| Objective:Screening the anti-burn effective extract of Periplaneta Americana(OPA)based on that Periplaneta Americana extracts exist the effect of promoting tissue repair,observing their burn protective effect on scald mice model, studying its anti-burn mechanism through the TGF-β1/Smads signal pathway, to reveal the anti-burn effects and material basis of PA.Methods:(1) Screening the effective extraction:The system solvent extraction was used to prepare each extraction part of the PA. Scald mice model was used to screen anti-burn effective extract of PA through detect the content of Transforming growth factor-β1 (TGF-β1),Interleukin-6 (IL-6) in serum and the rate of wound healing. (2) Validation study the anti-burn of the water extraction from PA:Healthy female ICR mouse were randomly divided into control group, model group, Kangfuxin group, water extract of Periplaneta Americana(WEoPA) low-dose group (0.327 g/kg), middle-dose group (0.490 g/kg) and high-dose group (0.653 g/kg) group according to body weight. The scald model mice were established by scalding the shaved back with hot Weights(82±2℃) for 7 seconds,dermal topical administration of WEoPA at the dosage0.327,0.490,0.653 g/kg were given to mice for 10days, respectively. The rate of wound healing on 5th and 10th day were detected,and the Histopathology of skin was examined to evaluate the effects of WEoPA against scalding in mice. (3) Investigationof mechanisms: ①The levels of TGF-β1 and IL-6 in serum, and the basic fibroblast growth factor (bFGF) and Epidermal Growth Factor (EGF) in skin tissue were detected by ELISA kits, the content of hydroxyproline in skin tissue was detected by alkaline hydrolysis, activity of Superoxide Dismutase (SOD) and content of Methane dicarboxylic aldehyde (MDA) in serum were detected by biochemical method. ②The expression levels of TGF-β1,Smad2 and Smad3 mRNA in skin tissue of scald mice were detected by RT-PCR. ③The protein expression of Smad2 and Smad3 in skin tissue of scald mice skin tissue was detected by Western-Blot.Results:(1) Compared with model group, WEoPA could significantly increase wound healing rate at at 5th,10th and 15th day as well as the content of TGF-β1 at 5th day, and it also can decrease the content of TGF-β1 at 10th,15th day (p< 0.05, p< 0.01) in serum; the Petroleum ether part, Ethyl acetate parts and Chloroform parts of PA could significantly improve wound healing rate at5th,10th day;Ethyl acetate parts significantly reduced the level of TGF-β1 at 15thday, Chloroform parts, waterextracts and 95%alcohol extraction parts from PA could significantly reduce the level of TGF-β1 at 10th day in serum; the Petroleum ether part, Ethyl acetate parts and 95% alcohol extraction parts from PA could significantly improved the content of IL-6 at 5th day,while reducing the content of IL-6 at 15thday (p< 0.05); (2) Compared with the model group,WEoPA significantly improve the scalding wound healing rate.Pathological test results show that WEoPA promote proliferation of skin fibrob-lasts,fibroblast, and capillary vessels in skin of scalded mice as well as promoting the repairation of epidermis, dermis and muscular tissue, and also could improve the disorder of dermal collagen fiber bundles,especially the WEoPA middle-dose group (0.490 g/kg) obviously repair the scalded skin. (3) Compared with model group;WEoPAat the dosage of 0.490 g/kg significantly increased the content or activity of TGF-β1 and SOD in serum at 5th day (p< 0.05,p< 0.01) and EGF and hydroxyproline content of skin tissue at 10th day (p< 0.05,p< 0.01),while decreasing MDA content at 5th day (p< 0.01) and the contents of TGF-β1 and IL-6 in serum at 10thday (p< 0.05); WEoPAat the dosage of 0.327 g/kg significantly improved the activity of SOD in serum(p< 0.05)and contents of EGF and bFGF of skin tissue at 5th day (p< 0.05),while reducingthe contentof TGF-β1 in serum at 10thday (p< 0.05); WEoPA at the dosage of 0.653 g/kg significantly increased the activity of SOD in serum (p< 0.05)and EGF content of skin tissue at 5th day (p< 0.05). (4) Compared with model group, WEoPAat the dosage of 0.490 g/kg,0.653 g/kg significantly increased the expression of Smad2 mRNA in skin tissue (p< 0.05),while at the dosage of 0.490 g/kg significantly increased the expressions of TGF-β1 and Smad3 mRNA in skin tissue at 5thday (p< 0.05); but when it at the dosage of 0.490 g/kg and 0.653 g/kg significantly decreased the expression of TGF-β1, Smad2 mRNA in skin tissue at 10thday (p< 0.05), WEoPA at the dosage of 0.327 g/kg significantly reduced the Smad2 mRNA expressionin skin tissue (p< 0.01).(5) Compared with model group, WEoPAat the dosage of 0.653 g/kg significantly increased the Smad2 protein expressionin scald skin tissue (p< 0.01), while it at the dosage of 0.490 g/kg significantly increasing the Smad3 protein expression level in scald skin tissue at 5th day (p< 0.05).Conclusion:WEoPA is the effective extract part of anti-burn, it has a good protection effects of promoting the rate of wound healing and improving the epidermis and dermisetc damage on scalding in mice. Antioxidation by increasing SOD activity in serum, tissue repairation by increasing hydroxyproline,EGF and bFGF levels in skin and anti-inflammation by reducing the contents of MDA and IL-6 in serum maybe its mechanisms of protecting mice from scalding and TGF-β1/Smads maybe an important signal transduction pathway regulating tissue repairation (promotion at the early time, inhibition in the late). |
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