The Role Of CGAS-STING Signaling Pathway In Zearalenone-induced Apoptosis Of PAM Cells And The Protective Effect Of N-acetylcysteine

Zearalenone(ZEA)is a mycotoxin produced by a variety of Fusarium graminearum,Fusarium trilinear and Fusarium nivalis.Animals can experience a variety of hazards such as hyperstrogenic symptoms and immunosuppression after eating feed contaminated with ZEA.Studies have shown that ZEA can inhibit the multiplication of T and B lymphocytes or promote their apoptosis to exert immunostimulatory and immunosuppressive effects and then affect the mammalian immune response,alveolar macrophage(AM)is an important immune cell of the body against external pathogenic microorganisms.However,the mechanism of toxic damage to AM cells by ZEA exposure has not been fully elucidated.It has been confirmed that the cGAS-STING signaling pathway can induce apoptosis through the Caspase family or other pathways,but whether the cGAS-STING signaling pathway is involved in AM cytotoxic damage caused by ZEA exposure has not been studied.N-acetylcysteine(NAC),as a precursor of intracellular reducing glutathione(GSH),is widely used in the body to play antioxidant and apoptosis functions.Therefore,this study aims to explore the specific molecular mechanism of the effect of ZEA exposure on apoptosis in PAM cells and whether NAC can play a mitigating role in this process by establishing in vitro poisoning models of wild-type porcine alveolar macrophages(PAMWT)and STING knockout porcine alveolar macrophages(PAMSTING-/-)ZEA.1.Effects of ZEA exposure on apoptosis in PAM cellsFirstly,the cell viability state was evaluated by MTT method,and the time and concentration of action at 30%lethal dose(IC30)of ZEA were screened.Subsequently,flow cytometry was used to detect the effects of different concentrations of ZEA exposure on intracellular reactive oxygen species,mitochondrial membrane potential(MMP)level,ATP production and apoptosis rate after PAM cells.Western Blot was used to analyze the changes of apoptosis-related proteins in cells.At the same time,transmission electron microscopy was used to observe the ultrastructure of the nucleus and mitochondria.The results showed as follows,①The effect of ZEA on the viability of PAM cells was significantly enhanced at low dose for a short time(P<0.05),high dose significantly decreased cell activity for a long time(P<0.01).Finally,5,10,20 μmol/L and 24 h were selected as the exposure dose and exposure time for follow-up experiments.②By detecting the phagocytic index of alveolar macrophages,it was found that compared with the control group,5 μmol/L ZEA significantly promoted the phagocytic ability of macrophages(P<0.01),and 20 μmol/L ZEA significantly weakened the phagocytic ability of macrophages(P<0.05).③Compared with the control group,the 20μmol/L ZEA group has a significant increase in ROS production(P<0.01),ATP content decreased significantly(P<0.01),and MMP decreased significantly with the increase of ZEA concentration(P<0.05);The ultrastructure of cells observed by transmission electron microscopy showed that the nucleus crumpled,dented and ruptured in different degrees under the action of ZEA,the mitochondrial ridge was fractured and blurred,and the mitochondrial double-layer membrane was fractured and dissolved.④ The results of flow cytometry showed that ZEA led to a significant increase in apoptosis rate at 5 μmol/L(P<0.05);Western Blot showed the same results,20 μmol/L ZEA treatment significantly increased Caspase 3,Caspase 9 protein expression,and BAX/Bcl 2 ratio(P<0.01).In summary,ZEA can induce mitochondrial dysfunction of PAM cells,which in turn promotes apoptosis in PAM cells.2.The role of the cGAS-STING signaling pathway in PAM apoptosis caused by ZEA exposureFirst,ZEA was exposed to PAMWT cells and PAMSTING-/-cells,and the expression of key proteins of the cGAS-STING signaling pathway and the transcription level of downstream inflammatory chemokine mRNA were detected by Western Blot and PCR,respectively.The results showed that,① Compared with the control group,the expression of cGAS-STING signaling pathway,the expression of key proteins cGAS,STING,and the ratio of P-IRF 3/IRF 3 and P-TBK 1/TBK 1 in PAMWT cells were significantly increased(P<0.01)after 20 μmol/L ZEA treatment.The transcription levels of mRNA transcription of inflammatory chemokines TNF-α,IFN-β and IL-6 downstream of the cGAS-STING signaling pathway were also significantly upregulated(P<0.01),indicating that the cGAS-STING signaling pathway in PAM cells was activated under the action of ZEA.②Compared with the PAMWT cell control group,there was no significant change in the expression of cGAS upstream of the cGASSTING signaling pathway in the PAMSTING-/-cell control group(P>0.05),and the expression of downstream STING and the ratio of P-IRF 3/IRF 3 and P-TBK 1/TBK1 were significantly reduced(P<0.01).mRNA transcription levels such as inflammatory chemokines TNF-α and IFN-β were significantly reduced(P<0.05).③Under 20 μmol/L ZEA treatment,PAMSTING-/cells significantly decreased compared with PAMWT cells(P<0.01)in the expression of key proteins cGAS-STING signaling pathway cGAS、STING and the ratio of P-IRF 3/IRF 3 and P-TBK 1/TBK 1.The transcription levels of mRNA such as inflammatory chemokines TNFα,IFN-β and IL-6 were significantly reduced(P<0.01).④Compared with the control group,there was no significant change in the expression of cGAS-STING signaling pathway,cGAS,STING,the ratio of P-IRF 3/IRF 3,P-TBK 1/TBK 1,and the downstream inflammatory chemokines TNF-α,IFN-β,IL-6 and other mRNA transcription levels(P>0.05)after 20μmol/L ZEA treatment.The above results showed that knocking out STING can inhibit ZEA activation of the cGAS-STING signaling pathway.Finally,we studied the changing trend of apoptosis rate before and after STING knockout,and the results showed that compared with PAMWT cell control group,the apoptosis rate of PAMSTING-/-cell control group was significantly decreased(P<0.05).After 20 μmol/L ZEA treatment,the apoptosis rate of PAMSTING-/-cells was significantly decreased compared with PAMWT cells(P<0.01).The above results show that ZEA can activate the cGAS-STING signaling pathway in PAM cells and induce apoptosis.Knocking out STING significantly inhibits ZEA to activate the cGASSTING signaling pathway and mitigate the occurrence of apoptosis.This shows that ZEAinduced apoptosis in PAM is partially achieved by activating the cGAS-STING signaling pathway.3.Protective effect and mechanism of N-acetylcysteine on apoptosis induced by ZEA exposureIn order to further explore the protective effect and mechanism of NAC on apoptosis caused by ZEA exposure,the MTT method was first used to screen and determine the optimal concentration of 3 mmol/L NAC for subsequent experiments.Western Blot,flow cytometry,and real-time PCR were then used to analyze the expression of apoptosis-associated proteins and signaling pathway-critical proteins in PAM cells under the action of NAC,as well as the transcriptional levels of downstream inflammatory chemokine mRNA.The results showed that,①Compared with the ZEA group of PAM cells,the addition of NAC led to a significant decrease in ROS(P<0.01),a significant increase in MMP level(P<0.01),a significant increase in ATP level(P<0.01),and a significant improvement in mitochondrial structural damage.This suggests that NAC may significantly alleviate mitochondrial damage in PAM cells due to ZEA exposure.②Compared with ZEA group,the expression of key proteins cGAS and STING in the cGAS-STING signaling pathway and the ratio of P-IRF 3/IRF 3 and P-TBK 1/TBK 1 in PAM cells were significantly decreased by addition of NAC(P<0.01),the mRNA transcription levels of downstream inflammatory chemokines TNF-α and IL-6 were significantly decreased(P<0.01),IFN-β mRNA transcription levels were significantly decreased(P<0.05);③Compared with the ZEA group,the expression level of apoptosis-related protein Caspase 3 protein and BAX/Bcl 2 ratio in the NAC+ZEA co-treated group were significantly reduced(P<0.05),and the expression level of Caspase 9 protein was significantly reduced(P<0.01).The apoptosis rate of flow cytometry also showed a significant decrease(P<0.01).The above results indicate that mitochondrial damage in PAM cells caused by ZEA exposure is the main cause of apoptosis induced by cGAS-STING signaling pathway,and the addition of NAC can effectively alleviate mitochondrial damage and apoptosis.In summary,this study found that ZEA exposure can induce mitochondrial damage in PAM cells,leading to mitochondrial DNA entry into cytoplasm and activation of cGASSTING signaling pathway as a damage molecule-related mode,ultimately leading to apoptosis in PAM cells.The addition of NAC significantly alleviated mitochondrial damage and improved apoptosis mediated by the cGAS-STING signaling pathway activated by ZEA.