[Effect of "Zhibian" (BL 54)-to-"Shuidao" (ST 28) needle insertion on the expression of Fas/FADD/Caspase-8 of death receptor pathway in rats with primary ovarian insufficiency].

OBJECTIVE: To explore the effect of "Zhibian" (BL 54)-to-"Shuidao" (ST 28) needle insertion on the ovarian function in the rats with primary ovarian insufficiency (POI) and the potential effect mechanism based on the Fas/FADD/Caspase-8 of death receptor pathway. METHODS: Forty-eight female SD rats were randomly divided into a blank group, a model group, a medication group and an acupuncture group, with 12 rats in each group. Except in the blank group, the rats in the other groups were intraperitoneally injected with cyclophosphamide to establish the POI model. In the acupuncture group, after successful modeling, the intervention was given with "Zhibian" (BL 54)-to- "Shuidao" (ST 28) needle insertion, once daily, 30 min in each intervention; and the duration of intervention was 4 weeks. In the medication group, estradiol valerate tablets were administered intragastrically, 0.09 mg•kg-1•d-1, for 4 weeks. The general situation and the estrous cycle of the rats were compared among groups. Using ELISA, the levels of follicle-stimulating hormone (FSH), luteinizing hormone (LH) and estradiol (E2) in the serum were detected. HE staining was adopted to observe the morphological changes of ovarian tissue of rats. The protein expression of Fas, FADD and Caspase-8 in ovarian tissue was detected with immunohistochemistry and Western blot. RESULTS: After modeling, except the rats of the blank group, the rats of the other groups had dry fur, lost hair, low spirits, reduced food intake, increased urination and loose stool. After intervention, the stool became regular gradually in the acupuncture group and the medication group. The percentage of estrous cycle disturbance was increased in the rats of the model group when compared with the blank group (P<0.01); in comparison with the model group, the percentages of estrous cycle disturbance were reduced in the acupuncture group and the medication group after intervention (P<0.01). When compared with the blank group, the body mass and E2 content in the serum were lower (P<0.01), the levels of FSH and LH in the serum and the protein expression levels of Fas, FADD and Caspase-8 were increased (P<0.01) in the model group. Compared with the model group, the body mass and E2 contents in the serum were higher (P<0.01), the levels of FSH and LH in the serum and the protein expression levels of Fas, FADD and Caspase-8 were reduced (P<0.01) in the acupuncture group and the medication group. CONCLUSION: "Zhibian" (BL 54)-to-"Shuidao" (ST 28) needle insertion can effectively improve the ovarian function of POI rats, and its effect mechanism may be related to regulating the serum sex hormone levels, reducing the expression of Fas, FADD and Caspase-8 in ovarian tissue and retarding apoptosis of ovarian cells.

[Effect of acupuncture at "Zhibian" (BL 54) through "Shuidao" (ST 28) on the expression of apoptosis-related factors in rats with premature ovarian insufficiency based on oxidative stress].

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.

[Effect of penetrative needling of "Zhibian" (BL54) through "Shuidao" (ST28) on expression of TRAIL and its receptors in rats with premature ovarian insufficiency].

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, D2 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.