The Role Of SIRT1-regulated Mitochondrial Homeostasis In NaAsO₂-induced Neuronal Pyroptosis

Objective:Arsenic is a ubiquitous metalloid element found in many minerals in the earth’s crust.More than 50 countries worldwide are exposed to arsenic-contaminated groundwater,and many studies have shown that chronic arsenic exposure can cause skin damage,cardiovascular symptoms.Arsenic can cross the blood-brain barrier and induce peripheral neuropathy or neuroinflammation,resulting in neurological damage.Nowadays,there are many reports on the mechanisms of neurotoxicity caused by arsenic exposure,including apoptosis,oxidative stress,and mitochondrial damage,however,there are few reports on the specific mechanisms of neuronal scorch death caused by arsenic exposure.Scorch death is a cell death phenomenon caused by excessive inflammation,which is mainly characterized by the formation of inflammatory vesicles,Caspase-1 activation and the release of inflammatory factors.Some studies have shown that SIRT1（Sirtuin 1,SIRT1）is widely expressed in the brain and has neuroprotective functions,while the specific mechanisms involved remain to be further investigated.Therefore,this paper intends to investigate whether NaAsO₂ can activate TXNIP/NLRP3inflammatory vesicles by inhibiting SIRT1 expression,which in turn leads to mitochondrial dysfunction and ultimately triggers neuronal scorchingMethods:SPF-grade,3-week-old C57BL/6 mice were purchased from the China Medical University Laboratory Animal Center.The experimental groups were:control group,NaAsO₂（10,20,40 mg/L）treated group.The body weight,water consumption,activity and growth of mice were recorded during the poisoning period.The expression levels of SIRT1,mitochondrial biogenesis-related protein,mitochondrial fusion-related protein and cell scorch death-related protein were detected by Western blot.The mouse hippocampal neuron HT-22 cell line was purchased from Beijing Beina Chuanglian Institute of Biotechnology.CCK-8 method was used to examine the effect of Resveratrol（0-18μM）at various concentrations on the proliferation viability of HT-22cells after arsenic exposure;the cells were divided into two treatment groups:NaAsO₂（0,4,6,8μM）treated for 24 h;Resveratrol intervention group:Control group,Resveratrol（2μM）,NaAsO₂（8μM）,Resveratrol（2μM）+NaAsO₂（8μM）treatment for 24 h.JC-1detected the change of mitochondrial membrane potential;CO-IP detected TXNIP and NLRP3 binding;Western blot method detected the related The proteins were detected in the same section as animal experiments.Result:1.NaAsO₂ exposure in animal experiments induced a decrease in SIRT1 protein expression in C57BL/6 mice;this in turn led to a decrease in the expression of the mitochondrial biogenesis-related proteins PGC-1α,NRF1,TFAM protein;a decrease in the expression of the mitochondrial fusion proteins OPA1,MFN1,an increase in p-DRP1616 protein expression and a decrease in p-DRP1 637 protein expression.TXNIP protein expression level increased;scorch death-related protein NLRP3,ASC,Caspase-1 p20,GSDMD-N protein expression increased,GSDMD-F expression decreased;inflammatory factors IL-18,IL-1βexpression level increased;the above results compared with the 0 dose group,the differences are statistically significant（P<0.05）2.The trend of related proteins in the mid-dose group of the cell experiment was the same as that of the animal dose group.JC-1 results showed that the mitochondrial membrane potential decreased with increasing concentration of dyed arsenic;CO-IP results showed that the binding of TXNIP to NLRP3 was enhanced in the presence of arsenic,and the difference was statistically significant（P<0.01）3.The intervention group in cellular experiments found that after resveratrol pretreatment,compared with the NaAsO₂（8μM）group,Resveratrol pretreatment resulted in increased SIRT1 protein expression;increased mitochondrial membrane potential;increased mitochondrial biogenesis-related protein PGC-1α,NRF1,TFAM protein expression;mitochondrial fusion protein OPA1,MFN1 expression The expression of mitochondrial fusion proteins OPA1 and MFN1 increased,p-DRP1 616decreased and p-DRP1 637 increased;TXNIP decreased;CO-IP results showed that TXNIP binding to NLRP3 was diminished;NLRP3,ASC,Caspase-1 p20 and GSDMD-N decreased and GSDMD-F increased;inflammatory factors IL-18 and IL-1βdecreased;the above results Compared with the NaAsO₂（8μM）group,the differences were all statistically significant（P<0.05）.Conclusion:NaAsO₂ mediates SIRT1-induced mitochondrial dysfunction thereby activating TXNIP/NLRP3 inflammatory vesicles and triggering neuronal scorching.