| Objectives: To explore the role of oxidative stress mediated by Nrf2/HO-1pathway,apoptosis mediated by ASK1/MKK3 pathway as well as inflammation mediated by p38 MAPK and TSC1/e-IF-2α/ATF4 pathways in Curcumin combined with Baicalin in improving ethanol-induced liver injury and its mechanism in rats.Methods: 1.Rats were randomly divided into control group,ethanol group（50%v/v）,curcumin group（50 mg/kg）,baicalin group（50 mg/kg）and curcumin（50 mg/kg）+ baicalin（50 mg/kg）group.2.After 6 weeks of the experiment,the rats in each group were sacrificed,and the liver was collected to calculate the liver organ coefficient.HE staining was used to observe the pathological changes of liver tissue.Biochemical assay was employed to detect the levels of serum liver function indicators（ALT,AST,AKP,TG and T-CHO）.3.Biochemical assay was used to determine the levels of liver oxidative stress indicators（GSH-Px,SOD,·OH,T-AOC,LPO）.ELISA method was employed to detect serum 8-OHd G level.RT-q PCR and Western Blotting were used to detect the expression levels of genes and proteins（Nrf2,HO-1 and NQO1）related to the Nrf2/HO-1 pathway in rat liver.4.TUNEL assay was used to evaluate hepatocyte apoptosis and the apoptosis index was further calculated.RT-q PCR and Western Blotting were used to detect the levels of genes and proteins（CHOP,GRP78,Caspase 3,Caspase 12,ASK1 and MKK3）related to apoptosis.5.ELISA method was used to measure liver inflammatory cytokines levels（TNF-α,INF-γ,IL-1β,NO,i NOS and TNOS）.RT-q PCR and Western Blotting were employed to detect the levels of p38 MAPK and TSC1/e-IF-2α/ATF4 pathways related genes and proteins（p38,Beclin1,LC3 BI,LC3BII,P62,TSC1,e IF-2α and ATF4）.6.Factorial variance analysis was used to analyze the interactive effects of curcumin and baicalin on the above indicators.Results: 1.HE staining showed that the arrangement of hepatocyte cords was disordered,fatty infiltration increased,and inflammatory cell infiltration around central vein enhanced in the ethanol group.The liver organ coefficient and serum liver function indexes（ALT,AST,AKP,TG and T-CHO）of the ethanol group were higher than those of the control group（P<0.05）.In the combination group,the pathological damage of liver tissue basically returned to normal,and the liver organ coefficient and serum liver function indexes were lower than those of the ethanol group,and its protective effect was significantly better than that of the curcumin or baicalin group（P<0.05）.2.Compared with the control group,the contents of oxidative stress indicators（·OH scavenging ability,SOD,GSH-Px and T-AOC）,the levels of Nrf2 and HO-1 mRNA as well as the ratio of p-Nrf2/Nrf2 decreased in the ethanol group（P< 0.05）,while the contents of 8-OHd G and LPO,the levels of Nrf2,p-Nrf2 and NQO1 protein as well as the level of NQO1 mRNA increased in the ethanol group（P<0.05）.Compared with the ethanol group,·OH scavenging ability,SOD,GSH-Px and T-AOC contents,Nrf2,HO-1 and NQO1 mRNA levels,Nrf2,p-Nrf2,NQO1 and HO-1 protein expression as well as p-Nrf2/Nrf2 ratio increased in the combination group（P<0.05）,while the contents of 8-OHd G and LPO decreased in the combination group（P<0.05）.And the ·OH scavenging ability,SOD and GSH-Px contents,NQO1 mRNA level,p-Nrf2,NQO1 and HO-1 protein expression and p-Nrf2/Nrf2 ratio of combination group were higher than those of the curcumin or baicalin group（P<0.05）.3.Compared with the control group,the apoptosis index,the mRNA and protein levels of GRP78,CHOP,Caspase 3 and Caspase 12,the protein levels of p-ASK1 and p-MKK3 as well as the ratios of p-ASK1/ASK1 and p-MKK3/MKK3 increased in the ethanol group（P<0.05）.Compared with the ethanol group,apoptosis index,the mRNA and protein levels of GRP78,CHOP,Caspase 3 and Caspase 12,the protein levels of p-ASK1 and p-MKK3 as well as the ratios of p-ASK1/ASK1 and p-MKK3/MKK3 decreased in the combination group（P<0.05）.And the apoptosis index,GRP78,CHOP and p-MKK3 protein expression and p-MKK3/MKK3 ratio of the combination group were lower than those of the curcumin or baicalin group（P<0.05）.4.Compared with the control group,the contents of inflammatory cytokines（TNF-α,IL-1β,INF-γ,NO,i NOS and TNOS）,the levels of p-p38,p-TSC1,p-e IF-2α,p-ATF4 and P62 protein as well as the ratios of p-p38/p38,p-TSC1/TSC1,p-ATF4/ATF4 and p-e IF-2α/e IF-2α increased in the ethanol group,while the levels of LC3 B and Beclin1 mRNA,the contents of Beclin1 and LC3 BII protein as well as the ratio of LC3BII/I decreased in the ethanol group（P<0.05）.Compared with the ethanol group,the levels of inflammatory cytokines,the contents of p-p38,p-TSC1,p-e IF-2α,p-ATF4 and P62 protein as well as the ratios of p-p38/p38,p-TSC1/TSC1,p-e IF-2α/e IF-2α and p-ATF4/ATF4 decreased in the combination group,while the levels of LC3 B and Beclin1 mRNA,the contents of Beclin1 and LC3 BII protein as well as the ratio of LC3BII/I increased in the combination group（P<0.05）.And in the combination group,the content of NO,the levels of P62,p-TSC1 and p-ATF4 protein as well as the ratios of p-TSC1/TSC1 and p-ATF4/ATF4 were lower than those of the curcumin or baicalin group,while the levels of Beclin1 and LC3 BII protein as well as the ratio of LC3 BII /I were higher than those of the curcumin or baicalin group（P<0.05）.5.Factorial variance analysis showed that curcumin combined with baicalin had a synergistic interaction on ·OH scavenging ability,GSH-Px and NO contents,NQO1,GRP78 and Caspase 3 mRNA levels,p-Nrf2,HO-1,LC3 BII and Beclin1 protein expression as well as p-Nrf2/Nrf2,p-ASK1/ASK1,p-MKK3/MKK3 and LC3BII/I ratios（P<0.05）.Conclusion: Curcumin combined with baicalin can ameliorate ethanol-induced liver injury in rats,and the combination therapy was superior to monotherapy.The hepatoprotective mechanism of curcumin combined with baicalin may be related to Nrf2/HO-1 pathway-mediated oxidative stress,ASK1/MKK3 pathway-mediated apoptosis as well as p38 MAPK and TSC1/e-IF-2α/ATF4 pathways-mediated inflammation. |
