| At present,most people are working at a fast pace and in a high life pressure.Continuous stress response leads to irregular life,poor sleep quality or lack of sleep and other symptoms.And then,the body activates the hypothalamus-pituitary-adrenal axis(HPA axis)to maintain the homeostasis of the internal environment.Long-term excessive stress may lead to the disorder of circadian rhythm,resulting in dysfunction of nervous function,imbalance of hormone levels,and disturbance of material metabolism and energy metabolism.With the deepening of stress degree and the prolongation of stress time,it will lead to the decompensation of self-regulation and repair of the body,which will further induce the pathological changes and disorders of multiple systems and organs of the body,such as anxiety,insomnia,depression,cardiovascular and cerebrovascular diseases,diabetes and even malignant tumors.The liver,as the metabolic center of the body’s nutrients,may also suffer from decompensation in varying degrees due to stress,and then may lead to liver injury,which is called stress-induced liver injury(SLI).SLI is common in people’s daily life.If we don’t intervene it in time,it may cause liver function damage,fatty liver,liver fibrosis and even liver cancer.At present,the exact cause of stress-induced liver injury has not been fully elucidated and there is still a lack of specific drugs against SLI.Therefore,we should establish an animal model that can simulate the SLI in human body,screen out stress-related genes by differential gene expression to intervene or reverse gene expression,find biomarkers that can be used to diagnose SLI,or find new drugs that can be used to prevent and treat SLI,so as to provide theoretical basis for clinical diagnosis and treatment of SLI.Experimental thesis consists of four parts.Part one: The establishment and confirmation of stress-induced liver injury model in SD rats.(1)Model building method: The model of SLI was established by binding 14 rats for 10 hours a day and 21 days in a row.They were divided into two groups: Normal control group(NC)and Stress-induced liver injury group(SLI).(2)Confirmation of the model: Compared with NC group,SLI rats lost weight(P<0.001),while the liver coefficient(P<0.01)and wet/dry ratio of liver were increased(P<0.001),indicating that liver swelling occurred.In addition,the increase of serum CORT(P<0.001),AST(P<0.001),ALT(P<0.001)and ALP(P<0.01)showed that the liver function of the rats was abnormal.The results of HE staining showed that water binding could lead to the change of organic function of liver.The results above together confirmed that chronic water-immersion restraint stress induced stress and successful liver injury modeling and we successfully made the SLI model.Part two: Bioinformatics study on differential gene expression and the signal pathway enrichment of SLI.By sequencing the transcriptome of the liver of rats,to find out the difference gene between NC group and SLI group.Go and KEGG analyze pathways which may lead to differences,In order to find the key genes and biomarkers,the differential genes were visualized by the software of Cytoscape.Through GO,KEGG,and systematic pharmacological analysis,it was found that when SLI occurs:(1)the genes of circadian rhythm were destroyed,such as Arntl was up-regulated,Per1 and Cry1 were down-regulated;(2)The abnormal expression of circadian rhythm genes led to disorder of liver lipid metabolism,including the increase of lipid synthesis(such as the up regulation of Scd and Fasn),the decrease of lipid decomposition(such as the down regulation of Cpt1 a and Decr1)and the disorder of lipid transport(such as the down regulation of Pparα).The serum levels of TC(P<0.01)and TG(P<0.001)were measured and oil red O staining was used to verify liver lipid accumulation.(3)The accumulation of lipid synthesis which lead to inflammation would lead to the occurrence of inflammatory reactions and liver damage,such as the up-regulation of the acute response phase protein Orm1 and the cytokine Tnfaip3.Significant increases in serum inflammatory factors IL-1β(P<0.001)and TNF-α(P<0.001)validated the increased inflammatory response in SLI.Part three: Study on the abnormal expression of circadian rhythm genes and the protective effect of melatonin on SLI.Through the analysis of transcriptome sequencing and gene differential expression,it was found that genes involved in clock regulation,Arntl was highly expressed in SLI,while Per1 and Cry1 were down-regulated,while melatonin(MLT)played a role in regulating circadian rhythm.(1)This part used 2.5 mg/kg melatonin(MLTL)and 5 mg/kg melatonin(MLTH)for intervention,and observed whether it could regulate the circadian gene Arntl and to play a role in anti-SLI.(2)The results showed that:(1)Different doses of melatonin could increase the weight of stressed rats,reduce liver coefficient and wet-dry weight ratio of liver;MLTH reduced serum CORT level in rats(P<0.05);MLTL and MLTH significantly reduced serum ALT(P<0.01,P<0.05)and AST level(no statistical difference,P<0.01);H&E staining was used to observe the effect of melatonin on liver histology,indicating that melatonin may effectively resist SLI.(2)q PCR showed that,MLTH could reverse the increase of the m RNA exprssion of Arntl(P<0.05)compared with the SLI group.Both of MLTL and MLTH reversed the down-regulation of the m RNA expression of Cry1,although there were no significant statistical differences.The results was indicated that melatonin could improve the disorder of circadian rhythm on SLI rats.(3)Compared with SLI group,MLTL and MLTH significantly reduced the level of serum TC(P<0.01,P<0.001),MLTH reduced the level of serum TG(P<0.05),Oil Red O staining showed that lipid accumulation in the liver was improved.Immunohistochemistry showed that both MLTL and MLTH could increase the expression of Pparα and Cpt1 a,key genes of lipid metabolism in the liver,and the difference was more significant in the high-dose group(P<0.05,P<0.01).(4)MLTL reduced the levels of serum IL-1β and TNF-α(P<0.05),and the levels of MLTH decreased more significantly(P<0.001,P<0.001);At the same time,MLTL and MLTH reduced the m RNA expression of the acute phase protein Orm1 and the inflammatory factor Tnfaip3 in the liver compared with the SLI group,suggesting that melatonin administration coud inhibite the inflammatory response when SLI occurred.(5)Western blot showed that the levels of Ampk,Pparα,and Cpt1 a protein in the liver of rats decreased after stress while the expression of all three increased in varying degrees after melatonin administration.(3)Summary: After the administration of melatonin,the state of circadian rhythm disturbance in the stressed rats was improved,the serum levels of CORT,ALT,and AST in the stressed rats were reduced,the occurrence of liver steatosis was prevented,and the inflammatory cytokines were reduced.They all inhibited the inflammatory response when SLI occurs.The mechanism of action was related to preventing the decrease of protein contents of Ampk、Pparα and Cpt1 a.Part four: Study on the abnormal expression of Pparα and the protective effect of Pparα agonist WY14643 on SLI.Through the analysis of transcriptome sequencing and gene differential expression,it was found that Pparα was down-regulated in liver tissues,so this part used its agonist WY14643 to observe the effect on SLI.(1)Established SLI model and used low-dose WY14643(WYL)for0.5 mg/kg and high-dose WY14643(WYH)for 1 mg/kg for intervention.(2)The experimental results showed that: After administration of WY14643,the weight of rats increased and the liver coefficient and the weight of wet/dry in liver were lower than SLI group.Elevated serum CORT was improved;Compared with the SLI group,WYL and WYH dose-dependently reduced the levels of serum ALT and AST(P>0.05,P<0.05),and the levels of serum cytokine IL-1β(P<0.01,P<0.001)although there was no statistically significant change in TNF-α;In addtion,MLT reduced the levels of serum TC and TG(P>0.05),and oil red O staining showed that lipid accumulation in the liver was improved.H&E staining showed that WY14643 could significantly improved liver histological.It is suggested that WY14643 has pharmacological effects to anti-SLI.(3)The mechanism study:(1)Effect of WY14643 on circadian:Compared with the SLI group,WY14643 administration could reverse the m RNA expression of Arntl(P<0.05,P<0.001)and Cry1(P> 0.05,P<0.01),suggesting Pparα agonist also had the function of regulating the circadian rhythm.(2)After administration of WY14643,immunohistochemistry showed that the expression of Pparα(P<0.05,P<0.001)and Cpt1a(P>0.05,P<0.001)in liver tissues was higher than that in SLI;The m RNA expression of Orm1(P<0.01,P< 0.001)and Tnfaip3(P<0.001,P<0.001)were lower than the SLI group,indicating that WY14643 administration could prevent adipose metabolism and inflammatory response due to adipose metabolism.(3)WY14643 could effectively inhibit the reduction of Ampk,Pparα and Cpt1 a after SLI,while WYL and WLH could increase Ampk by 5.3% and 16.5%(P<0.01),Cpt1 a expression by 3.4% and 15.1%(P<0.01),low Dose WY14643 increased Pparα expression by 6.8%,but high dose administration did not increase Pparα expression.(4)Summary:The Pparα agonist WY14643 could also correct the abnormal lipid metabolism and inflammatory response during SLI by adjusting the circadian clock molecules Arntl and Cry1,and restore the signal molecules protein level of Ampk,Pparα and Cpt1 a.Overall: The occurrence of SLI is not a change in a single gene or a signal pathway,but an adaptive change in the main transformation system of the body,which can be summarized as: circadian rhythm-endocrine-lipid metabolism-immune axis.Through study of differential gene expression,it was found that Arntl and Per1 of circadian rhythm and Pparα,which metabolize metabolism,together participate in the occurrence of SLI.Administering the circadian rhythm regulator melatonin and the lipid metabolism regulator Pparα agonist WY14643 could anti-SLI,which may corrected the disorder of circadian rhythm,reduced liver lipid accumulation and inflammatory responses.The mechanism was related to the activation of the Ampk/Pparα/Cpt1 a signal pathway. |
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