Screening Of Liver Protecting Chinese Herbs And Study On The Alleviating Effect Of Oxidative Stress Of Lateolabrax Maculatus

1.Establishment of oxidative damage model of primary hepatocytes of L maculatusIn this study,the primary hepatocytes of L maculatus were cultured in vitro and treated with different concentrations of H₂O₂（50,100,200,400 and 800μM）for different time（2 h and 4 h）to establish the oxidative damage model of the primary hepatocytes of L maculatus.The activities of GPT,GOT and LDH in supernatant of cell culture were determined by CCK-8 assay.The results showed that the survival rate of hepatocytes could be decreased by different concentrations of H₂O₂ for different time,and the decreasing trend was consistent.The survival rate of hepatocytes reached 68.01%.when the concentration of H₂O₂ was 200μm and the treatment time was 2 h.The activities of GPT,GOT and LDH in the supernatant of hepatocytes increased first and then decreased with the increase of H₂O₂ concentration,and reached the maximum when the concentration of H₂O₂ was 200μM.In conclusion,200μm H₂O₂ treatment for 2 h is the best condition to establish the model of oxidative damage of hepatocytes in primary culture of L maculatus.2.Protective effect of Chinese herbal medicine on oxidative damage of hepatocytes of L maculatusIn this study,the protective effect of Chinese herbal medicine on the oxidative damage of hepatocytes of primary cultured L maculatus was explored through the established H₂O₂oxidative damage model.Ten kinds of single prescription and 10 groups of compound traditional Chinese medicine（M1,M2,M3,M4,M5,M6,M7,M8,M9 and M10）were selected and decocted into water extracts with a final concentration of 5 mg/m L.The primary cultured hepatocytes of L maculatus were treated with 20 kinds of water extracts for 4 h,and then treated with 200μm H₂O₂ for 2 h for oxidative damage.Afterwards,the activities of intracellular enzymes（GPT,GOT and LDH）and antioxidant indexes（T-AOC,SOD and MDA）in the culture medium were determined.The results showed that the activities of GPT,GOT and LDH in culture medium were significantly higher than those in control group（P<0.05）,the activities of T-AOC and SOD in liver cells were significantly lower than those in control group（P<0.05）,and the content of MDA in liver cells was significantly higher than that in control group（P<0.05）.After oxidative damage with H₂O₂,the activities of GPT,GOT and LDH in Chinese herbal treatment groups were decreased,and the activities of GPT,GOT and LDH in curcuma,Schisandra chinensis and M9 groups were significantly lower than those in other Chinese herbal treatment groups（P<0.05）.The activities of T-AOC and SOD in curcuma and M9 groups were significantly higher than those in other groups（P<0.05）,and the content of MDA was significantly lower than that in other groups（P<0.05）.In conclusion,among the 10 monotherapy and 10 groups of compound Chinese herbal medicines in this study,rhubarb,Salvia miltiorrhiza,ginkgo biloba,turmeric,licorice,paeonia lactiflora,porcine glabra,schisandra chinensis,M1,M4,M6,M7 and M9 groups can all alleviate oxidative damage of hepatocytes of primary cultured perch induced by H₂O₂,among which turmeric and M9 groups have the strongest protective effect.3.Effects of dietary Chinese herbal medicine on growth performance and liver antioxidant capacity of L maculatusL maculatus were fed diets supplemented with 0.2%and 0.4%turmeric water extract and0.2%and 0.4%Chinese herbal medicine water extract in M9 group.The breeding experiment lasted for 56 days.After the experiment,the growth performance indexes of seabbass were determined as follows:WGR,SGR,FR and SR;Serum biochemical indices:GPT,GOT and LDH;Liver antioxidant indexes:SOD,CAT,T-AOC,GSH-Px and MDA;And the relative expression levels of anti-oxidation-related genes in liver:Nrf2,Keap1,HO-1 and GCLC.The results showed that the diet supplemented with 0.4%turmeric significantly promoted the growth of L maculatus（P<0.05）,while the diet supplemented with M9 had no significant effect on the growth of L maculatus（P>0.05）.The activities of serum GPT and GOT in groups supplemented with 0.2%and 0.4%turmeric and 0.2%M9 were significantly decreased（P<0.05）.The activities of antioxidant enzymes（CAT,GSH-Px and T-AOC）in liver were significantly increased by adding different levels of curcuma and M9 groups（P<0.05）,and the content of MDA was significantly decreased by adding 0.4%of curcuma and M9 group（P<0.05）.The relative expression levels of Nrf2,HO-1 and GCLC in liver of L maculatus in different levels of turmeric and M9 group were significantly up-regulated（P<0.05）.In conclusion,both curcuma and M9 groups can improve the antioxidant capacity of liver of L maculatus,and 0.4%curcuma and 0.4%M9 groups have the best protective effect.4.Protective effect of Chinese herbal medicine on acute oxidative stress of L maculatusAt the end of the fourth chapter,H₂O₂ oxidation damage test was carried out.The liver oxidative damage of L maculatus was induced by intraperitoneal injection.The survival of seabass was observed 48 hours later.Serum biochemical indexes（GPT,GOT,LDH）,liver antioxidant indexes（SOD,CAT,T-AOC,GSH-Px,MDA）and relative expression levels of antioxidant related genes（Nrf2,Keap1,HO-1,GCLC）were analyzed.The results showed that after the injection of H₂O₂,the survival rate of Chinese herbal medicine in curcuma and M9group was significantly higher than that in the injured group（P<0.05）,and the morphology of liver tissue tended to the control group.The activities of GPT and GOT in serum in curcuma and M9 groups were significantly lower than those in injury group（P<0.05）,the activities of T-AOC and GSH-Px in liver were significantly higher than those in injury group（P<0.05）,and the content of MDA was significantly lower than that in injury group（P<0.05）.There was no significant difference in MDA content between 0.4%turmeric and 0.4%M9 group（P>0.05）.The relative expression levels of Nrf2,HO-1 and GCLC in liver of L maculatus were significantly up-regulated by different levels of turmeric and M9 groups（P<0.05）,and the effect was the best when the supplemental level of 0.4%of turmeric and M9 group.Therefore,the addition of 0.4%turmeric and 0.4%M9 group of Chinese herbal medicine can better protect the liver and reduce H₂O₂ oxidative damage.In conclusion,among the 10 single prescription and 10 groups of Chinese herbal medicine,turmeric and M9 group of Chinese herbal medicine can significantly enhance the antioxidant capacity of liver cells of L maculatus,and have a good protective effect on H₂O₂-induced oxidative damage of liver cells.Different levels of turmeric and M9 groups could increase the antioxidant capacity of liver,and the antioxidant capacity of 0.4%turmeric and 0.4%M9 group was the strongest.The addition of different proportions of turmeric and M9 group could significantly improve the survival rate after acute oxidative injury,in which the addition of 0.4%turmeric and 0.4%M9 groups could better protect the liver,the specific mechanism may be related to the activation of Nrf2-Keap1 signaling pathway.