The Study Of Sanwei Ganjiang San Of Experimental Liver Injury Animal Model And Components Of Ingested Into Blood

ObjectiveTo investigate the hepatoprotective mechanism and components absorbed into blood of Sanwei Ganjiang San(SWGJS),we study the protection of liver mitochondrial structure and function,establish an alcoholic liver injury model and a ConA immunological liver injury model,so as to make a foundation for the further research about SWGJS.Methods1.72 SD rats were randomly divided into normal group,alcohol model group,silymarin positive group,SWGJS high,medium and low dose intervention group.In addition to the normal group,the rats were fed with 55%alcohol in the morning and fed with the corresponding dose of distilled water in the normal group for 10 days.The rat model of alcoholic liver injury was prepared.After 12 hours of the last administration,2%sodium pentobarbital anesthesia,collection of abdominal aorta blood,separation of serum for detection of liver function indexes of ALT,AST,liver was used for HE staining for pathological observation,the extracted liver mitochondria were used to detect mitochondrial membrane potential,membrane permeability,SOD,8-OHdG,respiratory chain enzyme complex Ⅰ,Ⅲ;preparation of liver homogenate to detect MDA,GSH,GSH-Px,the expression of Nrf2,Bachl and HO-1 mRNA in hepatocytes was detected by RT-PCR,the expression of Nrf2,Bachl and HO-1 protein in liver tissue was detected by Western Blot.2.72 KM mice were randomly divided into normal group,ConA model group,silymarin positive group,SWGJS high,medium and low dose intervention group.In addition to the normal group,each group were administered corresponding drug Volume 1 times,continuous 7d,normal groups with distilled water of equal volume in the last administration after 12h,except the normal group,other groups were injected into the tail vein of ConA 25mg/kg,preparation of ConA immune liver injury model.After modeling 4h eye blood collection,the isolated serum was used to detect liver function ALT and AST,the isolated liver was used for HE staining for pathological examination,extraction of liver mitochondriawas detected by mitochondrial membrane potential,SOD,8-OHdG,preparation of liver homogenate for determination of MDA,GSH,GSH-Px.3.Through the determination of UPLC-MS and the establishment of the chemical composition database of SWGJS,the samples of SWGJS containing serum,blank serum and single or compound were screened by Peakview software.With 6-gingerol reference substance,looking for SWGJS into the blood composition.Results1.The levels of ALT and AST in serum of alcohol model group were increased after 10 days of injection with 55%alcohol for SD rats(P<0.01).HE staining showed that the boundaries between the liver lobules in the alcohol model group were not very clear,the hepatic cell cord was disturbed,the hepatic sinus range was narrowed,the liver cells were also swollen,and the cytoplasm was loose,Most of the liver cells appear swelling,showing a balloon-like lesions.The fluorescence intensity ratio of alcohol model group decreased,indicating that JC-1 monomer increased,membrane potential △Ψm decreased(P<0.05),Within 10min,the A540 value decreased rapidly,△A540 value increased significantly(P<0.05),indicating that the mitochondrial volume increased,light scattering decreased.SOD activity decreased significantly(P<0.01),GSH-Px activity decreased significantly(P<0.01),GSH activity decreased(P<0.05);MDA activity increased significantly(P<0.01),and the activity of 8-OHdG was significantly increased(P<0.01).The activity of respiratory chain complex Ⅰ and Ⅲ was significantly decreased(P<0.01).The expression levels of Nrf2,Bachl and HO-1 mRNA in liver tissue were increased,and the expression of Nrf2 and HO-1 mRNA(P<0.05)and Bach1 mRNA expression(P<0.01).The symptoms of hepatocellular edema in San wei Gan jiang powder were alleviated by different levels,and the high level of the three flavors could reduce the serum ALT level(P<0.05),High dose of SOD activity increased(P<0.05),increased GSH-Px enzyme activity(P<0.05),increased the activity of GSH(P<0.01),Within 10min,the A540 value decreased rapidly(P<0.05),reduced the 8-OHdG content.MDA activity(P<0.01),increased respiration chain complex Ⅰ(P<0.01),increased respiratory chain complex Ⅲ activity(P<0.05),The expression of Nrf2 and HO-1 mRNA was up-regulated(P<0.05);The median dose could decrease the levels of serum ALT and AST(P<0.05),increase GSH enzyme activity(P<0.01),which could significantly decrease the activity of 8-OHdG(P<0.01)and decrease the activity of MDA(P<0.05),and could significantly increase the activity of respiratory chain complex Ⅰ and Ⅲ(P<0.01);The activity of MDA and the activity of 8-OHdG(P<0.05)were decreased in low dose group,and the activity of respiratory chain complex Ⅲ was increased(P<0.05).2.The levels of ALT and AST in serum of ConA model group were significantly increased(P<0.01).HE staining showed that the model group had hepatocyte edema,congestion and necrosis,hepatic lobular structure destruction,small fissure necrosis and inflammatory cell infiltration,and a large number of inflammatory cell infiltration in the portal area(P<0.05),and the number of A540 decreased rapidly and the value of △A540 was significantly increased(P<0.05),indicating that the mitochondria(P<0.05),and the mitochondria(P<0.05),and the decrease of the fluorescence intensity ratio of the ConA model group,indicating that the JC-1 monomer increased,the membrane potential △Ψm decreased(P<0.05),GSH-Px activity decreased significantly(P<0.01),GSH activity decreased(P<0.01),MDA activity increased significantly(P<0.05),the activity of SOD decreased significantly(P<0.01),8-OHdG content was significantly increased(P<0.01);SWGJS intervention group hepatocellular congestion and necrosis,hepatic lobule structure damage,small fissure necrosis and inflammatory cell infiltration,portal area inflammatory cell infiltration and other symptoms are different Ease the degree(P<0.01),increased the activity of SOD(P<0.05),increased the activities of GSH-Px and GSH(P<0.01),and increased the levels of ALT and AST in serum(P<0.01),and the activity of JC-1 was increased and the membrane potential △Ψm increased(P<0.01);and the content of JC-1 was increased(P<0.01),and decreased the activity of GSH-Px(P<0.05),and decreased the activity of GSH-Px(P<0.05).3.Using 6-gingerol as a reference substance,to detecte the blank serum、SWGJS containing serum、compound and unilateral,found that 6-gingerol in the drug-containing serum,SWGJS compound and ginger unilateral retention time is basically the same,the characteristic fragment peaks in the secondary mass spectrum are basically the same,suggesting that the 6-gingerol may be in the form of blood transfusion.Conclusion1.The effect of SWGJS on liver protection is related to the protection of mitochondria in the liver,specifically to protect the mitochondrial membrane structure,increase the number of antioxidant enzymes,reduce oxidative damage to DNA and restore mitochondrial redox system,regulate endogenous anti-Oxidation system,to reduce the body lipid antioxidant response,up-regulation of Nrf2,Bachl and HO-1 gene and protein expression.2.The UPLC-MS test shows 6-gingerol may be the composition of SWGJS into the blood,and with the prototype into the blood.