摘要
目的验证受体相互作用蛋白激酶1/受体相互作用蛋白激酶3/混合系激酶区域样蛋白(RIPK1/RIPK3/MLKL)信号通路参与介导视网膜色素上皮(RPE)细胞坏死性凋亡的过程,探索黄芩苷调控RPE细胞与RIPK1/RIPK3/MLKL信号通路之间的关系。方法采用细胞计数试剂-8(CCK8)实验测定黄芩苷0~200.0μmol/L的安全浓度范围、最佳有效浓度和最佳给药时间。将RPE细胞ARPE-19分成3组,对照组(CG)正常培养,模型组(MG)给予含1 mg/mL碘酸钠的完全培养基刺激,黄芩苷组(BG)在MG组基础上给予25.0μmol/L黄芩苷培养,3组均培养48 h。CCK-8法检测ARPE-19细胞活力;免疫荧光法观察细胞形态;WesternBlot法检测各组细胞RIPK1/RIPK3/MLKL信号通路相关蛋白的表达水平。结果(1)安全浓度范围:与0μmol/L组比较,黄芩苷50.0、100.0、200.0μmol/L组的细胞活力均降低(t_(50.0)=3.236,P=0.032;t_(100.0)=6.165,P=0.004;t_(200.0)=7.840,P=0.001);与25.0μmol/L组比较,1.0、10.0、50.0μmol/L组的细胞活力差异无统计学意义(均P>0.05),0.1μmol/L组的细胞活力升高(t=4.848,P=0.008),100.0、200.0μmol/L组的细胞活力降低,差异有统计学意义(t_(100.0)=9.714,P=0.001;t_(200.0)=9.485,P=0.001)。因此,黄芩苷的安全浓度范围为0~25.0μmol/L。(2)最佳给药浓度:当黄芩苷浓度为25.0μmol/L时,细胞活性最高,与0.1、1.0、10.0μmol/L浓度组比较,差异均有统计学意义(t_(0.1)=8.987,P=0.001;t_(1.0)=6.809,P=0.002;t_(10.0)=3.320,P=0.029),故25.0μmol/L为黄芩苷最佳给药浓度。(3)最佳给药时间:与CG组比较,MG组24、48、72 h细胞活力均下降(t_(24 h)=8.118,P=0.001;t_(48 h)=12.330,t_(72 h)=26.873,均P=0.000);与MG组比较,BG组24、48、72 h细胞活力均升高(t_(24 h)=8.632,P=0.001;t_(48 h)=12.148,t_(72 h)=10.539,均P=0.000),差异均有统计学意义,故选择时间最短的24 h作为黄芩苷最佳给药时间。(4)细胞形态:在24、48、72 h时,CG组细胞核呈现强荧光,MG组细胞核呈现核浓缩,染色加深,部分细胞�
OBJECTIVE To investigate the role of receptorinteracting protein kinase 1/receptor-interacting protein kinase 3/mixed line kinase region lik protein (RIPK1/RIPK3/MLKL) signaling pathway in mediating the necrotizing apoptosis of retina pigment epithelium (RPE) cells. To explore the relationship between the regulation of baicalin on RPE cells and RIPK1/RIPK3/MLKL signaling pathway.METHODS Cell counting kit-8 (CCK8was used to determine the safe concentration range, the best effective concentration and the bes administration time of baicalin from 0 to 200.0 μmol/L. RPE cells ARPE-19 were divided int three groups: These cells were cultured normally in the control group (CG),stimulated with complete medium containing 1 mg/mL sodium iodate in the model group(MG),and cultured with 25.0μmol/L baicalin on the basis ofMG in the baicalin group(BG),all cells were cultured for 48 h.CCK-8 assay was used to detect the viability of ARPE-19 cells,immunofluorescence assay was used to observe the cell morphology,and Western Blot was used to detect the expression levels of RIPK1/RIPK3/MLKL signaling pathway related proteins.RESULTS(1)Safe concentration range:Compared with the 0μmol/L group,the cell viability of baicalin 50.0,100.0 and 200.0μmol/L groups was decreased(t50.0=3.236,P=0.032;t100.0=6.165,P=0.004;t200.0=7.840,P=0.001);Compared with 25.0μmol/L group,there was no significant difference in cell viability in 1.0,10.0 and 50.0μmol/L groups(all P>0.05),while there was significant difference in cell viability in 0.1μmol/L group(t=4.848,P=0.008).The cell viability of 100.0 and 200.0μmol/L groups was decreased,and the differences were statistically significant(t100.0=9.714,P=0.001;t200.0=9.485,P=0.001).Therefore,the safe concentration range of baicalin is 0-25.0μmol/L.(2)Optimal concentration:When the concentration of baicalin reached 25.0μmol/L,the cell activity was the highest.Compared with cell activity of 0.1,1.0 and 10.0μmol/L groups,the differences were statistically significant(t0.1=8.987,P=0.001;t1.0=6.809,P=0.00
作者
王珍
李潆
吕海江
王露
郑博
李亚静
WANG Zhen;LI Ying;LV Haijiang;WANG Lu;ZHENG Bo;LI Yajing(Eye Hospital,China Academy of Chinese Medical Sci-ences,Beijing 100040,China)
出处
《中国中医眼科杂志》
2022年第11期846-852,共7页
China Journal of Chinese Ophthalmology
