[Potential pharmacodynamic substances of Laportea bulbifera in treatment of rheumatoid arthritis based on serum pharmacochemistry and pharmacology].

The present study investigated the pharmacodynamic material basis of Laportea bulbifera in the treatment of rheumatoid arthritis. Firstly, human rheumatoid arthritis fibroblast-like synoviocyte line MH7A was cultured in vitro and treated with tumor necrosis factor alpha(TNF-α, 50 ng·mL~(-1)). The proliferation and the levels of inflammatory cytokines such as prostaglandin E2(PGE2), interleukin-1ß(IL-1ß), and interleukin-6(IL-6) of the MH7A cells exposed to the serum containing L. bulbifera were determined to evaluate the anti-rheumatoid arthritis effects of the serum. Furthermore, the ultra-performance liquid chromatography tandem mass spectrometry fingerprints of the L. bulbifera crude extract, the drug-containing serum, and the drug-free serum were compared to identify the compounds newly generated in the serum after oral administration of the extract. According to the peak areas of common peaks and the results of anti-rheumatoid arthritis effect test, the active components were identified. The serum containing L. bulbifera significantly inhibited the proliferation of the MH7A cells activated by TNF-α and the expression of PGE2, IL-6, and IL-1ß. Thirty newly generated compounds were detected in the drug-containing serum. Among them, neochlorogenic acid, cryptochlorogenic acid, chlorogenic acid, rutin, isoquercitrin, luteoloside, kaempferol-3-O-rutinoside, and quercitrin were also present in the crude extract. Twelve characteristic peaks(3, 7, 8, 14, 18, 19, 21, 23, 24, m6, m7, and m15) were significantly correlated with the pharmaceutical effect. According to the correlations, neochlorogenic acid, cryptochlorogenic acid, and chlorogenic acid had great contributions to the anti-rheumatoid arthritis activity. This study preliminarily clarified the potential pharmacodynamic substances of L. bulbifera in the treatment of rheumatoid arthritis, which laid a theoretical and experimental foundation for further development and application of the medicinal plant.

[Effect of shikonin on function of rheumatoid arthritis fibroblast like synoviocytes].

To observe the effect of shikonin on the proliferation, migration, adhesion and invasion of rheumatoid arthritis(RA) fibroblast like synoviocytes induced by tumor necrosis factor-α(TNF-α), and to explore its mechanism of action from aspects of protein kinase B(Akt) and mitogen activated protein kinase(MAPK) signaling pathways. TNF-α(20 ng·mL~(-1)) was used in this experiment to induce human RA fibroblast like synovial cell line(MH7 A). After addition of different concentrations of shikonin(0.025, 0.05, 0.1 pmol·L~(-1)), the proliferation, migration, adhesion and invasion ability of MH7 A cells were detected by MTT test, scratch test, adhesion test, Transwell invasion test, respectively. Protein expression of Akt and MAPK signaling pathway molecules in MH7 A cells was detected by Western blot. The results showed that as compared with the control group, TNF-α could significantly induce the proliferation, migration, adhesion and invasion of MH7 A cells, and increase the phosphorylation level of Akt, JNK, p38 and extracellular regulatory protein kinase(ERK). As compared with the TNF-α group, shikonin had no significant effect on TNF-α-induced proliferation of MH7 A cells after 24 h treatment, and it could reduce the TNF-α-induced proliferation of MH7 A cells in a concentration dependent manner after 48 h treatment. Shikonin also significantly reduced the TNF-α-induced migration, adhesion, invasion and phosphorylation levels of Akt, JNK, p38, ERK in MH7 A cells within 24 h. These results suggested that shikonin could reduce the proliferation, migration, adhesion and invasion ability of MH7 A cells induced by TNF-α, and its mechanism may be related to the down-regulation of Akt and MAPK signaling pathway activation.

Jatrorrhizine Hydrochloride Suppresses Proliferation, Migration, and Secretion of Synoviocytes In Vitro and Ameliorates Rat Models of Rheumatoid Arthritis In Vivo.

Jatrorrhizine hydrochloride (JH), an active component isolated from the traditional Chinese herb Coptis chinensis, has been reported to have antimicrobial, antitumor, antihypercholesterolemic, and neuroprotective activities. However, its antirheumatoid arthritis (RA) property remains unknown. In this study, a collagen-induced arthritis (CIA) rat model was used to evaluate the therapeutic effects of JH on RA by using arthritis score, radiological evaluation, and histopathological assessment. The in vitro effects of JH on proliferation, migration, and production of inflammatory mediators in RA-derived fibroblast-like synoviocyte MH7A cells were determined by the EdU incorporation assay, wound healing assay, real-time PCR, and ELISA, respectively. The in vivo studies showed that JH treatment significantly prevented the progression and development of RA in CIA rats through anti-inflammation and suppressing bone destruction. The in vitro studies revealed that JH could effectively attenuate the destructive phenotypes of MH7A cells, including inhibiting proliferation, migration, and production of inflammatory mediators. Further mechanistic analysis demonstrated that JH suppressed tumor necrosis factor alpha (TNFα)-stimulated activations of nuclear factor of kappaB (NF-κB) and mitogen-activated protein kinases (MAPKs) (ERK and p38) leading to the downregulation of proinflammatory cytokines, which might be beneficial to the antiproliferative and antimigratory activities of FLS cells. Collectively, our results demonstrated that JH has a great potential to be developed into a novel therapeutic agent for treating RA.

Effect of a combination of Atractylodes macrocephala extract with strychnine on the TLR4/NF-κB/NLRP3 pathway in MH7A cells.

Rheumatoid arthritis (RA) is now widely recognized as a chronic systemic inflammatory autoimmune disease characterized by swelling, pain and stiffness, which are often disabling. Although the number of drugs available for the treatment of RA has increased in recent years, they are generally expensive, leave patients prone to relapse and can result in severe effects when discontinued. Thus, there is a need for an inexpensive drug with fewer side effects that can be adhered to relieve pain and slow down the progression of the disease. Strychnine, a traditional Chinese medicine, was often used in ancient times to treat swollen and painful joints; however, because of its somewhat toxic nature, it is often combined with Atractylodes macrocephala to reduce its toxicity for safer therapeutic action. The present study performed high-performance liquid chromatography (HPLC)-tandem mass spectrometry (MS/MS) analysis to confirm whether the use of strychnine with Atractylodes macrocephala had the effect of reducing strychnine content. MH7A cells were induced using IL-1ß to study the effect of strychnine with Atractylodes macrocephala on the Toll-like receptor 4 (TLR4)/NF-κB/NLR family pyrin domain-containing 3 (NLRP3) pathway in order to verify its role in the treatment of RA. The results indicated that the combined application of HPLC-MS/MS strychnine and Atractylodes macrocephala had a reducing effect on the strychnine content. From the subsequent experimental results, it can be inferred that Strychnine combined with Atractylodes macrocephala extract could promote the apoptosis of synovial cells, and could inhibit the expression levels of TLR4, NF-κB and NLRP3 in the cells as well as reducing the MH7A-positive cells. The expression levels of TLR4, IκB kinase ß, NF-κB and NLRP3 were significantly reduced after treatment with each administration group, resulting in a decrease in the phosphorylation levels of TLR4 and NF-κB, indicating that the combination potently inhibited their phosphorylation. The combination of strychnine and atractylenolide II was also revealed to be the main active ingredient in the treatment of RA.

Magnoflorine attenuates inflammatory responses in RA by regulating the PI3K/Akt/NF-κB and Keap1-Nrf2/HO-1 signalling pathways in vivo and in vitro.

BACKGROUND: As a prolonged autoimmune disorder, rheumatoid arthritis (RA) is characterised by synovial hyperplasia and the erosion of bone and cartilage. Magnoflorine (MAG) is the main component purified from Clematis manshurica Rupr. Recent studies have shown that MAG has anti-inflammatory, antioxidant, and immunosuppressive effects, which are relevant to anti-RA activities. OBJECTIVE: The current investigation was conducted to explore the anti-RA effects of MAG and to discover the possible molecular mechanisms. METHODS: In vitro experiments, CCK-8, wound healing, and transwell assays were utilized to evaluate the anti-proliferative, anti-migratory, and anti-invasive activities of MAG, respectively. The rate of cell distribution and cell apoptosis were evaluated by flow cytometry. ROS generation was detected by DCFH-DA staining. Western blotting, quantitative real-time polymerase chain reaction assay, and immunofluorescent staining were employed to test the anti-RA effect of MAG as well as to explore the potential mechanisms by evaluating related gene and protein expression. For in vivo experiments, an adjuvant-induced arthritis (AIA) rat model was established. The related parameters were measured in rats. Then, rats were sacrificed, and ankle joints were collected for histopathological analysis and observation. RESULTS: MAG significantly decreased the proliferation, migration, invasion, and reactive oxygen species levels in IL-1ß-treated MH7A cells. Furthermore, MAG promoted cell apoptosis by increasing Bax levels and decreasing Bcl-2 levels. MAG also induced cell cycle arrest. Inflammatory cytokines (iNOS, COX-2, IL-6, and IL-8) and MMPs (MMP-1, 2, 3, 9, and 13) were reduced by MAG treatment. Molecular analysis revealed that MAG exerted anti-RA effects by partly inhibiting the PI3K/Akt/NF-κB signalling axis and activating the Keap1-Nrf2/HO-1 signalling pathway. In vivo studies have revealed that MAG treatment substantially improved severe symptoms in AIA rats, and these curative effects were linked to the attenuation of inflammatory responses. CONCLUSION: These results first suggested that MAG exhibits anti-arthritic effects in IL-1ß-treated MH7A cells and AIA rat models. Thus, MAG may be used as a new drug to treat RA clinically.

Anti-proliferation and anti-inflammation effects of corilagin in rheumatoid arthritis by downregulating NF-κB and MAPK signaling pathways.

ETHNOPHARMACOLOGICAL RELEVANCE: The dried aboveground part of Geranium Wilfordii Maxim. (G. Wilfordii) is a traditional Chinese herbal medicine named lao-guan-cao. It has long been used for dispelling wind-dampness, unblocking meridians, and stopping diarrhea and dysentery. Previous investigations have revealed that 50% ethanolic extract of G. Wilfordii has anti-inflammatory and anti-proliferation activities on TNF-α induced murine fibrosarcoma L929 cells. Corilagin (COR) is a main compound in G. Wilfordii with the content up to 1.69 mg/g. Pharmacology study showed that COR has anti-inflammatory, anti-tumor, anti-microorganism, anti-oxidant, and hepatoprotective effects. However, there is no any investigation on its anti-proliferation and anti-inflammation effects in rheumatoid arthritis (RA). AIM OF THE STUDY: The present study aimed to evaluate the potential pharmacological mechanisms of anti-proliferation and anti-inflammation effects of COR in RA. MATERIALS AND METHODS: In vitro, MH7A cells model induced by IL-1ß was used. The anti-proliferation activity of COR was assessed by Cell Counting Kit-8 (CCK-8) assay, and the anti-migration and anti-invasion activity of COR was determined by wound healing assay and transwell assay, respectively. Furthermore, apoptosis assay by flow cytometer was used to measure the pro-apoptotic effect of COR. The mRNA expressions of Bax, Bcl-2, IL-6, IL-8, MMP-1, MMP-2, MMP-3, MMP-9, COX-2, and iNOS were measured by qRT-PCR, and related protein were further verified by ELISA kits or Western blot. Moreover, protein levels associated with NF-κB and MAPK signaling pathways of p65, P-p65, IκBα, P-IκBα, ERK1/2, P-ERK1/2, JNK, P-JNK1/2/3, p38, and P-p38 were determined by Western blot. The nuclear translocation of NF-κB-p65 was detected by immunofluorescent staining. In vivo, adjuvant-induced arthritis (AIA) rat model was used, and the body weight, paw swelling, and arthritis score during the entire period were measured. Histopathological analysis of joints of synovial tissues was also determined. The expression of pro-inflammatory cytokines in serum including IL-6, TNF-α, IL-1ß, and IL-17 were measured. RESULTS: The in vitro results showed that COR could dose-dependently inhibit the proliferation, migration, and invasion of IL-1ß-induced MH7A cells, as well as promote its apoptosis. Moreover, it also suppressed the over-expression of Bcl-2, IL-6, IL-8, MMP-1, MMP-2, MMP-3, MMP-9, COX-2, and iNOS while up-regulated the level of Bax. Besides, the ratios of P-p65/p65, P-IκBα/IκBα, P-ERK/ERK, P-JNK/JNK, and P-p38/p38 were decreased, and the nuclear translocation of p65 induced by IL-1ß was blocked by COR. In vivo results indicated that COR significantly reduced the paw swelling and arthritis score in AIA rats, and inhibited synovial tissue hyperplasia and erosion, as well as inflammatory cells infiltration. It also decreased the serum pro-inflammatory cytokines (IL-6, TNF-α, IL-1ß, and IL-17) production. CONCLUSION: These results revealed that COR exerted anti-rheumatoid arthritis effect, and its underlying mechanisms may be related to inhibiting the proliferation, migration, and invasion of synovial fibroblasts, enhancing cell apoptosis, and suppressing inflammatory responses via downregulating NF-κB and MAPK signaling pathways.

MicroRNA-23a-5p regulates cell proliferation, migration and inflammation of TNF-α-stimulated human fibroblast-like MH7A synoviocytes by targeting TLR4 in rheumatoid arthritis.

Rheumatoid arthritis (RA) is a chronic inflammatory disease characterized by synovial joint inflammation. RA synovial fibroblasts (RASFs) constitute a major cell subset of the RA synovia. MicroRNAs (miRNAs/miRs) have been reported to serve a role in the activation and proliferation of RASFs. The present study aimed to investigate the effects and underlying mechanisms of miR-23a-5p on RA progression. Peripheral blood was collected from patients with RA (n=20) to analyze the expression levels of miR-23a-5p. The effects of miR-23a-5p on cell apoptosis, proliferation and migration in MH7A cells were determined in TNF-α-treated human fibroblast-like synoviocytes (MH7A cells) by flow cytometry, colony formation assay and Transwell assay, respectively. The cell cycle distribution was evaluated using flow cytometry. The binding relationship between miR-23a-5p and toll-like receptor (TLR) 4 was analyzed using a dual luciferase reporter gene assay. ELISA and reverse transcription-quantitative PCR assays were used to detect the levels of the inflammatory factors IL-6, IL-1ß and IL-10. The expression levels of apoptosis- and migration-related proteins were analyzed using western blotting. The results of the present study revealed that the expression levels of miR-23a-5p were significantly downregulated in the plasma of patients with RA and in MH7A cells. In addition, the TNF-α-induced increase in the cell proliferative and migratory rates and the production of IL-6 and IL-1ß were markedly inhibited following miR-23a-5p overexpression. The TNF-α-induced decreases in MH7A cell apoptosis were also reversed following miR-23a-5p overexpression. Additionally, transfection with miR-23a-5p mimics significantly inhibited the activation of the TLR4/NF-κB signaling pathway in TNF-α-treated MH7A cells by targeting TLR4. Notably, TLR4 overexpression weakened the effects of miR-23a-5p mimic on cell proliferation, apoptosis, migration, inflammation and the TLR4/NF-κB signaling pathway in TNF-α-induced MH7A cells. In conclusion, the findings of the present study indicated that the miR-23a-5p/TLR4/NF-κB axis may serve as a promising target for RA diagnosis and treatment.

Transcriptome-Wide High-Throughput m6A Sequencing of Differential m6A Methylation Patterns in the Human Rheumatoid Arthritis Fibroblast-Like Synoviocytes Cell Line MH7A.

INTRODUCTION: N6-methyladenosine (m6A) is the most frequent internal modification in eukaryotic mRNAs and is closely related to the occurrence and development of many diseases, especially tumors. However, the relationship between m6A methylation and rheumatoid arthritis (RA) is still a mystery. METHODS: Two high-throughput sequencing methods, namely, m6A modified RNA immunoprecipitation sequence (m6A-seq) and RNA sequence (RNA-seq) were performed to identify the differentially expressed m6A methylation in human rheumatoid arthritis fibroblast-like synoviocytes cell line MH7A after stimulation with TNF-α. Gene Ontology (GO) analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses were used to obtain enriched GO terms and significant KEGG pathways. Then, four candidate genes, Wilms tumor 1-associating protein (WTAP), receptor-interacting serine/threonine protein kinase 2 (RIPK2), Janus kinase 3 (JAK3) and tumor necrosis factor receptor SF10A (TNFRSF10A) were selected to further validate the m6A methylation, mRNA and protein expression levels in MH7A cells and synovial tissues of adjuvant arthritis (AA) rats by RT-qPCR and Western blot. RESULTS: Using m6A-seq, we identified a total of 206 genes with differentially expressed m6A methylation, of which 118 were significantly upregulated and 88 genes were significantly downregulated. Likewise, 1207 differentially mRNA expressed mRNAs were obtained by RNA-seq, of which 793 were upregulated and 414 downregulated. Further joint analysis showed that the m6A methylation and mRNA expression levels of 88 genes changed significantly, of which 30 genes displayed increased m6A methylation and decreased mRNA expression, 57 genes displayed decreased m6A methylation and increased mRNA expression increased, and 1 gene displayed increased m6A methylation and increased mRNA expression. GO and KEGG analyses indicated that these unique genes were mainly enriched in inflammation-related pathways, cell proliferation and apoptosis. In addition, the validations of WTAP, RIPK2, JAK3 and TNFRSF10A were in accordance with the m6A and RNA sequencing results. CONCLUSION: This study established the transcriptional map of m6A in MH7A cells and revealed the potential relationship between RNA methylation modification and RA related genes. The results suggested that m6A modification was associated with the occurrence and course of RA to some extent.

Effects of emodin on autophagy in fibroblast-like synoviocytes in rheumatoid arthritis / 中国比较医学杂志

Objective To study the cGAS/STING signaling pathway and investigate the potential effect of emodin(EMD)on autophagy of human rheumatoid arthritis fibroblast synovial cells(MH7A).Methods CCK-8 method was used to detect MH7A cell proliferation,and the experimental concentration of EMD was screened according to cell survival rate.Then,autophagy inhibitor 3-MA was added to further verify the effect of EMD on autophagy.Autophagy of MH7A cells was detected via the monodansylcadaverine staining method.Protein expression levels of cGAS,STING,p-STING,LC3-Ⅰ,LC3-Ⅱ,P62 and Beclin-1 were detected by Western blot.Results Monodansylcadaverine staining indicated that EMD enhanced the autophagy of MH7A cells.Western blot indicated that EMD decreased the expression of autophagy related proteins cGAS,STING,p-STING and P62,and increased that of LC3-Ⅱ and Beclin-1 in MH7A cells.After addition of the autophagy inhibitor 3-MA,the expression of P62 protein in MH7A cells increased,while that of LC3-Ⅱ and Beclin-1 decreased.Conclusions EMD may accelerate autophagy and inhibit MH7A cell proliferation by down-regulating cGAS/STING signaling pathway proteins.

Fatty oil from Securidaca inappendiculata exerted therapeutic effects on adjuvant-induced arthritis in mice via suppression on fibroblast-like synoviocyte.

Securidaca inappendiculata Hassk. (SI) is a medicinal plant used to treat rheumatoid arthritis (RA) in South China. A substantial amount of fatty oil was isolated from SI (SIF), however little knowledge about its chemical composition and medicinal potentials was obtained. In this study, we analyzed its chemical composition with methyl esterification based GC-MS method, and investigated the therapeutic potentials on adjuvant-induced arthritis (AA) in mice. MTT and western-blot methods were employed to investigate its effects on proliferation rate and protein expressions in MH7A cells, respectively. It was revealed SIF was mainly comprised of saturated and monosaturated fatty acids, and the two predominant compounds were palmitic acid (36.89%) and oleic acid (31.12%). Treatment with SIF at 100 mg/kg resulted in significant alleviation of AA severity in mice, together with reduced synovial hyperplasia and inflammatory infiltration in joints, and decreased levels of sialic acid, malondialdehyde and alkaline phosphatase in serum. Results from immunohistochemical assays hinted the protective effects of SIF on joints were associated to the inhibition on production of some pathological factors in synovium, including IL-1ß, TNF-α and MMP-9. SIF inhibited the proliferation of MH7A cells in a concentration dependent manner, and abrogated phosphorylation of p65 in vitro. These evidences collectively suggested SIF could suppress the pathological functions of fibroblast-like synoviocyte, and protect joints from destruction under AA conditions.

α-Mangostin, A Natural Xanthone, Induces Apoptosis and ROS Accumulation in Human Rheumatoid Fibroblast-Like Synoviocyte MH7A Cells.

BACKGROUND: Rheumatoid arthritis (RA) is a complicated and heterogeneous chronic disease with the characteristic of progressive joint destruction, deformity and disability. It is associated with not only genetic but also environmental factors. Many studies suggest that RA-derived fibroblast-like synoviocyte (RA-FLS) is involved in the pathogenic process of RA. The apoptosis and proliferation of RA-FLS is of great importance in the development and progression in RA. Nowadays, more and more traditional Chinese medicine (TCM) or natural products are studied in the treatment of RA. OBJECTIVE: To investigate the effects of several natural products and the apoptosisinduced effect of α-mangostin and its underlying mechanisms in RA-FLS MH7A cells. METHODS: The effects of natural products on MH7A cells were detected by MTT assay. Annexin V-FITC/PI double labeling and DAPI staining were adopted to observe the apoptosis induced by α-mangostin. The apoptosis related proteins were measured with western blotting analysis. ROS accumulation was determined by DHE staining. RESULTS: Xantones, including Garcinone C, α-mangostin and γ-mangostin, significantly inhibited the MH7A cell viability. And α-mangostin induces apoptosis in MH7A cells. Further study showed that α-mangostin increased the ratio of Bax/Bcl-2 and increased the ROS generation in MH7A cells. CONCLUSION: α-Mangostin induces the apoptosis of MH7A cells through increasing ROS accumulation and the ratio of Bax/Bcl-2, suggesting that α-mangostin should be benefit to the therapy of RA.

Effect of Fuzitang on Proliferation of Human Rheumatoid Arthritis Synovial Fibroblast Cell Line MH7A and Expression of miR-155 / 中国实验方剂学杂志

ObjectiveTo observe the effects of Fuzitang （FZT） on the proliferation of MH7A cells， the human rheumatoid arthritis synovial fibroblasts， and the expression of miR-155 and explore its anti-rheumatoid arthritis mechanism. MethodMH7A cells were cultured in vitro and divided into a blank group， high- （25 g·L-1） and low-dose （12.5 g·L-1） FZT groups， and a positive drug group （hydroxychloroquine， 0.006 25 g·L-1）. The cell proliferation was detected by cell counting kit-8（CCK-8） method， and the change in the MH7A cell cycle was detected by flow cytometry. The mRNA expression of miR-155 and its downstream genes， including SH2 domain-containing inositol 5-phosphatase-1（SHIP-1）， protein kinase B 3（Akt3）， and mammalian target of rapamycin（mTOR）， was detected by Real-time fluorescence quantitative polymerase chain reaction（Real-time PCR）， and the protein expression of phosphatidylinositol 3-kinase （PI3K）， Akt3， and mTOR was detected by Western blot. ResultFZT in vitro in a concentration of 6.25 g·L-1 above could inhibit the proliferation of MH7A cells in the significant dose- and time-effect manner. Compared with the blank group， the FZT groups showed increased proportions of cells in the G2/M phase （P<0.05）， and the high-dose FZT group showed a decreased proportion of cells in the G0/G1 phase （P<0.05）. The arresting effect of FZT on the cell cycle was in a significant dose-effect manner. Compared with the blank group， the FZT groups showed down-regulated miR-155 and mTOR mRNA expression （P<0.05）， and the high-dose FZT group showed up-regulated SHIP1 mRNA expression and down-regulated Akt3 mRNA expression （P<0.05）. Compared with the blank group， the FZT groups showed reduced protein expression of PI3K， Akt3， and mTOR （P<0.05）. ConclusionFZT can significantly inhibit the proliferation of MH7A cells， and the mechanism is related to the promotion of the expression of SHIP-1 and down-regulation of the gene expression of the PI3K/Akt3/mTOR signaling pathway by down-regulating the expression of miR-155.

Selective modulation of MAPKs contribute to the anti-proliferative and anti-inflammatory activities of 1,7-dihydroxy-3,4-dimethoxyxanthone in rheumatoid arthritis-derived fibroblast-like synoviocyte MH7A cells.

ETHNOPHARMACOLOGICAL RELEVANCE: 1,7-Dihydroxy-3,4-dimethoxyxanthone (XAN) is an antirheumatic agent isolated from traditional Chinese medicine Securidaca inappendiculata Hassk. This study was designed to investigate its anti-proliferative and anti-inflammatory activities on rheumatoid arthritis derived fibroblast-like synoviocyte cell line MH7A, and explore the underlying mechanism of action. METHODS: The anti-proliferative activity of XAN on MH7A cells was assessed by an MTT method. Its pro-apoptotic and cell cycle arrest activities were analyzed by flow cytometry. W-B method was employed to investigate hallmark kinases involved in the course. Pro-inflammatory cytokines in culture supernatant of MH7A cells were determined by an ELISA method. RESULTS: The results showed XAN efficiently suppressed the proliferation and secretion of IL-1ß and IL-6 of MH7A cells in a concentration-dependent manner. Co-treatment with MAPKs inhibitors U0126, SB202190 and SP600125 indicated JNK and p38 pathways were involved in the course. Up-regulation of p-p38, p-ERK, bax and p21, and down-regulation of p-JNK, cyclin D1 and bcl-2 were observed upon the treatment with XAN. SB202190 partly reversed the modulatory effects. The results suggested XAN inhibited the proliferation of MH7A cells mainly via cell cycle arrest at G1/S phase, and the activity was due to the up-regulation of p-p38, which led to the modulation of p21 and cyclin D1. The down-regulation of p-JNK by XAN suppressed the secretion of pro-inflammatory cytokines, which was beneficial to the anti-proliferative activity of MH7A cells. CONCLUSION: XAN selectively modulated MAPKs signaling, and exerted the subsequent anti-proliferative and anti-inflammatory activities on MH7A cells.

Licochalcone A Inhibits Proliferation and Induces Apoptosis of MH7A Cells by Regulating MAPK Signaling Pathway / 中国实验方剂学杂志

Objective:To study the effects of licochalcone A （LCA） on the proliferation and apoptosis of rheumatoid arthritis fibroblast-like synoviocytes （MH7A） as well as the related inflammatory factors， also to reveal the relevance between mitogen activated protein kinase （MAPK） signaling pathway and LCA regulation of MH7A cell proliferation and apoptosis. Method:MH7A cells were cultured and divided into blank group， LCA groups （10，20，40 μmol·L-1）. The proliferation of MH7A cells was detected by methylthiazolyldiphenyl-tetrazolium bromide（MTT）and immunofluorescence staining. The cell cycle of MH7A cells was determined by flow cytometry after PI staining and apoptosis was detected by flow cytometry after Annexin V/PI staining. The effect of LCA on interleukin-1β（IL-1β） mRNA was detected by Real-time fluorescence quantitative polymerase chain reaction（Real-time PCR）. Western blot was used to detect the effect of LCA on the key proteins of MAPK signaling pathway， meanwhile， PD98059， a specific ERK inhibitor， was used to observe the expression levels of p-ERK and IL-1β. Result:Compared with blank group， LCA could inhibit the proliferation of MH7A cells in a dose-dependent manner， and the number of living cells decreased significantly（P<0.01）， while the number of early apoptotic cells increased significantly（P<0.01）. Compared with the tumor necrosis factor-α （TNF-α，10 μg·L-1）group， LCA could reverse the expression of IL-1β mRNA induced by TNF-α（P<0.01）. and compared with the blank group， LCA also promoted the phosphorylation of ERK， JNK and p38 in a dose-dependent manner（P<0.01）. After ERK inhibitor PD98059 inhibited ERK phosphorylation， the inhibitory effect of LCA 10， 20 μmol·L-1 on IL-1β disappeared. Conclusion:LCA can inhibit the proliferation and induce apoptosis of MH7A cells， which may be related to the phosphorylation of MAPK pathway related proteins， and then inhibit the expression of inflammatory factors.