Lupeol Inhibits Apoptosis Of Rabbit Nucleus Pulposus Cells In High Glucose Environment Through Antioxidative Stress

Objective: Lupinol,a triterpene,originated from Lupinus polyphyllus.Experimental studies have shown that it has a wide range of medicinal values,and has strong antioxidant,hepatoprotective,antimutagenic,anti-inflammatory,anti-rheumatic and anti-tumor effects.In recent years,the incidence of diabetes has increased year by year,and it has become one of the major causes of the health of middle-aged people.The annual increase in the incidence of diabetes,followed by chronic complications have become an important cause of human health such as: chronic vascular injury,hyperlipidemia,limb gangrene and so on.Oxidative stress caused by high glucose environment has been found,and oxidative stress has obvious damage to various organs and tissues of the body.Studies have shown that oxidative stress-induced damage is one of the causes of disc degeneration.The purpose of this research project is: in high glucose environment,lupeol inhibits apoptosis of rabbit nucleus pulposus cells through anti-oxidative stress.Methods: The subject was divided into four groups: control group,high glucose group,lupeol group,high sugar + lupeol group;firstly,the nucleus pulposus cells were isolated and extracted from rabbit intervertebral disc,and cultured.The third generation nucleus pulposus was selected.Cells experiment.The lupolnol medium in a concentration range of 30-70 ?g/ml was divided into 5 groups according to the same concentration gradient,and the optimum concentration of lupeol was screened,and then the intracellular total of each group was further performed by a multi-function plate reader.ROS detection,flow cytometry was used to detect the apoptosis rate in each group,fluorescence quantitative PCR was used to detect the expression of Bcl-2,Bax,cytochrome C,and Caspase 9/3 genes.Finally,Western blot analysis was performed for Bcl-2,Bax and cytochrome C.Expression of Caspase 9/3 protein.Results: The study showed that when the concentration of lupeol was 60ug/mL,there was no obvious anti-apoptosis effect on luteinized nucleus pulposus cells,and no obvious pro-apoptotic effect,and minimal impact on the normal growth of nucleus pulposus cells.The lupolanol group,the high-sugarseed and the lupeol group were pretreated with 60 ug/mL lupolol for 2 hours.Total ROS detection in the cells was performed by a multi-well plate reader.The results showed that the total ROS in the lupeol group,the high-sugar group,and the lupeol group were significantly lower(P< 0.05)and the cells were withered.The mortality rate was also significantly lower in the high glucose group(P< 0.05).The results of apoptosis experiments showed that early apoptosis and late apoptotic rate of high glucose and lupeol in the high glucose group were significantly higher,and the high glucose group compared with the control group showed early apoptosis and late apoptotic rate.Also significantly higher.In addition,lubutol enhanced the mitochondrial anti-oxidative stress status,and the results of fluorescence quantitative PCR showed that the expression of Bcl-2 was up-regulated and the expression of Bax was decreased,and the release of cytochrome C and activation of Caspase 9/3 were inhibited.: The expression of Bcl-2 protein was increased,and the expression of Bax,cytochrome C,and Caspase 9/3 were significantly decreased.Conclusion: The results of this study show that the expression of Caspase-9 and Caspase-3 is significantly increased in nucleus pulposus cells in high glucose environment,and the apoptotic rate of nucleus pulposus cells is also increased.After pretreatment with Lupeol,it is high.In the nucleus pulposus cells in the glycogen environment,the expression of Caspase-9 and Caspase-3 was significantly downregulated.After the detection of cell activity and apoptosis,the nucleus pulposus cells in the high-glucose environment,after luteol pretreatment,the total ROS decreased significantly,oxidative stress injury was inhibited,and the activity of nucleus pulposus cells was significantly enhanced.Therefore,we believe that lubutol has the effect of removing reactive oxygen species and resisting oxidative stress.Lumpol can inhibit mitochondrial anti-oxidative stress and inhibit apoptosis of nucleus pulposus cells in high glucose environment.And it can be used as a potential therapeutic drug to inhibit disc degeneration.