To Explore The Protective Mechanism Of Lycopene Against Chronic Restraint Stress-induced Liver Injury In Rats Based On Keap1-Nrf2/ARE Signaling Pathway

Chronic stress is widespread in the process of livestock and poultry breeding,causing certain harm to the health status and production performance of animals,and is the focus of attention in the field of animal husbandry and veterinary medicine.As an important organ of metabolism,detoxification and immune function,liver is one of the main target organs of chronic stress.Damage to the liver can cause liver dysfunction,resulting in the animal metabolism of toxic substances decreased ability,endangers animal health,bring great economic losses to the breeding industry.Therefore,it has always been the focus of veterinary clinical research to search for effective protective drugs for chronic stress-induced liver injury.Lycopene(LYC)is a kind of natural carotenoid widely present in nature,which has potent antioxidant activity and various biological functions.Since LYC absorbed by the body is mainly stored in the liver tissue,LYC has the potential of targeted prevention and treatment of animal liver diseases.However,whether LYC has a protective effect of chronic stress-induced liver injury and its mechanism remain unclear.Therefore,in this study,the protective mechanism of LYC on chronic stress-induced liver injury was explored through the establishment of rat chronic stress model and the administration of LYC combined with Nrf2 inhibitor Brusatol,which provided the corresponding theoretical basis and basis for the prevention and treatment of chronic stress-induced liver injury in veterinary clinic in the future.Fifty-four healthy male Wistar rats were randomly divided into 6 groups(n=9): CON group,CRS group,CRS+LYC group,CRS+Vehicle group,CRS+LYC+Bru group,CRS+LYC+DMSO group.The rats of CRS group,CRS+LYC group,CRS+Vehicle group,CRS+LYC+Bru group,and CRS+LYC+DMSO group were subjected to restraint stress for 6 h per day for 21 consecutive days.The rats of CRS+LYC group and CRS+Vehicle group were administered by gavage 10 mg/kg LYC and 2.5 m L/kg corn oil 1 h before restraint,respectively.The rats of CRS+LYC+Bru group and CRS+LYC+DMSO group were administered by gavage 10 mg/kg LYC 1 h before restraint each day,and were intraperitoneally injected with 0.4 mg/kg Brusatol and 2 m L/kg inhibitor solvent every other day,respectively.After the modeling,weight analysis,behavioral tests,serum CORT content detection,liver function tests,liver histopathological observation,liver cell ultrastructure observation,endoplasmic reticulum stress index detection,apoptosis-related factors detection,oxidative stress index detection,Keap1-Nrf2/ARE signaling pathway related factors detection and LYC and Keap1 protein interaction verification were performed on the rats.Results:(1)The results of body weight measurement showed that compared with the CON group,the body weight of rats in CRS group increased slowly,and the average daily gain was significantly decreased(p < 0.01).The results of the open field test showed that compared with the CON group,the total distance,the number of rearing and crossing in the CRS group were significantly reduced(p < 0.01).Compared with the CON group,serum CORT content in CRS group was significantly increased(p < 0.01).LYC intervention significantly alleviated the above changes in CRS rats.However,the protective effect of LYC to CRS rats was significantly inhibited by the use of Nrf2 inhibitor Brusatol.(2)The results of liver function test showed that compared with the CON group,the activities of serum ALT and AST in CRS group were significantly increased(p < 0.01).The results of liver histopathology showed that the morphology and structure of liver tissue were normal in the CON group.While the liver tissue damage was obvious in the CRS group,and the structure of liver lobule was destroyed.LYC intervention significantly improved liver function and liver histopathological changes in CRS rats.But Brusatol significantly reversed the protective effect of LYC on the liver of CRS rats.(3)Ultrastructural observation showed that the morphology and structure of the endoplasmic reticulum of the liver cell in the CON group were normal,while that in CRS group was obviously swollen and disordered.Endoplasmic reticulum stress marker detection results showed that compared with the CON group,m RNA and protein expressions of GRP78,CHOP and Cleaved caspase-12 in liver tissue of rats in CRS group were significantly increased(p < 0.01).LYC intervention significantly improved the endoplasmic reticulum ultrastructural damage and decreased the expression of endoplasmic reticulum stress markers in CRS rats.However,Brusatol significantly reversed the inhibitory effect of LYC on the endoplasmic reticulum stress,and aggravated the injury of the liver endoplasmic reticulum stress in rats.(4)TUNEL assay results showed that compared with the CON group,liver cells apoptosis index in CRS group was significantly increased(p < 0.01).Compared with the CON group,Cleaved caspase-3 and Bax/Bcl-2 protein expressions were significantly increased in the CRS group(p < 0.01).LYC intervention significantly reduced the level of liver cells apoptosis in CRS rats.But Brusatol significantly reversed the inhibitory effect of LYC on liver cells apoptosis in CRS rats.(5)ROS and MDA contents in liver tissue of rats in the CRS group were significantly increased compared with the CON group(p < 0.01).The results of CAT,SOD and GSH detection showed that compared with the CON group,the levels of CAT,SOD and GSH in liver tissue of rats in CRS group were significantly decreased(p < 0.01).LYC intervention significantly reduced the level of oxidative stress in liver tissue of CRS rats.But Brusatol treatment significantly inhibited the antioxidant effect of LYC.(6)The results of Keap1-Nrf2/ARE signaling pathway related indicators showed that compared with the CON group,the expression of Keap1 protein in the liver tissue of CRS group was significantly increased(p <0.01),and the expression of Nrf2 nuclear protein,NQO1 and HO-1 protein was significantly decreased(p < 0.01).LYC intervention significantly increased Nrf2 translocation into the nucleus of CRS rat liver tissue and promoted the transcription of downstream antioxidant genes.However,Brusatol inhibited the activation of Nrf2 by LYC.(7)Molecular docking and CETSA results showed that LYC can occupy the binding site of Nrf2 and Keap1,increase the thermal stability of Keap1 protein,and has a good binding ability with Keap1.The results showed that chronic restraint stress caused endoplasmic reticulum stress,cell apoptosis and oxidative stress in rat liver.By occupying the action sites of Nrf2 and Keap1,LYC competes with Nrf2 to bind Keap1,thus promoting the activation of Nrf2,increasing the expression of downstream antioxidant factors,clearing ROS and reducing the level of oxidative stress in rat liver,and further alleviating endoplasmic reticulum stress and its mediated apoptosis,and alleviates the liver damage caused by chronic restraint stress in rats.