Study On The Protective Effect And Mechanism Of Pparα Agonist WY14643 On Stress-induced Liver Injury

Stress reaction refers to the nervous,endocrine and physiological reactions of the body to external environmental changes or internal stimulation.Appropriate stress can enhance the ability of body to escape or resist injury,but long-term excessive stress will cause damage to the body,resulting in organ ischemia and hypoxia,multiple organ failure and even death.Stress-induced liver injury(SLI)refers to liver injury caused by different degrees of decompensation due to stress which can occur in septicemia,surgery,trauma and shock.At present,the mechanism of stress-induced liver injury is not completely clear,and thus this paper analyzed the liver tissue of rats under water immersion and restraint stress by transcriptional sequencing to find out the differential expression genes and signal pathways with strong correlation with SLI.Agonists or inhibitors were used to interfere with the screened differentially expressed genes and signal pathways to study their role and mechanism in SLI.The purpose is to provide theoretical basis for clinical prevention and treatment of stress-induced liver injury.Study on SLI was carried out in the following aspects:Part 1: The establishment and confirmation of rat stress-induced liver injury model induced by water immersion restraint stressThe rat SLI model was established by the water immersion restraint method.Fourteen rats were divided into two groups: normal control group(NC)and stress-induced liver injury group(SLI).The successful establishment of the model was confirmed by observing the changes of body weight,general behavior,HE staining of liver tissues,serum corticosterone(CORT),alanine transferase(ALT)and aspartate transferase(AST)level.According to the results,the growth rate of body weight(P < 0.01)and average body weight(P < 0.001)decreased significantly,and the serum CORT level increased significantly(P < 0.01)in SLI group,indicating that the rats in SLI group were in a state of stress.Compared with NC group,the levels of ALT(P < 0.01)and AST(P < 0.01)were significantly higher in SLI group,indicating that water immersion restraint stress could cause liver damage in rats.On the basis of HE staining,there was disorder of hepatic cords arrangerment,enlarged gap between hepatocytes and visible inflammatory cell infiltration in SLI group,suggesting that there were organic functional changes in the liver.The above results showed the successful establishment of SLI model.Part 2: Analysis of differentially expressed genes in liver tissue of water immersion restraint stressed ratsTranscriptome sequencing was performed on the liver tissues of rats in NC group and SLI group to obtain the differentially expressed genes of each group.q RT-PCR was used to further verify the differentially expressed genes.At the same time,KEGG and GO were used to find stress potential therapeutic target of SLI.According to the results,among the differentially expressed genes in SLI,lipid metabolism-related genes had the largest number of expressions,suggesting that changes of genes related to lipid metabolism may be involved in the process of SLI.In the KEGG function enrichment analysis,the changes of PPAR in signal pathways related to lipid metabolism were the most significant,of which the key gene PPARα was down-regulated,indicating that there was a close correlation between PPARα and SLI.Clues were provided for the further use of PPARαagonists to study whether they have potential anti-stress liver injury effects.Part 3: Protective effect of PPARα agonist WY14643 on SLIThrough the analysis of differentially expressed genes,it was found that there was a close relation between PPARα and SLI,and that there was a significant down-regulation of PPAR α expression in SLI,so the PPAR α agonist Wy14643 was selected to intervene in rats with SLI.The protective effect of Wy14643 was verified by observing the changes of average body weight and general behavior state of rats,detecting the serological indexes,HE staining and Oil Red O staining of liver tissue.The molecular mechanism of its protective effect was further explored by Western Blot and immunohistochemistry.According to the results,compared with the SLI group,the average body weight and weight growth rate of the WY14643 administration group increased,and the levels of CORT,ALT(P < 0.05),AST,TC(P <0.05)and TG in the serum of rats were decreased.In the light of the results of HE staining and Oil Red O staining of liver tissue,after the intervention of WY14643,there was a orderly arrangement of liver cells,and reduction of inflammatory cell infiltration and red lipid droplets in liver cells.The above results suggest that WY14643 had a protective effect on stress liver injury.Western Blot results showed that WY14643 can reduce the protein levels of p-IκBα(P < 0.001),p-AKT(P < 0.001),p-p65(P < 0.001)and PI3K(P < 0.01).It was suggested that WY14643 may inhibit the PI3K/AKT/NF-κB/IκBα signaling pathway to produce a protective effect against stress liver injury.Part 4: The mechanism of PPARα agonist WY14643 on corticosterone-induced cell damageThe concentrations of corticosterone and Wy14643 were determined by MTT colorimetry.The protective effect of Wy14643 was verified by detecting the contents of ALT,AST,TC and TG in LO2 cells and oil red O staining.The molecular mechanism of WY14643 protective effect was explored by Western blot.According to the results,the levels of ALT(P < 0.01),AST(P <0.001),TC(P < 0.05)and TG(P < 0.001)levels were significantly increased in SLI group,and there were obvious red lipid droplets in LO2 cells.After the intervention with WY14643,the levels of ALT(P < 0.01),AST(P < 0.05),TC(P < 0.01)and TG(P < 0.001)were significantly decreased,and the red lipid droplets in LO2 cells were significantly reduced,indicating that corticosterone could induce LO2 cells damage and lipid deposition,while WY14643 could alleviate the damage and lipid deposition of LO2 cells induced by corticosterone.In line with the results of Western Blot,the expressions of p-Akt(P < 0.001)and PI3K(P< 0.001)protein were significantly up-regulated in SLI group,while after intervention by WY14643,the expressions of PI3K(P < 0.001)、p-IκBα(P < 0.001)、p-p65(P < 0.005)and p-Akt(P < 0.001)protein were down-regulated,indicating that WY14643 may alleviate the damage of LO2 cells induced by corticosterone by inhibiting the PI3K/AKT/NFκB/IκBα signal pathway,which was consistent with the changes of water immersion restraint stress animals.On the whole,WY14643 has a protective effect on stress-induced liver injury,which is related to down regulating the expression of PI3 K,P-IκBα,p-p65 and p-Akt protein and reducing liver lipid deposition.