淫羊藿总黄酮含药血浆对雄兔干眼泪腺上皮细胞凋亡模型Caspase-3、Caspase-8 mRNA表达的影响

目的观察淫羊藿总黄酮含药血浆对雄兔干眼泪腺上皮细胞Caspase-3、Caspase-8 mRNA表达的影响。方法取61只健康无眼疾1月龄雄性新西兰大耳白兔,随机取1只雄兔泪腺,培养泪腺上皮细胞,取第3代泪腺上皮细胞用于实验。60只雄兔随机分成淫羊藿总黄酮10.0倍组、5. 0倍组、2.5倍组和空白对照组,通过MTT法确定后续干预细胞的含药血浆的浓度。将所培养第3代泪腺上皮细胞随机分为淫羊藿总黄酮治疗组、含雄激素培养对照组和空白组(DMEM/F12低糖培养基中分别加入含淫羊藿总黄酮血浆、10^-6mol·L^-1丙酸睾酮和空白血浆)。干预48 h后各组均加入H_2O_2继续培养60 min诱导细胞凋亡,采用RT-PCR法检测各组细胞凋亡相关基因Caspase-3、Caspase-8 mRNA的表达。结果 MTT实验显示应选取淫羊藿总黄酮2. 5倍组作为干预细胞含药血浆的浓度。淫羊藿总黄酮10. 0倍组、5. 0倍组、2. 5倍组和空白对照组兔血浆中朝藿定A浓度分别为120. 5μg·L^-1、65. 7μg·L^-1、21. 9μg·L^-1、0μg·L^-1,淫羊藿苷浓度分别为130. 8μg·L^-1、45. 3μg·L^-1、18. 9μg·L^-1、0μg·L^-1。淫羊藿总黄酮治疗组、含雄激素培养对照组、空白组的Caspase-3 mRNA相对含量分别为0. 35±0. 07、0. 68±0. 18、1. 00±0. 19,Caspase-8 mRNA相对含量分别为0. 27±0. 21、0. 72±0. 10、1. 00±0. 15。空白组Caspase-3、Caspase-8mRNA的表达均高于含雄激素培养组,空白组和含雄激素培养组均高于淫羊藿总黄酮治疗组,差异均有统计学意义(均为P <0. 05)。结论淫羊藿总黄酮能够下调雄兔干眼泪腺上皮细胞Capase-3、Capase-8 mRNA的表达,这可能是其治疗干眼的关键机制之一。

Effect of plasma containing epimedium total flavonoids on caspase-3 mRNA and caspase-8 mRNA expression in cell apoptotic model of lacrimal gland epithelial cells of xeroma male rabbits

Objective　To observe influences of total flavonoids of herba epimedii medicated plasma on lacrimal epithelial cells Caspase-3 and Caspase-8 mRNA expression of male rabbits with xerophthalmia.Methods　Totally sixty-one healthy male New Zealand rabbits with one-month age were selected.Lacrimal gland of one male rabbit was chosen at random to cultivate lacrimal epithelial cells.The third generation of lacrimal epithelial cells was used for the experiment.Sixty male rabbits were divided into 10.0 times group,5.0 times group and 2.5 times group of total flavonoids of herba epimedii,as well as the blank control group at random.Meanwhile,MTT method was used to determine the medicated plasma concentration in subsequent cells with intervention.The third generation of lacrimal epithelial cells was randomly divided into the treatment group with total flavonoids of herba epimedii,control group with androgen and blank group,of which the DMEM/F12 low-sugar medium was added with total flavonoids of herba epimedii medicated plasma,10^-6 mol·L^-1 testosterone propionate and blank blood plasma,respectively.After intervening for 48 h,H₂O₂ was added in each group to continue cultivating for 60 min to induce cellular apoptosis.RT-PCR method was utilized to detect apoptosis-related genes Caspase-3 and Caspase-8 mRNA expression in each group.Results　MTT experiment showed that 2.5 times group of total flavonoids of herba epimedii was selected as the medicated plasma concentration of cells with intervention.Bepimedin A concentration in rabbit plasma in 10.0 times group,5.0 times group and 2.5 times group of total flavonoids of herba epimedii was 120.5 μg·L^-1,65.7 μg·L^-1,21.9 μg·L^-1 and 0 μg·L^-1,respectively;and icariin concentration was 130.8 μg·L^-1,45.3 μg·L^-1,18.9 μg·L^-1,and 0 μg·L^-1,respectively.Caspase-3 mRNA relative content in treatment group with total flavonoids of herba epimedii,control group with androgen and blank group was 0.35±0.07,0.68±0.18 and 1.00±0.19,respectively.And Caspase-8 mRNA relative content was 0.27±0.21,0.72±0.10 and 1.00±0.15,respectively.Caspase-3 and Caspase-8 mRNA expression in the blank group were higher than those of the control group with androgen.The blank group and control group with androgen were higher than treatment group with total flavonoids of herba epimedii,and there was the statistical difference (all P<0.05).Conclusion　Total flavonoids of herba epimedii can reduce lacrimal Caspase-3 and Caspase-8 mRNA expression of male rabbits with xerophthalmia.This may be one of key mechanisms to cure xerophthalmia.