| BackgroundOsteoporosis (OP) is a aging-process-related metabolic bone disease t hat is caused by a variety of factors and marked by a high incidence. Chi na’s rapidly aging society witnesses more and more people are suffering o steoporosis, which not only affects people’s living standard severely, but also imposes huge burdens on medical system economically and socially. Thus, the prevention of osteoporosis should be put high on the agenda wi th the disease’s highly popularity. Statistics indicate the incidence of osteoporosis among sexagenarians (people aging from60to69) is56%, wh ile the incidence among females sexagenarians can be as high as between60%and70%. Osteoporosis ranked the fourth most common chronic diseases in China, with its incidence tending toward a younger population.Oxidative stress (OS) is a major cause of aging body. During the agin g process, the body’s mechanics deteriorate, accompanied by the imbalance in the body’s antioxidant system, intensified oxidative stress in the b ody, thereby causing a range of diseases. When in a hypoxic environment, the body generates reactive oxygen; large accumulation of reactive oxyge n species (ROS) causes cell senescence, apoptosis, and tissue damage. Ag ing body renders mesenchymal stem cells (MSCs) to defunct and enervates t he bone cells’differentiation, which tends to differentiate into adipos ities instead of osteoblasts, leading to age-related bone loss. This cha nge is closely related to the bone marrow microenvironment as well as oxi dative stress. Therefore, new prevention and treatment of osteoporosis ca n be explored by researching how oxidative stress effects the aging of b one marrow MSCs, how we can improve the body’s oxidative stress and promo te osteogenic differentiationObjectives:Firstly, use hydrogen peroxide to induce oxidative stress injury into rats, and then establish in vitro cell aging model to simulate MSCs pat hological changes in bone marrow microenvironment so as to explore how H202effects oxidative aging of MSCs and how this effect relates to MSCs dysf unction in a in vitro environmentSecondly, use three types of kidney recipes (JianGuErXian Pill, LiuWe iDiHuang Pill, JingGuiShenQi Pill) with serum to intervene the aging of M SC model, to throw light on the mechanism of how kidney recipes inhibit MSCs aging, and to better prove the theory that kidney leads the bone and marrow. The method also provides experimental basis for clinical applica tion. Finally, prove that these three types of recipes significantly improve t he marrow microenvironment, suppression of MSCs aging and anti-osteoporosi s effectMethod:1. Culture MSCs in vitro using somatic bone marrow culture method. Af ter rats killed with cervical dislocation, the femur and tibia removed u nder sterile conditions, we cleansed out of the bone marrow with L-DMEM c omplete medium, which were seeded in cell culture flasks. Then we selec ted the most dynamic P3,4MSCs and gathered the cells before using cell viability analyzer to draw out the MSCs growth curve.2. Model MSCs oxidative stress aging with H2O2as inducer. After P3,4MSCs were seeded in96-well plates, we assayed how H2O2with different c oncentrations (100,200,300,400,500,600,700,800,1000μmol·L-1re spectively) affected the MSCs for different lengths of time (2,12,24,48h) with tetrazolium dye MTT. After analyzing MSCs absorbance values, we co mpared the data with the control group so as to select the appropriate mo deling time and concentration for subsequent experiments. Afterwards β-galactosidase (SA-β-gal) was utilized to track MSCs aging so that we cou Id test whether the modeling was successful.3. The influence of three serum-based kidney recipes on metabolism of MSCS when under oxidative stress aging process. First we induced the agin g to MSCs with H2O2:three kidney recipes were added to MSCs with low, med ium and high concentrations respectively(5%,10%,20%)for different le ngths of time (24,48,72h). Subsequently we detected the impact of the th ree serum-based recipes on the metabolism of the MSCs using MTT assay4.The impact of the three serum-based kidney recipes on apoptosis of MSCs duting oxidative stress aging. MSCs modeled24hrs later, we analyzed the apoptosis of the MSCs with flow cytometry analysis, in which the ana lysed MSCs were treated with the three10%serum-containing kidney respec tively.5. The influence of the three serum-based kidney recipes on the aging-related gene (p16INK4, p21CIPI, p53) expression. RT-q real-time PC was applie d to detect the aging-related gene (p16INK4,p21CIPI, p53) expression in the MSCs after the cells were modeled24hrs later.Results:1.The culture of and metaphoric changes in aging MSCs. MCSs growing i n good condition showed fibrous metaphor and vortex growth. After being H2O2-treated, the cells appeared flat and slightly larger, with signs of aging:their nuclear size increased.2.The construction of H2O2-stimulated MSCs oxidative aging model. MTT method analysis showed that after500μmol·L-1H2O2was added to the MSCs24h later, the viability decreased to approximately70%(p<0.05), where as β-galactosidase-positive cells reached to about90%(p<0.01), as co nfirming that MSCs were senescent.3.All the three kidney recipes contained5%of serum and the recipes were added to the cells for24h,48h,72h respectively. Compared with the model group, the differences were statistically significant (P<0.05) as regards to both the24h and48h groups; in contrast, the result of the72h group lacked significant difference (P>0.05). When the concentration was10%and reacting time was24h, each compared with the model group, a11three kidney recipes stimulated the proliferation of MSCs (the differe nces were statistically significant:P<0.05). When the reacting time was48h, compared with model group, the differences were statistically sign ificant (P<0.05), excluding JianGuErXian Pill. When the concentration wa s20%, LiuWeiDiHuang Pill group JingGuiShenQi Pill promoted the MSCs prol iferation (P<0.05), but when with reacting time extended, the drugs star ted to show inhibitory effect on MSCs proliferation (P<0.05). 4. Compared with the control group (apoptosis rate:3.9±3.8%; sur vival:95%±3.9%), the MSC apoptosis rate increased significantly (P<0.01) in the model group (apoptosis rate:69.5%±2.7%; survival rate:69.5%±2.7%). Compared with the model group, JianGuErXian Pill group (apoptosis rate:17.1%±5.4%) LiuWeiDiHuang Pill group (apoptosis ra te:21.3%±3.7%) and kidney JingGuiShenQi Pill group (apoptosis rate:18.9%±2.9%) inhibited MSCs apoptosis rate (the differences were st atistically significant:P<0.05)5.After MSCs were treated with hydrogen peroxide24h later, compared with the control group, the expression of model p21CIPI, p53senescence-as sociated genes had some increment, in which p21CIPI, p53differences showe d statistical significance (P<0.05); compared with the result of the mod el group, the three recipes boosted the expression of p16INK4, the differe nee of which was not statistically significant.(P>0.05). JianGuErXian P ill group, JingGuiShenQi Pill group down regulated the expression of p21c IPI (p<0.05), while LiuWeiDiHuang Pill group up regulated (p<0.05); compa red with the model group, all three kidney medicine dragged down the exp ression of p53(the difference was statistically significant:P<0.05)Conclusion:The three serum-based kidney recipes oxidative metabolic activity can promote MSC vitality, inhibit apoptosis and stymie their senescence by d ownsizing the expression of aging-related genes p21CIPIMSCs and p53. Overal1, the results confirmed the effects of the kidney recipes on improving t he bone marrow microenvironment, in which the kidney benefit recipe and t he kidney Yang recipe has more significant effects... |
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