| Background and objective:Nonalcoholic fatty liver disease（NAFLD）is the most common chronic liver disease and one of the main causes of hepatocellular carcinoma.It generally refers to a clinicopathological disease characterized by excessive lipid droplets and lipid deposition in liver cells in the absence of excessive alcohol consumption（more than 20g/d for men and more than 10g/d for women）or other causes of liver disease（including drug treatment of hereditary disease fat or viral hepatitis,etc.）.According to the study,the prevalence of NAFLD is 25.2%worldwide and 29.2%nationwide in China.The majority of NAFLD patients have simple steatosis,a form of liver steatosis without substantial inflammation or fibrosis.Lipotoxicity can promote the progression of hepatitis and fibrosis in NAFLD patients,leading to the occurrence of non-alcoholic steatohepatitis（NASH）,and even the progression of cirrhosis and hepatocellular carcinoma（HCC）.However,the underlying molecular mechanism is still unclear,and there is no effective treatment at present.In this study,non-alcoholic fatty liver model was established by using methionine-and choline-deficient diet（MCD）The role and molecular mechanism of LSS dysfunction in the occurrence and progression of NAFLD and the possibility of LSS as a drug target for the treatment of NAFLS and other diseases.Methods:（1）Since complete LSS knockout in mice would lead to homozygous death,LSS heterozygous knockout mice（LSS+/-）were established using CRISRP/CAS9system,and their genotypes were identified by PCR.（2）NAFLD mouse model was established by MCD feeding.Wide-type（Wt）and LSS+/-mice aged 8 weeks were randomly divided into 4 groups:n-WT group of wild-type mice fed with normal diet,N-KO group of knockout mice fed with normal diet,M-WT group of wild-type mice fed with MCD diet,and knockout mice fed with MCD diet Mice in M-KO group were weighed and blood samples were taken from eyeballs for two weeks.Serum was collected to detect ALT and AST levels in serum,which reflected the damage of liver cells.The liver of the dissected mice was taken and weighed.HE staining,oil red O staining,Sirius red staining,etc.were used to detect the degree of liver injury lipid deposition and liver fibrosis.Transcriptome analysis of liver tissue was used to detect the differential genes,and the genes of interest were selected for RT-PCR verification.（3）Endoplasmic reticulum stress was induced by intraperitoneal injection of Tunicamycin（TM）in mice.Eight-week-old wild-type C57BL/6 mice were randomly divided into four groups（n=6）:DMSO+MCD group TM+Con group TM+MCD group and DMSO+Con group.Three days before the experiment,the mice ate normal diet and received intraperitoneal injection of TM and DMSO.The TM group was divided into two doses（high dose concentration of 7.5ug/m L,low dose concentration of 0.75ug/m L）:the body weight and blood glucose of the mice were recorded every three days from the first day.Two weeks later,TM and DMSO were intraperitoneally injected with MCD feed and NCD feed for 17 days in total.（4）Eyeball blood was taken and liver tissue was sampled:serum AST and AST levels were detected to reflect liver cell damage.HE staining,oil red O staining and Sirius red staining were used to detect the degree of liver injury lipid deposition and liver fibrosis,and THE expression levels of related genes were detected by RT-PCR and Western blot.Results:（1）Genotypic identification of mice:wild-type mice with a 328bp DNA band as the PCR amplification product,and LSS+/-mice with 328bp and 282bp DNA bands.（2）Serological results of NAFLD mouse model:There was no significant difference in ALT AST between LSS+/-mice and wild-type mice under normal diet.Serological AST and ALT were significantly different between LSS+/-mice and wild-type mice after MCD induction.（3）Histological staining results of NAFLD model group:Under normal diet,liver tissue of wild-type and LSS+/-mice was normal in morphology and structure,arranged in rope shape,and no obvious lipid droplets were found in liver tissue by oil red staining.In contrast,the liver tissue of wild-type mice induced by MCD was vacuolated in large number and volume.Oil red staining showed the production of a large number of lipid droplets,and there were necrotic cells and inflammatory cells in the liver tissue.Compared with wild-type mice,HE staining and oil red staining showed smaller volume and number of lipid droplet vacuoles,lighter liver cell necrosis and inflammatory cell infiltration in LSS+/-mice,and no fibrosis was observed in liver Sirius red staining in all groups.（4）Transcriptome analysis of liver tissue in NAFLD model:there were a large number of differences in gene expression between each group,and significant differences in the expression of er stress-related genes were observed through ke GG-Pathway enrichment bubble map.（5）According to transcriptome results,m RNA expression levels of er stress-related genes in liver tissues of NAFLD model were analyzed by RT-PCR,and transcriptome results verified that there were 9 up-regulated genes and 1 down-regulated gene between n-KO group and N-WT group,and6 up-regulated genes between M-KO group and M-WT group.Four down-regulated genes.（6）Western blot analysis of er stress-related protein expression in NAFLD model showed that GRP78 protein expression level was significantly increased in LSS+/-mice induced by MCD diet;The expression levels of Syvn1 and HYOU1 proteins in liver of LSS+/-mice fed with NCD diet were significantly higher than those of wild-type mice,and the expression levels of HYOU1 in LSS+/-mice fed with MCD diet were also increased.These results suggest that loss of LSS function has a protective effect on Mc D-induced NAFLD.Er stress-related genes may be involved in the protection of Mc D-induced NAFLD by loss of LSS function Therefore,ers-specific inducer TM was injected into mice to induce er stress to investigate whether the loss of LSS function has a protective effect on NON-alcoholic fatty liver disease（NAFLD）in C57 mice through er stress pathway.（7）Effects of different doses of TM on Mc D-induced liver injury:Serological results showed that ALT level in L-TM+MCD group was significantly lower than that in DMSO+MCD group,while there was no significant difference in ALT serological level between H-TM+DMSO group and DMSO+MCD group.According to the histomorphology,the liver inflammatory response and steatosis and other liver injuries in l-TM+DMSO group were significantly lower than those in DMSO+MCD group,while the liver inflammatory response and other characteristics in H-TM+DMSO group were significantly higher than those in DMSO+MCD group.（8）Analysis of endoplasmic reticulum stress-related proteins in liver of mice induced by different doses of TM:Intraperitoneal injection of TM can significantly increase the expression of HYOU1 and GRP78 proteins in mice,and the expression level of HYOU1is higher in the case of low dose of HYOU1.The expression level of IER1a in THE L-TM+MCD group was not significantly different from that in the DMSO+MCD group,while there was a significant difference between the H-TM+MCD group and the DMSO+MCD group.After intraperitoneal injection of TM,the expression level of Syvn1 protein was significantly higher in the H-TM+DMSO group than in the L-TM+MCD group.Conclusion:Loss of LSS function may play a protective role on MCD-induced non-alcoholic fatty liver disease（NAFLD）in C57 mice through changes in er stress-related protein expression. |
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