| [Background/Purposes]Aging is the inevitable law of the gradual loss of cell function in organisms over time.This phenomenon can be concentrated in a certain tissue and organ,and it can cause aging diseases.Chinese medicine emphasizes the importance of kidney in aging.Chinese medicine theory believes that kidney qi is an internal factor that regulates the growth,development and senescence of the organism.In the kidney,aging is related to the structural and physiological changes of the kidney,and there is a linear relationship between kidney aging and the decline of renal function.In this study,we planned to use cisplatin(CDDP)to induce renal aging and injury model to investigate whether quercetin can protect renal cells through anti-aging and its mechanism.[Methods]The rat proximal renal tubular epithelial cells(NRK-52E)were selected as the research object,and the morphological changes of different concentrations of cisplatin(10,20,30,40μmol·L-1)on the cells were observed under a light microscope.Cell proliferation/toxicity detection(CCK-8)method was used to detect cell survival rate,and senescence associated-β-galactosidase(senescence associated-β-galactosidase,SA-β-gal)staining method was used to detect cell senescence,2.7’-Dichlorofluorescein yellow diacetate(DCFH-DA)fluorescent probe to detect intracellular reactive oxygen species(ROS)levels Screen the effects of different concentrations of quercetin on cell proliferation activity,and select the optimal concentration and dose for subsequent experiments.The situation is:normal group,CDDP group(40μmol·L-1),normal+quercetin group(10mmol·L-1),CDDP+quercetin group(40μmol L-1+10mmol·L-1)detection of CDDP against NRK-52E The effect of cell Klotho,p53,p21,p16,p27 protein expression level;observe the effect of CDDP and quercetin on the proliferation activity of NRK-52E cells;observe the effect of quercetin on cisplatin-induced NRK-52E cells Klotho,p53,p21,p16 protein expression level and changes in SA-p-gal staining.[Results]1.The results of CCK-8 showed that after CDDP at different concentrations(10,20,30,40μmol·L-1)intervened in NRK-52E cells,there was no significant difference in the proliferation rate of each group within 3-12 hours.The 40μmol·L-1 group showed obvious inhibition of cell proliferation after 24 hours of exposure,combined with the senescence of cells under light microscope:the number of cells was reduced,the gap was enlarged,the cell morphology was shrunk and deformed,and more than half of the damaged cells were suspended.2.SA-β-gal senescent cell staining showed that with the increase of CDDP concentration,the number of blue-green stained cells continued to increase,indicating that CDDP successfully induced the senescence of NRK-52E cells.3.Western blotting(Western blot)detects the decrease in the expression of the anti-aging protein Klotho,and the increase in the expression of the cell cycle regulatory proteins p53,p21 and p16.4.Compared with the control group,the inhibition of cell proliferation in the quercetin group was improved,the number of suspended cells decreased,the number of blue and green stained SA-β-gal cells decreased,the expression of Klotho protein increased,and the protein expression of p53,p21,and p16 decreased.[Conclusion]CDDP can induce the senescence and damage of NRK-52E cells,and quercetin can delay cell senescence by regulating the p53 signaling pathway to protect renal cells. |
