| Objective: γ-Oryzanol(ORY)was used as an intervention method to explore its protective effect and related molecular mechanisms in mouse liver ischemia/reperfusion(I/R)injury.Methods: The following will be divided into two parts to illustrate the research method of this experiment,divided into two parts: in vivo experiment and in vitro experiment.The first part is the in vivo experiment:(1)First,50 8-week-old C57 male mice were randomly divided into 5 groups:(1)sham operation group(Sham group),(2)ischemia reperfusion group(I/R group),(3)Low-concentration γ-Oryzanol pretreatment group(I/R+ORY10 group),(4)Medium-concentration γ-Oryzanol pretreatment group(I/R+ORY20 group),(5)High-concentration γ-Oryzanol pretreatment group(I/R+ORY40 group),Among them,the(2)-(5)group underwent partial I/R surgery on mouse liver,and the(3)-(5)group underwent ORY pretreatment.(2)After partial hepatic ischemia and reperfusion,serum and liver were collected from mice for follow-up experiments.A transaminase detection kit was used to detect the levels of aspartate aminotransferase(AST)and alanine aminotransferase(ALT)in serum of each group;The liver samples of each group were stained with H&E(Hematoxylin and eosin,hematoxylin & eosin);(4)The superoxide dismutase(SOD)and myeloperoxidase(MPO)in the liver tissue of each group of mice were detected by the kit),reduced glutathione(GSH)and lipid peroxidation product malondialdehyde(MDA)content;(5)Tunel staining method was used to detect cell apoptosis in the liver tissues of mice in each group,and evaluate the liver of mice The degree of tissue damage;(6)Immunohistochemical method was used to detect the expression of GRP78 in mouse liver tissues,and Western blotting was used to detect NLRP3 inflammation pathway-related proteins and damagerelated molecules HMGB1,GRP78/PERK/EIF2S1/CHOP Expression of endoplasmic reticulum stress pathway proteins and apoptosis-related proteins(Bcl-2 and Bax).The second part of the in vitro experimental results found that:(1)After AML12 mouse hepatocytes were treated with different concentrations of γ-Oryzanol solution for 24 hours,the γ-Oryzanol solution within the concentration range of 400μg/ml had no significant effect on the proliferation of mouse hepatocytes;(2)After treating AML12 mouse hepatocytes with different concentrations of cobalt dichloride solution for 24 hours,as the concentration of cobalt dichloride solution increases,the cell viability value of mouse hepatocytes gradually decreases.When it was 300 μmol/L,the cell viability value decreased to 57.80%±3.45,and the hypoxia model was subsequently established to select this concentration;(3)Different concentrations of γ-oryzanol solution were used for pretreatment of mouse liver cells at 300 μmol/L cobalt dichloride After 24 hours of incubation in the solution condition, compared with the control group,the cell viability value of the γ-oryzanol solution at 160 μg/ml and 240 μg/ml increased significantly;(4)the hepatocytes in the pretreatment group(ORY240+ Co Cl2300 group)The active oxygen content was significantly higher than the cobalt dichloride treatment group(Co Cl2300 group);(5)Compared with the cobalt dichloride treatment group(Co Cl2300 group),the pretreatment group(ORY240+ Co Cl2300 group)had significantly fewer apoptotic cells;(6)The expression levels of GRP78/PERK/EIF2S1/CHOP endoplasmic reticulum stress pathway proteins in the ORY240+ Co Cl2300 group were significantly reduced.(7)Compared with the cobalt dichloride treatment group,the expression level of the pro-apoptotic protein Bcl-2 of hepatocytes was significantly higher than that of the cobalt dichloride treatment group,and the apoptotic protein Bax expression level was significantly lower than that of the cobalt dichloride treatment group.Results: The first part of the in vivo experimental results showed that:(1)The model of C57 mouse liver I/R injury was successfully established;(2)The serum AST and ALT of the γ-oryzanol pretreatment group were significantly lower than that of the I/R group;(3)each The pathological damage of liver tissue structure in the γ-oryzanol pretreatment group was significantly lighter than that of the I/R group;(4)The SOD and GSH contents of liver tissue in the γ-oryzanol pretreatment group were significantly higher than those of the I/R group,and The contents of MDA and MOP were significantly lower than those in the I/R group;(5)Tunel staining results showed that the apoptosis of liver tissue in the γ-oryzanol pretreatment group was significantly less than that in the I/R group;(6)Compared with the I/R group Compared with the immunohistochemical detection of the various concentrations of γ-oryzanol pretreatment group,the expression of GRP78 in the liver tissue of mice was significantly decreased;(7)Compared with the I/R group,the liver tissues in the Sham group and each γ-oryzanol pretreatment group The expression of NLRP3 inflammation pathway-related proteins and injury-related molecules HMGB1 was significantly decreased;(8)Compared with the I/R group,the GRP78/PERK/EIF2S1/CHOP endoplasmic reticulum of the liver tissues in the Sham group and the γ-oryzanol pretreatment groups The expression level of stress pathway proteins was significantly reduced.(9)Compared with the I/R group,the expression of the anti-apoptotic protein Bcl-2 in the liver tissue of the Sham group and each γ-oryzanol pretreatment group was significantly increased,and the expression of the apoptotic protein Bax was significantly reduced;The second part of the in vitro experimental results found that:(1)After AML12 mouse hepatocytes were treated with different concentrations of γ-Oryzanol solution for 24 hours,the γ-Oryzanol solution within the concentration range of 400μg/ml had no significant effect on the proliferation of mouse hepatocytes;(2)After treating AML12 mouse hepatocytes with different concentrations of cobalt dichloride solution for 24 hours,as the concentration of cobalt dichloride solution increases,the cell viability value of mouse hepatocytes gradually decreases.When it was 300 μmol/L,the cell viability value decreased to 57.80%±3.45,and the hypoxia model was subsequently established to select this concentration;(3)Different concentrations of γ-oryzanol solution were used for pretreatment of mouse liver cells at 300 μmol/L cobalt dichloride After 24 hours of incubation in the solution condition,compared with the control group,the cell viability value of the γ-oryzanol solution at 160 μg/ml and 240 μg/ml increased significantly;(4)the hepatocytes in the pretreatment group(ORY240+Co Cl2300 group)The active oxygen content was significantly higher than the cobalt dichloride treatment group(Co Cl2300 group);(5)Compared with the cobalt dichloride treatment group(Co Cl2300 group),the pretreatment group(ORY240+Co Cl2300 group)had significantly fewer apoptotic cells;(6)The expression levels of GRP78/PERK/EIF2S1/CHOP endoplasmic reticulum stress pathway proteins in the ORY240+Co Cl2300 group were significantly reduced.(7)Compared with the cobalt dichloride treatment group,the expression level of the pro-apoptotic protein Bcl-2 of hepatocytes was significantly higher than that of the cobalt dichloride treatment group,and the apoptotic protein Bax expression level was significantly lower than that of the cobalt dichloride treatment group.Conclusion: γ-Oryzanol pretreatment can ameliorate liver damage aused by liver I/R.Its possible mechanism is related to oryzanol alleviating oxidative stress,inhibiting NLRP3 inflammation pathway,and reducing the expression of injury-related molecule HMGB1 to reduce inflammation and inhibit The activation of the GRP78/ PERK/EIF2S1/CHOP endoplasmic reticulum stress pathway reduces cell apoptosis.This study explored the protective effect of ORY on liver I/R injury,and provided evidence for the future use of ORY in the treatment of liver diseases. |
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