SelenoM Targets Nrf2 To Participate In NiCl₂-Induced Endoplasmic Reticulum Stress-Dependent Ferroptosis In The Liver Of Mice

Nickel is the fifth most abundant element discover ed after iron,oxygen,magnesium,and silicon,and the 24th most common element in the Earth’s cr ust.Nickel and its compounds have a wide range of applications due to their excellent physical and chemi cal properties.In daily life,humans can come into contact with nickel and its compounds through food pro-cessing,household products,and industrial p rocesses,and their accelerated accumulation in the environment during production,use,recycling,and di sposal inevitably poses a serious threat to human health.Nickel compounds have been classified as human carcinogens.In-creasing evidence shows that nickel has multiple toxic effects on various systems in the body,which can lead to immune sensitization,a bnormal epithelial cell development,asthma,fibro-sis,and lung cancer.The exact mechanisms underlying these pathological effects caused by nickel are not fully understood,but these mechanism s may involve changes in signaling pathways,regulation of gene expression,and alterations in chromatin.Most studie s suggest that the toxicity of nickel is most likely related to oxidative stress.Selenium（Se）is an essential trace element for human hea lth with extremely important biological functions.Selenium’s special functions are mainly executed by the genetic diversi-ty of selenoproteins.So far,25 types of selenoproteins have been found in the human prote-ome,of which only a few have functional characteristics.Selenoprotein M（SelenoM,Sel M）,which is located in the endoplasmic reticulum（ER）and is commonly referred to as th e ER-resident selenoprotein,plays an important physiological role in humans and ot her organisms.Previous studies by our research group have shown that SelenoM plays a core role in main-taining mitosis in high-fat diet（HFD）-induced non-alcoholic fatty liver disease（NAFLD）.SelenoM is significantly downregulated in the livers of HFD-fed mice,and SelenoM defi-ciency exacerbates HFD-induced liver steatosis and fibrosis,worsening liver dama ge in NAFLD.Furthermore,studies have shown that SelenoM has neuropr otective effects in the nervous system.In addition,SelenoM participates in biolog ical processes such as calcium ion regulation and synaptic transmission within cells.In order to investigate the role of SelenoM in nickel-induced liver injury in mice,the present study utilized SelenoM knockout(SelenoM^-/-)mice previously constructed by the re-search group.Forty 8-week-old C57BL/6N wild-type mice and SelenoM^-/-mice were random-ly selected and divided into four groups（n=20）:C57BL/6N wild-type mouse control group（NC group）,C57BL/6N wild-type mouse nickel exposure group（Nigroup）,Sel eno M^-/-mouse group（KO group）,and SelenoM^-/-mouse nickel exposure group（KO+Nigroup）.During the experiment,mice were allowed to freely feed and drink,and a NiCl ₂ solution was adminis-tered via gavage to establish a mouse liver injury model induced by 21 days of NiCl₂ expo-sure.Liver tissue and serum were collected,and HE staining,Prussian blue staini ng,liver function tests,antioxidant function tests,and ultrastruct ural observation were performed to confirm the successful establishment of the C57B L/6N wild-type and SelenoM^-/-mouse nick-el exposure model.In vitro cultured liver cells（AML12 cells）w ere used to construct the fol-lowing models:（1）SelenoM knockdown models,which were divided into the following four groups:AML12 cell group（NC group）,AML12 cells exposed to NiCl₂（Nigroup）,SelenoM knockdown AML12 cell group（KO group）,and SelenoM knockdown AML12 cell group ex-posed to NiCl₂（KO+Nigroup）.（2）SelenoM overexpression models were also constructed and divided into the following four gro ups:AML12 cell group（NC group）,AML12 c ells ex-posed to NiCl₂（Nigroup）,SelenoM overexpression AML12 cell group（SelenoM group）,and SelenoM overexpression AML12 cell group exposed to NiCl₂（SelenoM+Nigroup）.（3）After demonstrating that Nrf2 is an interacting protein of SelenoM,Nrf2 overexp ression models were constructed.RSL3（a ferroptosis inducer）was used to construct an AML12 cell ferrop-tosis model,which was then divided into the following four groups:AML12 cell group（NC group）,RSL3-induced AML12 cell group（RSL3 group）,RSL3-induced SelenoM overexpres-sion AML12 cell group（RSL3+SelenoM group）,and RSL3-induced Nrf2 overexpression AML12 cell group（RSL3+Nrf2 group）.（4）In addition,4-PBA（an endoplasmic reticulum stress inhibitor）was used in combination with NiCl₂ to construct a weakened NiCl₂-induced endoplasmic reticulum stress model,which was divided into the following five groups:AML12 cell group（NC group）,AML12 cells exposed to NiCl₂（Nigroup）,AML12 cells ex-posed to NiCl₂ and treated with 4-PBA（Ni+4-PBA group）,SelenoM overexpression AML12cell group exposed to NiCl₂（Ni+SelenoM group）,and SelenoM overexpression AML12 cell group exposed to NiCl₂ and treated with 4-PBA（Ni+SelenoM+4-PBA group）.By measuring oxidative stress levels,mitochondrial function,endoplasmic reticulum stress levels,and fer-roptosis levels,this study aimed to investigate the toxic effects of NiCl₂ on the liver or liver cells（AML12 cells）.The primary experimental results are summarized as follows:（1）The pathological histology and ultrastructure observations of the liver showed that in the Nigroup,the liver lobules were arranged uniformly but without clear boundaries,and the central vein congestion was severe.There was significant nuclear aggregation a nd displace-ment,indicating that the liver cells were damaged.In the KO+Nigro up,no obvious liver lobule structure was observed,and the central vein congestion was irregu lar.The liver cells were non-radial and showed nuclear aggregation and vacuolizati on,and even a large amount of nuclear aggregation near the portal vein or part ial absence of nuclei,indicating severe damage to the liver cells.The results of ultrastruct ure observation showed that the mitochon-dria in the liver of the mice in the Nigro up had significant deformation with uncle ar bounda-ries and reduced volume and mitochondrial cristae density,which was even more severe in the KO+Nigroup,indicating that S eleno M knockout exacerbated the damage caused by NiCl₂.（2）The results of liver function tests showed that compared with th e Nigroup,the KO+Nigroup of mice had increased levels of serum AST and ALT（P<0.05）,decreased levels of ALB（P<0.05）,and increased levels of GLB（P<0.05）,TBIL and DBIL（P<0.05）,as well as increased levels of ALP and CHOL（P<0.05）.The results of antioxidant capacity testing showed that the MDA content increased（P<0.05）while the activities of CAT,GSH,GSH-Px,SOD,and T-AOC decreased（P<0.05）after NiCl₂ treatment,indicating that NiCl₂ can cause liver dysfunction and oxidative stress,and that the knockout of SelenoM exacerbates the t ox-ic effects of NiCl₂.（3）The present study investigated the effects of NiCl ₂ exposure and SelenoM knockout on oxidative stress and mitochondrial function in mouse liver.The r esults showed that com-pared with the Nigroup,the m RNA and protein expression of HO-1 and NQO1 in the KO+Nigroup were decreased,while the m RNA and protein expression of K eap1 were increased（P<0.05）.Moreover,the m RNA expression of Tfam,VDAC2,Nrf1,M fn1,and Mfn2 were decreased（P<0.05）,and the protein expression of Tfam,VDAC 2,and VDAC3 were also de-creased（P<0.05）.In addition,the effects of NiCl2 exposure and Sele no M knockout on endo-plasmic reticulum stress and ferroptosis in mouse liver were st udied.Compared with the Nigroup,the m RNA and protein expression of SelenoM w ere decreased（P<0.05）in the KO+Nigroup.Meanwhile,the m RNA and protein expression of endop lasmic reticulum stress-related genes ATF4,ATF6,GRP78,PERK,and CHOP were increa sed（P<0.05）,and the m RNA and protein expression of GPX4,Xc-,and Nrf2 were decreased（P<0.05）,indicating that SelenoM plays an important role in the toxic effects of NiC l₂.（4）In this study,the effects of NiCl₂ exposure and SelenoM knockdown on oxida tive stress and mitochondrial function of AML12 cells were examined.After SelenoM knockdown,mitochondrial volume decreased,mitochondrial cristae reduced or disappeared in Niand KO+Nigroups of AML12 cells.Compared to the Nigroup,the m RNA and protein expression of HO-1 and NQO1 were decreased（P<0.05）,while the m RNA and protein expression of Keap-1 were increased（P<0.05）,and ROS level was increased（P<0.05）in the KO+Nigroup.In addition,compared to the Nigroup,the m RNA and protein expression o f Tfam,VDAC2,and VDAC3 were decreased（P<0.05）,and mitochondrial membrane potential was reduced in the KO+Nigroup（P<0.05）.The study also investigated the effects of NiCl₂ exposure and Se-leno M knockdown on endoplasmic reticulum stress and ferroptosis in AML12 cells.Com-pared to the Nigroup,the calcium ion level was increased（P<0.05）,and the m RNA and pro-tein expression of endoplasmic reticulum stress-related genes,such as ATF4,ATF6,GRP78,PERK,and CHOP,were upregulated（P<0.05）,while the m RNA and protein expression of SelenoM were decreased（P<0.05）in the KO+Nigroup.Additionally,compared to the Nigroup,the levels of iron ions and lipid peroxides were increased（P<0.05）,and the m RNA and protein expression of Nrf2,Xc^-,and GPX4 were decreased（P<0.05）in the KO+Nigroup.These results indicate that SelenoM knockdown further increases oxidat ive stress,endoplas-mic reticulum stress,ferroptosis,and impairs mitochondrial function in AML12 cells after NiCl₂ exposure.（5）SelenoM and Nrf2 were overexpressed in AML12 cells,respectively,and a model of SelenoM and Nrf2 overexpression in AML12 cells was established,which was verified by CO-IP assay,confirming that one of the reciprocal proteins of SelenoM is Nrf2,with good molecular docking score,which can also demonstrate the targeting regulation relationship between SelenoM and Nrf2.This experiment investigated the effects of NiCl₂ exposure and SelenoM overexpression on oxidative stress and mitochondrial function regulation in AML12cells.Compared to the Nigroup,overexpression of SelenoM in the SelenoM+Nigroup re-sulted in increased m RNA and protein ex pression of HO-1 and NQO1（P<0.05）,decreased m RNA and protein expression of Keap-1（P<0.05）,and decreased ROS levels（P<0.05）.Compared to the Nigroup,the SelenoM+Nigroup had decreased mitochondrial membrane potential（P<0.05）and increased m RNA and protein expression of Tfam,VDAC2,and VDAC3（P<0.05）.Additionally,this experiment investigated the effects of NiCl₂ exposure and SelenoM overexpression on endoplasmic reticulum（ER）stress and ferroptosis in AML12cells.Compared to the Nigroup,the Sel eno M+Nigroup had decreased calcium levels（P<0.05）,decreased m RNA and protein expression of ER stress-related genes ATF4,ATF6,GRP78,PERK,and CHOP（P<0.05）,and increased m RNA and protein expression of SelenoM（P<0.05）.Furthermore,the SelenoM+Nigroup had decreased levels of iron ions and lipid peroxides（P<0.05）,and increased m RNA and protein expression of Nrf2,Xc^-,and GPX4（P<0.05）compared to the Nigroup.Taken together,these results indicate that SelenoM over-expression attenuates oxidative stress,ER stress,and ferroptosis in AML1 2 cells following NiCl₂ exposure,and enhances mitochondrial fu nction.（6）In this study,a model of ferroptosis with reduced downstream GPX4 of Nrf2 was es-tablished using RSL3 as an inducer to investigate the effects of SelenoM and Nrf2 overex-pression on oxidative stress and mitochondrial function in AML12 cells.Compared to the RSL3 group,the RSL3+SelenoM group and RSL3+Nrf2 group showed a decrease in m RN A and protein expression levels of HO-1 and NQO1（P<0.05）,an increase in m RNA and protein levels of Keap-1（P<0.05）,and a decrease in ROS levels（P<0.05）.Compared to the RSL3group,the RSL3+SelenoM group and RSL3+Nrf2 group showed a decrease in mitocho ndrial membrane potential（P<0.05）and a decrease in m RNA and protein expression levels of Tfam,VDAC2,and VDAC3（P<0.05）.The study also examined the effects of SelenoM and Nrf2overexpression on endoplasmic reticulum stress and ferroptosis in AML12 cell s.Compared to the RSL3 group,the RSL3+SelenoM group and RSL3+Nrf2 group showed a decrease in cal-cium ion levels,a decrease in m RNA and protein expression levels of endoplasmic reticulum stress-related genes ATF4,ATF6,GRP78,PERK,and CHOP（P<0.05）,and an increase in m RNA and protein expression levels of SelenoM（P<0.05）.Compared to the RSL3 group,th e RSL3+SelenoM group and RSL3+Nrf2 group showed a decrease in iron ion and lipid perox-ide levels（P<0.05）and an increase in m RNA and protein expression levels of Nrf2,Xc^-,and GPX4（P<0.05）.These findings suggest that SelenoM and Nrf2 overexpression can effective-ly alleviate RSL3-induced ferroptosis.（7）This experiment investigated the effe cts of 4-PBA（a CHOP inhibitor）and SelenoM overexpression on oxidative stress and mitochondrial function in AML12 cells exposed to NiCl₂,by suppressing endoplasmic reticulum stress.Compared to the Nigroup,the Ni+4-PBA and Ni+SelenoM groups showed incr eased m RNA and protein expression of HO-1 and NQO1（P<0.05）,and decreased m RNA and protein expression of Keap-1（P<0.05）.The Ni+4-PBA+SelenoM group showed even more significant changes in the expression of these genes compared to the Ni+4-PBA and Ni+SelenoM groups.In addition,compared to the Nigroup,the Ni+4-PBA and Ni+SelenoM groups showed decreased mitochondrial membrane potential（P<0.05）and decreased m RNA and protein expression of Tfam,VDAC2,and VDAC3（P<0.05）,with the Ni+4-PBA+SelenoM group showing even more significant changes.The effects of 4-PBA and SelenoM overexpression on endoplasmic reticulum stress and ferropto-sis were also examined.Compared to the Nigroup,the Ni+4-PBA and Ni+SelenoM groups showed decreased calcium ion levels and d ecreased m RNA and protein expression of ATF4,ATF6,GRP78,PERK,and CHOP（P<0.05）,with the Ni+4-PBA+SelenoM group showing even more significant changes.Furthermore,compared to the Nigroup,the Ni+4-PBA and Ni+SelenoM groups showed decreased iron ion a nd lipid peroxide levels（P<0.05）,and de-creased m RNA and protein expression of Nrf2,Xc^-,and GPX4（P<0.05）,with the Ni+4-PBA+SelenoM group showing even more signifi cant changes in the expression of these genes.In summary,this study demonstrated that S eleno M deficiency exacerbates nickel-induced liver injury in mi ce,leading to impaired liver function,reduced antioxidant capacity,decreased liver mitochondrial volu me,and decreased mitochondrial cristae density.In AML12 cells,SelenoM knockdown and ov erexpression affected oxidative stress,mitochon-drial dysfunction,endoplasmic reticulum stress,and ferroptosis levels induced by nickel ex-posure.To explore its specific mechanism,this study applied the ferroptosis inducer RSL3and endoplasmic reticulum stress inhibitor 4-PBA.The results showed that SelenoM target ed Nrf2 to regulate ATF4/GRP78 and Xc^-/GPX4 pathways involved in nickel-induced endoplas-mic reticulum stress-dependent ferroptosis in the mouse liver.