Effect Of Sinomenine On The Expression Of A2A In AA Rats And The Relationship With Targeting ?7nAChR

Objective?7 nicotinic acetylcholine receptor(?7n ACh R)is a receptor that has been identified to play a key role in the cholinergic anti-inflammatory pathway(CAP).Adenosine receptor A2 A is a major subtype of adenosine receptors that exert anti-inflammatory and immunosuppressive effects.Sinomenine(SIN)is a commonly used drug for the treatment of rheumatoid arthritis(RA).Previous laboratory studies suggest that SIN exerts anti-arthritis effects on the rat model of Adjuvant-induced Arthritis(AA)by acting on ?7n ACh R.In a bacterial endotoxemia(LPS)-stimulated model of mouse endotoxemia,it was found that sinomenine can modulate A2 A expression when exerting anti-inflammatory effects.In this study,the rat model of AA was used to analyze the effect of SIN on the expression of A2 A and the relationship between the expression of A2 A and the pathogenesis of AA.The relationship between anti-arthritis and the change of A2 A expression was studied.Further study of SIN on fibroblasts was conducted.The effect of anti-proliferative and anti-inflammatory effects of Fibroblast-like synoviocytes(FLS)on the expression of A2 A and its relationship with targeted ?7n ACh R provide experimental basis for clarifying the mechanism of SIN anti-arthritis.Methods1.Effect of SIN on A2 A expression in AA model ratsMale Sprague-Dawley rats,100-120 g,were injected intradermally with complete Freund's adjuvant to establish AA models.The rats were divided into control group,model group,SIN group,nicotine(Nic)group,and methotrexate.In the(Methotrexate,MTX)group,liver,spleen,and synovium were taken after intragastric administration(one time/day)for 30 consecutive days.Western blot was used to detect the expression of A2 A protein in liver of each group.Immunohistochemistry was used to detect the expression of A2 A in spleen and synovial tissue.The effect of SIN on the expression of A2 A in AA model rats was initially observed.2.Changes of A2 A expression in the pathogenesis of AA modelThe modeling methods were the same as the above,grouped into control group,model group,SIN group,MTX group,administered intragastrically,once/day,and Western blot was used to detect the rats of the control group and model group at different time points(0,12,18,24,30d)Synovial,liver A2 A expression changes.At the peak time of A2 A expression change,the effect of SIN and MTX on A2 A expression was examined.3.Preliminary observation of the effect of SIN on the expression of A2 A in FLS by immunofluorescenceThe synovial tissues of rats in the model group were separated 30 days after model establishment.The primary FLS was cultured using the tissue block method.FLS,tumor necrosis factor-?(TNF-?)and LPS-induced FLS induction were detected by flow cytometry.In vitro proliferation or inflammation model,immunofluorescence initially observed the effect of SIN on the expression of A2 A protein in FLS.4.The effect of SIN on the proliferation of FLS induced by TNF-? and the effect of ?-BTX on the regulation of SIN-mediated A2 A expressionMTT assay was used to observe the effects of different concentrations of SIN(100?M,200?M and 400?M)on the proliferation of FLS induced by TNF-?;RT-PCR was used to observe the change of A2 A m RNA expression;ELISA was used to detect the proinflammatory cytokine monocyte chemotaxis in cell culture supernatants.Monocyte chemotactic protein 1(MCP-1)levels;Immunofluorescence observation of the antagonistic effect of ?7n ACh R-specific antagonist ?-BTX on SIN-mediated A2 A expression.5.Observing the effect of SIN on the anti-inflammatory effect of LPS-induced FLS and the effect of A2 A expression by silencing ?7n ACh RLPS-induced FLS inflammation models were used to compare the effects of si RNA silencing between ?7n ACh R and non-silencing groups on inflammatory cytokine release and A2 A expression.The expression of ?7n ACh R protein was detected by WB and the silencing effect was verified.The changes of pro-inflammatory cytokines MCP-1 and IL-6 in cell culture supernatant were detected by ELISA,and the expression of A2 A protein was detected by WB.Results:1.Effect of SIN on A2 A expression in AA model ratsCompared with the control group,the expression of A2 A in the liver tissue model group was decreased,and SIN,Nic and MTX could significantly up-regulate the expression of A2 A.The results of immunohistochemistry showed that the expression of A2 A in the spleen and synovial tissues was consistent with that in the liver.2.Changes of A2 A expression in the pathogenesis of AA modelIn the pathogenesis of AA rats,the expression of A2 A in the 0-30 d model group firstly decreased and then gradually increased.The 18 th day was the highest peak of AA model inflammation in rats,and the lowest expression of A2 A protein was negatively correlated with the development of RA.At 18 days,compared with the model group,both SIN and MTX could significantly increase A2 A expression.3.The effect of SIN on the expression of A2 A in FLS and its relationship with targeting ?7n ACh RCompared with the control group,TNF-? and LPS stimulation both down-regulated A2 A expression,and SIN up-regulated A2 A expression.In FLS,SIN targeting ?7n ACh R exerted anti-inflammatory effects,and after silencing ?7n ACh R,the up-regulation of SIN on A2 A was significantly attenuated.Conclusion:1.In vivo experiments demonstrated that SIN exerts anti-inflammatory effects while up-regulating A2 A expression.2.The expression of A2 A was negatively correlated with the pathogenesis of AA in rats.3.In vitro experiments,TNF-? and LPS promote FLS inflammation should occur,with A2 A downregulation,while SIN play an anti-inflammatory effect upregulated A2 A expression,and ?-BTX intervention can weaken the upregulation of SIN on A2 A.4.SIN targeting ?7n ACh R exerts anti-inflammatory effects and may upregulate A2 A expression through ?7n ACh R.