| Objective: To investigate the protective mechanism of paeonol on Alcoholic liver disease(ALD)and clarify the effects of CYP2E1/Nrf2 and TLR4/NF-κB signaling pathways in the alleviation of ALD by paeonol.Methods: In vivo experiments: Forty-two C57BL/6J male mice were randomly and equally divided into: control group,model group,silybin(36.8 mg/kg)group and high(480 mg/kg),medium(240 mg/kg),low(120 mg/kg)dose group of Paeonol.The liver index was measured,and HE staining was used to observe the pathological changes in liver and ileum.Oil red O staining was used to observe the fatty lesions in liver tissue.The levels of TG,T-CHO,ALT and AST in serum,the contents of CAT,GSH,T-SOD,MDA and MPO in liver were determined.The contents of TNF-α,IL-1β,IL-6 and LPS in serum were determined by ELISA.The immunofluorescence assay was used to detect the expression and distribution of Nrf2,TLR4 and NF-κB proteins.The immunohistochemistry was used to detect the expression and distribution of CYP2E1,TLR4,Nrf2,NF-κB and ZO-1 and Claudin-1 proteins in ileum.Western blot was used to detect the expression of CYP2E1/Nrf2,TLR4/NF-κB related pathway proteins in liver tissue.The q RT-PCR method was used to detect the expression of Cyp2e1,Nrf2,Tlr4 and Nf-κb genes in liver tissue.In vitro experiments: Macrophages RAW264.7were cultured and divided into control group,LPS(1 μg/m L)group,Paeonol(240μmol/m L),TAK242(10 μmol/m L)group and ML385(5 μmol/m L).The CCK8 method was used to detect cell activity,and the cell morphology was observed under an inverted microscope.The contents of GSH and MDA in cell culture medium were determined by colorimetric method,and the contents of TNF-α and IL-6 in cell culture medium were determined by ELISA.The mitochondrial membrane potential of macrophage 264.7was detected by JC-1 method,the expression and distribution of F4/80 and TLR4 proteins in macrophage 264.7 were detected by immunofluorescence assay,and the expressions of Nrf2,HO-1,TLR4,IκBα and NF-κB proteins in macrophage 264.7 were detected by Western blot.Results: In vivo experiments: Compared with the model group,Paeonol improved the liver index of mice(P<0.001),reduced the contents of ALT,AST,T-CHO and TG in serum of mice(P<0.001),and improved the pathological changes and fat particle accumulation in liver and ileum tissues of mice.The contents of MDA and MPO in mouse liver tissue were decreased(P<0.05),the contents of CAT,GSH,and T-SOD in mouse liver tissue were increased(P<0.05),and the contents of IL-6,IL-1β,TNF-α,and LPS in mouse serum were decreased(P<0.01).Immunofluorescence showed upregulated the expression and distribution of Nrf2 protein(P<0.05),and down-regulated the expression and distribution of TLR4 and NF-κB proteins(P<0.05).The immunohistochemical results showed that the expression and distribution of Nrf2 protein in liver tissue and ZO-1 and Claudin-1 protein in ileum were increased(P<0.01),and the expression and distribution of CYP2E1,TLR4 and NF-κB proteins in liver tissue were decreased(P<0.05).Western blot results showed that the expression levels of CYP2E1,TLR4,My D88,IκBα and NF-κB proteins in the mouse liver tissue were down-regulated(P<0.05),and the expression levels of Nrf2 and HO-1 proteins were upregulated(P<0.05).q RT-PCR results showed up-regulation of Nrf2 gene expression(P<0.001)and down-regulation of Cyp2e1,Tlr4,and Nf-κb gene expression(P<0.001).In vitro experiment: Compared with the blank group,the cell viability induced by 1μg/m L LPS was 0.497± 0.061(P<0.01),close to half the inhibition rate.Compared with the LPS group,the cells pretreated with 240 μmol/m L Paeonol down-regulated the expression of NF-κB protein most significantly(P<0.01),and the cells pretreated with10 μmol/m L TAK242 inhibited the expression of TLR4 protein most significantly(P<0.01).Compared with the control group,5 μmol/m L ML385 significantly inhibited the expression of Nrf2 protein(P<0.01).Compared with the LPS group,the cell morphology of the Paeonol group was restored.The content of MDA in cell culture medium was decreased and the content of GSH was increased(P<0.01).Reduce that expression of inflammatory factors in cell culture medium(P<0.01).In the mitochondrial membrane potential results,the red light was significantly enhanced and the green light was significantly weakened in the Paeonol group(P<0.001).The protein expression profiles of F4/80 and TLR4 in the Paeonol group were significantly decreased(P<0.01),and the protein expression profiles of TLR4,IκBα,and NF-κB were down-regulated(P<0.05),and the protein expression profiles of Nrf2 and HO-1were up-regulated(P<0.05).Conclusion: Paeonol can improve alcoholic liver injury,and its mechanism may be related to the CYP2E1/Nrf2 and TLR4/NF-κB signaling pathways,to reduce the fatty lesions,inflammatory damage and oxidative stress damage of hepatocytes. |
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