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周芳, 曾晶, 王永连, 陈嘉盈, 刘牧涯.基于相关细胞凋亡途径探讨补肾健脾方调控大鼠卵巢储备功能减退的机制研究[J].湖南中医药大学学报英文版,2024,44(7):1167-1174.[Click to copy
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| 基于相关细胞凋亡途径探讨补肾健脾方调控大鼠卵巢储备功能减退的机制研究 |
| 周芳,曾晶,王永连,陈嘉盈,刘牧涯 |
| (湖南中医药大学第一附属医院, 湖南 长沙 410007) |
| 摘要: |
| 目的 观察补肾健脾方对环磷酰胺诱导的卵巢储备功能减退大鼠模型卵巢功能的影响，并基于肿瘤坏死因子诱导相关细胞凋亡途径探讨补肾健脾方调控大鼠卵巢储备功能减退的机制研究。方法 60只动情周期正常的健康雌性SD大鼠，随机分成正常组10只和造模组50只，造模组采用环磷酰胺75 mg/kg单次腹腔注射造模。造模成功的大鼠再随机分为模型组、西药组[戊酸雌二醇0.103 mg/（kg·d）+黄体酮胶囊2.575 mg/（kg·d）]、补肾健脾方低剂量组[3.21 g/（kg·d）]、补肾健脾方中剂量组[6.43 g/（kg·d）]、补肾健脾方高剂量组[12.85 g/（kg·d）]，每组10只。正常组及模型组均予等体积蒸馏水灌胃，每组干预1次/d，干预14 d。检测各组大鼠动情周期、体质量、卵巢脏器指数、子宫脏器指数；ELISA法检测大鼠血清肿瘤坏死因子-α（tumor necrosis factor-α, TNF-α）、γ干扰素（interferon-γ, IFN-γ）含量；免疫荧光法检测大鼠卵巢颗粒细胞内B细胞淋巴瘤-2（B-cell lymphoma-2, Bcl-2）、Bcl-2相关X蛋白（Bcl-2 associated X protein, Bax）蛋白表达情况；HE染色检测卵巢组织病理情况；Western blot法检测卵巢磷酸化蛋白激酶Ｂ（posphorylated-proteinkinase B, p-AKT）、磷酸化哺乳动物雷帕霉素靶蛋白（p-mammalian target of rapamycin, p-mTOR）、自噬蛋白苄氯素-1（Beclin-1）表达情况。结果 与正常组比较，模型组大鼠动情周期紊乱，可见卵巢结构紊乱、各级卵泡数量不同程度的减少、未见成熟卵泡，卵巢间质中见到明显炎细胞浸润；大鼠体质量、卵巢脏器指数和子宫脏器指数无明显变化；血清TNF-α、IFN-γ水平升高（P<0.05，P<0.01）；卵巢颗粒细胞Bcl-2、Bax、Beclin-1蛋白表达水平升高（P<0.05，P<0.01），p-AKT、p-mTOR蛋白表达水平降低（P＜0.01）。与模型组比较，西药组及补肾健脾方低、中剂量组血清TNF-α、IFN-γ水平降低（P<0.05，P<0.01）；补肾健脾方中剂量组Bax蛋白表达水平降低（P<0.05）；补肾健脾方高剂量组Bcl-2蛋白阳性表达升高（P<0.05）；西药组、补肾健脾方低剂量组Beclin-1蛋白表达水平降低（P<0.05，P＜0.01），p-AKT、p-mTOR蛋白表达水平升高（P＜0.01）。与西药组比较，补肾健脾方高剂量组TNF-α水平升高（P<0.01）；补肾健脾方低、高剂量组IFN-γ水平降低（P<0.05）；补肾健脾方低剂量组Bax蛋白表达水平升高（P<0.05），补肾健脾方中剂量组Bcl-2蛋白水平表达升高（P<0.05）；补肾健脾方中、高剂量组Beclin-1蛋白表达水平升高（P<0.05，P＜0.01），p-AKT、p-mTOR蛋白表达水平降低（P＜0.01）。结论 补肾健脾方可改善卵巢病理变化、提高卵巢储备功能，其机制可能通过抑制肿瘤坏死因子TNF-α、IFN-γ表达，增强Bcl-2蛋白表达水平，降低Bax蛋白表达水平，激活mTOR通路抑制大鼠卵巢颗粒细胞的自噬与凋亡，从而改善环磷酰胺造模所致的大鼠卵巢储备功能减退的影响。 |
| 关键词: 卵巢储备功能减退 补肾健脾方 肿瘤坏死因子 细胞自噬 细胞凋亡 |
| DOI：10.3969/j.issn.1674-070X.2024.07.004 |
| Received:December 26, 2023 |
| 基金项目:长沙市自然科学基金项目（Kq2202452）；湖南省卫生健康委员会重点指导项目（C202305017704）；湖南省中医药管理局委托项目（D2023002）；湖南中医药大学校级重点项目（2019XJJJ036）；湖南中医药大学研究生创新课题校级项目（2023CX96）。 |
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| The mechanism of Bushen Jianpi Formula regulating diminished ovarian reserve in rats based on related apoptosis pathways |
| ZHOU Fang, ZENG Jing, WANG Yonglian, CHEN Jiaying, LIU Muya |
| (The First Hospital of Hunan University of Chinese Medicine, Changsha, Hunan 410007, China) |
| Abstract: |
| Objective To observe the effects of Bushen Jianpi Formula (BSJPF) on ovarian function in rats with cycloph-osphamide-induced decreased ovarian reserve function, and to investigate the mechanism of BSJPF regulating diminished ovarian reserve (DOR) in rats based on tumor necrosis factor-induced apoptosis pathway. Methods A total of 60 healthy female SD rats with normal estrous cycle were randomly divided into normal group (10 rats) and modeling group (50 rats). The modeling group was treated with a single intraperitoneal injection of cyclophosphamide at 75 mg/kg to establish models. The successful modeling rats were then randomly divided into model group, western medicine control group (estradiol valerate 0.103 mg/kg·d + progesterone capsule 2.575 mg/kg·d), and low- (3.21 g/kg·d), medium- (6.43 g/kg·d), and high-dose (12.85 g/kg·d) BSJPF groups, with 10 rats in each group. Normal group and model group were given equal volume of distilled water by gavage once a day, for 14 consecutive days. Estrous cycle, body mass, ovarian organ index, and uterine organ index were checked. The content of tumor necrosis factor-α (TNF-α) and interferon-γ (IFN-γ) in serum of rats were determined by ELISA. The expressions of B-cell lymphoma-2 (Bcl-2) and Bcl-2 associated X protein (Bax) in ovarian granulosa cells were measured by immunofluorescence assay. HE staining was used to examine the pathological status of ovarian tissues. Western blot analysis was used to check the ovarian posphorylated-proteinkinase B (p-AKT), p-mammalian target of rapamycin (p-mTOR), and Beclin-1 expressions. Results Compared with the normal group, the estrous cycle of the model group was disturbed, the ovarian structure was disturbed, the number of follicles at all levels was reduced to different degrees, no mature follicles were seen, and obvious inflammatory cell infiltration was seen in the ovarian stroma. There were no significant changes in body mass, ovarian organ index, or uterine organ index. Serum levels of TNF-α and IFN-γ were higher (P<0.05, P<0.01). The protein expressions of Bcl-2, Bax, and Beclin-1 in ovarian granulosa cells were higher (P<0.05, P<0.01), while the protein expressions of p-AKT and p-mTOR were lower (P<0.01). Compared with model group, serum levels of TNF-α and IFN-γ were reduced in western medicine control group, and low- and medium-dose BSJPF groups (P<0.05, P<0.01). The expression level of Bax protein in medium-dose BSJPF group decreased (P<0.05); the expression level of Bcl-2 protein in high-dose BSJPF group increased (P<0.05); Beclin-1 protein expressions in western medicine control group and low-dose BSJPF group decreased (P<0.05, P<0.01), and P-AKT and p-mTOR protein expression levels increased (P<0.01). Compared with western medicine control group, the level of TNF-α in high-dose BSJPF group increased (P<0.01); the levels of IFN-γ in the low- and high-dose BSJPF groups decreased (P<0.05); the expression level of Bax protein in low-dose BSJPF group increased (P<0.05), and the expression levels of Bcl-2 protein in medium-dose BSJPF group increased (P<0.05). In medium- and high-dose BSJPF groups, Beclin-1 protein expression levels increased (P<0.05, P<0.01), and p-AKT and p-mTOR protein expression levels decreased (P<0.01). Conclusion BSJPF can decrease the pathological changes of ovary and strengthen ovarian reserve function. The mechanism may involve inhibiting the expressions of TNF-α and IFN-γ of tumor necrosis factor, increasing the expression of Bcl-2 and lowering the expression of Bax, and activating mTOR pathway to inhibit the autophagy and apoptosis of rat ovarian granulosa cells. The effects of cyclophosphamide modeling on DOR in rats were improved. |
| Key words: diminished ovarian reserve Bushen Jianpi Formula tumor necrosis factor autophagy apoptosis |
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