| OBJECTIVE Stem cell aging is the key reason for the latest theory of aging and age-related diseases, looking for ways to regulate stem cell senescence delaying aging and preventing critical diseases in the elderly.Hematopoietic stem/progenitor cells of aging and the aged diseases are closely related.We have demonstrated that Angelica Sinensis Polysaccharide is an important anti-aging ingredients of Angelica sinensis, the "blood and Qi" way to control and delay the senescence of HSC/HPCs; antagonistic and repair agent on HSC/HPCs damage induced by D-galactose; can effectively protect the immune system and hematopoietic system. So far, the research showed the mechanism of ASP regulation of HSC/HPCs aging is not clear. The stem cell aging theories and traditional Chinese medicine theory of aging closely together, through the construction of HSC/HPCs aging model in vitro and in vivo.The study of ASP delay HSC/HPCs senescence and the molecular mechanism of ASP control key target of Wnt/p-catenin signaling pathwayMETHODS1. Sca-1+ HSC/HPCs were separated and purified by immune magnetic-activated cell sorting, MACS) technology. The cell purity was measured by flow cytometry.2.Separation and purification of Sca-1+HSC/HPCs were randomly divided into control group, aging model group, signal pathway blocker group,antioxident group,ASP anti-aging group.①Detection of related biological indicators of aging:the ratio of the SA-β-Gal staining positive cell were counted; the capability of colony formation was examined by CFU-Mix cultivation; the content of advanced glycosylation end products(AGEs) were detected in cell culture supernatant by Elisa;The mRNA expression of p16, p21 and the protein expression of P16、P21、P53 and Rb was detected.②Oxidation test:the distribution of ROS levels was analyzed by FCM and LSCMA; the total antioxidant capacity (T-AOC) of the cell were detected by enzymatic assay;The content of 8-OHDG and 4-HNE of DNA and lipid oxidative damage markers were detected.③Detection of Wnt/β-catenin signaling pathway related proteins and genes:The mRNA expression of β-catenin、c-myc and the protein expression of β-catenin、GSK-3β、Phospho-GSK-3p and TCF-4 were detected. 3.Male C57BL/6J mice aging 6 to 8 weeks were randomly divided into normal group aging model group、ASP aging model group and vitamin E group. Detection index:the same.RESULTS 1. The purity of Sca-1+ HSC/HPCs was only (89.83±4.26)% before MACS. But the percentage of separated Sca-1+HSC/HPCs from MNCs was (11.83±3.05)% after MACS. These results suggest that the separated Sca-1+HSC/HPCs have a higher purity.2.The experimental results in vitro:①In the aging model group,the percentage of SA-β-Gal and the content of AGEs in cell culture supernatant were significantly increased. The colony formation of CFU-Mix was markedly decreased. The expression of p 16、p21、β-catenin、c-myc mRNA were markedly increased. The expression of P16、P21、P53、Rb、β-catenin in cytoplasm、β-catenin in nucleus、Phospho-GSK-3β and TCF-4 were evidently up-regulated. But the expression of GSK-3β was evidently down-regulated. The product of ROS、8-OHDG and 4-HNE were significantly reduced.However,the T-AOC of the cell was significantly promoted.②In the signal pathway blocker group, the percentage of SA-P-Gal and the content of AGEs in cell culture supernatant were significantly decrease. The colony formation of CFU-Mix was markedly increased. The expression of p16、p21、β-catenin、c-myc mRNA were markedly increased. The expression of P16、P21、P53、Rb、β-catenin in cytoplasm、β-catenin in nucleus、Phospho-GSK-3β and TCF-4 were evidently down-regulated. But the expression of GSK-3P was evidently up-regulated. The product of ROS、8-OHDG and 4-HNE were significantly droped.However,the T-AOC of the cell was significantly rised.③In the antioxident group, the result of indices of oxidation and key targets of Wnt/β-catenin signaling pathway conformed to signal pathway blocker group. However,the result of indices of related biological indicators of aging had no significant change,except the percentage of SA-P-Ga1、the content of AGEs、the expression of p16、p21 mRNA and the expression of P21、Rb.④In the ASP anti-aging group, the result of indices of related biological indicators of aging、Oxidation and key targets of Wnt/p-catenin signaling pathway conformed to signal pathway blocker group.3. The experimental results in vivo:①In the aging model group,the percentage of SA-β-Gal and the content of AGEs in cell culture supernatant were significantly increased. The colony formation of CFU-Mix was markedly decreased. The expression of p16、p21、β-catenin、c-myc mRNA were markedly increased. The expression of P16、P21、P53、Rb、 β-catenin in cytoplasm、β-catenin in nucleus、Phospho-GSK-3β and TCF-4 were evidently up-regulated. But the expression of GSK-3β was evidently down-regulated. The product of ROS、8-OHDG and 4-HNE were significantly reduced.However,the T-AOC of the cell was significantly promoted.②In the ASP aging model group,the percentage of SA-β-Gal and the content of AGEs in cell culture supernatant were significantly decrease The colony formation of CFU-Mix was markedly increased. The expression of p16、p21、β-catenin、c-myc mRNA were markedly increased.The expression of P16、P21、P53、Rb、β-catenin in cytoplasm、β-catenin in nucleus、Phospho-GSK-3β and TCF-4 were evidently down-regulated. But the expression of GSK-3β was evidently up-regulated. The product of ROS、8-OHDG and 4-HNE were significantly droped.However,the T-AOC of the cell was significantly rised.③In the vitamin E group,The product of ROS、8-OHDG and 4-HNE were significantly droped.However,the T-AOC of the cell was significantly rised. The result of indices of related biological indicators of aging and Wnt/p-catenin signaling pathway related proteins and genes had no significant change.CONCLUSIONSl.Ox-LDL can be used as an effective aging agent, which activates the Wnt/β-catenin signaling pathway through combined with low density lipoprotein receptor related protein 5/6 (LRPS5/6) of the co receptor of Wnt/β-catenin signaling pathway, and then triggers the p16INK4a/Rb and p53/p21 pathways, eventually leading to senescence of Sca-1+HSC/HPCs.2.The results showed that ASP can effect the activation Wnt/β-catenin signaling pathway by inhibition of oxidative stress, delay the senescence of Sca-1+HSC/HPC in vitro and in vivo.Objective To explore the effects and the relative mechanism of angelica sinensis polysaccharide (ASP) on hematopiesis in aging model rats, offer theoretical and experimental evidences for seeking effective natural medicine of antiaging or protecting hematopoietic function. Methods male SD rats (n=40) aging 6 to 8 weeks were randomly divided into normal group(NG, n=10), ASP normal group(ANG, n=10), aging model group(AMG,n=10) and ASP aging model group(AAMG,n=10). After 2 days of finishing the treatment, the eyeball blood was collected to detect peripheral blood routine; The femur was taken to count the number of each femur BMNCs, the proliferation of BMNCs was detected by CCK-8;the distribution of cell cycles and ROS levels was analyzed by flow cytometry(FCM);the capability of colony formation was examined by CFU-Mix cultivation; the ratio of the SA-β-Gal staining positive BMNCs were counted; the total antioxidant capacity of the cell were detected by enzymatic assay;the aging related proteins of P53 and P21 were detected by Western blotting. Results:Compared with the aging model group, in the ASP aging model group,the decrease in the amounts of the peripheral blood RBC、PLT and WBC was evidently inhibited; the proliferation of the BMNCs was enhanced;the colony formation of CFU-Mix was markedly increased; the percentage of SA-β-Gal, the ratio of G1 stages and the product of ROS positive cells was significantly reduced; the ratio of S stages was markedly promoted; the total antioxidant capacity of the cell was significantly promoted; the expression of the aging related protein of P53 and P21 was evidently down-regulated. Conclusions:ASP can protect hematopoietic function from decline by antagonize D-galactose that induced oxidative stress, suggesting the mechanism of ASP protecting hematopiesis may be regulate p53/p21 pathway. |
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