基于PERK/eIF2α/ATF4/CHOP通路探讨健骨颗粒对UMR-106细胞内质网应激凋亡的影响

Jiangu Granules Regulates Endoplasmic Reticulum Stress-Induced Apoptosis in UMR-106 Cells by Modulating the PERK/eIF2α/ATF4/CHOP Pathway

目的探讨健骨颗粒含药血清对UMR-106成骨样细胞内质网应激ERS凋亡的影响和作用机制。方法选用UMR-106成骨样细胞,采用0、0.01、0.02、0.03、0.04、0.05μmol/L不同浓度的GSK2606414(PERK抑制剂)对细胞进行干预,采用CCK8法筛选GSK2606414最佳干预浓度。将生长状态较好的UMR-106细胞随机分为阴性对照组(NC组)、模型组(H_(2)O_(2)组)、健骨颗粒组(H_(2)O_(2)+JG组)和阳性对照组(H_(2)O_(2)+GSK2606414组)4组。NC组和H_(2)O_(2)组采用10%生理盐水血清干预12 h,H_(2)O_(2)+JG组采用10%健骨颗粒含药血清干预12 h,H_(2)O_(2)+GSK2606414组采用0.03μmol/L的GSK2606414和10%生理盐水血清干预12 h,除NC组外,其余各组在不更换新培养基的情况下,再分别加入10μmol/L H_(2)O_(2)干预12 h。采用激光共聚焦显微镜观察细胞内NC组和H_(2)O_(2)组GRP78和Caspase-12荧光表达情况,DCFH-DA检测活性氧(ROS)含量,激光共聚焦显微镜观察细胞内钙离子实时动态变化判断ROS/ERS模型是否成立。再采用An⁃nexin V-FITC/PI检测4组细胞晚期凋亡率,qPCR和Western blot检测4组ERS相关标志指标GRP78、PERK、eIf2α、ATF4和CHOP mRNA转录水平和蛋白相对表达量。结果与0μmol/L组比较,0.03μmol/L的GSK2606414是干预UMR-106细胞12 h后对细胞活力没有影响的最大浓度,故使用该浓度作为后续H_(2)O_(2)+GSK2606414组的实验干预条件。与NC组相比,H_(2)O_(2)组ROS含量显著增高(P<0.01),GRP78和Caspase-12的蛋白荧光表达量明显增加,细胞内钙离子流动速度加快并持续增高,表明H_(2)O_(2)诱导UMR-106细胞ROS/ERS模型的成功建立。与NC组相比,H_(2)O_(2)组凋亡率显著增高(P<0.05),GRP78、PERK、eIf2α、ATF4、CHOP mRNA转录水平和蛋白相对表达量均显著增高(P<0.01)。与H_(2)O_(2)组相比,H_(2)O_(2)+JG组和H_(2)O_(2)+GSK2606414组ROS含量显著降低(P<0.01),凋亡率显著降低(P<0.05),GRP78、PERK、eIf2α、ATF4、CHOP mRNA转录水平(P<0.05)和蛋白相对表达量(P<0.01)均显著降低。结论健骨颗粒可通过PERK/eIF2α/ATF4/CHOP信号通路缓解内质网过度应激,降低成骨细胞凋亡率,发挥防治绝经后骨质疏松症的作用。

Objective To observe and analyze the mechanism by which Jiangu Granules containing serum alleviate endoplasmic reticulum stress(ERS)-induced apoptosis in UMR-106 osteoblast-like cells.Methods UMR-106 osteoblast-like cells were selected for the study,and various concentrations(0,0.01,0.02,0.03,0.04,0.05μmol/L)of GSK2606414(PERK inhibitor)were employed to intervene in the cells.The optimal intervention concentration of GSK2606414 was determined using the CCK8 method.UMR-106 cells exhibiting favorable growth status were randomly allocated into four groups:negative control group(NC group),model group(H_(2)O_(2) group),Jiangu Granules group(H_(2)O_(2)+JG group),and positive control group(H_(2)O_(2)+GSK2606414 group).The NC and H_(2)O_(2) groups were intervened with 10%saline serum for a duration of 12 hours.The H_(2)O_(2)+JG group was intervened with 10%Jiangu Granules containing serum for 12 hours,while the H_(2)O_(2)+GSK2606414 group underwent intervention with 0.03μmol/L GSK2606414 and 10%normal saline serum,for 12 hours.The last three groups were supplemented with 10μmol/L H_(2)O_(2) solution without altering the new culture medium for 12 hours.The fluorescence expression of GRP78 and Caspase-12 in cells of the NC and H_(2)O_(2) groups was observed using laser confocal microscopy.Intracellular ROS content was detected using DCFH-DA.The real-time dynamic changes of intracellular calcium ions were observed using laser confocal microscopy to determine whether the ROS/ERS model was constructed.Late apoptosis rate was determined using Annexin V-FITC/PI in the four groups.Additionally,mRNA transcription level and protein expression of ERS related markers GRP78,PERK,eIf2α,ATF4,and CHOP were detected using qPCR and Western blot methods.Results In comparison to the 0μmol/L group,it was found that 0.03μmol/L of GSK2606414 had no impact on cell viability after 12 hours of intervention in UMR-106 cells,and this concentration was chosen for subsequent experiments.Compared with the NC group,the H_(2)O_(2) group showed a significant increase in ROS content(P<0.01).The protein fluorescence expression level of GRP78 and Caspase-12 exhibited a significant increase.Additionally,the intracellular calciumion flow rate demonstrated an accelerated and continuous increase,indicating the successful establishment of the H_(2)O_(2) induced ROS/ERS model in UMR-106 cells.In comparison to the NC group,the apoptosis rate in the H_(2)O_(2) group displayed a significant increase(P<0.05).Furthermore,the mRNA transcription level and protein expression level of ATF4 and CHOP,as well as GRP78,PERK,and eIf2α,exhibited a significant increase(P<0.01).Compared with the H_(2)O_(2) group,the H_(2)O_(2)+JG group and the H_(2)O_(2)+GSK2606414 group showed a significant decrease in the ROS content(P<0.01)and apoptosis rate(P<0.05).The mRNA transcription level(P<0.05)and protein expression(P<0.01)of GRP78,PERK,eIf2α,ATF4,CHOP were significantly down-regulated.Conclusion These findings suggest that Jiangu Granules can alleviate endoplasmic reticulum stress and reduce apoptosis in osteoblast-like cells through the PERK/eIF2α/ATF4/CHOP signaling pathway,thereby potentially playing a role in the prevention and treatment of postmenopausal osteoporosis.