| Objective:Explore the effect of Wenyang Zhenshuai Granules on the directional differentiation and proliferation of mesenchymal stem cells(BMSCs),and observe the promotion of stem cell survival,proliferation,and differentiation into endogenous cardiac stem cells after MSCs transplantation by Wenyang Zhenshuai Granules;Study the mechanism of action of Wenyang Zhenshuai Granules combined with BMSCs on myocardial hypoxic injury,and reveal the effect and mechanism of Wenyang Zhenshuai Granules on ischemic heart disease based on the theory of "Yang Hua Qi,Yin Shaping" Yin Yang Qi theory.Methods:Experiment 1:10 SD rats were selected to prepare the medicated serum of Wenyang Zhenshuai Granule and the normal control serum.After decapitation,bone marrow mesenchymal stem cells(BMSCs)were isolated and cultured,and the BMSCs were adjusted.According to the different treatment methods,they were divided into blank group(normoxia,DMEM containing20% normal serum,1% P/S),model group(hypoxia,DMEM containing 20% normal serum,1%P/S)Medicated serum-model group(hypoxia,containing 20% medicated serum+20% normal serum,1% P/S DMEM),serum-free model group(hypoxia,containing 1% P/S DMEM),continued to culture under hypoxia for 6 hours,the expression and location of CXCR4 in BMSCs were detected by ICC,the level of SDF-1 in cell supernatant was detected by ELISA,the ultrastructural changes of BMSCs were observed by transmission electron microscopy,the changes of surface structure were observed by scanning electron microscopy,and the cytoskeleton was observed by ghost pen cyclic peptide staining,CCK8 was used to detect proliferation activity,Brd U was used to detect proliferation,and Transwell was used to detect migration ability.Experiment 2: Rat cardiomyocytes H9C2 were maintained and cultured in DMEM medium containing 10% FBS and 1% P/S under conventional conditions.Grouping: A.H9C2+normoxia+normal serum group: DMEM containing 20% normal serum and 1% P/S;B.H9C2+hypoxia+normal serum group: DMEM containing 20% normal serum and 1% P/S;C.H9C2+BMSCs+hypoxia+normal serum group: DMEM containing 20% normal serum and 1%P/S;D.H9C2+BMSCs+hypoxia+drug-containing serum group: DMEM containing 20%drug-containing serum and 20% normal serum and 1% P/S.After 6 hours of cultivation under hypoxic conditions,Western blot was used to detect the expression levels of SDF-1 and CXCR4 in BMSCs of groups C and D;Western blot was used to detect the expression levels of AKT1,p-AKT1,m TOR,p-m TOR,PI3 K,and p-PI3 K in H9C2 of each group.CCK8 was used to detect proliferation activity,and Hoechst staining was used to detect the apoptosis of H9C2 in each group.Results:Experiment 1: CXCR4 was highly expressed in the drug-containing serum-model group,which was higher than that in the model group and serum-free model group(P<0.05);The level of SDF-1 in the cell supernatant of the drug-containing serum-model group was significantly higher than that of the model group and the drug-containing serum-model group(P<0.05);The drug-containing serum-model group had mitochondrial edema,vacuolation,muscle filament thinning and thinning,and the overall inflammatory condition was lighter than that of the model group and the serum-free model group;There were a large number of intercellular substance in the drug-containing serum-model group;The cytoskeletal fibrillary actin in the drug-containing serum-model group showed stronger fluorescence,lighter rearrangement,more stress fibers,and more orderly cytoskeleton;The proliferative activity of drug-containing serum-model group was stronger than that of model group and serum-free model group(P<0.05);The proliferation rate of drug-containing serum-model group was higher than that of model group and serum-free model group(P<0.05);The migration activity of drug-containing serum-model group was stronger than that of model group and serum-free model group(P<0.05);The expression of cyclin D1 in the drug-containing serum-model group was higher than that in the model group and the serum-free model group(P<0.05).Experiment 2: The expression of SDF-1 and CXCR4 in BMSCs of H9C2+BMSCs+hypoxia+drug-containing serum group was significantly higher than that of H9C2+BMSCs+hypoxia+normal serum group(P<0.05);Compared with H9C2+hypoxia+normal serum group and H9C2+BMSCs+hypoxia+normal serum group alone,the expression levels of p-AKT1S473,p-m TOR and p-PI3 K in H9C2 of H9C2+BMSCs+hypoxia+drug-containing serum group were higher(P<0.05),the proliferation activity of H9C2 was higher(P<0.05),and the apoptosis rate of H9C2 was lower(P<0.05).Conclusion:(1)Wenyang Zhenshuai Granule can repair myocardium by regulating the homing and proliferation of BMSCs transplantation by regulating SDF-1/CXCR4.It plays an important role in the chemotaxis,localization and homing of stem cell transplantation in the treatment of heart related diseases.(2)Wenyang Zhenshuai Granule can improve the morphology and structure of BMSCs in hypoxic rats,and improve the proliferation and migration ability of BMSCS.(3)Wenyang Zhenshuai Granule can inhibit the expression of p16ink4 a by inhibiting the expression of Cyclin D1,and finally achieve the effect of inhibiting the cell cycle progression and promoting the proliferation of BMSCs.(4)The high expression of SDF-1 and CXCR4 in combination with Wenyang Zhenshuai Granule and BMSCs under hypoxia may achieve the anti-apoptosis effect of cardiac myocytes by activating PI3K-AKT-m TOR signal pathway.(5)Wenyang Zhenshuai Granule combined with BMSCs can improve the apoptosis of hypoxic damaged cardiomyocytes H9C2 and enhance the proliferation activity of hypoxic damaged cardiomyocytes H9C2.(6)Based on the theory of "Yang Hua Qi,Yin Shaping",the pathogenesis of ischemic heart disease in traditional Chinese medicine is related to the insufficient function of "Yang Hua Qi" leading to excessive "Yin Shaping".Wen Yang Zhen Fai Granules improve the damaged "Yang Hua Qi" function by warming and nourishing Yang Qi,eliminating the Yin evil caused by "Yin shaping",thereby enhancing the ability to induce MSCs to proliferate and differentiate myocardial cells,and improving myocardial hypoxia damage. |
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