Protective Effect Of Txnrd3 On Renal Cell Apoptosis Induced By Nickel And Alleviating Effect Of Melatonin

Nickel is a heavy metal with toxic effects on animals,widely used in metallurgy and daily necessities.Txnrd3 takes thioredoxin as the main reaction substrate and participates in various processes such as cell proliferation,metabolism,redox,and apoptosis.It plays a biological role in antagonizing metals in the liver,heart,and spleen.Melatonin is also recognized as an antioxidant with good antioxidant and anti-apoptotic functions.This study aims to investigate the effect of Txnrd3 on nickel induced kidney damage in mice and the relief effect of melatonin（Mel）by establishing a three-dimensional model of nickel（Ni）exposure induced kidney damage in mice,as well as the effect of Txnrd3 on nickel induced kidney damage.The experimental mice were divided into eight groups:wild-type control group（Wild-NC）,wild-type nickel administration group（Wild-Ni）,wild-type melatonin group（Wild-Mel）,wild-type nickel and melatonin group（Wild-Ni+Mel）,Txnrd3^-/-control group(Txnrd3^-/--NC),Txnrd3^-/-Nickel treatment group(Txnrd3^-/--Ni),Txnrd3^-/-melatonin treatment group(Txnrd3^-/--Mel),Txnrd3^-/-Nickel and melatonin treatment group(Txnrd3^-/--Ni+Mel).The nickel administration concentration was 10 mg/kg,and the Mel concentration was 2 mg/kg.Both were administered by gavage with an 8-hour interval.Nickel chloride solution was administered in the morning,Melatonin was administered in the evening,and the control group was administered physiological saline.The administration period was 21 days.Ultrastructural observation of mice kidney tissue was conducted,and combined with TUNEL staining results,renal apoptosis was analyzed.q RT-PCR and Western Blot were used to analyze the expression of mitochondrial apoptosis related genes to explore the role of Txnrd3 in nickel induced mice kidney apoptosis and the improvement of melatonin.The research results are as follows:（1）Ultrastructural observation shows that nickel exposure can cause significant atrophy of the nuclei of mice kidney cells.In the Wild-Ni group,the kidneys of mice exhibit symptoms such as mitochondrial cristae rupture and obvious vacuolization,leading to significant mitochondrial apoptosis.However,after Txnrd3 was knockdown,the phenomenon in Txnrd3^-/--Ni group became worse,the nuclear shrinkage became more serious,chromatin gathered,the degree of mitochondrial vacuolization increased,and the mitochondrial apoptosis further deepened.There was no significant difference in pathological changes between the Txnrd3^-/--NC group and the Wild-NC group;The H&E staining results showed that the kidneys in the Wild-Ni group exhibited glomerular degeneration and tubular atrophy;There was no significant change in H&E staining results between the Wild-Mel group and the Wild-NC group.The situation of glomerular atrophy and tubular degeneration in the Wild-Ni+Mel group was slower than that in the Wild Ni group;Txnrd^3-/--NC group showed no significant pathological changes in the kidneys,and there was no difference in H&E staining results compared to Wild-NC;The Txnrd3^-/--Ni group showed further glomerular atrophy and significant tubular degeneration,which was more severe than the Wild-Ni group;The degeneration of glomeruli and renal tubules in the Txnrd3^-/--Ni+Mel group was alleviated compared to the Txnrd3^-/--Ni group,but the degree of relief was not as good as in the Wild-Ni+Mel group.（2）The TUNEL results also showed that in Wild-Ni group,the green fluorescence area increased and apoptosis occurred.The phenomenon intensified in the Txnrd3^-/--Ni group,and the green fluorescence area increased and apoptosis further intensified.There was no significant difference between the Txnrd3^-/--NC group and the Wild-NC group.This result also confirms the important role of Txnrd3 in mice kidneys and the ability of melatonin to alleviate damage.（3）The results of oxidative stress kit showed that the activities of T-AOC,SOD and GSH in Wild-Ni group were significantly decreased,and the content of MDA was significantly increased（p<0.01）,The activities of T-AOC,SOD and GSH in the Wild-Ni+Mel group were significantly higher than those in the Wild-Ni group,but still lower than those in the Wild-NC group,and the MDA content was significantly lower than that in the Wild-Ni group（p<0.01）,In Txnrd3^-/--Ni group,the activities of T-AOC,SOD and GSH were significantly decreased,and the content of MDA was significantly increased（p<0.01）,The above oxidative stress indexes were reversed in Txnrd3^-/--Ni+Mel group,T-AOC,SOD and GSH in Txnrd3^-/--Ni+Mel group were significantly lower than those in Wild-Ni+Mel group,while MDA was significantly increased（p<0.01）.（4）The m RNA expression of mitochondrial related genes Bax,Bcl-2,Caspase-3,Caspase-9,and Cyt-c in mice kidney tissue was detected.The results showed that the m RNA content of Bax,Caspase-3,Caspase-9,and Cyt-c in the Wild-Ni group significantly increased,while the m RNA content of Bcl-2significantly decreased（p<0.01）.The expression of these genes in the Wild-Ni+Mel group was reversed（p<0.01）,There was no significant difference in the expression levels of the aforementioned genes between the Wild-NC group and the Wild-Mel group（p>0.05）.The m RNA expression of Bax,Caspase-3,Caspase-9,and Cyt-c in the Txnrd3^-/--Ni group was significantly higher than that in the Txnrd3^-/--NC group（p<0.01）,while the m RNA content of Bcl-2 was significantly reduced（p<0.01）.The above genes in the Txnrd3^-/--Ni+Mel group were reversed compared to the Txnrd3^-/--Ni group,with significant differences in changes（p<0.01）;There was no significant difference in the m RNA content of Bcl-2,Cyt-c,and Caspase-3 between the Txnrd3^-/--Mel group and the Txnrd3^-/--NC group（p>0.05）;The m RNA expression levels of Bax,Cyt-c,Caspase-3,and Caspase-9 in the Txnrd3^-/--Ni+Mel group were higher than those in the Wild-Ni+Mel group,while the Bcl-2 content was significantly reduced compared to the Wild Ni+Mel group（p<0.01）.（5）The protein expression of mitochondrial related genes Bax,Bcl-2,Caspase-3,Caspase-9,and Cyt-c in mice kidney tissue was detected.The results showed that the protein content of Bax,Caspase-3,Caspase-9,and Cyt-c in the Wild-Ni group was upregulated,while the protein expression of Bcl-2 was significantly downregulated（p<0.01）.The expression of these proteins in the Wild-Ni+Mel group was reversed,with a significant difference from the Wild-Ni group（p<0.01）,there was no significant difference in the expression levels of the aforementioned genes between the Wild-NC group and the Wild-Mel group（p>0.05）.The expression of Bax,Caspase-3,Caspase-9,and Cyt-c in the Txnrd3^-/--Ni group was significantly higher than that in the Txnrd3^-/--NC group,while the protein expression of Bcl-2 was significantly reduced（p<0.01）;The above genes in the Txnrd3^-/--Ni+Mel group were reversed compared to the Txnrd3^-/--Ni group,with significant differences（p<0.01）;The protein expression of Bax,Cyt-c,Caspase-3,and Caspase-9 in the Txnrd3^-/--Mel group was significantly reduced compared to the Txnrd3^-/--NC group（p<0.01）;The protein expression of Bax,Cyt-c,Caspase-3,and Caspase-9 in the Txnrd3^-/--Ni+Mel group was higher than that in the Wild-Ni+Mel group,and the content of Bcl-2 was significantly reduced compared to the Wild-Ni+Mel group（p<0.01）.In conclusion,nickel exposure can cause kidney damage in mice,resulting in oxidative stress and mitochondrial apoptosis,and the oxidative stress and apoptosis of mice were further intensified after Txnrd3 knockout,and melatonin can alleviate such damage.Our results revealed that Txnrd3 has an antagonistic effect on mouse kidney against heavy metals,and the damage caused by nickel exposure will be further aggravated after Txnrd3 knockout,while the function of melatonin against heavy metals is inhibited after Txnrd3 knockout,and its effect is weakened.After knocking out Txnrd3,the effect of melatonin on nickel expose-induced injury was significantly weaker than that of wild-type mice.This lays a foundation for further research on the effects of Txnrd3 on organisms.