基于PI3K/Akt/eNOS信号通路探究孙氏助孕方治疗肾虚血瘀薄型子宫内膜大鼠的作用机制

Investigation of the Mechanism of Action of Sun’s Pregnancy-assisting Formula for the Treatment of Kidney Deficiency and Blood Stasis in Thin Endometrial Rats Based on the PI3K/Akt/eNOS Signaling Pathway

目的:观察孙氏助孕方治基于磷酸肌醇-3-激酶(PI3K)/蛋白激酶B(Akt)/内皮一氧化氮合成酶(eNOS)信号通路的研究机制。方法:选取广州市固堡生物科技有限公司提供的40只3个月月龄斯泼累格•多雷(SD)农场雌性大鼠进行研究,将所有大鼠按随机数表法分为正常组、模型组、对照组和观察组,每组各10只。观察组大鼠每天饮食自由摄入,其他组大鼠均予以造模,模型组未应用药物;对照组给予戊酸雌二醇疗法;观察组给予孙氏助孕方治疗。治疗后采集两组大鼠子宫内膜组织后,使用实时荧光定量逆转录-多聚酶链反应(RT-PCR)检测PI3K、Akt、eNOS的mRNA基因表达水平及蛋白表达情况。对比两组大鼠子宫内膜厚度以及PI3K、Akt以及eNOS的mRNA基因表达水平及蛋白图像,分析孙氏助孕方对肾虚血瘀薄型子宫内膜的PI3K/Akt/eNOS信号通路的影响。结果:与对照组比较,观察组大鼠的子宫内膜厚度差异不明显,差异无统计学意义(t=0.74,P>0.05);与模型组比较,观察组的大鼠子宫内膜厚度增高明显,差异有统计学意义(t=22.61,P<0.05);与正常组比较,观察组大鼠子宫内膜厚度稍低,差异无统计学意义(t=0.43,P>0.05);观察组大鼠的P13K、Akt、eNOS的mRNA基因表达水平、蛋白表达显著高于空白组,差异有统计学意义(t=10.10、21.71、18.13,P<0.05);而观察组大鼠的P13K、Akt、eNOS的mRNA基因表达水平、蛋白表达与对照组相比,差异无统计学意义(t=0.38、0.52、0.51,P>0.05);与对照组相比,模型组大鼠P13K、Akt、eNOS的mRNA基因表达、蛋白表达水平明显下降,差异有统计学意义(t=10.56,29.36,30.56,P<0.05)。结论:中药经验方“孙氏助孕方”可从血管生成的信号通路方面改善子宫内膜厚度的作用机制。

Objective:To observe the mechanism of phosphatidylinositol-3-kinase(PI3K)/protein kinase B(Akt)/endothelial nitric oxide synthase(eNOS)signaling pathway-based study by applying Sun’s pregnancy-assisting formula to kidney-deficient and blood-stasis-thin endometrial rats.Methods:40 female rats of 3-month-old Sprague-Dorey(SD)farms provided by Guangzhou Solid Castle Biotechnology Co.,Ltd.were selected for the study,and all the rats were divided into normal group,model group,controlgroup and observation group according to the randomized number table method,with 10 rats in each group.The rats in the blank group were fed and watered freely every day,the rats in other groups were modeled,no drugs were applied to the model group,the control group was given estradiol valerate therapy,and the observation group was given Sun’s formula for assisting pregnancy.The mRNA gene expression levels and protein expression of PI3K,Akt,and eNOS were detected using real-time fluorescence quantitative reverse transcription-polymerase chain reaction(RT-PCR)after collection of endometrial tissues from rats in both groups after treatment.The endometrial thickness as well as the mRNA gene expression levels and protein images of PI3K,Akt,and eNOS were compared between the two groups of rats to analyze the effects of Sun’s pregnancy-assisting formula on the PI3K/Akt/eNOS signaling pathway in the kidney-deficient-blood-stasis-thin endometrium.Results:Compared with the control group,the endometrial thickness of the rats in the observation group did not differ significantly and the difference was not statistically significant(t=0.74,P>0.05).Compared with the model group,the endometrial thickness of rats in the observation group increased significantly and the difference was statistically significant(t=22.61,P<0.05).Compared with the normal group,the endometrial thickness of rats in the observation group was slightly lower,and the difference was not statistically significant(t=0.43,P>0.05).The P13K,Akt,eNOS mRNA gene expression levels and protein expression of rats in the observation group were significantly higher compared with those in the blank group,and the differences were statistically significant(t=10.10,21.71,18.13,P<0.05).There was no statistically significant difference in P13K,Akt,eNOS mRNA gene expression levels,protein expression in the observation group of rats compared with the control group(t=0.38,0.52,0.51,P>0.05).Compared with the control group,the levels of P13K,Akt,and eNOS mRNA gene expression and protein expression of rats in the model group decreased significantly,and the differences were statistically significant(t=10.56,29.36,30.56,P<0.05).Conclusion:The Chinese herbal formula“Sun’s formula for assisting pregnancy”can improve the mechanism of endometrial thickness in terms of angiogenic signaling pathways.