Protective Effect And Potential Mechanism Of Betulinic Acid On Alcoholic Liver Injury Based On Endoplasmic Reticulum Stress

Background:Betulinic acid（BA）is a pentacyclic triterpenoid compound extracted from birch bark and has a variety of biological activities such as anti-oxidative stress and immunity enhancement.Our previous research found that BA could prevent alcohol liver injury through the regulation of the mitochondrial signaling pathway.Meanwhile,alcoholic liver injury is closely related to apoptosis which caused by endoplasmic reticulum stress（ERs）.However,hepatoprotective activity and molecular mechanisms of BA through regulating ERs has not been reported in alcoholic liver injury.Objective:The objective of this study was to explore the underlying mechanism of BA on alcoholic liver injury in mice based on ERs which may provide a certain scientific basis for its clinical application and commercial development of BA.Methods:（1）60 KM mice were randomly divided into 6 groups（n=10）,namely the control group;0.5 mg/kg BA group;the alcohol group（50%ethanol,10 ml/kg）;the low,medium,high dosages of BA（0.25,0.5 and 1 mg/kg）and alcohol groups.After intragastric administration of BA for 14 days,the blood and liver were collected for the following experiments:Protective effect of BA on alcoholic liver injury in mice:The levels of triglyceride（TG）,total cholesterol（TC）,aspartate aminotransferase（AST）,alanine aminotransferase（ALT）in serum as well as the contents of TG,TC,nitric oxide（NO）and Ca²⁺in liver were detected;The morphological changes of liver was observed by H&E staining,and the ultrastructure of liver was observed by transmission electron microscopy;and the cell apoptosis was determined by Tunel.Effect of BA on ERs of alcoholic liver injury in mice:m RNA expressions of Grp78,Grp94,Caspase-3,Caspase-7 and Caspase-12 in ER signaling pathway were detected by PCR.Protein expressions of Grp78,Grp94,ATF6,IRE1α,p-IRE1α,PERK,p-PERK,CHOP,Caspase-12 and Caspase-7 in ER signaling pathway,and protein expressions and phosphorylation of JNK,ERK and p38 in MAPK signaling pathway were determined by Western blot.（2）50 KM mice were randomly divided into 5 groups（n=10）,namely the control group,alcohol group（50%ethanol,10 ml/kg）,BA（1 mg/kg）+alcohol group,4-phenylbutyric acid（4-PBA）+alcohol group,and BA+4-PBA+alcohol group.After intragastric administration of BA for 14 days,blood and liver of mice were collected for the following experiments:BA alleviated alcoholic liver injury in mice based on ERs:The levels of Ca²⁺and reactive oxygen species（ROS）were detected in the liver;The morphological changes of liver was observed by H&E staining,and the cell apoptosis was determined by TUNEL;the protein expressions of ATF6,IRE1α,p-IRE1α,PERK,p-PERK,CHOP and Caspase-7 in the ER signaling pathway were determined by Western blot.Result:（1）Pretreatment with BA reduced the levels of AST,ALT,TC and TG in serum,and decreased the contents of TC,TG,NO and Ca²⁺in liver induced by alcohol;Morphological observations found that hepatic fat deposition and organelle lesions were reduced,and Tunel detection found that hepatocyte apoptosis was inhibited in alcoholic liver injury.The results showed that BA has a protective effect on alcoholic liver damage by reducing the activities of serum enzymes,inhibiting liver lipid deposition,restoring liver morphology,and alleviating hepatocyte apoptosis.（2）Pretreatment with BA inhibited the alcoholic liver injury induced ERs in mice.BA pretreatment decreased the m RNA expressions of Grp78,Grp94,Caspase-3,Caspase-7 and Caspase-12,as well as reduced the protein expression of Grp78,Grp94,ATF6,p-IRE1α/IRE1α,p-PERK,CHOP,Cleaved-caspase-12/Pro-caspase-12 and Cleaved-caspase-7/Pro-caspase-7 in the ER pathway.At the same time,pretreatment with BA inhibited the MAPK signaling pathway via reducing protein phosphorylation of JNK,ERK and p38 in liver.The results showed that BA protects alcohol liver injury by inhibiting ERs pathway and MAPK signaling pathway.（3）Pretreatment with BA and/or 4-PBA lessened the level of Ca²⁺,inhibited the protein expression of ATF6,p-IRE1α/IRE1α,p-PERK/PERK,CHOP and Cleaved-caspase-7/Pro-caspase-7 in liver induced by alcohol.In addition,BA reduced the increase of ROS in liver caused by alcohol.This indicated that inhibition of ROS generation and ERs play an important role in the alleviation of alcohol-induced liver injury by pretreatment with BA.Conclusion:BA has a protective effect on alcohol liver injury in mice by decreasing ROS production and inhibiting MAPK and ERs signaling pathway.