Study Of ATF4/CHOP Axis Mediated Mitochondrial Unfolded Protein Response In Neuronal Apoptosis Induced By Methylmercury

Objective:Methylmercury（Me Hg）is an environmental contaminant that is prevalent in water bodies and can transcend the blood-brain barrier and cause serious damage to the central nervous system.Humans are exposed to Me Hg mainly by serving contaminated fish and shellfish and rice,which causes serious health problems.Studies have shown that Me Hg induced mitochondrial damage and apoptosis are important processes of its neurotoxic effects.The buildup of Me Hg disrupts the structure and function of mitochondria,which triggers mitochondrial stress and apoptosis,ultimately causing cell death.The mitochondrial unfolded protein response(UPR^mt)is a mitochondrial stress response that is activated during states of stress to maintain the stability of proteins within the mitochondria.In order to revitalize UPR^mt,the involvement of activating transcription factor 4（ATF4）and C/EBP-homologous protein（CHOP）is required in mammals.However,the molecular mechanism of Me Hg-induced UPR^mt has not been clarified.The study sought to elucidate the induction of the mitochondrial apoptotic pathway by Me Hg and the regulation of UPR^mt by the ATF4/CHOP axis in this process,thus furnishing experimental and theoretical support for the prevention of Me Hg neurotoxicity.Methods:1.Kunming mice were used to establish an in vivo model of Me Hg exposure.Forty SPF-grade adult Kunming mice were randomly divided into four groups,including control,4μmol/kg Me Hg,8μmol/kg Me Hg and 12μmol/kg Me Hg groups,with 14 mice in each group,half male and half female.After 28 days of poisoning by gavage,and the changes of body weight were monitored daily.The neurobehavioral damage of the mice was detected by claw grasping force and gait test.The mice were then executed and the cerebral cortex was taken for subsequent experiments.Determination of mercury content in mouse cerebral cortex by cold atomic absorption spectrometry.Nissl staining was used to detect the pathological changes of nerve cells in the cerebral cortex of mice.Mitochondrial membrane potential was detected by flow cytometry,and mitochondrial DNA copy number was detected by q PCR.The protein expressions of apoptosis related proteins Bax,Bcl-2,cleaved caspase-3 and Cyt C in cerebral cortex were detected by Western blot.The m RNA and protein expression levels of ATF4、CHOP and UPR^mt-related indicators HSP70,HSP60,CLPP,Lon P1 were detected by RT-q PCR and Western Blot.The positive cell ratio of ATF4 and CHOP was detected by immunohistochemistry,and the interaction of ATF4 and CHOP was detected by co-immunoprecipitation（Co-IP）.So as to explore the inducing effects of Me Hg exposure on mitochondrial damage and apoptosis in cerebral cortex.2.The HT22 cell line was used to establish the Me Hg exposure model in vitro,and the cells were pretreated with ATF4 and CHOP small interfering RNA（si RNA）.The experiment was split into four distinct groups:the control,4μM Me Hg,control si RNA,ATF4 si RNA plus Me Hg,and CHOP si RNA plus Me Hg.Fluorescence microscopy revealed si RNA transfection,and the efficiency of the transfection was more than 70%,which provides a reliable basis for subsequent experiments.The activation of CHOP by ATF4 was observed by detecting CHOP fluorescence intensity by immunofluorescence.Flow cytometry revealed the rate of apoptosis,while Western Blot revealed the expression levels of apoptosis-related proteins cleaved caspase-3 and Cyt C,thereby demonstrating the effect of ATF4/CHOP axis on apoptosis.The protein expression changes of ATF4,CHOP,HSP70,HSP60,CLPP and Lon P1 were detected by Western Blot to investigate the regulatory role of ATF4/CHOP axis on UPR^mt during Me Hg-induced neuronal apoptosis and further study the neurotoxic mechanism of Me Hg.Results:1.Compared with the control group,the claw grasping force of mice exposed to Me Hg was decreased in a dose-dependent manner.In the gait analysis experiment,the body speed and coordination of mice exposed to Me Hg were significantly reduced.The cerebral cortex of mice in each group exhibited a marked augmentation in mercury content.Staining of Nissl revealed a decrease in the morphology of neurons in the cerebral cortex of mice exposed to Me Hg,as well as a decrease in the number of Nissl bodies and volume.The expression of apoptosis related proteins Bax,cleaved caspase-3 and Cyt C increased with the increase of the dose of Me Hg,while Bcl-2 showed a downward trend.Compared with the control group,the mitochondrial DNA copy number and mitochondrial membrane potential were gradually decreased in the Me Hg exposure groups.With the increase of exposure dose,the protein and m RNA expressions of ATF4,CHOP and UPR^mt-related proteins HSP70,HSP60,CLPP and Lon P1 were up-regulated.The surface area stained by ATF4 and CHOP-positive cells gradually showed an increase,and the protein interaction between ATF4 and CHOP increased significantly.2.The Me Hg exposure group saw a marked increase in the fluorescence intensity of CHOP,whereas the ATF4/CHOP si RNA pretreatment group experienced a significant decrease.The Me Hg exposed group displayed an augmented rate of apoptosis and a heightened expression of apoptosis-related proteins cleaved caspase-3 and Cyt C,yet these were significantly reduced after pre-treatment with ATF4/CHOP si RNA.Elevated expression levels of UPR^mt-related proteins HSP70,HSP60,CLPP and Lon P1 were significantly decreased after pretreatment with ATF4/CHOP si RNA.Conclusion:1.Me Hg exposure induces UPR^mt and apoptosis in nerve cells,leading to functional damage to the nervous system in mice.2.Me Hg exposure regulates UPR^mt by activating the ATF4/CHOP axis and induces neuronal apoptosis.