Objective To explore the effect and mechanism of rapamycin and deferoxamin on wound healing after ischemia and hypoxia. Methods The model of ischemia and hypoxia wound was made on the back of 40 SPF male adult Sprague Dawley rats, weight (300±20) g; they were randomly divided into 4 groups (n=10): the control group (group A), deferoxamine intervention group (group B), rapamycin intervention group (group C), and deferoxamine+rapamycin intervention group (group D). At 3, 6, and 9 days after model preparation, rats of groups A, B, C, and D were intra-peritoneally injected with normal saline, deferoxamin (10 mg/kg), rapamycin (3 mg/kg), deferoxamin (10 mg/kg)+rapamycin (3 mg/kg) respectively. The wound healing was observed and the healing time was recorded in each group; the wound healing tissue was harvested to test the mRNA and protein expressions of mammalian target of rapamycin (mTOR), hypoxia inducible factor 1α (HIF-1α), and vascular endothelial growth factor (VEGF) by real-time fluorescence quantitative PCR and Western blot at 2 days after wound healing. Results All rats survived to the end of the experiment, and wounds healed; the healing time of groups A, B, and D was significantly shorter than that of group C (P<0.05), but there was no significant difference between groups A, B, and D (P>0.05). Real-time fluorescence quantitative PCR showed that the expression of mTOR mRNA in groups C and D was significantly decreased when compared with the expressions in groups A and B (P<0.05); there was significant difference between groups A and B (P<0.05), but no significant difference between groups C and D (P>0.05). The expressions of HIF-1α mRNA and VEGF mRNA were signi-ficantly higher in groups B and D than groups A and C, and in group A than group C (P<0.05), but there was no signifi-cant difference between groups B and D (P>0.05). Western blot showed that the relative expressions of mTOR protein in groups C and D were significantly decreased when compared with the expressions in groups A and B (P<0.05), but there was no significant difference between groups C and D (P>0.05). The relative expressions of HIF-1α protein in groups A, B, and C were significantly increased when compared with expression in group D (P<0.05), but there was no significant difference between groups A, B, and C (P>0.05). The relative expression of VEGF protein were significantly lower in groups B, C, and D than group A, in group D than groups B and C, and in group C than group B (P<0.05). Conclusion Defe-roxamin can promote the wound healing of rats after ischemia and hypoxia, and the effect of rapamycin is opposite. It may be related to the existence of mTOR and HIF-1 signaling pathway in chronic ischemia-hypoxia wound.
ObjectiveTo investigate the expression and correlation of hypoxia inducible factor 1α (HIF-1α) and autophagy related molecules (Beclin1 and LC3B) in rat nucleus pulposus cells under hypoxia in vitro.MethodsThe nucleus pulposus cells were extracted from the nucleus pulposus of healthy adult Sprague Dawley rats and passaged. The 3rd generation cells were identified by HE staining and collagenase type Ⅱ immunofluorescence staining and randomly divided into 4 groups. The cells in group A were cultured for 8 hours under normal oxygen condition (37℃, 5%CO2, 20%O2); the cells in group B were cultured for 8 hours under hypoxia condition (37℃, 5%CO2, 1%O2); the cells in group C were transfected with HIF-1α-small interfering RNA and cultured for 8 hours under hypoxia condition; and the cells in group D were cultured with autophagy inhibitor 3-MA for 8 hours under hypoxia condition. Western blot and real-time fluorescence quantitative PCR (qRT-PCR) were used to detect the expressions of HIF-1α and autophagy related molecules (Beclin1 and LC3B) in all groups.ResultsHE staining of the 3rd generation nucleus pulposus cells showed that the cytoplasm was light pink and the nucleus was blue black, and the collagenase type Ⅱ immunofluorescence staining was positive. Western blot and qRT-PCR results showed that the relative expressions of HIF-1α, Beclin1, and LC3B proteins and genes in group B were significantly higher than those in group A (P<0.05); the relative expressions of HIF-1α, Beclin1, and LC3B proteins and genes in group C were significantly lower than those in group B (P<0.05). There was no significant difference in the relative expression of HIF-1α protein and gene between groups B and D (P>0.05); while the relative expressions of Beclin1 and LC3B proteins and genes in group D were significant lower than those in group B (P<0.05).ConclusionHypoxia can induce the expressions of HIF-1α and autophagy related molecules (Beclin1 and LC3B) in rat nucleus pulposus cells, and HIF-1α in rat nucleus pulposus cells under hypoxia is related to the expression of autophagy related molecules, that is, down-regulation of HIF-1α can significantly reduce the expression of autophagy related molecules, while the down-regulation of autophagy levels under hypoxia has no or little effect on the expression of HIF-1α.
ObjectiveTo investigate the effects of hypoxia inducible factor 1α (HIF-1α) overexpression on the differentiation of stem cells derived from human exfoliated deciduous teeth (SHED) into vascular endothelial cells.MethodsSHED was isolated from the retained primary teeth donated by healthy children by using collagenase digestion method. The third generation cells were identified by flow cytometry and alizarin red and alkaline phosphatase (ALP) staining after osteogenic differentiation culture. The SHED were divided into blank control group (SHED without any treatment), empty group (SHED infected with empty lentivirus), HIF-1α overexpression group (SHED infected with HIF-1α overexpression lentivirus), Wnt inhibitor group (SHED interfered by IWR-1), and combination group (HIF-1α overexpressed SHED interfered by IWR-1). Real-time fluorescence quantitative PCR (qRT-PCR) and Western blot were used to analyze the expressions of HIF-1α mRNA and protein in the SHED of blank control group, empty group, and HIF-1α overexpression group. Then the SHED in 5 groups were induced differentiation into vascular endothelial cells for 14 days. The expressions of cell surface marker molecule [von Willebrand factor (vWF) and CD31] were detected by flow cytometry. The mRNA expressions of vascular cell adhesion protein 1 (VCAM-1), KDR (Kinase-inserted domain containing receptor), and VE-cadherin (VE) were analyzed by qRT-PCR. The protein expressions of phosphate-glycogen synthasc kinase 3β (p-GSK3β) and β-catenin were analyzed by Western blot. The tube forming ability of induced cells was detected by Matrigel tube forming experiment. The ability of endothelial cells to phagocytic lipid after differentiation was detected by DiI-labeled acetylated low density lipoprotein (DiI-Ac-LDL) phagocytosis.ResultsAfter identification, the cells were SHED. After lentivirus transfection, compared with the blank control group and the empty group, the expressions of HIF-1α mRNA and protein in the HIF-1α overexpression group increased significantly (P<0.05). Compared with the blank control group and the empty group, the expressions of VCAM-1, KDR, and VE mRNA, the percentages of vWF positive cells and CD31 positive cells, and the relative expression of β-catenin protein were significantly higher (P<0.05), the relative expression of p-GSK3β protein was significantly lower (P<0.05), the number of tubules formed and the ability to phagocytic lipids significantly increased (P<0.05) in the HIF-1α overexpression group; while the indicators in the Wnt inhibitor group were opposite to those in the HIF-1α overexpression group (P<0.05). Compared with the HIF-1α overexpression group, the expressions of VCAM-1, KDR, and VE mRNA, the percentages of vWF positive cells and CD31 positive cells, and the relative expression of β-catenin protein were significantly lower (P<0.05), the relative expression of p-GSK3β protein was significantly higher, and the number of tubules formed and the ability of phagocytosis of lipids significantly reduced, showing significant differences between groups (P<0.05).ConclusionOverexpression of HIF-1α can promote SHED to differentiate into vascular endothelial cells by activating Wnt/β-catenin signaling pathway.