四君子汤含药血清抑制晶状体上皮细胞凋亡的实验研究

Inhibitory effects of sijunzi decoction on apoptosis of lens epithelial cells

目的观察四君子汤含药血清对人晶状体上皮细胞(human lens epithelial cell,HLEC)凋亡的抑制作用,从而探讨四君子汤防治老年性白内障的机制。方法 30只SD大鼠随机分为2组,分别用四君子汤(1.725 g·kg^(-1)·d^(-1))和生理盐水灌胃,用药7 d后经腹主动脉采血,离心提取四君子汤含药血清和空白血清冻存备用。细胞实验分为4组:阴性对照组、过氧化氢组(300μmol·L^(-1)过氧化氢作用4 h)、空白血清组(300μmol·L^(-1)过氧化氢作用4 h后加入体积分数20%空白血清作用12 h)和四君子汤含药血清组(300μmol·L^(-1)过氧化氢作用4 h后加入体积分数20%四君子汤含药血清作用12 h),流式细胞仪检测HLEC细胞周期;透射电子显微镜观察各组HLEC超微结构的改变。结果流式细胞仪检测结果示:过氧化氢组G1期细胞比例为(67.127±2.615)%,较阴性对照组明显增加,S期细胞比例为(24.603±2.547)%、G2期细胞比例为(8.270±1.149)%,均较阴性对照组减少,差异均具有统计学意义(均为P<0.05)。四君子汤含药血清组G1期细胞比例为(41.517±20.008)%,较过氧化氢组明显减少,S期细胞比例为(38.423±10.213)%、G2期细胞比例为(20.060±11.004)%,均较过氧化氢组明显增加,差异均具有统计学意义(均为P<0.05)。透射电子显微镜结果显示:阴性对照组胞膜完整,各细胞器结构正常;过氧化氢组细胞表面微绒毛消失,染色质凝集成块分布于核膜下,核膜欠完整,细胞质内可见溶酶体;空白血清组细胞质内空泡增多,线粒体、内质网等细胞器结构仍可见;四君子汤含药血清组胞膜完整,细胞质内可见少许空泡,线粒体、内质网等细胞器结构清晰。结论四君子含药血清能有效抑制过氧化氢诱导的HLEC凋亡,这可能是四君子汤防治老年性白内障的机制之一。

Objective To observe the inhibitory effect of Sijunzi decoction on apoptosis of human lens epithelial cells(HLECs)and to explore the mechanism of Sijunzi decoction in preventing and treating senile cataract.Methods A total of 30 adult Sprague-Dawley rats were randomly divided into Sijunzi decoction group(n=15),and the rats were given Sijunzi decoction(1.725 g·kg-1·d-1)and control group(n=15),while the rats received 0.9%sodium chloride injection(0.5 mL·kg-1·d-1).After one week,the blood was collected from abdominal aorta of these rats,then extracted by centrifugation for preparation of cell experiment.Four groups were set in the cell experiment,and they were negative control group,hydrogen peroxide group,blank serum group and Sijunzi decoction containing serum group.All the HLECs except those in negative control group were treated with 300μmol·L-1 hydrogen peroxide for 4 h firstly.Then the cells in the blank serum group were incubated with 15%volume fraction of the blank serum for 12 h,and those in Sijunzi decoction containing serum group with 20%volume fraction of the Sijunzi decoction containing serum equally.Finally,the cell cycle of HLECs was detected by flow cytometry,and the ultrastructural changes were observed by transmission electron microscope.Results According to the flow cytometry,the proportion of G1 phase cells in hydrogen peroxide group was(67.127±2.615)%,which was significantly higher than that in the negative control group.But the proportions of S and G2 phase cells were significantly lower than those in the negative control group(all P<0.05),with(24.603±2.547)%and(8.270±1.149)%respectively.The percentage of G1 phase cells in Sijunzi decoction containing serum group was(41.517±20.008)%,which was significantly lower than that in the hydrogen peroxide group,while the percentages of G2 phase cells were significantly higher than those in hydrogen peroxide group(all P<0.05),with(38.423±10.213)%and(20.060±11.004)%,respectively.The results of transmission electron microscopy showed that the cell membrane was intact and the organelle structure was normal in the negative control group.But in the hydrogen peroxide group,microvilli disappeared,and chromatin clumps were distributed under the incomplete nuclear membrane with some lysosomesin cytoplasm.More vacuoles and some organelles such as mitochondria and endoplasmic reticulum were observed in the blank serum group,while intact cell membrane,a few vacuoles in cytoplasm,and mitochondria,endoplasmic reticulum with clear structure were observed in Sijunzi decoction containingserum group.Conclusion It is possibility that Sijunzi decoction prevents and treats senile cataract,which is partly proved by the result of effective inhibition on the apoptosis of HLECs induced by hydrogen peroxide.