Study On The Effect Of Water Extract Of Daphne Genkwa On The Repair Of Wounds In Patients With Anal Fistula

Background:Anal fistula is an anorectal common diseases,including high-level anal fistula surgical trauma,healing longer,anal function is affected,and even lead to serious incontinence.How to speed up the wound healing of patients with anal fistula has become one of the important goals of anal fistula treatment.There are records of treatment of hemorrhoids and fistulas in ancient literature,most of which are combined with drug treatment of hemorrhoids and fistulas.Anal fistula line and hemorrhoids Department of hemorrhoid therapy(ligation therapy)are used in the method of Dwarf roots.There is no other modern research literature on why Dwarf roots should be used without using other drugs for cooking lines,and what effect the Dwarf roots have when they are taken.Studies have found that chemicals extracted from the roots of Daphne genkwa by ethanol can play an anti-inflammatory,edema and analgesic effect.Recent studies have shown that:(1)TGF-?,MMP-3,Timp-1,COL1A1,COL3A1,MEK1/2 and ERK1/2 are important factors affecting wound healing.(2)HSF cells,as the main constituent cells of granulation tissue during wound healing,are the key to promote wound healing.(3)The MEK/ERK signaling pathway plays an important role in cell growth and proliferation.MEK1/2 and ERK1/2 are the activation factors of MEK/ERK signaling pathway.This study was divided into clinical observation,cell model and animal model three parts,using real-time quantitative PCR,Western Blot and immunohistochemistry and other research methods to observe the whole body,cells and molecular level of the treatment of anal fistula water extract,To explore its effective role of components and possible mechanism of action for its application in the treatment of anal fistula to provide the appropriate theoretical basis.The first part Effect of water extract of Daphne genkwa on wound healing and related gene expression in patients with anal fistulaOBJECTIVE:To determine the efficacy of water extract of Daphne genkwa by comparing the duration of wound healing,the quality and speed of granulation growth of the treated group and the control group.Methods:Sixty cases of high fistula with operation indications were enrolled in the clinical trial.The patients were randomly divided into control group and medication group.The patients in both groups were treated with thread-hanging operation.The wounds were treated with water extract of Daphne genkwa For 7 days,the control group was treated with skullcap ointment wound dressing.The wound healing of the two groups were observed and compared.The fresh granulation tissues of the patients were collected and the expression of COL1A1,COL3A1,Timp-1,MMPs in the granulation tissue were detected by real-time PCR,Western Blot and immunohistochemistry-3,MEK1/2 and ERK1/2 expression.Results:1.The wound healing time(23.1 ± 2.8d)in the group treated with water extract of Daphne genkwa was significantly lower than that in the control group(28.7 ± 3.4d)(P<0.05).2.The expression of COL1A1,COL3A1,Timp-1,TGF-?,MEK1/2 and ERK1/2 in granulation tissue of the Daphne genkwa root water extract group was significantly increased,while the expression of MMP-3 was significantly decreased(P<0.05).Conclusion:The water extract of Daphne genkwa can effectively shorten the wound healing time and promote the growth of granulation tissue.The second part Effects of water extract of Daphne genkwa on scar and injured human fibroblastsOBJECTIVE:To investigate the effect of water extract of Daphne genkwa on the proliferation of HSF cells treated with water extract of different concentrations of Daphne genkwa,and to detect the expression of related genes in HSF cells.Methods:Human skin fibroblasts(HSF)were induced by TNF-?(10?g/ml)in vitro.The cells were treated with appropriate concentration of water extract and treated with CCK-8 Method and flow cytometry analysis of water extract of Daphne genkwa on impaired HSF cell viability,cell cycle and apoptosis;In addition,real-time quantitative PCR and Western Blot and other methods to detect damaged HSF model TGF-?,COL1A1,COL3A1,Timp-1,MMP-3 and MEK/ERK and other signaling pathways.Results::1.The water extract of Daphne genkwa could promote the growth of HSF,and the effect was most obvious when the concentration reached 25?g/ml.Flow cytometry showed that the water extract of Daphne genkwa could significantly increase the proportion of S phase cells,The proportion of cells in GO/G1 and G2/M phase decreased(P<0.05);2.In HSF cell line,the COL1A1,COL3A1,Timp-1,c-Fos and Cyclin D1 expression were significantly higher than the blank control group,while the expression of MMP-3 was significantly lower than the blank control group(P<0.05).3.In HSF injury model,TNF-a-induced apoptosis was significantly inhibited by water extract of Daphne genkwa(early apoptosis rate from 27.4%to 19.4%,advanced apoptosis rate from 15.7%to 9.4%)The downregulation of COL1A1,COL3A1,Timp-1,Cyclin D1 and c-fos induced by TNF-awere reversed significantly(P<0.05).Conclusion:1.The water extract of Daphne genkwa can significantly increase the proportion of S phase cells and promote the growth of HSF;2.The water extract of Daphne genkwa can activate MEK/ERK signaling pathway in HSF cell lines,inhibiting apoptosis;The third part The water extract of Daphne genkwa Wound infection in rats with wound repairOBJECTIVE:To observe the wound healing and the expression of related genes in the wound model of rats infected by human fecal contamination with water extract of Daphne genkwa.Methods:Wound rat model was established by spraying E.coli suspension with human excrement.After successful modeling,the rats in the treatment group were treated with water extract of Daphne genkwa,Sterile water was used instead of water.After 12 days,wound healing of rats in control group and Daphne genkwa was observed.Tissue samples of wounds were collected.The expression of TGF-?,MMP-3,Timp-1,COL1A1,COL3A1,MEK1/2 and ERK1/2 expression changes.Results:1.The wounds of the 4 groups were basically healed or scarified.The expression of COL1A1,COL3A1 and Timp-1 were significantly up-regulated while the expression of MMP-3 was down-regulated.2.The water extract of Daphne genkwa increased the protein levels of MEK1/2 and ERK1/2 in rat wound tissue.Conclusion:The aqueous extract of Daphne genkwa can effectively promote wound healing in rats.The mechanism may be related to up-regulating the expression of TGF-? and activating MEK/ERK signal pathway.