Mechanism of Qishen Decoction inhibition of macrophage M1 type polarization by targeting TGR5-mediated NLRP3 inflammasome

Objective:To observe the effect of Qishen decoction on TGR5-mediated activation of NLRP3 inflammasome,so as to clarify the molecular mechanism of its inhibition of macrophage M1-type polarisation to ameliorate non-alcoholic steatohepatitis;Methods:Mouse macrophage cell line RAW264.7 was randomly divided into a control group,model group,Qishen decoction group,TGR5 agonist group and Qishen decoction+TGR5 agonist group.Except for the control group,the remaining groups were constructed the macrophage NLRP3 activation model by palmitic acid induction,and the corresponding drugs were given to intervene.ELISA was used to detect the levels of TNF-α,IL-6,IL-1βand CXCL2 in macrophage supernatants,flow cytometry was used to detect the expression levels of macrophage polarisation marker molecules CD86 and iNOS,and Western blot was used to detect the expression of the TGR5/STAT1/STAT6 signaling pathway and the expression of NLRP3 inflammasome-associated proteins,respectively.Results:Compared with the control group,the contents of macrophages TNF-α,IL-6,IL-1β,CXCL2 and the proportion of macrophages with positive expression of CD86 and iNOS were significantly increased in the model group,and the differences were all statistically significant(P<0.01).Compared with the model group,the contents of TNF-α,IL-6,IL-1β,CXCL2 and the proportion of macrophages with positive expression of CD86 and iNOS were significantly decreased in the Qishen decoction group,and the differences were all statistically significant(P<0.01).In addition,the expression of NLRP3 and Pro-IL-1βproteins in the macrophage lysate and the expression of Caspase-1 p10,Caspase-1 p20 and IL-1βp17 proteins in the cell supernatant of the model group were significantly increased when compared with the control group,and the differences were all statistically significant(P<0.01).Compared with the model group,the expression of NLRP3 and Pro-IL-1βproteins in macrophage lysate and the expression of Caspase-1 p10,Caspase-1 p20 and IL-1βp17 proteins in cell supernatant of the Qishen decoction were significantly reduced,and the differences were all statistically significant(P<0.01);Conclusion:Qishen decoction can inhibit the activation of NLRP3 inflammasome in macrophages by inhibiting the TGR5/STAT1/STAT6 signaling pathway,thereby inhibiting macrophage M1 polarization and improving inflammatory response.