Jiangu Granules Regulates Endoplasmic Reticulum Stress-Induced Apoptosis in UMR-106 Cells by Modulating the PERK/eIF2α/ATF4/CHOP Pathway
Objective To observe and analyze the mechanism by which Jiangu Granules containing serum alleviate endoplas-mic reticulum stress(ERS)-induced apoptosis in UMR-106 osteoblast-like cells.Methods UMR-106 osteoblast-like cells were se-lected for the study,and various concentrations(0,0.01,0.02,0.03,0.04,0.05 μmol/L)of GSK2606414(PERK inhibitor)were em-ployed 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(H2O2 group),Jiangu Granules group(H2O2+JG group),and positive control group(H2O2+GSK2606414 group).The NC and H2O2 groups were intervened with 10%saline serum for a duration of 12 hours.The H2O2+JG group was intervened with 10%Jiangu Granules containing serum for 12 hours,while the H2O2+GSK2606414 group underwent intervention with 0.03 μmol/L GSK2606414 and 10%normal saline serum,for 12 hours.The last three groups were supplemented with 10 μmol/L H2O2 solution without altering the new culture medium for 12 hours.The fluorescence expression of GRP78 and Caspase-12 in cells of the NC and H2O2 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 tran-scription 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 0 μmol/L group,it was found that 0.03 μmol/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 experi-ments.Compared with the NC group,the H2O2 group showed a significant increase in ROS content(P＜0.01).The protein fluores-cence 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 H2O2 induced ROS/ERS model in UMR-106 cells.In comparison to the NC group,the apoptosis rate in the H2O2 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 H2O2 group,the H2O2+JG group and the H2O2+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 postmenopaus-al osteoporosis.
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维普
