| [Objective]To examine the protective effect and ameliorative effect of electroacupuncture (EA) on ovarian in primary ovarian insufficiency (POI) model rat induced by 4-vinylcyclohexene diepoxide (VCD), and try to elucidate the possible mechnisms.[Methods]1 The protective effect and mechanism of EA on ovarian in POI model rat induced by 4-vinylcyclohexene diepoxide (VCD).A total of 32 Sprague-Dawley female rats (10-12 weeks) with normal estrous cycle were randomly divided into four groups:control group(n=8), model group(n=8), EA group(n=8) and grabbing group (n=8). Model rats, EA rats and grabbing rats were all intraperitioneally (i.p.) injected with sesame oil plus VCD(160 mg/kg) for 15 consecutive days. At the same time, EA group rats were treated by electroacupuncture on the acupoints of bilateral BL33 and ST25 in alternate days. The needles were inserted vertically at BL33 to a depth of 7-lOmm, and at ST25 to a depth of 3-5mm. Bilateral BL33 (or ST25) were connected to an electroacupuncture device (Huatuo, model SDZ-V). EA stimulation lasted for 20 minutes with a continuous wave of 1-3Hz and current intensity of 1 mA-10 mA. The skin around the acupoints shivering mildly indicated the proper dose. The EA rats were treated once a day, five times a week for the first two weeks. After the final VCD injection, the EA rats were continued to treat once a day, three times a week for two weeks. Grabbing rats were binded by a small sack. The rat position when bondage, the duration, the frequency and the total number of bind were all same as the EA group. During the treatment, rats were monitored daily for the survival situation, general condition, body weight, and the estrous cycle. Disordered estrous cycle indicated the success of primary ovrian insufficiency model establishment. When POI model were estabished among model group, all rats were anesthetized by intraperitoeal administration of 0.35ml/100g body weight of a mixture of 10% chloral hydrate prior to euthanasia. We took the weight of sexual gland on electronic balance and calculated the sexual gland index. We detected the level of serum sex hormone (AMH, inbibinB, FSH, LH, E2, P and T) with ELISA and RIA. We stained the paraffin sections of ovarian with HE, and counted the number of ovarian follicles. We detected the expression of AMH mRNA in ovarian with RT-PCR, and the expression of AMH protein, Bax protein and Bcl-2 protein with Western blotting.2 The ameliorative effect and mechanism of EA on ovarian in POI model rat induced by 4-vinylcyclohexene diepoxide (VCD).A total of 58 Sprague-Dawley female rats (10-12 weeks) with normal estrous cycle were divided into six groups:control group(n=8), model group(n=10), EA group(n=10), grabbing group (n=10), tamoxifen group (n=10), and corn oil group (n=10). Model rats, EA rats, grabbing rats, tamoxifen (TA) rats and corn oil were all intraperitioneally (i.p.) injected with sesame oil plus VCD(160 mg/kg) for 15 consecutive days. When POI model were estabished, the intervention groups were treated for 1 month. EA group rats were treated by electroacupuncture on the acupoints of bilateral BL33 and ST25 in alternate days (the EA method, frequency and parmeters were the same as the EA group in experement 1), Grabbing group rats were binded by a small sack (the grabbing method, frequency were the same as grabbing group in the experement 1). TA group rats were subcutaneously injected with corn oil plus tamoxifen(1 mg/kg) 5 times for the first two weeks, and 3 times for the next two weeks. Corn group rats were subcutaneously injected with corn oil (0.3ml/times), the injection frequency was the same as the TA group. Rats were monitored daily for the survival situation, general condition, body weight, and the estrous cycle.24 hours after the final intervention, all rats were anesthetized to be taken femoral artery blood, ovary and uterus. We took the weight of sexual gland on electronic balance and calculated the sexual gland index. We detected the level of serum sex hormone (AMH, inbibinB, FSH, LH, E2, P and T) with ELISA and RIA. We stained the paraffin sections of ovarian with HE, and counted the number of ovarian follicles. We detected the expression of AMH mRNA, IGF-1 mRNA, IGF-1R mRNA in ovarian with RT-PCR, and the expression of AMH protein with Western blotting.[Results]1 The protective effect and possible mechanism1.1 Survival situationBefore sacrifice, control group, model group, EA group and grabbing group were left 8,7, 8 and 7 rats, respectively.1.2 General conditionThe general condition of control group rats was normal (normal eating and drinking, good activtiy, and bright body hair color) during the whole experiment. The general condition of model group, EA group and grabbing group was less normal than that in control group during the period of VCD injection, however, their condition gradually recovered after ceasing VCD injection, and EA group rats’ condition were much better than model and grabbing group.1.3 Body weightThe control group rats’ body weight gradually increased week by week. The model group, the EA group and the grabbing group rats’body weight gradually decreased during the period of VCD injection, and to a minimum at 15d. The latter three groups rats’ body weight gradually increased after ceasing VCD injection, and of which EA group rats grew fastest. After that, the grow rate of model group, EA group and grabbing group were similar to control group. At the end of experiment, EA group rats’body weight were heavier than that in model group and grabbing group, while it was lighter than control group.1.4 The estrous cycleThe estrous cycle of control group rats were regular during the whole experiment.0 of 8 (0%) EA rats had disordered estrous cycle during the period of VCD injection from Oth day to 15th day,compared with 1 of 7 (14.29%) model rats, and 0 of 7 (0%) grabbing rats.1 of 8 (12.5%) EA rats had disordered estrous cycle from 15th day to 30th day,compared with 1 of 7 (14.29%) model rats, and 2 of 7 (28.57%) grabbing rats. At 70th day, all rats in model group, EA group and grabbing group had disordered estrous cycle.1.5 Sexual gland weight and sexual gland indexa. ovary weight and ovary index:70 days after the initial dose of VCD (160mg/kg,15 d), ovary weight and ovarian index were lower in model group than that in control group (left: 15.43±3.21 vs 52.12±13.80, right:16.43±3.60 vs 48.50±8.85; ovary index:0.12±0.02 vs 0.35±0.06) in ovary, ovary weight and ovary index were higher in EA group than that in model group(left:29.13±7.20 vs 15.43v3.21, right:28.63±6.43 vs 16.43±3.60, ovary index: 0.20±0.04 vs 0.12±0.02). ovary index were higher in EA group than that in grabbing group(0.20±0.04 vs 0.16(0.02)).b. Uterus weight and uterus index:70 days after the initial dose of VCD (160mg/kg,15 d), uterus weight was lower in model group than that in control group (535.43±121.38 vs 756.75±142.31). Uterus weight was higher in EA group than that in model group and grabbing group (694.38±160.79 vs 535.43±121.38 vs 541.14±85.27). There is no statistical difference of uterus index among four groups.1.6 Serum sex hormoneSeventy days after the initial dose of VCD (160mg/kg,15 d), serum AMH level was higher in EA group than that in model group and grabbing group, while it was lower than that in control group (6.10±1.43 vs 3.25±1.71 vs 2.96±1.78 vs 12.03±4.29). Serum inhibinB level was higher in EA group than that in model group and grabbing group, while it was lower than that in control group (20.09±3.10 vs 14.57±2.46 vs 14.06 (2.43) vs 29.13±6.73). Serum FSH level was lower in EA group than that in model group and grabbing group, while it was higher than that in control group (3.17±0.87 vs 4.98±1.08 vs 4.83±1.05 vs 2.04±0.64). Serum LH level was lower in EA group than that in model group and grabbing group, while it was higher than that in control group (7.04±1.62 vs 10.46±1.73 vs9.88±1.38 vs 5.25±1.68). Serum E2 level was higher in EA group than that in model group and grabbing group, while it was lower than that in control group (19.94±4.64 vs12.44±6.16 vs 13.02±5.24 vs 28.47±6.76). Serum P level was higher in EA group than that in model group, while it was no difference with grabbing group, and is lower than that in control group (2.76±1.23 vs 1.0(0.97) vs 2.06±1.60 vs 6.22±1.43). Serum T level showed no significant differences among EA group, model group and grabbing group. However, Serum T level was higher in EA groups than that in control group (0.108(0.19) vs 0.269±0.171 vs 0.230±0.198 vs 0.066±0.045).1.7 Histology and follicle countingCompared with control group, the ovary in model group, EA group and grabbing group were deep dark, small size and atrophic. The damage degree of follicle was lesser in EA group than that in model group and grabbing group. Primordial follicle was higher in EA group than that in model group and grabbing group, while it was lower than that in control group (6.50±1.20 vs 4.00±1.41 vs 3.29±1.11 vs 9.50±1.60). Primary follicle was higher in EA group than that in model group and grabbing group, while it was lower than that in control group (5.13±1.89 vs 2.86±1.35 vs 4.00±2.45 vs 5.75±1.39). Antral follicles in EA group showed no statistical difference with model group and grabbing group, while it was lower than that in control group (2.38±1.30 vs 2.57±1.51 vs 2.14±1.07 vs 6.63±2.50). Atresia follicles was higher in EA group than that in model group and grabbing group, while it was lower than that in control group (5.13±1.89 vs 2.86±1.35 vs 4.00±2.45 vs 5.75±1.39).1.8 AMH mRNA expression in ovarySeventy days after the initial dose of VCD (160mg/kg,15 d), AMH mRNA expression in ovary was higher in EA group than that in model group and grabbing group, while it was lower than that in control group (2-ΔΔCT value:0.590±0.021 vs 0.383±0.098 vs 0.293±0.129 vs 1.073±0.120).1.9 AMH protein, Bax protein and Blc-2 protein expression in ovarySeventy days after the initial dose of VCD (160mg/kg,15 d), AMH protein expression in ovary was higher in EA group than that in model group, while it was no difference with grabbing group, but is lower than that in control group (the grey value of protein expression: 0.289±0.26 vs 0.134±0.020 vs 0.219±0.048 vs 0.390±0.066). Bax protein expression in ovary was lower in EA group than that in model group, while it was no difference with grabbing group, but is higher than that in control group (the grey value of protein expression: 0.271±0.078 vs 0.398±0.025 vs 0.299±0.036 vs 0.139±0.011). Bcl-2 protein expression in ovary was higher in EA group than that in model group, while it was no difference with grabbing group, but is lower than that in control group (the grey value of protein expression: 0.264±0.090 vs 0.120±0.022 vs 0.225±0.036 vs 0.414±0.087).2 The ameliorative effect and possible mechanism2.1 Survival situationBefore sacrifice, control group, model group, EA group, grabbing group, TA group and corn oil group were left 8,7,7,7,7 and 7 rats, respectively.2.2 General conditionThe general condition of control group rats was normal (normal eating and drinking, good activtiy, and bright body hair color) during the whole experiment. The general condition were less normal in other groups than control group during the period of VCD injection, however, their condition gradually recovered while waiting for the success of the POI model. During the intervention, the condition were much better in EA group than model group and grabbing group, while it is the same as TA group.2.3 Body weightThe control group rats’ body weight gradually increased week by week, and other groups rats’body weight gradually decreased during the period of VCD injection, and to a minimum at 15d. The latter five groups rats’body weight gradually increased after ceasing VCD injection, and the grow rates were similar among them. At the end of experiment, the body weight were heavier in EA group and TA group than that in model group, grabbing group and corn oil group, while it is lighter than control group.2.4 The estrous cycleThe estrous cycle of control group rats were regular during the whole experiment.0 of 7 (0%) EA rats had disordered estrous cycle during the period of VCD injection from Oth day to 15th day,compared with 1 of 7 (14.29%) model rats,0 of 7 (0%) grabbing rats,0 of 7 (0%) TA rats, and 1 of 7 (14.29%) com oil rats. At 70th day, all rats in model group, EA group,grabbing group,TA rats and corn oil rats had disordered estrous cycle. From 70th day to 98th day, the estrous cycle of control group rats were regular, only 1 of 7 (14.29%) EA rats had regular estrous cycle (2 cycles), the other rats in model group, EA group,grabbing group,TA group and corn oil group remained disordered estrous cycle.2.5 Sexual gland weight and sexual gland indexa. ovary weight and ovary index:after one month intervention, there was no statistical difference of ovary weight and ovary index between EA group and model group (left: 16.71±5.31 vs 14.86±4.06, right:18.71±5.31 vs 17.00±6.16; ovary index:0.126±0.042 vs 0.118±0.039). There was no statistical difference of ovary weight and ovary index among EA group, grabbing group and TA group.(left:16.71±5.31 vs 13.43±8.01 vs 20.00±6.08, right: 18.71±5.31 vs 15.00±6.56 vs 21.86±12.71, ovary index:0.126±0.042 vs 0.103±0.047 vs 0.147±0.055).b. Uterus weight and uterus index:after one month intervention, There were no statistical difference of uterus weight and uterus index between EA group and model group (ovary weight:605.71±121.60 vs 541.00± 122.72, ovary index:2.26±0.36vs 1.99±0.51). There were no statistical difference of uterus weight and uterus index among EA group, grabbing group and TA group (694.38±160.79 vs 535.43±121.38 vs 541.14±85.27).2.6 Serum sex hormoneAfter one month intervention, serum AMH level was higher in EA group than that in model group and grabbing group, while it showed no difference with TA group, but was lower than that in control group (8.63±2.87 vs 2.73±1.34 vs 2.57±0.86 vs 9.40±3.29 vs 12.23±3.96). Serum inhibin B level was higher in EA group than that in model group and grabbing group, while it showed no difference with TA group, but was lower than that in control group (19.75±3.31 vs 13.29±3.6 vs 12.78±2.23 vs 20.09±2.72 vs 24.45±6.80). Serum FSH level was lower in EA group than that in model group and grabbing group, while it showed no difference with TA group, but was higher than that in control group (4.30(0.72)vs 46.08±0.74 vs 5.92±0.72 vs 4.18±0.94 vs 2.77±1.16). Serum LH level was lower in EA group than that in model group and grabbing group, while it showed no difference with TA group, but was higher than that in control group (7.29±1.62 vs 12.50±1.09 vs 13.34±1.42 vs 7.33±1.48 vs 5.39(1.34)). Serum E2 level was higher in EA group than that in model group and grabbing group, while it showed no difference with TA group, but was lower than that in control group (17.83±4.71 vs 10.93±4.71 vs 9.74±3.66 vs 18.36±5.42 vs 26.02±2.72).2.7 Histology and follicle countingCompared with control group, the ovary in model group, EA group, grabbing group, TA group and corn oil group were deep dark, small size and atrophic. The damage degree of follicle was lesser in EA group than those in model group and grabbing group, while it showed no difference with TA group. Primordial follicles in EA group showed no statistical difference with model group, grabbing group and TA group, while it was lower than that in control group (2.29±0.76 vs 1.86±0.69 vs 1.0(1.0) vs 2.14±1.21 vs 8.88±2.10). Primary follicles in EA group showed no statistical difference with model group, grabbing group and TA group, while it was lower than that in control group (3.86±1.35 vs 2.86±1.07 vs 2.71±1.25 vs 3.14±1.21 vs 5.63±1.41). Antral follicles in EA group showed no statistical difference with model group, grabbing group and TA group, while it was lower than that in control group(2.43±1.40 vs 2.57±1.27 vs 2.14±0.90 vs 2.0(2.0) vs 4.50±1.19). Atresia follicle was lower in EA group than that in model group and grabbing group, while it showed no difference with TA group, but was higher than that in control group(7.14±2.41 vs 10.14±1.68 vs 971±2.29 vs 6.57±1.72 vs 3.63±1.41).2.8 AMH protein expression in ovaryAfter one month intervention, AMH protein expression in ovary was higher in EA group than that in model group and grabbing group, while it showed no difference with TA group, but was lower than that in control group (the grey value of protein expression:0.304±0.059 vs 0.119±0.039 vs 0.140±0.073 vs 0.328±0.050 vs 0.432±0.0571).2.9 AMH, IGF-1, IGF-1R mRNA expression in ovaryAfter one month intervention, AMH mRNA expression in ovary was higher in EA group than that in model group and grabbing group, while it showed no difference with TA group, but was lower than that in control group (2-ΔΔCT value:0.69±0.59 vs 0.13±0.02 vs 0.15±0.08 vs 0.71±0.31 vs 1.26±0.21). IGF-1 mRNA expression in ovary in EA group showed no difference with model group, grabbing group, TA group and control group(2-ΔΔCT value: 1.38±0.64 vs 1.96±0.52 vs 1.40±0.16 vs 1.10±0.20 vs 0.95±0.08).IGF-1R mRNA expression in ovary was lower in EA group than that in model group and grabbing group, while it showed no difference with grabbing group and control group (2-ΔΔCT value:0.78±0.13 vs 1.86±0.30 vs 1.02±0.11 vs 0.80±0.20 vs 0.73±0.25).[Conclusion]1 Electroacupuncture could protect and improve ovarian function in POI model rat induced by 4-vinylcyclohexene diepoxide (VCD).2 The protective and ameliorative effect on ovarian function of electroacupuncture is the effect of electroacupuncture itself, while it isn’t the stress reaction during the binding process.3 One of the possible mechanism accounting for the protective effect of electroacupuncture for ovarian is that it could up-regulate Blc-2 protein expression, and down-regulated Bax protein expression in granulosa cell in ovarian.4 One of the possible mechanism accounting for the ameliorative effect of electroacupuncture for ovarian is that it could regulate the expression of IGF-1/IGF-1R pathway by down-regulating IGF-1R mRNA expression in ovarian. |
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