少腹逐瘀汤调控PTEN/Akt/mTOR信号通路减轻子宫内膜异位症纤维化

Shaofu Zhuyu Decoction attenuates fibrosis in endometriosis through regulating PTEN/Akt/mTOR signaling pathway

探讨少腹逐瘀汤对子宫内膜异位症纤维化模型小鼠的保护作用,并从第10号染色体缺失的磷酸酶和张力蛋白同源物(phosphatase and tensin homolog deleted on chromosome ten, PTEN)/蛋白激酶B(protein kinase B,Akt)/哺乳动物雷帕霉素靶蛋白(mammalian target of rapamycin, mTOR)信号通路探讨其作用机制。将85只BALB/c雌性小鼠随机分为空白组、模型组、孕三烯酮(YT)组以及少腹逐瘀汤高(SFZY-H)、中(SFZY-M)、低(SFZY-L)剂量组,采用异体移植腹腔注射法诱导小鼠子宫内膜异位症模型,造模后第14天起,SFZY-H、SFZY-M、SFZY-L和YT组分别给予高、中、低剂量的少腹逐瘀汤和孕三烯酮混悬液灌胃,空白组和模型组给予等容积的蒸馏水灌胃,共给药14 d。比较各组小鼠不同时间段的体质量及热痛潜伏期、剥离的异位灶总质量,采用苏木精-伊红(hematoxylin-eosin, HE)染色、马松三色(Masson trichrome, Masson)染色对异位灶组织进行病理评价。采用实时荧光定量PCR法测定异位灶组织中α-平滑肌肌动蛋白(α-smooth muscle actin,α-SMA)、Ⅰ型胶原蛋白(collagen typeⅠ,collagen-Ⅰ)mRNA的表达,蛋白免疫印迹法测定异位灶组织中PTEN、Akt、mTOR、p-Akt、p-mTOR等蛋白的表达水平。结果显示,与空白组相比,模型组小鼠体质量呈先下降后增加趋势,异位灶总质量增加,热痛潜伏期明显缩短;与模型组相比,治疗后小鼠体质量增加缓慢,热痛潜伏期延长,异位灶总质量减轻,差异具有统计学意义。各组药物干预后异位灶HE染色病理损害明显改善,Masson染色胶原沉积面积较模型组减少,其中以SFZY-H、YT组改善显著(P<0.01)。与空白组相比,模型组异位灶α-SMA、collagen-ⅠmRNA表达量增加;药物干预后表达量降低,以SFZY-H、YT组改善显著(P<0.05,P<0.01)。与空白组相比,模型组中PTEN蛋白表达降低,Akt、mTOR、p-Akt、p-mTOR等蛋白的表达水平显著升高(P<0.01,P<0.001);与模型组相比,各给药组PTEN蛋白表达增加,而Akt、mTOR、p-Akt、p-mTOR等蛋白的表达水平下降,以SFZY-H、YT组改善显著(P<0.01)。综上,少腹逐瘀汤干预可显著减轻子宫内膜异位症模型小鼠病灶纤维化,其作用机制可能与调控PTEN/Akt/mTOR信号通路有关。

The present study aimed to investigate the protective role of Shaofu Zhuyu Decoction(SFZY)against endometriosis fibrosis in mice,and decipher the underlying mechanism through the phosphatase and tensin homolog deleted on chromosome ten(PTEN)/protein kinase B(Akt)/mammalian target of rapamycin(mTOR)pathway.Eighty-five BALB/c female mice were randomly assigned into a blank group,a model group,high-,medium,and low-dose SFZY(SFZY-H,SFZY-M,and SFZY-L,respectively)groups,and a gestrinone suspension(YT)group.The model of endometriosis was induced by intraperitoneal injection of uterine fragments.The mice in different groups were administrated with corresponding groups by gavage 14 days after modeling,and the blank group and model group with equal volume of disilled water by gavage.The treatment lasted for 14 days.The body weight,paw withdrawal latency caused by heat stimuli,and total weight of dissected ectopic focus were compared between different groups.The pathological changes of the ectopic tissue were observed via hematoxylin-eosin(HE)and Masson staining.Real-time PCR was employed to measure the mRNA levels ofα-smooth muscle actin(α-SMA)and collagen type I(collagen-I)in the ectopic tissue.The protein levels of PTEN,Akt,mTOR,p-Akt,and p-mTOR in the ectopic tissue were determined by Western blot.Compared with the blank group,the modeling first decreased and then increased the body weight of mice,increased the total weight of ectopic focus,and shortened the paw withdrawal latency.Compared with the model group,SFZY and YT increased the body weight,prolonged the paw withdrawal latency,and decreased the weight of ectopic focus.Furthermore,the drug administration,especially SFZY-H and YT(P<0.O1),recovered the pathological and reduced the area of collagen deposition.Compared with the blank group,the modeling upregulated the mRNA levels ofα-SMA and collagen-I in the ectopic focus,and such up-regulation was attenuated after drug intervention,especially in the SFZY-H and YT groups(P<0.05,P<0.01).Compared with the blank group,the modeling downregulated the protein level of PTEN and up-regulated the protein levels of Akt,mTOR,p-Akt,and p-mTOR(P<0.01,P<0.001).Drug administration,especially SFZY-H and YT,restored such changes(P<0.01).SFZY may significantly attenuate the focal fibrosis in the mouse model of endometriosis by regulating the PTEN/Akt/mTOR signaling pathway.