The Role Of Melatonin In Antagonizing Nickel Induced Spleen Pyroptosis In Mice Through Selenoprotein M And Its Mechanism

Nickel is a heavy metal element and a pollutant that threatens the health of organisms.Melatonin（Mel）is an antioxidant substance that the body can secrete and has a protective effect on the body.Sele-noprotein M（Sel M）is a widely distributed selenium protein that has antioxidant,neuroprotective,and intracellular calcium regulation effects.To investigate the role of Sel M in immune organs of mice ex-posed to nickel（Ni）and whether melatonin can be used as a related antagonist,this study established a nickel poisoning model and a melatonin antagonizing model in Sel M gene knockout mice.80Sel M^+/+wild-type and 80 Sel M^-/-homozygous（KO）mice were divided into 8 groups with 20 mice in each group,including Control group mice（9:00 AM and 5:00 PM normal saline）,Ni group mice（9:00AM nickel chloride,5:00 PM normal saline）,Mel group mice（9:00 AM normal saline,5:00 PM mela-tonin）,Ni+Mel group mice（9:00 AM nickel chloride,5:00 PM melatonin）,KO+C group mice（9:00 AM and 5:00 PM normal saline）,KO+Ni group mice（9:00 AM nickel chloride,5:00 PM normal saline）,KO+Mel group mice（9:00 AM normal saline,5:00 PM melatonin）,KO+Ni+Mel group mice（9:00 AM nickel chloride,5:00 PM melatonin）.The Ni group was given a concentration of 10 mg/kg by gavage,while the Mel group was given a concentration of 2 mg/kg by gavage at a weight of 0.1 m L/10 g for 21days.By using H&E staining electron microscopy,oxidative stress kit,q RT-PCR,and Western Blot,the histopathological changes and ultrastructure of spleen tissue cells were observed,and the antioxidant capacity and expression levels of pyroptosis related genes m RNA and protein in each group were detect-ed.（1）The results of H&E staining showed that the spleen tissue in the control group was neatly ar-ranged,the structures of lymphocytes,splenic cords,and splenic sinuses were complete,and the white and red pulp were clearly colored;In the Ni group,white pulp was scattered,lymphocytes were partially stained,splenic sinuses were enlarged,and a small amount of red cells were present in the red pulp,which was more severe than in the control group;In the Ni+Mel group,the white pulp boundary was unclear and lymphocytes were heavily stained,which reduced the damage compared to the Ni group;The performance of KO+C group was normal,similar to that of the control group;In the KO+Ni group,the white pulp was scattered,the red pulp was atrophic,the splenic cord was ruptured,and there were many scattered red blood cells in the splenic sinuses.Compared with the Ni group and the KO+C group,the damage was aggravated;In the KO+Ni+Mel group,the white pulp atrophy,lymphocyte concentra-tion,splenic sinus enlargement,and a small amount of red blood cells in the splenic sinus were more severe than in the Ni+Mel group and KO+C group,but the injury was lighter than in the KO+Ni group.Melatonin alleviated the pathological damage of mice spleen caused by nickel exposure,while Sel M knockout aggravated the damage.The ultrastructural observation showed that the morphology of the nuclei and mitochondria of the spleen in the control group was normal,the nuclear membrane was intact,and the nucleoli and mito-chondrial cristae were obvious;Nickel caused the disorder of mitochondrial cristae and formation of vacuoles in spleen cells,resulting in the formation of pyrolytic bodies;In the KO+Ni group,the shape of spleen nucleus was irregular,nucleolus disappeared,chromatin in the nucleus was concentrated,cell membrane was broken,mitochondria swelled,mitochondrial crista disappeared disorderly,some vacu-olized,and a large number of scorched bodies appeared.Nickel exposure leads to splenic pyroptosis in mice,and Sel M knockout increases the level of pyroptosis.（3）The results of oxidative stress kit showed that compared with the control group,nickel caused a decrease in GSH and GSSG content in spleen tissue（P<0.05）,a decrease in T-AOC and SOD activity,and an increase in MDA content（P<0.05）;Compared with the KO+C group,the content of GSH and GSSG in the KO+Ni group decreased,and the content of MDA increased,the activities of T-AOC and SOD decreased（P<0.05）,which explains the inhibitory effect of nickel exposure on antioxidant en-zymes in the spleen of mice.Compared with the normal group,the contents of GSH and GSSG as well as the activities of T-AOC and SOD in the Mel group increased,while the content of MDA decreased;Compared with the KO+C group,the KO+Mel group had an increase in GSSG content,an increase in T-AOC and SOD activity（P<0.05）,and a decrease in GSH and MDA content（P<0.05）,which ex-plained the antioxidant effect of melatonin.Compared with Ni group,the contents of GSH and GSSG,as well as the activities of T-AOC and SOD（P<0.05）in Ni+Mel group increased,while the content of MDA decreased.Melatonin weakened the inhibitory effect of nickel exposure on antioxidant enzymes in mouse spleen;Compared with the KO+Ni group,the content of GSH,GSSG,T-AOC,and SOD activity in the KO+Ni+Mel group increased,while the content of MDA decreased,but the change was not sig-nificant,indicating that melatonin can alleviate but cannot eliminate the impact of Sel M deficiency.Compared with the control group,the content of MDA and GSSG,as well as the activities of T-AOC and SOD in the KO+C group decreased,while the content of GSH increased（P<0.05）;Compared with Ni group,the content of GSH and GSSG,T-AOC and SOD activities in KO+Ni group significantly de-creased,while the content of MDA further increased（P<0.05）;Compared with Mel group,T-AOC ac-tivity increased and MDA content decreased in KO+Mel group,while other levels were similar;Com-pared with the Ni+Mel group,the KO+Ni+Mel group had a lower T-AOC activity,lower GSH and GSSG contents（P<0.05）,and a similar MDA content,which explained the antioxidant effect of Sel M,while the absence of Sel M reduced the antioxidant ability.In summary,melatonin alleviated oxidative stress levels in the spleen of mice exposed to nickel,while Sel M knockout increased oxidative stress levels.（4）The m RNA and protein expressions of pyroptosis related genes in mouse spleen tissue were de-tected by q RT-PCR and Western Blot methods,respectively.The results showed that nickel caused Nox2,Txnip,Trx1,Asc,Nlrp3,Caspase-1,Aim2,IL-1β,IL-18 in spleen tissue,the m RNA expression level of IL-18 increases,including ASC,NLRP3,Caspase-1,and IL-1βThe protein expression level of the pro-tein increased;Compared with the KO+C group,the m RNA and protein levels of pyroptosis related genes in the KO+Ni group increased（P<0.05）,indicating that nickel induced pyroptosis in the spleen tissue.Compared with the normal group,the m RNA and protein levels of pyroptosis related genes in the Mel group decreased（P<0.05）;Compared with the KO+C group,the m RNA level of pyroptosis related genes in the KO+Mel group decreased and the protein level was similar,indicating that melatonin re-duced the occurrence of pyroptosis in spleen tissue.Compared with the Ni group,the m RNA and protein levels of pyroptosis related genes in the Ni+Mel group decreased;Compared to the KO+Ni group,the m RNA and protein levels of pyroptosis related genes in the KO+Ni+Mel group decreased（P<0.05）,but compared to the KO+C group,the m RNA and protein levels of pyroptosis related genes increased,indicating that melatonin reduced the level of nickel induced pyroptosis,but did not completely alleviate it.The levels in the KO+C group were similar to those in the control group;Compared with Ni group,the m RNA and protein levels of pyroptosis related genes in KO+Ni group increased（P<0.05）;Com-pared with the Mel group,the m RNA and protein levels of pyroptosis related genes in the KO+Mel group increased;Compared with the Ni+Mel group,the m RNA and protein levels of pyroptosis related genes in the KO+Ni+Mel group increased,indicating that Sel M knockout increased the level of spleen pyroptosis.Results indicate that melatonin alleviates pyroptosis in mice spleen caused by nickel expo-sure,while Sel M knockout increases pyroptosis.However,in the absence of Sel M,the relief of melato-nin is limited.In summary,Sel M deficiency aggravates nickel induced splenic pyroptosis through activation of oxidative stress,while melatonin alleviates this condition.However,when Sel M deficiency occurs,the effect of melatonin is not significant,reflecting the role of Sel M in nickel induced cell pyroptosis.Our research aims to explore the role and mechanism of nickel exposure induced spleen damage and the an-tagonistic effect of melatonin on nickel through various molecular biology and histomorphology exper-iments,and clarify the effect of Sel M on nickel induced spleen tissue pyroptosis in mice,with a view to providing references for clinical medication and disease treatment through the above experiments.