Objective To investigate the protective effect of carvacrol(CAR)on high glucose(HG)-induced retinal ganglion cell(RGC)damage by regulating the protein kinase B(PKB,also known as Akt)/nuclear factor erythroid 2-relat-ed factor 2(Nrf2)signaling pathway.Methods The RGC-5 cells in the logarithmic growth phase were inoculated into 96-well cell culture plates with 4 × 104 cells/well and then treated with CAR(5.0 μmol·L-1,10.0 μmol·L-1,20.0 μmol·L-1,40.0 μmol·L-1,80.0 μmol·L-1,and 160.0 μmol·L-1)together with 30.0 mmol·L-1 glucose for 24 hours.Cell viability was detected by the 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide(MTT)assay to screen the ex-perimental concentration of CAR.Then,these RGC-5 cells were inoculated into 6-well plates and randomly assigned into the Control group,30.0 mmol·L-1 glucose(HG)group,low-,medium-,and high-concentration CAR groups(CAR-L group,CAR-M group,and CAR-H group),and high-concentration CAR+Nrf2 pathway inhibitor(ML385)group(CAR-H+ML385 group).Flow cytometry was used to detect the apoptosis of RGC-5 cells.The fluorescent probe was used to de-tect the level of oxidative stress in RGC-5 cells.The enzyme-linked immunosorbent assay(ELISA)was employed to detect the level of superoxide dismutase(SOD),lactate dehydrogenase(LDH),malondialdehyde(MDA),glutathione peroxidase(GSH-PX),and reactive oxygen species(ROS)in the supernatant of RGC-5 cells.Western blot was used to detect the ex-pression of proteins related to the Akt/Nrf2 signaling pathway.Results Compared with the HG group,the survival rate of RGC-5 cells increased significantly when the concentration of CAR was above 20.0 μmol·L-1(all P＜0.05).Therefore,20.0 μmol·L-1,40.0 μmol·L-1,and 80.0 μmol·L-1 of CAR were used for subsequent experiments.Compared with the Control group,the apoptosis rate of RGC-5 cells in the HG group increased significantly,the protein expression of Bax and Caspase-3 was up-regulated significantly,the level of interleukin-1β(IL-1β),interleukin-6(IL-6),tumor necrosis factor-α(TNF-α),ROS,LDH,and MDA increased significantly,and the level of SOD and GSH-PX decreased significantly(all P＜0.05).Compared with the HG group,the apoptosis rate of RGC-5 cells in the CAR-L,CAR-M,and CAR-H groups de-creased significantly,the protein expression of Bax and Caspase-3 was down-regulated significantly,the level of IL-1β,IL-6,TNF-α,ROS,LDH,and MDA decreased significantly,and the level of SOD and GSH-PX increased significantly(all P＜0.05).Compared with the CAR-H group,the apoptosis rate of RGC-5 cells in the CAR-H+ML385 group increased signifi-cantly,the protein expression of Bax and Caspase-3 was up-regulated significantly,the level of IL-1β,IL-6,TNF-α,ROS,LDH,and MDA increased significantly,and the level of GSH-PX and SOD decreased significantly(all P＜0.05).Compared with the Control group,the p-Akt/Akt expression ratio and the relative expression level of Nrf2 in RGC-5 cells decreased significantly in the HG group(both P＜0.05).Compared with the HG group,the p-Akt/Akt expression ratio and the rela-tive expression level of Nrf2 in RGC-5 cells increased significantly in the CAR-L,CAR-M,and CAR-H groups(all P＜0.05).Compared with the CAR-H group,the p-Akt/Akt expression ratio and the relative expression level of Nrf2 decreased signifi-cantly in the CAR-H+ML385 group(both P＜0.05).Conclusion CAR may reduce the inflammatory response level in RGC-5 cells by activating the Akt/Nrf2 signaling pathway,thus inhibiting oxidative stress-related injury and apoptosis.

Objective To investigate the protective effect of carvacrol(CAR)on high glucose(HG)-induced retinal ganglion cell(RGC)damage by regulating the protein kinase B(PKB,also known as Akt)/nuclear factor erythroid 2-relat-ed factor 2(Nrf2)signaling pathway.Methods The RGC-5 cells in the logarithmic growth phase were inoculated into 96-well cell culture plates with 4 × 104 cells/well and then treated with CAR(5.0 μmol·L-1,10.0 μmol·L-1,20.0 μmol·L-1,40.0 μmol·L-1,80.0 μmol·L-1,and 160.0 μmol·L-1)together with 30.0 mmol·L-1 glucose for 24 hours.Cell viability was detected by the 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide(MTT)assay to screen the ex-perimental concentration of CAR.Then,these RGC-5 cells were inoculated into 6-well plates and randomly assigned into the Control group,30.0 mmol·L-1 glucose(HG)group,low-,medium-,and high-concentration CAR groups(CAR-L group,CAR-M group,and CAR-H group),and high-concentration CAR+Nrf2 pathway inhibitor(ML385)group(CAR-H+ML385 group).Flow cytometry was used to detect the apoptosis of RGC-5 cells.The fluorescent probe was used to de-tect the level of oxidative stress in RGC-5 cells.The enzyme-linked immunosorbent assay(ELISA)was employed to detect the level of superoxide dismutase(SOD),lactate dehydrogenase(LDH),malondialdehyde(MDA),glutathione peroxidase(GSH-PX),and reactive oxygen species(ROS)in the supernatant of RGC-5 cells.Western blot was used to detect the ex-pression of proteins related to the Akt/Nrf2 signaling pathway.Results Compared with the HG group,the survival rate of RGC-5 cells increased significantly when the concentration of CAR was above 20.0 μmol·L-1(all P＜0.05).Therefore,20.0 μmol·L-1,40.0 μmol·L-1,and 80.0 μmol·L-1 of CAR were used for subsequent experiments.Compared with the Control group,the apoptosis rate of RGC-5 cells in the HG group increased significantly,the protein expression of Bax and Caspase-3 was up-regulated significantly,the level of interleukin-1β(IL-1β),interleukin-6(IL-6),tumor necrosis factor-α(TNF-α),ROS,LDH,and MDA increased significantly,and the level of SOD and GSH-PX decreased significantly(all P＜0.05).Compared with the HG group,the apoptosis rate of RGC-5 cells in the CAR-L,CAR-M,and CAR-H groups de-creased significantly,the protein expression of Bax and Caspase-3 was down-regulated significantly,the level of IL-1β,IL-6,TNF-α,ROS,LDH,and MDA decreased significantly,and the level of SOD and GSH-PX increased significantly(all P＜0.05).Compared with the CAR-H group,the apoptosis rate of RGC-5 cells in the CAR-H+ML385 group increased signifi-cantly,the protein expression of Bax and Caspase-3 was up-regulated significantly,the level of IL-1β,IL-6,TNF-α,ROS,LDH,and MDA increased significantly,and the level of GSH-PX and SOD decreased significantly(all P＜0.05).Compared with the Control group,the p-Akt/Akt expression ratio and the relative expression level of Nrf2 in RGC-5 cells decreased significantly in the HG group(both P＜0.05).Compared with the HG group,the p-Akt/Akt expression ratio and the rela-tive expression level of Nrf2 in RGC-5 cells increased significantly in the CAR-L,CAR-M,and CAR-H groups(all P＜0.05).Compared with the CAR-H group,the p-Akt/Akt expression ratio and the relative expression level of Nrf2 decreased signifi-cantly in the CAR-H+ML385 group(both P＜0.05).Conclusion CAR may reduce the inflammatory response level in RGC-5 cells by activating the Akt/Nrf2 signaling pathway,thus inhibiting oxidative stress-related injury and apoptosis.