机构地区:[1]河南中医药大学第二临床医学院,河南郑州450003 [2]河南省中医院,河南郑州450002
出 处:《中药材》2024年第9期2314-2319,共6页Journal of Chinese Medicinal Materials
基 金:国家自然科学基金项目(82274536);河南省中医药科学研究专项课题(2022ZY2030);河南省中医药科学研究专项课题(20-21ZY2060);河南省科技攻关计划(192102310159)。
摘 要:目的:探索益肾通络方对人精索静脉曲张内皮细胞的作用及其机制,以期为该方治疗精索静脉曲张提供理论依据。方法:50只雄性SD大鼠分为空白组、迈之灵片(0.054 g/kg)组及益肾通络方低(0.675 g/kg)、中(1.35 g/kg)、高(2.7 g/kg)剂量组,各给药组灌胃给药,空白组灌胃相应体积生理盐水,连续给药2 w后收集各组大鼠血清。手术采集精索静脉曲张(VC)患者曲张及附近0.5 cm正常的精索静脉并分离、培养,经鉴定人精索静脉内皮细胞(HSVECs)和人精索静脉曲张内皮细胞(HSVVECs),将HSVECs设为正常对照组,将HSVVECs随机分为曲张模型组、迈之灵片含药血清组、LY294002(PI3K抑制剂)组及益肾通络方低、中、高剂量含药血清组。Western Blot、CCK-8、Transwell、体外成血管试剂盒、AnnexinV-FITC/PI双染法分别检测各组细胞增殖、迁移、成管能力及凋亡情况,Western Blot检测p-Akt/Akt、p-PI3K/PI3K、p-mTOR/mTOR水平及HIF-1α、VEGF蛋白表达。结果:与正常对照组比较,模型组细胞增殖、迁移能力及成管节点数、交叉点数、血管长度、血管分支数显著升高,凋亡率显著降低,HIF-1α、VEGF蛋白表达及p-Akt/Akt、p-PI3K/PI3K、p-mTOR/mTOR水平显著升高。与模型组比较,益肾通络方含药血清组细胞增殖、迁移能力及成管节点数、交叉点数、血管长度、血管分支数显著降低,凋亡率显著升高,HIF-1α、VEGF蛋白表达及p-Akt/Akt、p-PI3K/PI3K、p-mTOR/mTOR水平显著降低。结论:益肾通络方可改善精索静脉内皮细胞缺氧环境和抑制精索静脉内皮细胞增殖、迁移、血管生成并促进凋亡从而改善精索静脉损伤,其作用机制与抑制PI3K/AKT/mTOR和HIF-1α/VEGF信号通路的过度激活有关。Objective:To explore the effect and mechanism of Yishen tongluo formula on human varicocele endothelial cells,so as to provide theoretical basis for its treatment of varicocele.Methods:Fifty male SD rats were divided into blank group,Maizhiling tablet(0.054 g/kg)group and Yishen tongluo formula low(0.675 g/kg),medium(1.35 g/kg)and high(2.7 g/kg)dose groups.Each administration group was administrated intragastrically,and the blank group was administrated intragastrically with corresponding volume of normal saline.The serum of each group was collected after 2 weeks continuous administration.Varicocele(VC)patients with varico-cele and nearby 0.5 cm normal spermatic veins were collected surgically,and human varicocele endothelial cells(HSVECs)and human varicocele endothelial cells(HSVVECs)were isolated,cultured,identified,the HSVECs were used as normal control group,the HSV-VECs were randomly divided into varicose model group,Maizhiling tablet drug-containing serum group,LY294002(PI3K inhibitor)group and Yishen tongluo formula low,medium and high dose drug-containing serum groups.Western Blot,CCK-8,Transwell,in vitro Angiogenesis kit and AnnexinV-FITC/PI double staining were used to detect the proliferation,migration,tube-forming ability and apop-tosis in each group.Western Blot was used to detect the levels of p-Akt/Akt,p-PI3K/PI3K,p-mTOR/mTOR and HIF-1α,VEGF pro-tein expressions.Results:Compared with the normal control group,cell proliferation,migration ability,node number,cross number,vessel length and vascular branch number in model group were significantly increased,apoptosis rate was significantly decreased,HIF-1α,VEGF protein expressions and p-Akt/Akt,p-PI3K/PI3K,p-mTOR/mTOR levels were significantly increased.Compared with the model group,the cell proliferation,migration ability,node number,cross number,vessel length and vascular branch number in Yishen tongluo formula drug-containing serum groups were significantly decreased,the apoptosis rate was significantly increased,HIF-1αand VEGF protein expressions
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