Role Of Nupr1 In Methamphetamine-induced Hepatotoxicity

Backgroud:Methamphetamine(METH),which is called methyl-amphetamine,deoxidation ephedra in Chinese,for its pure white crystal appearance,the exterior glittering and translucent and acute toxicity,it is also called "crystal METH".It is wildly spread all over the world due to its easy access to raw materials,simple processing technology,and absorb very fast after use with long lasting exciting time.People will have a relatively strong feelings with an exciting experience after the drug abuse.In recent years,despite the strictly control of the Chinese government,the abuse of methamphetamine has been increasing rapidly because of the lower price compared with other drugs(such as marijuana)and stronger feelings of excitement and addiction.The ratio of younger ages and the amount numbers of people is on the rise.The abuse of METH not only brings physical and mental damage to the abusers themselves,but also brings huge social and public safety problem to the public,and it turns out to be a great challenges for social governance power control.In forensic practice,it is common to see lots of cases in which an abuser dies from an overdose of drug abuse.In recent years,the research on methamphetamine mainly focuses on the central nervous system,and the research on the injury outside the central nervous system is rare.Liver is the most important metabolic site of methamphetamine,so our study focus on the methamphetamine-induced hepatotoxicity.with a large dose.Transcriptional regulation factor nuclear protein(nuclear protein,Nupr1)is a kind of cell stress protein,which plays an essential role under complicated stress and environment,Our previous findings shows that Nupr1 can be mediated by Chop and induced autophagy in METH related toxicology with a higher dose of METH,but its role in methamphetamine-induced hepatotoxicity remain to be investigated.Our present study establish a model of methamphetamine subacute poisoning in wild type C57 group and Nupr1 knockout C57 mice group respectively.The aim to discuss the role of Nupr1 deficiency in methamphetamine-induced hepatotoxicity on autophagy by detection of beclinl,LC3 ?,ATG5 expression.Then the Morphologic observation was also performed and the expression of Chop was also detected by using RT-qPCR and western blot.We preliminarily discussed the role of Nupr1 in methamphetamine-induced hepatotoxicity and ite mechanism,in order to provide more theoretical basis for hepatotoxicity caused by methamphetamine with a relatively high dose.Objectives:The C57 wild-type mouse model and the Nupr1 knockout mouse model with methamphetamine poisoning were established,and the expression levels of autophagy-related proteins were detected by RT-qPCR and Western Blot respectively.The changes in liver injury after administration were detected from the molecular level,followed by HE staining,and the changes in liver injury after administration were observed from the morphological perspective.Finally,changes in the expression level of Chop were detected to explore the molecular mechanism.The effects of Nupr1 knockout on hepatotoxicity caused by methamphetamine poisoning were verified from the morphological and molecular levels,and then the relevant molecular mechanisms were discussed.The project intends provide more theoretical basis for the hepatotoxicity caused by methamphetamine.Methods:1.We devided the all the mice in this experiment into four groups:the wild-type C57 mice,the METH-injected wild type C57 mice,the Nupr1 KO C57 mice and the Nupr1 KO C57 mice with METH injected.2.Establish subacute poisoning model group of METH by using wild-type C57 mice according to previous study of our research group:15mg/kg,one injection every 12h,a total of 8 injections;Establish subacute poisoning model group of METH by using Nupr1 KO C57 mice according to previous study of our research group:15mg/kg,one injection every 12h,a total of 8 injections.Other groups injected with the same dose of physiological saline.3.The interval time from the last injection was 12h,the mice were anesthetized with pentapenobarbital,then the mice were given cardiac perfusion to drain as much blood as possible from the liver tissues.The extracted mouse liver tissues were divided into three parts for next examination.4.A part of the extracted liver tissues was fixed,dehydrated,embedded,sectioned,stained and sealed for HE staining.5.Rt-qPCR was performed on the other part of the liver tissues(>100mg).6.The remaining liver tissues should be extracted about 20mg:frozen in liquid nitrogen,and put into a tissue grinding machine for grinding.RIPA lysate was cracked on ice and cracked by ultrasound,then use the Western blot to detect protein level.Results:1.METH caused liver damage and increased autophagy in mice,2.Western blot shows a higher expression of autophagy-related proteins(LC3 ?,ATG5,beclin1)in METH poison wild type C57 mice group compared with METH poison Nupr1 knockout C57 mice group3.RT-qPCR shows a higher expression of autophagy-related genes(LC3B,beclin1)in METH poison wild type C57 mice group compared with METH poison Nupr1 knockout C57 mice group4.METH poison Nupr1 knockout C57 mice group shows an allevate hepatotoxicity compared with METH poison wild type C57 mice group5.Western blot and RT-qPCR shows a higher expression of CHOP in METH poison wild type C57 mice group compared with METH poison Nupr1 knockout C57 mice group6.Western blot shows an increase of phosphorylation of T-mTOR in Nupr1 knockout C57 mice group with METH injection.compared with METH poison wild type C57 mice group.Conclusions:1..METH induced hepatotoxicity and increase the level of autophagy2.Nupr1 knockout reduced the highly expressed autophagy level of the liver after methamphetamine treatment3.Nupr1 knockout alleviated the liver injury caused by METH.4.Nupr1 alleviates the toxic effect of methamphetamine on the liver by inhibiting Chop induced autophagy.