The Mechanism Of TGF-Β1 On Collagen Anabolism And Its Application In The Weakening Fascia Of Abdominal Wall Hernia

Background: Inguinal hernia is a common disease in surgery.With increasing incidence per year,and the population with inguinal hernia has a tendency for aging.The causes of inguinal hernia include increased abdominal pressure and weakened abdominal wall strength.Among them,the frail abdominal wall fascia which caused by collagen metabolism disorder,often lead abdominal wall strength decreasing.In the mechanism of collagen metabolism disorder in the inguinal hernia,some early studies have suggested that the enzymatic hydrolysis of matrix metalloproteinases(MMPs)causes excessive degradation of the extracellular matrix including collagen in the inguinal region fascia,resulting in weak fascia;In recent years,studies have shown that the abnormal expression of transforming growth factor β1(TGF-β1)is closely related to the occurrence of inguinal hernia and even abdominal wall hernia.Moreover,there is a correlation between TGF-β1 and MMPs,and TGF-β1 is involved in physiological and pathological activities such as organ fibrosis,wound healing,and scar formation,and plays a role in promoting cell proliferation,migration,andpromoting collagen synthesis and regulation of fibrosis.Therefore,we used these functions of TGF-β1,and explore the improvement of weak fascia,which promoted the increase of weak fascia fibrosis by overexpressed TGF-β1 and with the purpose of increasing abdominal wall strength.So we may explore a new research for reducing or delaying inguinal hernia.Objective: The aim of this study was to explore the effects of synthesis or metabolism of collagen in weak fascia,and the improvement of fascia weakness by overexpression of TGF-β1.Methods:(1)Firstly,the transversalis fascia specimens of normal,direct hernia and indirect hernia patients were obtained.The morphological features of transversalis fascia specimens in each group were analyzed by HE staining and Masson staining,and the collagen area was statistically analyzed.Secondly,Immunohistochemical staining was used to detect the positive expression of TGF-β1 in each group,and we could analysis the relationship between TGF-β1 and inguinal hernia qualitatively.For understanding the relationship between TGF-β1 and inguinal hernia.The protein expression level of TGF-β1,Smad,MMPs were detected by Western blot.(2)In vitro,fibroblasts as the research objects,constructing lentivirus carrying overexpressing TGF-β1,then transfecting into fibroblasts,observing the effect of TGF-β1 on the growth and migration of fibroblasts.The expression of Smad,MMPs and Collagen genes and protein levels incollagen anabolic pathways were analyzed by RT-PCR and Western blot.The expression signals of type I and type III collagen were detected by immunofluorescence.(3)In vivo,SD rats were selected and craping about one-half of the thickness of the left and right inguinal fascia tissues for constructing an animal model with weak fascia in the inguinal region.And injected with TGF-β1 lentivirus,no-load virus and PBS,respectively.To vertify the genes level and protein level expression changes of Smad,MMPs and Collagen which involved in in the collagen anabolic pathway with the vitro test by RT-PCR and Western-blot.For analysis the changes of collagen synthesis,we detected the expression signals of type I and type III collagen by immunofluorescence.Otherwise,to understand the effect of TGF-β1 on the mechanical properties of the fascia,we detected the rupture strength(tensile resistance strength)of the fascia was measured by mechanical properties.Results:(1)The results of first part indicated that the fibers were loosely arranged and disordered,and the spacing between the fibers increased,while the normal group was neatly arranged of the transverse fascia specimens with the inguinal hernia patients in the HE staining.In immunohistochemical staining,the expression of TGF-β1 in inguinal hernia was lower than that in normal group.There was significant lower protein expression of TGF-β1 and Smad 2,3,and higher expression ofMMP-2,9 compared with normal group(P<0.05)in Western blot.(2)Among the vitro results,there was no relationship between TGF-β1 and apoptosis of fibroblasts,compared with the control group,the apoptotic rate was not statistically increased(P>0.05).And promotes the migration of fibroblasts in some extent.In addition,we found that TGF-β1regulates collagen synthesis through Smad2/3 signaling pathway by upgrated of Smad 2 and 3,meanwhile inhibits MMP-9,and its mRNA and protein expression levels are decreased,the difference is statistically significant(P<0.05),but MMP-2 increased in the same direction as the expression of TGF-β1 increased(P<0.05).In terms of collagen synthesis,COL-1 and COL-3 showed different degrees of expression enhancement,mainly COL-1(P<0.05).The expression signals of COL-1 and COL-3were significantly enhanced in immunofluorescence compared with the control group.(3)In vivo experiments,a weak animal model of the inguinal region was successfully constructed.Wsetern and PCR detection showed that TGF-β1 and Smad 2,3 were enhanced in different degrees in each group,and more obvious in the lentivirus carrying overexpressing TGF-β1 group.(P<0.05),while MMP-9 expression dereased.There was no statistical difference in MMP-2 between groups.Among the COL test results,the expression of COL-1 in the experimental group was significantly higher than that in the no-load and PBS groups(P<0.05).There was no significantdifference in the expression of COL-3 between the two groups(P>0.05).The mechanical properties test showed that the rupture strength of fascia specimens was 2.00±0.66 MPa,which was significantly enhanced compared with the control group(P<0.05).Conclusion: This study confirmed the inevitable correlation between TGF-β1 and inguinal hernia inevitablity,and TGF-β1 plays a significant role in the regulation of collagen synthesis and metabolism through Smad signaling pathway,and reduce the degradation of collagen by inhibits the matrix metalloproteinase in some extent.The use of TGF-β1 to promote cell proliferation,migration and collagen synthesis increases the collagen synthesis at the weak fascia and enhances its strength.