Mechanism Study Of Dachengqi Decoction Alleviates Sepsis-related Acute Lung Injury By Regulating Pyroptosis And Autophagy Through VDR

Objective:To investigate the protective mechanism of dachengqi decotion(DCQD)on sepsis-related acute lung injury by regulating the expression of vitamin D receptor(VDR),so as to provide scientific basis for DCQD treatment of sepsis-related acute lung injury.Methods:1.Animal Experiment: Cecal ligation and puncture(CLP)method was used to establish the model of sepsis-related acute lung injury.The mice were pretreated with DCQD(2.5/5/10g/kg)and VDR inhibitor(ZK16821).Pathological changes of lungs were evaluated by HE staining,lung tissue injury score,lung wet/dry weight ratio and total protein content of bronchoalveolar lavage fluid(BALF).The levels of diamine oxidase(DAO),LPS(lipopolysaccharide)in mice serum and the expression levels of tight junction protein 1(ZO-1)and Occludin in the intestinal tissues of mice were detected to evaluate the permeability and integrity of intestinal mucosal barrier.The expression levels of interleukin-6(IL-6),IL-8,IL-10,tumor necrosis factor-α(TNF-α),IL-18 and IL-1β in BALF were detected by enzyme linked immunosorbent assay(ELISA).The expression levels of VDR in lungs and intestine of mice,the pyroptosis-related molecules nucleotidebinding oligomerization domain-like receptor family pyrin domain containing 3(NLRP3),aoptosis-associated speck-like protein containing a CARD(ASC),gasdermin D(GSDMD),cysteinyl aspartate specific proteinase-1(CASP1)and autophagy-related molecules icrotubule associated protein 1 light chain 3(LC3),autophagy related protein16 like protein 1(ATG16L1),autophagy-related gene(Beclin1)in lungs of mice were detected by western blot(WB)and real-time quantitative polymerase chain reaction(RT-PCR).2.Cell experiments: The rat alveolar macrophage cell line NR8383 were stimulated with LPS or LPS+ATP to establish the inflammatory model or pyroptotic model,then were treated with DCQD(25/50/100μg/ml).The small interfering RNA(si RNA)technology was used to silence the expression of VDR in NR8383 cells.The levels of proptosis-related indexes,autophagy-related indexes and cytokines were detected by WB,RT-PCR,ELISA and Flow cytometry.Results:1.DCQD pretreatment could decrease the contents of DAO and LPS in serum and increase the expression of VDR,ZO-1 and Occludin in intestinal tissues of mice,decrease the lung injury score,lung wet/dry weight ratio,protein concentration and the contents of IL-6,IL-8,TNF-α,IL-18 and IL-1β in BALF,inhibit the expression of NLRP3,ASC,CASP1 and GSDMD in lung tissues of mice,increase the number of autophagosomes and the expression of VDR,LC3,ATG16L1 and Beclin1 in lung tissues of mice.Moreover,the inhibition of VDR could significantly weaken the effects of DCQD on the above indexes.2.DCQD could decrease the contents of Il-18,IL-1β and LDH in the supernatant,promote the expression of VDR,and inhibit the expression of NLRP3,ASC,CASP1 and GSDMD in NR8383 cells induced by LPS and ATP.DCQD could decrease the contents of IL-6,IL-8,TNF-α and increase the content of Il-10 in the supernatant,increase the number of autophagosomes and the expression of VDR,LC3,ATG16L1,Beclin1 in NR8383 cells induced by LPS.Moreover,the effects of DCQD on the above indexes were remarkablely counteracted by the silence of VDR.Conclulsions: This study confirmed that DCQD could improve intestinal mucosal barrier permeability,reduce intestinal the LPS into the blood,regulate the pyroptosis and autophagy levels in lung tissues by promoting VDR expression,and then alleviated the inflammatory responses and pathological damage in the lung tissues of sepsis-related ALI mice.Meanwhile,DCQD could also modulate the levels of pyroptosis and autophagy in NR8383 cells by promoting VDR expression,thereby reducing its inflammatory responses.