Mechanism Of Platelet Racl Regulating PI3K/AKT Pathway In DSS-induced Colitis

Background and aimsUlcerative colitis(UC)is an inflammatory bowel disease(IBD).The chronic relapsing intestinal inflammation of UC seriously affects the quality of life.Unfortunately,the incidence of UC in both developed and developing countries still undergoes sustained growth.Therefore,it is of great significance to further clarify the pathogenesis of UC.The degree of neutrophil aggregation in the lesion of UC patients is closely related to the severity of the disease.Besides,reducing the aggregation of neutrophils by inhibition of specific adhesion molecules or chemokines can ameliorate the intestinal mucosal injury.Platelets,one of important factors in inflammatory response,have showed its crucial role in the pathogenesis of UC in both animal and clinical study.In the mouse model of UC,depletion of platelets can significantly ameliorate colonic inflammation and reduced neutrophils infiltration in intestinal mucosa,indicating that platelets may play a role in aggregation of neutrophils.Nevertheless,the specific mechanism of platelet activation in UC is still poorly understood.Platelets are rich in chemokines such as CCL5 which can induce neutrophil aggregation.And the release of CCL5 is regulated by PI3K/AKT pathway through platelet a granules,the majority storage site of CCL5.Rac1 in platelets has been widely regarded as a multi-effect regulator in inflammation and tumor.It also plays an important role in platelet function.The purpose of this study was to elucidate the role of Racl and PIK/AKT signaling pathways in DSS-induced colitis.MethodsIn vivo animal experiment,30 C57BL6 mice were randomly assigned to 5 groups:normal control group,DSS group,DSS+Vehicle group,DSS+Wortmannin group and DSS+NSC23766 group.The mice in the normal control group were free to drink pure water,and the other four groups were free to drink 2%DSS solution for six consecutive days.The intervention drugs were given by intraperitoneal injection.The disease activity index(DAI)was evaluated daily and the mice were sacrificed on the 7th day.Before sacrifice,platelet was isolated from peripheral blood to detect the expression of P-AKT in platelets by Western blot.Colonic tissue was collected,colonic length was measured,colorimetric method was used to detect MPO activity,and ELISA method was used to detect CCL5 in plasma and CCL5,CXCL2,IL-6 in colon.In vitro platelet experiment,thrombin was used to activate platelets and Wortmannin and NSC23766 were used to intervene in advance to observe their effects on platelet P-AKT expression.Results1.There was a high expression of platelet P-AKT in DSS group and DSS+Vehicle group,and the DAI score,pathological score,colonic MPO activity,plasma CCL5,colonic CCL5,CXCL2,IL-6 were significantly higher than those in normal control group(P<0.05).The colon length of the mice in DSS group and DSS+Vehicle group was significantly shorter than that in normal control group(P<0.05).There was no significant difference in the above-mentioned indexes between DSS group and DSS+Vehicle group(P>0.05).2.There was a low expression of platelet P-AKT in DSS+NSC23766 group and DSS+Wortmannin group,and the DAI score,pathological score,colonic MPO activity,plasma CCL5,colonic CCL5,CXCL2,IL-6 were significantly lower than those in DSS group and DSS+Vehicle group(P<0.05).3.In vitro platelet experiments,there was a high expression of platelet P-AKT in the thrombin activating group.NSC23766 and Wortmannin could lower the expression of P-AKT in platelets.Conclusion1.The PI3K/Akt pathway in the platelets of DSS induced colitis mice is activated;2.Racl in platelets can regulate the release of CCL5 through PI3K/AKT pathway and regulate the severity of colitis.