Preliminary Study On The Role And Mechanism Of Pyroptosis In Mice Acute Lung Injury Induced By Endotoxin

Background Acute lung injury(ALI)is a non-cardiogenic acute progressive hypoxic respiratory failure caused by a variety of extrapulmonary or pulmonary exogenous factors.Acute Respiratory Distress Syndrome(ARDS)is the serious stage of acute lung injury.As a common type of critical disease,ARDS is diagnosed in approximately 150 000 individuals in the United States each year.The characteristic of ARDS is progressive respiratory failure,accompanied by ferocious and intractable symptom of hypoxemia with the rapid onset,the deterioration of which is difficult to be reversed by conventional treatment.Therefore,it is important to further understand the pathogenesis of ARDS in order to search for effective therapeutic targets.Pyroptosis is an inflammatory programmed cell death discovered in recent years,and it is usually accompanied by the release of inflammatory factors and the up-regulation of inflammatory responses.Studies have found that gasdermin D(GSDMD)protein is a common effector of canonical and noncanonical cell pyroptosis pathway.The expression of GSDMD protein can not only induce cell death,but also can regulate the release of inflammatory factors.Although some upstream regulatory proteins of GSDMD have been involved in the pathogenesis of ARDS induced by LPS,there has been little research about GSDMD participating in it.Therefore,we hypothesized that in the pathogenesis of ARDS induced by endotoxin,the synthesis and activation of GSDMD protein may play an important role in promoting the aggravation of lung lesions.Inhibition of GSDMD may attenuate the inflammatory reaction and lung tissue damage in ARDS induced by lipopolysaccharide(LPS).Methods In the first chapter,mouse alveolar macrophage MH-S cell pyroptosis model induced by LPS was established in the first place.Followed by the performance of 6 different assays identifying the pyroptosis of MH-S cells which were stimulated by LPS with or without nigericin and Cholera toxin B(CTB).Details of 6 assays were as follows: mortality of MH-S cells was determined by lactate dehydrogenase(LDH)release assay,PI staining,and flow cytometry analysis(FCM);IL-1? level in cell culture supernatant was detected by enzyme-linked immunosorbent assay(ELISA);the m RNA transcriptional level of GSDMD was evaluated by real-time quantitative RT-PCR;the expression of GSDMD protein level was evaluated by western blot.In the second chapter,the GSDMD knockdown MH-S cell line was established using RNA interference(RNAi)technique.After LPS stimulation,the LDH level,PI positive cell proportion,IL-1 ? level and activated GSDMD expression level were detected.The aforementioned results showed the difference of cell pyroptosis and the IL-1? secretion level between the wild type(WT)cells and the knockdown(KD)cells,which further indicated the role of GSDMD protein in LPS induced MH-S cell pyroptosis.In the third chapter,the role of GSDMD in vivo was investigated in GSDMD-/-C57/B6 mice.The ARDS animal model induced by intratracheal injecting LPS was established,and followed by the comparation of bronchoalveolar lavage fluid(BALF)inflammatory factor concentrations and lung injury level between WT mice and GSDMD knockout(KO)mice via LPS inducing ARDS using ELISA and HE staining,respectively.Results 1.The mouse alveolar macrophage MH-S pyroptosis model was established successfully by LPS induction.Stimulating by LPS+nigericin and LPS+CTB separatly,the level of LDH and IL-1? expression in MH-S cell supernatant were both significantly increased(p<0.01),and FCM showed that the proportion of positive PI staining cells was significantly increased(p<0.05).In the meantime,the expression of caspase-1 and caspase-11 increased(p<0.01)compared to the control group,and the GSDMD protein was activated in the MH-S cell.The abovementioned results suggested that LPS could induce MH-S cell pyroptosis via canonical and noncanonical pathways.2?The GSDMD KD MH-S cell line was successfully established by RNAi technique.Stimulating with LPS+Nigericin and LPS+CTB separately,the expression of LDH and IL-1? and the proportion of positive PI staining cells were all significantly lower in the GSDMD KD MH-S cell line compared with WT cells(p<0.05).3?The ARDS mice model by intratracheal LPS instillation was established and was set as the foundation of the following results.Compared with the WT-ARDS group,the lung injury pathological score,wet/dry ratio and total BALF protein level were all significantly lower in KO-ARDS group(p<0.01).In addition,TNF-?,IL-1? and IL-6 levels in BALF were significantly lower in KO-ARDS group(p<0.05).Conclusion 1.The pyroptosis of the MH-S cells can be induced by LPS,which is achieved via canonical and noncanonical pathways.2.The pyroptosis of MH-S cells by LPS induction is accompanied by the release of IL-1?.3.Down regulation of GSDMD protein expression could inhibit MH-S pyroptosis and the release of IL-1? induced by LPS.4.The expression and activation of GSDMD protein in the lung tissue of mice could be induced by intratracheal instillation of LPS.5.In the LPS induced mice ARDS model,knockout of GSDMD can inhibit the inflammation level of the lung tissue and reduce its damage degree in mice.