Effects Of HB-EGF In Collagen Metabolism And Cell Migration Of Hepatic Stellate Cell

Liver fibrosis is a response and curing reaction to chronic liver damagedue to a virus, metabolism, alcohol, chemical toxins, genetic and othercauses.This process leads to extracellular matrix (ECM) excessivedeposition.If the cause of persistent, liver fibrosis will eventually develop intocirrhosis even liver cancer. The activation and proliferation of hepatic stellatecells (HSC) causes the synthesis and abnormal deposition of ECM, which isthe key link to the process of liver fibrosis.HSCs are nonparenchymal in the form of quiescent cells which exist innormal liver. Numerous studies find that, when the pathogenic factors causeliver injury, HSCs transformed into an activation process and constantlyvalue-added, in addition, secrete a mass of ECM, such as TypeⅠ collagen,thus promote the occurrence and development of liver fibrosis. The activationof HSC is the main source of matrix metalloproteinases (MMPs) and tissueinhibitors of metalloproteinases (TIMPs), which can adjust the degradation ofECM. A variety of cytokines can lead to HSC value-added, collagen synthesisand migration, such as the epidermal growth factor (EGF), platelet-derivedgrowth factor (PDGF), etc.Heparin-binding epidermal growth factor-like growth factor (HB-EGF)which have a specific heparin binding domain is a member of the ErbB familythat binds to and activates two ErbB receptors, epidermal growth factorreceptor (EGFR/ErbB1) and EebB4. The activation of ErbB receptorsprovokes mitogen-activated kinase (MAPK), participating in multiple cellproliferation and migration. Studies show that HB-EGF can promote cellvalue-added and inhibit apoptosis, while the effect in collagen metabolism andcell migration is unclear.The purpose is to research the effects of HB-EGF in collagen metabolism and cell migration of HSC, further explore the role of HB-EGF in the processof hepatic fibrosis to provide theoretical basis for effective treatment.Objectives: To study the effects of HB-EGF in collagen metabolism andcell migration of HSC and explore the mechanism of the impact in cellbiology behavior.Methods： Human hepatic stellate cell line HSC LX-2is used inexperiments in vitro. HSCs were preincubated with or without CRM197for24h, and then stimulated with or without HB-EGF for24h. The proteinexpressions of α-SMA and type Ⅰcollagen were determined by Western blotand immunocytochemistry. The mRNA levels of α-SMA, typeⅠ collagen,MMP-1and TIMP-1were measured by R-T real-time PCR. The proteinexpression of type Ⅰ collagen, MMP-1and TIMP-1were detected by ELISA.Cell migration was detected by the wound-healing assay.Results:①The α-SMA expression were detected by immunocyto-chemistry: the cell number with positive expression, the HB-EGF group wasmore than control group, the CRM197group was less than control group.②The Western blot was used in my project, to measure the protein expression ofα-SMA, the HB-EGF group(0.74±0.04) increased than control group(0.43±0.02), the CRM197group compared with the control group(0.33±0.01vs0.43±0.02), the HB-EGF+CRM197group compared with the HB-EGFgroup(0.44±0.02vs0.74±0.04), the expression reduced(P<0.01); to test theprotein expression of type Ⅰ collagen, the HB-EGF group(0.57±0.06)increased than control group(0.36±0.02)(P<0.05), the CRM197groupcompared with the control group (0.14±0.02vs0.36±0.02), theHB-EGF+CRM197group compared with the HB-EGF group (0.17±0.02vs0.57±0.06), the expression reduced (P<0.01).③The Real time-PCR wasused to test the mRNA expression, about the α-SMA, the result of controlgroup, HB-EGF group, HB-EGF+CRM197group and CRM197group were1、4.37±0.43、1.97±0.26、0.32±0.12, the result was agreed with the Westernblot; about the mRNA expression of type Ⅰc ollagen, the HB-EGF group(4.77±0.62) increased obviously than control group(1±0)(P<0.01), the CRM197 group compared with the control group(0.15±0.05vs1±0), theHB-EGF+CRM197group compared with the HB-EGF group(0.49±0.18vs4.77±0.62), the mRNA expression reduced (P<0.05); to test the TIMP1、MMP1expression, HB-EGF group compared with control group, theexpression of TIMP1increased(5.90±0.34vs1±0)(P<0.01), but theexpression of MMP1had no difference(1.27±0.20vs1±0)(P＞0.05), theMMP1/TIMP1reduced(0.22±0.04、1±0)(P<0.05).④The ELISA was usedto measure the expression of α-SMA, type Ⅰc ollagen, TIMP1and MMP1, theresult were agreed with the Real time-PCR(P<0.05).⑤Wound-healing assaywas used to detect cell migration, to measure the residual area, the HB-EGFgroup(1.23±0.1) was smaller than control group(1.78±0.3), and it increasedin CRM197group(2.49±0.38) compared with control group(1.78±0.3)(P<0.05).Conclusions: HB-EGF has potent to add α-SMA expression whichproved the further activation of HSC. HB-EGF promotes analysis of collagen.HB-EGF up-regulates TIMP1mRNA and protein expression. It enlarge thedisbalance between MMP1and TIMP1. As a result, HB-EGF inhibitsdegradation and promotes sedimentation of ECM which has composite.HB-EGF accelerates cell migration.