Mechanism Of FlaA N/C Inhibit Radiation Induced Pyroptosis Via Inducing Autophagy Of NAIP-NLRC4 Inflammasome

Research background and purpose:Radiation lung injury is a common complication of patients with malignant tumors of the chest treated with ionizing radiation,which seriously affects the treatment progress and quality of life of patients,and currently lacks specific preventive drugs for this.The research team independently developed the recombinant flagellin FlaAN / C.Previous studies have shown that it can produce highly effective radiation protection,but the protection mechanism is still unknown.Pyroptosis is a newly discovered pro-inflammatory programmed cell death method,which has been widely proven to play an important role in the occurrence and development of various inflammatory diseases,and has become a new target for clinical treatment.The purpose of this study is to clarify the involvement of Inflammasome in radiation injury,to explore the effective targets of FlaAN / C in inhibiting radiation cell scoring,and to provide experimental evidence and potential strategies for the development of drugs for the prevention and treatment of radiation damage.Methods:1.FlaA N / C inhibits radiation-induced human lung epithelial cell injuryConstruct human lung epithelial cells(BEAS-2B)radiation injury model,evaluate cell damage by CCK-8 cell viability test and lactate dehydrogenase(LDH)release test,and detect inflammatory factor interleukin in cell culture medium by ELISA method 1?(IL-1?),interleukin 18(IL-18)levels.2.The role of pyroptosis in FlaA N / C in reducing radiation-induced BEAS-2B cell injuryBEAS-2B cells were treated with caspase-1(caspase-1)specific inhibitor(Z-YVAD-FMK,YVAD)and FlaA N / C and irradiated after 2h.CCK-8 activity test and LDH release level detection experiment were used to evaluate the damage of the treated cells.The caspase-1 activity detection kit was used to detect the caspase-1activity of the treated cells.Western blot was used to detect the expression levels of IL-1?,IL-18 and casp1 p20 protein in the treated cells,and ELISA was used to detectthe secretion of cytokines IL-1? and IL-18 in the cell culture supernatant.3.FlaA N / C induces NLRC4 inflammasome autophagyAfter BEAS-2B cells were treated with FlaA N / C,fluorescence colocalization and co-immunoprecipitation methods were used to detect the formation of NAIP-NLRC4 inflammatory bodies and the binding of NAIP,Beclin-1 and NLRC4.Immunofluorescence was used to detect the aggregation of LC3.Western Blot method was used to detect the expression of LC3 protein in cells.The website predicts the binding sites of NLRC4 inflammatory body components and LC3;After BEAS-2B is treated with FlaA N / C,immunofluorescence detects NAIP,NLRC4,and co-localized expression with LC3,and Western blot detects NAIP,NLRC4,ASC,and LC3 in cells Protein expression level.4.Regulatory effect of NLRC4 inflammasome autophagy on the occurrence of radiation-pyroptosisAutophagy inhibitor(spautin1),FlaA N / C and si-NAIP pre-treated BEAS-2B and irradiated.Western blot was used to detect NLRC4 inflammasome components and expression levels of scorch and autophagy related proteins.8 Cell viability test and LDH release test to evaluate cell damage after irradiation,caspase-1 activity detection kit to detect caspase-1 activity after cell irradiation,and ELISA method to detect inflammatory factors IL-1? and IL-18 secretion level.Result:1.When BEAS-2B cells were irradiated with a radiation dose greater than 10 Gy for 48 h,the cells were significantly damaged.FlaA N / C pretreatment can significantly inhibit the secretion of IL-18 and IL-1? in BEAS-2B cells caused by radiation,and reduce the occurrence of radiation damage in BEAS-2B cells.2.The expression of casp1 p20,IL-1?,and IL-18 scorch protein increased in BEAS-2B cells with radiation dose greater than 10 Gy,and the secretion levels of IL-1? and IL-18 in cell culture medium increased;co-immunoprecipitation The experimental results showed that the complexes of NLRP3,AIM2 and ASC protein increased after irradiation;after treatment with FlaA N / C for 2 h,the activity of caspase-1 in BEAS-2B cells decreased,and casp1 p20,IL-1?,and IL-18 were the3.focal death markers.Protein expression was reduced;secretion levels of IL-1? and IL-18 in cell culture media were reduced;damage to BEAS-2B cells was reduced.4.After FlaA N / C treatment,the expression of casp1 p20,IL-1?,and IL-18 scoring marker protein increased in the FlaA N / C(500pg / mL)treatment group;after FlaA N / C(50pg / mL)treatment,The results of co-immunoprecipitation showed that the level of NLRC4 inflammasome complex was significantly increased.The results of immunofluorescence showed that LC3 had obvious aggregation after FlaA N / C treatment,and Western blot results showed that LC3 II protein expression increased.5.Compared with FlaA N / C treatment,the protein expression levels of NLRC4,ASC,and NAIP in the FlaA N / C + spautin1 group and the FlaA N / C + si-NAIP group were significantly increased;the website predicts that LC3 has multiple binding sites;immunofluorescence in the FlaA N / C treatment group showed that LC3 had significant co-localized expression with NLRC4 and NAIP.Compared with the pre-radiation after FlaA N / C pretreatment,spautin1 and si-NAIP pre-treatment radiation increased the levels of scorch marker proteins,increased IL-18 and IL-1?secretion levels,decreased cell viability,caspase1 activity and LDH release levels increased significantly.Conclusion:1.NLRP3,AIM2 inflammasome-mediated pyroptosis plays an important role in BEAS-2B cells injury induced by radiation.2.FlaA N / C exerts radiation protection by promoting autophagy of NLRC4 and inhibiting the occurrence of radiation-pyroptosis.