Protective Effect Of Tetrastigma Hemsleyanum Diels Et Gilg Or Intestinal Mucosal Injury Induced By Sepsis And Its Effect On TLR4/MyD88/NF-κB Signaling Pathway

Objective Sepsis is a clinical syndrome defined by a systemic response to infection that,over the course of the disease,causes excessive systemic inflammation and leads to multiple organ dysfunction syndrome(MODS).The gut plays a key role in the development and progression of sepsis.Therefore,from the perspective of anti-inflammatory and intestinal mucosal barrier protection,this study explored the effect of Tetrastigma hemsleyanum Diels et Gilg(SYQ)on sepsis intestinal mucosal injury through in vivo experiments and verified the predicted targets and mechanisms of network pharmacology.It provides new ideas for more effective clinical treatment of sepsis.Methods 1.First,search the Chinese National Knowledge Infrastructure(CNKI)and PubMed databases to collect the active ingredients and targets of the cloverleaf,and then search the TCMSP database to find oral bioavailability(OB)≥30%,drug likeness(DL)≥0.18,collect the active ingredients and summarize and integrate the action targets of each ingredient.Sepsis disease targets were collected from the Gene Cards and OMIM databases,and a cloverleafcompound-target-sepsis network was constructed;The STRING database was used to construct a protein interaction network,and the DAVID database was used for GO functi on and KEGG pathway enrichment to analyze and predict the target and mechanism of cloverleaf in the treatment of sepsis.2.Then 72 SPF grade ICR male mice were randomly divided into normal group,model group,SYQ high,medium,and low dose groups(SYQ-G,Z,D),dexamethasone group(DSMS),and Bificon group(PFK).The treatment group was continuously administered for 7 days before modeling,and the normal group and the model group were given equal volumes of distilled water.After the last intragastric administration for 1 h,LPS(10 mg/kg)was injected intraperitoneally to establish a mouse model of intestinal mucosal injury in sepsis,and the materials were collected after 3 hours.The change of IL-6,TNF-α,IL-1β,IL-10,CRP,PCT,and iFABP in blood and colon tissues of septic mice were detected by enzyme-linked immunosorbent assay(ELISA).A lactate dehydrogenase(LDH)kit was used to determine intestinal LDH levels;The inflammatory related proteins of IL-6,TNF-α,and IL-1β in intestinal tissue were detected by WB.Hematoxylin-eosin staining(HE)was used to compare the histopathological changes of ileum and cecum mucosa of mice in each group and to evaluate the intestinal injury degree.The above methods were used to explore the effect of SYQ on the degree of intestinal inflammation and injury in sepsis.3.In order to evaluate the effect of SYQ on intestinal mucosal barrier function in sepsis,the tight junctions of intestinal mucosal epithelial cells were observed by transmission electron microscopy;The changes in β-GD content were determined by β-glucuronidase(β-GD)kit method;The expression of tight junction Claudin-5 and ZO-1 was detected by protein immunofluorescence method;the expression of tight junction protein(Occludin)in intestinal tissue was detected by WB;The mRNA expression of Occludin,Claudin-5 and ZO-1 was detected by the qRT-PCR method.The expression of apoptotic proteins Bax/Bcl2 and Cleaved caspase3 in intestinal epithelial cells were observed by immunohistochemistry or WB assay;The apoptosis of intestinal epithelial cells was detected by TUNEL assay;4.Finally,WB was used to detect the changes in the expression of TLR4/MyD88/NF-κB signaling pathway in intestinal tissue,and qRT-PCR was used to determine the mRNA expression of MLCK and MLC,to further explore the mechanism of cloverleaf in intervening sepsis.Results 1.According to the results of network pharmacology analysis,10 potentially active components in SYQ can act on sepsis,and 124 key targets are common between Trifolium repens and sepsis.It mainly involves key targets such as TNF,IL-6,IL-1β,IL-10,and Caspase3.GO functional enrichment analysis obtained a total of 837 items,mainly related to apoptosis,inflammation,etc.KEGG pathway enrichment analysis showed 120 signaling pathways,including PI3K-Akt,TLR,and NF-κB signaling pathways related to inflammation.2.Compared with the normal group,CRP,PCT,iFABP,and LDH in the colon tissue of the model group were significantly increased(P<0.05,0.01),and decreased after SYQ intervention(P<0.05,0.01),so as to play an anti-sepsis role;In the intestinal injury model of sepsis mice,SYQ could reduce the content and protein expression of IL-6,TNF-α and IL-1β in serum or colon tissue(P<0.05,0.01),and significantly increase IL-10(P<0.05,0.01),thereby reducing the inflammatory response of sepsis.Intestinal epithelial cell shedding,intestinal mucosal edema,inflammatory cell infiltration,and villus passivation occurred in the model group,and intestinal pathological damage was alleviated after SYQ intervention.3.SYQ reduces the destruction of tight junctions between intestinal mucosal cells,ultrastructural damage,and severe epithelial cell damage in the ileum tissue and reduces the content of β-GD in the colon tissue(P<0.05);Significantly increased the protein and mRNA expressions of tight junction proteins Occludin,Claudin-5 and ZO-1 in the ileum and colon tissues of septic mice(P<0.05,0.01),thereby repairing intestinal mucosal barrier damage;Compared with the normal group,the expressions of Bax/Bcl2 and Cleaved caspase3 in the model group were increased(P<0.05,0.01),and SYQ was decreased(P<0.05,0.01).In order to further verify the effect of SYQ on intestinal tissue apoptosis,compared with the model group,SYQ intervention reduced ileal tissue apoptosis and reduced intestinal tissue apoptosis.4.Compared with the normal group,the expression levels of TLR4,MyD88 and pNF-κB p65/NF-κB p65 protein in the colon tissue of mice in the model group were significantly increased(P<0.05,0.01),and the mRN A expression levels of MLCK and MLC were increased(P<0.01).After SYQ intervention,the expression levels were significantly decreased(P<0.05,0.01).It was found that SYQ could inhibit the activation of TLR4/MyD88/NF-κB signaling pathway,prevent the expression of MLCK and MLC,and reduce the damage of intestinal mucosa in sepsis.Conclusion This study confirmed that SYQ could effectively control the progression of sepsis by inhibiting the occurrence of inflammatory reactions,thereby further reducing the permeability of intestinal mucosa and maintaining the integrity of intestinal mucosal barrier function,thereby effectively alleviating the injury of intestinal mucosa in sepsis.The mechanism may be related to the inhibition of TLR4/MyD88/NF-κB signaling pathway activation.