| Objective:To investigate the scientific hypothesis that Tuolixiaodu powder can promote diabetic skin ulcer healing by regulating the inflammatory response.By establishing a diabetic skin ulcer(DSU)model,the experimental effect of Tuolixiaodu powder in treating DSU was examined on the one hand;on the other hand,the mechanism of Tuolixiaodu powder modulating the inflammatory response and promoting the DSU wounds healing was initially investigated.This study aimed to provide a theoretical basis for the use of Tuolixiaodu powder in intreating DSU and to provide a new strategy of Chinese medicine for the regulation of high glucose-induced inflammation in DSU by oral traditional Chinese medicine decoction.Methods:Part Ⅰ:A type 2 diabetic SD rat model was constructed by high-fat diet plus intraperitoneal injection of 1%plastid ratio of streptozotocin solution,on which a diabetic skin ulcer model was further constructed by surgery.All rats were randomly divided into a blank group(normal rats with skin ulcers),model group,positive control group,LPS(Lipopolysaccharide,LPS)group,LPS Chinese medicine group(medium-dose Chinese medicine),low-dose Chinese medicine group,medium-dose Chinese medicine group,and high-dose Chinese medicine group,8 rats in each group.The low,medium,and high-dose Chinese medicine groups were given 1.575g/(kg·d),3.15g/(kg·d),and 6.3g/(kg·d)Tuolixiaodu powder,respectively.In the LPS group and the LPS Chinese medicine group,1 mg/(kg·d)LPS caudal vein injection and 1 mg/(kg·d)LPS caudal vein injection+3.15 g/(kg·d)traditional Chinese medicine gavage were administered,respectively.The blank group and the model group were gavaged with the corresponding doses of distilled water,and the positive control group was gavaged 0.4 μg/kg beraprost sodium tablet solution.The blank group was fed with an ordinary diet and the rest groups were fed with the high-fat diet.The wound dressing was changed every other day and covered with Vaseline gauze and bound with a sterile dressing.Four rats were sampled from each group on the 10d and 14d after the intervention.The mental activity,hair color,uniformity,water drinking,urination,and body weight of the rats in each group were observed during the experiment.The body weight and random caudal apex blood glucose of the rats were measured regularly.The ulcers of 4 rats in each group were taken for examination on the 10th and 14th day after treatment,respectively.The wound healing rate was calculated on 10d and 14d,and the tissue morphology of the wounds was also performed by HE staining.Part Ⅱ:Animal modeling and experimental grouping were the same as in Part 1.The infiltration of M1 and M2 macrophages was observed by homologous double-label immunofluorescence assay,and the expression of Toll-like receptor 4(TLR4)and Macrophage colony-stimulating factor(M-CSF)was detected by Western blot,and the mRNA expressions of TLR4 and M-CSF in the wound tissue were detected by RT-qPCR.The expressions of Tumor necrosis factor-α(TNFα)and Interleukin-6(IL-6)in the wound tissue were detected by Elisa.Results:In experiment part I,the general situation among the rats ware better in the blank group than in type 2 diabetic rats.The wound healing in the blank group was significantly higher than in other groups,the difference was statistically significant(P<0.05).Compared with the model group,the healing speed of low,medium,and high-dose Chinese medicine groups was significantly faster,and the difference was statistically significant(P<0.05).Compared with the blank group,HE staining showed that neutrophils and macrophages increased to varying degrees on the wound surface of the other groups,accompanied by tissue necrosis and exudation;neutrophils increased significantly in the model group,fibroblasts decreased significantly,and inflammation and necrosis tissue in some groups increased significantly into bands;low,medium and high-dose Chinese medicine groups showed less inflammation while fibroblasts increased significantly.In experiment part II,On the 10th and 14th day after wound modeling,homologous double standard immunofluorescence shows that there were differences inthe M1 and M2 macrophage and ratio of M1/M2 between groups,which was statistical significance(P<0.05).the protein contents of M-CSF and TLR4 tested by WB were significantly different between groups on the 10th day after wound modeling(P<0.05).On the 14th day after wound modeling,there were no significant differences in M-CSF,TLR4 protein content among most groups,with no statistical significance(P>0.05).On the 10th day after wound modeling,the relative expression levels of M-CSF mRNA and TLR4 mRN A tested by RT-qPCR showed that there were significantly different between groups(P<0.05).On the 14th day after wound modeling,there was no significant difference in the relative expression levels of M-CSF mRNA and TLR4 mRNA among all groups(P>0.05).On the 10th and 14th day after wound modeling,Elisa showed the expression of TNF-α and IL-6 between groups had statistical significance(P<0.05).Conclusion:The results of this research suggest that the high glucose microenvironment induced excessive inflammation and the abnormally active inflammatory response in the wound,which may cause the failure of skin ulcer healing in type 2 diabetic rats,and effectively regulate the inflammatory response of the wound microenvironment can promote wound healing.Traditional Chinese medicine compound Tuolixiaodu powder can increase the expression of M-CSF,inhibit the M1-polarization of macrophages,promote the M2-polarization of macrophages,and reduce the expression of the inflammatory regulatory protein(TLR4)and inflammatory factors(IL-6,TNF-α)to achieve the effect of expelling toxi".At the same time,it can promote the growth of fibroblasts by reducing the inflammatory response to achieve the role of "promoting tissue regeneration",and the two work together can accelerate the diabetic skin ulcer wounds healing. |
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