| Objective:Previous studies have shown that a7nAChR expressing on the surface of RAW264.7 is a key target for the anti-inflammatory effect of sinomenine,but the relationship between arthritis and a7nAChR is unclear.This study first investigated whether sinomenine can act on a7nAChR to play an anti-arthritis effect in adjuvant-induced arthritis(AIA)rats,and further study of sinomenine on rats in different tissues and organs a7nAChR expression and the relationship between a7nAChR and the pathogenesis of AIA were investigated.Finally,the effects of sinomenine on the proliferation of fibroblast-like synoviocytes and the expression of a7nAChR were studied.From the above aspects to explore that sinomenine plays an anti-arthritis immune pharmacological effect through a7nAChR.Methods:1.Relationship between anti-arthritis effect of sinomenine and a7nAChRThe AIA rats were established by intradermal injection of complete Freund's adjuvant.The relationship between the anti-arthritis effect of sinomenine and_a7nAChR was observed.First determine the modeling dose of the AIA rats,the anti-arthritis dose of nicotine,and the antagonistic dose of alpha-bungarotoxin.The effect of a7nAChR-specific antagonist a-bungarotoxin on the anti-arthritis effect of sinomenine was observed.The rats were divided into control group,model group,antagonist(5?g/mL ?-BTX)group,sinomenine(120mg/kg/d)with or without a-BTX group,nicotine(1 mg/kg/d)with or without a-BTX group,indomethacin(1 mg/kg/d)with or without a-BTX group,and methotrexate(7.8mg/kg/w)with or without a-BTX group.The administration period was 30 days,and hindpaw swelling,erythrocyte sedimentation rate,arthritis score and serum TNF-? production were measured on day 0,6,12,18,24 and 30 to study whether a-BTX can block the anti-arthritis effect of sinomenine.2.Effect of sinomenine on expression of a7nAChR in AIA ratsAt the 30th day,the rats peritoneal macrophages were extracted;the synovium,lung,liver,spleen,thymus,kidney and lymph nodes were removed,fixed with 4%paraformaldehyde or frozen at-70?.The expression of a7nAChR in peritoneal macrophages was detected by immunofluorescence.RT-PCR and immunohistochemistry were used to detect the expression of a7nAChR in synovium,lung,liver,spleen,thymus and kidney.Western blot was used to detect the expression of a7nAChR in lymph nodes.The above experiment was used to study the effect of sinomenine on a7nAChR expression.3.Relationship between the progression of AIA rats and a7nAChRThe swelling,erythrocyte sedimentation rate(ESR),arthritis score and TNF-? were measured at 0,6,12,18,24 and 30d after injection of CFA.Also,the expression of a7nAChR in the synovium,lung,liver,spleen,thymus,kidney and lymph nodes of rats were measured by western blot.In addition,sinomenine and methotrexate were given to observe the changes of the above indexes.The above experiment study the relationship between the progression of AIA model and a7nAChR.4.Effects of sinomenine on the proliferation of fibroblast-like synoviocytes and the expression of a7nAChRPrimary FLS was cultured using tissue explant method.Then,CD90 detection of FLS was measured by flow cytometry.FLS were divided into vehicle,TNF-? and sinomenine groups(the concentration was 100,200,400pmol·L-1,respectively).Sinomenine groups was treated with sinomenine 30 minutes later,TNF-? group and sinomenine groups were stimulated by 20ng·mL-1 TNF-? for 24 hours.Then,the proliferation of FLS of each group was measured by MTT assay,the release of serum IL-6 was measured by ELISA,and the expression of a7nAChR,TLR2 and TLR4 was measured by RT-PCR or western blot.Results:1.Relationship between anti-arthritis effect of sinomenine and a7nAChRThe results showed that sinomenine,nicotine,indomethacin and methotrexate all inhibited the hindpaw swelling,arthritis score,erythrocyte sedimentation rate and serum TNF-?.?-bungarotoxin blocks the above effects of sinomenine and nicotine,but can not reverse the above effects of indomethacin and methotrexate.The results show that the anti-arthritis effect of sinomenine can be blocked by ?-bungarotoxin,which indicate that sinomenine has a similar target with nicotine,which is acting on ?7nAChR to play an anti-arthritis effect.2.Effect of sinomenine on expression of a7nAChR in AIA ratsThe expression of a7nAChR in rats peritoneal macrophages,liver and synovium was down-regulated by sinomenine and nicotine,whereas the expression of a7nAChR in thymus,spleen,lung,kidney and lymph nodes was different.Nicotine showed an up-regulation effect on a7nAChR of thymus,spleen,lung,kidney and lymph nodes,while sinomenine showed down-regulation.Indomethacin,methotrexate had no significant effect on the expression of a7nAChR.3.Relationship between the progression of AIA rats and a7nAChRThe results showed that there was no difference between control group and AIA rats on day 0 to 11.The related indexes of the process of RA in AIA rats(hindpaw swelling,arthritis score,erythrocyte sedimentation rate and serum TNF-?)were significantly increased on day 12,peak on day 18,and decreased during day 18 to day30.However,the relevant indicators were still higher than the control group rats.The expression of a7nAChR in AIA rats was highly correlated with the indexes of hindpaw swelling and arthritis.SIN not only reduces the RA-related indicators,but also reduces the a7nAChR expression.MTX can only reduce the relevant indicators of RA process,whereas has no effect on a7nAChR expression.4.Effects of sinomenine on the proliferation of fibroblast-like synoviocytes and the expression of ?7nAChRFLS were significantly increased after stimulating by TNF-?,and the release of supernatants IL-6 was also increased distinctly.Furthermore,both the expression of mRNA and protein of ?7nAChR,TLR2 and TLR4 was upregulated.However,the proliferation of FLS and the release of supernatants IL-6 was reversed by sinomenine(200,400?mnol·L-1).In addition,the expression of mRNA and protein of ?7nAChR,TLR2 and TLR4 was downregulated by sinomenine.Conclusion:1.The anti-arthritis effect of sinomenine in AIA rats depends on a7nAChR.?7nAChR is an important target of anti-arthritis effect of sinomenine in vivo.2.Sinomenine inhibited the expression of a7nAChR in different tissues and organs of AIA rats.3.The expression of ?7nAChR was positively correlated with the hindpaw swelling and arthritis score.4.The proliferation of FLS is related with the expression of ?7nAChR.Sinomenine can inhibit the proliferation of FLS,and downregulate the expression of ?7nAChR. |
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