| Backgrand Chronic liver disease is a serious threat to human health.A variety of factors,such asviral hepatitis,alcohol,drugs and autoimmune liver disease,can lead to chronic liver disease which caused the body to produce a compensated repair response to injury,which is defined as liver fibrosis.The research on the pathogenesis of liver fibrosis is helpful to the early detection and diagnosis of liver fibrosis.After different damage factors acted on the liver,there are existing difference of the pathophysiological proces and cytokines involved in liver fibrosis,but the activation of hepatic stellate cells(HSC)and extracellular matrix(ECM)deposition,eventually lead to liver fibrosis which is the common pathological physiological process.Therefore,different aetiological agent which lead to liver fibrosis animal model will be adopted for research,that can be helpful to elucidate the pathogenesis of early liver fibrosis from different angles.The ECM synthesis and degradation are regulated by matrix metalloproteinases(MMPs)and tissue inhibitors of metalloproteinases(TIMPs)in the liver.When the damage factors act on the liver,a series of pathological processes are initiated,such as HSC activation,ECM deposition which is given priority to with Collagen ?,expression of ?-SMA.After a series of pathological processes,the MMPs/TIMPs balance is damaged,also,synthesis and degradation of the ECM is imbalance and excessively deposited in the liver.Liver fibrosis and cirrhosis are developing.Insulin-like growth factor binding protein related proteins 1(IGFBPr P1)is found by tutor team as a new factor leading to liver fibrosis,which was supported by the national natural science foundation for more than ten years.A lot of research work was performed on the mechanism of liver fibrosis induced by IGFBPr P1.In the development of liver fibrosis,IGFBPr Pl play an important role on HSC activation,ECM synthesis,degradation and reshape.Transforming growth factor beta 1(TGF?1)is recognized as the most powerful cytokines leading to liver fibrosis,IGFBPr P1 interact with TGF?1,which likely accelerates the development of liver fibrosis.Whether IGFBPr P1 induced the ECM deposition by adjusting the MMPs/TIMPs balance?There were not reported at present.Hedgehog(Hh)signaling pathway is one of the classical pathway involved in early development.The Hh pathway controls growth and proliferation of the cells and plays a regulatory role in liver fibrosis.IGFBPr P1-inducible genes of the hepatic fibrosis related pathway were assessed by PCR array by tutor team.IGFBPr P1 can lead to HSC activation,ECM synthesis and promote the development of liver fibrosis by activating Hh pathway.Therefore,IGFBPr P1,MMPs/TIMPs balance and Hh signaling pathway,they were how to regulate and interact with each other? There were not reported at present.Therefore,this topic proposed to determine whether the development of IGFBPr Pl induced liver fibrosis by adjusting the MMPs/TIMPs balance.In addition,the relationships between IGFBPr Pl on the regulation of MMPs/TIMPs balance with the Hh signaling pathway are also discussed.Part 1 The significance and dynamic changes of IGFBPr P1 and MMPs/TIMPs balance in the development of liver fibrosis in miceExperiment 1 The dynamic changes of IGFBPr P1 and MMPs/TIMPs balance in the development of liver fibrosis induced by TAA or BDL in miceObjective:To investigate the dynamic changes of IGFBPr P1 and MMPs/TIMPs balance in the development of liver fibrosis induced by TAA or BDL.Methods:(1)C57BL/6 wild-type mice were randomly divided into normal group: normal breeding;control group: intraperitoneal injection of normal saline 0.1ml/10 g,three times a week;TAA group: intraperitoneal injection of TAA100mg/Kg,three times a week.Liver tissues and serum were obtained at week 2,4 and 6.We examined the pathological changes of livers by HE and Sirius red staining.and assessed the level of IGFBPr P1 in serum.The protein expression of IGFBPr P1,?-SMA,TGF?1,MMPs and TIMPs were examined by immunohistochemical techniques and western blot.The correlations were evaluated by Pearson analysis.(2)C57BL/6 wild-type mice were randomly divided into normal group: normal breeding;control group: only free bile duct;BDL group: bile duct ligation.Specimen collection and testing are same as the former.Results:(1)liver function index in serum:when TAA administration to mice,at 2 w,ALT,AST and LDH level were increased significantly,while decreased at 4 w,6 w(P< 0.05).After BDL administration to mice,at 2 w,ALT,AST level were increased significantly,while decreased at 4 w,6 w(P< 0.05);ALP and DBIL/TBIL ratio were also gradually increased(P< 0.05);while,GGT,LDH,TBIL level were increased significantly at 2w,reached peak at 4 w and decreased at 6w(P< 0.05).(2)HE and Sirius red staining: after TAA administration to mice,with disease progress,inflammatory cells infiltration,liver cells degeneration and necrosis and hyperplasia of fibrous tissue induce the development of liver fibrosis.After BDL administration to mice,with disease progress,the portal area expanded,bile duct expansion,inflammatory cells infiltration,a large number of small bile duct proliferation and collagen deposition induce the development of liver fibrosis.(3)ELISA,immunohistochemistry and western blot analysis the protein expression of IGFBPr P1,?-SMA,TGF?1,Collagen ? in serum or liver: after TAA or after BDL administration to mice,the serum and liver IGFBPr P1 expression was increased with the degree of liver fibrosis(P< 0.05),?-SMA,TGF?1,Collagen ? expressed in liver were also increased(P< 0.05).(4)Immunohistochemistry and western blot analysis the protein expression of MMPs and TIMPs in liver: MMPs and TIMPs protein expression at different time point was analysed.In TAA-induced liver fibrosis group,the MMP2/TIMP2 ratio was no change at 2 w and increased at 4 w and 6 w(P< 0.05);while the MMP9/TIMP1 ratio decreased significantly at every time point(P< 0.05).In BDL-induced liver fibrosis group,the MMP2/TIMP2 ratio was not changed at 4 w and decreased at 2 w and 6 w(P< 0.05);while the MMP9/TIMP1 ratio was decreased significantly at every time point(P< 0.05).(5)There were statistically significant positive correlations between the expression of IGFBPr P1 with MMP2,TIMP2,MMP9,TIMP1 in mice with liver fibrosis induced by TAA or BDL.Conclusion: Liver fibrosis was aggravated by TAA or BDL on time dependence.HSC activation,the increased expression of IGFBPr Pl and TGF?1,MMP2/TIMP2 and MMP9/TIMP1 unbalances played an important role in the development of liver fibrosis.Part 2 The regulation of MMPs/TIMPs balance in IGFBPr P1-induced liver fibrosis in miceExperiment 2 Gene transfer of IGFBPr P1 contributes to hepatic fibrosis and regulates the MMP2/TIMP2 and MMP9/TIMP1 balances in miceObjective: To investigate the dynamic changes of MMPs/TIMPs balance in the development of liver fibrosis induced by IGFBPr Pl.Methods: C57BL/6 wild-type mice were randomly divided into normal group: normal breeding;CAd group: injected with CAd 2×109 pfu per mouse via the tail vein;Ad IGFBPr P1 group: injected with Ad IGFBPr P1 2×109 pfu per mouse via the tail vein.Liver tissues and serum were obtained at week 1,2,4,8 and 12.Specimen collection and testing are same as Experiment 1.Results:(1)The bright green fluorescence of EGFP was observed by fluorescence microscope and expression levels of EGFP in liver peaked at 2 week after adenoviral infection.The green fluorescence of EGFP gradually weakened at 1 weeks and dispeared by 4 weeks after the infection.(2)liver function index in serum:when Ad IGFBPr P1 transfer to mice,at 1 w,ALT,AST level were increased significantly,while decreased at other time point(P< 0.05).while LDH level were increased and reached peak at 2 w and then decreased(P< 0.05).(3)HE and Sirius red staining: after Ad IGFBPr P1 transfer to mice,with disease progress,a large number of inflammatory cells infiltration,hyperplasia of collagen fibers,liver cells degeneration and necrosis,the destroyed structure of hepatic lobule,these induced the development of liver fibrosis.(4)ELISA,immunohistochemistry and western blot analysis the protein expression of IGFBPr P1,?-SMA,TGF?1,Collagen ? in serum or liver: after Ad IGFBPr P1 transfer to mice,the serum and liver IGFBPr P1 expression was increased and reached peak at 2 w(P< 0.05),?-SMA,TGF?1,Collagen ? expressed in liver were increased with the degree of liver fibrosis(P< 0.05).(5)Immunohistochemistry and western blot analysis the protein expression of MMPs and TIMPs in liver: MMPs and TIMPs protein expression at different time point was analysed.after Ad IGFBPr P1 transfer to mice,the MMP2/TIMP2 and MMP9/TIMP1 ratio were decreased significantly at every time point(P< 0.05).Conclusion:Gene transfer of IGFBPr P1 induced hepatic fibrosis in mice.HSC activation,the increased expression of TGF?1,MMP2/TIMP2 and MMP9/TIMP1 unbalances played an important role in this progress.Part 3 The influence of MMPs/TIMPs balance in the process of liver fibrosisby sh RNA-IGFBPr P1Experiment 3 Sh RNA-IGFBPr P1 construction and its influence of MMPs/TIMPs balance in vitroObjective: Constructing and screening the most efficient sh RNA-IGFBPr P1 plasmid with interference,down-regulating the expression of IGFBPr P1,whether could reverse the MMPs/TIMPs balance to reduce the generation of the ECM in NIH/3T3 cells.Methods:(1)Constructing three sh RNA-IGFBPr P1 plasmid with different interference sequence and sh RNA-NC,respectively transfect to NIH/3T3 cells,screening the most efficient sh RNA-IGFBPr P1 plasmid with interference.(2)NIH/3T3 cells were transfected with the most efficient sh RNA-IGFBPr P1 plasmid with interference or sh RNA-NC,all group cells were gathered at 24 h,48h,72 h,96h.?-SMA,TGF?1,MMPs,TIMPs m RNA and protein were examined by quantitative RT-PCR and western blot.Results:(1)RT-PCR and western blot analysis the m RNA and protein expression of IGFBPr P1 in cells:compared with control and sh RNA-NC transfected,sh RNA-IGFBPr P1 plasmid with different interference sequence inhibited the m RNA and protein expression of IGFBPr P1 in different degree,of which sh RNA-IGFBPr P1-1 has the highest inhibition rate(P< 0.05).(2)RT-PCR and western blot analysis the m RNA and protein expression of TGF?1,?-SMA,Fibronectin in cells:after sh RNA-IGFBPr P1 transfected,the m RNA and protein expression of TGF?1,?-SMA,Fibronectin were decreased significantly at every time point(P< 0.05).(3)RT-PCR and western blot analysis the m RNA and protein expression of MMPs,TIMPs in cells: MMPs and TIMPs m RNA and protein expression at different time point was analysed.After sh RNA-IGFBPr P1 transfected,the MMP2/TIMP2 and MMP9/TIMP1 ratio were increased significantly at every time point(P< 0.05).Conclusion: Down-regulating the expression of IGFBPr P1 of NIH/3T3 cells exerted inhibitory effects against liver fibrosis by significantly inhibiting cell activation,reducing the expression of TGF?1,reversing MMP2/TIMP2 and MMP9/TIMP1 balance to reduce the ECM synthesis and secretion.Experiment 4 The influence of MMPs/TIMPs balance in the process of liver fibrosis in mice by UTMD-mediated sh RNA-IGFBPr P1 delivery with CMBObjective: Down-regulating the expression of IGFBPr P1 exert inhibitory effects against liver fibrosis whether by reversing the MMPs/TIMPs balance to reduce the generation of the ECM in mice induced with TAA or BDL.Methods:(1)C57BL/6 wild-type mice were randomly divided into TAA group: intraperitoneal injection of TAA 100mg/Kg,three times a week;TAA+sh RNA-NC group: one week following TAA,the CMB-sh RNA-NC solution was infused into the tail vein,simultaneously,an ultrasound beam was delivered to ultrasound targeted microbubble destruction(UTMD);TAA+sh RNA-IGFBPr P1 group: one week following TAA,the CMB-sh RNA-IGFBPr P1 solution was infused into the tail vein,simultaneously,an ultrasound beam was delivered to UTMD.Liver tissues and serum were obtained at week 2,4 and 6.Specimen collection and testing are same as Experiment 1.(2)C57BL/6 wild-type mice were randomly divided into BDL group: bile duct ligation;BDL+sh RNA-NC group: one week following BDL,the CMB-sh RNA-NC solution was infused into the tail vein,simultaneously,an ultrasound beam was delivered to UTMD;BDL+sh RNA-IGFBPr P1 group: one week following BDL,the CMB-sh RNA-IGFBPr P1 solution was infused into the tail vein,simultaneously,an ultrasound beam was delivered to UTMD.Liver tissues and serum were obtained at week 2,4 and 6.Specimen collection and testing are same as Experiment 1.Results:(1)Immunohistochemistry and western blot analysis the protein expression of IGFBPr P1,?-SMA,TGF?1,Collagen ? in liver: after sh RNA-IGFBPr P1 transfected to mice,the protein of IGFBPr P1,TGF ?1,?-SMA,Collagen ? expressed in liver were decreased with the degree of liver fibrosis(P< 0.05).(2)Immunohistochemistry and western blot analysis the protein expression of MMPs and TIMPs in liver: In liver fibrosis mice induced by TAA or BDL,MMPs and TIMPs protein expression at different time point was analysed,after sh RNA-IGFBPr P1 transfected,the MMP2/TIMP2 and MMP9/TIMP1 ratio were increased significantly at every time point(P< 0.05).Conclusion: Down-regulating the expression of IGFBPr P1 by UTMD-mediated gene delivery with CMB exert inhibitory effects against liver fibrosis by inhibiting HSC activation,reducing the expression of TGF?1,reversing MMP2/TIMP2 and MMP9/TIMP1 balance in mice induced with TAA or BDL.Part 4 The relationship between the regulation of MMPs/TIMPs balance by IGFBPr P1 with Hh pathway during hepatic fibrosisExperiment 5 The dynamic changes of Hh pathway in the development of liver fibrosis in miceObjective:To invesgate the changes of Hh pathway in the development of liver fibrosis induced by TAA or BDL or Ad IGFBPr P1.Methods: The experimental groups and treatments in mice are similar with the experiment 1 and 2.The protein expression of Shh and Gli1 in liver were examined by immunohistochemical techniques and western blot.The correlations were evaluated by Pearson analysis.Results:(1)Immunohistochemistry and western blot analysis the protein expression of Shh and Gli1 in liver: after TAA or after BDL administration to mice,the liver Shh and Gli1 expression was increased with the degree of liver fibrosis(P < 0.05);after Ad IGFBPr P1 transfected to mice,the liver Shh and Gli1 expression was also increased with the degree of liver fibrosis(P< 0.05).(2)There were statistically significant positive correlations between the expression of IGFBPr P1 with Shh or Gli1 in mice with liver fibrosis induced by TAA or BDL or Ad IGFBPr P1.Conclusion: IGFBPr P1 contributes to hepatic fibrosis by the activation of Hh pathway.Experiment 6 The influence of Hh pathway in the process of liver fibrosis in mice by sh RNA-IGFBPr P1Objective:To invesgate the changes of Hh pathway after down-regulating the expression of IGFBPr P1 in mice induced by TAA or BDL and in NIH/3T3 cells.Methods: The experimental groups and treatments in cells are similar with the experiment 3,while in mice are similar with the experiment 4.The m RNA and protein expression of Shh and Gli1 were examined by quantitative RT-PCR and western blot.Results:(1)RT-PCR and western blot analysis the m RNA and protein expression of Shh and Gli1 in cells:after sh RNA-IGFBPr P1 transfected,the m RNA and protein expression of Shh and Gli1 were decreased significantly at 24 h,48h,72 h,96h(P< 0.05).(2)Immunohistochemistry and western blot analysis the protein expression of Shh and Gli1 in liver: In liver fibrosis mice induced by TAA or BDL,after sh RNA-IGFBPr P1 transfected,the liver Shh and Gli1 expression was decreased(P < 0.05).Conclusion:Down-regulating the expression of IGFBPr P1 exert inhibitory effects against liver fibrosis by inhibiting the activation of Hh pathway in mice induced with TAA or BDL and in NIH/3T3 cells.Experiment 7 Inhibiting Hh pathway by cyclopamine affected the regulation of MMP2/TIMP2 and MMP9/TIMP1 balance by IGFBPr P1 in miceObjective: Inhibiting Hh pathway by cyclopamine exert inhibitory effects against liver fibrosis whether by inhibiting the activation of Hh pathway,reversing MMPs/TIMPs balance in mice induced with TAA or BDL or Ad IGFBPr P1.Methods:(1)C57BL/6 wild-type mice were randomly divided into TAA group: intraperitoneal injection of TAA 100mg/Kg,three times a week;TAA+Vehicle group: one week following TAA,intraperitoneal injection of Vehicle 0.15ml/10g/d for 2 weeks;TAA+cyclopamine group: one week following TAA,intraperitoneal injection of cyclopamine 15mg/Kg/d for 2 weeks.Liver tissues and serum were obtained at week 2,4 and 6.Specimen collection and testing are same as Experiment 1 and 5.(2)C57BL/6 wild-type mice were randomly divided into BDL group: bile duct ligation;BDL+Vehicle group: one week following BDL,intraperitoneal injection of Vehicle 0.15ml/10g/d for 2 weeks;BDL+cyclopamine group: one week following BDL,intraperitoneal injection of cyclopamine 15mg/Kg/d for 2 weeks.Liver tissues and serum were obtained at week 2,4 and 6.Specimen collection and testing are same as Experiment 1 and 5.(3)C57BL/6 wild-type mice were randomly divided into Ad IGFBPr P1 group: injected with Ad IGFBPr P1 2×109 pfu per mouse via the tail vein;Ad IGFBPr P1+Vehicle group: one week following Ad IGFBPr P1,intraperitoneal injection of Vehicle 0.15ml/10g/d for 2 weeks;Ad IGFBPr P1+cyclopamine group: one week following Ad IGFBPr P1,intraperitoneal injection of cyclopamine15mg/Kg/d for 2 weeks.Liver tissues and serum were obtained at week 1,2,4,8and 12.Specimen collection and testing are same as Experiment 1 and 5.Results:(1)Immunohistochemistry and western blot analysis the protein expression of Shh and Gli1 in liver: In liver fibrosis mice induced by TAA or BDL or Ad IGFBPr P1,after intraperitoneal injection of cyclopamine,the liver Shh and Gli1 expression was decreased(P< 0.05).(2)Immunohistochemistry and western blot analysis the protein expression of IGFBPr P1,?-SMA,TGF?1,Collagen?in liver: In liver fibrosis mice induced by TAA or BDL or Ad IGFBPr P1,after intraperitoneal injection of cyclopamine,the TGF?1??-SMA?Collagen ? expression was decreased(P< 0.05),The expression of IGFBPr P1 was not changed in the early state of liver fibrosis and reduced later(P< 0.05).(3)Immunohistochemistry and western blot analysis the protein expression of MMPs and TIMPs in liver: In liver fibrosis mice induced by TAA or BDL or Ad IGFBPr P1,MMPs and TIMPs protein expression at different time point was analysed,after intraperitoneal injection of cyclopamine,the MMP2/TIMP2 and MMP9/TIMP1 ratio were increased significantly at every time point(P< 0.05).Conclusion: Inhibiting Hh pathway by cyclopamine exert inhibitory effects against liver fibrosis by inhibiting the activation of Hh pathway,inhibiting HSC activation,reducing the expression of TGF?1,reversing MMPs/TIMPs balance in mice induced with TAA or BDL or Ad IGFBPr P1. |
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