The Role Of Gut Microbiome In The Pathogenesis Of Severe Acute Pancreatitis-Associated Acute Lung Injury And The Intervention Of Qingyi Decoction

Background: The etiology of Severe acute pancreatitis-associated acute lung injury(SAP-ALI)has not yet been fully elucidated.Previous studies have shown that disruption of the gut microbiota,damage to the gut barrier,and bacterial translocation play an essential role in the pathogenesis of SAP-ALI and affect its prognosis.The role of small molecule metabolites derived from the gut microbiota along the gut-lung axis in the remote regulation of SAP-ALI has become an emerging research topic.Qingyi decoction(QYD)is an experienced traditional Chinese medicine formula commonly used in the clinical treatment of SAP,which has various pharmacological effects such as antiinflammatory,enhancing intestinal barrier function and reducing oxidative stress.However,its specific mechanism has yet to be fully explored.This project proposes to investigate the role of gut microbiota and its derived shortchain fatty acids(SCFAs)in the pathogenesis of SAP-ALI and QYD interventions in a mouse model of SAP-ALI induced by caerulein combined with lipopolysaccharide(LPS)and a pseudo-germ-free mice model induced by cocktail-antibiotics,using various histological techniques and experimental methods.These findings provide new insights into treating SAP-ALI through modulation of the gut microbiota and have prospective implications for future primary research.Part I: The role of the gut microbiome during SAP-ALI and the intervention of QYD.Objective: To investigate the effect of gut microbiota on the severity of SAP-ALI and the therapeutic role of QYD.Methods: All C57BL/6 mice were randomized into a sham operation group(SO),a disease model group(SAP),a pseudo-germ-free model treated with cocktail-antibiotics before molding group(Abx+SAP)and a QYD treatment group(QYD+SAP).HE staining was used to assess the pathological changes in pancreatic,intestinal and lung tissues and to calculate the pathological damage score;transmission electron microscopy was used to detect the ultrastructural changes in gut and lung tissues;ELISA was used to detect the serum levels of amylase,IL-1β,IL-6,TNF-α,D-lactate,LPS and diamine oxidase(DAO).Immunofluorescence staining for myeloperoxidase(MPO)in pancreatic and lung tissues.Western blot(WB)assay and immunofluorescence were used to detect the expression of intestinal barrier proteins(ZO-1 and Occludin)in every group.Results:(1)Compared with the SO group,the expression of MPO in the pancreatic and lung tissues was increased in the SAP group(P<0.001),the histopathological damage was severe,and the pathological score was significantly higher(P<0.001).The alveolar epithelium in the lung tissues was damaged,associated with disruption of the air-blood barrier.In addition,the serum levels of AMY,IL-1β,IL-6,and TNF-α were significantly increased in the SAP group relative to the SO group(P<0.001).(2)Significant intestinal damage and increased pathology scores were observed in the SAP group compared to the SO group(P<0.05).The intestinal epithelium was damaged,and the expression of intestinal barrier proteins(ZO-1,Occludin)was reduced(P<0.01).In addition,serum levels of D-lactate,LPS,and diamine oxidase were significantly increased in the SAP group(P<0.001).(3)Compared to the SAP group,the QYD+SAP group showed reduced pancreatic and lung histopathological damage and significantly lower pathological scores(P<0.001),restoration of barrier function was observed in intestinal and lung tissues,and expression of intestinal barrier proteins(ZO-1,Occludin)was significantly increased(P<0.01).(4)Compared with the SAP group,the pathological damage to the pancreas and lung tissue was significantly reduced,the pathological score was lower in the Abx+SAP group(P<0.001),and the damage to the intestine was partially restored(P<0.05).In addition,serum levels of AMY,IL-1β,IL-6,TNF-α,D-lactate,LPS,and DAO were significantly lower in the Abx+SAP and QYD+SAP groups compared to the SAP group(P<0.001).Part II: Alterations in the gut microbiome and metabolism of SCFAs during SAPALI and the intervention of QYD.Objective: To investigate the alteration of gut microbiota and metabolism of SCFAs during SAP-ALI and the intervention of QYD.Methods: The experimental animals,experimental groups,model preparation,and sampling methods are the same as in Part I.16 S r DNA sequencing was used to detect the bacteria composition,diversity,and function of the cecum contents in different groups and liquid chromatography-tandem mass spectrometry(LC-MS/MS)was used to quantify SCFAs in feces,intestinal,plasma and lung tissues.Partial least squares discriminant analysis(PLSDA)models and variable importance in projection(VIP)scores were used to screen for markers in different groups of SCFAs;Spearman’s rank correlation analysis was applied to study the association between gut microbiota and SCFAs in various parts.Results:(1)Compared to the SO group,the SAP group showed significant differences in the composition of the bacteria and reduced species richness(P<0.01).At the phylum level,the proportion of the Firmicutes was significantly higher in the SAP group,and the ratio of the Firmicutes to the Bacteroidetes increased(P<0.001).At the genus level,the SAP group showed an increased relative abundance of Escherichia,Enterococcus,Enterobacter,Peptostreptococcus,Helicobacter,Porphyromonas,and decreased relative abundance of SCFAs-producing bacteria such as Bacteroides,Roseburia,Parabacteroides,Akkermansia,Prevotella.In addition,the synthesis of fatty acids by bacteria in the SAP group was reduced,and Escherichia fergusonii and Enterococcus faecium were the most representative species in the SAP group.(2)The concentration of SCFAs in feces was highest in all groups,followed by intestinal,plasma,and lung tissues,and showed a gradual decreasing trend.Compared with the SO group,propionate and butyrate in feces,plasma,colon,and lung tissues in the SAP group showed a down-regulation trend,especially in feces(P<0.01)and lung tissues(P<0.05);whereas acetate in plasma,intestine and lung tissues was significantly higher(P<0.001).In addition,the SCFAs with higher concentrations in the SO group were butyrate and propionate,while the most representative SCFAs in the SAP group were acetate.(3)Compared with the SAP group,the microbial community composition of the QYD+SAP group was similar to that of the SO group,and the QYD-treated group had a significantly higher proportion of Bacteroidetes and a lower ratio of Firmicutes to Bacteroidetes(P<0.001).At the genus level,the abundance of SCFAs-producing bacteria and the ability to synthesize fatty acids increased in the QYD-treated group(P<0.001).In addition,butyrate was the most representative SCFA for the QYD+SAP group.(4)The relative abundance of SCFAs-producing bacteria was significantly positively correlated with butyrate and propionate levels and negatively correlated with acetate by Spearman’s rank correlation analysis(P<0.05).Escherichia,Enterococcus,and Enterobacter were positively correlated with acetate in plasma and gut(P<0.05).Part III: Role of SCFAs-mediated AMPK/NF-κB/NLRP3 signalling pathway in SAPALI and the intervention of QYD.Objective: To investigate the expression of AMPK/NF-κB/NLRP3 signalling pathway targeted and regulated by SCFAs in SAP-ALI and the intervention of QYD.Methods: The experimental animals,experimental groups,model preparation and sampling methods are the same as in Part I.WB assay to detect the expression of AMPK/NF-κB/NLRP3 signalling pathway related proteins;RT-q PCR analysis of AMPK/NF-κB/NLRP3 signalling pathway m RNA transcription;immunofluorescence staining for NF-κB and NLRP3 proteins.Sample gene enrichment analysis(ss GSEA)was used to explore the association between SCFAs and AMPK/NF-κB/NLRP3 signalling pathway in SAP patients;Spearman’s rank correlation analysis was used to explore the association between SCFAs and AMPK/NF-κB/NLRP3 signalling pathway in mouse samples.Results:(1)Compared with healthy individuals,the gene abundance of FFAR2,FFAR3,CD36 and ACSS2 was increased in peripheral blood mononuclear cells(PBMCs)and the expression of HDAC9 was decreased in SAP patients(P<0.001).ss GSEA results showed that the AMPK/NF-κB/NLRP3 signalling pathway showed a significant positive correlation with FFAR2,FFAR3,ACSS2(P<0.001),while a significant negative correlation with HDAC9(P<0.001).(2)Compared with the SO group,the expression of p-AMPK was down-regulated and the expression of NF-κB and NLRP3 was up-regulated in the ileum and lung tissues of mice in the SAP group(P<0.01).In addition,the expression level of AMPK m RNA was decreased in the SAP group(P<0.01),while the m RNA levels and immunofluorescence intensity of NF-κB and NLRP3 were increased(P<0.05).(3)Compared with the SAP group,the levels of p-AMPK were increased in ileal and lung tissues in the QYD+SAP group,while the levels of NF-κB and NLRP3 were decreased(P<0.05).Besides,the AMPK m RNA expression level was increased in the QYD+SAP group and approached that of the SO group,especially in lung tissue(P<0.01),while the m RNA expression levels of NF-κB and NLRP3 were decreased(P<0.05).(4)Propionate and butyrate in feces were positively correlated with AMPK expression(P<0.05)and significantly negatively correlated with NF-κB and NLRP3 expression(P<0.01),while acetate in lung tissue was positively correlated with NF-κB and NLRP3 and negatively correlated with AMPK expression.In addition,AMPK was positively correlated with the intestinal barrier proteins ZO-1 and Ocludin(P<0.05)and negatively correlated with pathological injury scores and systemic inflammatory indicators in pancreatic,intestinal and lung tissues(P<0.05),while NF-κB and NLRP3 showed the opposite trend.Conclusions:(1)A mouse model of SAP-ALI can be successfully constructed using caerulein combined with LPS intraperitoneal injection.(2)Gut microbiota affects the severity of SAP-ALI and intestinal barrier function.(3)The abundance and diversity of intestinal bacteria were altered during SAP,mainly by an increase in the relative abundance of pathogenic bacteria such as Escherichia,Enterococcus,Enterobacter,and a decrease in the relative abundance of SCFAsproducing bacteria such as Bacteroides,Roseburia,Parabacteroides.QYD treatment reversed the trend and increased the capacity for fatty acid synthesis.(4)The highest concentrations of SCFAs were found in the stool of all groups,followed by intestinal,plasma,and lung tissue.The composition of SCFAs in the QYD-treated and SO groups was similar,with acetate and butyrate being the most representative SCFAs distinguishing the SAP and QYD+SAP groups,respectively.(5)The AMPK/NF-κB/NLRP3 signalling pathway mediated by SCFAs along the gutlungs axis may play a crucial role in the mechanism of which QYD treats SAP-ALI by modulating gut microbiota.