Protective Effect And Mechanism Of Lycopene On Chronic Restraint Stressinduced Hippocampal Injury In Rats

Chronic stress is ubiquitous in livestock production and life.If not treated in time,it can seriously damage the health of animals and human beings.Chronic stress can lead to hippocampal damage,induce abnormal behavior in animals,and even causing dysfunction in other tissues and organs.The pathogenesis of chronic stress induced-hippocampal damage is complex and easy to relapse.Therefore,to clarify the pathogenesis of hippocampal injury caused by chronic stress and to find safe and effective prevention and treatment drugs have always been the focus and difficulty of veterinary and medical research.Recent studies have shown that overactivation or death of microglia in the hippocampus is an important mechanism in the pathogenesis of neurodegenerative diseases and depression.However,the role and mechanism of microglia programmed cell death in hippocampal injury induced by chronic stress remain unclear.Lycopene(LYC)has strong antioxidant and neuroprotective effects and can inhibit the abnormal activation of microglia.Therefore,it is speculated that LYC can protect hippocampal injury induced by chronic stress via inhibiting microglia programmed cell death.In this study,72 male Wistar rats were randomly divided into control(CON)group,chronic restraint stress(CRS)group,LYC intervention(50 mg/kg LYC+CRS)group and vechicle group(Vehicle+CRS)group.The stress procedure was restraint stress for 6 h per day for 21 consecutive days.In order to verify whether the chronic stress rat model was successfully replicated,after the modeling,the behavioral test was carried out,bodyweight and hippocampal weight were weighed,the hippocampal coefficient was calculated,and the serum CORT content was detected.The microscopic and ultrastructural changes of hippocampal tissues were observed.Oxidative stress,microglia activation,inflammatory cytokines,ferroptosis,apoptosis,pyroptosis,and necroptosis were detected.The expression and localization of GPX4,Cleaved caspase-3,NLRP3,Cleaved Caspase-1,MLKL and Iba-1 were observed by immunofluorescence multiple staining.The ASK1/JNK signaling pathway related proteins and genes expression were detected.To further reveal the role of microglia programmed cell death in hippocampal injury induced by chronic stress and the protective mechanism of LYC on microglia programmed cell death in chronic stress rat hippocampus,in this study,rat HAPI microglia cells were selected and divided into CON group,corticosterone(10?M CORT)group,LYC pretreatment(4?M LYC+10?M CORT)group,LYC control(4?M LYC)group,ASK1 inhibitor intervention(10?M GS-4997+10?M CORT)group,JNK inhibitor intervention(30?M SP600125+10?M CORT)group,and then incubated for 24 h.Cell viability,LDH release,changes in cell morphology and ultrastructure,oxidative stress,inflammatory cytokines,ferroptosis,apoptosis,pyroptosis,and the ASK1/JNK signaling pathway related proteins and genes were observed and detected.The experimental results are as follows:(1)CRS led to slow weight gain in rats,increased serum CORT content,reduced the autonomous activity and spatial exploration ability,caused anxiety,insecurity,anhedonia and despair in rats,indicating that the chronic stress rat model was successfully replicated.However,LYC pretreatment significantly restored the trend of weight gain,decreased the serum CORT content,and improved the depression-like behavior in CRS rats,suggesting that LYC has a certain anti-chronic stress effect.(2)LYC preconditioning can prevent CRS from causing microscopic and ultrastructural damage and loss of nerve cells in the hippocampus.Similarly,in vitro experiments,LYC preconditioning significantly alleviated the morphological changes and ultrastructural damage of HAPI cells caused by CORT.These results indicate that LYC has a good protective effect on hippocampal damage caused by chronic stress in rats.(3)LYC pretreatment inhibited the production of ROS,MDA and H₂O₂,and increased the activities of T-AOC,CAT and GSH-Px in the hippocampus of CRS rats.Consistent with in vivo results,LYC preconditioning inhibited the production of ROS and MDA induced by CORT,and enhanced the activities of T-AOC and GSH-Px in HAPI cells.These results suggest that LYC can improve CRS-induced hippocampal oxidative damage by scavenging oxygen free radicals through enhancing antioxidant capacity.(4)LYC preconditioning inhibited the activation and proliferation of microglia induced by CRS,thereby reducing the release and genes expression of IL-1?,IL-18,IL-6 and TNF-?,and then alleviating the inflammatory response in CRS rat hippocampus.In accordance with in vivo results,LYC preconditioning inhibited the levels of IL-1?,IL-18,IL-6 and TNF-?in HAPI cells supernatant after CORT stimulation.These results suggest that LYC can inhibit inflammatory cytokines released by hippocampal microglia in CRS rats.(5)CRS reduced the proteins expression of FPN1 and Ferritin,leading to iron metabolism disorder and iron accumulation,increased LPO content,up-regulated the gene expression of ferroptosis high confidence genes ATP5G3,IREB2,RPL8 and CS,and weakened the activity of GSH-Px in the hippocampus.Immunofluorescence double staining results showed that CRS reduced the expression of GPX4 in activated microglia in the hippocampus,suggesting that CRS caused microglia ferroptosis,and aggravated oxidative damage in the hippocampus.However,LYC pretreatment improved the trend of ferroptosis related indexes.Consistent with in vivo results,LYC preconditioning also inhibited CORT-induced HAPI cells ferroptosis.These results suggest that the protective effect of LYC on hippocampal injury in CRS rats is at least partially attributed to the inhibitory effect of LYC on microglia ferroptosis,and that LYC can alleviate the hippocampal oxidative damage induced by CRS by inhibiting microglia ferroptosis.(6)LYC pretreatment increased the expression ratio of Bcl-2 and Bax in the mitochondria,decreased the cytoplasmic Cyt-c,and weakened the activities of Caspase-9 and Caspase-3 in CRS rat hippocampus.TUNEL staining results showed that LYC pretreatment could significantly reduce the number of TUNEL positive cells in the hippocampus of CRS rats.Immunofluorescence double-staining results showed that LYC preconditioning decreased Cleaved Caspase-3 expression of activated microglia in the hippocampus of CRS rats.Consistent with in vivo results,LYC preconditioning inhibited CORT-induced HAPI cells apoptosis.These results suggest that LYC can inhibit microglia apoptosis in the hippocampus of CRS rats.(7)CRS increased the protein and gene expressions of NLRP3,ASC,Cleaved Caspase-1 and GSDMD,and increased the levels of IL-1?and IL-18 in the hippocampus of rats.Immunofluorescence tri-staining results showed that CRS resulted in decreased expression of NLRP3 and Cleaved Caspase-1 in activated microglia of the hippocampus.These results indicated that CRS led to microglia pyroptosis in the hippocampus of rats,which further aggravated the inflammation of the hippocampus.LYC pretreatment improved the change trend of the above pyroptosis related indexes caused by CRS.In accordance with in vivo results,LYC preconditioning reversed CORT-induced microglia pyroptosis.These results suggest that the protective effect of LYC on hippocampal injury in CRS rats is at least partially attributed to the inhibitory effect of LYC on microglia pyroptosis,and that LYC can alleviate CRS-induced hippocampal inflammatory damage by inhibiting microglia pyroptosis.(8)CRS increased the content and gene expression of TNF-?,and up-regulate the proteins and genes expression of RIP1,RIP3 and MLKL in rat hippocampus.However,the localization and expression of MLKL in CRS-activated hippocampal microglia were not observed in the results of the double-staining immunofluorescence experiment.These results suggested that CRS might not trigger microglia necroptosis in rat hippocampus.However,LYC pretreatment reduced the content and gene expression of TNF-?,inhibited the proteins and genes expression of RIP1 RIP3 and MLKL,and reduced the fluorescence intensity of MLKL in the hippocampus of CRS rats.These results suggest that the protective effect of LYC on hippocampal injury in CRS rats may be partially attributed to the inhibitory effect of LYC on other cells necroptosis in the hippocampus,other than microglia.(9)CRS promoted the phosphorylation and genes expression of ASK1 and JNK,while LYC preconditioning inhibited the phosphorylation and genes expression of ASK1 and JNK in the hippocampus of CRS rats.Consistent with in vivo results,LYC preconditioning significantly inhibited CORT-induced the phosphorylation and genes expression of ASK1 and JNK in HAPI cells.These results suggested that the ASK1/JNK signaling pathway might be involved in the neuroprotective effect of LYC on CRS-induced hippocampal injury.The inhibition of ASK1 and JNK by GS-4997 and SP600125 significantly improved CORT-induced ferroptosis,apoptosis,pyrotosis,oxidative stress,inflammatory cytokines,cell morphological changes,and LDH release.These results suggest that the protective effect of LYC on microglia programmed cell death is at least partially attributed to the inhibitory effect of LYC on the ASK1/JNK signaling pathway.In summary,CRS can cause an increase in serum CORT,which leads to oxidative stress in the hippocampus,activates microglia,and causes inflammation,leading to microglia programmed cell death,thereby further aggravating oxidative and inflammatory damage in the hippocampus of rats.LYC can reduce serum CORT content,enhance antioxidant capacity and eliminate ROS,thus alleviating oxidative stress.Meanwhile,LYC can improve microglia programmed cell death by inhibiting the ASK1/JNK signaling pathway,thus further inhibiting oxidative and inflammatory damage in the hippocampus,and then playing a neuroprotective role.This study can provide a new target for the treatment of chronic stress-related encephalopathy,and provide new ideas and means for the prevention and treatment of chronic stress-related encephalopathy in medicine and veterinary clinic.