Study On The Effects And Mechanisms Of Different Compatibility Of Envoy Herb In Xuefu Zhuyu Decoction On NAFLD-related Neutrophil Chemotaxis

Objective: Based on the preliminary research results of the effect of Xuefu Zhuyu Decoction on non-alcoholic fatty liver disease(NAFLD)and its mechanism,in the in vitro culture system,observe the different compatibility forms of medicated serum in Xuefu Zhuyu Decoction.The chemotaxis of neutrophils in C57BL/6J mice was stimulated by LPS,and the effect of different compatible forms of medicated serum of Xuefu Zhuyu Decoction on the chemotaxis of NAFLD-related neutrophils was evaluated,and Explore its mechanism of action,provide molecular support for the mechanism of Xuefu Zhuyu Decoction in the treatment of non-alcoholic fatty liver,and provide experimental basis for the theory of prescriptions and meridians.Methods: 25 SD rats were given 0.9% sodium chloride,Xuefu Zhuyu Decoction,Xuefu Zhuyu minus Niuxi Decoction,Xuefu Zhuyu minus Jiegeng Decoction,and Xuefu Zhuyu minus Niuxi and Jiegeng by Gavage 3 day.After intragastric administration for 3 days,blank serum and each drug-containing serum were obtained.10 C57BL/6J mice were selected aseptically extracted bone neutrophils for in vitro culture.The blank serum was divided into a Model group(M group)and a Positive Control group(PC group),and each corresponding medicine was divided into Xuefu Zhuyu Decoction(XFZY group).Xuefu Zhuyu decoction minus Niuxi group(XFZY-N group),Xuefu Zhuyu decoction minus Jiegeng group(XFZY-J group),Xuefu Zhuyu decoction minus Niuxi and Jiegeng group(XFZY-NJ),PC group was given p38 MAPK inhibitor SB203580 Intervention.After each group was given100ng/m L LPS intervention culture,Transwell Tissue Culture Systems measured neutrophil migration ability,Western-Blot method was used to detect p38 MAPK,phospho-p38 MAPK,MKP7 protein expression in each group,and ELISA method was used to detect MPO in cells.After cells in each group were cultured for 24 hours without LPS stimulation,flow cytometry was used to determine the apoptosis rate of neutrophils in each group.Western-Blot method was used to detect Bcl-xl,Bad,Bak,Bax,Cyt c,Caspase-9 and Caspase-3,AIF,Smac protein expression levels.Results:(1)Neutrophil chemotaxis: Compared with group PC and XFZY group,XFZY-NJ group neutrophils increased significantly(P<0.05).(2)Neutrophil apoptosis rate: Compared with group M,the apoptosis rate of each group was significantly increased(P<0.05);compared with group PC and XFZY,group XFZY-N,XFZY-J The apoptosis rate of the XFZY-NJ group and XFZY-NJ group was significantly reduced(P<0.05).(3)p38MAPK: Compared with the group M,the relative expression of p38 MAPK in the cells of each group was significantly reduced(P<0.05);compared with the group PC,the XFZY group,XFZY-N group,XFZY-J and XFZY-NJ group Significantly increased(P < 0.05);compared with XFZY group,XFZY-J group increased(P<0.05).(4)phospho-p38MAPK: Compared with group M,the relative expression of phospho-p38 MAPK in XFZY group,XFZY-N group and XFZY-J group decreased significantly(P < 0.05);compared with XFZY group,XFZY-NJ group increased significantly(P<0.05)).(5)MKP7: Compared with the group PC,the relative expression of MKP7 in the cells of the XFZY-N,XFZY-J,and XFZY-NJ groups was significantly reduced(P <0.05);compared with the XFZY group,the XFZY-J group and the XFZY-NJ group Increased significantly(P <0.05).(6)Bcl-xl:Compared with the XFZY group,the relative expression of Bcl-xl in the cells of the XFZY-N,XFZY-J and XFZY-NJ groups all increased(P <0.05).(7)Bad: Compared with group M and group PC,the relative expression of Bad in the cells of XFZY group and XFZY-N group was significantly increased(P <0.05).(8)Bak: Compared with the group PC and the XFZY group,the relative expression of Bak in the XFZY-NJ group was significantly increased(P <0.05).(9)Bax: Compared with the XFZY group,the relative expression level of Bax in the XFZY-J group and XFZY-NJ group was significantly reduced(P <0.05).(10)Cyt.c: Compared with the group PC and the XFZY group,the relative expression of Cyt.c in the XFZY-J group and the XFZY-NJ group was significantly reduced(P <0.05).(11)Caspase-9: Compared with the group PC and the XFZY group,the relative expression of Caspase-9 in the XFZY-J group and the XFZY-NJ group was significantly reduced(P <0.05).(12)Caspase-3: Compared with the XFZY group,the relative expression of Caspase-3 in the cells of the XFZY-NJ group was significantly reduced(P <0.05).(13)AIF:Compared with group M,the relative expression of AIF in XFZY group cells decreased statistically(P <0.05).(14)Smac: There was no statistical difference between Smac groups(P >0.05).(15)MPO: Compared with the group PC and XFZY group,the XFZY-N group,XFZY-J and XFZY-NJ The group was significantly higher(P<0.05).Conclusion: 1.Xuefu Zhuyu Decoction can inhibit the migration of neutrophils induced by LPS,there by playing a certain preventive and therapeutic effect on NAFLD;2.Xuefu Zhuyu Decoction can reduce the relative expression of p38 MAPK in neutrophils and inhibit LPS /p38 MAPK signaling pathway,inhibits neutrophil chemotaxis and reduces the expression of inflammatory factor MPO;3.Xuefu Zhuyu Decoction can promote neutrophil apoptosis;4.Xuefu Zhuyu Decoction can effectively down-regulate apoptosis inhibitor protein The expression of Bcl-xl up-regulates the expression of pro-apoptotic proteins Bax,Bak,Bad,Cyt.c,Caspase-9,Caspase-3;5.The compatibility of the meridian-inducing drugs Achyranthes bidentata and Platycodon grandiflorum contributes to the above-mentioned functions of Xuefu Zhuyu Decoction Play a synergistic effect.