Protective Effect Of Naringin On D-galactose Induced Bmscs Senescence

Objective Our study focused on observing the protective effect ofNaringin against senescence induced by D-galactose on BMSCs and theprotective mechanism, discussing Aging related genes(P16，P21，P53andtgfb1mRNA) how to the important role in naringin anti-aging, andproviding theoretical and experimental basis for naringin as an anti-agingtherapy in the future.Methods （1）BMSCs from rats were isolated, cultured and purifiedby the whole bone marrow adherence method, for morphologyobservations, the growth curve was drawn and BMSCs were induced todifferentiate into osteoblasts and adipocytes.（2）The third generation ofBMSCs were treated with different D-galactose concentrations(4﹑8﹑16﹑32﹑64g L)．To investigate the aging effect of D-galactose on damagesof BMSCs by MTT and SA-β-galactosidase staining. Determined byMTT experiment and SA-β-galactosidase staining method,D-galatosecan inducted bone mesenchymal stem cells senescence，and16g L wasthe best．（3）BMSCs were divided into normal control group, aging groupinduced by16g L of D-galactose. The senescent cells were treated witll different Naringin concentrations(10﹣7、10﹣6、10﹣5、10﹣4、10﹣3、10﹣2mmol／L).To observe the protective effect of Naringin on senescentBMSCs induced by D-galactose，the viability of BMSCs were detectedby MTT, the BMSCs aging degree were detected by SA-β-galactosidasestaining, the activity of superoxide dismutase(SOD)in cells weremeasured by kits．And RT-PCR was used to detect the mRNA expressionof P16，P21，P53and tgf‐β1in aging cells．Results （1）BMSCs from rats were spindle cell-based, showingradial colony arrangement. Cells kept strong growth and could passage incontinuous and stable manner over10passages. The growth curve andcell cycle demonstrated that BMSCs were consistent with the growthcharacteristics and good activity of normal cells. Following induction, oilred O staining, alkaline phosphatase staining and alizarin red stainingproduced a strong reaction in cells.（2）The levels of cell viability inaging group(4﹑8﹑16﹑32﹑64g L) were significantly decreased，andthe dyeing rates of SA-β-galactosidase staining in aging group(4﹑8﹑16﹑32﹑64g L) were significantly increased，compared with the normalcontrol group.（3）The level of cell viability in Naringin protection groupand Naringin pretreatment group(10﹣7、10﹣6、10﹣5、10﹣4、10﹣3、10﹣2mmol／L) were significantly increased, and the dyeing rates ofSA-β-galactosidase staining in Naringin protection group (10﹣7、10﹣6、10﹣5、10﹣4、10﹣3、10﹣2mmol／L) were significantly decreased，compared with the aging group (P<0.05). Different dosage groups ofthe Naringin((10﹣7、10﹣6、10﹣5、10﹣4、10﹣3、10﹣2mmol／L) can improvethe SOD activity in senescent BMSCs (P<0.05)，Compared with theaging group，the expression of P16were significantly reduced in Naringin protection group(10﹣7、10﹣6、10﹣5、10﹣4mmol／L), the expression of p21were significantly reduced in Naringin protection group(10﹣7、10﹣6、10﹣5、10﹣4、10﹣3mmol／L),the expression of p21were significantly reduced inNaringin protection group(10﹣7、10﹣6、10﹣5、10﹣4、10﹣3、10﹣2mmol／L).Conclusion Naringin can slow BMSCs aging process, themechanism may be related to the inhibition of P16, P21,P53and tgf‐β1mRNA expression.