| Objective:In this study,D-galactose(D-gal)-induced mouse cardiac aging model was used to detect myocardial mitochondrial function and oxidative stress levels in order to study the regulation of h UC-MSCs in mouse cardiac aging process.With peroxisome proliferator-activated receptor-γcoactivator-1α(PGC-1α)as the target,the potential mechanism of h UC-MSCs delaying cardiac aging was explored under the intervention of LV-PGC-1αsi RNA.Methods:1.D-gal was used to establish a mouse heart aging model.The mice were40 healthy 8-week-old C57BL/6J male mice,which were randomly divided into 5 groups with 8 mice in each group:Control group,D-gal group,D-gal+h UC-MSCs group,D-gal+h UC-MSCs+LV-GFP group and D-gal+h UC-MSCs+LV-PGC-1αsi RNA group.Except for the Control group,the other groups were injected with D-gal(200mg/kg/d)subcutaneously into the neck every day for 8 weeks to induce the mouse heart aging model.The Control group was injected with the same dose of normal saline.2.h UC-MSCs delays D-gal-induced cardiac senescence in mice.In the above process of D-gal-induced cardiac aging in mice,the D-gal+h UC-MSCs group,D-gal+h UC-MSCs+LV-GFP group and D-gal+h UC-MSCs+LV-PGC-1αsi RNA group,h UC-MSCs dissolved in normal saline(1×10~6/head)were injected through the tail vein at the 5th and 7th weeks respectively,and the remaining two groups were injected with the same amount of normal saline.After the experiment,the changes of mouse heart structure and function were observed by cardiac ultrasound;the Hematoxylin-eosin(HE)staining was used to observe histological changes of mouse heart;the progress of myocardial fibrosis was evaluated by Masson staining;Western blot was used to detect the expression of aging marker p53 protein and fibrosis key signaling molecule transforming growth factor-β1(TGF-β1)in mouse myocardial tissue.3.h UC-MSCs delays D-gal-induced cardiac senescence in mice by regulating PGC-1α.D-gal+h UC-MSCs+LV-GFP group and D-gal+h UC-MSCs+LV-PGC-1αsi RNA group were respectively injected with LV-GFP(1×10~7TU)and LV-PGC-1αsi RNA(1×10~7TU)dissolved in 1×PBS at the fifth week through the tail vein.The remaining groups were injected with the same amount of 1×PBS.After the experiment,Western blot method was used to detect the expression of PGC-1αprotein in mouse myocardial tissue;RT-PCR was used to detect the level of PGC-1αm RNA in mouse myocardial tissue.The content of MDA in serum and myocardial tissue homogenate was detected using a malondialdehyde(MDA)assay kit.Western blot was used to detect the expression of oxidase p47-phox,p67-phox,gp91-phox and antioxidant enzymes catalase(CAT),superoxide dismutase2(SOD2),superoxide dismutase1(SOD1)in mouse myocardial tissue.Results:1.h UC-MSCs ameliorate D-gal-induced cardiac aging in mice.After 8weeks of D-gal intervention,compared with the Control group mice,the left ventricular ejection fraction(EF)and left ventricular fraction shortening(FS)of the D-gal group mice were significantly reduced(P<0.01),and h UC-MSCs inhibited the damaging effect of D-gal on mouse heart EF and FS(P<0.01);after LV-PGC-1αsi RNA silences PGC-1αgene expression,the EF and FS of the mouse heart in the D-gal+h UC-MSCs+LV-PGC-1αsi RNA group decreased(P<0.01).HE results showed that the morphology of myocardial cells in the control group was normal and arranged neatly;the cardiomyocytes of the mice in the D-gal group were hypertrophy and irregularly arranged(P<0.01);after h UC-MSCs treatment,D-gal-induced cell hypertrophy and arrangement disorder were significantly improved(P<0.01);after further intervention by LV-PGC-1αsi RNA,the therapeutic effect of h UC-MSCs was significantly reduced(P<0.01).Masson staining showed that the collagen accumulation in the myocardial interstitium and perivascular area of the D-gal group mice was significantly higher than that in the Control group(P<0.01);h UC-MSCs improved the D-gal-induced myocardial interstitial fibrosis in mice(P<0.01);in contrast,the application of LV-PGC-1αsi RNA once again enhanced the fibrosis of the mouse myocardial interstitium(P<0.01).Western blot results showed that the expression of aging-related protein p53 and the key signaling molecule TGF-β1 in fibrosis increased in myocardial tissue of mice treated with D-gal(P<0.01),while h UC-MSCs inhibited the induction of D-gal on the expression of aging-related protein p53 and the key signaling molecule TGF-β1 in fibrosis in the myocardium of mice(P<0.01);compared with D-gal+h UC-MSCs+LV-GFP group,LV-PGC-1αsi RNA was significantly up-regulated the expression level of aging-related protein p53 and fibrosis key signaling molecule TGF-β1(P<0.01).2.h UC-MSCs ameliorates cardiomyocyte mitochondrial biological function and oxidative stress by up-regulating the expression of PGC-1αto delay D-gal-induced cardiac senescence in mice.RT-PCR and Western blot analysis showed that compared with the control group,the expression of PGC-1αm RNA and PGC-1αprotein in the myocardial tissue of the mice in the D-gal group was significantly reduced(P<0.01);while h UC-MSCs could significantly inhibit the D-gal-induced decrease in the expression of PGC-1αm RNA and PGC-1αprotein;compared with the D-gal+h UC-MSCs+LV-GFP group,PGC-1αm RNA and PGC-1αprotein was significantly decreased in the D-gal+h UC-MSCs+LV-PGC-1αsi RNA group(P<0.01).The detection results of MDA content in serum and myocardial tissue of mice showed that the D-gal group was significantly increased compared with the control group(P<0.01),and the MDA content was significantly reduced after h UC-MSCs treatment(P<0.01);compared with the D-gal+h UC-MSCs+LV-GFP group,the MDA content in the serum and myocardial tissue of the D-gal+h UC-MSCs+LV-PGC-1αsi RNA group increased significantly(P<0.01).Western blot analysis showed that compared with the control group,the expressions of oxidase p47-phox,p67-phox and gp91-phox in the myocardial tissue of the D-gal group were significantly increased(P<0.01),and the expressions of antioxidant enzymes CAT,SOD2,SOD1 were significantly decreased(P<0.01);while h UC-MSCs can significantly improve the D-gal-induced increased expression of oxidase p47-phox,p67-phox,gp91-phox and decreased expression of antioxidant enzymes CAT,SOD2,SOD1(P<0.01);compared with D-gal+h UC-MSCs+LV-GFP group,the expression of oxidase p47-phox,p67-phox and gp91-phox in D-gal+h UC-MSCs+LV-PGC-1αsi RNA group was significantly increased(P<0.01),while the expression of antioxidant enzymes CAT,SOD2,SOD1 was significantly decreased(P<0.01).Conclusion:h UC-MSCs can delay D-gal-induced cardiac senescence in mice.The mechanism may be related to h UC-MSCs increasing the expression of PGC-1αin myocardial tissue,improving the biological functions of mitochondria,and inhibiting oxidative stress. |
PDF Full Text Request