Objective: To explore the possible mechanism of acupuncture at "Zhibian" (BL 54) through "Shuidao" (ST 28) on premature ovarian insufficiency (POI) from the perspective of oxidative stress.
Methods: Sixty female SD rats were randomly divided into a blank group, a model group, a sham acupuncture group, a medication group, and an acupuncture group, 12 rats in each group. Except the blank group, the rats in the remaining groups were intraperitoneally injected with cyclophosphamide to establish the POI model. After the model was successfully established, the rats in the acupuncture group were treated with acupuncture at "Zhibian" (BL 54) through "Shuidao" (ST 28), with a depth of about 12 mm, and the needle was retained for 30 min; the acupuncture was given once a day, for a total of 4 weeks. The rats in the sham acupuncture group were treated with blunt-head needle to tap the skin surface of "Zhibian" (BL 54), without penetrating the skin, once a day for 4 weeks. The rats in the medication group were treated with estradiol valerate by gastric gavage for 4 weeks. After the intervention, the level of reactive oxygen species (ROS) in the ovarian tissue was detected by fluorescence probe; the expression of c-Jun N-terminal kinase (JNK), forkhead box O1 (FoxO1), tumor suppressor gene protein 53 (p53) and p53 up-regulated modulator of apoptosis (Puma) mRNA and protein in ovarian tissue were detected by real-time fluorescence quantitative PCR and Western blot.
Results: Compared with the blank group, the level of ROS and the expression of JNK mRNA, p-JNK protein, FoxO1, p53, Puma mRNA and protein in the ovarian tissue in the model group were increased (P<0.01). Compared with the model group, the level of ROS and the expression of p-JNK protein, FoxO1, p53, Puma mRNA and protein in the ovarian tissue in the sham acupuncture group were slightly reduced, but the difference was not statistically significant (P>0.05). The level of ROS and the expression of JNK mRNA, p-JNK protein, FoxO1, p53, Puma mRNA and protein in the ovarian tissue in the acupuncture group and the medication group were reduced (P<0.01).
Conclusion: Acupuncture at "Zhibian" (BL 54) through "Shuidao" (ST 28) could improve the level of oxidative stress, down-regulate the expression of apoptosis-related factors JNK, FoxO1, p53 and Puma induced by oxidative stress, and inhibit the premature failure of ovarian reserve function caused by apoptosis of ovarian granulosa cells in POI rats.
目的：从氧化应激角度探讨“秩边透水道”针法改善早发性卵巢功能不全（POI）的可能机制。方法：将60只SD雌性大鼠随机分为空白组、模型组、假针刺组、药物组、秩边透水道组，每组12只。除空白组外，其余大鼠采用腹腔注射环磷酰胺的方式制备POI大鼠模型。造模成功后，秩边透水道组针刺大鼠双侧“秩边”，并刺向同侧“水道”方向，深约12 mm，留针30 min，每日1次，共4周；假针刺组用钝针头在“秩边”穴皮肤表面进行点按，不刺入皮下，每日1次，共4周；药物组给予戊酸雌二醇灌胃，共4周。干预结束后，采用荧光探针检测大鼠卵巢组织中活性氧自由基（ROS）水平；实时荧光定量PCR法和Western blot法检测大鼠卵巢组织中c-Jun氨基末端激酶（JNK）、叉头状转录因子1（FoxO1）、肿瘤抑制基因p53（p53）和p53上调凋亡调控因子（Puma）mRNA及蛋白表达情况。结果：与空白组比较，模型组大鼠卵巢组织ROS水平，JNK mRNA、p-JNK蛋白和FoxO1、p53、Puma mRNA及蛋白表达升高（P<0.01）；与模型组比较，假针刺组大鼠卵巢组织ROS水平，p-JNK蛋白和JNK、FoxO1、p53、Puma mRNA及蛋白表达略低，但差异无统计学意义（P>0.05），秩边透水道组、药物组大鼠卵巢组织ROS水平，JNK mRNA、p-JNK蛋白和FoxO1、p53、Puma mRNA及蛋白表达降低（P<0.01）。结论：“秩边透水道”针法可以改善POI模型大鼠体内氧化应激水平，下调氧化应激引发的促凋亡相关因子JNK、FoxO1、p53、Puma表达，抑制卵巢颗粒细胞凋亡引发卵巢储备功能过早衰竭。.
Keywords: JNK/FoxO1; Zhibian (BL 54) through Shuidao (ST 28); acupuncture; oxidative stress; premature ovarian insufficiency (POI).