Effect And Mechanism Of Tanshinone ⅡA On Intestinal Injury In Septic Mice Based On NETs

Objective: To explore the effect and mechanism of Tanshinone IIA(Tan IIA)on intestinal injury in septic mice based on neutrophil extracellular traps(NETs).Methods: Twenty-four healthy male C57BL/6 mice were randomly divided into Control group,lipopolysaccharide group(LPS group),LY294002 group(LPS+LY group)and Tanshinone ⅡA group(LPS+TanⅡA group),six mice in each group.Except for the Control group,which was injected with equal amount of phosphate buffer saline(PBS),the remaining groups were injected intraperitoneally with LPS10mg/kg to establish the sepsis model.In the LPS+LY group,LY294002 30mg/kg was intraperitoneally injected 30 min before modeling.In the LPS+TanⅡA group,TanⅡA 30mg/kg was injected intraperitoneally 30 min before modeling.In the remaining two groups,equal amount of PBS [containing 0.1% dimethyl sulfoxide(DMSO)] was injected intraperitoneally at the above time points.Specimens were collected 12 h after modeling.Hematoxylin-eosin(HE)staining was used to observe pathological changes in the small intestine and Chiu’s score was performed.The serum D-lactate(D-Lac),small intestine myeloperoxidase-DNA complex(MPO-DNA)and citrullinated histone H3(Cit H3)were detected by enzyme-linked immunosorbent assay(ELISA).Small intestine Claudin-1,phosphorylated PI3K(p-PI3K),phosphorylated Akt(p-Akt)protein expression were detected by western blot.Result:(1)The mice in the Control group were in good mental state and active in daily behaviors;the mice in the LPS group were depressed,curled up,shortness of breath,increased secretions in the corners of the eyes,and had significantly reduced daily behavioral activities;the overall state of mice in LPS+LY group and LPS+Tan IIA group was significantly improved,the secretion of the corner of the eye was reduced,and the number of daily activities was increased,and the respiratory rate tended to be normal.(2)In the Control group,the intestinal villi were closely arranged,the structure was complete,and there was no pathological change;in the LPS group,the intestinal villi were severely edematous and the integrity was destroyed,with massive inflammatory cell infiltration;in LPS+LY group and LPS+Tan IIA group,the pathological damage of small intestine was alleviated,the integrity of small intestinal villi was partially recoverd,and the infiltration of inflammatory cells was lessen.Compared with the Control group,the Chiu’s score of the LPS group increased(P<0.001);compared with LPS group,the Chiu’s score of LPS+LY group and LPS+Tan IIA group decreased(P<0.001).(3)Compared with the Control group,the serum D-Lac levels in the LPS group were increased(P<0.001);compared with the LPS group,the serum D-Lac levels in both LPS+LY and LPS+TanⅡA groups were decreased(P<0.001).(4)Compared with the Control group,the MPO-DNA and Cit H3 contents in small intestine of the LPS group were increased(P<0.001);compared with the LPS group,the MPO-DNA and Cit H3 contents in small intestine of LPS+LY group and LPS+Tan IIA group were decreased(P<0.001).(5)Compared with the Control group,Claudin-1 protein expression in small intestine was decreased in the LPS group(P<0.001);compared with the LPS group,Claudin-1 protein expression in small intestine was increased in both LPS+LY group and LPS+Tan IIA group(P<0.05 or P<0.001).(6)Compared with the Control group,the expression of p-PI3 K and p-Akt protein were increased in small intestine of the LPS group(P<0.001);compared with the LPS group,the expression of the p-PI3 K and p-Akt protein were decreased in both LPS+LY and LPS+TanⅡA group(P<0.01).Conclusion: Tan IIA may play a protective role in intestinal injury in septic mice by inhibiting PI3K/Akt signaling pathway,reducing NETs production,increasing tight junction protein expression,and reducing intestinal permeability.