Study On The Regulation Of TLR Signaling Pathway From Ubiquitin Proteasome Pathway To Study The Mechanism Of Bushen Recipe Against Liver Injury

Objective:To study the mechanism of Bushen Recipe against liver injury by regulating the TLR signaling pathway from the ubiquitin proteasome pathway.Methods:The research is divided into two parts.The first part is the research on the effect of Bushen Recipe on liver injury,and the second part is the research on the mechanism of Bushen Recipe against liver injury.The first part of the study of Bushen Recipe on anti-liver injury:35 normal mice and HBV transgenic mice were randomly divided into normal group,model group,Bushenquanfang group and disassembled group(complementing kidney and yin group,tonifying kidney yang)Group,Qinghua Damp-heat group,and drug-induced group).The drug was administered once a day at the same time for one week,and after 4 hours at the end of the seventh day,each group of mice(except the normal group)was injected with 15 mg/kg of concanavalin A in the tail vein.After 18 hours,the mice were sacrificed by eyeball removal.Detection of liver function,liver histopathology,peripheral blood related inflammatory factors.The second part of the mechanism of Bushen Recipe against liver injury:35 normal mice,Nrdp1 knockout mice,male and female,randomly divided into normal group,model group,Bushenquanfang group and split group(tonic kidney)Yin group,Bushenyang group,Qinghua damp heat group,and medicine group).The model administration method is the same as the first part.ELISA was used to detect the related inflammatory factors mediated by TLR signaling pathway.Westrn Blot was used to detect the expression of key proteins TBK1,IRF3,TLR9,TLR4,TLR3,TRAF6,NF-kB,TRIP and MyD88 in TLR pathway.Results:The first part of the study on the effect of Bushen Recipe on liver injury:1.The effect on liver function indicators.The ALT levels in the normal mice,the Bushenyin group and the Qinghua group were significantly decreased(P<0.001).The AST(P<0.05),TBIL(P<0.05)and ALT in the Bushenyang group were all in the tonifying kidney group.P<0.05),AST(P<0.05)and TBIL(P<0.05)in the Qinghua group and ALT(P<0.05)in the medicated group significantly decreased;DBIL(P<0.01)and Bushenyang in the tonifying kidney group The DBIL of the group(P<0.01)and the DBIL of the Qinghua group(P<0.01)decreased significantly,and the ChE of the kidney group was significantly increased(P<0.01).The ALT of the whole kidney group of HBV transgenic mice(P<0.01)TBIL(P<0.01)and ALT(P<0.01)in the Bushenyang group and ALT and DBIL(P<0.01)in the Qinghua group were significantly decreased,and the ChE(P<0.01)in the Bushenyin group was significantly increased.AST(P<0.05),DBIL(P<0.05)and ALT(P<0.05),TBIL(P<0.05)in the Bushen Yin group and DBIL(P<0.05)in the Bushenyang group were significantly decreased.ChE(P<0.05)was significantly elevated.2 effects on liver histopathology.The area of the necrotic area in the tonifying kidney group and the Bushenyang group in the normal mice and the HBV transgenic mice was significantly reduced compared with the model group,and the area of the necrotic area in the other administration groups was reduced.3.The influence of peripheral blood related inflammatory factors.After administration,the expression of TNF-α(P<0.01)was significantly decreased in normal mice with IL-1(P<0.05),IL-6(P<0.05),IFN-γ(P<0.05).The expressions of IL-1(P<0.05)and IFN-y(P<0.05)in Bushenyang group were significantly decreased.The expressions of IL-6(P<0.05)and IFN-γ(P<0.05)in Bushenyin group were significantly decreased.The expression of IL-1(P<0.01),TNF-α(P<0.05)and IL-6(P<0.05)in HBV transgenic mice was significantly decreased.The expression of IL-1(P<0.01)and TNF-α(P<0.05)was significantly decreased in the yang group;the expression of IL-1(P<0.05)and IL-6(P<0.05)was significantly decreased in the spleen-negative group;The expression of IL-1(P<0.05)and IL-1(P<0.05)in the drug-induced group was significantly decreased.The second part of the mechanism of Bushen Recipe against liver injury:1.The effect on liver function indicators.In normal mice(Nrdp1 not knocked out),the expression of ALT(P<0.001)in the tonifying kidney group was significantly decreased.The AST(P<0.01)and TBIL(P<0.01)in the tonifying kidney group and the ALT in the tonifying kidney group(P<0.01),the expression of ALT(P<0.01)in the Bushenyin group was significantly decreased;the ChE(P<0.01)in the tonifying kidney group and the ChE(P<0.01)in the tonifying kidney group were significantly increased;DBIL(P)in the tonifying kidney group<0.05),AST(P<0.05)and TBIL(P<0.05)in Bushenyang group,AST(P<0.05),TBIL(P<0.05)and DBIL(P<0.05)in Bushen Yin group,Qinghua group The expressions of ALT(P<0.05),AST(P<0.05),TBIL(P<0.05),DBIL(P<0.05)and ALT(P<0.05)in the menstrual group were significantly lower;ChE in the Bushenyang group<0.05),the expression of ChE(P<0.05)in the Qinghua group was significantly increased.The expression of ALT(P<0.001)in the spleen-negative group was significantly decreased in Nrdpl knockout mice,TBIL in the tonifying kidney group(P<0.01),ALT in the tonifying kidney group(P<0.01),and ALT in the tonifying kidney group(P<0.01),the expression of TBIL(P<0.01)in the Qinghua group was significantly decreased;AST(P<0.05),DBIL(P<0.05)in the tonifying kidney group,AST(P<0.05)and TBIL in the Bushenyang group(P<0.05),DBIL(P<0.05),AST(P<0.05),TBIL(P<0.05),DBIL(P<0.05)in the Bushen Yin group,ALT(P<0.05)and AST in the Qinghua group(P<0.05)The expressions of 0.05),DBIL(P<0.05)and ALT(P<0.05)were significantly decreased;ChE in the kidney group(P<0.05),ChE in the Bushenyang group(P<0.05),and ChE in the Bushenin group(P<0.05),the expression of ChE(P<0.05)in the Qinghua group was significantly increased.2.Expression of related inflammatory factors mediated by TLR signaling pathway.TNF-α(P<0.0001),IL-6(P<0.0001),IL-1(P<0.0001)and TNF-β in normal kidney(Nrdpl not knockout)and Nrdp1 knockout mice(P<0.0001)was significantly decreased in IL-6(P<0.0001),IL-1(P<0.0001),TNF-β(P<0.0001)and TNF of Qinghua group in Ningp1 knockout mice.The expressions of-a(P<0.0001),IL-6(P<0.0001),and IL-1(P<0.0001)were significantly decreased.The expression of TNF-α(P<0.001),IL-6(P<0.001)and TNF-β(P<0.001)in the Bushenyang group was significantly lower in normal mice(Nrdp1 not knocked out).3.Effects on key proteins in the TLR signaling pathway.TLR9(P<0.001),TLR3(P<0.001),NF-kB(P<0.0001),and TLR4(P<0.0001)were significantly decreased in normal mice(Nrdp1 not knocked out).TLR9 in Bushenyang group(P<0.001)and MyD88(P<0.0001)were significantly decreased;TLR3(P<0.001),MyD88(P<0.001),and NF-kB(P<0.01)were significantly decreased in the Bushen Yin group.TLR4(P<0.001),MyD88(P<0.001),TLR3(P<0.01)and NF-kB(P<0.01)were significantly decreased in the Ndp1 knockout mice.TLR4 in the Bushenyang group(P<0.001)MyD88(P<0.01)and TRAF6(P<0.05)decreased significantly;MyD88(P<0.01)and NF-kB(P<0.01)decreased significantly in the Bushen Yin group.Conclusion:1.Bushen Recipe has significant anti-liver damage effects.2.The dissection study showed that the Bushenyang group had a better effect on lowering the levels of ALT,AST,IL-1 and TNF-α.The Bushen Yin group had a better increase in ChE levels and decreased IL-6 and IFN-y levels.The role of the Qinghua group has a better effect on reducing the levels of TBIL and DBIL,indicating that different disassembled parties have synergistic effects to play a role in anti-liver injury.In Nrdp1 knockout mice,Bushen Recipe has an inhibitory effect on the expression of key proteins in TLR signaling pathway.In normal mice(Nrdp1 not knocked out),Bushen Recipe has a stronger inhibitory effect on the expression of key proteins in TLR signaling pathway.It can be shown that Bushen Recipe has a ubiquitin ligase-like effect,and regulates the inflammatory signaling pathway of TLR through the ubiquitin proteasome pathway,and finally achieves its anti-liver injury effect.