Protective Effects Of Hybrids Of Betulinic Acid And Nucleoside Against Hepatic Injury And The Mechanism Of Anti-alcoholic Liver Injury

BackgroundThe development of chronic liver disease is a complex process including from the initial necrosis of liver cells and steatosis to fatty liver and liver fibrosis(some may be accompanied by hepatitis).It develops into cirrhosis and eventually evolves into liver cancer or liver failure.Liver injury is the initial pathological condition of all chronic liver diseases.During the development of liver from the initial hepatocyte necrosis to liver fibrosis,people do not show too many symptoms.Most patients with chronic liver disease have developed liver cirrhosis or even liver failure when they find problems.At present,the clinical treatment methods for chronic liver diseases such as cirrhosis and liver failure are only targeted at some patients to improve the severity of their liver diseases.There are no specific drugs that can effectively treat such chronic liver diseases.Therefore,people must pay attention to the treatment and prevention of early liver injury.There are many types of liver injury,which can be divided into alcoholic liver injury,immune liver injury,and chemical liver injury according to inducers.Stopping exposure to substances that can cause liver damage,such as alcohol and certain drugs,is very helpful for the early treatment of chronic liver disease.But sometimes we have to come into contact with these substances.For example,sometimes you have to drink and you have to take medicine when you are sick.At this time we often choose to ignore the damage to the liver caused by these substances.If accompanied by hepatitis,chronic liver disease will not alleviate the condition by stopping exposure to inducers.Therefore,it is necessary to find a medicine that can protect the liver and protect the liver when people have to come into contact with alcohol and certain drugs.anti-oxidative stress and autophagy are two targets for liver injury.Antioxidants have been widely used in liver protection drugs,and the mechanism has been studied to some extent.It is understood that the nuclear receptor FXR can activate Nrf2 and increase the expression of the antioxidant enzymes HO-1,NQO1,etc.,thereby acting as an anti-liver injury.Autophagy is a means of self-protection for cells.When liver damage occurs,autophagy can fight liver damage by engulfing the damaged mitochondrial membrane and Reactive oxygen species.Betulinic acid has certain antioxidant functions,and has the advantages of low-toxicity and high safety.It is considered to be a promising therapeutic drug.However,the-use ofbetulinic acid for anti-hepatic injury requires a higher drug concentration,and the water solubility of betulinic acid is-very low,which affects the application of betulinic acid for anti-liver injury.On the bases of previous studies,we synthesized hybrds of betulinic acid and nucleoside and investigated its potential application as a novel molecule against liver damages.The research results fully suggest that this hybrids of betulinic acid and nucleoside can play an anti-hepatic role by increasing antioxidant activity and increasing autophagy.ObjectiveIn this-study the effects of hybrids of betulinic acid and nucleoside on serum biochemical indicators of various liver injuries were determined by constructing mouse alcoholic liver injury,immune liver injury,and chemical liver injury models.Pick which type of liver injury has the best effect.We detect the mouse liver to detect the degree of liver damage in mice,And the expression of FXR,Nrf2,HO-1,NQO1 and other antioxidant proteins,And the expression of Beclinl,LC-3,ATG14 proteins to provide experimental evidence for the molecular mechanism of hybrids of betulinic acid and nucleoside against liver injury.Methods1.Alcohol(70%)and CCl4 were administered to Kunming mice by gavage,and ConA was injected into the tail vein to establish a model of alcoholic liver injury,immune liver injury,and chemical liver injury in mice.2.Take the serum of the mice and use the biochemical analyzer to detect the levels of ALT and AST in the serum of the mice to verify the success of the liver injury model.Preliminary detection of hybrids of betulinic acid and nucleoside has anti-hepatic effects and screening which model works best for it.After selecting the best model,the serum TG and CHO levels of the best model mice were further tested to further verify the anti-hepatic effect.3.The kit was used to detect liver damage index MDA,antioxidant indexes SOD,GSH-Px,and CAT on a UV spectrophotometer in a kit to prove whether the new drug has anti-oxidant effect and provide clues for the subsequent research on liver damage mechanisms.4.Making HE stained sections of mouse liver can directly explain the degree of mouse liver damage.5.Using QPCR and Western Blot technology to detect the expression of HO-1,NQO1,Nrf2,Keap1,FXR in mouse liver to validate whether new drugs can fight liver damage through antioxidants;Detection of Beclin,LC-3,ATG14 expression in mouse liver to validate whether new drugs can fight liver damage by up-regulating autophagy.;Detection of the expression of apoptosis-related proteins Caspase3 and Bcl2 in mice to verify whether new drugs have anti-liver damage effects at the molecular level.6.LC-3Ⅱimmunofluorescence sections were prepared from mouse livers to further verify the effect of the new drug on autophagy.Results1.The Results of serum ALT and AST in three mouse models show that model group is higher than control group,the positive drug group was lower than the model group,dosage group was lower than the positive drug group.Also,the effects of alcoholic liver injury models are better than the other two.Therefore,the alcoholic liver injury model was selected to futher verify the mechanism of action of hybrids of betulinic acid and nucleoside on liver injury.2.Mouse-serum TG and CHO results show that serum TG and CHO in model group were higher than those in control group,the positive drug group was lower than the model group,dosage group was lower than the positive drug group.3.Mouse liver MDA,SOD,GSH-Px,CAT test results show that compared with the control group,MDA levels in the model group increased significantly,while SOD,GSH-Px,and CAT levels significantly decreased.The MDA level of the positive drug group decreased,and the level of the drug group was lower than-that of the positive drug group.The levels of SOD,GSH-Px,and CAT in the positive drug group increased,and the levels of SOD,GSH-Px,and CAT in the drug administration group were higher than those in the positive drug group.4.The H&E staining reveals widespread periportal inflammations and evenly spread hepatic cell death in the model group.Within the BAN treated group,the periportal inflammations and hepatic cell death are significantly reduced,especially in the high hybrids of betulinic acid and nucleoside dosage treatment group.5.Western Blot results show that compared with the control group,the levels of model histones FXR,Nrf2,HO-1,NQO1,Beclin,LC-39 ATG14,and Bcl2 decreased significantly,while the levels of these proteins in each administration group increased significantly,showing a certain amount of dependence.The levels of proteins Keap1 and Caspase3 increased significantly in the model group,but decreased significantly in the administration group and showed a certain amount of dependence.6.QPCR results showed that the-mRNA levels of FXR,Nrf2,HO-1,NQO1,Beclin,LC-3,ATG14,and Bcl2 in the model group decreased-significantly.The levels of Keap1 and Caspase3 mRNA-increased significantly in the model group,but decreased significantly in the administration group.7.The results of immunofluorescence showed that the amount of LC-3Ⅱ in mouse liver increased significantly.Conclusion1.hybrids of betulinic acid and nucleoside can play a role in resisting alcoholic liver injury,imrpune liver injury,chemical liver injury,and has better effects on alcoholic liver injury than the other two.2.hybrids of betulinic-acid and nucleoside can increase the expression of anti-reactive oxygen enzymes in liver cells by-activating FXR,thereby anti-liver injury.3.hybrids of betulinic acid and nucleoside can increase-the level of autophagy during liver-injury,and activate the cell’s self-protection function,which can play a role in anti-liver injury.4.hybrids of betulinic acid and nucleoside can reduce the expression of hepatocyte apoptosis-related proteins,inhibit hepatocyte apoptosis,and protect hepatocytes.