Limonin Reduces the Damage of High Glucose to Nucleus Pulposus Cells by Regulating Apoptosis and Oxidative Stress
Objective Exploring the protective mechanism of Limonin against high glucose(HG)induced damage to nucleus pulposus(NP)cells.Methods Divide human NP cells into control group(0 μmol·L-1,Ctl group),HG group(200 nmol·L-1),HG(200 mmol·L-1)+Limonin(50 μmol·L-1)group(Limonin+HG group).CCK-8 was used to detect cell survival rates after 24 hours of treatment with different concentrations of HG(50,100,200,300 mmol·L-1),and different concentrations of Limonin(12.5,25,50,100,200 μmol·L-1)after 24 and 48 hours of treatment;Western blot was used to detect the expression levels of apoptosis related indicators Bcl-2 and Bax,oxidative stress related indicators Nrf2 expression levels,as well as AKT and p-AKT protein expression levels;Nrf2 was used to detection of nuclear translocation levels;TUNEL was used to detect the level of cell apoptosis.Results CCK-8 assay showed that HG had different effects on the viability of NP cells at different concentrations(50,100,200,300 mmol·L-1).Compared with the Ctl group,NP cell viability decreased at a HG concentration of 200 mmol·L-1(P＜0.05),and a large number of deaths occurred at 300 mmol·L-1(P＜0.01).Therefore,200 mmol·L-1 HG were selected for subsequent experiments.Compared with the Ctl group,different concentrations(12.5,25,50,100,200 μmol·L-1)of Limonin showed different cell viability when co-cultured with NP cells for 24 hours,at 200 μmol·L-1 the activity significantly decreased(P＜0.05);At 48 hours,when the concentration of Limonin were 100 μmol·L-1 the activity of NP cells showed the decreased trend(P＜0.05),When the concentration were 200 pmol·L-1,the vitality significantly decreased(P＜0.05),so 50 pmol·L-1 were selected for further experiments.Western blot results showed that compared with the Ctl group,with nucleus pulposus cells HG treated were damaged,mainly manifested by upregulation of apoptosis related protein Bax/Bcl-2 expression(P＜0.01),downregulation of oxidative stress related protein Nrf2 expression(P＜0.05),downregulation of signaling pathway related protein p-AKT/AKT expression(P＜0.05),and downregulation of Bax/Bcl-2 expression after Limonin pretreatment(P＜0.01);Nrf2 expression was upregulated(P＜0.05);The expression of p-AKT/AKT was upregulated(P＜0.05).The Nrf2 staining results showed that the Nrf2 level in the nucleus of NP cells in the Ctl group remained relatively high in the cytoplasm.After HG induced injury,the Nrf2 positive level in the NP cells were lower and the total number of cells decreased(P＜0.05).Limonin intervention could significantly enhance the significant nuclear translocation of Nrf2 after HG induced injury(P＜0.01).The TUNEL staining results showed that compared with the Ctl group,the number of positive cells in the HG group increased(P＜0.01);Compared with the HG group,after Limonin treatment,the induction of NP cells apoptosis by HG were weakened,and the number of positive cells decreased(P＜0.01).Conclusion Limonin could alleviate the damage of HG to human NP cells by activating the AKT/Nrf2 signaling pathway,and inhibit apoptosis and oxidative stress induced by HG in human NP cells.
万方数据
维普
