Si Nomenine Inhibits Inflammation Through ? 7nAChR In Endotoxemia Mice

Objective:A large body of study have existed on the anti-inflammation effect of sinomenine,but which target that sinomenine binds to on the immune cells is not known.Our previous study has demonstrated that macrophages RAW264.7 expresses a 7 nicotine acetylcholine receptor,which is one of the essential target of sinomenine that has an immunization effects of inflammation.Firstly,this study investgate whether sinomenine has an anti-inflammation effect on endoxemia in mice via a 7 nicotine acetylcholine receptor,then we investgate the expression of a 7 nicotine acetylcholine receptor on different organs in mice after treatment with sinomenine.Base on the above,we explain the mechanism of immunopharmacology of sinomenine exerting anti-inflammation effects,which lay a basis for the mechanism of immunopharmacology of sinomenine exerting anti-inflammation effects.Methods:1.The effects of sinomenine on the endotoxemia model in mice and the relationship between sinomenine and a 7nAChRIn this study,we established the endotoxemia models in mice through the intraperitoneal injection of lipopolysaccharide(LPS).The nicotine used as the positive control and the berberine as the negative.Firstly,we confirmed the IC50 dose of LPS before we had found out the effective anti-inflammation dose of sinomenine,nicotine and berberine.Meanwhile,we investgated the effects of mortality after treatment with sinomenine or nicotine.Then we set various timings,and found out the timing of peak value through the level of serum TNF-a and IL-6 measured by ELISA(enzyme-linked immuno sorbent assay)with the time goes by on the endotoxemia model in mice.Further,we used the nicotine as the positive control and observed the anti-inflammation effects of sinomenine after treatment with mecamylamine,the specific antagonist of nAChR.To study whether the specific antagonist of nAChR can inhibit the anti-inflammation effects of sinomenine,the level of serum TNF-? and IL-6 was measured by ELISA.Finally,we used the nicotine as the positive control and the berberine as the negative one and observed the anti-inflammation effects of sinomenine after treatment with a-bungarotoxin(a-BTX),the specific antagonist of a 7nAChR.To study whether the specific antagonist of a 7nAChR can inhibit the anti-inflammation effects of sinomenine,the level of serum TNF-? and IL-6 was measured by ELISA.2.The effects of sinomenine on the expression of a 7nAChR on the endotoxemia model in miceBALB/c mice were used in this study.Mice were injected intraperitoneally with sinomenine or nicotine or berberine 30min ago before the endotoxemia models were established through the intraperitoneal injection of LPS.A patr of mice' peritoneal macrophages were extracted at 3h after injection with LPS.Another part of mice' liver,spleen,thymus,brain and the superior mesenteric lymph nodes were taken at 6h after injection with LPS,and were fixed in 4%paraformaldehyde or kept at-70?.The expression of a 7nAChR on peritoneal macrophages was measured by immunofluorescence assay;The expression of a 7nAChR on liver,spleen and thymus was measured by Immunohistochemistry and RT-PCR;The expression of a 7nAChR on brain and the superior mesenteric lymph nodes was measured by wesrern blot and RT-PCR.The effects of sinomenine on the expression of a 7nAChR were study in these experiments.Results:1.The effects of sinomenine on the endotoxemia model in mice and the relationship between sinomenine and a 7nAChRAs the data shown,the mortality was 66.7%in mice after injection with 40 mg/kg LPS during 72h,whereas both 80mg/kg sinomenine and 2mg/kg nicotine pre-injected 30min ago could significantly reduce the mortality on the endotoxemia models of mice.The mortality of mice reduced to 0%in sinomenine group,and 22.20%in nicotine group,respectively.The endotoxemia models of mice were successfully established after injection with 15mg/kg LPS,and the timing peak value of the serum TNF-a and IL-6 in mice was 1h and 3h,respectively.However,among the dose of 40mg/kg,80mg/kg and 160mg/kg of sinomenine could suppress the serum inflammatory cytokine on this models with dose-dependent manner effectively.The serum inflammatory cytokine on this models was effectively reduced after treatment with 2mg/kg nicotine and 20mg/kg berberine.The anti-inflammatory effects of 40mg/kg sinomenine and 2mg/kg nicotine were reversed by 2mg/kg mecamylamine effectively.The anti-inflammatory effects of 40mg/kg sinomenine,2mg/kg nicotine and 20mg/kg berberine were reversed by 8 ?g/kg a-BTX effectively.2.The effects of sinomenine on the expression of a 7nAChR on the endotoxemia model in miceTo establish the endotoxemia models,mice were injected 15mg/kg LPS.Mice were injected intraperitoneally with drugs before models had established 30min ago.As the results shown that LPS could increase the expression of a 7nAChR of peritoneal macrophage,liver,spleen,thymus,superior mesenteric lymph nodes and brain in mice,whereas sinomenine could reduce the LPS-induced increased expression of a 7nAChR,Ber had no significantly effect on the expression of a 7nAChR.However,the expression of a 7nAChR of peritoneal macrophage,liver and brain was decreased by nicotine,while it had an adverse effect on spleen,thymus and superior mesenteric lymph nodes.Conclusion:1.Sinomenine plays an important role in anti-inflammation effects on the endotoxemia model in mice via a 7nAChR,and a 7nAChR is an essential target of sinomenine exerting anti-inflammation effects in vivo.2.Sinomenine inhibits the expression of a 7nAChR in various tissues and organs on the endotoxemia model in mice.