| Objective: To investigate the mechanism of Aloe vera polysaccharides(AP) regulates proliferation in human vascular endothelial cellmediated by Ras signaling pathways of VEGFR-2 and promotes deep partial burn wound healing in mice.Method: 1 In vitro study, the human umbilical vein endothelial cells(HUVECs) cultured in Dulbecco’s modified Eagle medium with 10% fetal bovine serum were randomly divided into two groups –AP groups and control group. The HUVECs were treated with different doses of AP concentration(100, 200, 400 mg/L) in AP groups, and without AP as control group. Subsequently, the cells viability and proliferation were assayed by 3-(4, 5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide(MTT), and the morphoses of cells were observed under inverted phase-contrast microscope, and the cell cycle was determined by flow cytometry. The acridine orange-ethidium bromide(AO-EB) double staining was performed to evaluate the toxic effect of AP, and the lactate dehydrogenase(LDH) assay was used in detection AP on the degree of injury HUVECs. 2 The HUVECs of AP groups and control group were blocked the signal pathway by turns: ⑴ Added the half maximal inhibitory concentration(IC50) of SU5416 to block VEGFR-2; ⑵ Added IC50 of farnesyl pyrophosphate(FPP) to block Ras after completely blocked PLC-γ by U73122; ⑶ Without inhibitor as inhibition control group. The cells viability and proliferation were assayed by MTT in each group, and the VEGF and VEGFR-2 levels of the cell supernatant were measured by enzyme-linked immunosorbent assay(ELISA). The signaling protein(VEGFR-2, Ras, Raf, p-Ras and p-Raf) expressions were determined by Western Blot. 3 In Vivo study, one hundred and fifty C57BL/6 male mice were randomly divided into two groups: control group(n=6) and experimental groups(n=144), each mice in the experimental group received one areas of deep-partial thickness burn wounds with 2cm diameter in the dorsolateral regions. The experimental groups were randomly subdivided into 4 treated groups(36 mouse in each treated group). In treated groups, each wound respectively treated with once-daily application of 0.5% AP cream, 1% SD-Ag, blank bases and saline. In control group the sham burn wounds received no medications. The treatments were initiated on day 1 and were applied daily for 21 days. To investigate the process of wound healing, the animals in each group were sacrificed on days 1, 3, 7, 10, 14 and 21d(n=6), respectively, the area of wounds was measured at the same time and the wound closure rates were counted. The VEGF and VEGFR-2 levels in wound tissues were detected by ELISA.Result: 1 The MTT assay showed that the proliferation of HUVECs treated with AP were more active than control group, reaching their highest level on 5th day, but there were no difference in the morphology of the HUVECs among the groups under inverted phase-contrast microscope. Compared with the control group, G0/G1 phase cells decreased significantly as a percentage, and G2/M phase and S phase of the cell occupied the percentage increased significantly(P <0.05); There were no variation observed in the apoptosis or death of HUVECs by AO-EB double staining in each group. Additionally, the LDH leakage rate in each group did not change significantly. 2 ⑴ Compared with control group, the VEGF levels in the culture supernatant were significantly increased in 200 and 400mg/L AP groups(P <0.05-0.01), and the Ras phosphorylation expression of HUVECs was significantly increased(P<0.05). The VEGFR-2, Ras and Raf expression were also significantly enhanced in 100, 200 and 400mg/L AP groups(P <0.05-0.01). ⑵Compared with AP group and control group without inhibitors, the proliferation of HUVECs decreased significantly in corresponding group after IC50 of SU5416 to block VEGFR-2 at 48h(P <0.01), but the VEGF levels in the culture supernatant were clearly increased(P<0.05-0.01), and the VEGFR-2, Ras and Ras phosphorylation expression were significantly decreased(P <0.05-0.01). However, compared with the inhibition control group, the proliferation of HUVECs increased significantly in the inhibition 200 and 400mg/L AP groups(P<0.01), and the VEGF levels in the culture supernatant were significantly increased in the inhibition 100, 200 and 400mg/L AP groups(P <0.05), but only 200mg/L AP group the VEGFR-2 levels were remarkably elevated(P<0.05); The VEGFR-2 expression were significantly enhanced in inhibition 100, 200 and 400mg/L AP groups(P<0.01); The Ras and its phosphorylation expression were clearly enhanced in inhibition 200 and 400mg/L AP groups(P <0.05). ⑶Compared with AP group and control group without inhibitors, the proliferation of HUVECs decreased significantly in corresponding group after IC50 of FPP to block Ras at 48h(P <0.01), and the Ras, Raf and their phosphorylation expression were significantly decreased(P<0.05-0.01). However, compared with the inhibition control group, the proliferation of HUVECs increased significantly in the inhibition 200 and 400mg/L AP groups(P<0.01), and the Ras and Raf expression were significantly enhanced in inhibition 100, 200 and 400mg/L AP groups(P <0.01), and their phosphorylation expression were rising trend. 3 Compared with SD-Ag, blank bases and saline group, AP cream group showed faster wound closure by day 10,14 and 21(P<0.05). At post burn 1, 3, 7, 10 and 21 d, the VEGF levels of wound tissues in AP cream group were higher than that in saline group, and there were the same results at post burn 3, 7 and 21 d compared with SD-Ag and blank bases groups(P <0.05-0.01). However, compared with saline group, the VEGFR-2 levels of wound tissues in AP cream group were significantly increased at post burn 7, 10, 14 and 21 d, but there were only distinguished higher at post burn 14 and 21 d compared with SD-Ag and blank bases groups(P <0.05-0.01).Conclusion: AP is the non-toxic extract which promotes HUVEC proliferation. AP promotes HUVECs to secrete VEGF, VEGFR-2, and enhances the Ras signaling pathway mediated by VEGFR-2, Ras, p-Ras and Raf signaling proteins to promote HUVEC proliferation. AP promotes wound tissue to produce VEGF and increases the VEGFR-2 expression of wound tissue, thereby accelerating wound healing. This indicates that the Aloe vera polysaccharides might play an important role in regulating vascular endothelial cell proliferation mediated by Ras signaling pathways of VEGFR-2 during wound healing. |
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