目的研究驻景丸加减方含药血清对过氧化氢(H 2O 2)诱导的人视网膜色素上皮(ARPE)-19细胞上皮－间质转化(EMT)的作用及其机制。
方法选取2月龄SPF级雌性SD大鼠30只，采用随机数字表法将其随机分为空白对照组和驻景丸加减方组，每组15只，分别给予生理盐水和驻景丸加减方连续灌胃7 d，以制备空白血清和含药血清。采用SD大鼠制备驻景丸加减方含药血清。将ARPE-19细胞分为正常对照组，模型对照组，空白血清组，2.5%、5.0%、10.0%含药血清组，SB216763组和SB216763+含药血清组；其中前2个组均于正常培养基中培养，后6个组分别于含空白大鼠血清培养基、相应浓度含药血清培养基、10 μmol/L GSK-3抑制剂SB216763培养基以及10 μmol/L SB216763+10.0%的含药血清培养基中培养；正常对照组常规培养，其他各组先常规培养24 h，后加入终浓度200 μmol/L的H 2O 2继续培养24 h。采用细胞计数试剂盒-8(CCK-8)法检测细胞活性；Transwell法检测细胞迁移能力；DCFH-DA法检测细胞内活性氧簇(ROS)含量；硫化巴比妥酸法检测细胞内丙二醛(MDA)含量；Western blot法检测细胞中Nrf2通路相关蛋白核因子E2相关因子2(Nrf2)、血红素加氧酶1(HO-1)、醌氧化还原酶1(NQO-1)和EMT相关蛋白转化生长因子β2(TGF-β2)、蛋白激酶B(AKT)、糖原合成酶激酶3β(GSK-3β)、snail家族锌指转录因子1(SNAIL1)、α平滑肌肌动蛋白(α-SMA)、上皮钙黏蛋白(E-cadherin)的表达。
结果空白血清组、2.5%、5.0%、10.0%组细胞存活率总体比较差异无统计学意义( F＝0.163， P>0.05)。正常对照组、模型对照组、2.5%含药血清组、5.0%含药血清组、10.0%含药血清组细胞存活率分别为(100.50±5.91)%、(60.87±4.30)%、(73.27±4.46)%、(80.73±5.67)%和(89.90±4.97)%，正常对照组、模型对照组、空白血清组、2.5%含药血清组、5.0%含药血清组和10.0%含药血清组细胞迁移数分别为(84.67±8.33)、(222.33±13.58)、(215.67±10.02)、(174.67±10.60)、(143.67±8.02)和(107.67±6.66)个/视野，总体比较差异均有统计学意义( F＝26.628、99.289，均 P<0.01)。模型对照组细胞内ROS和MDA含量较正常对照组显著增加，差异均有统计学意义(均 P<0.01)；各含药血清组细胞内ROS和MDA含量均较模型对照组显著减少，差异均有统计学意义(均 P<0.01)。模型对照组细胞内SNAIL1、α-SMA、TGF-β2、p-AKT及p-GSK-3β蛋白相对表达量明显多于正常对照组和各浓度含药血清组，E-cadherin蛋白相对表达量明显少于正常对照组和各浓度含药血清组，差异均有统计学意义(均 P<0.05)。与正常对照组比，模型对照组细胞质Nrf2蛋白相对表达量降低，细胞核Nrf2、HO-1和NQO-1蛋白相对表达量升高，差异均有统计学意义(均 P<0.05)；与模型对照组比，各含药血清组细胞质Nrf2蛋白相对表达量降低，细胞核Nrf2、HO-1和NQO-1蛋白相对表达量升高，差异均有统计学意义(均 P<0.01)。与模型对照组相比，SB216763组细胞质Nrf2蛋白相对表达量降低，细胞核Nrf2蛋白相对表达量升高，差异均有统计学意义(均 P<0.05)；与SB216763组相比，SB216763+含药血清组细胞质Nrf2、SNAIL1和α-SMA蛋白相对表达量降低，细胞核Nrf2和E-cadherin蛋白相对表达量升高，差异均有统计学意义(均 P<0.05)。
结论驻景丸加减方含药血清抑制H 2O 2诱导的ARPE-19细胞EMT，可能与AKT/GSK-3β通路的抑制及Nrf2信号通路的激活有关。
ObjectiveTo study the effect of ZhuJing pill variant formula medicated serum on hydrogen peroxide (H 2O 2)-induced epithelial-mesenchymal transition (EMT) of human retinal pigment epithelial (ARPE-19) cells and its mechanism.
MethodsThirty female SPF grade SD rats aged 2 months old were selected.The rats were randomized into blank control group and Zhujing pill variant formula group according to random number table method, with 15 in each group, which were intragastrically administered with normal saline and ZhuJing pill variant formula solution for 7 days accordingly to prepare blank control serum and medicated serum.ZhuJing pill variant formula medicated serum was prepared with SD rats.ARPE-19 cells were divided into normal control group, model control group, blank serum group as well as 2.5%, 5.0% and 10.0% medicated serum groups, SB216763 group and SB216763+ medicated serum group.Normal and blank control groups were cultured in normal culture medium, while the other six groups were cultured in blank rat serum medium, medicated serum medium of corresponding concentration, 10 μmol/L SB216763 medium and 10 μmol/L SB216763+ 10.0% medicated serum medium, respectively.Normal control group was routinely cultured, while the other groups were routinely cultured for 24 hours, and then added with H 2O 2 with the final concentration of 200 μmol/L for 24 hours.Cell viability was assessed by cell counting kit-8 (CCK-8) assay, and cell migration ability was detected by Transwell assay.Intracellular reactive oxygen species (ROS) level was detected by dichloro-dihydro-fluorescein diacetate (DCFH-DA) assay, and MDA level was identified by sulfhydryl barbituric acid assay.The expression levels of Nrf2 pathway related proteins including nuclear factor erythroid 2-related factor 2 (Nrf2), heme oxygenase-1 (HO-1), quinone oxidoreductase 1 (NQO-1) and EMT-related proteins including transforming growth factor-β2 (TGF-β2), protein kinase B (AKT), glycogen synthase kinase-3β (GSK-3β), snail family zinc finger 1 (SNAIL1), α-smooth muscle actin (α-SMA), epithelial cadherin (E-cadherin) in cells were measured by western blot assay.The use and care of animals complied with Regulations for the Administration of Affairs Concerning Experimental Animals.
ResultsThere was no significant difference in cell survival rate among blank serum group, 2.5%, 5.0% and 10.0% medicated serum groups ( F=0.163, P>0.05). The cell survival rates were (100.50±5.91)%, (60.87±4.30)%, (73.27±4.46)%, (80.73±5.67)% and (89.90±4.97)% in normal control group, model control group, 2.5%, 5.0% and 10.0% medicated serum groups, and the number of migrating cells was (84.67±8.33), (222.33±13.58), (215.67±10.02), (174.67±10.60), (143.67±8.02) and (107.67±6.66) pcs/visual field in normal control group, model control group, blank serum group, 2.5%, 5.0% and 10.0% medicated serum groups, respectively, with significant differences among the groups ( F=26.628, 99.289; both at P<0.01). The contents of ROS and MDA in model control group were significantly increased in comparison with normal control group (both at P<0.01). The contents of ROS and MDA of 2.5%, 5.0% and 10.0% medicated serum groups were significantly decreased in comparison with model control group (all at P<0.01). The relative expression levels of SNAIL1, α-SMA, TGF-β2, p-AKT and p-GSK-3β proteins were significantly higher and the relative expression level of E-cadherin protein was significantly lower in model control group compared with normal control group, 2.5%, 5.0% and 10.0% medicated serum groups (all at P<0.05). Compared with normal control group, the relative expression level of cytoplasmic Nrf2 in model control group was decreased, while the relative expression levels of nuclear Nrf2, HO-1 and NQO-1 were increased, and the differences were statistically significant (all at P<0.05). Compared with model control group, the relative expression levels of cytoplasmic Nrf2 in 2.5%, 5.0% and 10.0% medicated serum groups were reduced, and the relative expression levels of nuclear Nrf2, HO-1 and NQO-1 were enhanced, and the differences were statistically significant (all at P<0.01). Compared with model control group, the relative expression level of cytoplasmic Nrf2 in SB216763 group was decreased, and the relative expression level of nuclear Nrf2 was increased, and the differences were statistically significant (both at P<0.05). Compared with SB216763 group, the relative expression levels of cytoplasmic Nrf2, SNAIL1 and α-SMA in SB216763+ medicated serum group were decreased, and the relative expression levels of nuclear Nrf2 and E-cadherin protein were increased, and the differences were statistically significant (both at P<0.05).
ConclusionsZhuJing pill variant formula medicated serum can inhibit H 2O 2-induced EMT in ARPE-19 cells.The mechanism may be related to the inhibition of AKT/GSK-3β pathway and the activation of Nrf2 signaling pathway.
邵雁,张元钟,杨宁,等. 驻景丸加减方含药血清对过氧化氢诱导的人RPE细胞上皮－间质转化的抑制作用及其机制[J]. 中华实验眼科杂志,2022,40(11)：1013-1022.
DOI:10.3760/cma.j.cn115989-20210907-00501版权归中华医学会所有。
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