硒蛋白M敲除在褪黑素拮抗镍诱导的小鼠心脏细胞凋亡和内质网应激中的作用

Role of selenoprotein M knockdown in the melatonin antagonism of nickel-induced apoptosis and endoplasmic reticulum stress in mouse heart 硒蛋白M敲除在褪黑素拮抗镍诱导的小鼠心脏细胞凋亡和内质网应激中的作用 1 equal first-author 2 equal 1 1 1 1 1 1 corresp Jingzeng CAI, caijingzeng@neau.edu.cn Jingzeng CAI, caijingzeng@neau.edu.cn caijingzeng@neau.edu.cn https://orcid.org/0000-0001-6929-5431 1 3 corresp Ziwei ZHANG, zhangziwei@neau.edu.cn Ziwei ZHANG, zhangziwei@neau.edu.cn zhangziwei@neau.edu.cn https://orcid.org/0000-0002-5705-4611 东北农业大学动物医学学院， 中国哈尔滨市， 150030 东北农业大学动物医学学院， 中国哈尔滨市， 150030 College of Veterinary Medicine, Northeast Agricultural University, Harbin 150030, China College of Veterinary Medicine, Northeast Agricultural University, Harbin 150030, China 绥化市农业农村局， 中国绥化市， 152000 绥化市农业农村局， 中国绥化市， 152000 Suihua Agricultural and Rural Bureau, Suihua 152000, China Suihua Agricultural and Rural Bureau, Suihua 152000, China 东北农业大学黑龙江省教育厅动物常见病防治重点实验室， 中国哈尔滨市， 150030 东北农业大学黑龙江省教育厅动物常见病防治重点实验室， 中国哈尔滨市， 150030 Key Laboratory of the Provincial Education, Department of Heilongjiang for Common Animal Disease Prevention and Treatment, Northeast Agricultural University, Harbin 150030, China Key Laboratory of the Provincial Education, Department of Heilongjiang for Common Animal Disease Prevention and Treatment, Northeast Agricultural University, Harbin 150030, China The aim of this study was to investigate the role of selenoprotein M (SelM) in endoplasmic reticulum stress and apoptosis in nickel-exposed mouse hearts and to explore the detoxifying effects of melatonin. At 21 d after intraperitoneal injection of nickel chloride (NiCl 2 ) and/or melatonin into male wild-type (WT) and SelM knockout (KO) C57BL/6J mice, NiCl 2 was found to induce changes in the microstructure and ultrastructure of the hearts of both WT and SelM KO mice, which were caused by oxidative stress, endoplasmic reticulum stress, and apoptosis, as evidenced by decreases in malondialdehyde (MDA) content and total antioxidant capacity (T-AOC) activity. Changes in the messenger RNA (mRNA) and protein expression of genes related to endoplasmic reticulum stress (activating transcription factor 4 (ATF4), inositol-requiring protein 1 (IRE1), c-Jun N-terminal kinase (JNK), and C/EBP homologous protein (CHOP)) and apoptosis (B-cell lymphoma-2 (Bcl-2), Bcl-2-associated X protein (Bax), Caspase-3, Caspase-9, and Caspase-12) were also observed. Notably, the observed damage was worse in SelM KO mice. Furthermore, melatonin alleviated the heart injury caused by NiCl 2 in WT mice but could not exert a good protective effect in the heart of SelM KO mice. Overall, the findings suggested that the antioxidant capacity of SelM, as well as its modulation of endoplasmic reticulum stress and apoptosis, plays important roles in nickel-induced heart injury. 本研究旨在研究硒蛋白M（SelM）在由镍诱导的小鼠心脏内质网应激和细胞凋亡中的作用，并探索褪黑素的解毒作用。在对雄性野生型（WT）和SelM敲除型（KO）C57BL/6 J小鼠腹腔注射氯化镍（NiCl 2 ）和/或褪黑素21天后，我们发现NiCl 2 能诱发WT和SelM KO小鼠心脏的微观结构和超微结构的变化，并通过丙二醛（MDA）含量和总抗氧化能力（T-AOC）下降证明这些变化是由氧化应激、内质网应激和细胞凋亡引起的。同时，我们观察到与内质网应激（激活转录因子4（ATF4）、肌醇需要酶1（IRE1）、c-Jun N-端激酶（JNK）和C/EBP同源蛋白（CHOP））和细胞凋亡（B细胞淋巴瘤2型蛋白（Bcl-2）、Bcl-2相关蛋白X（Bax）、半胱氨酸-天冬氨酸蛋白酶3（Caspase-3）、Caspase-9和Caspase-12）相关基因的信使RNA（mRNA）和蛋白表达的变化。值得注意的是，这种损伤在SelM KO小鼠中更严重。此外，褪黑素减轻了WT小鼠由NiCl 2 引起的心脏损伤，但对SelM KO小鼠的心脏却不能产生良好的保护作用。综上所述，本研究结果表明SelM的抗氧化能力以及它对内质网应激和细胞凋亡的调节在镍引起的心脏损伤中起着重要作用。 硒蛋白M（SelM） 氯化镍 褪黑素 细胞凋亡 小鼠心脏 Selenoprotein M (SelM) Nickel chloride (NiCl 2 ) Melatonin Apoptosis Mouse heart 1 Introduction 1 Nickel, as a transition metal, is a product of industrial, liquid, and solid fuel use, which has been widely distributed in the environment including the air, water, and soil ( Cameron et al., 2011 Cameron et al., 2011 ). Exposure to nickel can cause cardiovascular and kidney diseases, lung fibrosis, lung cancer, and nasal cancer in humans ( Genchi et al., 2020 Genchi et al., 2020 ). Clinical cases have shown that drinking water contaminated with nickel chloride (NiCl 2 ) causes shortness of breath and dizziness, and the accidental consumption of nickel sulfate (NiSO 4 ) can result in cardiac arrest and death. Animal experiments have indicated changes in the heart mass of rats after exposure to NiCl 2 ( Das et al., 2018 Das et al., 2018 ). In terms of mechanisms of action, the generation of reactive oxygen species (ROS) was thought to be involved in the underlying mechanism of heavy metal-induced toxicity ( Rehman et al., 2018 Rehman et al., 2018 ). Previous studies have confirmed that nickel sulfide increases ROS formation in rat lymphocytes in a concentration-dependent manner ( Chakrabarti and Bai, 1999 Chakrabarti and Bai, 1999 ; Zhao et al., 2021 Zhao et al., 2021 ). Nickel induces apoptosis in the kidney, liver, as well as the nervous and immune systems by regulating ROS, mitochondria, and the endoplasmic reticulum ( Guo et al., 2016 Guo et al., 2016 ; Liu JB et al., 2022 Liu JB et al., 2022 ). In addition, previous research has shown that nuclear factor of activated T cells (NFAT), nuclear factor-‍κB (NF-‍κB), activator protein-1 (AP-1), and other transcription factors were activated after exposure to nickel, and triggered the increased expression of inflammatory factors, including tumor necrosis factor-α (TNF-α) and cyclooxygenase-2 (COX-2), thereby causing lung inflammation, eventually leading to lung cancer ( Lu et al., 2014 Lu et al., 2014 ). Selenoprotein M (SelM), located in the endoplasmic reticulum and containing a redox group CXXU, has attracted great interest since its discovery in 2002. Because of this unique motif, SelM can bind toxic metal ions, such as Zn 2+ , Cd 2+ , Hg 2+ , and Cu 2+ , to maintain a dynamic balance of metal ions in the organism and antagonize the damage caused by toxic metal ions ( Pitts and Hoffmann, 2018 Pitts and Hoffmann, 2018 ). In our preliminary study, it was demonstrated that SelM was significantly downregulated in the liver of high-fat diet mice; furthermore, levels of steatosis, inflammation, oxidative stress, and fibrosis were higher in the liver of SelM knockdown mice fed with a high-fat diet ( Cai et al., 2022 Cai et al., 2022 ). SelM overexpression caused a significant rise in extracellular signal-regulated protein kinase (ERK) phosphorylation in the brain, heart, liver, and intestine, so as to promote the regulation of various functions ( Lee et al., 2009 Lee et al., 2009 ). Melatonin ( N -acetyl-5-methoxytryptamine, MT) is a hormone produced by the pineal gland and other tissues. It is involved in physiological processes, including sleep promotion, circadian regulation, immune regulation, and thermoregulation, which has also been investigated for its anti-microbial, anti-cancer, and anti-inflammatory properties, as well as its potential benefits for neurodegenerative, periodontal, and cerebrovascular diseases ( Roohbakhsh et al., 2018 Roohbakhsh et al., 2018 ). MT relieved 2,2,4,4-tetra-brominated diphenyl ether (PBDE-47)‍-induced necroptosis and the secretion of inflammatory factors through the miR-140-5p/Toll-like receptor 4 (TLR4)/NF-‍κB axis ( Li XY et al., 2022 Li XY et al., 2022 ). Previous experiments have shown that MT has a significant protective effect against ischemia/reperfusion (I/R) injury, atherosclerosis, and hypertension ( Roohbakhsh et al., 2018 Roohbakhsh et al., 2018 ). Moreover, it was demonstrated that MT prevented ultraviolet (UV)‍-induced skin damage caused by ROS, apoptosis, and mitochondrial dysfunction ( Kleszczyński et al., 2011 Kleszczyński et al., 2011 ; Wang CX et al., 2021 Wang CX et al., 2021 ). According to a growing body of literature, MT may exert its antioxidant effects by modulating the endoplasmic endoplasmic reticulum stress response ( Fernandez et al., 2015 Fernandez et al., 2015 ). In this study, we established a SelM knockout (KO) mice model to evaluate nickel-induced myocardial injury, analyze the role of SelM in this model, and assess the alleviating impact of MT. 2 Materials and methods 1 2.1 Grouping of test animals and sample collection 2 Male wild-type (WT) and SelM KO C57BL/6J mice were purchased from Cyagen Biosciences (Cyagen Inc., USA). A total of 80 mice, both WT and SelM KO, were divided into eight groups, including WT control group (control), WT+NiCl 2 group (Ni), WT+MT group (MT), WT+NiCl 2 +MT group (Ni+MT), KO control group (KO control), KO+NiCl 2 group (KO Ni), KO+MT group (KO MT), and KO+NiCl 2 +MT group (KO Ni+MT), with ten mice per group. The mice were gavaged with 2 mg/kg MT (MedChemExpress, USA) at 9:00 a.m. and 10 mg/kg NiCl 2 (Sigma, St. Louis, MO, USA) at 5:00 p.m. daily; the control group was treated with saline. Modeling was euthanized for 21 d. Each mouse had free access to water and food during the test. The mice were euthanized on Day 22, and the hearts were quickly removed after soaking in alcohol. A portion of the heart tissue was placed in 4% (volume fraction) paraformaldehyde solution for hematoxylin and eosin (H & E) staining, another portion was stored at 4 ℃ in 2.5% (volume fraction) glutaraldehyde solution for transmission electron microscopy (TEM), a third portion was used for kit assays, and the remaining heart tissue was stored at -‍80 ‍°‍C for later messenger RNA (mRNA) and protein measurements. 2.2 H&E staining 2 The microstructure of the heart was evaluated by H & E staining. Hearts fixed in 4% paraformaldehyde were removed, sequentially dehydrated in a series of 75%, 85%, 90%, and 95% (volume fraction) alcohol, followed by embedding and deparaffinization ( Zheng et al., 2021 Zheng et al., 2021 ). After H & E staining, sections were sequentially placed in an ascending alcohol gradient for dehydration. Next, sections were placed in xylene and finally sealed with neutral resin. Observation and image acquisition were performed under a microscope at 2000× magnification (Olympus Corporation, IX53, Japan). 2.3 TEM 2 A transmission electron microscope (HITACHI, Japan) was used to examine the heart tissue. In brief, samples were removed from a solution containing 2.5% glutaraldehyde (Merck KGaA, Darmstadt, Germany), washed with phosphate-buffered saline (PBS), fixed for 90 min in a solution containing 1% (volume fraction) tetraoxomic acid (Aladdin, Shanghai, China), dehydrated with various concentrations of ethanol (Shandong Lierkang Disinfection Technology Co., Ltd., China), and then replaced with acetone. Finally, resin was employed for embedding and the tissue was cut into sections of 50‍–‍60 nm thickness ( Liu Q et al., 2022a Liu Q et al., 2022a ) by employing lead citrate and uranyl acetate for double-staining experiments. TEM at a magnification of 20 ‍000× was performed to examine the ultrastructure ( Miao et al., 2022b Miao et al., 2022b ). 2.4 Malondialdehyde kit and total antioxidant capacity kit 2 Malondialdehyde (MDA) kit (A003-1-2, Nanjing Jiancheng Bioengineering Institute, Nanjing, China) and total antioxidant capacity (T-AOC) kit (A015-1-2, Nanjing Jiancheng Bioengineering Institute) were used to determine the antioxidant capacity of the heart. The mouse heart tissue was ground into a homogenate and processed according to the kit instructions ( Song et al., 2021 Song et al., 2021 ). MDA kit: firstly, the homogenate and reagents were added to centrifuge tubes in sequence and mixed, then heated in warm water at 95 ℃ for 40 min, cooled, and centrifuged at 3500 r/min for 10 min; finally, the absorbance of the solutions in each tube was determined at 532 nm. T-AOC kit: after mixing heart tissue homogenate and reagents, the solution was left for 10 min, and the absorbance values were determined at 520 nm. 2.5 RNA isolation and quantitative real-time PCR analysis 2 Total RNA was separated from lymphocytes in both the control and NiCl 2 treatment groups by TRIzol reagent at 18 h. Complementary DNA (cDNA) was synthesized according to the instructions provided with a reverse transcription kit (Transgene, Beijing, China). Apoptosis-specific primers (B-cell lymphoma-2 (Bcl-2), Bcl-2-associated X protein (Bax), Caspase-3, Caspase-9, and Caspase-12) and endoplasmic reticulum stress-specific primers (glucose-regulated protein 94 (GRP94), GRP78, C/EBP homologous protein (CHOP), c-Jun N-terminal kinase (JNK), protein kinase RNA-like endoplasmic reticulum kinase (PERK), activating transcription factor 4 (ATF4), ATF6, X-box-binding protein 1 (XBP1), inositol-requiring enzyme 1 (IRE1)) ( Table 1 Table 1 ) were designed based on known primer sequences using Primer BLAST at the National Center for Biotechnology Information (NCBI) ( Gong et al., 2022 Gong et al., 2022 ). Quantitative real-time polymerase chain reaction (qPCR) was performed using a BIOER detection system (Hangzhou, China). All reactions were performed in a 10-‍μL reaction mixture containing 5 μL of 2× SYBR Green I PCR MasterMix (BIOER Technology, Hangzhou, China), 1 μL of cDNA, 0.3 μL of each primer (10 μmol/L), and 3.4 μL of PCR grade water ( Li JH et al., 2022 Li JH et al., 2022 ). The relative abundance of each mRNA was calculated according to the <math id="M1"><msup><mrow><mn mathvariant="normal">2</mn></mrow><mrow><mo>-</mo><mi mathvariant="normal">Δ</mi><mi mathvariant="normal">Δ</mi><msub><mrow><mi mathvariant="normal">C</mi></mrow><mrow><mi mathvariant="normal">T</mi></mrow></msub></mrow></msup></math> http://html.publish.founderss.cn/rc-pub/api/common/picture?pictureId=43461918&type= http://html.publish.founderss.cn/rc-pub/api/common/picture?pictureId=43461917&type= 7.78933382 2.96333337 method, and values were normalized to the mean expression of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) ( Liu XJ et al., 2022 Liu XJ et al., 2022 ; Cao et al., 2023 Cao et al., 2023 ). GRP: glucose-regulated protein; PERK: protein kinase RNA-like endoplasmic reticulum kinase; ATF: activating transcription factor; IRE1: inositol-requiring enzyme 1; XBP1: X-box-binding protein 1; Cyt c : cytochrome c ; Bcl-2: B-cell lymphoma-2; Bax: Bcl-2-associated X protein; JNK: c-Jun N-terminal kinase; CHOP: C/EBP homologous protein; GAPDH: glyceraldehyde-3-phosphate dehydrogenase. Gene name Forward (5'→3') Reverse (5'→3') GRP94 CTTCTACCAGACACCAAGGCGTATG GTTCTTCCTCCACCTGTGCTTCAG GRP78 ATGATGAAGTTCACTGTGGTGG CTGATCGTTGGCTATGATCTCC PERK GAGATCTGGCTCAAAGACGAAA AAGGAGCTATGACTTCGATCTG ATF4 AGTTTAGAGCTAGGCAGTGAAG CATACAGATGCCACTGTCATTG IRE1 TTACACATAGTACACCGTGACC CAGAATGGAGACTGAAGCTACA XBP1 TTGCCTCTTCAGATTCTGAGTC CCAGGTGATGCCTTTGTTCTTGTTG Cyt c CCAAATCTCCACGGTCTGTTCGG AAGGAGCTATGACTTCGATCTG Bax AGGCCTCCTCTCCTACTTCG CCTTTCCCCTTCCCCCATTC Bcl-2 AGCATGCGACCTCTGTTTGA GCCACACGTTTCTTGGCAAT Caspase-3 GAAACTCTTCATCATTCAGGCC GCGAGTGAGAATGTGCATAAAT Caspase-9 TGTGAATATCTTCAACGGGAGC GAGTAGGACACAAGGATGTCAC Caspase-12 TGGCCCATGAATCACATCTAAT TGGACAAAGCTTCAGTGTATCT JNK CGCCTTATGTGGTGACTCGCTAC CTCCCATGATGCACCCAACTGAC CHOP CTCCAGATTCCAGTCAGAGTTC ACTCTGTTTCCGTTTCCTAGTT ATF6 TGCCTTGGGAGTCAGACCTATGG CTGTGGACCGAGGAGAGGAGATG GAPDH GGCAAATTCAACGGCACAGTCAAG TCGCTCCTGGAAGATGGTGATGG 2.6 Protein extraction and western blot analysis 2 Each group of hearts was collected, lysed on ice with radio immunoprecipitation assay (RIPA) buffer and phenylmethylsulfonyl fluoride (PMSF), and then centrifuged at 12 ‍000 r/min and 4 ℃ for 25 min. Sodium dodecyl sulfate (SDS) was added to the supernatant, and samples were boiled for 10 min and preserved at -‍20 ℃ for detection ( Chen et al., 2021 Chen et al., 2021 ). Proteins were isolated and transferred to nitrocellulose membranes after SDS-polyacrylamide gel electrophoresis (PAGE). Next, immunoblotting was performed, and membranes were blocked with 5% (0.05 g/mL) skimmed milk at 37 ℃ for 2 h and then incubated with primary antibodies overnight. The protein expression of apoptosis-specific primers (Bax, Bcl-2, Caspase-3, Caspase-9, and Caspase-12) and endoplasmic reticulum stress-specific primers (ATF6, ATF4, CHOP, IRE1, JNK, and GRP78) was detected. The dilution ratios of primary antibodies are shown in Table 2 Table 2 . Subsequently, membranes were washed three times with 1× PBS-Tween-20 (PBST), incubated with a secondary antibody at room temperature for 1 h, and washed three times with 1× Tris-buffered saline-Tween-20 (TBST) ( Zhang WY et al., 2023 Zhang WY et al., 2023 ). The protein bands were visualized using enhanced chemiluminescence detection reagents (Applygen Technologies Inc., Beijing, China) and X-ray films (TransGen Biotech Co., Beijing, China). The relative abundance of proteins was standardized using β‍-actin as control ( Qiao et al., 2022 Qiao et al., 2022 ; Zhang YH et al., 2023 Zhang YH et al., 2023 ). * Volume ratio. ATF: activating transcription factor; CHOP: C/EBP homologous protein; IRE1: inositol-requiring enzyme 1; JNK: c-Jun N-terminal kinase; Bcl-2: B-cell lymphoma-2; Bax: Bcl-2-associated X protein; GRP78: glucose-regulated protein 78; GAPDH: glyceraldehyde-3-phosphate dehydrogenase. Antibody name Dilution ratio * Size (kDa) Company information ATF6 1:2000 75 Proteintech, Wuhan, China ATF4 1:1500 39 Cell Signaling Technology, Inc., Boston, USA CHOP 1:750 19 Wanlei, Shenyang, China IRE1 1:1500 110 ABclonal, Wuhan, China Caspase-3 1:500 32 Proteintech, Wuhan, China Caspase-9 1:500 42 Proteintech, Wuhan, China Caspase-12 1:500 42 Proteintech, Wuhan, China JNK 1:500 46 Abmart, Shanghai, China Bax 1:1000 23 Abmart, Shanghai, China GRP78 1:1500 78 Proteintech, Wuhan, China Bcl-2 1:500 26 Proteintech, Wuhan, China GAPDH 1:10 000 37 Proteintech, Wuhan, China 2.7 Data analysis 2 Statistical analysis was carried out by one-way analysis of variance (ANOVA) using GraphPad Prism Version 8.0 software, and Tukey’s multiple comparison test was conducted to analyze the differences ( Liu Q et al., 2022b Liu Q et al., 2022b ; Zhang et al., 2022 Zhang et al., 2022 ). All experiments were performed at least six times. The results were expressed as mean±standard deviation (SD). Significant differences between two or more groups were indicated by P < 0.05; in case no significant differences between different groups were observed, P > 0.05. 3 Results 1 3.1 Microscopic observation of the heart 2 Fig. 1 Fig. 1 shows that the microstructures of mouse cardiomyocytes in the control and KO control group were neatly shaped, the myocardial fibers were evenly aligned, and staining was uniform. In contrast, mice in the Ni group had more disorganized myocardial fibers with wavy bending, crumpling, and even lysis. In addition, gaps and inflammatory cell infiltration were visible between cells, and myocardial fibers in the KO Ni group were significantly disorganized with increased gaps. 3.2 Ultrastructural observation of the heart 2 TEM was employed to evaluate the ultrastructure of mouse heart tissue, and the results are shown in Fig. 2 Fig. 2 . The nuclei of mouse heart tissue of the control group were intact and a large number of clear mitochondria were visible. Meanwhile, mice in the Ni group exhibited nuclear wrinkling, mitochondrial enlargement, and loss of mitochondrial cristae. Cardiomyocyte nuclei in the KO Ni group were more wrinkled compared to the Ni group, and the endoplasmic reticulum and mitochondria were fragmented. 3.3 Determination of heart antioxidant capacity 2 The antioxidant capacity of the mouse heart was assessed by measuring the MDA content and T-AOC activity. The results are depicted in Fig. 3 Fig. 3 . The activity of T-AOC in the mouse heart of the Ni group was significantly reduced compared to the control group ( P < 0.05), while the content of MDA was significantly increased ( P < 0.05). However, after MT treatment, the cardiac MDA content and T-AOC activity were reversed ( P < 0.05), which was also the case in KO mice. It should be noted that the MDA content of the Ni group was significantly lower than that of the KO Ni group ( P < 0.05). 3.4 mRNA and protein expression of endoplasmic reticulum stress-associated genes 2 The protective effect of MT against nickel-induced endoplasmic reticulum stress was investigated using mRNA and protein expression assays on cardiac tissues ( Fig. 4 Fig. 4 ). The PCR results showed that Ni significantly increased the mRNA expression of GRP94 , IRE1 , CHOP , ATF4 , XBP1 , PERK , and JNK ( P < 0.05), while the mRNA expression of GRP78 or ATF6 was not significant ( P > 0.05). The results of western blot analysis showed that the protein expression of ATF4, ATF6, IRE1, GRP78, CHOP, and JNK was significantly higher in the Ni group compared to the control group ( P < 0.05). The protein expression was significantly lower in the Ni+MT group compared with the Ni group ( P < 0.05). Notably, except for GRP78, the mRNA and protein levels were significantly increased in KO Ni mice compared with the Ni group ( P < 0.05). 3.5 mRNA and protein expression of apoptosis-related genes 2 Fig. 5 Fig. 5 a presents the effects of NiCl 2 exposure on the mRNA expression levels of apoptotic genes Bax , Bcl-2 , cytochrome c ( Cyt c ), Caspase-3 , Caspase-9 , and Caspase-12 in mouse hearts. The PCR results revealed that the mRNA expression of Bax , Cyt c , Caspase-3 , Caspase-9 , and Caspase-12 was increased ( P < 0.05) in the Ni group, while the mRNA expression of Bcl-2 was decreased ( P < 0.05). These findings were alleviated by treatment with MT. Fig. 5 Fig. 5 b shows the protein results of apoptosis-related genes by western blot analysis. In the Ni group, the protein expression of Bax, Caspase-3, Caspase-9, and Caspase-12 increased while Bcl-2 expression decreased ( P < 0.05). Furthermore, MT significantly reduced the Ni-induced increase in apoptosis genes ( P < 0.05). Notably, when compared to the Ni group, levels of apoptosis-related mRNAs and proteins were significantly increased in the KO Ni group ( P < 0.05), whereas Bcl-2 expression was significantly decreased ( P < 0.05). Taken together, these findings indicated that apoptotic damage was more severe in SelM KO mice compared to WT mice, and Ni-induced apoptosis was alleviated by MT. Fig. 6 Fig. 6 a shows the correlation of expression of different genes determined in mouse hearts: apoptosis-related genes positively correlated with endoplasmic reticulum stress-related genes, except for Bcl-2. The protein‍–‍protein interaction (PPI) network ( Fig. 6 Fig. 6 b) shows gene interactions; the interacting genes included ATF4 , ATF6 , Caspase-3 , Caspase-9 , Bax , Bcl-2 , and XBP1 , suggesting that these genes play key roles in the MT antagonism of nickel-induced apoptosis and endoplasmic reticulum stress, in which SelM is involved. 4 Discussion 1 Previous studies have highlighted that Sec-to-Cys mutant selenoprotein M (SelM') is capable of binding and modulating Zn 2+ -mediated ROS production and neurotoxicity. That is, SelM may be involved in regulating the redox balance as well as metal homeostasis ( Du et al., 2013 Du et al., 2013 ). It has previously been observed that costimulation of SelM overexpression and selenium treatment may modulate multiple functions in the heart by inducing a significant change in ERK phosphorylation ( Lee et al., 2009 Lee et al., 2009 ; Miao et al., 2022a Miao et al., 2022a ). In addition, recent work has demonstrated that following nickel exposure, glutathione peroxidase (GPX) activity decreases and superoxide dismutase (SOD) activity increases in the brain and heart of goldfish ( Kubrak et al., 2014 Kubrak et al., 2014 ). In our study, after exposure to nickel, myocardial fibers in WT mice displayed wavy bending, crinkling, and lysis phenomena, as well as a significant infiltration of inflammatory cells between cells. Myocardial fibers in SelM KO mice displayed even greater disorganization, with clear gaps and more severe damage. Ultrastructural examination revealed that the nuclei of the control group were unharmed and the mitochondria were clear. In the nickel-exposed group, the nucleus was wrinkled and the mitochondria were expanded, while the endoplasmic reticulum was ruptured in the nuclei of SelM KO mice. Overall, these findings showed that nickel severely damaged mouse cardiac tissue, and the damage was worse in SelM KO mice. SelM is a protein containing selenocysteine, has redox activity, and is involved in antioxidant reactions ( Lu et al., 2012 Lu et al., 2012 ). It was previously found that the endoplasmic reticulum stress inducer dithiothreitol (DTT) alters the expression of selenoproteins (SelS, SelK, SelT, SelM, and SelN) in MCF7 breast cancer cells ( Selvik et al., 2013 Selvik et al., 2013 ). In chickens with cartilage damage and selenium deficiency, miR-138-5p participated in the mitochondrial apoptosis pathway by targeting SelM in chicken chondrocytes ( Chi et al., 2019 Chi et al., 2019 ). Furthermore, nano-selenium protected rat kidney cells exposed to NiSO 4 by inhibiting endoplasmic reticulum stress and mitochondrial pathway apoptosis ( Wang Y et al., 2021 Wang Y et al., 2021 ). In sesamin prevention experiments against nickel-induced hepatotoxicity, NiCl 2 increased the levels of ROS and lipid peroxidation, decreased the activity of antioxidant enzymes (total SOD (T-SOD), catalase (CAT), and GPX), and promoted apoptosis by modulating the phosphatidylinositol 3-kinase (PI3K)‍-protein kinase B (Akt) signaling pathway ( Liu et al., 2013 Liu et al., 2013 ; Li XJ et al., 2022 Li XJ et al., 2022 ). Our data showed that nickel exposure increased the mRNA and protein expression of IRE1, ATF6, ATF4, CHOP, JNK, BAX, Caspase-3, Caspase-12, and Caspase-9 in mouse heart tissue and decreased that of Bcl-2. Thus, these findings imply that too much nickel may cause stress on the endoplasmic reticulum and apoptosis in mouse hearts. Notably, the expression was more obvious in the hearts of SelM KO mice, indicating that SelM KO influences nickel-induced apoptosis through controlling endoplasmic reticulum stress. MT is an indole derivative produced by the pineal gland that antagonizes the toxic effects of heavy metals. Recent studies have shown that MT prevents cadmium-induced nephrotoxicity by alleviating oxidative stress, restoring the mitochondrial membrane potential, and inhibiting mitochondrial fission mediated by dynamin-related protein 1 (Drp1) and mitochondrial fission protein 1 (Fis1) ( Dong et al., 2022 Dong et al., 2022 ). Lead toxicity caused neurotoxicity and oxidative stress in the rat cerebellum at specific developmental stages, whereas MT alleviated motor deficits and oxidative stress by antagonizing lead toxicity ( Suresh et al., 2006 Suresh et al., 2006 ; Fan et al., 2021 Fan et al., 2021 ). In this study, MT alleviated myocardial injury caused by Ni by alleviating oxidative stress and regulating the IRE1/JNK/Caspase-3 pathway. Compared with the Ni+MT group, heart injury in the KO Ni+MT group was more severe. Data obtained from mRNA and western blot analysis showed that, except for the mRNA of GRP78 , the mRNA and protein expression of stress and apoptosis genes in the endoplasmic reticulum of the two groups of mice was statistically different. Compared with the KO Ni group, the mRNAs of endoplasmic reticulum stress-related genes ( GRP94 , GRP78 , IRE1 , CHOP , XBP1 , and JNK ) in the KO Ni+MT group did not show significant changes, and the same was observed for mRNAs of apoptosis-related genes ( Bax , Bcl-2 , Caspase-3 , Caspase-9 , and Caspase-12 ). Thus, while MT alleviated some of the myocardial damage caused by nickel, it did not play a sufficient antagonistic role in SelM KO mice, indicating that SelM was very important in the process of Ni-induced myocardial damage in mice. 5 Conclusions 1 In this study, a NiCl 2 intoxication model was established in WT and SelM KO mice. MT alleviated the occurrence of oxidative stress, endoplasmic reticulum stress, and apoptosis in the hearts of WT mice, but not in the hearts of SelM KO mice, indicating that the expression of SelM in the heart played a protective role. References Cai JZ , Huang JQ , Yang J , et al. , 2022 . The protective effect of selenoprotein M on non-alcoholic fatty liver disease: the role of the AMPKα1-MFN2 pathway and Parkin mito phagy. Cell Mol Life Sci , 79 ( 7 ): 354 . https://doi.org/10.1007/s00018-022-04385-0 https://doi.org/10.1007/s00018-022-04385-0 The protective effect of selenoprotein M on non-alcoholic fatty liver disease: the role of the AMPKα1-MFN2 pathway and Parkin mito Cameron KS , Buchner V , Tchounwou PB , 2011 . Exploring the molecular mechanisms of nickel-induced genotox icity and carcinogenicity: a literature review. Rev Environ Health , 26 ( 2 ): 81 - 92 . https://doi.org/10.1515/reveh.2011.012 https://doi.org/10.1515/reveh.2011.012 Exploring the molecular mechanisms of nickel-induced genotox Cao JW , Xu R , Wang FH , et al. , 2023 . Polyethylene microplastics trigger cell apoptosis and inflammation via inducing oxidative stress and activation of the NLRP3 inflammasome in carp gills . Fish Shellfish Immunol , 132 : 108470 . https://doi.org/10.1016/j.fsi.2022.108470 https://doi.org/10.1016/j.fsi.2022.108470 Polyethylene microplastics trigger cell apoptosis and inflammation via inducing oxidative stress and activation of the NLRP3 inflammasome in carp gills Chakrabarti SK , Bai CJ , 1999 . Role of oxidative stress in nickel chloride-induced cell injury in rat renal cortical slices . Biochem Pharmacol , 58 ( 9 ): 1501 - 1510 . https://doi.org/10.1016/s0006-2952(99)00232-4 https://doi.org/10.1016/s0006-2952(99)00232-4 Role of oxidative stress in nickel chloride-induced cell injury in rat renal cortical slices Chen Y , Jing HY , Chen MY , et al. , 2021 . Transcriptional profiling of exosomes derived from Staphylococcus aureus -infected bovine mammary epithelial cell line MAC-T by RNA-seq analysis . Oxid Med Cell Longev , 2021 : 8460355 . https://doi.org/10.1155/2021/8460355 https://doi.org/10.1155/2021/8460355 Transcriptional profiling of exosomes derived from Staphylococcus aureus-infected bovine mammary epithelial cell line MAC-T by RNA-seq analysis Chi QR , Luan YL , Zhang YM , et al. , 2019 . The regulatory effects of miR-138-5p on selenium deficiency-induced chondrocyte apoptosis are mediated by targeting SelM . Metallomics , 11 ( 4 ): 845 - 857 . https://doi.org/10.1039/c9mt00006b https://doi.org/10.1039/c9mt00006b The regulatory effects of miR-138-5p on selenium deficiency-induced chondrocyte apoptosis are mediated by targeting SelM Das KK , Reddy RC , Bagoji IB , et al. , 2018 . Primary concept of nickel toxicity‍—‍‍an overview. J Basic Clin Physiol Pharmacol , 30 (2 ) :141 -152 . https://doi.org/10.1515/jbcpp-2017-01 https://doi.org/10.1515/jbcpp-2017-01 71 Primary concept of nickel toxicity‍—‍‍an overview. J Basic Clin Physiol Pharmacol Dong WX , Yan LQ , Tan Y , et al. , 2022 . Melatonin improves mitochondrial function by preventing mitochondrial fission in cadmium-induced rat proximal tubular cell injury via SIRT1-PGC-1α pathway activation . Ecotoxicol Environ Saf , 242 : 113879 . https://doi.org/10.1016/j.ecoenv.2022.113879 https://doi.org/10.1016/j.ecoenv.2022.113879 Melatonin improves mitochondrial function by preventing mitochondrial fission in cadmium-induced rat proximal tubular cell injury via SIRT1-PGC-1α pathway activation Du XB , Li HP , Wang Z , et al. , 2013 . Selenoprotein P and selenoprotein M block Zn 2+ -mediated Aβ 42 aggregation and toxicity . Metallomics , 5 ( 7 ): 861 - 870 . https://doi.org/10.1039/c3mt20282h https://doi.org/10.1039/c3mt20282h Selenoprotein P and selenoprotein M block Zn2+-mediated Aβ42 aggregation and toxicity Fan RF , Tang KK , Wang ZY , et al. , 2021 . Persistent activation of Nrf2 promotes a vicious cycle of oxidative stress and autophagy inhibition in cadmium-induced kidney injury . Toxicology , 464 : 152999 . https://doi.org/10.1016/j.tox.2021.152999 https://doi.org/10.1016/j.tox.2021.152999 Persistent activation of Nrf2 promotes a vicious cycle of oxidative stress and autophagy inhibition in cadmium-induced kidney injury Fernandez A , Ordóñez R , Reiter RJ , et al. , 2015 . Melatonin and endoplasmic reticulum stress: relation to autophagy and apoptosis . J Pineal Res , 59 ( 3 ): 292 - 307 . https://doi.org/10.1111/jpi.12264 https://doi.org/10.1111/jpi.12264 Melatonin and endoplasmic reticulum stress: relation to autophagy and apoptosis Genchi G , Carocci A , Lauria G , et al. , 2020 . Nickel: human health and environmental toxicology . Int J Environ Res Public Health , 17 ( 3 ): 679 . https://doi.org/10.3390/ijerph17030679 https://doi.org/10.3390/ijerph17030679 Nickel: human health and environmental toxicology Gong ZG , Zhao Y , Wang ZY , et al. , 2022 . Epigenetic regulator BRD4 is involved in cadmium-induced acute kidney injury via contributing to lysosomal dysfunction, auto phagy blockade and oxidative stress. J Hazard Mater , 423 : 127110 . https://doi.org/10.1016/j.jhazmat.2021.127110 https://doi.org/10.1016/j.jhazmat.2021.127110 Epigenetic regulator BRD4 is involved in cadmium-induced acute kidney injury via contributing to lysosomal dysfunction, auto Guo HR , Chen L , Cui HM , et al. , 2016 . Research advances on pathways of nickel-induced apoptosis . Int J Mol Sci , 17 ( 1 ): 10 . https://doi.org/10.3390/ijms17010010 https://doi.org/10.3390/ijms17010010 Research advances on pathways of nickel-induced apoptosis Kleszczyński K , Hardkop LH , Fischer TW , 2011 . Differential effects of melatonin as a broad range UV-damage preventive dermato-endocrine regulator . Dermatoendocrinol , 3 ( 1 ): 27 - 31 . https://doi.org/10.4161/derm.3.1.14842 https://doi.org/10.4161/derm.3.1.14842 Differential effects of melatonin as a broad range UV-damage preventive dermato-endocrine regulator Kubrak OI , Poigner H , Husak VV , et al. , 2014 . Goldfish brain and heart are well protected from Ni 2+ -induced oxidative stress . Comp Biochem Physiol C Toxicol Pharmacol , 162 : 43 - 50 . https://doi.org/10.1016/j.cbpc.2014.03.011 https://doi.org/10.1016/j.cbpc.2014.03.011 Goldfish brain and heart are well protected from Ni2+-induced oxidative stress Lee YK , Yim SY , Jung SE , et al. , 2009 . The costimulation of selenium treatment and selenoprotein M overexpression significantly induced the up- and down-regulation of ERK MAPK signaling pathway in various tissues . Lab Anim Res , 25 ( 3 ): 201 - 205 . The costimulation of selenium treatment and selenoprotein M overexpression significantly induced the up- and down-regulation of ERK MAPK signaling pathway in various tissues Li JH , Zhang WY , Zhou P , et al. , 2022 . Selenium deficiency induced apoptosis via mitochondrial pathway caused by oxidative stress in porcine gastric tissues . Res Vet Sci , 144 : 142 - 148 . https://doi.org/10.1016/j.rvsc.2021.10.017 https://doi.org/10.1016/j.rvsc.2021.10.017 Selenium deficiency induced apoptosis via mitochondrial pathway caused by oxidative stress in porcine gastric tissues Li XJ , Bai RC , Bai YC , et al. , 2022 . ROS-mediated PPAR/RXR inhibition contributes to acetochlor-induced apoptosis and autophagy in Ctenopharyngodon idella hepatic cells . Fish Shellfish Immunol , 128 : 684 - 694 . https://doi.org/10.1016/j.fsi.2022.08.053 https://doi.org/10.1016/j.fsi.2022.08.053 ROS-mediated PPAR/RXR inhibition contributes to acetochlor-induced apoptosis and autophagy in Ctenopharyngodon idella hepatic cells Li XY , Zhang HR , Qiao SQ , et al. , 2022 . Melatonin administration alleviates 2, 2 ,4,4-tetra-brominated diphenyl ether (PBDE-47)‍-induced necroptosis and secretion of inflammatory factors via miR-140-5p/TLR4/NF-κB axis in fish kidney cells. Fish Shellfish Immunol, 128 : 228 - 237 . https://doi.org/10.1016/j.fsi.2022.08.004 https://doi.org/10.1016/j.fsi.2022.08.004 Melatonin administration alleviates Liu CM , Zheng GH , Ming QL , et al. , 2013 . Sesamin protects mouse liver against nickel-induced oxidative DNA damage and apoptosis by the PI3K-Akt pathway . J Agric Food Chem , 61 ( 5 ): 1146 - 1154 . https://doi.org/10.1021/jf304562b https://doi.org/10.1021/jf304562b Sesamin protects mouse liver against nickel-induced oxidative DNA damage and apoptosis by the PI3K-Akt pathway Liu JB , Li ZF , Lu L , et al. , 2022 . Glyphosate damages blood-testis barrier via NOX1-triggered oxidative stress in rats: long-term exposure as a potential risk for male reproductive health . Environ Int , 159 : 107038 . https://doi.org/10.1016/j.envint.2021.107038 https://doi.org/10.1016/j.envint.2021.107038 Glyphosate damages blood-testis barrier via NOX1-triggered oxidative stress in rats: long-term exposure as a potential risk for male reproductive health Liu Q , Sun Y , Zhu Y , et al. , 2022a . Melatonin relieves liver fibrosis induced by Txnrd3 knockdown and nickel expos ure via IRE1/NF‍-‍‍κB/NLRP3 and PERK/TGF‍-‍‍β1 axis activation. Life Sci, 301 :1206 22 . https://doi.org/10.1016/j.lfs.2022.1206 .https://doi.org/10.1016/j.lfs.2022.1206 22 Melatonin relieves liver fibrosis induced by Txnrd3 knockdown and nickel expos Liu Q , Du PY , Zhu Y , et al. , 2022b . Thioredoxin reductase 3 suppression promotes colitis and carcinogenesis via activating pyroptosis and necrosis . Cell Mol Life Sci , 79 ( 2 ): 106 . https://doi.org/10.1007/s00018-022-04155-y https://doi.org/10.1007/s00018-022-04155-y Thioredoxin reductase 3 suppression promotes colitis and carcinogenesis via activating pyroptosis and necrosis Liu XJ , Wang YQ , Shang SQ , et al. , 2022 . TMT induces apoptosis and necroptosis in mouse kidneys through oxidative stress-induced activation of the NLRP3 inflammasome . Ecotoxicol Environ Saf , 230 : 113167 . https://doi.org/10.1016/j.ecoenv.2022.113167 https://doi.org/10.1016/j.ecoenv.2022.113167 TMT induces apoptosis and necroptosis in mouse kidneys through oxidative stress-induced activation of the NLRP3 inflammasome Lu JY , Zhang M , Jin HL , et al. , 2014 . Advances on molecular mechanism of chronic inflammation-driven lung cancer induced by environmental carcinogens . Prog Biochem Biophys , 41 ( 1 ): 41 - 51 . https://doi.org/10.3724/sp.J.1206.2013.00386 https://doi.org/10.3724/sp.J.1206.2013.00386 Advances on molecular mechanism of chronic inflammation-driven lung cancer induced by environmental carcinogens Lu W , Li WW , Jin XK , et al. , 2012 . Reproductive function of Selenoprotein M in Chinese mitten crabs ( Eriocheir sinesis ) . Peptides , 34 ( 1 ): 168 - 176 . https://doi.org/10.1016/j.peptides.2011.04.022 https://doi.org/10.1016/j.peptides.2011.04.022 Reproductive function of Selenoprotein M in Chinese mitten crabs (Eriocheir sinesis) Miao ZR , Miao ZY , Shi X , et al. , 2022a . The antagonistic effect of selenium on lead-induced apoptosis and necroptosis via P38/JNK/ERK pathway in chicken kidney . Ecotoxicol Environ Saf , 231 : 113176 . https://doi.org/10.1016/j.ecoenv.2022.113176 https://doi.org/10.1016/j.ecoenv.2022.113176 The antagonistic effect of selenium on lead-induced apoptosis and necroptosis via P38/JNK/ERK pathway in chicken kidney Miao ZR , Miao ZY , Wang SC , et al. , 2022b . Exposure to imidacloprid induce oxidative stress, mitochondrial dysfunction, inflammation, apoptosis and mitophagy via NF-kappaB/JNK pathway in grass carp hepatocytes . Fish Shellfish Immunol , 120 : 674 - 685 . https://doi.org/10.1016/j.fsi.2021.12.017 https://doi.org/10.1016/j.fsi.2021.12.017 Exposure to imidacloprid induce oxidative stress, mitochondrial dysfunction, inflammation, apoptosis and mitophagy via NF-kappaB/JNK pathway in grass carp hepatocytes Pitts MW , Hoffmann PR , 2018 . Endoplasmic reticulum-resident selenoproteins as regulators of calcium signaling and homeostasis . Cell Calcium , 70 : 76 - 86 . https://doi.org/10.1016/j.ceca.2017.05.001 https://doi.org/10.1016/j.ceca.2017.05.001 Endoplasmic reticulum-resident selenoproteins as regulators of calcium signaling and homeostasis Qiao SQ , Sun Y , Jiang YY , et al. , 2022 . Melatonin amelior Melatonin amelior ates nickel induced autophagy in mouse brain: diminution of oxidative stress . Toxicology , 473 : 153207 . https://doi.org/10.1016/j.tox.2022.153207 https://doi.org/10.1016/j.tox.2022.153207 ates nickel induced autophagy in mouse brain: diminution of oxidative stress Rehman K , Fatima F , Waheed I , et al. , 2018 . Prevalence of exposure of heavy metals and their impact on health consequences . J Cell Biochem , 119 ( 1 ): 157 - 184 . https://doi.org/10.1002/jcb.26234 https://doi.org/10.1002/jcb.26234 Prevalence of exposure of heavy metals and their impact on health consequences Roohbakhsh A , Shamsizadeh A , Hayes AW , et al. , 2018 . Mela tonin as an endogenous regulator of diseases: the role of autophagy. Pharmacol Res , 133 : 265 - 276 . https://doi.org/10.1016/j.phrs.2018.01.022 https://doi.org/10.1016/j.phrs.2018.01.022 Mela Selvik LKM , Fjeldbo CS , Flatberg A , et al. , 2013 . The duration of gastrin treatment affects global gene expression and molecular responses involved in ER stress and anti-apoptosis . BMC Genomics , 14 : 429 . https://doi.org/10.1186/1471-2164-14-429 https://doi.org/10.1186/1471-2164-14-429 The duration of gastrin treatment affects global gene expression and molecular responses involved in ER stress and anti-apoptosis Song N , Wang W , Wang Y , et al. , 2021 . Hydrogen sulfide of air induces macrophage extracellular traps to aggravate inflammatory injury via the regulation of miR-15b-5p on MAPK and insulin signals in trachea of chickens . Sci Total Environ , 771 : 145407 . https://doi.org/10.1016/j.scitotenv.2021.145407 https://doi.org/10.1016/j.scitotenv.2021.145407 Hydrogen sulfide of air induces macrophage extracellular traps to aggravate inflammatory injury via the regulation of miR-15b-5p on MAPK and insulin signals in trachea of chickens Suresh C , Dennis AO , Heinz J , et al. , 2006 . Melatonin protection against lead-induced changes in human neuroblastoma cell cultures . Int J Toxicol , 25 ( 6 ): 459 - 464 . https://doi.org/10.1080/10915810600959576 https://doi.org/10.1080/10915810600959576 Melatonin protection against lead-induced changes in human neuroblastoma cell cultures Wang CX , Zhang R , Wang S , et al. , 2021 . Protective effects of nano-selenium on nickel-induced renal cell apoptosis in rats . J Toxicol , 35 ( 3 ): 193 - 197 (in Chinese) . https://doi.org/10.16421/j.cnki.1002-3127.2021.03.004 https://doi.org/10.16421/j.cnki.1002-3127.2021.03.004 Protective effects of nano-selenium on nickel-induced renal cell apoptosis in rats Wang Y , Zhao HJ , Liu YC , et al. , 2021 . Environmentally relevant concentration of sulfamethoxazole-induced oxidative stress-cascaded damages in the intestine of grass carp and the therapeutic application of exogenous lycopene . Environ Pollut , 274 : 116597 . https://doi.org/10.1016/j.envpol.2021.116597 https://doi.org/10.1016/j.envpol.2021.116597 Environmentally relevant concentration of sulfamethoxazole-induced oxidative stress-cascaded damages in the intestine of grass carp and the therapeutic application of exogenous lycopene Zhang HR , Zhao FQ , Gai XX , et al. , 2022 . Astilbin attenuates apoptosis induced by cadmium through oxidative stress in carp ( Cyprinus carpio L.) head kidney lymphocyte . Fish Shellfish Immunol , 125 : 230 - 237 . https://doi.org/10.1016/j.fsi.2022.05.021 https://doi.org/10.1016/j.fsi.2022.05.021 Astilbin attenuates apoptosis induced by cadmium through oxidative stress in carp (Cyprinus carpio L.) head kidney lymphocyte Zhang WY , Sun XY , Shi X , et al. , 2023 . Subacute cadmium exposure induces necroptosis in swine lung via influencing Th1/Th2 balance . Biol Trace Elem Res , 201 ( 1 ):‍ 220 - 228 . https://doi.org/10.1007/s12011-022-03133-6 https://doi.org/10.1007/s12011-022-03133-6 Subacute cadmium exposure induces necroptosis in swine lung via influencing Th1/Th2 balance Zhang YH , Xu S , Li K , et al. , 2023 . TBBPA induced ROS overproduction promotes apoptosis and inflammation by inhibiting autophagy in mice lung . Ecotoxicol Environ Saf , 252 : 114607 . https://doi.org/10.1016/j.ecoenv.2023.114607 https://doi.org/10.1016/j.ecoenv.2023.114607 TBBPA induced ROS overproduction promotes apoptosis and inflammation by inhibiting autophagy in mice lung Zhao HJ , Wang Y , Liu YC , et al. , 2021 . ROS-induced hepatotoxicity under cypermethrin: involvement of the crosstalk between Nrf2/Keap1 and NF‍-‍κB/iκB-‍α pathways regulated by proteasome . Environ Sci Technol , 55 ( 9 ):‍ 6171 - 6183 . https://doi.org/10.1021/acs.est.1c00515 https://doi.org/10.1021/acs.est.1c00515 ROS-induced hepatotoxicity under cypermethrin: involvement of the crosstalk between Nrf2/Keap1 and NF‍-‍κB/iκB-‍α pathways regulated by proteasome Zheng YY , Guan HY , Yang J , et al. , 2021 . Calcium overload and reactive oxygen species accumulation induced by selenium deficiency promote autophagy in swine small intestine . Anim Nutr , 7 ( 4 ): 997 - 1008 . https://doi.org/10.1016/j.aninu.2021.05.005 https://doi.org/10.1016/j.aninu.2021.05.005 Calcium overload and reactive oxygen species accumulation induced by selenium deficiency promote autophagy in swine small intestine Xintong ZHANG: writing ‍– ‍original draft; Xiaoxue GAI: data curation; Lihua XU: software; Wenxue MA: validation; Qiaohan LIU: visualization; Bendong SHI: methodology; Cheng FANG and Jingzeng CAI: writing ‍–‍ review & editing; Ziwei ZHANG: funding acquisition. All authors have read and approved the final manuscript, and therefore, have full access to all the data in the study and take responsibility for the integrity and security of the data. The work was supported by the Heilongjiang Provincial Natural Science Foundation for Outstanding Youth (No. YQ2021C021), China. 10.1631/jzus.B2200694 1673-1581（2023）05-0406-12 张鑫彤,盖晓雪,徐礼华等.硒蛋白M敲除在褪黑素拮抗镍诱导的小鼠心脏细胞凋亡和内质网应激中的作用[J].浙江大学学报（英文版）（B辑：生物医学和生物技术）,2023,24(05):406-417. 张鑫彤,盖晓雪,徐礼华等.硒蛋白M敲除在褪黑素拮抗镍诱导的小鼠心脏细胞凋亡和内质网应激中的作用[J].浙江大学学报（英文版）（B辑：生物医学和生物技术）,2023,24(05):406-417. The work was supported by the Heilongjiang Provincial Natural Science Foundation for Outstanding Youth (No. YQ2021C021), China. 2022-12-29 2023-01-27 2023-05-15 2023-05-15 2023-03-31 Copyright © Zhejiang University and Springer-Verlag GmbH Germany, part of Springer Nature 2022 2023 浙江大学出版社 37538392 Xintong ZHANG, Xiaoxue GAI, Lihua XU, Wenxue MA, Qiaohan LIU, Bendong SHI, Cheng FANG, Jingzeng CAI, and Ziwei ZHANG declare that they have no conflict of interests. All procedures were carried out following the directive of the Council of the European Communities (No. 86/609/EEC) and approved by the Committee on Animal Management and Use of Northeast Agricultural University (No. SRM-11), China. Compliance with ethics guidelines The data underlying this article will be shared on reasonable request to the corresponding author. Data availability 浙江大学学报（英文版）（B辑：生物医学和生物技术） 5 24