| Cold stress is an important cause of serious damage to the economic benefit of livestock and poultry breeding.Our previous study found that cold exposure can lead to liver injury,inflammation and oxidative stress,etc.However,what is the mechanism through which cold exposure causes liver injury? Through literature review,it is found that cell apoptosis signaling pathway is significantly activated in liver diseases,and inhibition of cell apoptosis signaling pathway can alleviate the disease and become a way of prevention and treatment.So,can cold stress cause liver injury by activating the cell apoptosis signaling pathway? In this study,Western Blot and other experimental methods were used to construct cold stress models of mice in vivo and in vitro,aiming to clarify the mechanism of cold exposure activated cell apoptosis signaling pathway to induce liver injury in mice,and provide a new direction for the prevention and treatment of damage caused by cold stress.To investigate the effect of chronic cold exposure on liver injury in mice,4-week-old male C57BL/6 mice were randomly divided into two groups(Control group: Control;Chronic Cold exposure group: Cold)with 6 rats in each group.Mice in the Cold group were exposed to 4 ℃ daily for 3 h for 4 weeks.Then ELISA,HE staining and other experiments were conducted.The results showed that Alanine aminotransferase(ALT),Aspartate aminotransferase(AST),and serum Lactate dehydrogenase(LDH)levels were significantly increased.The results of HE staining showed inflammatory cell infiltration and other pathological changes in liver cells of cold-exposed mice.Western Blot and q PCR results showed that Heat Shock protein70(HSP70)and Heat Shock protein 90(HSP90)levels were significantly higher than those in the Control group,respectively.It shows that the cold stress model is successfully constructed.The expression of inducible nitric oxide synthase(i NOS),cyclooxygenase-2(COX-2),tumor necrosis factor-α(TNF-α)and high mobility group box 1 protein(HMGB1)were significantly increased after chronic cold exposure,which further suggested that chronic cold exposure could lead to increased expression of inflammatory cytokines.In addition,the expressions of nuclearfactor erythroid derived 2-like 2(Nrf2)and its downstream proteins and genes were significantly increased compared with the control group,indicating that chronic cold exposure could cause oxidative stress in liver of mice.In addition,B-cell lymphoma-2(Bcl-2)and Bcl-2-associated X(Bax)ratio and cleaved cysteine-aspartic acid protease 3(Cleaved caspase-3)expression were also significantly increased,suggesting that chronic cold exposure could induce apoptosis in liver of mice.Nucleotide binding domain(NOD)-like receptor(NLR)protein 3,Caspase-1,gasdermin D(GSDMD),interleukin-1β(IL-1β),interleukin-18(IL-18)protein levels were significantly increased.These results indicate that chronic cold exposure can induce activation of pyroptosis signaling pathway.In addition,the immunohistochemical results showed that the expressions of NLRP3,GSDMD,caspase-1 and Nrf2 were increased after cold exposure,which further indicated that cold exposure could induce pyroptosis in mouse liver,and it may be related to NLRP3 and antioxidant signaling pathway.In order to explore the effect of mild hypothermia on mouse hepatocytes AML12,light microscope observation and MTT test showed that the morphology of AML12 cells would change after 32 ℃ Mild hypothcrmia(MH)for different time,and the phenomenon of "drawing" would appear.The number of cells decreased in a time-dependent manner,indicating that mild hypothermia could inhibit the growth of AML12 cells.Hoechst 33258 staining results showed that the cells presented fragmented dense staining after different times of mild hypothermia treatment,suggesting that mild hypothermia induced apoptosis of AML12 cells,which was further confirmed by the increased expression of apoptotic protein.The results of reactive oxygen species(ROS)probe staining showed that the ROS level in AML12 cells and the expression of antioxidant proteins and genes were significantly increased after mild hypothermia treatment for different time,which indicated that mild hypothermia could cause oxidative stress in AML12 cells.The protein expression levels of HSP70 and HSP90 were increased,which indicated that the mouse liver cell model of mild hypothermia was successfully constructed for subsequent experiments.The expression levels of NLRP3,ASC,Caspase-1,GSDMD,IL-1β,IL-18 protein and gene were significantly increased,and the expression levels of inflammatory cytokines i NOS,COX-2,TNF-α and HMGB1 were significantly increased,all indicating that mild hypothermia treatment could activate the pyroptosis signaling pathway of AML12 cells.Causing inflammatory damage to the liver.To verify the relationship between mild hypothermia mediated pyroptosis of AML12 cells and the antioxidant signaling pathway,AML12 cells were treated with NLRP3 inhibitor MCC950 and GSDMD inhibitor Necrosulfonamide for 3 h,respectively,and then placed in an incubator at 32 ℃ for 6 h.The expression levels of NLRP3,Caspase-1,GSDMD,IL-1β,Nrf2 and Heme oxygenase-1(HO-1)proteins were detected by Western Blot.The results showed that 100 μM MCC950 and 20 μM Necrosulfonamide could effectively inhibit the activation of NLRP3 and GSDMD in AML12 cells induced by mild hypothermia,as well as the expression of caspase-1,IL-1β,Nrf2 and downstream HO-1 proteins.It is proved that mild hypothermia activates the pyroptosis and oxidative stress signaling pathways of AML12 cells through the NLRP3/Caspase-1 pathway,and inhibition of the pyroptosis signaling pathway may reduce the oxidative stress injury to a certain extent.The relevant mechanism still needs further experimental proof.Conclusion: Cold stress mediates NLRP3/Caspase-1 pathway to activate AML12 cell pyroptosis and oxidative stress signaling pathway to induce liver tissue damage in mice.Therefore,regulating cell pyroptosis signaling pathway may be a preventive measure against cold stress. |
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