| Animals in a low temperature environment are prone to cold stress,and young animals are more susceptible to sudden cold air,which in turn induces hypothermia and leads to death.Cold stress has a serious impact on animal behavior,learning and memory ability.As an important part of advanced neural activities such as learning and memory,the development of the hippocampus is continuously related to the establishment of learning and memory abilities in animals during childhood,and may be affected by a variety of stressors.At present,there are few reports on the cognitive performance of juvenile animals and the mechanism of hippocampal injury after acute cold exposure.Therefore,clarifying the pathogenic mechanism of acute cold stress and exploring the damage of hippocampus in juvenile animals after cold exposure are of great significance for the research and development of anti-cold stress drugs for juvenile animals and the sustainable development of animal husbandry in northern alpine regions.In order to clarify the injury effect and mechanism of acute cold exposure on the hippocampus of young mice,this study randomly divided 3-week-old weaned C57BL/6 male mice into 5 groups:Room Temperature(RT)and acute cold exposure 3 h,6 h,9 h and 12 h(Cold Exposure,CE)groups,the CE group was continuously exposed to cold for 3 h,6 h,9 h and 12 h to build the acute cold stress model of young mice.The results showed that compared with RT group,hippocampal dopamine(DA)and serum DA levels were significantly increased after acute cold exposure(P<0.05),and the content of tyrosine hydroxylase(TH)in hippocampus of CE-9h group was significantly increased(P<0.001),indicating that acute cold exposure induces an increase in dopamine content in the hippocampus of young mice;The expression levels of hippocampal heat shock proteins HSP90 and HSP70 were significantly increased after acute cold exposure(P<0.05),indicating that the acute cold stress model was successfully constructed;The effects of acute cold exposure on hippocampal neurons of young mice were evaluated by immunohistochemistry,Nissl staining and Western blot.The results showed that compared with RT group,the expression of microtubule-binding protein MAP2 protein in hippocampus was extremely significant after acute cold exposure.decreased(P<0.01);the number of Nissl bodies in CE-9h group was significantly decreased(P<0.001);the results of ROS staining test of young mice brain slices showed that the content of ROS in hippocampus of CE-9h group was significantly increased(P<0.001);the expressions of antioxidant-related proteins GCLC,GCLM,NQO1,HO-1 and GPX-4 were significantly increased after acute cold exposure(P<0.05);the expressions of inflammation-related proteins i NOS,IL-6,TNF-α and HMGB1 Both were significantly increased after acute cold exposure(P<0.05);the expressions of pyroptosis-related proteins NLRP3,ASC,CleavedCaspase-1,GSDMD-N,m-IL-1β and m-IL-18 were significantly increased after acute cold exposure(P<0.05);The expressions of thioredox protein(Trx1)and thioredoxin-interacting protein(TXNIP)were significantly increased after acute cold exposure(P<0.05).The above results indicate that acute cold exposure leads to damage to the hippocampus of young mice,which may be related to the oxidative stress and activation of the pyroptotic signaling pathway in the hippocampus of young mice after acute cold exposure.In addition,according to the experimental results,it is speculated that the content of DA in the hippocampus is up-regulated after acute cold exposure,which leads to oxidative stress and pyroptosis,resulting in neuronal damage in the hippocampus of young mice,but the role of Trx1 in this is not fully understood.To confirm the hypothesis that DA causes neuronal damage by inducing oxidative stress and activating pyroptosis,different concentrations of DA hydroxy derivative 6-hydroxydopamine(6-OHDA)were used to treat human neuroblastoma cells(SH-SY5Y)to construct a neuronal injury model in vitro,and the effects of 6-OHDA on SH-SY5 Y cells were detected by CCK-8,Western blot,ROS probe staining,transmission electron microscopy and immunofluorescence.The results showed that compared with the control group(0 μM),in the 200 μM 6-OHDA treatment group,the cell morphology was changed and the cell viability was significantly decreased(P<0.001);LDH content was significantly increased(P<0.001);ROS content was significantly increased;the cell membrane was perforated and damaged;the expressions of antioxidant-related proteins GCLC,GCLM,NQO1,HO-1 and GPX4 were significantly decreased(P<0.05);the expressions of inflammation-related proteins i NOS,IL-6,TNF-α and HMGB1 were significantly up-regulated(P<0.01);the expression of pyroptosis-related proteins NLRP3,Cleaved-Caspase-1,ASC,GSDMD-N,m-IL-1β,m-IL-18 was significantly up-regulated(P<0.01);the expression of Trx1 protein was significantly increased decreased(P<0.05),and the expression of TXNIP protein was significantly up-regulated(P<0.01).The above results indicated that 6-OHDA caused neuronal cell damage by promoting oxidative stress and activating the pyroptotic signaling pathway,and Trx1 protein may play an important function.In order to explore the role and mechanism of Trx1 in 6-OHDA-induced SH-SY5 Y cell damage,this study constructed Trx1 overexpressing adenovirus and interfering si RNA.The results showed that overexpression of Trx1 alleviated 6-OHDA-induced SH-SY5 Y oxidative stress and pyroptosis.by reducing the ROS content and increasing the expression of antioxidant proteins GCLC,GCLM,NQO1,HO-1 and GPX4,and inhibiting the expression of pyroptosis-related proteins NLRP3,Cleaved-Caspase-1,ASC,GSDMD-N,m-IL-1β and m-IL-18,as well as reducing cell membrane perforation damage and accumulation of inflammatory mediators.Silencing Trx1 aggravated 6-OHDA-induced SH-SY5 Y oxidative stress and pyroptosis by reducing the expression of antioxidant proteins GCLC,GCLM,NQO1,HO-1,and GPX4,and increasing the pyroptosis-related protein NLRP3,Cleaved-Caspase-1,ASC,GSDMD-N,m-IL-1βand m-IL-18 expression and accumulation of inflammatory mediators.The above results indicated that Trx1 alleviated 6-OHDA-induced SH-SY5 Y cell damage by increasing antioxidant levels and inhibiting pyroptosis.Taken together,acute cold exposure leads to excessive accumulation of DA in the hippocampus of young mice,and induces oxidative stress and pyroptosis,which eventually leads to neuronal damage in the hippocampus of young mice;6-OHDA caused damage to SH-SY5 Y cells by inducing oxidative stress and pyroptosis,while Trx1 played a protective role in 6-OHDAinduced damage of SH-SY5 Y cells by enhancing antioxidant capacity and inhibiting pyroptosis. |
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