| Objective:To establish a photoaging model of human immortalized keratinocytes(HaCaT cells)irradiated by ultraviolet B(UVB),study the protective effect of Choerospondias axillaris fruit extracts on oxidative stress damage of HaCaT cells,and provide experimental basis for the treatment of skin photoaging with Choerospondias axillaris fruit extracts.Methods:1.Thin layer chromatography was used to identify the extract of Choerospondias axillaris fruit.2.Conduct in vitro culture of HaCaT cells.3.The CCK8 method was used to screen the dose of photoaging cell model and the optimal drug concentration of Choerospondias axillaris fruit extract on photoaging cells.Different doses(0m J/cm~2,15m J/cm~2,30m J/cm~2,45m J/cm~2,60m J/cm~2)of UVB were used to irradiate HaCaT cells,and the cell morphology was observed under the microscope.The OD value was measured by an enzyme-linked immunosorbent assay to calculate cell survival rate and determine the modeling dose;Apply a series of concentration gradients of Choerospondias axillaris fruit extract(100μg/ml、200μg/ml、300μg/ml、400μg/ml、500μg/ml、600μg/ml、700μg/ml、800μg/ml、900μg/ml)acts on HaCaT cells and observes whether the drug has a toxic effect on the cells;Select a series of concentration gradient drugs with no toxic effects on cells(200μg/ml、400μg/ml、600μg/ml、800μg/ml),acting on photoaging cells,to screen the optimal drug concentration.4.Set up groups:blank control group,UVB irradiation group,and Choerospondias axillaris fruit extract+UVB irradiation group.5.TBA and WST-8 methods were used to detect the changes in MDA and SOD expression in HaCaT cells between different groups.6.ROS stainig was used to detect the changes of oxygen free radicals in cells of each group 7.RT q PCR method was used to detect the changes in the expression of Nrf2,HO-1,and GPX4m RNA in each group of cells.Result:1.The color rendering of the Choerospondias axillaris fruit extract and the control sample was at the same level,which can be determined.2.The morphological changes of HaCaT cells were observed under a microscope under different irradiation doses of UVB.As the irradiation dose increased,the cells gradually became round and smaller,and the number of cells significantly decreased;The analysis of CCK8 detection results showed that based on the OD values measured in the Enzyme marker,there were statistical differences between each group and the blank control group.The calculated IC50 value determined that the modeling dose was30m J/cm~2;Through the action of different concentrations of Choerospondias axillaris fruit extract on HaCaT cells in each group,it was found that Choerospondias axillaris fruit extract had a concentration of 900μg/ml,it has a toxic effect on cells(P<0.05),so this concentration is not selected;Different concentrations of jujube extract without cytotoxicity were applied to the photoaging cell model,and it was found that when the concentration of Choerospondias axillaris fruit extract was 400μg/ml,the photoaging cytoprotection is the most obvious,so this concentration is selected for subsequent experiments.3.The Enzyme marker results showed that400μg/ml was given in advance in HaCaT cells irradiated with Choerospondias axillaris fruit extract,the MDA value was significantly lower than that of the UVB group,and the SOD expression was significantly higher than that of the UVB group.4.ROS staing was used to detect the change of oxygen free radicals in the cells.Compared with the normal group,the ROS content in the cells in the model group was significantly up-regulated by irradiation stimulation of ROS production.Compared with the model group,the ROS in the HaCaT cells treated with Axillary choerospondias fruit extracts was significantly decreased in the treatment group.5.RT-q PCR was used to detect the expression changes of Nrf2,HO-1,and GPX4m RNA in each group of cells.The experimental results showed that the Nrf2,HO-1,and GPX4m RNAs in the Choerospondias axillaris fruit extract+UVB irradiation group were higher than those in the UVB group and the normal group,with a statistically significant difference(p<0.05).Conclusion:1.Establish a photoaging model of HaCaT cells under UVB irradiation,and determine the irradiation dose as 30m J/cm~2.2.The optimal drug concentration for screening the protective effect of Choerospondias axillaris fruit extract on photoaging cells is 400μg/ml。3.Choerospondias axillaris fruit extract can reduce the expression of oxidative stress indicator MDA and ROS,increase the expression of SOD in UVB irradiated photoaging cells.4.Choerospondias axillaris fruit extract may have a protective effect on photoaging cells by upregulating the Nrf2、HO-1 and GPX4m RNA. |
