基于“治未病”理论探讨穴位注射对卵巢储备功能减退大鼠PI3K、AKT、FOXO3a表达的影响

Discussion on the effect of acupoint injection on the expression of PI3K, AKT, FOXO3a in rats with decreased ovarian reserve function based on the preventive treatment theory

目的:基于中医“治未病”理论探讨穴位注射胎盘多肽早期预防对卵巢储备功能减退(DOR)模型大鼠的保护作用和可能机制。方法:将有正常动情周期的SPF级12周龄雌性大鼠随机分为空白组、模型组、生肌组、生穴组、肽肌组、肽穴组,每组8只。除空白组外,其余5组以雷公藤多苷混悬液造模,造模同时,行相应干预,共2周。干预结束后,检测各组大鼠体质量、卵巢指数、卵巢组织的病理变化,ELISA检测血清中血清卵泡刺激素(FSH)、雌二醇(E2)、抑制素B(INHB)水平,免疫组化检测磷脂酰肌醇3-激酶(PI3K)、蛋白激酶B(AKT)、叉头状转录因子O亚族3(FOXO3a)蛋白的表达。结果:与空白组比较,模型组大鼠血清中E2、INHB水平降低、FSH水平显著升高(P<0.01);卵巢中各级卵泡数量减少,闭锁卵泡不同程度增多,PI3K、AKT、FOXO3a蛋白含量显著升高(P<0.01)。与模型组比较,肽穴组血清中E2、INHB水平显著升高,FSH水平显著降低(P<0.01,P<0.05),卵巢中成熟卵泡增加,黄体数量增多,PI3K、AKT、FOXO3a蛋白含量显著降低(P<0.01);生穴组、肽肌组血清中E2水平显著升高(P<0.05),AKT蛋白含量显著降低(P<0.05)。结论:穴位注射胎盘多肽注射液可以早期预防大鼠卵巢储备功能减退,其作用机制可能与调节PI3K、AKT、FOXO3a蛋白的表达,改善血清FSH、E2、INHB水平有关。

Objective:To explore the protective effect and possible mechanism of early intervention of placental polypeptide acupoint injection on rats with diminished ovarian reserve(DOR)based on the preventive treatment theory.Methods:The 12-week-old female rats of SPF grade with normal estrous cycle were randomly divided into blank control group,model control group,normal saline intramuscular injection group,normal saline acupoint injection group,placental polypeptide muscle injection group,and placental polypeptide acupoint injection group,8 rats in each group.Except for the blank group,the other five groups were intragastrically treated with Tripterygium Wilfordii Polyglycosides suspension to establish the DOR rat model and intervened for 2 weeks.After the intervention,the body mass,ovarian index and pathological changes of ovarian tissue were detected,the levels of FSH,E_(2),and INHB in serum were detected by ELISA;and the expression of PI3K-AKT and FOXO3a protein was detected by immunohistochemistry method.Results:Compared with the blank group,the contents of E2 and INHB in the serum of the model group were significantly decreased,while the content of FSH was significantly increased(P<0.01),the number of follicles at all levels in the ovary was significantly decreased,the atretic follicles increased in varying degrees,and the protein contents of PI3K,AKT and FOXO3a were significantly increased in the model group(P<0.01).Compared with the model group,placental polypeptide acupoint injection group could significantly increase the contents of E_(2) and INHB and decrease the content of FSH in serum(P<0.01,P<0.05),increase the number of mature follicles and corpus luteum and decrease the contents of PI3K,AKT and FOXO3a protein in ovary(P<0.01).The level of serum E_(2) increased and the content of AKT protein decreased in normal saline acupoint injection group and placental polypeptide intramuscular injection group(P<0.05).Conclusion:Acupoint injection of placental polypeptide can prevent the further decline of ovarian reserve function in rats in early stage,and its mechanism may be related to regulating the expression of PI3K,AKT and FOXO3a protein and improving the levels of serum FSH,E_(2) and INHB.