The Repair Effect Of Betulinic Acid On Liver Injury Induced By Zearalenone In Mice

Zearalenone(ZEA),also known as F-2 toxin,has been shown in previous studies to produce hepatotoxicity and induce oxidative damage and apoptosis of liver cells.Betulinic acid(BA)is a class of pentacyclic triterpenes with anti-inflammatory,anti-oxidation and other biological activities.Currently,many signaling pathways,such as endoplasmic reticulum stress(ERS),Nrf2 and MAPK,have been proved to be closely related to oxidative stress and cell apoptosis.So are the liver injury caused by ZEA and the repair effect of BA closely related to the regulation of these signaling pathways? There has been no specific reports.Objective: From the perspective of molecular biology,this paper discussed the effect and mechanism of the liver damage caused by ZEA and the repair effect of BA,so as to provide theoretical and experimental basis for the clinical prevention and treatment of ZEA poisoning and the drug development of BA..Methods: Sixteen±1 g 4-week-old male Kunming rats were randomly divided into 6groups(n=10): blank control group,ZEA group(20 mg/kg· BW),ZEA+ low and high dose BA groups(0.25 and 0.5 mg/kg· BW),ZEA+VE group(100 mg/kg· BW)and high dose BA group(0.5 mg/kg· BW).In the first week of the study,the control group and the high-dose BA group were gavaged with 5% ethanol solution,while the other groups were gavaged with ZEA suspension mixed with 5% ethanol(20 mg/kg· BW)once a day for consecutive 7 days to model liver injury.In the second week of the study,the blank group was given starch paste solution,while the other groups were given the corresponding dose of BA and VE suspended by starch paste,once a day,for consecutive 7 days.After that,the mice were dissected and the blood and liver were collected for the following studies:We evaluated the change of liver organ coefficient and the changes of liver tissue morphology were observed by HE staining while the number and structure of various organelles in liver were observed by transmission electron microscope;Biochemical indexes such as Glutamic-oxalacetic transaminase and glutamic-pyruvic transaminase in serum and calcium ion content in liver of mice were detected;The levels of Reactive oxygen species(ROS),Superoxide dismutase(SOD),Glutathione(GSH),Glutathione peroxidase(GSH-Px),Catalase(CAT),Malondialdehyde(MDA)and other enzymes involved in oxidative stress were detected;The expressions of interleukin-1β(IL-1β),interleukin-6(IL-6),Tumor necrosis factor-α(TNF-α)and interleukin-10(IL-10)were detected by Q-PCR;TUNEL assay was used to detect the Hepatocyte apoptosis;The expression of related proteins in the ERS/MAPK/Nrf2 signaling pathway in mice liver was detected by Western blot and the expressions of Caspase-3,CHOP and Nrf2 in the liver of mice were detected by immunohistochemistry.Results: 1)ZEA processing after a week in mice,the organ index of mice was significantly increased(P < 0.01),while the liver organ index was significantly decreased after BA treatment(P < 0.01);2)HE staining showed that the hepatocytes of mice were swollen and the hepatic cord arrangement was disordered after treatment with ZEA,and the pathological changes of liver cell morphology and structure caused by ZEA were effectively improved after treatment with BA;3)The results of electron microscopy showed that ZEA caused moderate liver edema,mitochondria edema in the cytoplasm,and the matrix in the membrane weakened.BA treatment could improve the edema of liver cells to a certain extent and repair the damage of organelles;4)mice serum biochemical index and the content of calcium ion in the liver did not change significantly,but the total protein content,total cholesterol content and urea content were increased in BA treatment group,while the ratio of albumin to globulin,glutamic oxalacetic transaminase and total bilirubin content were decreased(P < 0.05);5)In terms of oxidative stress,the production of ROS and MDA in liver was significantly increased after ZEA treatment(P < 0.01),and the corresponding antioxidant enzyme levels were significantly down-regulated;while in BA treatment group,the contents of ROS and MDA in liver were significantly decreased(P < 0.01),and the activity of the corresponding antioxidant enzymes was up-regulated(P < 0.05);6)Q-PCR assay showed that the expression of IL-6 and TNF-α was significantly increased by ZEA(P< 0.01).After corresponding BA treatment,IL-1β,IL-6 and TNF-α were significantly decreased,and IL-10 was significantly increased(P < 0.01);7)In the Tunel apoptosis test,ZEA significantly up-regulated the apoptosis rate of hepatocytes,and after BA treatment,the apoptosis rate of hepatocytes was significantly decreased(P < 0.01);8)Western blot detection the MAPK/Nrf2 /ERS signaling pathways in the corresponding protein changes,the results showed that ZEA significantly increased phosphorylation of P38,JNK,ERK and PERK protein levels(P < 0.01,P < 0.05),increase the Keap1,CHOP,BIP,ATF6,IRE1,Bax,Caspase-9 and Caspase-3 protein expression,cut the Nrf2,HO-1 and Bcl-2 protein expression(P < 0.01,P < 0.05),After BA treatment,the changes of protein expression caused by ZEA were significantly improved and restored to the level of control group;9)The results of immunohistochemistry showed that the protein level of Nrf2 was significantly decreased after ZEA treatment(P < 0.01),while the CHOP and Caspase-3 were significantly increased(P < 0.01).After BA treatment,the expression of Nrf2 was up-regulated,but the CHOP and Caspase-3 were down-regulated(P < 0.01).Conclusion: BA can alleviate the morphological and structural damage of liver cells caused by ZEA,enhance the antioxidant capacity of mice liver,reduce the production of ROS,and reduce the apoptosis rate of liver cells.Meanwhile,BA may further repair the damage caused by ZEA to mice liver by regulating the expression of related proteins in ERS/MAPK/Nrf2 signaling pathway.