Molecular Mechanism Of Flagellin A N/C Against NLR Pyrin Domain-containing 3 Inflammasome-dependent Radiative Pyroptosis

PurposeRadiation-induced enteropathy is among the most prevalent dose-limiting radiation toxicities.Despite its prevalence and burden,there are currently no effective treatments for radiation-induced intestinal injury,and understanding of this pathology remains poor.It is an urgent need to study the pathogenesis of radionitis and seek effective means of prevention and control.pyroptosis as inflammatory necrosis,has been found to be widely involved in many inflammatory tissue injuries,and has become an important target for clinical treatment.Flagellin A(FlaA)N/C is a self-developed radiation-protective protein with patent intellectual property,our previous research shows that FlaA N/C can inhibit radiation induced injury and prolong the survival time of mice by regulating a variety of inflammatory factors.The purpose of this study was to explore the mechanism of FlaA N/C inhibiting the NLRP3-dependent pyroptosis in intestine injury,and to provide a new perspective for clinical prevention.Methods1.In order to study the extent of lesion of intestine after subcutaneous injection of FlaA N/C into model mice,a mouse model of radiation enteritis was built.And athological tissue sections were observed by HE staining and TUNEL.Western blot was used to detect γ-histone 2AX(γ-H2AX),while the levels of IL-6,IL-8,TNF-α,MCP-1in intestine tissue were detected by real-time fluorescence quantitative PCR.2.After the mice were injected with caspase-1 and IL-1β inhibitors,the activity of caspase-1 was detected by caspase-1 activity assay kit.Real-time quantitative PCR,Western blot and immunohistochemical staining were used to detect the expression of pyrin-containing NOD-like receptor family 3(NLRP3),caspase-1,CARD-containing domain apoptosis-associated speckle-like protein(ASC / TMS1)in intestine tissue.The levels of IL-1β and IL-18 in serum were detected by RT-PCR and ELISA.The effect of pyroptosis on FlaA N/C in inhibiting intestinal radiation injury was explored.3.The expression of superoxide dismutase(SOD),glutathione peroxidase(GST),glutathione reductase(GSHG)and glutathione(GSSG)and gsh(GSSG + gsh)activity were detected to understand the role of ROS pathway after radiation;After the modelmice were injected with ROS inhibitor subcutaneously,the activity of caspase-1 was detected by caspase-1 activity assay kit.Real-time quantitative PCR,Western blot and immunohistochemical staining were used to detect the expression of pyrin-containing NOD-like receptor family 3(NLRP3),caspase-1,CARD-containing domain apoptosis-associated speckle-like protein(ASC / TMS1)in intestine tissue.The levels of IL-1β and IL-18 in serum were detected by RT-PCR and ELISA.The effect of ROS pathway on FlaA N/C in inhibiting intestinal radiation injury was explored.Results1.FlaA N/C attenuated the radiation-induced intestinal inflammatory response in mice and inhibited the DNA damage of intestine cells,thereby alleviating radiation-induced intestinal tissue damage.2.The expression of NLRP3,caspase-1 and TMS1 in intestine tissue of mice after radiated were increased and the levels of IL-1β and IL-18 in serum were also increased.After treatment with FlaA N/C,caspase-1 and IL-1β inhibitors,the activity of caspase-1was decreased and the expression of NLRP3,caspase-1 and TMS1 were also decreased in the intestine.Moreover,the levels of IL-1β and IL-18 in serum and intestinal tissue were decreased.In results,the degree of intestinal damage was weakened and DNA damage was partially inhibited in intestine cells of mice after radiated,thus confirming pyroptosis involved in radiation injury in the intestine and FlaA N/C can reduce the intestinal injury caused by radiation by inhibiting pyroptosis.3.The activity of ROS pathway in the mice after radiated was enhanced.After treated with FlaA N/C and ROS pathway inhibitors,the activity of caspase-1 in intestinal tissue of mice was decreased,while the expression of NLRP3,caspase-1 and TMS1 were also decreased.IL-1β and IL-18 in serum and intestinal tissue were decreased.The extent of intestinal lesion was weakened and the DNA damage of intestinal cells was partially inhibited,which confirmed the involvement of ROS pathway in the process of radiation injury of the causal intestine and FlaA N/C can reduce the intestinal damage caused by radiation by inhibiting the generation of ROS.ConclusionROS-induced NLRP3 inflammasomes mediated radiation-induced pyroptosis of the intestinal cells,and FlaAN/C suppressed pyroptosis to protect the intestinal tissue.