Effects Of Shengji Xiangpi Mastic On Wound Healing In Rats With Diabetic Ulcer Through BFGF/Akt/Caspase Pathway

Objective:To observe the expressions of basic fibroblast growth factor(b FGF),serine/threonine kinase(Akt)and cystein-asparate protease-9(Caspase-9)in diabetic rat ulcer granulation tissues,in order to investigate the mechanism of Shengji Xiangpi mastic in promoting the ulcer healing in diabetic rats through b FGF/Akt/Caspase pathway.Methods: We used 120 healthy SD male rats.SD rats were divided into two experiment groups randomly according to the weight,the diabetic group(100 rats)and normal control group(20 rats).Rats in normal control group were fed with normal diet.The other rats were fed with high fat diet.After 8 weeks,diabetic rat model was induced by tail vein injection of STZ(30mg/kg)in diabetic group rats,and the normal control group was injected comparable amount of citrate buffer solution(PH 4.2,0.1mmol/L).One week after the injection,the random blood glucose of model rats more than 16.7mmol/L was induced successfully,and the death and failure rats were excluded.All rats were anaesthetised by phenobarbital after adaptive observation about 2 days.Then the diabetic rats skin ulcer model prepared by the punch(2.5cm in diameter)in back.According to the blood sugar levels,the diabetic group rats were randomly divided into three groups: sheng ji xiang pi mastic group(A,22 rats),vaseline group(B,23 rats)and normal saline group(C,22 rats).Normal group is the blank control group(D,20 rats).Four groups of rats were fed adopting single cage free.Replaceing the bedding 2 times a day,changing the gauze 1 time a day,removing the wound crusts,and disinfecting by conventional alcohol.The rats in group A were treated with Shengji Xiangpi mastic gauze compress,those in group B treated with vaseline gauze compress,and those in group C and D treated with physiological saline.The Wound healing condition in each group were observed after trauma for 1,3,7,14,21 d,and reserve the ulcer tissue.Use hematoxylin-eosin(HE)staining to observe the wound granulation tissue morphological changes.Real-time PCR and Western Blot was used to analyze the expression of b FGF,ELISA was used to analyze the expression of p-Akt,and immunohistochemistry was used to analyze the expression of Caspase-9.Results:(1)We observed the morphological change of the skin ulcer under the light microscope.Compared with the normal control group,inflammatory response of diabetic group rats was delayed and lasted for a long time.To sheng ji xiang pi mastic group,wound granulation tissue matured faster,new blood vessels and fibroblasts appeared earlier and more than other groups.Those provides premise for the accumulation of collagen fiber,reticular fiber and elastic fiber,which were wound repair material,auxiliary to wound repair.(2)Real-time PCR and Western Blot was used to analyze the expression of b FGF.The expression level of b FGF of sheng ji xiang pi mastic group on 3d,7d aftert the trauma were obviously higher than that of saline group,vaseline group(P<0.05).It is 3d,7d that the peak expression of b FGF in sheng ji xiang pi mastic group,and the expression trend was consistent with the normal control group.(3)ELISA was used to analyze the expression of p-Akt.The content of p-Akt of all groups was increased first,but falling later,and the peak expression is at 3d.1d,the expression of p-AKT in diabetic groups was lower than the normal control group(P<0.05).On 3d,7d,14 d,the expression of p-AKT in sheng ji xiang pi mastic group was significant increaseing than that in saline group and vaseline group(P<0.05).(4)Immunohistochemistry was used to analyze the expression of Caspase-9.1d,the expression of Caspase-9 in diabetic groups was incresing than the normal control group(P<0.05).On 3d,7d,14 d,21d,the expression of Caspase-9 in sheng ji xiang pi mastic group was significant decreaseing than that in saline group and vaseline group(P<0.05),which have no difference with the normal group.Conclusion:(1)Diabetes can reduce the activity of b FGF.But sheng ji xiang pi mastic can raise the level of b FGF,which can combine with its receptor,and promotes the division,proliferation and migration of fibroblasts cell,then promotes wound healing.(2)At the same time,b FGF can active PI3K/Akt signaling pathway,promote the expression of p-AKT,promote proliferation and migration of fibroblasts and endothelial cell,then promotes angiogenesis and wound healing.(3)Sheng ji xiang pi mastic can reduce the Caspase-9 activation through the raising p-AKT.While decreasing Caspase–9,suppress cascade amplification of caspases,reducing the apoptosis of cells and promoting wound healing.