Intervention Effect And Mechanism Of Qingjiehuagong Decoction On The Inflammatory Response And Intestinal Mucosal Barrier Damage In Rats With Acute Pancreatitis

Objective:Acute pancreatitis(AP)is one of the common acute and critical diseases of the digestive system that requires hospitalization.The mortality rate of moderate severe and severe AP is still high.However,there is no specific drug for its treatment at present.Before now,the research group found that Qingjiehuagong decoction can reduce the inflammatory response and protect the intestinal mucosal barrier damage in AP patients,but the research on animal experiments and mechanism of intervention has not been carried out.Therefore,this study intends to verify the intervention effect of this prescription on the inflammatory response and intestinal mucosal barrier damage in the AP rat model,and explored the effect and mechanism of this prescription on inflammatory response and intestinal mucosal barrier injury in AP rats by regulating the TLR4/NF-κB pathway.Methods:(1)The pathogenesis and treatment of AP were discussed from the perspective of modern medicine and traditional Chinese medicine,and on this basis,the academic thought of the tutor Professor Chen Guozhong on the use of Qingjiehuagong decoction in the treatment of AP was expounded.(2)The TCMSP database was used to screen the active ingredients of Qingjiehuagong decoction and the target of Chinese medicine,and the OMIM,GeneCards and Disgenet databases were used to screen the disease targets of acute pancreatitis and intestinal mucosal barrier damage.By taking the intersection of the drug and the disease target,the intersection target of the traditional Chinese medicine and the intestinal mucosal barrier damage in acute pancreatitis can be obtained.The STRING data platform and Cytoscape software were used to construct the protein interaction network analysis of the intersecting target genes and screen the core targets,and the DAVID database was used for GO and KEGG enrichment analysis.(3)SD rat AP model was established by intraperitoneal injection of caerulein and lipopolysaccharide.The experimental animals were randomly divided into 4 groups:sham group,model group,western medicine group(intervention with ulinastatin)and Chinese medicine group(intervention with Qingjiehuagong decoction).Intervention was begun at 0h after the model completion,once every 6h,a total of 4 times.Serum,pancreas and ileum tissues were collected at 7h,13h and 19h after modeling.Serum amylase was detected by the kit,serum TNF-α,IL-1β and IL-10 levels were detected by enzymelinked immunosorbent assay(ELISA),and the pathological changes and pathological scores of pancreatic tissue were observed by HE staining.(4)Animal grouping,modeling and sampling were the same as in part(3),serum D-lactic acid and endotoxin levels were detected by ELISA,ileum tissue pathological changes and pathological scores were observed by HE staining,and TLR4,p-NF-κB and Occludin in ileum tissue were detected by immunohistochemistry.The expressions of TLR4,p-NF-κB and Occludin in ileum tissue were also detected by Western Blot.Results:1.The study of Qingjiehuagong decoction in the treatment of intestinal mucosal barrier damage in acute pancreatitis based on network pharmacology(1)Qingjiehuagong decoction screened 255 active ingredients,involving 301 potential targets.8388 AP disease targets and 2905 intestinal mucosal barrier damage disease targets.There are 209 intersection targets directly acting on drugs and diseases.(2)The network analysis of traditional Chinese medicine-effective pharmaceutical active ingredients-target of action,obtained 10 main effective pharmaceutical active ingredients such as quercetin,luteolin and kaempferol.11 core targets including transcription factor p65(RELA),interleukin 6(IL-6)and signal transducer and activator of transcription 3(STAT3)were selected for the interaction analysis of the intersection target proteins.(3)The top 15 GO enrichment analysis involves biological processes such as inflammation.The KEGG pathway enrichment analysis found that 97 pathways were involved after screening.The signaling pathways involved in inflammation include:Toll-like receptor(TLR)signaling pathway,tumor necrosis factor(TNF)signaling pathway,mitogen-activated protein kinase(MAPK)signaling pathway,interleukin 17(IL-17)signaling pathway,phosphatidylinositol 3-kinase-protein kinase B(PI3K-Akt)signaling pathway,NOD-like receptor There are 7 signaling pathways in total,including the tyrosine protein kinase JAK-signal transducer and activator of transcription(JAK-STAT)signaling pathway.2.Effects of Qingjiehuagong decoction on pancreatic pathological damage and inflammatory cytokine expression in rats with acute pancreatitis(1)Serum amylase content:in 7h,13h and 19h,compared with the sham group,the model group was significantly increased(P<0.01);in 7h,compared with the model group,the Chinese medicine group decreased significantly(P<0.01);in 13h and 19h,compared with the model group,the western medicine group and the Chinese medicine group decreased significantly(P<0.01);in 19h,compared with the western medicine group,the Chinese medicine group had a greater decrease(P<0.05).(2)In terms of inflammatory cytokine expression:Serum TNF-α:in 7h,13h and 19h,compared with the sham group,the model group was significantly increased(P<0.01);in 7h,compared with the model group,the Chinese medicine group decreased significantly(P<0.01);in 13h and 19h,compared with the model group,the western medicine group and the Chinese medicine group all decreased significantly(P<0.01),and compared with the western medicine group,the Chinese medicine group decreased more significantly(13h:P<0.05;19h:P<0.01).Serum IL-10:in 7h,13h and 19h,compared with the sham group,the model group was significantly increased(P<0.01);in 7h,13h and 19h,compared with the model group,the western medicine group and the Chinese medicine group both increased(P<0.01);in 19h,compared with the western medicine group,the Chinese medicine group increased more significantly(P<0.05).Serum IL-1β:in 7h,13h and 19h,compared with the sham group,the model group was significantly increased(P<0.01);in 7h,13h and 19h,compared with the model group,the western medicine group and the Chinese medicine group were significantly decreased(7h:P<0.05;13h and 19h:P<0.01);and in 19h,compared with the western medicine group,the Chinese medicine group decreased more significantly(P<0.05).(3)Histopathological changes of pancreas:in 7h,13h and 19h,compared with the sham group,the pancreas pathological scores of the model group were significantly increased(P<0.01);in 13h,compared with the model group,the Chinese medicine group decreased significantly(P<0.05);in 19h,compared with the model group,the western medicine group and the Chinese medicine group were significantly decreased(P<0.01),and compared with the western medicine group,the Chinese medicine group decreased more significantly(P<0.05)3.Research on the effect and mechanism of Qingjiehuagong decoction on the intestinal mucosal barrier damage in rats with acute pancreatitis based on TLR4/NF-κB pathway(1)Serum D-lactic acid and endotoxin expression:serum D-lactic acid:in 7h,13h and 19h,compared with the sham group,the model group was significantly increased(P<0.01);in 7h,13h and 19h,compared with the model group,the western medicine group and the Chinese medicine group both decreased in different degrees(P<0.01),and in 13h and 19h,compared with the western medicine group,the Chinese medicine group significantly decreased more(13h:P<0.05;19h:P<0.01).Serum endotoxin:in 7h,13h and 19h,compared with the sham group,the model group was significantly increased(P<0.01);in 7h,13h and 19h,compared with the model group,the western medicine group and the Chinese medicine group decreased in different degrees(P<0.01);and in 13h and 19h,compared with the western medicine group,the Chinese medicine group significantly decreased more(13h:P<0.05;19h:P<0.01).(2)Histopathological changes of the ileum:in 7h,13h and 19h,compared with the sham group,the ileum histopathological scores of the model group were significantly increased(P<0.01);in 7h,there was no diference among the scores of the model group,the western medicine group and the Chinese medicine group(western medicine group vs model group:P=0.804;Chinese medicine group vs model group:P=0.622;western medicine group vs Chinese medicine group:P=0.804);in 13h,compared with the model group,the Chinese medicine group decreased slightly,but the difference was not statistically significant(P=0.054);in the 19h,compared with the model group,the scores of the western medicine group and the Chinese medicine group were all decreased,but there was no significant difference between the western medicine group and the Chinese medicine group(western medicine group vs model group:P=0.107;Chinese medicine group vs model group:P<0.05),while compared with the western medicine group,the Chinese medicine group had a greater decrease,but the difference was not statistically significant(P=0.231)(3)Immunohistochemical detection of protein expression in ileum mucosa:for TLR4:in 7h,13h and 19h,compared with the sham group,the mean optical density(MD)of TLR4 in the ileum mucosa of rats in the model group was significantly increased(P<0.01);in 7h,compared with the model group,the Chinese medicine group decreased significantly(P<0.05);in 13h and 19h,compared with the model group,the western medicine group and the Chinese medicine group were significantly decreased(P<0.01);in 19h,compared with the western medicine group,the Chinese medicine group decreased more significantly(P<0.05).For p-NF-κB:in 7h,13h and 19h,compared with the sham group,the MD value of p-NF-κB in the ileum mucosa of rats in the model group was significantly increased(P<0.01);in 7h,compared with the model group,the MD values of the western medicine group and the Chinese medicine group were significantly decreased(P<0.01),and compared with the western medicine group,the Chinese medicine group had a greater decrease,but the difference was not statistically significant(P=0.089).In 13h and 19h,compared with the model group,the MD values of the western medicine group and the Chinese medicine group were significantly decreased(P<0.01).For Occludin:in 7h,13h and 19h,compared with the sham model,the MD value of Occludin in the ileum mucosa of rats in the model group decreased significantly(7h:P<0.05,13h and 19h:P<0.01);in 13h,compared with the model group,the western medicine group and the Chinese medicine group were slightly higher,but the difference was not statistically significant(western medicine group:P=0.231;Chinese medicine group:P=0.066);in 19h,compared with the model group,the western medicine group and the Chinese medicine group were significantly increased(P<0.01).(4)Western Blot detection of ileum tissue protein expression:for TLR4:in 7h,13h and 19h,compared with the sham group,the relative gray value of the model group was significantly increased(P<0.01);in 13h and 19h,compared with the model group,the western medicine group and the Chinese medicine group both decreased significantly(P<0.01),and in 19h,compared with the western medicine group,the Chinese medicine group decreased more significantly(P<0.01).For p-NF-κB:in 7h,13h and 19h,compared with the sham group,the relative gray value of the model group was significantly increased(P<0.01);in 13h and 19h,compared with the model group,the relative gray value of the western medicine group and the Chinese medicine group were significantly decreased(P<0.01).For Occludin:in 7h,13h and 19h,compared with the sham group,the relative gray value of the model group decreased significantly(P<0.01);in 13h and 19h,compared with the model group,the western medicine group and the Chinese medicine group were significantly increased(western medicine group vs model group in 19h:P<0.05;the rest:P<0.01),and in the 19h,compared with the western medicine group,the increase in the Chinese medicine group was more significant(P<0.05)Conclusion(s):(1)The intervention effect of Qingjiehuagong decoction on AP intestinal mucosal barrier injury may be closely related to reducing the inflammatory response of AP by regulating the TLR4/NF-κB pathway.(2)Qingjiehuagong Decoction can alleviate the pathological damage of the pancreas of AP rats and reduce the level of serum amylase,and has a protective effect on the pancreas of AP rats.(3)Qingjiehuagong Decoction can inhibit the expression of serum inflammatory cytokines in AP rats,and can up-regulate the expression of antiinflammatory cytokines,which can significantly reduce the infiltration and damage of inflammatory cells in the pancreas and ileum of AP rats.The inflammatory response in rats was significantly reduced.(4)Qingjiehuagong Decoction can reduce the pathological damage of ileum mucosa in AP rats,and can down-regulate the levels of serum D-lactic acid and endotoxin,which can protect the damage of intestinal mucosal barrier in AP rats.(5)Qingjiehuagong Decoction can reduce the inflammatory response and protect the intestinal mucosal barrier damage in AP rats,which may be achieved by inhibiting the activation of TLR4/NF-κB pathway.