Cistanche needs to be emphasized properly.

The work in the manuscript is novel and contains a large amount of data from RNA sequencing. However, the data representation can be organized and improved in both result and discussion section to show the novelty and importance of the work.

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Reviewer #1: No

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30 Dec 2021

Dear Editor and Reviewers,

Thank you for your letter and for the reviewers’ comments concerning our manuscript entitled “Cistanche promotes the adipogenesis of 3T3-L1 preadipocytes” (PONE-D-21-25814). The comments are all valuable and very helpful for revising and improving our paper, as well as the important guiding significance to our researches. We have read the comments carefully and made corrections which we hope meet with approval. Revised portions are marked in shadow in our revised manuscript.

Editor comments：

1. There are not any mRNA level of key trancription factors for adipogenesis such as PPAR aP2, LPL, were shown with any treatment only Nile Red staining and Oil Red O staining were shown in the result. Also there are not any validation of RNA-seq data by qPCR which should be included in the results.

We appreciate constructive comments from the associate editor. We have examined the mRNA level of key transcription factors for adipogenesis such as PPAR, AP2 and LPL (Fig 8). Additionally, to validate changes in gene expression patterns, we identified and verified the expression level of Bnip3, a gene related to adiopogenesis by using RT-PCR. The results indicated that cistanche promoted adipogenesis, which was accompanied by up-regulated level of Bnip3 and PPAR in Boiled group. We tried our best to improve the manuscript and made some changes in the manuscript. These changes will not influence the content and framework of the paper. And here we did not list the changes but marked in shadow in our revised manuscript. We hope that the correction will meet with approval. Once again, thank you very much for your comments and suggestions.

Reviewer Comments:

1. The English needs to be improved throughout the text. The figure numbers need to be uniform. The figure legends should be detailed and explanatory. Example Figure 2A should mention the details of all the stains used. Figure 3 showing RNA sequencing result should be detailed.

Response: The English has been improved throughout the text. We really hope that the flow and language level have been substantially improved. The figure numbers are uniformed and the figure legends are enriched in revised manuscript. Additionally, Fig 2A was the Oil Red O staining of differentiated 3T3-L1 adipocytes. The lipid drops were stained in red and nucleus was stained in blue, and the more red dots in the figure, the more lipid accumulation. Fig 3 was the Pearson correlation coefficients of all gene expression. Pearson correlation coefficients of all gene expressions between each two samples were compared. The higher the correlation coefficient was, the more similar the gene expression level was. And the correlation coefficient values above 0.995 are presented as red and yellow, the correlation coefficient values below 0.995 are indicated in blue. These samples were highly correlated and did not have larger difference in the same group, the cistanche experimental group were separated from control. These figure legends were enriched in the revised manuscript.

2. Figure are in both Figure 2 A and 2B representing the normal group shows very less cell numbers as compared to the treated group. As the cell number is different in each group, the quantification of lipid accumulation in terms of absorbance will not confirm the increase in lipid content. Either fixed cell number or amount of protein needs to be considered during final calculation. Further, figure legend or material section doesn’t show the time of incubation in different treatments.

Response: Thank you for your careful review and constructive suggestion regarding our manuscript. Firstly, the number of cells is uniformed in a 6-well culture plate at a density of 1×105 cells/ml in normal and treated group following previously established standard culture system [1]. Additionally, the number of cells in the Differ group is appropriate, and the comparison between the experimental group and Differ control group will be more reasonable. Moreover, Oil red O and Nile red stainings were performed to exhibit the amount of lipid droplets rather than the cell number, The triglyceride, lipoprotein and lipid of intracellular could be stained into jacinth and red by ORO and Nile red staining. Thus, the quantification of triglyceride, lipoprotein and lipid are included in terms of absorbance in final calculation. We have enriched the figure of pre-staining cells in supplement materials, which could present the cell status and number (S2 Fig). As for the time of incubation, two days after cells reached confluence, cell differentiation was then induced with 10% FBS DMEM contain MDI (0.5μM isobutylmethylxanthine IBMX, 5μM dexamethasone and 0.5 μg/ml insulin) with cistanche for 2 days. Next, the medium was replaced with 10% FBS DMEM containing 5 μg/ml insulin with cistanche for 2 days. Next, the differentiation medium was replaced with 10% FBS DMEM contains cistanche for another 2 days. We added this information in the material section in the revised manuscript.

3. Both RNA sequencing results along with the Enrichment analysis needs to be explained properly in the result section.

Response: We have found this suggestion very useful to confirm the experimental results. However, the analysis results we got from the sequencing HuaDa Gene Company is about the differential genes (DEGs) of the experimental groups. The company did not provide us the Enrichment data. Moreover, in many research articles, differential gene RNA transcriptome sequencing is mainly used to analyze DEGs in different groups [2, 3]. Thus, the Enrichment analysis could not be provided in the manuscript.

4. As the authors mentioned about the expression of Ghrp, Bnip3 and Gipr genes were significantly increased, representation of selected genes with a heatmap can emphasize the difference.

Response: We thank the reviewer for this comment. We have re-analysis the data among these DEGs, and the important DEGs were presented in heatmap (S1 Fig). We re-analysed the comparison between Differ and cistanche treated groups. The level of Bnip3 was significantly increased in all comparison of Differ and cistanche treated group. To validate changes in gene expression patterns, we identified and examined the expression level of Bnip3, a gene related to adiopogenesis by using RT-qPCR. The data showed that the level of Bnip3 was significantly increased in Boiled group. Additionally, the results of key transcription factors for adipogenesis such as PPAR, aP2 and LPL were measured. The results indicated that cistanche promoted adipogenesis, which was accompanied by up-regulated levels of Bnip3 and PPAR in Boiled group. We added these results in the revised manuscript.

5. Authors should rewrite the discussion while properly discussing the outcomes and the significance of the outcomes. The pathways which significantly changed with the treatment of Cistanche needs to be emphasized properly.

Response: We have enriched the outcomes and the significance of the outcomes in the discussions in the revised manuscript. Cistanche is a traditional herb with a wide range of medicinal properties. Previous studies has showed that cistanche has a wide range of possible effects on lipid peroxidation and lipid accumulation, but its regulatory effects and mechanism on 3T3-L1 adipocytes are unknown. We expounded the mechanism of cistanche promoted the adipogenesis from the droplet formation and genetic level, which may help to bridge this gap. Moreover, in the cistanche-treated group, the mRNA levels of key transcription factors for adipogenesis such as PPAR, AP2, LPL and DEGs Bnip3 were up-regulated. These findings provide new insight into the mechanism by which cistanche promote the adipogenesis of 3T3-L1 preadipocyte and identify Bnip3 as a new molecular target for adipogenesis.

In additionally, our results showed that the mRNA levels of Bnip3 were up-regulated in cistanche treated group. Studies have reported that Bnip3 encodes the mitophagy signaling pathway, which is associated with the lipid metabolism in the liver [4]. Additionally, Bnip3 is essential for mitochondrial bioenergetics during adipocyte remodeling and relates with adipose storage capacity [5]. Moreover, the latest studies have indicated that Bnip3 is a key effector of PPAR mediated adipose expression, and Bnip3-/- mice lead to systemic metabolic dysfunction [6]. Our findings suggested that cistanche promotes adipogenesis in 3T3-L1 cells, which was accompanied by up-regulated mRNA expression of Bnip3 and PPAR signaling pathway. Moreover, the Biosynthesis of secondary metabolites pathway and the Galactose metabolism pathway were primarily enriched in the top 20 significant DEGs pathways. These signaling pathways are closely related to the accumulation of lipids. We have added these discussions in the revised manuscript.

References

1. Han JH, Jang KW, Park MH, Myung CS. Garcinia cambogia suppresses adipogenesis in 3T3-L1 cells by inhibiting p90RSK and Stat3 activation during mitotic clonal expansion. Journal of cellular physiology. 2021;236(3):1822-39. Epub 2020/07/28. doi: 10.1002/jcp.29964. PubMed PMID: 32716094.

2. Xin Y, Li C, Guo Y, Xiao R, Zhang H, Zhou G. RNA-Seq analysis reveals a negative role of MSMO1 with a synergized NSDHL expression during adipogenesis of 3T3-L1. Biosci Biotechnol Biochem. 2019;83(4):641-52. Epub 2018/12/26. doi: 10.1080/09168451.2018.1559719. PubMed PMID: 30582412.

3. Yun J, Jin H, Cao Y, Zhang L, Zhao Y, Jin X, et al. RNA-Seq Analysis Reveals a Positive Role of HTR2A in Adipogenesis in Yan Yellow Cattle. International journal of molecular sciences. 2018;19(6). Epub 2018/06/15. doi: 10.3390/ijms19061760. PubMed PMID: 29899319; PubMed Central PMCID: PMCPMC6032390.

4. Zhang Y, Wang Y, Wang X, Ji Y, Cheng S, Wang M, et al. Acetyl-coenzyme A acyltransferase 2 promote the differentiation of sheep precursor adipocytes into adipocytes. Journal of cellular biochemistry. 2018. Epub 2018/11/30. doi: 10.1002/jcb.28080. PubMed PMID: 30485515.

5. Choi JW, Jo A, Kim M, Park HS, Chung SS, Kang S, et al. BNIP3 is essential for mitochondrial bioenergetics during adipocyte remodelling in mice. Diabetologia. 2016;59(3):571-81. Epub 2015/12/24. doi: 10.1007/s00125-015-3836-9. PubMed PMID: 26693709.

6. Tol MJ, Ottenhoff R, van Eijk M, Zelcer N, Aten J, Houten SM, et al. A PPARγ-Bnip3 Axis Couples Adipose Mitochondrial Fusion-Fission Balance to Systemic Insulin Sensitivity. Diabetes. 2016;65(9):2591-605. Epub 2016/06/22. doi: 10.2337/db16-0243. PubMed PMID: 27325287; PubMed Central PMCID: PMCPMC5001173.

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17 Feb 2022

Cistanche promotes the adipogenesis of 3T3-L1 preadipocytes

PONE-D-21-25814R1

Dear Dr. Zhao,

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 but I highly recommend to provide better microscopic images for the final publication which also a reviewer concern.

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,

Abdul Qadir Syed, PhD

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

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 #1: All comments have been addressed

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Reviewer #1: Yes

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

Reviewer #1: Yes

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Reviewer #1: Yes

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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 #1: Yes

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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 #1: Authors have answered all the comments raised. High Res microscopic images can be provided for better visualization.

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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.

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Reviewer #1: No

21 Feb 2022

PONE-D-21-25814R1

Cistanche promotes the adipogenesis of 3T3-L1 preadipocytes

Dear Dr. Zhao:

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.

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on behalf of

Dr. Abdul Qadir Syed

Academic Editor

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