摘要
目的:观察护卵汤对雷公藤多苷诱导早发性卵巢功能不全模型小鼠卵巢组织中凋亡相关蛋白沉默信息调节子1(SIRT1),肿瘤抑制基因p53,乙酰化p53(Ac-p53),细胞周期蛋白依赖性抑制剂p21,核转录因子-κB(NF-κB)p65表达情况及组织结构变化。方法:50只雌性C57BL/6J小鼠随机分正常组、模型组、护卵汤低、高剂量组、戊酸雌二醇组,采用雷公藤多苷80 mg·kg^(-1)·d^(-1)连续灌胃,第15天给予护卵汤低、高剂量组1.6,6.2 g·kg^(-1)·d^(-1)灌胃,戊酸雌二醇组给予戊酸雌二醇0.13 mg·kg^(-1)·d^(-1),连续21 d,正常组予等量蒸馏水。末次给药后取血和卵巢,苏木素-伊红(HE)染色观察组织形态学变化,ELISA检测血清中抗苗勒激素(AMH),促卵泡激素(FSH),黄体生成素(LH)水平含量,实时荧光定量聚合酶链式反应(Realtime PCR)法检测p53,p21 mRNA表达水平,蛋白免疫印迹法(Western blot)检测SIRT1,p53,p21,Ac-p53,NF-κB蛋白表达水平。结果:与正常组比较,模型组小鼠动情周期紊乱,卵巢体积减小,各级卵泡数明显减少,AMH含量降低,FSH,LH含量升高,p53,p21 mRNA表达显著升高,p53,p21,Ac-p53,NF-κB蛋白表达显著升高,SIRT1蛋白表达显著降低(P<0.01);与模型组比较,护卵汤高剂量组卵泡增多,AMH升高,FSH,LH降低,SIRT1蛋白表达升高(P<0.01),p53,p21 mRNA表达水平显著下调(P<0.01),p53,p21,Ac-p53,NF-κB蛋白表达明显降低(P<0.01)。结论:护卵汤的治疗可明显扭转衰老进程,其改善卵巢功能的机制可能与提高了卵巢细胞中SIRT1/NF-κB/p53/p21通路活性,改变细胞凋亡状态相关,从而增加了生长卵泡及成熟卵泡含量,降低了闭锁卵泡的含量。
Objective:To observe the effects of Huluan decoction on the expression of apoptosis-related protein silence information regulator 1(SIRT1),tumor suppressor gene(p53),acetylated p53(Ac-p53),cyclin-dependent kinase inhibitor(p21),and nuclear factor-κB(NF-κB)p65 and changes in ovarian tissue structure of a mouse model with premature ovarian insufficiency(POI)induced by tripterygium glycosides.Method: Fifty female C57 BL/6 J mice were randomly divided into the blank group,model group,low-and high-dose Huluan decoction groups, and western medicine(estradiol valerate) group. After intragastric administration of tripterygium glycosides at 80 mg·kg^(-1)·d^(-1) for 14 successive days,mice in the low-and highdose Huluan decoction groups and western medicine group were treated with Huluan decoction at 1.6 g·kg^(-1)·d^(-1) and 6.2 g·kg^(-1)·d^(-1) and estradiol valerate at 0.13 mg·kg^(-1)·d^(-1),respectively,by gavage since the 15 thday,while those in the blank group were provided with an equal amount of distilled water for 21 consecutive days.Following the last administration,the blood was sampled for detecting the levels of serum anti-Müllerian hormone(AMH),follicle-stimulating hormone(FSH),and luteinizing hormone(LH)by ELISA and the ovary was harvested for observing the morphological changes by HE staining. The mRNA expression levels of p53 and p21 were measured by Real-time polymerase chain reaction(Real-time PCR),and the protein expression levels of SIRT1,p53,p21,Ac-p53,NF-κB were assayed by Western blot. Result: Compared with the blank group,the model group exhibited a disordered estrous cycle,diminished ovarian volume,decreased number of follicles at various developmental stages,reduced AMH,and elevated FSH and LH,and elevated p53,p21,Ac-p53,NF-κB protein expression and p53,p21 mRNA,reduced SIRT1 protein expression(P<0.01). As revealed by the comparison with the model group,each medication group displayed an increased number of follicles,elevated AMH,reduced FSH and LH(P<0.01),up-regulated SIRT1 protein expression(P<0.01),and significantly down-regulated mRNA and protein expression of aging-related genes p53,p21,and down-regulated Ac-p53,NF-κB protein expression(P<0.01). Conclusion: Huluan decoction significantly reverses the aging process and improves ovarian function possibly by boosting the activity of SIRT1/NF-κB/p53/p21 pathway in ovarian cells,changing the apoptotic state,increasing the growing and mature follicles,and reducing the atretic follicles.
作者
邓敦
吴阳
申思楠
刘秀
雷磊
DENG Dun;WU Yang;SHEN Si-nan;LIU Xiu;LEI Lei(College of Integrated Traditional Chinese and Western Medicine,Hunan University of Chinese Medicine,Changsha 410208,China;The Second Military Medical University,Shanghai 200433,China;Hunan Key Laboratory of Traditional Chinese Medicine Prescription and Syndromes Translational Medicine,Hunan University of Chinese Medicine,Changsha 410208,China)
出处
《中国实验方剂学杂志》
CAS
CSCD
北大核心
2021年第15期36-42,共7页
Chinese Journal of Experimental Traditional Medical Formulae
基金
湖南省教育厅科学研究项目(19A365)
湖南中医药大学中西医结合一流学科开放基金项目(2018ZXYJH07)。