| ObjectiveD-gal was injected subcutaneously into rats to establish a rat liver injury model.Using extract of Wen Wang Yi Zhi Bi to interfere with this model.By observing the expression of liver injury related proteins and the changes in pathological morphology,we explored the protective effect and the possible mechanism of Wen Wang Yi Zhi Bi on the liver of rats with liver injury,and provided theoretical and experimental basis for the treatment of liver injury by traditional Chinese medicine.MethodsAccording to random number table method,sixty male Sprague-Dawley(SD)rats were selected and divided into normal group,model group,Wen Wang Yibi low?medium and high dose group and positive control group(n=10).The model group,each dose group of Wen Wang Yi Zhi Bi and the positive control group were injected with 100mg/kg D-gal subcutaneously to establish a rat model of liver injury.At the same time,rats in thethe Wen Wang Yi Zhi Bi low?medium and high dose group were gavaged with 50?100?and 200mg/kg Wen Wang Yi Zhi Bi solution for 42 days,and rats in the positive control group were gavaged with 0.05mg/kg sodium selenite for 42 days.The normal group and the model group were given an equal volume of distilled water for 42 days.After the animal was anesthetized,we began to perfuse the material,collect cardiac blood for serum ALT?AST?DBIL testing;HE staining to detect the morphology of liver tissue;immunohistochemical detection of BDNF and Trkb expression in liver tissue;TUNEL staining to detect hepatocyte apoptosis;WB detection of Bax?Bcl-2?Caspase-3?BDNF?Trk Bexpression;the thiobarbituric acid method were used to determine the content of MDA and xanthine oxidase method were used to determine the activity of SOD in liver tissues.Results1 General condition of liver injury in rats:The rats in the normal group had agile and flexible behavior,bright hair,normal eating and drinking,and increased body weight.Compared with the normal group,the rats in the model group had less activity,thinner hair,hair loss,and less food.Compared with the model group,after Wen Wang Yi Zhi Bi and sodium selenite intervention,the above-mentioned conditions were significantly improved,and the weight also increased relatively.2 Results of serum testing:Compared with the normal group,the activity of ALT and AST and the content of DBIL in model rats were significantly increased(P<0.05),indicating that D-gal has a significant effect on rat liver function,suggesting that D-gal induced Liver injury modeling was successful.Compared with the model group,the activity of ALT and AST in the sodium selenite group(positive control group)and Wen Wang Yi Zhi Bi low and high dose groups decreased(P<0.05),and the content of DBIL also showed a decreasing trend(P<0.05).Compared with the model group,the activity of ALT and AST in the middle-dose group of Wen Wang Yi Zhi Bi was significantly reduced(P<0.05),and the content of DBIL also showed a trend of significant decrease(P<0.05).3 Changes in morphology:(1)Anatomical changes of rat liver:The liver of the rats in the normal group is ruddy,the surface is smooth,full of elasticity,and there is no lesion;compared with the normal group,the liver of the model group is pale yellow;Compared with the model group,the liver injury of rats in the low,medium and high dose groups of Wen Wang Yi Zhi Bi and the sodium selenite group(positive control group)was improved.(2)HE staining results of liver tissue sections:In the normal group,the liver lobule structure is clear,the liver cells are neatly arranged,and the cell nucleus is large and round,without inflammatory infiltration and pathological changes.In the model group,the hepatic lobule structure of the rats with liver injury was incomplete,the arrangement was disordered,and the cell volume swelling increased,degeneration,necrosis and other phenomena,and a little lymphocyte infiltration was seen.After drug intervention,it was found that Wen Wang Yi Zhi Bi and sodium selenite can improve liver damage to varying degrees.Compared with the model group,Wen Wang Yi Zhi Bi low,medium and high dose groups and sodium selenite group showed clearer liver lobular structure,the cells were arranged relatively neatly,and the cell volume swelling increased,degeneration,necrosis and other conditions lighten.(3)Immunohistochemical results of liver tissue BDNF and Trk B:In the normal group,there was no expression of BDNF and Trk B in the rat liver tissue;compared with the normal group,the positive expression particles of BDNF and Trk B in the rat liver tissue of the model group were significantly increased,showing specific yellow or brown yellow.Compared with the model group,the BDNF and Trk B positive expression particles in the liver tissue of rats in the low and high dose group of Wen Wang Yi Zhi Bi and the sodium selenite group were reduced,and they appeared light yellow.Compared with the model group,the positive expression particles of BDNF and Trk B in the liver tissues of rats in the middle dose group of Wen Wang Yi Zhi Bi were significantly reduced,and appeared yellowish.(4)Results of TUNEL test:Compared with the normal group,the hepatocyte apoptosis of the model group was significantly increased(P<0.05);compared with the model group,the apoptotic cells of the low and high dose group of Wen Wang Yi Zhi Bi and positive control group were reduced(P<0.05);Compared with the model group,the apoptotic leaves in the middle dose group of Wen Wang Yi Zhi Bi were significantly reduced(P<0.05).4 Effects of SOD and MDA levels in rat serum:Compared with the normal group,the SOD activity in the liver tissue of the model group was significantly reduced(P<0.05),and the MDA content in the liver tissue of the model group was significantly increased(P<0.05).Compared with the model group,the low and high dose group of Wen Wang Yi Zhi Bi and positive control liver tissues had higher SOD activity(P<0.05)and lower MDA content(P<0.05);compared with the model group,the low and high dose group of Wen Wang Yi Zhi Bi and positive control liver tissues was significantly increased(P<0.05),and the MDA content was significantly reduced(P<0.05).5 Effects of Western Blot:Compared with the normal group,the Bcl-2 expression in the liver tissue of the model group was significantly decreased,and the expression levels of Bax and Caspase-3 were significantly increased(P<0.05),indicating that hepatocyte apoptosis in rats with liver injury was significant.Compared with the model group,the expression levels of Bcl-2 in the low,medium and high dose groups of Wen Wang Yi Zhi Bi and positive control group increased significantly(P<0.05),and the expressions of BAX and Caspase-3 decreased significantly(P<0.05).There was no expression of BDNF and Trk B in the normal group;compared with the normal group,the expression of BDNF and Trk B protein in the model group was significantly increased(P<0.05);compared with the model group,the expression levels of BDNF and Trkb in the low,medium and high dose groups of Wen Wang Yi Zhi Bi were significantly reduced(P<0.05).Conclusion:Wen Wang Yi Zhi Bi can increase the expression of Bcl-2 in liver tissue,inhibit the expression of Bax and Caspase-3,increase the activity of SOD,reduce the content of MDA,and inhibit the apoptosis of liver cells.The protection of the extract of Wen Wang Yi Zhi Bi on the liver may be related to the reduction of the expression of BDNF and Trk B in hepatocytes. |
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