| Pyrrolizidine alkaloids(PAs)are a common group of chemical constituents and were found in about 3% of the world`s flowering plants.More than 660 PAs and PA N-oxides have been identified and most of them were hepatotoxicity.As reported,PAs distribute in over 6000 plants and Fabaceae,Leguminosae and Boraginaceae are the dominant species.Monocrotaline(MCT),an 11-membered macrocyclic diester without α,β-unsaturated double bond,occurs in plants of the genus Crotalaria.Species diversity and wide distribution are the features of Crotalaria.Therefore,they are probably the most common poisonous plants affecting livestock.Liver is the target organ of MCT.Long-term intake by livestock can cause liver injury and even death.MCT caused serious economic losses due to livestock poisoning and restricts the sustainable development of grassland animal husbandry.Therefore,elucidating the mechanism of liver injury caused by MCT and revealing the key proteins for regulating the damage can provide a scientific basis for the further development of detoxification drugs.Studies have confirmed that endoplasmic reticulum stress and its mediated autophagy and apoptosis play an important role in the occurrence of many liver diseases.When endoplasmic reticulum stress continues to occur,it will induce cell apoptosis.At the same time,excessive endoplasmic reticulum stress can cause the autophagy to be over-activated,then a large number of cells to be degraded to stimulate cell apoptosis.Therefore,this study used rats as the research object to establish monocrotaline liver injury model,and to explore monocrotaline-induced liver pathological changes and the effects on endoplasmic reticulum stress,autophagy and apoptosis.Meanwhile,in order to further explore the relationship between endoplasmic reticulum stress,autophagy and apoptosis,we used primary rat hepatocytes as the research object.Finally,we want to clarify the mechanism of endoplasmic reticulum stress-mediated autophagy and apoptosis in monocrotaline-induced liver toxicity,the results of this project are as follow:(1)This stduy confirmed that monocrotaline-induced liver injury in rats and induced endoplasmic reticulum stress,autophagy and apoptosis.In this study,rats were oral administration of different dose of MCT(45 mg/kg and 90 mg/kg)for 48 h.Collecting blood samples to detect biochemical indicators.Compared with the control group,the total bilirubin(T-Bil),alanine aminotransferase activity(ALT)and aspartate aminotransferase activity(AST)increased with a dose-dependent manner.Collecting liver tissue to calculate liver coefficient.Compared with the control group,the liver coefficient of the 45 mg/kg dose group did not change significantly,while the 90 mg/kg dose group increased significantly.We also found that the 45 mg/kg dose group showed that mild sinus hemorrhage,sinus dilatation,and hepatocyte nuclear pyknosis,and the 90 mg/kg dose group showed disorder of hepatic,sinusoidal dilatation,sinusoidal hemorrhage,coagulative necrosis of hepatocytes and endothelial damage of the central vein.The results of western blot and immunohistochemistry showed that the related proteins of ERs were increased,including GRP78,p-e IF2α,ATF4,CHOP,p-IRE1,Caspase-12 and ATF6.In addition,MCT can inhibit the PI3K/AKT/m TOR pathway to active autophagy,which showed that the expression of proteins of LC3 II,Becline-1 and Atg5 were increased and p62 was decreased.Thus,it further induced the occurrence of caspase-dependent apoptosis,which causes liver damage.(2)The effect of ERs in MCT induced apoptosis in primary rat hepatocytes.In this study,primary rat hepatocytes were isolated using the standard two-step perfusing procedure according to modified Slegen.The viability of liver cells was calculated by trypan blue staining,the CK18 protein in the liver cells was identified by immunofluorescence staining,and the purity of the liver cells was analyzed by PAS staining,which proved that the viability and purity of the isolated rat primary liver cells were available for further research use.After treating liver cells with different concentrations of MCT(100,200,300,and 400 μM)for 6,12,24,36,and 48 h,the cell viability was tested by CCK-8.The results showed that 300 μM MCT was used to treat hepatocytes for 36 h inhibited cell viability significantly.We used flow cytometry and western blot detection found that MCT has a dose-dependent effect on hepatocyte apoptosis.The results of transmission electron microscopy showed that endoplasmic reticulum expanded when exposed by MCT.Meanwhile,the expression of ERs related proteins increased with a dose-dependent manner.Pre-treatment hepatocytes with 4-PBA founded that ERs was inhibited significantly,and alleviated apoptosis.CHOP is an upstream regulator of apoptosis.We used si RNA to interfere with the expression of CHOP protein,it was found that si CHOP inhibited hepatocyte apoptosis.Collectively,ERs mediated apoptosis by MCT in primary rat hepatocytes,and CHOP play a key role between ERs and apoptosis.(3)The effect of ERs-mediated autophagy in MCT induced apoptosis in primary rat hepatocytes.In this study,transmission electron microscopy and western blot showed that autophagy was induced upon and the expression of autophagy related protein LC3 II,Becline-1 and Atg5 were increased and p62 was decreased in hepatocytes treated by MCT,indicating that MCT can induce autophagy in hepatocytes.In order to analyze the regulation effect of ERs-mediated autophagy,4-PBA was used to pretreat hepatocytes and found that ERs can enhance the phosphorylation of PI3K/AKT/m TOR pathway proteins to inhibit the occurrence of autophagy.In order to further explore the regulation effect of CHOP protein on autophagy,the expression of autophagy-related proteins was analyzed after transfecting si CHOP.The result showed that si RNA targeting CHOP can inhibit the expression of LC3 II,Becline-1 and Atg5 and enhanced the expression of p62.In addition,it also enhanced the phosphorylation of AKT and m TOR.In order to analyze the role of autophagy in hepatotoxicity of MCT,we used autophagy agonist(rapamycin)and autophagy inhibitor(baflumycin and chloroquine)to pretreated hepatocytes to detect apoptosis caused by MCT.The results showed that Rapamycin increased the rate of hepatocyte apoptosis and the expression of cleaved caspase-3,while Bafilomycin and Chloroquine decreased the rate of hepatocyte apoptosis and the expression of cleaved caspase-3.It indicated that MCT-induced autophagy promotes cell apoptosis.All this indicated that ERs-mediated autophagy can enhance apoptosis induced by MCT,and CHOP is the key factor.In conclusion,we used MCT as the research object,rat and primary rat hepatocytes as the models to study the relationship between ERs,autophagy,apoptosis and liver injury in vivo and in vitro.In vivo studies confirmed that MCT can cause ERs,autophagy and apoptosis in rat liver.In order to further clarify the relationship between ERs,autophagy and apoptosis,firstly,in vitro study revealed that MCT induced hepatocyte apoptosis through endoplasmic reticulum stress.Meanwhile,we found that CHOP was the key protein of MCT regulating ERs induced apoptosis.Secondly,we used 4-PBA to inhibite ERs and si RNA to interfere the expression of CHOP protein.ERs promoted MCT induced autophagy,and CHOP was the key factor of ERs to mediate autophagy.Finally,we confirmed that ERs-mediated autophagy enhanced MCT induced hepatocyte apoptosis.This study lay a molecular basis for the hepatotoxicity of MCT,and give important theoretical basis for the prevention and treatment of poisoning by MCT.In order to provide scientific guidance for the research and development of MCT detoxification drugs... |
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