| Objective:To investigate the mechanism of Aloe vera polysaccharides(AP) regulates proliferation in human vascular endothelial cells(HUVECs) mediated by PKC signaling pathways of VEGFR-2 and promotes deep partial burn wound healing in mice.Methods:1 In vitro study, the HUVECs cultured in Dulbecco’s modified Eagle medium with10% fetal bovine serum were randomly divided into four groups–0(control group),100, 200 and 400mg/L groups according to the concentration of aloe polysaccharide.The cells ultrastructrue were observed by electron microscope and the proliferation assayed by 3-(4, 5-dimethylthiazolyl-2)-2,5-diphenyltetrazoliumbromide(MTT).The levels of EGFL7, Ang-2, TGF-β1, b FGF and SDF-1α in the supernatant of cultured HUVECs were measured by enzyme-linked immunosorbent assay(ELISA).2 After completely blocked Ras by farnesyl pyrophosphate(FPP), HUVECs randomly divided into control group, AP groups(100, 200 and 400mg/L), inhibitor control group(0+mg/l, Go6983, 10 n M) and AP treated inhibitors groups(100+, 200+, 400+mg/L, Go6983,10 n M) according to the concentration of aloe polysaccharide and PKC inhibitors. The signaling protein(PKC, Erk1/2, p-PKC and p-Erk1/2)expressions were determined by Western Blot and the m RNA(PKC and ERK2)were detected by quantitative PCR.3 One hundred and two male c57bl/6 mouse were randomized into control group(n=6) and four experiment groups(24 mouse in each group) which received scald wound followed by topical application of saline solution, silver sulfadiazine1%(SD-Ag), blank bases and aloes burn cream. The histopathologic changes of wound tissue were observed by hematoxylin and eosin(HE) staining. The TGF-β1and EGFL7 levels of wound tissues were detected on days 3, 7, 10 and 14 by ELISA. The expression of PKC in wound tissue was assayed by immunohistochemistry.Results:1 The cellular structure of HUVECs remained intact among each group and the cells treated with AP were seen to have proliferated actively under the transmission electron microscopy(TEM). The proliferation of HUVECs treated with AP were more active than control group by the MTT assay. The levels of EGFL7,Ang-2and TGF-β1in the supernatant of cultured HUVECs treated with AP were significantly higher than those in control groups by ELISA, and there were no significant change in the levels of b FGF and SDF-1α.2 Compared with control group, the PKC, Erk1/2, p-PKC and p- Erk1/2 expressions of HUVECs were significantly increased in AP groups, while in the blank inhibitor group, they were significantly declined(P<0.05).The expressions of PKC and Erk1/2 protein and their tyrosine phosphorylation were significantly increased in the AP treated inhibitor group than those in the blank inhibitor group(P<0.05).Compared with the control group, the m RNA levels of PKC and ERK2 were significantly increased in the AP group(P<0.05), but they were significantly declined in the blank inhibitor group(P<0.05). The m RNA levels of PKC and ERK2 were significantly increased in the AP treated inhibitor group compared with the blank inhibitor group(P<0.05).3 Re-epithelialization and angiogenesis were significantly improved in aloes burn cream group compared with the other treatments and control groups on post injury day(PID) 10 and 14 with HE staining. The TGF-β1 levels of wound tissue in aloes burn cream group on PID 3 and 7 were significantly higher than those in other treatments and control groups(P< 0.05), as well as the EGFL7 levels on PID 3 and7 in aloes burn cream group(P <0.05). On PID 10, the EGFL7 levels maintained at higher levels in aloes burn cream group compared with SD-Ag and blank bases groups(P <0.05). On PID 7, 10 and 14,the expressions of PKC in aloes burn cream group were significantly higher than those in other treatments and control groups(P <0.05).Conclusion:Aloe Vera polysaccharides can effectively stimulate HUVECs proliferation in vitro and promote to secrete EGFL7、Ang-2and TGF-β1. Its mechanisms are that Aloe Vera polysaccharides can increase PKC expression and phosphorylation and regulate HUVECs proliferation through MAPK cascades, and accelerate tissue microvascular formation and wound healing. |
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