Mechanism of Jiawei Shaofu Zhuyutang in treatment of endometriosis by regulating platelet activation via PI3K/Akt/GSK-3β signaling sathway

Objective To investigate the potential molecular mechanism of Jiawei Shaofu Zhuyutang (JWSFZYT) in the treatment of endometriosis through regulating platelet activation via the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt)/glycogen synthase kinase 3β (GSK-3β) signaling pathway.Methods Seventy- two female SD rats were randomly divided into a sham group（n=12）and a modeling group（n=60）.The rats in the sham group underwent exploratory laparotomy,while the rats in the modeling group were used to establish an endometriosis model through the autotransplantation method. Sixty rats with successful modeling were randomly divided into the model group,JWSFZYT-H group, JWSFZYT-M group,JWSFZYT-L group (30, 15, 7.5g/kg) and Dienogest group (0.18mg/kg) , with 12 rats in each group. The therapeutic effect was evaluated based on the number of writhing episodes in rats and the volume of heterotopic lesion tissues. Hematoxylin and eosin (HE) staining was used to compare pathological changes,flow cytometry was used to detect the expression levels of P-selectin (CD62p) and lysosomal membrane glycoprotein 3 (CD63). Immunofluorescence(IF) was used to detect the relative fluorescence intensity of integrin αIIb (CD41) in endometrial tissue. The protein expression levels of PI3K, phosphorylated phosphoinositide 3-kinase (p-PI3K), Akt, phosphorylated protein kinase B (p-Akt), GSK-3β, phosphorylated glycogen synthase kinase 3β (p-GSK-3β) and the mRNA expression levels of PI3K, Akt and GSK-3β in heterotopic lesion tissues were detected by Western blot and quantitative real-time polymerase chain reaction (Real-time PCR). Enzyme-linked immunosorbent assay (ELISA) was employed to quantify tumor necrosis factor-α (TNF-α),interleukin-6(IL-6),interleukin-1β(IL-1β),platelet-derived growth factor (PDGF) and vascular endothelial growth factor (VEGF) in serum.Results In contrast to the sham group, model rats displayed a characteristic writhing reaction, and heterotopic lesions tissues was observed in the abdominal wall, the HE result showed mesenchymal hyperplasia and glandular dilatation;the relative fluorescence intensity of CD41 was enhanced;the protein expression of PI3K, p-PI3K, Akt, p-Akt, GSK-3β and p-GSK-3β was increased;and mRNA expression of PI3K, Akt , GSK-3β was increased; the serum levels of IL-6,IL-1β , TNF-α ,Conclusion The mechanism of Jiawei Shaofu Zhuyutang in the treatment of endometriosis may be related to its inhibition of platelet activation mediated by the PI3K/Akt/GSK-3β pathway, reduction of inflammatory response and inhibition of angiogenesis.