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.

Significance and successful construction of stable transfection of MH7A cells with lower TRAF2 expression using lentiviral vector / 安徽医科大学学报

Objective To construct a stable synovial cell line MH7A from rheumatoid arthritis(RA)patients using lentiviral vectors that interfere with the expression of tumor necrosis factor receptor associated factor 2(TRAF2),and to study the role of TNF-α-TRAF2 signaling in MH7A abnormal proliferation.Methods Based on the design principles of human TRAF2 gene sequence and shRNA sequence,three pairs of TRAF2 shRNA interference se-quences were designed and synthesized.The primers were annealed by PCR,and a linear vector was obtained by double enzyme digestion PLKO.1-puro.The linearized vector was connected to the annealed primers through Solu-tion I,and the connected products were introduced into receptive cells.The plates were coated,and positive colo-nies were selected for sequencing.Three different recombinant plasmids of PLKO.1-TRAF2-shRNA lentivirus were constructed,and lentivirus packaging plasmids was used to package logarithmic growth phase HEK 293T cells.Vi-rus solution was collected to infect MH7A cells.At the same time,puromycin was used to screen MH7A stable transgenic strains with low TRAF2 expression.CCK-8 method,Western blot,and qPCR were used to detect the proliferation function of MH7A induced by TNF-α and low expression of TRAF2,as well as downstream signal TRAF2,P65 protein expression and mRNA levels.Results PLKO.1-TRAF2-shRNA(1),PLKO.1-TRAF2-shR-NA(2),and PLKO.1-TRAF2-shRNA(3)lentivirus vector plasmids and control group lentivirus vector plasmids PLKO.1-puro were successfully constructed.The three TRAF2-shRNA lentivirus vector plasmids and control group lentivirus vector plasmids PLKO.1-puro were respectively introduced into the lentivirus packaging plasmid of HEK 293T to obtain virus solution.After infecting MH7A cells with the virus solution,they were treated with puromycin(2.00 μ G/mL)screening and obtaining MH7A stable transgenic plants after 2 days.Through qPCR and Western blot results,it was found that the expression of TRAF2 mRNA and protein in PLKO.1-TRAF2-shRNA(1)MH7A stably transfected cells was significantly reduced compared to the negative control group.The results of CCK-8 and Western blot showed that after knocking down TRAF2 in MH7A,the proliferation of MH7A cells with low TRAF2 expression induced by TNF-α and the phosphorylation level of P65 were significantly reduced.Conclusion A sta-ble transgenic strain of PLKO.1-TRAF2-shRNA(1)MH7A cells was successfully constructed to investigate the role of TNF-α-TRAF2 signal activation in mediating abnormal proliferation of RA synovial cells.

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.

Targeted inhibition of GRK2 kinase domain by CP-25 to reverse fibroblast-like synoviocytes dysfunction and improve collagen-induced arthritis in rats

Rheumatoid arthritis (RA) is an autoimmune disease and is mainly characterized by abnormal proliferation of fibroblast-like synoviocytes (FLS). The up-regulated cellular membrane expression of G protein coupled receptor kinase 2 (GRK2) of FLS plays a critical role in RA progression, the increase of GRK2 translocation activity promotes dysfunctional prostaglandin E4 receptor (EP4) signaling and FLS abnormal proliferation. Recently, although our group found that paeoniflorin-6'-

The inhibitory effect of compound Q-l on proliferation of MH7A induced by TNF-α and its significance / 中国药理学通报

Aim To investigate the effect of compound Q-L on MH7A based on NF-KB and MAPK signaling pathways and its mechanism. Methods Human rheumatoid arthritis fibroblast synovial cell line (MH7A) was selected as the experimental object. The effect of compound Q-l on the proliferation of MH7A cells was determined by CCK-8 method. The effect of compound Q-l on the migration ability of MH7A cells was detected by Transwell assay. TNF-α solution was used as inducer, and the content of TNF-α and IL-6 in cell supernatant was determined by ELISA. The protein expressions of p65, p-p65, IκBα, P-IκBα, p38, p-p38, ERK, p-ERK, JNK and p-JNK in the cells were determined by Western blot. Results Compound Q-l at different concentrations significantly inhibited the activity of MH7A cells. Compound Q-l significantly inhibited the migration of MH7A cells. Compound Q-l significantly reduced the contents of TNF-α and IL-6 in cell supernatant. Compound Q-l could significantly down-regulate the protein expression levels of p65, p-p65, P-IκBα, p38, p-p38 induced by TNF-α, but had no marked effects on IKBCX, ERK, p-ERK, JNK and p-JNK proteins. Conclusion Compound Q-l can significantly inhibit the proliferation of MH7A cells, reduce the expression of inflammatory cytokines TNF-α and IL-6 in cell supernatant, and down-regulate the protein expressions of p65, p-p65, IKBCX, p-LKBA, p38, p-p38 induced by TNF-α. The possible mechanism of action is related to NF-κB and p38MAPK sig-naling pathways.

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.

Effect of Fengshi Qutong Capsule on Function of Human Synovial Cells in Rheumatoid Arthritis Induced by TNF-α / 中国实验方剂学杂志

Objective: To study the effects of Fengshi Qutong capsule (FSQTC) on proliferation, migration, adhesion, invasion and secretion of human synovial cells in rheumatoid arthritis (RA) induced by tumor necrosis factor-α (TNF-α) and explore its mechanism. Method: Human synovial cells (MH7A) in RA patients were induced in vitro by using TNF-α (20 μg·L-1). After treatment with different concentrations of FSQTC (0.02,0.1,0.5 μg·L-1), MTT colorimetric assay, transwell migration, adhesion and invasion tests were used to detect the proliferation, migration, adhesion and invasion of the MH7A, respectively. The expression levels of vascular endothelial growth factor (VEGF) and interleukin-1β (IL-1β) in MH7A supernatant were detected by enzyme linked immunosorbent assay (ELISA). Result: As compared with blank control group, TNF-α (20 μg·L-1) significantly increased the proliferation, migration, adhesion, invasion and secretion of IL-1β and VEGF of MH7A cells (P-1) had no significant effect on proliferation of TNF-α-induced MH7A cells after treatment for 24 hours. After 48 hours of treatment, proliferation of MH7A cells induced by TNF-α was decreased in a concentration-dependent manner (PPPPConclusion: FSQTC can inhibit the proliferation, migration, adhesion, invasion and secretion of IL-1β and VEGF in MH7A cells.

Steroidal alkaloids ameliorate cell proliferation, oxidative stress, inflammation and histology outcome in vitro and in vivo

Objective:To investigate the effects of steroidal alkaloids and sarcovagine D isolated from Sarcococca hookeriana var. digyna on cell-proliferation and secretion of inflammatory cytokines in TNF-α-induced human RA-FLS MH7A in vitro, and against complete Freund’s adjuvant (CFA)-induced arthritis rats in vivo.Methods:CCK-8 assay was utilized to evaluate the anti-proliferation activity in vitro. In in-vivo study, rats were randomly divided into control group, model group (CFA), steroidal alkaloids (STA) groups (5.0, 2.5 and 1.25 g/kg BW), and sarcovagine D (SD) groups (50, 100 and 200 mg/kg BW), 10 rats for each group. To evaluate the anti-inflammation effect, the histology, biochemical parameters and expression of inflammatory cytokines were detected.Results:Steroidal alkaloids and sarcovagine D showed strong anti-proliferative activity during MH7A cell culture proliferation and downregulated NO levels, and inflammatory cytokines (IL-1ß, IL-6 and PGEConclusions:The results suggest that Sarcococca hookeriana var. digyna has anti-inflammatory effect. Steroidal alkaloids and sarcovagine D has the potential to cure RA.

Steroidal alkaloids ameliorate cell proliferation, oxidative stress, inflammation and histology outcome in vitro and in vivo

Objective: To investigate the effects of steroidal alkaloids and sarcovagine D isolated from Sarcococca hookeriana var. digyna on cell-proliferation and secretion of inflammatory cytokines in TNF-α-induced human RA-FLS MH7A in vitro, and against complete Freund's adjuvant (CFA)-induced arthritis rats in vivo. Methods: CCK-8 assay was utilized to evaluate the anti-proliferation activity in vitro. In in-vivo study, rats were randomly divided into control group, model group (CFA), steroidal alkaloids (STA) groups (5.0, 2.5 and 1.25 g/kg BW), and sarcovagine D (SD) groups (50, 100 and 200 mg/kg BW), 10 rats for each group. To evaluate the anti-inflammation effect, the histology, biochemical parameters and expression of inflammatory cytokines were detected. Results: Steroidal alkaloids and sarcovagine D showed strong anti-proliferative activity during MH7A cell culture proliferation and downregulated NO levels, and inflammatory cytokines (IL-1β, IL-6 and PGE

Effects of Caffeoylquinic Acid Derivative Fractions in Miao Medicine Periploca forrestii on Proliferation and Secretion of Inflammatory Cytokines in Human Rheumatoid Arthritis Fibroblast-like Synoviocytes MH7A / 中国药房

OBJECTIVE:To study the effects of caffeoylquinic acid derivative fractions (CADF) in Miao medicine Periploca forrestii on proliferation and secretion of inflammatory cytokines in tumor necrosis factor α(TNF-α)-induced human rheumatoid ar-thritis(RA)fibroblast-like synoviocytes MH7A,and explore its mechanism on anti-RA. METHODS:MH7A cells were divided in-to blank group,TNF-α model group,methotrexate group (positive control,20 mg/L) and CADF different mass concentrations groups(50,100,200,400 mg/L). Except for blank group,other groups received 50 μg/L of TNF-α to stimulate MH7A cells. Af-ter treated by suspension with TNF-α and related medicines for 24 h,the cell proliferation and contents of nitric oxide(NO),pros-taglandin E2 (PGE2),interleukin 1β(IL-1β),interleukin 6 (IL-6) in culture medium were detected. RESULTS:Compared with blank group,cell proliferation activity in TNF-αmodel group was significantly enhanced(P<0.01),contents of NO,PGE2,IL-1β, IL-6 in culture medium were significantly increased(P<0.01). Compared with TNF-α model group,cell proliferation in each ad-ministration group were significantly inhibited(P<0.05 or P<0.01),contents of NO,PGE2,IL-1β,IL-6 in culture medium weresignificantly decreased (P<0.01),showing certain dose-effect relationship with CADF. CONCLUSIONS:CADF can play the role in anti-RA by inhibiting the TNF-α-induced prolifera-tion of MH7A cells and reducing the secretion of inflammatory cytokines NO,PGE2,IL-1β,IL-6.

Effects of Caffeoylquinic Acid Derivative Fractions in Miao Medicine Periploca forrestii on Proliferation and Secretion of Inflammatory Cytokines in Human Rheumatoid Arthritis Fibroblast-like Synoviocytes MH7A / 中国药房

OBJECTIVE:To study the effects of caffeoylquinic acid derivative fractions (CADF) in Miao medicine Periploca forrestii on proliferation and secretion of inflammatory cytokines in tumor necrosis factor α(TNF-α)-induced human rheumatoid ar-thritis(RA)fibroblast-like synoviocytes MH7A,and explore its mechanism on anti-RA. METHODS:MH7A cells were divided in-to blank group,TNF-α model group,methotrexate group (positive control,20 mg/L) and CADF different mass concentrations groups(50,100,200,400 mg/L). Except for blank group,other groups received 50 μg/L of TNF-α to stimulate MH7A cells. Af-ter treated by suspension with TNF-α and related medicines for 24 h,the cell proliferation and contents of nitric oxide(NO),pros-taglandin E2 (PGE2),interleukin 1β(IL-1β),interleukin 6 (IL-6) in culture medium were detected. RESULTS:Compared with blank group,cell proliferation activity in TNF-αmodel group was significantly enhanced(P<0.01),contents of NO,PGE2,IL-1β, IL-6 in culture medium were significantly increased(P<0.01). Compared with TNF-α model group,cell proliferation in each ad-ministration group were significantly inhibited(P<0.05 or P<0.01),contents of NO,PGE2,IL-1β,IL-6 in culture medium weresignificantly decreased (P<0.01),showing certain dose-effect relationship with CADF. CONCLUSIONS:CADF can play the role in anti-RA by inhibiting the TNF-α-induced prolifera-tion of MH7A cells and reducing the secretion of inflammatory cytokines NO,PGE2,IL-1β,IL-6.