Study On The Molecular Mechanism Of Anti-inflammation Effect Of SiNi Decoction In Vitro Based On P38 MAPK And ERK1/2 Signaling Pathway

Objective:To establish an inflammatory model of RAW264.7 cells induced by lipopolysaccharide(LPS),to study the anti-inflammatory effect of Sini Decoction in vitro,and to explore whether the anti-inflammatory effect of Sini Decoction can play a role through P38 MAPK and ERK1/2 signaling pathway.Method:1.Sini decoction was prepared according to the method recorded in Chinese pharmacopoeia in 2010 and packed separately for use;MTT method was used to detect cell viability,so as to determine the best concentration of Sini decoction;2.Lipopolysaccharide(LPS)induces RAW264.7 cells to establish inflammatory model.The content of nitric oxide(NO)in cell supernatant was detected by Griess method,and the content of lactate dehydrogenase(LDH)in supernatant was detected by biochemical enzyme method;3.RT-q PCR was used to detect the transcription level of IL-6,IL-10,i NOS and COX-2 m RNA,and ELISA was used to detect the secretion of IL-6 and IL-10 inflammatory factors;4.The expression levels of P38,p-P38,ERK and p-ERK were detected by Western blot;5.After the action of pathway inhibitors(SB203580 and PD98059),the release level of nitric oxide(no)in the cell supernatant was detected according to certain experimental groups(normal group,model group,inhibitor group,LPS + inhibitor group,LPS + drug group,LPS + drug + inhibitor group,a total of six groups),and the change of protein expression corresponding to each inhibitor was detected by Western blotting.Result:1.The optimal concentration of Sini decoction was determined as 2.1mg/m L,2.4mg/m L,2.7mg/m L and 3.0mg/m L by MTT method.The results showed that there was no statistical difference between Sini Decoction group and normal group by detecting LDH secretion in cell supernatant;2.The optimal concentration of Sini Decoction inhibited the no level(P<0.05)and LDH level(P<0.05)of RAW264.7 cells induced by LPS in varying degrees;3.SND inhibited the secretion of IL-6 and IL-10 to a certain extent(P<0.05),and significantly reduced the transcription levels of IL-6,IL-10,i NOS and COX-2 m RNA(P<0.01);4.SND adjusted the phosphorylated protein expression of P38 and ERK1/2 induced by LPS(P<0.05);5.After using the pathway inhibitor(SB203580 and PD98059),the NO secretion in the supernatant of the cells in the inhibitor + Sini Decoction group,LPS + inhibitor group and LPS + Sini Decoction + inhibitor group decreased(P<0.05),and the NO secretion in the LPS + Sini Decoction + inhibitor group decreased most significantly(P<0.01);after using the same pathway inhibitor,the NO secretion in the inhibitor + Sini Decoction group and LPS + inhibitor group was compared with that in the LPS group The protein phosphorylation level of the two groups in the preparation group decreased(P<0.05),and the inhibition of P38 and ERK1/2 protein phosphorylation level in the LPS + Sini Decoction + inhibitor group was the most significant(P<0.01).Conclusion:Sini decoction has a certain anti-inflammatory effect in LPS induced RAW264.7 macrophage inflammatory model in vitro.The mechanism of its anti-inflammatory effect is related to P38 MAPK and ERK1/2 signaling pathway.It can activate the signaling pathway through phosphorylation of some key proteins,such as P38 and ERK,and regulate the expression of inflammatory factors and m RNA transcription,so as to finally exert the anti-inflammatory effect.