Astragalus polysaccharide alleviates LPS-induced inflammation injury by regulating miR-127 in H9c2 cardiomyoblasts.

Astragalus polysaccharide (APS) has been widely reported to play an important role in inflammatory response. In this study, we aimed to explore the effects and underlying mechanisms of APS on lipopolysaccharide (LPS)-induced inflammation injury in H9c2 cardiomyoblasts. H9c2 cells were treated with different concentrations of APS, and cell viability was detected by the Cell Counting Kit-8 (CCK-8) assay. Then, the effect of APS on cell viability and apoptosis induced by LPS was determined by CCK-8, flow cytometry, and western blot. The expression and release of inflammatory cytokines were evaluated by quantitative real-time polymerase chain reaction (qRT-PCR), western blot, and enzyme-linked immunosorbent assay (ELISA). Furthermore, expression of miR-127 in H9c2 cells was analyzed by qRT-PCR, and knocked down by transfection with miR-127 inhibitor. Western blot was used to analyze signaling pathway molecules. APS had no effect on H9c2 cells viability. However, APS could alleviate LPS-induced inflammation injury by increasing cell viability, reducing apoptosis, and inhibiting release of inflammatory cytokines in H9c2 cells ( P < 0.05). Additionally, we found that APS increased toll-like receptor 4 (TLR4) expressions in LPS-treated H9c2 cells. Mechanistically, we found that APS exerted the protective effect by down-regulating LPS-increased expression of miR-127 ( P < 0.05), inhibiting nuclear factor kappa B (NF-κB), JNK and promoting phosphoinositide 3-kinase/protein kinase B (PI3K/AKT) signaling pathways in LPS-treated H9c2 cells. The results demonstrated that APS could protect H9c2 cells against LPS-induced inflammation injury, which might be partially due to miR-127 down-regulation and regulation of NF-κB, JNK, and PI3K/AKT signaling pathways. These findings indicated that APS might be a potential therapeutic drug for treatment of myocarditis.

Introduction

Myocarditis, also known as inflammatory cardiomyopathy, is a kind of limited or diffused inflammatory lesion of the myocardium with a wide range of symptoms in children and adults.[1] Viral infection is the most common cause of myocarditis in developed countries, and other etiologies include bacterial infections, toxins, drug reactions, and autoimmune diseases.[2] Clinically, the mild patients have no obvious symptoms; however, severe patients may suffer from heart failure or even sudden death.[3] Currently, medications, intravenous immunoglobulin, implantable cardiac defibrillator, and heart transplant were used for treatment of myocarditis in the different phases.[4] More importantly, recent studies have reported that several herbal medicines could improve symptoms, ventricular premature beat, level of myocardial enzymes, and cardiac function in myocarditis.[5,6] These findings have aroused strong interest to explore the effect of traditional herbal medicine on myocarditis.

Traditional herbal medicine as an alternative and supplemental medicine has been widely used to treat various diseases in China, Japan, and other Asian countries.[7] Astragalus membranaceus, a commonly used Chinese medicinal plant, contains polysaccharides, saponins, flavonoids, amino acids, and other components to promote antibody production and immune response.[8] Astragalus polysaccharide (APS) is one of the components derived from Astragalus membranaceus. It has been reported to exert anti-tumor immune response and confer sensitivity of tumor cells to chemotherapy with fewer side effects.[9] Additionally, APS could ameliorate muscle wasting and increase the expression of phosphorylated protein kinase B (AKT) in insulin-resistant skeletal muscle.[10] Recently, accumulating evidences indicate that APS has anti-viral and anti-bacterial properties which enable to be used as an immune booster.[11] Furthermore, several studies demonstrated that APS could inhibit the expression of inflammatory factors and inflammation injury. For example, Lu et al.[12] reported that APS effectively ameliorated palmitate-induced pro-inflammatory responses through AMP-activated protein kinase (AMPK) activity. Wang et al.[13] showed that APS had anti-inflammatory and structure protective properties for lipopolysaccharide (LPS)-infected Caco2 cells. However, the effects and underlying mechanisms of APS on LPS-induced inflammation injury in H9c2 cardiomyoblasts remain elusive.

In this study, we aimed to explore the effect of APS on myocarditis. A model of LPS-induced inflammation injury in H9c2 cells was constructed. We found that APS had no effect on H9c2 cells viability. However, APS could attenuate LPS-induced impairment of H9c2 cells. The protective functions of APS on H9c2 cells might be through suppressing miR-127 expression and regulating nuclear factor kappa B (NF-κB), c-Jun NH2-terminal protein kinase (JNK), and phosphoinositide 3-kinase (PI3K)/AKT signaling pathways. The findings suggested that APS possibly acted as a potential therapeutic drug for treatment of myocarditis.

Material and methods

Cell culture and treatment

A rat embryonic-heart derived cell line H9c2 obtained from the American Type Culture Collection (ATCC, Rockville, MD, USA) was cultured in Dulbecco’s Modified Eagle medium (DMEM; Gibco BRL, Gaithersburg, MD, USA) containing 10% (v/v) fetal bovine serum (FBS, Gibco BRL) at 37°C in a humidified 5% CO2 atmosphere. APS and LPS were purchased from Sigma-Aldrich (St Louis, MO, USA). H9c2 cells were first treated with 10 µM of LPS for 12 h to construct the inflammation injury model. Then, different concentrations (0, 50, 100, 150, and 200 µg/mL) of APS were used to treat H9c2 cells for 24 h again.

Cell viability assay

H9c2 cells were seeded in a 96-well plate with 5000 cells/well, and treated with the following conditions: fresh culture medium alone (control), fresh culture medium with different concentrations (0–200 µg/mL) of APS (Sigma-Aldrich), and/or fresh culture medium with 10 µM LPS (Sigma-Aldrich). Cell viability was assessed by a Cell Counting Kit-8 (CCK-8; Dojindo Molecular Technologies, Gaithersburg, MD, USA) according to manufacturer’s instructions. Briefly, after treatment, the CCK-8 solution was added to the culture medium and incubated at 37°C for 1 h. The absorbance was read at 450 nm with a microplate reader (Bio-Rad, Hercules, CA, USA). Cell viability was calculated by (experimental group absorbance value/control group absorbance value) × 100%.

Apoptosis assay

The apoptosis ratios of H9c2 cells undergoing various treatments were measured using Annexin V-fluorescein (AV) and propidium iodide (PI) apoptosis detection kit (Invitrogen, Carlsbad, CA, USA) by flow cytometry. Briefly, after stimulation, cells were washed with PBS and incubated with 10 µL of Annexin V-FITC and 5 µL of PI for 15 min at room temperature in the dark. Flow cytometry analysis was done by a FACScan flow cytometer (Beckman Coulter, Fullerton, CA, USA), and the data were analyzed by using FlowJo software (Tree Star, Ashland, OR, USA).

Cell transfection

MiR-127 inhibitor and negative control (NC) were designed and synthesized by GenePharma (Shanghai, China). Cell transfection was performed using Lipofectamine 3000 (Invitrogen) according to the manufacturer’s instructions. After transfection for 48 h, the efficiency of transfection was monitored by quantitative real-time polymerase chain reaction (qRT-PCR).

Enzyme-linked immunosorbent assay

H9c2 cells were seeded in a 24-well plate and exposed to various treatments for 24 h. After incubation, culture supernatants were collected and concentrations of inflammatory cytokines interleukin-6 (IL-6), interleukin-8 (IL-8), and tumor necrosis factor-alpha (TNF-α) were measured by rat ELISA kits (TaKaRa, Dalian, China) according to manufacturer’s instructions, respectively.

qRT-PCR

Total RNA was isolated from H9c2 cells using TRIzol reagent (Invitrogen) according to manufacturer’s instructions. Reverse transcription was performed by the Multiscribe RT kit (Applied Biosystems, Foster, CA, USA). Expression levels of miR-127 were amplified using a TaqMan microRNA assay (Applied Biosystems) by normalizing to U6. For analysis of IL-6, IL-8, and TNF-α, a SYBR Green PCR kit (TaKaRa) was used to quantify the messenger RNA (mRNA) levels of IL-6, IL-8, and TNF-α. β-actin was amplified as control. The relative expression levels of miR-127, IL-6, IL-8, and TNF-α were calculated using the 2−ΔΔCT method.[14]

Western blot

Protein used for western blot was extracted from H9c2 cells using RIPA lysis buffer (Beyotime Biotechnology, Shanghai, China) and quantified using the Bicinchoninic Acid (BCA) Protein Assay Kit (Pierce, Appleton, WI, USA). Equal amount of protein was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore, Billerica, MA, USA). The membranes were incubated with the indicated primary and secondary antibodies. The primary antibodies used in this study included anti-Bax (ab32503, 1:1000), anti-pro-Caspase3 (ab44976, 1:500), anti-cleaved-Caspase3 (ab13847, 1:500), anti-Caspase9 (ab25758, 1:200), anti-IL-6 (ab9770, 1:5000), anti-IL-8 (ab7747, 1:10), anti-TNF-α (ab6671, 1:1000), anti-p65 (ab16502, 1:2000), anti-toll-like receptor 4 (anti-TLR4, ab13556, 1:500), anti-p-p65 (ab86299, 1:2000), anti-IκBα (ab7217, 1:2000), anti-JNK (ab179461, 1:1000), anti-p-JNK (ab124956, 1:5000), anti-c-Jun (ab32137, 1:1000) and anti-p-c-Jun (ab32385, 1:1000), anti-PI3K (ab191606, 1:1000), anti-p-PI3K (ab182651, 1:1000), anti-AKT (ab32505, 1:2000), anti-p-AKT (ab131443, 1:1000), all purchased from Abcam (Cambridge, UK). Anti-p-IκBα (#5209, 1:1000) was purchased from Cell Signaling Technology (Boston, USA). β-actin (ab8226, 1:1000; Abcam) was used as the loading control. Immunoreactive bands were visualized using ECL detection reagent (Millipore). The intensity of the bands was quantified by using Image LabTM Software (Bio-Rad, Shanghai, China).

Statistical analysis

Data from this study were presented as mean ± standard deviation (SD) and statistically analyzed by one-way analysis of variance (ANOVA) using SPSS 19.0 statistical software (IBM Analytics, New York, USA). A P-value of <0.05 was considered to indicate a statistically significant result. All experiments were repeated at least three times.

Results

APS had no effect on H9c2 cells viability

H9c2 cells were first treated with different concentrations (0–200 µg/mL) of APS for 24 h, and then the effect of APS on cell viability was examined using the CCK-8 assay. As shown in Figure 1, cell viability of H9c2 cells treated with various concentrations of APS had no significant difference compared with control group. The results indicated that APS had no effect on H9c2 cells viability.

Figure 1.

Effect of APS on the cell viability of H9c2 cardiomyoblasts. H9c2 cells were treated with different doses (0, 50, 100, 150, and 200 µg/mL) of APS for 24 h. After treatment, cell viability of H9c2 cells was detected by CCK-8 assay. Each experiment was repeated at least three times.

APS: Astragalus polysaccharide; CCK-8: Cell Counting Kit-8.

APS alleviated LPS-induced impairment of H9c2 cells

In order to explore the effect of APS on LPS-induced inflammation injury in H9c2 cells, we observed cell viability by CCK-8 assay, and determined the rate of apoptotic cells by flow cytometry in the presence or absence of APS treatment. Consistent to previous findings,[15,16] our results demonstrated that LPS could inhibit H9c2 cells viability. However, the inhibitory effect of LPS on cell viability of H9c2 cells was markedly elevated by APS in dose-dependent manner (P < 0.05, Figure 2(a)). In addition, significant increases in the percentage of apoptotic cells were observed in LPS-treated group compared with control group, but the promoting effect was significantly declined by APS in a dose-dependent manner (P < 0.05, Figure 2(b)). To further confirm our results, we performed western blot to detect the protein expression levels of specific markers of apoptosis (Bax, Caspase-3, and Caspase-9). As shown in Figure 2(c) and (d), the protein levels of Bax, cleaved-Caspase-3, and cleaved-Caspase-9 in the LPS-treated group were drastically up-regulated compared with control group, but the promoting effect was markedly decreased in the co-treatment with LPS and APS groups. Taken together, these data indicated that APS alleviated LPS-induced impairment of H9c2 cells by increasing cell viability and reducing apoptosis in a dose-dependent manner.

Figure 2.

Effects of APS on LPS-induced inflammation injury in H9c2 cells. H9c2 cells were treated with LPS or co-treated with APS and LPS for 24 h. (a) Cell viability was evaluated by CCK-8 assay. (b) Cell apoptosis of H9c2 cells was detected by flow cytometry. (c and d) The apoptosis-related protein levels were examined by western blot. Different letters above the bars (a, b, c, d) indicate that the means of different groups were significantly different (P < 0.05) by ANOVA. Each experiment was repeated at least three times.

APS: Astragalus polysaccharide; LPS: lipopolysaccharide; CCK-8:Cell Counting Kit-8; ANOVA: one-way analysis of variance.

***P < 0.001 vs control group; #P < 0.05, ##P < 0.01, ###P < 0.001 vs LPS group.

APS inhibited LPS-induced inflammatory cytokines production in H9c2 cells

The effects of APS on the production of inflammatory cytokines IL-6, IL-8, and TNF-α induced by LPS in H9c2 cells were evaluated by qRT-PCR, western blot, and ELISA. qRT-PCR and western blot analysis revealed that the mRNA and protein levels of IL-6, IL-8, and TNF-α were markedly elevated in LPS-treated H9c2 cells compared with control group (P < 0.05). However, the increased expression levels of IL-6, IL-8, and TNF-α induced by LPS decreased in the presence of APS in a dose-dependent manner (P < 0.05, Figure 3(a) and (b)). These results were consistent with data obtained from ELISA assay; significant increases in the release of IL-6, IL-8, and TNF-α were observed in LPS-treated group, but the promoting effects of LPS-induced IL-6, IL-8, and TNF-α releases decreased in the co-treatment with LPS and APS groups (P < 0.05), and the release of inflammatory cytokines was reduced as the concentration of APS increased, except the slight up-regulation of IL-6 at 50 µg/mL APS (Figure 3(c)–(e)). These data suggested that APS could inhibit LPS-induced overproduction of IL-6, IL-8, and TNF-α in H9c2 cells in a dose-dependent manner.

Figure 3.

Effects of APS on LPS-induced the expression and release of inflammatory cytokines in H9c2 cells. (a)–(b) The mRNA and protein expression levels of IL-6, IL-8, and TNF-α in H9c2 cells were measured by qRT-PCR and western blot after treatment with LPS or co-treatment with APS and LPS. (c)–(e) The release of IL-6, IL-8, and TNF-α was measured by ELISA assay in H9c2 cells. Different letters above the bars (a, b, c, d, e) indicate that the means of different groups were significantly different (P < 0.05) by ANOVA. Each experiment was repeated at least three times.

APS: Astragalus polysaccharide; LPS: lipopolysaccharide; IL-6: interleukin-6; IL-8: interleukin-8; TNF-α: tumor necrosis factor-alpha; qRT-PCR: quantitative real-time polymerase chain reaction; ELISA: enzyme-linked immunosorbent assay; ANOVA: one-way analysis of variance.

***P < 0.001 vs control group; #P < 0.05, ##P < 0.01, ###P < 0.001 vs LPS group.

APS increased TLR4 expression in LPS-treated H9c2 cells

To explore the effect of APS on TLR4 expression, H9c2 cells were treated with LPS or co-treated with APS (50, 100, 150, and 200 µM) and LPS for 24 h. The protein level of TLR4 was examined by western blot. As shown in Figure 4, the result revealed that LPS notably reduced the expression level of TLR4 in H9c2 cells (P < 0.001). However, APS remarkably induced TLR4 expression (P < 0.05, P < 0.01 or P < 0.001). These data demonstrated that APS could induce TLR4 expression in H9c2 cells. It indicated that TLR4 might play a vital role in APS regulating LPS-induced cell injury.

Figure 4.

APS increased TLR4 expression in LPS-treated H9c2 cells. H9c2 cells were treated with LPS or co-treated with APS and LPS for 24 h. Then, the protein level of TLR4 was examined by western blot assay. Each experiment was repeated at least three times.

APS: Astragalus polysaccharide; TLR4: toll-like receptor 4; LPS: lipopolysaccharide.

***P < 0.001 vs control group; #P < 0.05, ##P < 0.01, ###P < 0.001 vs LPS group.

LPS activated NF-κB and JNK signaling pathways via regulation of miR-127

Recently, the importance of microRNAs (miRNAs) in determining inflammatory disorders has been increasingly appreciated.[17,18] MiR-127 is found to be highly expressed in embryos and implicated in the cellular apoptosis and inflammation.[19] NF-κB and JNK signaling pathways are known to play important roles in many physiological functions.[20,21] Based on these previous studies, we further investigated the effect of miR-127 on LPS-affected NF-κB and JNK signaling pathways in H9c2 cells. First, we analyzed expression levels of miR-127 in the presence or absence of LPS by qRT-PCR and we found that the expression of miR-127 in H9c2 cells treated with LPS was higher than in control group (P < 0.05, Figure 5(a)). To examine whether LPS was involved in regulation of NF-κB and JNK signaling pathways, the expression levels of p-p65, p-IκBα, p-JNK, and p-c-Jun were detected by western blot assay. As shown in Figure 5(b) and (c), treatment with LPS caused a moderate increase in the expression levels of p-p65, p-lκBα, p-JNK, and p-c-Jun, indicating a promoting effect of LPS on NF-κB and JNK signaling pathways. To demonstrate whether LPS promoted NF-κB and JNK signaling pathways by regulation of miR-127, the expression of miR-127 in H9c2 cells was knocked down by transfection with miR-127 inhibitor, and the efficiency of transfection was confirmed by qRT-PCR (P < 0.01, Figure 5(d)). Then, we performed western blot assay after H9c2 cells were treated with LPS and miR-127 inhibitor. We found that the significant up-regulations of p-p65, p-lκBα, p-JNK, and p-c-Jun induced by LPS were reduced by miR-127 suppression (Figure 5(e) and (f)). Taken together, these data suggested that LPS promoted NF-κB and JNK signaling pathways by regulation of miR-127.

Figure 5.

LPS promoted NF-κB and JNK signaling pathways by regulation of miR-127. (a) Expression levels of miR-127 in H9c2 cells were measured by qRT-PCR after treatment with LPS. (b)–(c) Protein levels of the main factors of NF-κB and JNK signaling pathways were detected by western blot after treatment with LPS. (d) The expression of miR-127 knocked down by miR-127 inhibitor was examined by qRT-PCR. (e)–(f) Protein levels of the main factors of NF-κB and JNK signaling pathways were examined by western blot again after being treated with LPS or co-treated with LPS and miR-127 inhibitor. Each experiment was repeated at least three times.

LPS: lipopolysaccharide; NF-κB: nuclear factor kappa B; JNK: c-Jun NH2-terminal protein kinase; miR: microRNA; qRT-PCR: quantitative real-time polymerase chain reaction.

*P < 0.05, **P < 0.01, ***P < 0.001 vs control group; ##P < 0.01 vs LPS + NC group.

APS reduced LPS-induced inflammation injury by down-regulating miR-127 and inhibiting NF-κB and JNK and promoting PI3K/AKT signaling pathways in H9c2 cells

Having determined that LPS was able to promote NF-κB and JNK signaling pathways by regulating miR-127, we further studied whether APS exerted its relieving effects on LPS-induced inflammation injury through inhibiting these ways. As shown in Figure 6(a), qRT-PCR analysis revealed that the elevated expressions of miR-127 induced by LPS were markedly decreased in the co-treatment with LPS and APS groups (P < 0.05). In addition, APS reversed the up-regulations of p-p65, p-lκBα, p-JNK, and p-c-Jun induced by LPS (Figure 6(b) and (c)). Furthermore, the protein levels of p-PI3K and p-AKT were down-regulated by LPS, whereas the inhibitory effect of LPS on PI3K/AKT was abolished by APS in H9c2 cells (Figure 6(d)). These data indicated that the relieving effects of APS on LPS-induced inflammation injury might be through down-regulating miR-127, inhibiting NF-κB and JNK, and promoting PI3K/AKT signaling pathways in H9c2 cells.

Figure 6.

APS reduced LPS-induced inflammation injury by down-regulating miR-127 and inhibiting NF-κB and JNK and promoting PI3K/AKT signaling pathways in H9c2 cells. (a) H9c2 cells were treated with LPS or co-treated with APS and LPS, and then expression levels of miR-127 in H9c2 cells were measured by qRT-PCR. (b)–(d) Protein expression levels of p65/p-p65, lκBα/p-lκBα JNK/p-JNK, c-Jun/p-c-Jun, PI3K/p-PI3K, and AKT/p-AKT were detected by western blot. Different letters above the bars (a, b, c) indicate that the means of different groups were significantly different (P < 0.05) by ANOVA. Each experiment was repeated at least three times.

APS: Astragalus polysaccharide; miR: microRNA; NF-κB: nuclear factor kappa B; JNK: c-Jun NH2-terminal protein kinase; PI3K: phosphoinositide 3-kinase; AKT: protein kinase B; qRT-PCR: quantitative real-time polymerase chain reaction; ANOVA: one-way analysis of variance.

***P < 0.001 vs control group; #P < 0.05, ##P < 0.01, ###P < 0.001 vs LPS group.

Discussion

Majority of cases of myocarditis were caused by viral infection, bacterial infection, drug reaction, and autoimmune disease.[22] Persistent myocardial inflammation leads to myocyte damage and eventually leads to heart failure or death. Therefore, it is important to find an efficient drug for treatment of myocarditis by inhibition of inflammation injury. In this study, LPS was used to induce inflammation injury in H9c2 cardiomyocytes to simulate a model of myocarditis in vitro. Consistent with previous findings, our results demonstrated that LPS significantly inhibited cell viability, enhanced apoptosis, and led to the overproduction of inflammatory cytokines IL-6, IL-8, and TNF-α in H9c2 cells, indicating the model of myocarditis was successfully established.

More and more studies have demonstrated that traditional herbal medicine has become an effective and alternative approach for clinical treatment of various diseases.[23] APS extracted from the root of Astragalus membranaceus is a water-soluble macromolecular compound with various biological effects.[24] A recent study revealed that APS could inhibit cell viability of C2C12 myoblasts in a dose-dependent manner.[25] Additionally, it has been reported that APS exerted inhibitory effects on apoptosis and inflammation injury.[26] Interestingly, the result in our study showed that APS had no effect on H9c2 cells viability, but it could dramatically increase cell viability, decrease apoptosis, and reduce the release of inflammatory cytokines in LPS-damaged H9c2 cells. It seems that APS had no impact on normal cardiomyocytes, but exerted protective functions on LPS-damaged cardiomyocytes. In recent years, accumulated evidences indicated that APS could increase TLR4 expression, and TLR4 could recognize LPS and induce related signaling pathways to play important roles in host defense against LPS.[27,28] Thus, we further explore the effect of APS on TLR4 expression. We found that the expression level of TLR4 was remarkably promoted by APS in LPS-treated H9c2 cells. These data indicated that APS exhibited selectivity for LPS-induced cell damage might be due to the increased expression level of TLR4.

It is well known that miRNAs are small endogenous single-stranded non-coding RNAs, which completely or incompletely bind to target mRNAs, block translation, or lead to degradation of mRNAs.[29] It has been proved that miRNAs function as oncogenes or tumor suppressors and play an essential role in the initiation and progression of certain cancer types.[30] Recent studies have begun to unravel the roles of miRNAs in regulation of the inflammatory factors.[17,31] MiR-127 is previously found to be involved in cell proliferation, migration, and apoptosis.[32] Famously, one study indicated that miR-127 was a key mediator in the inflammation-related disorders, such as bleomycin, or immunocomplex-induced lung injury and inflammation,[33] suggesting the regulatory role of miR-127 in inflammatory response. Here, we found that LPS could increase miR-127 expression, but the elevated expression level of miR-127 induced by LPS was markedly decreased in the presence of APS. These data suggested that APS might decrease LPS-induced inflammation injury by down-regulating miR-127.

Furthermore, we investigated the related signaling pathways to reveal the potential mechanisms. NF-κB, an important signaling pathway, is known to be associated with many physiological functions, including cellular proliferation, malignant transformation, and inflammation.[34] Inhibition of this pathway might increase cell viability and decrease apoptosis and inflammation injury. Phosphorylation of p65 and lκBα, which are key molecules of NF-κB signaling pathway, usually increases apoptosis and inflammatory response. Here, we found that the elevated expression levels of p-p65 and p-lκBα induced by LPS were suppressed by miR-127 suppression, as well as suppressed by APS. Therefore, these data indicated that the relieving effects of APS on LPS-induced inflammation injury might be through down-regulating miR-127 and inhibiting NF-κB signaling pathway in H9c2 cells.

Recent evidences have demonstrated that JNK signaling pathway, is also linked to cell proliferation, apoptosis, and inflammation.[35,36] Here, we investigated whether APS could also decrease LPS-induced inflammation injury by regulating miR-127 and inhibiting JNK signaling pathway in H9c2 cells. JNK and its downstream molecule c-Jun (key molecules in JNK pathway) are proved to be activated after injury.[37] In this study, western blot analysis indicated that the expressions of p-JNK and p-c-Jun were increased in LPS-treated group, but the increases were reversed after transfection with miR-127 inhibitor, as well as after APS treatment. Overall, these data indicated that APS could inhibit JNK signaling pathway in H9c2 cells by down-regulating miR-127.

PI3K/AKT is an important signaling pathway, which participates in various cell biological processes such as proliferation, apoptosis and transformation, and inflammation.[38,39] In our study, we further investigated the effect of APS on PI3K/AKT in LPS-treated H9c2 cells. The results revealed that APS remarkably reversed the down-regulations of p-PI3K and p-AKT reduced by LPS in H9c2 cells. These data suggested that APS could promote PI3K/AKT signaling pathway in H9c2 cells by down-regulating miR-127.

In summary, APS could protect H9c2 cells against LPS-induced inflammation injury by increasing cell viability, reducing apoptosis, and suppressing the production of inflammatory cytokines. APS exerted protective functions maybe through down-regulation of miR-127 and inhibition of NF-κB and JNK pathways. These findings provide evidences that APS might be a potential therapeutic drug for the treatment of myocarditis in vitro. Further study still needs to clarify the hypothesis.