补肾活血中药血清对加压纯化培养视网膜神经节细胞PI3K/AKT信号转导通路的影响

Effects of Chinese medicine bushenhuoxue on PI3K/Akt signal transduction pathway of pressurized and cultured retinal ganglion cells in vitro

目的研究补肾活血中药血清对加压纯化培养视网膜神经节细胞(retinal ganglion cells,RGCs)凋亡模型PI3K/Akt信号转导通路主要成员PI3K及Akt表达的影响,探索补肾活血法保护RGCs的机制。方法制备补肾活血中药含药血清,体外纯化SD大鼠RGCs,采取开放式压力控制培养系统建立体外加压培养RGCs凋亡模型,以50g·L^(-1)、100 g·L^(-1)、200 g·L^(-1)血清浓度梯度补肾活血中药血清分别处理。将RGCs分为5组,分别为正常培养组(N组)、对照组(C组)、50 g·L^(-1)补肾活血中药血清组(50 g·L^(-1)BSHX组)、100 g·L^(-1)补肾活血中药血清组(100 g·L^(-1)BSHX组)、200g·L^(-1)补肾活血中药血清组(200 g·L^(-1)BSHX组),Annexin V-FITC/PI双染检测细胞凋亡率,实时荧光定量PCR(qRT-PCR)检测补肾活血中药血清对RGCs PI3K及Akt mRNA表达水平的影响,Western blot检测各组PI3K、Akt蛋白表达量。结果Q-PCR检测各组mRNA结果:C组(0.04±0.01)与N组(1.00±0.04)相比,RGCs中PI3K、Akt的mRNA表达水平下降,差异均有统计学意义(均为P<0.05),而50 g·L^(-1)、100 g·L^(-1)、200 g·L^(-1)BSHX组(0.18±0.01、0.21±0.02,0.22±0.01、0.36±0.01,0.84±0.10、1.07±0.17)与C组相比,PI3K、Akt mRNA含量逐渐升高,差异均有统计学意义(均为P<0.05)。Western blot检测各组蛋白表达,C组与N组相比,细胞PI3K、Akt的蛋白表达水平下降,差异有统计学意义(P<0.05),而50 g·L^(-1)、100 g·L^(-1)、200 g·L^(-1)BSHX组与C组相比,PI3K、Akt蛋白表达量逐渐升高,差异均有统计学意义(均为P<0.05)。结论补肾活血中药血清抑制加压诱导的RGCs凋亡,其机制可能与激活PI3K/Akt信号转导通路有关。

Objective To investigate the influence of bushenhuoxue drug-contai- ning serum on PI3K and Akt signaling pathway in purified retinal ganglion cell （RGCs） in vitro of Sprague-Dawley （SD） rats, and to explore the protective mechanisms of bushenhuoxue recipe on RGCs. Methods At first,bushenhuoxue drug-containing ser- um was prepared, and the RGCs of SD rots were purified;after the apoptotic model of pressurized and purified RGCs was established successfully in vitro using open pressure control system,RGCs were dealt with 50 g · L^-1 ,100 g · L^-1 ,200 g · L^-1 concentration gradient of bushenhuoxue drug-containing serum. Then the subjected cells were divided into normal culture group （N group）, control group （ C group） ,50 g · L^-1 bushenhuox- ue group （50 g · L^-1 BSHX group） ,100 g · L^-1 bushenhuoxue group （ 100 g · L^-1BSHX group） ,200 g · L^-1 bushenhuoxue group （200 g · L^-1 BSHX group）. Finally, cell apop- totic rate was detected by Annexin V-FITC/PI staining,while real-time quantitative PCR （qRT-PCR） and Western blot were used to detect the mRNA and protein expression of PI3K and Akt in each group respectively. Results The results of qRT-PCR detection showed that PI3K,Akt mRNA expression level in C group （0.04±0.01 ） was decreased compared with N group （ 1.00 ± 0.04 ）, and the difference was statistically significant （all P 〈0.05 ）, while PDK,Akt mRNA levels in 50 g · L^-1 ,100 g · L^-1 and 200 g · L^-1 BXHX group （0.18±0.01,0.21 ±0.02,0.22±0.01,0.36±0.01,0.84±0. 10,1.07 ± 0. 17） were increased compared with the C group, and the difference was statistically significant （ all P 〈0.05 ）. The Western blot results of each group showed that PI3K,Akt protein expression level in C group was decreased compared with N group, with statisti- cal difference （ all P 〈 0.03 ）, while PDK,Akt protein expression levels in 50 g L^-1,100 g · L^-1 and 200 g ·L^-1 BSHX group were increased compared with C group, with statis- tical difference （ all P 〈 0. 05 ）. Conclusion Bushenhuoxue drug-containing serum may inhibit the RGCs apoptosis induced by pressure, which may be related to the activa- tion of PI3K/Akt signal transduction pathway.