| Purpose: To explore the protective effect of DL-3-n-butylphthalide(NBP)on oxidative stress damage induced by high glucose in rat retinal müller cells and investigate its specific mechanism.Materials and Methods:(1)The rat retinal müller cells was co-incubate with different concentrations of NBP(0.1μM,1μM,10μM,20μM,50μM,100μM)or glucose(5.5 mM,15 mM,25 mM,35 mM,45 mM,55 mM,65 mM)for 24 hours,and the safe concentration of NBP and the optimal high glucose-induced concentration were screened by CCK-8reagent.(2)According to the experimental design,the cells were divided into the following 5 groups: a control group(group C)cultured in 5.5mM low glucose medium;a butylphthalide group(group BP)cultured in 10 μ M butylphthalide and 5.5mM low glucose medium;a high glucose group(group HG)cultured in 35 mM high glucose medium;a high glucose combined with butylphthalide group(group HB),cultured in10μM butylphthalide and 35 mM high glucose medium;a brusatol group(group BT),in which cells were pretreated with 300 nM brusatol(Nrf2 inhibitor)for 2h before high glucose combined with butylphthalide intervene.(3)Cell viability was detected by CCK-8 assay,and changes in cell morphology were observed by optical microscope.Cell apoptosis changes was detected by Hoechst 33342 staining assay.ROS levels were quantitatively detected by reactive oxygen kit.The mRNA expressions of Bax,Bcl-2,Nrf2 and HO-1 were detected by PCR.The expressions of Nrf2 and HO-1 were detected by Western blot,and the expression and localization of Nrf2 were detected by cellular immunofluorescence.Results:(1)Compared with the group C,there was no significant difference(all P > 0.05)in cell survival rate when NBP concentration was ≤100μM.When glucose concentration was≥25mM,cell survival rate decreased with increasing glucose concentration(all P <0.05),and a significant decrease was observed starting at 35 mM(P < 0.001).Therefore,10 μ M concentration of NBP was selected as the drug concentration for subsequent experiments,and 35 mM concentration of glucose was selected as the high glucose-induced concentration for subsequent experiments.(2)There was no significant difference in cell survival rate between group BP and group C(P > 0.05).The cell survival rate in group HG was significantly lower than that in group C(P < 0.05).The cell survival rate in group HB was significantly improved compared with group HG(P < 0.05).Microscopic observation of cell morphology showed that the cells in group C and group BP grew well and exhibited an elongated spindle shape with abundant cytoplasm.Compared with group C,the cell state of group HG was worse,with abnormal cell morphology and more elongated shape.Compared with group HG,cells in the group HB showed significant improvement in cell state,and the cell morphology was close to normal cell.(3)Hoechst 33342 staining showed that the nucleus in group BP and group C appeared round and dark blue,while nucleus in the group HG appeared bright blue with nuclear condensation and deformation.In contrast,nucleus in the group HB appeared mostly dark blue,and the nuclear morphology was close to normal.The results of PCR detection of apoptotic genes showed that there was no significant difference in the Bax/Bcl-2 mRNA ratio between group BP and group C(P > 0.05),the Bax/Bcl-2 mRNA ratio in group HG increased compared with group C(P < 0.05),and the Bax/Bcl-2 mRNA ratio in group HB decreased compared with group HG(P < 0.05).(4)ROS detection showed that there was no significant difference in intracellular ROS level between the group BP and group C(P > 0.05).The intracellular ROS level in group HG was significantly up-regulated compared with that in group C(P < 0.05).The intracellular ROS level in group HB was significantly down-regulated compared with group HG(P <0.05).(5)The mRNA and protein expressions of Nrf2 and HO-1 in group BP markedly increased compared with those in group C(all P < 0.05).The protein expression of Nrf2 in group HG increased compared with that in group C(P < 0.05),the mRNA and protein expressions of HO-1 increased(all P < 0.05).The mRNA and protein expressions of Nrf2 and HO-1 further increased in group HB compared with group HG(all P < 0.05).(6)Immunofluorescence showed that Nrf2 protein fluorescence in the group C was weak and mainly concentrated in the cytoplasm.In the group BP,Nrf2 protein fluorescence was significantly enhanced compared to the group C and mainly concentrated in the nucleus.In the group HG,Nrf2 protein fluorescence was enhanced compared to the group C,with partial expression in the nucleus.In the group HB,Nrf2 protein fluorescence was further enhanced in the nucleus compared to the HG group.(7)After BT pretreatment,compared with group HB,the mRNA and protein expressions of Nrf2 and HO-1 prominently decreased(all P < 0.05),the fluorescence of intracellular apoptosis staining was prominently enhanced,with an increased proportion of nuclear condensation and deformation.Additionally,Bax/Bcl-2 mRNA ratio prominently increased(P < 0.05),and the intracellular ROS level was prominently elevated(P < 0.05).Conclusions:(1)The level of apoptosis and oxidative stress in retinal müller cells increased under high glucose environment.(2)NBP alleviated apoptosis and oxidative stress in high glucose-induced retinal müller cells.(3)NBP exerted a protective effect on high glucose-induced retinal müller cells through the Nrf2/HO-1 signaling pathway. |
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