摘要
目的 探讨滋阴奠基汤对排卵障碍大鼠卵巢炎症及凋亡因子的影响及其作用机制。方法 将48只SPF级8周龄雌性SD大鼠按随机数字表法分为空白组、模型组、芬吗通组和滋阴奠基汤低、中、高剂量组,每组各8只。按照环磷酰胺75 mg/kg腹腔注射制造排卵障碍大鼠模型,14 d后,空白组和模型组各以1 ml/100 g体重灌服生理盐水。芬吗通组按照0.5 ml/100 g、雌激素2 d+孕激素3 d循环灌服3个周期,滋阴奠基汤低、中、高剂量组分别给予滋阴奠基汤5.7 g/kg、11.4 g/kg、22.8 g/kg灌服,连续15 d。根据阴道涂片结果,选择D期时处死,采集腹主动脉血,用于检测血清激素水平,采集双侧卵巢组织进行检测,观察指标包括大鼠一般情况、体重变化、阴道涂片、动情周期变化、卵巢、子宫的重量系数;酶联免疫吸附测定法(Enzyme-linked immunosorbent assay, Elisa)检测血清卵泡刺激素(Follicle stimulating hormone, FSH)、黄体生成素(Luteinising hormone, LH)、雌二醇(Estradiol, E2)、抗苗勒管激素(Anti-mullerian hormone, AMH)水平;卵巢组织Tunnel染色和Ki67免疫组化染色;实时荧光定量聚合酶链式反应(RT-PCR)检测大鼠卵巢组织Bcl-2相关死亡促进因子(Bad)、Bcl-2相关X蛋白(Bax)、B淋巴细胞瘤-2(BCL-2)、活化型半胱氨酸天冬氨酸蛋白酶-3(Caspase-3)、半胱氨酸天冬氨酸蛋白酶-9(Caspase-9)、肿瘤坏死因子-α(Tumor necrosis factor-α,TNF-α)、白介素-1β(Interleukin-1β,IL-1β)、白介素-6(Interleukin-6,IL-6)、白介素-10(Interleukin-10,IL-10)mRNA表达水平;采用蛋白免疫印迹法(Western blot)检测大鼠卵巢组织蛋白活化型半胱氨酸天冬氨酸蛋白酶-3(Caspase-3)、肿瘤因子-53(Tumor Protein 53)、IL-6、TNF-α的蛋白表达水平。结果 应用滋阴奠基汤干预后,大鼠体重缓慢增长,动情周期逐渐规律,卵巢系数较模型组和芬吗通对照组升高,Elisa检测血清激素水平,可见滋阴奠基汤干预后血清LH、FSH下降,E2、AMH升高,卵巢组织Tunnel染色显示滋阴奠基汤可改善环磷酰胺引起的颗粒细胞的凋亡,Ki67染色显示可促进颗粒细胞的增殖,促进卵泡生长,效果优于对照组。滋阴奠基汤可降低促凋亡指标Bad、Bax、Caspase-3、Caspase-9 mRNA表达水平,Caspase-3、P53蛋白表达,升高抗凋亡BCL-2表达,降低炎症因子TNF-α、IL-1β、IL-6 mRNA水平及TNF-α、IL-6蛋白表达水平,升高抗炎因子IL-10 mRNA。结论 滋阴奠基汤可通过降低卵巢组织炎症水平、抑制颗粒细胞凋亡促进卵泡发育。
Objective To investigate the effects of Ziyin Dianji Decoction on ovarian inflammation and apoptoticfactors in the rat model of ovulation failure and decipher the underlying mechanism.Methods A total of 48 eight-week-old SPF female rats were assigned into normal,model,femoston,and low-,medium-,and high-dose(5.7,11.4,and22.8 g/kg,respectively)Ziyin Dianji Decoction groups(n=8)according to the random number table.The rat model of ovulation failure was established by intraperitoneal injection of 75 mg/kg cyclophosphamide.After 14 days,the blank and model groups were administrated with normal saline at I ml/100 g by gavage.The femoston group received cyclic administrationof estrogen for 2 days followed by progestogen for 3 days,at a dose of 0.5 ml/100 gfor three consecutive cycles.The low-,medium-,and high-dose Ziyin Dianji Decoction groups were administrated with Ziyin Dianji Decoction at correspondingdoses for 15 consecutive days.According to the results of vaginal smear,rats were subjected to abdominal aortic blood collection at stage D for measuring serum levels of hormones.The bilateral ovarian tissue was collected for determination of related indicators.The general conditions,body weight changes,vaginal smear,and estrous cycle changes were observed,andthe ovary index and uterus index were calculated.The serum levels of luteinizing hormone(LH),follicle-stimulating hormone(FSH),estradiol(E2),and anti-Mullerian hormone(AMH)were measured by enzyme-linked immunosorbent assay(ELISA).Terminal deoxynucleoitidyl transferase mediated nick-end labeling(TUNEL)and Ki67 staining were performedfor the ovarian tissue.RT-PCR was employed to determine the mRNA levels of B-cell lymphoma-2(Bcl-2),Bcl-2-associated death promoter(Bad),Bcl-2-associated X protein(Bax),cysteinyl aspartate-specific proteinase-3(caspase-3),caspase-9,tumor necrosis factor(TNF)-α,interleukin(IL)-1β,IL-6,and IL-10 in the ovarian tissue.Western blotting was employed to determine the protein levels of caspase-3,tumor protein 53(P53),IL-6,andTNF-αin the ovarian tissue.Results Compared with the model group and the femoston group,after the intervention withZiyin Dianji Decoction,the rats showed slowly increased body weights,recovering estrous cycle,increased ovarian index,lowered serum levels of LH and FSH,and elevated serum levels of E2 and AMH.TUNEL staining of the ovarian tissueshowed that Ziyin Dianji Decoction reduced the granulosa cell apoptosis induced by cyclophosphamide.Ki67 stainingshowed that the decoction promoted granulosa cell proliferation and follicular growth,with the effects outperforming femoston.Moreover,Ziyin Dianji Decoction down-regulated the mRNA levels of Bad,Bax,caspase-3,and caspase-9 and theprotein levels of caspase-3 and P53,and up-regulated the mRNA level of Bcl-2.In addition,the decoction down-regulated the mRNA levels of TNF-α,IL-1β,and IL-6 and the protein levels of TNF-αand IL-6,and up-regulatedthe mRNA level of IL-10.Conclusion Ziyin Dianji Decoction can promote follicular development by reducing ovarianinflammation and inhibiting granulosa cell apoptosis.
作者
冯雪
周惠芳
王光辉
FENG Xue;ZHOU Hui-fang;WANG Guang-hui(Tai'an Hospital of Traditional Chinese Medicine,Tai'an Shandong 271000;Affiliated Hospital of Nanjing Universityof Chinese Medicine,Nanjing Jiangsu 210000)
出处
《世界中西医结合杂志》
2025年第7期1325-1336,共12页
World Journal of Integrated Traditional and Western Medicine
基金
山东省中医药科技项目(M-2022072)。
关键词
滋阴奠基汤
排卵障碍
凋亡
炎症因子
Ziyin Dianji Decoction
Ovulation Failure
Apoptosis
Inflammatory Factors
