| Objective: To investigate the mechanism of MyD88-NF-κBp65 signaling pathway in myocardial injury of sepsis and the effect of Gentiopicroside on myocardial injury in sepsis by building an animal model.Methods: 1.Grouping: Thirty-two male Wistar rats were randomly divided into sham operation group(n=8),sepsis group(n=8),gentiopicin group(n=8)and MyD88 inhibitor group(TJ-M2010-5)(n=8,hereinafter referred to as inhibitor group).2.Grouping treatment:The rats in the sham operation group were given intraperitoneal injection of the same amount of normal saline control variables 3 days before the operation.During the operation,only the intestinal tube was opened,the cecum was not ligation and perforation,and the abdomen was closed by intermittent suture.3 days before modeling,the rats in sepsis group were given intrapitoneal injection of the same amount of normal saline control variables,and the myocardial injury model of sepsis was established by cecal ligation and perforation(CLP).Gentiopicroside group rats were pretreated with intraperitoneal injection of Gentiopicroside(50mg/kg·d)3 days before modeling,and then the model was constructed by CLP method.The inhibitor group rats were pretreated by intrabitoneal injection of TJ-M2010-5(25mg/kg·d)3 days before modeling,and were modeled by CLP method.3.Observation and detection indicators: The general state and local changes in abdominal cavity of rats in each group were observed;The myocardial tissue changes stained by hematoxylin-eosin(HE)were observed under light microscope,and the pathological changes of myocardial tissue were scored.The content of troponin(c Tn T)and the levels of tumor necrosis factor(TNF)-α and interleukin-6were determined by enzyme-linked immunosorbent assay(ELISA).The expressions of MyD88 and NF-κBp65 proteins in myocardial tissue were detected by Immunocytochemistry(IHC),and semi-quantitative analysis of these proteins were performed.Results: 1.General status of rats and local changes in abdominal cavity during sampling:(1)General status: rats in the sham operation group had stable breathing,normal activity and formed stool;In the sepsis group,the rats suffered from depression,shortness of breath,poor reaction,erect hair,mutual aggregation,and almost no food or water.Gentiopicroside group was characterized by mental lethargy,slight shortness of breath,less activity,and decreased intake and intake of water.In the inhibitor group,the rats had slight shortness of breathing,erect hair,less activity,mutual gathering and decreased water consumption.(2)Local changes in the abdominal cavity during sampling: In the sham operation group,no obvious signs of bleeding and infection were observed when the abdominal cavity was opened.In the sepsis group,pus and blood exudate were found in the peritoneal cavity,with foul odor.The cecum showed signs of infection such as blackness and necrosis,and intestinal adhesion.In gentiopicroside group,pus and blood exudate were found in the abdominal cavity,cecum necrosis and other signs of infection were observed.In the inhibitor group,intestinal dilatation,purulent and bloody exudate were found in the opening of the abdominal cavity,and signs of necrotic infection appeared in the cecum.2.Pathological changes of myocardial tissue:(1)Sham operation group: myocardial cells were arranged neatly,no nuclear swelling,no obvious inflammatory cell infiltration.(2)Sepsis group: myocardial cells were disordered,the space between muscle fibers was widened,some of them were broken,the nuclei were aggregated,and inflammatory cells were infiltrated.(3)Gentiopicroside group: the myocardial fiber gap widened,the arrangement was still in line,a small amount of nuclear swelling,inflammatory cell infiltration can be observed.⑷ In the inhibitor group,the myocardial fiber void widened,a little nuclear swelling,vascular dilation and a little inflammatory cell infiltration were observed.Myocardial histopathological scores: Compared with sham operation,the pathological scores of sepsis group,Gentiopicroside group and inhibitor group were significantly higher(P < 0.05);The pathological scores of Gentiopicroside and inhibitor groups were significantly lower than those of sepsis group(P <0.05);There was no significant difference in myocardial histologic score between Gentiopicroside group and inhibitor group(P < 0.05).3.Serum c Tn T,TNF-α and IL-6 levels were detected by ELISA: serum c Tn T,TNF-α and IL-6 levels were detected,and the levels in sepsis group,Gentiopicroside group and inhibitor group were significantly higher than those in sham operation group,with statistical difference(P < 0.05).Gentiopicroside group and inhibitor group were lower than sepsis group,with statistical difference(P < 0.05).The cytokine TNF-αIL-6 in Gentiopicroside group was higher than that in inhibitor group(P <0.05),but there was no significant difference in c Tn T level between the two groups(P <0.05).4.Detection of MyD88 and NF-κBp65 levels in myocardial tissue by IHC: Detection of the expression levels of protein molecules MyD88 and NF-κBp65 in myocardial tissue,sepsis group,Gentiopicroside group and inhibitor group were significantly higher than those in sham operation group,with statistical difference(P < 0.05);Gentiopicroside group and inhibitor group were lower than sepsis group,with statistical difference(P < 0.05).The expression of Gentiopicroside group was higher than that of inhibitor group,and the difference was statistically significant(P < 0.05).Conclusion: 1.In this study,the myocardial injury model of sepsis was successfully established in rats by cecal ligation and perforation(CLP).2.The uncontrolled inflammatory response mediated by the high expression of inflammatory factors activated by MyD88-NF-κBp65 signaling pathway may be one of the mechanisms of myocardial injury in sepsis.3.Gentiopicroside may inhibit the activation of MyD88-NF-κBp65 signaling pathway,reduce the release of inflammatory factors,and play the role of anti-inflammatory and myocardial protection. |
