The Effects And Mechanism Of Overexpression Of Heparin Binding Epidermal Growth Factor On Liver Fibrosis In Transgenic Mice

Various etiologies including hepatitis B and C,alcohol abuse,,drug damage,genetic metabolic,autoimmune damage may lead to chronic inflammation,and then cause liver fibrosis,which is characterized by the deposition of extracellular matrix?ECM?mainly type I collagen,and distortion of normal architecture.Liver fibrosis will eventually lead to cirrhosis,associated with nodule formation and Porto-systemic shunts,the patients with liver cirrhosis appear clinical complications such as alimentary tract hemorrhage,ascites,hepatic encephalopathy,hepatocellular carcinoma,even lead to death.The quiescent hepatic stellate cells?HSCs?are located inthe perisinusoidal space of Disse,storing rich retinoids?vitamin A?.But following fibrogenic stimuli,HSCs are rapidly activated,losing lipid droplet,acquired the ability to proliferate,producing a large number of ECM.The injured hepatocytes,infiltrated inflammatory cells and kuffer cells secrete a variety of cytokines and activate HSCs.It was identified that a lot of cytokines such as transforming growth factor-??TGF-??,platelet-derived growth factor?PDGF?,interleukin-1?IL-1?,tumor necrosis factor?TNF-??,interfer-??INF-??can activate HSCs.Until recently,the currently accepted view is that liver fibrosis is irreversible,HSCs are a major source of collagen producing cells in fibrotic liver,Clearance of activated HSCs is vital for achieving resolution of fibrosis.In recent decades,the pathogenesis of hepatic fibrosis and HSC activation have always been a hot topic.A lot of clinical and animal experimental studies have shown that epidermal growth factor family play a certain role in liver fibrosis and HSCs activation.Heparin-binding epidermal growth factor?HB-EGF?is a member of the epidermal growth factor?EGF?superfamily,a new type of strong mitogen.HB-EGF is expressed in lung,heart,skeletal muscle,but the expression of HB-EGF in the healthy liver is very low or undetectable.however,it is significantly elevated during acute damage and in liver cirrhosis.HB-EGF can bind to epidermal growth factor receptor?EGFR?and ErbB4,activate the downstream signaling pathways,promote cell proliferation,inhibite cell apoptosis,promote the synthesis of ECM.HB-EGF participates in the early repair response of tissue injury,closely associated with multiple organ fibrosis.Studies had shown that HB-EGF promotes pancreatic fibrosis and cardiac fibrosis.Previously our team had found that the levels of HB-EGF and its receptor increased in the activated HSC,and exogenous HB-EGF promoted the proliferate and suppressed the apoptosis of HSCs.These findings suggest that HB-EGF has profibrotic effects.Paradoxically,HB-EGF knockout mice develop increased liver fibrosis induced by CCl₄ or DMN.To date,the function of HB-EGF in liver fibrosis is still not well addressed.In this study liver fibrosis in HB-EGF transgenic mice induced by CCL₄and common bile duct ligation was investigated,at the same time the proliferation and apoptosis of primary HSCs isolated from Tg and WT mice were evaluated.This paper is divided into three parts as following.Part 1 The effects of overexpression of HB-EGF on experimental liver fibrosisObjective:To evaluate the degree of liver fibrosis in HB-EGF transgenic mice induced by CCl₄ and common bile duct ligation,Our aim is to evaluate the pro-fibrogenic effect of HB-EGF.Methods:We select HB-EGF transgenic?Tg?mice and the same age,sex,nest,non-Tg wild type?WT?mice as the research objects.Using the method of carbon tetrachloride?CCl₄?intraperitoneally injected and bile duct ligation?BDL?to establish two kinds of liver fibrosis model.CCl₄ model mice were divided into four groups:WT control group,Tg control group,WT CCl₄ group and Tg CCl₄ group.All animals were euthanized 48 hours after the final injection of CCl₄ and their livers were excised.BDL model mice were divided into four groups:WT sham group,Tg sham group,WT BDl group and Tg BDL group.The mice were euthanized 2 weeks after surgery.The sections were stained with hematoxylin and eosin?H&E?to observe morphology of the liver,and Sirius red staining to assesse collagen deposition in livers,Hepatic hydroxyproline content was detected with a hydroxyproline detection kit?alkaline hydrolysis?,Immunohistochemical Analysis to assay the protein expression level of HB-EGF and?-SMA in liver tissue,Western blot and Real time PCR method to determine the protein and gene expression level of HB-EGF,?-SMA,type I collagen,MMP-13 and TIMP-1 in liver tissue,Zymography method to determine the the activity of MMP-13.Results:1 Overexpression of HB-EGF in HB-EGF Tg mice was confirmed by Real time PCR,western blot and IHC.2 As examined by H&E and Sirius red staining,both fibrotic models induced by CCl₄ and BDL were successfully established.3 Overexpression of HB-EGF prompted the ECM deposition in the liver.As shown by H&E and Sirius red staining,BDL and CCl₄ treated HB-EGF Tg mice show more extensive fibrotic area than that in WT model group.4 HB-EGF TG mice exhibited a marked increase in total amount of collagen compared with WT mice by hydroxyproline analysis.In the liver fibrosis model induced by CCl₄ and BDL,the content of hydroxyproline in HB-EGF Tg group was obviously higher than that of WT model group.5 Overexpression of HB-EGF prompted the synthesis of type I collagen in liver.Western blot and Real time PCR detection results show that the content of type I collagen in HB-EGF Tg mice was obviously higher than that of WT.6 The expression of?-SMA in fibrotic liver.As shown by IHC,CCl₄injection resulted in an increased?-SMA accumulation around the fibrotic portal tracts and fibrotic septa in in fibrotic liver induced by CCl₄ and BDL,the?-SMA stained areas more extensive in the HB-EGF Tg mice than in the WT mice by IHC.The results of Western blot and Real time PCR show that the protein and gene expression level of?-SMA in WT model group and Tg model group was obviously higher than that of control group,the protein and gene expression level of?-SMA in Tg model group was obviously higher than that of WT model group.7 HB-EGF down-regulate the ratio of MMP-13/TIMP-1 in two kinds of liver fibrosis mice.The results of Real time PCR showed that the expression level of TIMP-1 mRNA in two kinds of models mice were significantly higher than that of control group,the expression of TIMP-1 mRNA in Tg model group was obviously increased than that of WT modle group.The expression level of MMP-13 mRNA has not obviously change between the two groups,the ratio of MMP-13/TIMP-1 was decreased obviously in two kinds of liver fibrosis mice.The results of western blot showed that HB-EGF promotes the ratio of MMP-13/TIMP-1 decreased.The results of Zymography showed that the over expression of HB EGF inhibit the activity of MMP-13,which further confirmed the above results.Conclusions:HB-EGF aggravated CCl₄ and BDL induced liver fibrosis,this may be provoked by down-regulation of ratio of MMP-13/TIMP-1,it also was associated with increased?-SMA positive cells.Part 2 Overexpression of HB-EGF increased proliferation and collagen synthesis,reduced apoptosis in primary HSCsObjective:To evaluate the effects of overexpression of HB-EGF on proliferation,collagen synthesis and apoptosis in primary HSCs.Methods:Mouse HSCs were extracted from HB-EGF Tg and WT mice by a traditional method with minor modification.Quiescent stellate cells were examined by 0.4%trypan blue and fluorescence microscope to detecte the purity of isolated cells.HSCs were subsequently cultured in Dulbecco's minimum essential medium?DMEM?/F-12 and passaged.The HSCs were divided into two groups:HB-EGF Tg HSCs and WT HSCs.The level of?-SMA in HB-EGF Tg HSCs and WT HSCs was evaluated by immunocy-tochemistry staining,Western blot and Real time PCR methods to determine the protein and gene expression level of HB-EGF,?-SMA,type I collagen,MMP-13 and TIMP-1 HB-EGF Tg HSCs and WT HSCs,zymography method to determine the the activity of MMP-13.Proliferative capacity of Primary cultured HB-EGF Tg HSCs and WT HSCs were measured by CCK8 assay and EdU incorporation assay.Apoptosis of Primary cultured TG HSCs and WT HSCs were tested using TUNEL assay and flow cytometric analysis of Annexin V FITC propidium iodide.Results:1 The purity and survival rate of Primary HSCs were more than 90%,and the pick-up rate was 1.0×105¹.3×105,unde the inverted fluorescence microscope,the newly extracted HSCs appear round or oval,emitting blue-green light at the 328nm wavelength fluorescence excitation.The majority of cells were adherent,the shape changed into a flat after 24 h growth in the plastic bottle,then gradually extended,showing stellate and spindle.Immunocytochemical staining showed that the activated HSCs cells were positive for?-SMA staining.2 HB-EGF promotes the synthesis of intracellular fibrosis markers in HSCs.The result of Real time PCR and western blot had shown that HSCs cells isolated from transgenic mice had high expression of HB-EGF;along with the expression of HB-EGF increased,compared with the HSCs isolated from wild type mice,the expression level of fibrosis markers?-SMA and collagen type I increased significantly in HB-EGF Tg HSCs.3 The ratio of MMP-13/TIMP-1 was decreased significantly in HB-EGF Tg HSCs cells.The result of Real time PCR and western blot had shown that the protein and mRNA expression levels of TIMP-1 in HB-EGF Tg HSCs cells were significantly higher than those in WT HSCs,but the expression of MMP-13 was slightly up-regulated,and the ratio of MMP-13/TIMP-1 was down regulated.Gelatin zymography assay showed that the activity of MMP-13 was inhibited in HB-EGF Tg HSCs cells.4 HB-EGF promotes proliferation of HSCs cells.The results of CCK-8assay showed that the proliferation rate of HB-EGF Tg HSCs was significantly increased,which was consistent with the results.The data of EDU incorporation showed that DNA synthesis in HB-EGF Tg HSCs cells increased.5 HB-EGF inhibits the apoptosis of HSCs.TUNEL positive cells were counted by TUNEL staining,and the apoptotic index of HSCs extracted from HB-EGF Tg mice was significantly lower than that obtained from WT mice.HB-EGF Tg V-FITC/PI and WT HSCs were labeled with Annexin HSCs,and then detected by flow cytometry,it was found that over expression of HB-EGF significantly reduced the number of apoptotic cells.Conclusions:Overexpression of HB-EGF in primary HSCs increased proliferation and collagen synthesis,reduced the ratio of MMP-13/TIMP-1,reduced apoptosis.Part 3 The mechanism of HB-EGF on increased proliferation and collagen synthesis,reduced apoptosis in primary HSCsObjective:To study the regulatory effect of HB-EGF on its downstream molecules EGFR and ERK in primary HSCs cells.Methods:HB-EGF Tg and WT mice were injected intraperitoneally with CCl₄,Injections were administered every third day,12 times altogether.The primary HSCs was obtained by livers perfused with collagenase-pronase in situ and Percoll gradient centrifugation.The primary HSCs were divided into4 groups:WT control group,Tg control group,WT CCl₄ group,and Tg CCl₄.The protein expression level of?-SMA,type I collagen,EGFR,ERK,pEGFR and pERK were determined by Western blot,and the mRNA expression level of HB-EGF,?-SMA,type I collagen,EGFR,ERK,pEGFR and p ERK were determined by Real time PCR.Results:1 HB-EGF enhanced the activation of HSCs stimulated by CCl₄.Western blot analysis showed that after treatment with CCl₄,the expression levels of?-SMA and type I collagen in HSCs isolated from HB-EGF Tg mice were significantly higher than those from WT mice.2 Compared with WT mice,the mRNA expression levels of HB-EGF,?-SMA and type I collagen were significantly increased in HSCs from HB-EGF Tg mice induced by CCl₄.3 HB-EGF up-regulated phosphorylation level of the MAPK pathway.the MAPK pathway plays a key role in hepatic fibrosis.Western blot analysis showed that the phosphorylation level of EGFR was significantly increased in HSCs isolated from HB-EGF Tg mice,which further activated its downstream effect factor ERK.Conclusions:HB-EGF promotes the phosphorylation level of EGFR and its downstream factor ERK,which may be one of the mechanisms of which promote the proliferation and inhibit apoptosis of HSCs.