| Objective The diabetic skin ulcer model established by Streptozotocin(STZ) tail vein injection and dorsal perforating method and the human brain microvascular cells was used as the research object in this study. The mechanism of effect of Toli Xiaodu powder on the wound healing of diabetic skin ulcer was discussed by the whole prescription group intervention and the disassembled prescriptions treatment(tuo disassembled prescription group, tou disassembled prescription group).Methods1. 200 healthy male SD rats were randomly divided into two groups, the control group(40 rats) and the diabetic group(160 rats) after feeding for 7d. Diabetic model was induced by tail vein injection of 50mg/kg(STZ) in diabetic group rats, and the model random blood glucose more than 16.7mmol/L was induced successfully. Death and failure model rats were excluded. Phenobarbital anesthesia after feeding four weeks, and preparation of diabetic skin ulcer model by using the diameter 1cm punch in the back, Death and failure model rats were excluded. The diabetic rats were randomly divided into four groups: model group, tuo disassembled prescription group,tou disassembled prescription group, and the whole prescription group. The whole prescription intervention group were given tuolixiaodu powder decoction(1.5g/kg of Angelica sinensis, Astragalus membranaceus 1.5g/kg, Angelica dahurica 1.2g/kg,Gleditsia sinensis 1.2g/kg) by gavage. The tuo disassembled prescription group were administered with the method of administration(Angelica sinensis 1.5g/kg,Astragalus membranaceus 1.5g/kg) to fill the stomach. The tou disassembled prescription group were given through the method of prescription drugs(Angelica dahurica 1.2g/kg, Gleditsia sinensis 1.2g/kg) by gavage. Rats in the control group and model group were given equal volume of normal saline, with the control of the amount of 2ml in each group. Rats in each group were administered once a day,continuous administration of 14 d. Wound were observed after trauma for 5d, 8d, 11 d,14d to evaluate the healing of ulcer, calculate the rate of wound healing, and to kill and take the ulcer tissue. HE staining was used to observe the structural changes of granulation tissue, and the expression of Ang-1, Ang-2 and Tie-2 were detected by RT-PCR method, Western Blotting method and immunohistochemical method.2. Human microvascular pericytes were cultured in vitro. And the cells were treated with different concentrations of Angelica sinensis, Astragalus membranaceus,Angelica dahurica, Gleditsia sinensis extract to observe the effects of different drug extracts on cell proliferation, migration and apoptosis.Results1.in vivo experiments1.1 The model state: The blood glucose level of 8 rats in the diabetic group(160rats) were not reach the standard of diabetes,and 4 rats died in the diabetic group; so the model forming rate was 92.5%.1.2 The healing rate of each treatment group was higher than that of diabetic group at all time points(P?0.05). 5d after trauma, the healing rate of the tou group was higher than that of the tuo group and the whole group(P?0.05). 8, 11, 14 d after trauma, the healing rate of the whole group was higher than that of the tuo group and the tou group(P?0.05).1.3 The expression levels of Ang-1 and Tie-2 were higher in the whole group than in the diabetic group at different time points, and the expression level of Ang-2 was lower than that of diabetic group(P?0.05). 5, 8d after trauma, the expression level of Ang-2 in the tou group was significantly lower(P?0.05); 11, 14 d after trauma, the expression level of Ang-1 and Tie-2 in the tuo group was upregaulation(P?0.05).2. in vivo experiments2.1 Different concentrations of Angelica sinensis, Astragalus membranaceus,Angelica dahurica can stimulated cell proliferation under high glucose condition(P?0.05); Gleditsia sinensis stimulation has no obvious effect on cell proliferation on the condition of high glucose.2.2. Under high glucose conditions, the migration of the pericytes was significantly inhibited. Angelica dahurica could obviously promote the migration of pericytes inhigh glucose condition(P ? 0.05); Angelica sinensis, Astragalus membranaceus,Gleditsia sinensis promoting effect was not obvious.2.3. Western Blot showed that, under high glucose conditions, the activation of Caspase3 in pericytes was significantly increased, and Astragalus membranaceus could significantly inhibit the activation of Caspase3 in pericytes in high glucose conditions(P?0.05), inhibit the apoptosis of pericytes; Angelica sinensis, Angelica dahurica, Gleditsia sinensis effect is not obvious. RT-PCR showed that, under high glucose condition, the expression of anti apoptotic protein bcl-2 decreased, and the expression of bax was significantly increased. Astragalus membranaceus extract significantly reduced the expression of bax in pericytes in high glucose conditions(P?0.05), increased expression of bcl-2(P?0.05), inhibited the apoptosis of pericytes;the expression of bax and bcl-2 in Angelica sinensis, Angelica dahurica, Gleditsia sinensis intervention group showed no significant difference compared with the hing glouse group.2.4 Coculture of endothelial cells and pericytes in vitro, Angelica sinensis, Astragalus membranaceus, Angelica dahurica could promote the chemotaxis of pericytes to endothelial cells under high glucose condition.The effect of Gleditsia sinensis on promoting the chemotaxis of pericytes to endothelial cells under high glucose condition were not obvious.Conclusions1. The model of diabetic skin ulcer was successfully prepared by the tail vein injection of STZ combined with the back hole drilling method. And the wound healing was delayed in diabetic rats.2. Both the whole tolixiaodu powder and its disassembled prescription can promote the wound healing of diabetic skin ulcer rats, and the effect of the whole group was the most significant.3. The expression of Ang-1 and Tie-2 in the wound of diabetic group was decreased,and the expression of Ang-2 was increased, which resulted in the impairment of the maturation of the new blood vessels, and the wound healing was impaired. The Tou disassembled prescription can cut down the expression of Ang-2 in the early stage ofwound healing, and the Tuo disassembled prescription can increase the expression of Ang-1 and Tie-2 in the late stage of wound healing, which can promote the maturation of the new blood vessels and promote the healing of diabetic wound.4. Human brain vascular pericytes and Human umbilical vein endothelial cells were successfully cultured in vitro. Under high glucose condition, the pericytes proliferation, migration were decreased, and apoptosis were increased, and the chemotaxis of endothelial cells to peripheral blood cells was decreased.5. The four kinds of traditional Chinese medicine extracts in Toli Xiaodu powder can promote the proliferation of pericytes; In addition, Angelica dahurica can significantly promote the migration of pericytes; Astragalus membranaceus can significantly inhibit the apoptosis of pericytes; Angelica sinensis, Astragalus membranaceus, Angelica dahurica can promote chemotaxis of endothelial cells on pericytes. |
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