“秩边透水道”针法对早发性卵巢功能不全大鼠肿瘤坏死因子相关凋亡诱导配体及其受体表达的影响

Effect of penetrative needling of“Zhibian”(BL54)through“Shuidao”(ST28)on expression of TRAIL and its receptors in rats with premature ovarian insufficiency

目的:通过观察“秩边透水道”针法对早发性卵巢功能不全(POI)大鼠肿瘤坏死因子相关凋亡诱导配体(TRAIL)及其受体死亡受体4(DR4)、死亡受体5(DR5)、诱骗受体1(DcR1)、诱骗受体2(DcR2)、死亡受体结构域(FADD)表达的影响,探讨“秩边透水道”针法治疗POI的可能机制。方法:雌性SD大鼠随机分为空白组、模型组、戊酸雌二醇组、秩边透水道组,每组10只。除空白组外,其余大鼠均以腹腔注射环磷酰胺的方式复制POI大鼠模型。造模成功后,秩边透水道组针刺大鼠双侧“秩边”并向同侧“水道”方向透刺,深约12 mm,留针30 min,每日1次,共4周;戊酸雌二醇组给予戊酸雌二醇(0.09 mg/kg)灌胃,每日1次,共4周。干预结束后,用ELISA法检测大鼠血清中卵泡刺激素(FSH)、黄体生成素(LH)、雌二醇(E2)、血管内皮生长因子(VEGF)含量;计算大鼠卵巢系数并用HE染色法观察卵巢组织病理形态及各级卵泡数量变化;实时荧光定量PCR法检测卵巢组织TRAIL、DR4、DR5、DcR1、DcR2、FADD的mRNA表达;免疫组织化学法检测卵巢组织TRAIL、DR4、DR5的阳性表达。结果:与空白组比较,模型组大鼠血清FSH、LH含量升高(P<0.01),E2和VEGF含量、卵巢系数降低(P<0.01),各级生长卵泡数量减少(P<0.01),闭锁卵泡数量及卵巢组织中TRAIL、DR4、DR5阳性表达增加(P<0.01),卵巢组织中TRAIL、DR4、DR5、FADD mRNA表达水平升高(P<0.01);与模型组比较,戊酸雌二醇组、秩边透水道组大鼠血清VEGF含量、卵巢系数升高(P<0.01),各级生长卵泡数量增加(P<0.01),闭锁卵泡数量及卵巢组织中TRAIL、DR4、DR5阳性表达减少(P<0.01),卵巢组织中TRAIL、DR4、DR5、FADD mRNA表达水平降低(P<0.01,P<0.05);戊酸雌二醇组大鼠卵巢组织中初级卵泡数量多于秩边透水道组(P<0.01)。结论:秩边透水道针法可增加POI大鼠卵巢质量,改善局部血液循环,促进卵泡发育,其机制可能是通过下调死亡受体途径促凋亡蛋白TRAIL、DR4、DR5、FADD的表达,抑制卵巢颗粒细胞凋亡,改善大鼠的卵巢功能实现的。

Objective To observe the effect of penetrative needling of“Zhibian”(BL54)through“Shuidao”(ST28)on the expressions of death receptor pathway-related protein tumor necrosis factor-related apoptosis-inducing ligand(TRAIL)and its receptors,as death receptor 4(DR4),death receptor 5(DR5),decoy receptor 1(DcR1)and decoy receptor 2(DcR2)in premature ovarian insufficiency(POI)rats,so as to explore its mechanisms underlying improvement of POI.Methods Forty female SD rats were randomly divided into blank control,model,penetrative needling and medication(estradiol valerate)groups,with 10 rats in each group.The POI model was established by intraperitoneal injection of cyclophosphamide(D1:50 mg·kg-1·d-1,D 2 to D15:8 mg·kg-1·d-1,for a total of 15 d).After successful modeling,the rats in the penetrative needling group received penetrative needling of BL54 through ST28,with the needle retained for 30 min,once a day for a total of 4 weeks.Rats of the medication group received gavage of estradiol valerate(0.09 mg·kg-1·d-1)once daily for 4 weeks.After the intervention,the content of serum follicles of stimulation hormone(FSH),lateinizing hormone(LH),estradiol(E2)and vascular endothelial growth factor(VEGF)were assayed using enzyme-linked immunosorbent assay,and histopathological changes of ovarian tissue and the number of follicles were observed under light microscope after H.E.staining.The expression levels of TRAIL,DR4,DR5,DcR1,DcR2,and Fas-associated death domain(FADD)in ovarian tissues were detected using quantitative real-time PCR,and the immunoactivity of ovarian TRAIL,DR4 and DR5 detected using immunohistochemistry.The body weight and the damp weight of ovary were measured for calculating the ovarian coefficient.Results Compared with the blank control group,the E2 and VEGF contents,ovarian coefficient,and the number of the primary,secondary and sinus follicles were significantly decreased(P<0.01)in the model group,whereas FSH and LH contents,the atretic follicle number,TRAIL,DR4 and DR5 immunoactivity,and the expression levels of TRAIL,DR4,DR5 and FADD mRNAs considerably increased in the model group(P<0.01).In comparison with the model group,the decrease of the VEGF content,ovarian coefficient,and the number of the primary,secondary and sinus follicles,and the increase of the atretic follicle number,TRAIL,DR4 and DR5 immunoactivity,and expression levels of TRAIL,DR4,DR5 and FADD mRNAs were reversed in both penetrative needling and medication groups(P<0.01,P<0.05).The number of primary follicles was significantly more in the medication group than in the penetrative needling group(P<0.01).Conclusion Penetrative needling of BL54 and ST28 can improve ovarian weight and promote follicular development in POI rats,which may be associated with its function in down-regulating the expression of pro-apoptotic proteins TRAIL,DR4,DR5 and FADD of the death receptor pathway to inhibit apoptosis of ovarian granulosa cells.