| Acute alcoholic liver injury refers to a series of changes such as liver cell damage caused by excessive alcohol intake within a short period of time,resulting in the blood alcohol concentration exceeding the liver’s metabolic capacity.The molecular events involved in acute alcoholic liver injury are complex,previous studies have elucidated mechanisms that may be involved in the process of acute alcoholic liver injury,however,its molecular mechanism in terms of gene expression remains to be elucidated.A series of related investigations have concluded that endoplasmic reticulum stress(ERS)plays an important part in the formation of acute and chronic liver injury.It is supposed to be a self-protection mechanism of the body,but when the stress is too long or duration is too long,it may cause inflammation and apoptosis,etc.,could lead to pathological changes.However,the mechanism of ERS involvement in chronic and acute liver injury,especially in the mechanism of acute alcoholic liver injury remains to be elucidated.A single alcohol gavage-induced acute liver injury model is one of the common models of alcoholic liver injury.In this study,we first observed the suitable reference genes in the model of acute alcoholic liver injury and further studied the effect of ERS on the expression of exosomal mi R-122.The main contents are as follows:1.Selection of reference genes in mouse models of acute alcoholic liver injurySince a single common reference gene may not be suitable under all conditions,the utility of different reference genes must be experimentally validated for specific experimental design.In order to further study the molecular mechanism of alcohol-induced acute liver injury,it is important to select a stable and reliable reference gene.1.1 Evaluation of the stability of reference gene expression in mouse models of acute alcoholic liver injury using the ge Norm,Best Keeper and Norm Finder toolsWe used a single alcohol gavage to establish acute alcoholic liver injury with mouse model,a small portion of liver tissue of equal size was taken for HE and TUNEL staining,the serum was obtained for the detection of alanine aminotransferase(ALT)after centrifugation of the mouse blood and calculated mouse liver weight index(liver weight / body weight)was detected to verified the model was successfully established.The expression levels of reference genes(Actb,Gapdh,Gusb,Hprt1,18 S,Tbp and B2m)in liver tissues were detected by RT-q PCR.The ge Norm,Best Keeper and Norm Finder tools were used to evaluate the stability of the reference gene expression in a mouse model of acute alcoholic liver injury.The results showed that the Hprt1 and Gapdh were validated as the optimal reference gene pair in mice model of acute alcoholic liver injury,and Hprt1 expression was most stable.1.2 Effects of different reference genes on relative expression of ER stress-related geneIn acute alcoholic liver injury with mouse model,the most stable gene Hprt1 and the most unstable gene 18 S were selected as the reference genes to detect the expression level of ERS-related genes(GRP78,CHOP,GRP94,XBP1,XBP1 t,ERdj4,PDI).The results showed that using Hprt1 as the reference gene,the levels of GRP78,CHOP and PDI were significantly increased in the livers of the ethanol 12 h group,compared with the control group.While using 18 S as the reference gene,no statistically significant differences in gene expression were found between the ethanol and control groups.The results indicated that Hprt1 was a suitable conforming gene in the model of acute alcoholic liver injury in mice,which was selected by three statistical methods.2.Effects of ERS on expression of mi R-122 in mouse model of acute alcoholic liver injuryExosomes have recently become a research hotspot,which is a small vesicle that contains a lipid bilayer structure.Exosomes contain different types of active substances,such as mi RNAs,which are fused with similar membranes,transfer active substances to the receptor cells,regulate intercellular signaling transduction,and play a biological role.Studies have shown that exosomes are rich mi RNAs,their membrane structure can enhance the stability of mi RNAs molecules,and could be used as a reliable carrier of mi RNAs research.In the acute alcoholic liver injury,alcohol can directly or indirectly lead to the occurrence of ERS.4-Phenylbutyrate(PBA)is widely used as a ERS inhibitor in the study of ERS-related diseases.Studies have shown that PBA can reduce the degree of hepatocyte necrosis and apoptosis by alleviating ERS.Another study shows that the expression of exosome mi R-122 is upregulated in alcoholic liver injury.Therefore,this experiment further investigated the effect of ERS on the expression of serum exosomal mi R-122 in acute alcoholic liver injury.2.1 Effect of PBA on the endoplasmic reticulum stressTo further investigate the effect of ERS on the expression level of serum exosomal mi R-122 in acute alcoholic liver injury.We used a single alcohol gavage to establish a acute alcoholic liver injury with mouse model,PBA intervention group mice were injected intraperitoneally with different doses of PBA(75 mg / kg,150 mg / kg)at 12 h and 24 h before modeling,a small portion of liver tissue of equal size was taken for HE staining,the serum was obtained for the detection of ALT after centrifugation of the mouse blood and calculated mouse liver weight index was detected to verified the model was successfully established.Detection of the ERS-associated protein GRP78,p IRE1α and p e IF2α expression in mouse liver tissue by Western blot.The results showed that PBA could inhibit the expression of ERS-associated proteins,the inhibitory effect of 150 mg / kg dose of PBA on the expression of ERS-associated protein was more obvious.2.2 Effect of endoplasmic reticulum stress on the expression of exosomal mi R-122In acute alcoholic liver injury model group and PBA intervention group,detection of the ERS-associated protein GRP78,p IRE1α and p e IF2α expression by Western blot,q PCR detection of exosomal pri-mi R-122,mi R-122 expression in serum.The results showed that ERS could lead to the up-regulation of exosomal pri-mi R-122 and mi R-122 expression level,while the ERS inhibitor PBA could down regulated the expression level of pri-mi R-122 and mi R-122.The results suggest that ERS could affect the expression of exosomal mi R-122. |
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