Function And Mechanism Of Micheliolide Mediated Autophagy Of NLRP3 In Radiation Enteritis

Objective:Radiation therapy is used as one of the main treatments for malignant tumors.Although there have been significant improvements in precision radiotherapy technologies,the radiation treatment process can still cause varying degrees of normal tissue damage.The small intestine tissue is extremely sensitive to radiotherapy,and prolonged exposure to radiation fields can lead to the development of radiation enteritis.Patients may experience abdominal pain,diarrhea,mucous bloody stools,intestinal obstruction,and perforation,which not only greatly reduces the patient’s quality of life,but also limits the further implementation of clinical radiation therapy protocols.Currently,there are no clinically applied drugs that can effectively treat or prevent radiation enteritis.As a form of inflammatory cell death,literature and our previous studies have suggested that pyroptosis may play a key role in radiation-induced inflammatory diseases.Those finding suggests that intervention of pyroptosis may be a new potential intervention strategy for radiation enteritis.Therefore,further research is needed to clarify the role of pyroptosis in radiation enteritis and develop novel protective drugs targeting pyroptosis.Detailed molecular regulatory mechanisms are also crucial to fully understand the potential clinical application prospects and strong economic and social values of such interventions.Methods:（1）In intestinal epithelial cells,interference with NLRP3 followed by radiation treatment through multiple assays which includes CCK-8 assay;LDH release assay;ELISA for IL-1βand IL-18 secretion;Western blotting to detect the expression levels of pyroptosis-related proteins such as Caspase-1 p20 active end,NLRP3 and GSDMD N termini.In addition,PI fluorescence staining and flow cytometry analysis of PI/Annexin V was performed to evaluate the role of NLRP3 inflammasome-mediated cell pyroptosis in intestinal epithelial cell injury.In Nlrp3 wild-type and Nlrp3^-/-mice,HE staining of intestinal tissues was performed.Western blotting was used to detect the expression levels of pyroptosis-related proteins such as Caspase-1p20 active end,NLRP3 and GSDMD N termini in intestinal tissues.Immunohistochemistry and ELISA were used to detect the release of IL-1β,IL-18,TGF-β1,TNF-αand IFN-γin intestinal tissues and serum.Lastly,immunofluorescence was used to detect the co-localization of NLRP3 with ASC and to evaluate the role of NLRP3-mediated pyroptosis in radiation enteritis.（2）In cell model of radiation-induced intestinal epithelial injury,a small molecule compound library targeting cell pyroptosis was used to screen and obtain a potential therapeutic drug for radiation-induced enteritis:Micheliolide.At the level of intestinal epithelial cells,Micheliolide was used for pretreatment followed by radiation treatment.Multiple assays were performed,including the CCK-8 assay,LDH release assay,and ELISA to detect the secretion of IL-1βand IL-18.Western blotting was performed to detect the expression levels of pyroptosis-related proteins such as Caspase-1 p20,NLRP3,and GSDMD N termini.PI fluorescence staining and PI/Annexin V flow cytometry analysis were also performed to evaluate the protective effect of Micheliolide on radioactive intestinal epithelial cell injury.At the animal level,radiation treatment was performed,followed by HE staining of intestinal tissues.Western blotting was performed to detect the expression levels of pyroptosis-related proteins such as Caspase-1 p20,NLRP3 and GSDMD N termini in intestinal tissues.Immunohistochemistry and ELISA assays were performed to detect the release of IL-1β,IL-18,TGF-β1,TNF-α,and IFN-γ.Immunofluorescence was used to detect the co-localization of NLRP3 and ASC,and to evaluate the preventive value of Micheliolide in radiation enteritis.（3）On the level of intestinal epithelial cells and animal models,Micheliolide pretreatment was followed by radiation,and LC3 aggregation was detected by immunofluorescence.Autophagosome formation was detected through transmission electron microscopy（TEM）,and autophagy-related protein expression was detected by immunohistochemistry and western blotting.Co-localization of NLRP3,LC3 and LAMP1 was detected through immunofluorescence to determine the promoting effect of Micheliolide pretreatment on autophagy.The protective effect of Micheliolide on radioactive intestinal epithelial cell injury was evaluated at the level of intestinal epithelial cells by pretreatment with Micheliolide followed by radiation,and blocking of autophagy by the autophagy inhibitor chloroquine.Assays performed include CCK-8 assay,LDH release assay,and ELISA for IL-1βand IL-18 secretion.The protein expression levels of pyroptosis-related proteins Caspase-1 p20,NLRP3,and GSDMD N termini were also detected through western blotting.On Beclin1^-/+wild-type and Beclin1^-/+mice,pretreatment with Micheliolide before radiation treatment,followed by HE staining through the intestinal tissues.Western blotting was performed to detect the expression levels of focal pyroptosis-related proteins such as Caspase-1 p20 active end and NLRP3 and GSDMD N termini in intestinal tissues.Immunohistochemistry and ELISA were performed to detect the release of IL-1β,IL-18,TGF-β1,TNF-αand IFN-γ.Immunofluorescence was used to detect the co-localization of NLRP3 with ASC and to clarify the effect of autophagy blockage on the protective effect of Micheliolide-mediated radiation enteritis.Results:Through NLRP3 interference at the cellular level and NLRP3 silencing at the animal level,we have identified the role and intervention value of NLRP3inflammasome-mediated cell pyroptosis in the occurrence of radiation enteritis.Following that,through the screening of small molecule compounds library targeting pyroptosis and subsequent cellular level validations,we obtained a potential radio-protective small molecule drug for radiation enteritis,called Micheliolide.Further study showed that Micheliolide treatment significantly improved the occurrence of radiation-induced pyroptosis,tissue damage,inflammatory cell infiltration,and pro-inflammatory cytokine release in small intestinal tissues.Additionally,it was found that Micheliolide treatment downregulates NLRP3expression.Combined with the existing studies,we hypothesized and validated that Micheliolide inhibits radiation-induced cell pyroptosis by promoting NLRP3autophagy.Through the study of chloroquine,an autophagy inhibitor,and the autophagy-associated gene Beclin1^-/+mice,we further clarified that the protective effect of Micheliolide is dependent on NLRP3 autophagy.Conclusion:The present study demonstrated that Micheliolide achieves its protective effect against radiation enteritis by inducing NLRP3 autophagic degradation and inhibiting the occurrence of NLRP3 inflammasome-mediated radiation-induced cell pyroptosis.Our results suggest that Micheliolide may have a good application potential as a protective drug against radiation enteritis.