Effect of Electroacupuncture on the Immune Balance of Th1/Th2 in Diminished Ovarian Reserve Rats Based on the Preventive Treatment Theory
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摘要:
目的 基于“治未病”理论, 探讨电针对雷公藤多苷诱导的卵巢储备功能减退(Diminished ovarian reserve, DOR)模型大鼠卵巢功能和Th1/Th2免疫平衡的影响。 方法 将27只动情周期正常的雌性SPF级SD大鼠随机分为空白组、模型组和电针组, 每组9只。模型组和电针组采用雷公藤多苷片混悬液按50 mg·kg-1·d-1连续灌胃14 d, 每日1次, 空白组每日给予1次等体积的生理盐水灌胃, 连续14 d。电针组大鼠在灌胃1 h后, 进行电针干预, 选取“中脘” “关元”或双侧“肾俞”穴, 2组穴位隔日交替, 每日1次, 每次10 min, 连续14 d。电针参数设置为2/100 Hz疏密波(疏波与密波交替持续的时间各约1.5 s), 电流强度为1.0 mA。每日采用阴道脱落细胞涂片观察大鼠动情周期; 干预结束后, 采用HE染色观察卵巢组织学形态; ELISA检测大鼠血清FSH、E2、IL-4、IFN-γ含量; 免疫组化、Western blot和qPCR分别检测大鼠卵巢组织中T-bet、GATA3蛋白及mRNA的表达情况; 流式细胞术检测大鼠脾脏Th1、Th2细胞比例并计算Th1/Th2比值。 结果 与空白组相比, 模型组大鼠原始卵泡减少(P < 0.05)、闭锁卵泡增加(P < 0.01), 卵巢指数、脾脏指数降低(P < 0.01), 血清FSH、IFN-γ水平升高(P < 0.05), E2、IL-4水平降低(P < 0.05), 卵巢组织中T-bet蛋白及mRNA表达增多(P < 0.05), GATA3蛋白及mRNA表达减少(P < 0.05, P < 0.01), T-bet/GATA3比值升高(P < 0.05), Th1细胞比例及Th1/Th2比值升高(P < 0.01), Th2细胞比例降低(P < 0.05);与模型组相比, 电针组大鼠原始卵泡增多, 闭锁卵泡减少(P < 0.01), 卵巢指数、脾脏指数升高(P < 0.01), 血清FSH、IFN-γ水平降低(P < 0.01), E2、IL-4水平升高(P < 0.05, P < 0.01), 卵巢组织中T-bet蛋白及mRNA表达减少(P < 0.05, P < 0.01), GATA3蛋白及mRNA表达增多(P < 0.05, P < 0.01), T-bet/GATA3比值降低(P < 0.01), Th1细胞比例及Th1/Th2比值降低(P < 0.05, P < 0.01), Th2细胞比例升高(P < 0.05)。 结论 电针对DOR大鼠卵巢储备功能具有保护作用, 能阻抑雷公藤多苷所致的卵巢功能损伤, 其机制可能与调节Th1/Th2免疫平衡有关。 -
关键词:
- 卵巢储备功能减退 /
- 电针 /
- Th1/Th2 /
- T-bet/GATA3
Abstract:OBJECTIVE To explore the effect of electroacupuncture on ovarian reserve function and Th1/Th2 immune balance in DOR rats based on the preventive treatment theory. METHODS A total of 27 female SPF Sprague-Dawley rats with normal estrus cycle were randomly divided into control group, DOR group, and electroacupuncture group, with 9 rats in each group. The DOR group and the electroacupuncture group were treated with intragastric administration of tripterygium glycosides(TGs) tablet suspension at a dose of 50 mg·kg-1·d-1 for 14 days to establish the DOR models. An equal volume of normal saline was given to the control group once a day for 14 days. One hour after intragastric administration, rats in the electroacupuncture group had a daily 10-minute electroacupuncture treatment at bilateral "Shenshu" (BL23) or "Guanyuan" (CV4) and "Zhongwan" (CV12). The two groups of acupoints were alternated every other day. The electroacupuncture parameters were as follows: 2/100 Hz, 1.0 mA. Vaginal smears were obtained every day starting the first day of intervention to monitor the estrus cycle of the rats. After the intervention, HE staining was used to observe the histological morphology of ovaries; the level of estradiol (E2), follicle-stimulating hormone (FSH), interleukin-4 (IL-4) and interferon-γ (IFN-γ) were measured through ELISA kits; the expression of T-bet and GATA3 mRNA was detected by qPCR; the protein levels of T-bet and GATA3 in ovarian tissue were detected by immunohistochemistry and Western blot; the ratio of Th1 and Th2 cells in the spleen was detected by flow cytometry. RESULTS Compared with the control group, the number of primordial follicles in the DOR group decreased (P < 0.05), and atretic follicles increased (P < 0.01); both the ovarian index and spleen index were reduced in the DOR group (P < 0.01); serum E2 and IL-4 levels decreased (P < 0.05), while the levels of FSH and IFN-γ increased in the DOR group (P < 0.05); GATA3 protein and mRNA expression lessened (P < 0.05, P < 0.01), while T-bet protein and mRNA expression were upregulated in ovarian tissues (P < 0.05); the T-bet/GATA3 ratio was elevated (P < 0.05); the proportion of Th1 cells as well as Th1/Th2 ratio increased (P < 0.01), and the proportion of Th2 cells decreased (P < 0.05). In comparison with DOR group, the number of primordial follicles in the electroacupuncture group increased notably, which was accompanied by decreased atretic follicles (P < 0.01); the ovarian index and spleen index increased (P < 0.01); the levels of E2 and IL-4 both increased(P < 0.05, P < 0.01), while FSH and IFN-γ concentrations decreased in DOR group (P < 0.01); GATA3 protein and mRNA were expressed higher in ovarian tissues (P < 0.05), while the expression of T-bet protein and mRNA was downregulated (P < 0.05, P < 0.01); the T-bet/GATA3 ratio is reduced (P < 0.01); the proportion of Th1 cells alone with Th1/Th2 ratio decreased (P < 0.05, P < 0.01), and the proportion of Th2 cells was higher than that in DOR group (P < 0.05). CONCLUSION Electroacupuncture has a protective effect on ovarian reserve in DOR rats, and its mechanism may be related to the regulation of Th1/Th2 immune balance. -
Key words:
- diminished ovarian reserve /
- electroacupuncture /
- Th1/Th2 /
- T-bet/GATA3
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图 1 大鼠动情周期各阶段阴道脱落涂片(×200, 标尺=100 μm)
注: A.动情前期; B.动情期; C.动情后期; D.动情间期; 绿色箭头示核上皮细胞, 红色箭头示角化无核上皮细胞, 黑色箭头示白细胞
Figure 1. Vaginal smears at each stage of the estrous cycle (×200, scale bar=100 μm)
图 2 各组大鼠动情周期变化(n=9)
注: P.动情前期; E.动情期; M.动情后期; D.动情间期
Figure 2. Changes in the estrus cycle of rats in each group(n=9)
图 3 各组大鼠卵巢组织形态比较(HE染色, ×200, 标尺=100 μm)
注: “▲”表示原始卵泡; “△”表示初级卵泡; “”表示次级卵泡; “→”表示闭锁卵泡
Figure 3. Comparison of ovarian histomorphological structure of rats in each group (HE staining, ×200, scale bar=100 μm)
图 4 流式细胞术检测各组大鼠脾脏Th1、Th2细胞比例及其比值
注: 与空白组比较, *P < 0.05, * *P < 0.01;与模型组比较, #P < 0.05, ##P < 0.01。 x±s, n=6。
Figure 4. The proportion and ratio of spleen Th1 and Th2 cells in each group by flow cytometry
图 5 各组大鼠卵巢T-bet、GATA3蛋白表达情况(免疫组化, ×400, 标尺=50 μm)
Figure 5. Expression of T-bet and GATA3 protein in ovary of rats in each group (Immunohistochemistry, ×400, scale bar=50 μm)
图 6 各组大鼠卵巢T-bet、GATA3阳性细胞平均光密度比较
注: 与空白组比较, *P < 0.05;与模型组比较, ##P < 0.01。 x±s, n=6。
Figure 6. Comparison of average optical density of T-bet and GATA3 positive cells in rats of each group
图 7 各组大鼠卵巢T-bet、GATA3蛋白表达情况
注: 与空白组比较, *P < 0.05;与模型组比较, #P < 0.05。 x±s, n=3。
Figure 7. Expression of T-bet and GATA3 protein in ovary of rats in each group
表 1 各组大鼠原始卵泡数和闭锁卵泡数比较( x±s, n=6)
Table 1. Comparison of number of primordial follicles and atretic follicles of rats in each group ( x±s, n=6)
组别 原始卵泡数 闭锁卵泡数 空白组 938.33±348.45 30.00±10.95 模型组 371.67±56.00* 81.67±7.53** 电针组 1 076.67±468.13## 41.67±13.29## 注: 与空白组比较, *P < 0.05, * *P < 0.01;与模型组比较, ##P < 0.01。 
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表 2 各组大鼠卵巢指数和脾脏指数比较( x±s, n=6)
Table 2. Comparison of ovarian index and spleen index of rats in each group( x±s, n=6)
组别 卵巢指数/‰ 脾脏指数/% 空白组 0.34±0.02 2.98±0.36 模型组 0.27±0.02** 2.20±0.29** 电针组 0.36±0.02## 3.18±1.97## 注: 与空白组比较, * *P < 0.01;与模型组比较, ##P < 0.01。
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表 3 各组大鼠血清FSH、E2水平比较( x±s, n=6)
Table 3. Comparison of serum levels of FSH, E2 of rats in each group( x±s, n=6)
组别 FSH/(ng·mL-1) E2/(pg·mL-1) 空白组 97.26±14.97 8.35±3.92 模型组 123.06±21.14* 4.87±1.42* 电针组 90.07±4.74## 9.91±2.51## 注: 与空白组比较, *P < 0.05;与模型组比较, ##P < 0.01。
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表 4 各组大鼠血清IFN-γ、IL-4水平比较( x±s, n=6)
Table 4. Comparison of serum levels of IFN-γ, IL-4 of rats in each group( x±s, n=6)
组别 IFN-γ/(pg·mL-1) IL-4/(pg·mL-1) 空白组 34.06±11.17 145.06±45.57 模型组 49.76±11.20* 86.80±20.66* 电针组 21.06±5.92## 134.04±25.48# 注: 与空白组比较, *P < 0.05;与模型组比较, #P < 0.05, ##P < 0.01。
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表 5 各组大鼠卵巢T-bet、GATA3 mRNA相对表达水平及其比值比较( x±s, n=6)
Table 5. Comparison of ovarian T-bet and GATA3 mRNA expression levels and ratios in each group( x±s, n=6)
组别 T-bet GATA3 T-bet/GATA3 空白组 0.94±0.047 1.16±0.13 0.81±0.075 模型组 1.14±0.14* 0.69±0.15** 1.33±0.33* 电针组 0.77±0.12## 0.94±0.76# 0.50±0.043## 注: 与空白组比较, *P < 0.05, * *P < 0.01;与模型组比较, #P < 0.05, ##P <0.01。
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