Qingchang Mixture Targets TLR4/NF-κB Pathway In The Treatment Of Intestinal Ischemia-reperfusion Injury

Objectives:In this study,we established a rat model of intestinal ischemia/reperfusion(IR)to investigate the therapeutic effects of the active ingredients of intestinal cleansing compound on rats with intestinal ischemia/reperfusion injury,and explored the possible mechanisms of the therapeutic effects of Qingchang Mixture based on TLR4/NF-κB signaling pathway,aiming to provide theoretical studies for the clinical application of traditional Chinese medicine.Methods:A rat model of intestinal ischemia-reperfusion injury was established,and 50 SD rats were divided into control group(sham),model group(IR),and experimental group(QIR,IR+TAK,IR+PDTC).The sham group was opened and only the superior mesenteric artery(SMA)was isolated without clamping,and the remaining rats in each group were isolated and intermittently clamped The SMA was isolated and intermittently clamped in the remaining groups,and the ischemia-reperfusion model was manufactured.The modeling was successful when the intestinal color changed to pale and the pulsation was reduced.After successful modeling,intraperitoneal titration was performed,saline was given to the sham group,and TAK242,and PDTC were given to the experimental group,respectively.after reperfusion,cardiac arterial blood and some damaged small intestinal tissues were collected for testing.Pathological changes in the rat intestine were observed under the microscope using hematoxylin-eosin staining(HE).ELISA could be used to detect the expression levels of IL-6,TNF-α and IL-1β in serum,and biochemical analysis was performed to determine the serum malondialdehyde(MDA),the Biochemical analysis was used to determine the expression levels of Malondialdehyde(MDA),Superoxide dismutase(SOD)and Glutathione peroxidase(GSH-Px)in serum,and Western biot(WB)can be used to investigate the expression of TLR4,NF-κB,ZO-1,claudin-1 and ocludin in intestinal tissues.Results:In this study,we successfully established a rat model of intestinal ischemia-reperfusion and investigated the effect of Qingxin in rats with intestinal ischemia-reperfusion by clearing heat and activating blood.1.Results of intestinal histopathological sections of Intestinal ischemia-reperfused rats: The results of HE staining and Chiu scoring indicated that the structure and morphology of intestinal mucosa were basically normal in the sham group.the IR group had heavier intestinal mucosal damage,significantly higher intestinal wall tissue damage,and significantly higher intestinal wall tissue damage than the control group(P<0.05);compared with the IR group,the mice in the QIR group had less intestinal tissue damage,less intestinal tissue damage,and less intestinal tissue damage(P<0.05).2.Results of intestinal tissue biochemical analysis in Intestinal ischemia-reperfused rats: compared with Sham group,the results of IR group showed upregulation of MDA and downregulation of SOD and GSH-Px(P<0.05).Compared with the IR group,the QIR group after pretreatment with cleansing compound showed down-regulation of MDA,up-regulation of SOD and GSH-Px(P<0.05),and reduced oxidative stress status.3.Expression of IL-6,IL-1β and TNF-α in rat intestinal tissues: The contents of IL-6,IL-1β and TNF-α in the intestine of the rats in the IR group were significantly higher than those in the control group(P<0.05);while the differences between the QIR group and the IR group were statistically significant(P<0.05).4.Expression levels of claudin-1,Occludin,and ZO-1 in rat intestinal tissues: Compared with Sham group,the expression of claudin-1,ZO-1,ocludin in IR group was obviously lower than that in Sham group(P<0.05).These proteins were significantly higher in QIR group than in IR group(P<0.05).5.TLR4/NF-κB signaling pathway expression in rat intestinal tissues:The expression of TLR4 and NF-κB was significantly upregulated in the IR group compared with the Sham group,and downregulated in the QIR group compared with the IR group(P<0.05).To further demonstrate the role of TLR4/NF-κB signaling pathway in intestinal ischemia-reperfusion,rats were intervened using specific small molecule inhibitors of TLR4 and NF-κB(TAK242 and PDTC),and the results showed that the expression levels of TLR4 and NF-κB were decreased in the IR+TAK group and IR+PDTC group.In addition,Chiu score was decreased,expression of MDA,IL-6,IL-1βand TNF-α was down-regulated,and expression of SOD,GSH-Px,ZO-1,claudin-1,occludin protein was significantly increased compared with the IR group(P<0.05),but the effects were not significantly different from those of the QIR group.Conclusion:Qingchang mixture has the effect of clearing away heat and activating blood circulation,and can improve the intestinal mucosal injury in rats with ischemia and reperfusion.The mechanism may be that TLR4/NF-KB signaling pathway is inhibited,TNF-α,IL-6 and IL-1Β are decreased,ZO-1,Occludin and Claudin-1 are up-regulated,and intestinal inflammation and intestinal barrier damage are improved.