Effect And Mechanism Of Clostridium Butyricum And Butyrate On Severe Acute Pancreatitis Complicated With Liver Injury

Background:Acute pancreatitis is the local inflammation of the pancreas caused by pancreatic enzyme activation,which can be divided into mild,moderate and severe,among which severe AP is also called severe acute pancreatitis.SAP develops from initial local inflammation to release a large number of inflammatory mediators into the circulatory system,leading to Multiple organ dysfunction syndrome(MODS)and Systemic inflammatory response syndrome(SIRS).During SIRS and MODS,the release of a large number of inflammatory mediators throughout the body may lead to intestinal barrier dysfunction and the transfer of a large number of pathogenic bacteria and Endotoxin(ET)in the intestines,resulting in sepsis and aggravating SIRS and MODS.At the same time,liver is the earliest and most prone organ in SAP secondary multiple organ injury,with a high probability of 88.9%.The release of a large amount of inflammatory mediators throughout the body is the main factor causing liver damage.Secondly,liver removal of ET and secondary attack of enterogenic infection can lead to liver function damage,which is mainly manifested as the increase of Aspartate Transaminase(AST),Alamine Aminotransferase(ALT)and Total bilirubin(TBil).The degree of liver injury can affect the prognosis of SAP and is the main cause of death in SAP patients with MODS.In recent years,studies found that High Mobility Group box 1 protein(HMGB1),as a non-histone protein in the nucleus,plays an important role in SAP and liver injury.During SAP,HMGB1 is released by monocytes/macrophages under the stimulus of inflammatory mediators such as Tumor necrosis factor alpha(TNF-α)or the main component of ET,Lipopolysaccharide(LPS).It also releases into the extracellular level through the necrotic cells of various tissues.The released HMGB1 binds to the toll-like receptor Toll(TLR4)receptor.Nuclear factor of kappa B(NF-κB)activation mediates the expression of inflammatory mediators such as Interleukin 6(IL-6)and TNF-α.Clinically,the treatment methods for SAP patients are mostly symptomatic treatment,such as early humoral resuscitation,maintenance of organ functions,blood purification treatment,use of preventive antibiotics,nutritional support.There are few therapeutic methods to reduce HMGB1 level and improve liver function.C.butytyricum is a common probiotic in the intestinal tract.It can decompose intestinal contents in the intestinal tract to produce various short-chain fatty acids.Some studies have suggested that butyric acid,its main product,can maintain the stability of intestinal barrier,inhibit the expression of inflammatory mediators,regulate intestinal microecology and intestinal immunity in intestinal inflammatory diseases.Through previous experiments,our team proved that clostridium caseae and its butyric acid could protect intestinal barrier function,reduce inflammatory mediators and endotoxin entering circulatory system through intestinal mucosa,and reduce systemic inflammatory response.But can clostridium caseate maintain liver injury by protecting intestinal mucosal barrier and reducing systemic inflammation? In this study,clofusium caseinate and butyric acid are pretreated in the rat model of SAP with liver injury to observe the effect of clofusium caseate and butyric acid on liver injury,and to explore whether the effect could be played by inhibiting the activation of HMGB1/TLR4/NF-κB pathway.1.The SAP and liver injury rat model is established to observe the effects of clostridium caseinate and its metabolite butyrate on systemic inflammatory response,liver injury,pancreatic injury,intestinal mucosa,capillary permeability and local inflammation of liver,pancreas and intestinal tissues.Model and Methods:1.Establishment of animal model: 40 SPF grade Sprague-Dawley rats,using random number table method,divided into 4 groups(n=10): normal control group(Control group,CON group),severe acute pancreatitis with liver injury Group(SAP group),severe acute pancreatitis with liver injury treated with Clostridium butyricum group(SAP+ C.butyricum group,SAP+CB group),severe acute pancreatitis with liver injury treated with sodium butyrate group(SAP+ Sodium butyrate group,SAP+SB group).Rats in 4 groups are intragastrically administered with normal saline,normal saline,Clostridium butyricum and butyric acid for 10 days,and on the 11 th day they are modeled with 4.5% taurocholic acid retrograde injection method.CON group is replaced by sham operation.2.Material selection: After 24 hours of modeling,the material is collected,and the intra-abdominal and ascites pressures are measured before the laparotomy,and the blood,pancreas,liver,and ileum tissues of the abdominal aorta of rats are taken.3.Detection indicators: check blood amylase,lipase content and pancreatic tissue pathology score to assess pancreatic injury;check blood AST,ALT,TBil content and liver tissue pathology score to assess liver injury;measure blood LPS content,ileal tissue pathology score Using Western Blot to detect the ileal tight junction protein expression to evaluate the intestinal mucosal injury,blood TNF-α,IL-6 to assess systemic inflammation.Check the blood HMGB1 content,use immunohistochemistry and Western Blot method to detect the expression of HMGB1,TLR4,NF-κB protein in the ileum and liver tissue,use RT-qPCR method to detect the expression of HMGB1 mRNA,TLR4 mRNA,NF-κB mRNA,and evaluate The expression of HMGB1/TLR4/NF-κB pathway in intestinal and liver tissues.Result:1.The content of blood amylase,lipase,AST,ALT,TBil,pancreatic pathology score and liver pathology score of SAP group are significantly higher than that of CON group(P<0.05),the pancreas and liver injury are obvious,and the modeling is successful.After treatment with Clostridium butyricum or sodium butyrate,the SAP+CB group and SAP+SB group are significantly reduced compared with the SAP group(P<0.05).There is no significant difference between the SAP+CB and SAP+SB groups(P>0.05).2.The blood LPS content and small intestinal pathology score of SAP group are significantly higher than that of CON group.After treatment with Clostridium butyricum or sodium butyrate,SAP+CB group and SAP+SB group are significantly reduced compared with SAP group(P<0.05).There is no significant difference between the SAP+CB and SAP+SB groups(P >0.05).The expression of tight junction proteins(ZO-1,Claudin-1,Occludin)in the SAP group is significantly reduced in the ileum compared with the CON group(P <0.05).The SAP+CB group and SAP+SB group are significantly higher than the SAP group(P<0.05).There is no significant difference between the SAP+CB and SAP+SB groups(P>0.05).3.The levels of TNF-α and IL-6 in the SAP group are significantly higher than those in the CON group(P<0.05).After treatment with Clostridium butyricum or sodium butyrate,the SAP+CB and SAP+SB groups are significantly reduced compared to the SAP group(P <0.05).There is no significant difference between the SAP+CB and SAP+SB groups(P>0.05).4.The content of blood HMGB1 in SAP group,the expression of HMGB1,TLR4,NF-κB protein and genes in ileum and liver tissue are significantly higher than those in control group(P<0.05).After treatment with Clostridium butyricum or sodium butyrate,SAP+CB group Compared with SAP group,SAP+SB group decreased significantly(P<0.05).There is no significant difference between the SAP+CB and SAP+SB groups(P>0.05).Conclusion1.When SAP is accompanied by liver injury,a large number of inflammatory mediators are released throughout the body,resulting in varying degrees of damage to organs such as the pancreas,liver and intestinal mucosa.The intervention of the probiotics closteum caseinate and its metabolite butyric acid can,to a certain extent,inhibit the release of inflammatory mediators,protect the intestinal mucosa,and alleviate the injury of organs such as the pancreas and liver.2.Clostridium caseinate and butyrate may play an anti-inflammatory role in the intestine and liver when SAP is associated with liver injury,possibly by inhibiting the activation of the HMGB1/TLR4/NF-κB pathway.