| Objective:through the contrast with the water extract of Daphne genkwa blank and baicalin ointment topical in rats after operation of anal fistula and perianal edema dressing changes and wound healing of wound edema,inflammatory reaction.In order to verify the validity of the relevant pharmacological effects of anti-inflammatory,detumescence and treatment of hemorrhoid and fistula of the roots of Daphne genkwa,and provide experimental basis for the study of animal research to further explore the roots of Daphne genkwa.Methods:the first part:experimental model preparation wound after operation of anal fistula in rats on the back of the successful model of the rats were divided into A group(saline group),B group(group C,root of Daphne genkwa)group(baicalin ointment group),respectively,using saline water extract,skullcap root of Daphne genkwa ointment soaked gauze to wound dressing in rats.In 3,7 and 14 days of medication,wound were taken for HE staining and MASSON staining,observe the changes of granulation tissue,inflammatory cell infiltration and fibrosis changes,the proportion of Treg/Th17 expression and application of quantitative detection of PCR in serum of IL-6,IL-12 and the use of flow cytometry in rat peripheral in the blood.The second part:to establish a rat model of croton oil anal swelling,after the success of modeling,the rats were divided into group A(normal group),group B(saline group),C group(group D,root of Daphne genkwa)group(baicalin ointment group),A group without any treatment,B,C,D group every 4 and 48 hours respectively,24 saline,the roots of water extract and apply the ointment baicalin swelling anus,after 48.5 hours,were taken and perianal swelling tissues of rats by HE staining and MASSON staining,to observe rat perianal swelling,angiogenesis and inflammatory cell infiltration the situation changes and fibrosis,the expression of immunohistochemical detection of SM22 protein and immunofluorescence localization of SM22 protein expression,and the application of PCR and ELISA IL-17 and TNF-alpha in serum were examined.Data were analyzed using SPSS 18.0.Results:in the first part,PCR quantitative results showed that after 3 days of treatment,the expression of A,B,C group IL-12 with X ± S were 1.02 ± 0.28,2.52 ± 1.01,2.78 ± 1.83,through the statistical analysis of P<0.05(P=0.011),there was significant difference between the C group and B,.P>0.05,(P=0.648),no significant difference.The expression of IL-6 in the three groups was X ± S,which was 1.04 ± 0.46,0.84 ± 0.29,0.69 ± 0.33,P>0.05(P=0.157),and there was no significant difference between the two groups.After 7 days of medication,the expression of IL-12 in the three groups were respectively at the rate of 0.80 ±0.24,2.75 ± 1.13,3.02 ±2.21,and there were significant differences in P<0.05(P=0.006)by statistical analysis,and B,C group,P>0.05(P=0.700),no significant difference.The expression of IL-6 in the three groups was 1.86 +0.56,0.62 ± 0.21,0.53 ±0.23,and there was significant difference between B and C group,P>0.05(P=0.589),P>0.05,no significant difference.After 14 days of medication,the expression of IL-12 in the three groups were respectively at the rate of 0.49 ±0.21,3.48 ± 1.45,4.19 ± 1.48,and there was significant difference between B and C group,P>0.05(P=0.237),P<0.05,no significant difference.The expression of IL-6 in the three groups was 2.84 ± 0.99,0.49 ± 0,23,0.31 ± 0.14,and there was significant difference between the P>0.05 and C groups(P=0.516),and there was no significant difference between the B and P>0.05 groups.HE staining showed that after 3 days of treatment A,B,C three groups have fiber in adipose tissue showed a large number of acute and chronic inflammatory cell infiltration,local muscle tissue degeneration and necrosis,vascular proliferation,wall thickening,A group also see interstitial edema and vascular occlusion,A group inflammation is more important,B,C the A group is slightly lighter,two groups of the degree of inflammation.After 7 days of treatment,three groups of fibers in adipose tissue is still a large number of acute and chronic inflammatory cell infiltration,A,C group of local muscle tissue degeneration and necrosis,calcification,B,C group of local formation of granulation tissue,B and C group began to repair wound.After 14 days of treatment,inflammatory cell infiltration in B group and C group was significantly lower than that in group A,and there were hyaline changes.Masson staining showed:3 days after treatment,there were a lot of mucus and collagen fibers in the wound tissue,and B and C groups were more than A group.After 7 days of treatment,the collagen fibers in wound tissue increased,and the B and C groups were more than those in A group.After 14 days of treatment,the collagen fibers in the wound tissue of A group decreased,and the collagen fibers increased in B and C groups.The proportion of Th17/Treg in peripheral blood were detected by flow cytometry and the results with X ± S said:after 3 days of treatment,A,B,C groups were 0.663 ± 0.042,0.770 ± 0.009,0.926 ± 0.002,through the statistical analysis of P<0.05(P=0.004),there was significant difference.7 days after treatment,respectively,was 0.565 ±0.001,1.077 ± 0.033,1.372 ± 0.065,(P<0.05),there was significant difference(P=0.001).14 days after treatment,respectively,was 0.295±0.011,1.722 ±0.074,2.362 ±0.187,(P<0.05),there was significant difference(P=0.001).The second part of the experiment,quantitative detection of PCR TNF-alpha and IL-17 index showed that the expression of A,B,C,D,TNF-in the four groups with X ± S alpha were 0.88 ± 0.21,5.03 ± 1.31,2.63 ± 0.96,1.57 ± 0.27,P<0.05,and C,there are significant differences,D group,P>0.05(P= 0.073),no significant difference.The expression of IL-17 in the four groups was 0.92 ±0.26,3.37 ±0.85,2.15 ±0.45,1.48± 0.39,P>0.05,and there was significant difference between the C group and D group(P=0.072),and there was no significant difference between the P>0.05 and groups.The results of ELISA quantitative detection of TNF-and IL-17 were consistent with the results of PCR test,and the P value was less than 0.05.HE staining showed that A group had normal mucosa,muscle and other tissues.B,C,D group of anal mucosa and muscle in both acute and chronic inflammatory cell infiltration,C,D group than in A group,C group than in D and there was no inflammatory cell infiltration and less muscle tissue in the C group and accompanied by necrosis,vascular proliferation,C and D group after treatment with A group of inflammatory edema,inflammation,edema and C were the lightest.The results of MASSON staining to observe the degree of fibrosis of anal margin were as follows:in the A group,the normal anal margin tissue was observed.In B group,a large number of mucus and collagen fibers were observed.C group and D group were treated with a small amount of mucus and collagen fibers.Immunohistochemistry was used to detect the expression of SM22 protein that results with IOD value,A,B,C,D were 8841.39 ±49841.88,4339.75 ± 24700.33,7211.28 ± 29230.33,7702.81 ± 39702.19,four group P<0.05,with significant difference,but C,D two groups P>0.05(P=0.166),no statistically significant the difference.Immunofluorescence staining showed that SM22 protein and STIM1 protein were located in the cytoplasm,and red fluorescence was located in the nucleus,so the Orai1 protein was located in the cytoplasm.Conclusion:the water extract of the roots of Daphne genkwa reduce inflammatory reaction after anal fistula surgery and postoperative edema.To reduce the inflammatory cell infiltration in the early stage of wound healing,increased collagen production,increase the expression of SM22 protein,and then promote the healing of the wound after anal fistula surgery. |
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