Preliminary Study Of The Effects Of Nerve Growth Factor And P75^NTR Protein On Hepatocytes

The liver plays a central role in biotransformation and detoxification. Factors likevirus, endotoxin, metabolism, and immune could induce cell injury,necrosis/apoptosis,which are the basic pathological process of acute and chronic liver disease includingfulminant hepatitis, hepatic fibrosis, and so on. The liver fibrogenesis is due to excessiveextracellular matrix (ECM) deposition, which is produced by activated myofibroblastsderived from portal fibroblasts and hepatic stellate cells (HSCs). The damaged hepatocytesrelease apoptotic bodies, which are engulfed by resident kupffer cells and HSCs, releasingchemokines and cytokines, which then can promote HSC activation and transdifferentiationinto myofibroblast. Therefore, taking effective measures to protect the hepatocyte andreduce its apoptosis is an important therapeutic strategy that could be adopted in curingliver fibrosis.Nerve growth factor (NGF) is produced by the nervous or non-nervous system cells,which in turn regulates the cells differentiation and survival or apoptosis,and actuallyrecognized as a pleiotropic factor. It could modulate the tropomyosin trkA tyrosine kinasereceptor (TrkANGFR), and the p75pan-neurotrophin receptor (p75^NTR) expression byautocrine/paracrine pathway, and then regulates the survival/apoptosis of fibroblasts,inflammatory cells and immunocytes, finally participates in inflammatory-allergic response,wound-healing, and tissue repair-remodeling process. NGF expressed by regeneratinghepatocytes and activated HSC regulates HSCs differentiation/apoptosis by NGF/p75^NTRsignal pathway during hepatic injury-repair process. Since there is little information ofNGF and p75^NTRprotein on hepatocytes, this study is to investigate the effects of NGF andp75^NTRprotein on L-02cells.The current study aims to observe NGF, TrkANGFR, and p75^NTRexpression in liverfibrosis tissue and L-02cells, investigate the effects of ectogenesis recombinant humanNGF-β and p75^NTRprotein on L-02cells. The study includes four parts as following. The First Part: The Protective Effects of Nerve Growth Factor-β against D-Galactosamine-induced L-02Cells Injury in Primary CultureOBJECTIVE: To investigate the hepatoprotective effects of nerve growth factor-β (NGF-β)against D-galactosamine (D-GalN)-induced L-02cells injury in vitro.METHODS: The L-02cells were pretreated with different concentration NGF-β for30min,Then L-02was incubated by D-GalN (40mmol/L) for24h, silibinin (100mg/L) as positivecontrol,without treatment as negative control. The morphological changes of L-02cells inculture were examined using inverted microscope (Olympus CK40). Culture medium andcell lysate were collected for biochemical assays. Lactate dehydrogenase (LDH), Malondial-dehyde (MDA), Glutathione (GSH), and Albumin (ALB) were measured by colorimetrictechnique. The cell viability and mitochondrial membrane potential were evaluated by XTTassays and Flow Cytometry with JC-1kit respectively.RESULTS: D-GalN caused severe injury to L-02cells in primary culture after24h ofincubation. L-02cells incubated with D-GalN were associated with discontinuities of thecytoplasm membrane, spherical shape, highly granular cytoplasm and marked reduction inbrightness contrast between the nucleus and cytoplasm in comparison with intact cells.Ectogenesis NGF-β considerably reduced cell morphological changes and promoted theproliferation of L-02damaged by D-GalN. The signifcant reduction of LDH and MDArelease was observed in the culture medium of L-02cells incubated by NGF-β. Content ofALB and GSH was dramatically increased in L-02cells treated with NGF-β, and mitochon-drial membrane permeability was dramatically improved by NGF-β.CONCLUSION: NGF-β can protect L-02cells from injury induced by D-GalN, which isassociated with reduction of plasma membrane damage, increased synthesis of glutathionepreventing of lipid peroxidant, and recovered mitochondrial membrane potential. The Second Part: To Study the Expression of NGF, TrkANGFRand p75^NTRin Human Liver Fibrosis Tissue and L-02CellsOBJECTIVE: To observe nerve growth factor (NGF) and its high-affinity tyrosine kinase A receptor (TrkANGFR), low-affinity p75neurotrophin receptor (p75^NTR) expression in humanliver fibrosis tissue and L-02cells.METHODS: Immunohistocytochemistry was performed in formalin-fixed, paraffin-embedded human liver fibrosis sections and L-02cells primary cultured in vitro for NGF,TrkANGFR, and p75^NTRprotein. NGF, TrkANGFR, and P75NTRmRNA transcripts were shown byreal-time Polymerase Chain Reaction.RESULTS: Immunohistocytochemistry staining with antibody of NGF, TrkANGFRandp75^NTRwere immunoreactive in human liver fibrosis tissue and L-02cells. L-02cellsexpressed NGF, TrkANGFRand p75^NTRprotein and mRNA transcripts. NGF mainly localizedin the cytoplasm and nuclei of L-02cells. TrkANGFRhighly and p75^NTRweakly were detectedon cell membrane and cytoplasm.CONCSLUSION: NGF, TrkANGFRand p75^NTRare immunoreactive in human liver fibrosistissues, and L-02cell line also expresses NGF, TrkANGFR, and p75^NTR. The Third Part: Nerve Growth Factor–β (NGF-β) Promotes Proliferationof L-02Cells in Vitro via TrkANGFRSignal PathwayOBJECTIVE: To observe the influence of exogenous recombinant human nerve growthfactor-β (NGF-β) on NGF and its high-affinity tyrosine kinase A receptor (TrkANGFR),low-affinity p75neurotrophin receptor (P75NTR) expression in L-02cells, and investigatethe effects of exogenous recombinant human NGF-β in L-02cells.METHODS: L-02Cell line was used in the experiment. The expression and intracellulardistribution of NGF, TrkANGFR, and p75^NTRwere detected by immunocytochemistry andfluorescent quantitation Polymerase Chain Reaction. The cell proliferation, apoptosis, andthe change of the cell cycle of L-02cells after exposed to exogenous NGF-β, anti-NGF,anti-TrkANGFR, anti-p75^NTRwere detected by XTT (2,3-bis(2-methoxy-4-nitro-5-sulfo-phenyl)-5-[(phenylamino)carbonyl]-2H-tetrazolium hydroxide) assay and flow cyto-metry with Annexin-V-FITC and Propidium iodide (PI) staining. RESULTS: Ectogenesis NGF-β promoted L-02cell expression of NGF, TrkANGFR, andp75^NTR. NGF mainly localized in the cytoplasm and nuclei of L-02cells, TrkANGFRhighlyand p75^NTRweakly were detected on cell membrane and cytoplasm. Low dose exogenousNGF-β (12.5-200μg/L) promoted L-02cells proliferation, and had an inhibition apoptosistendency by promoting cell mitosis from G1to S phase. High dose NGF-β (>400μg/L) didnot promote L-02cells proliferation. Specific neutralizing antibody of NGF and TrkANGFRdecreased the cell proliferation of L-02cells induced by exogenous NGF-β. However,blocking the binding of NGF to p75^NTRby anti-p75^NTRdid not affect the proliferation ofL-02cells.CONCSLUSION: Exogenous recombinant human NGF-β at optimal dose promotes L-02cell expression of NGF, TrkANGFRand p75^NTR, and promotes L-02cells proliferation in adose-dependent manner via NGF/TrkANGFRsignal pathways possibly. The Fourth Part: p75^NTRProtein Promotes Proliferation of L-02Cells inVitro via TrkANGFR/p75^NTRHeterodimer Signal PathwayOBJECTIVE: To observe the influence of exogenous recombinant human NGFR/TNFRSF16Fc Chimera (p75^NTR) on NGF and its receptor TrkANGFR, and p75^NTRexpression in L-02cells, and to investigate the effects of exogenous recombinant humanp75^NTRin L-02cells.METHODS: L-02Cells were cultured in vitro, intracellular distribution and expression ofNGF, TrkANGFR, and p75^NTRwere assessed by immunocytochemistry and fluorescentquantitation Polymerase Chain Reaction. The cell proliferation, apoptosis, and the changeof the cell cycle of L-02cells after exposed to exogenous p75^NTR, NGF-β, NGF-β+p75^NTR, anti-TrkANGFR, and anti-p75^NTRwere detected by XTT (2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino) carbonyl]) assay and Flow Cytometry withAnnexin-V-FITC and Propidium iodide (PI) staining.RESULTS: Ectogenesis p75^NTRprotein promoted L-02cell expression of NGF, TrkANGFR,and p75^NTR. NGF was mainly localized in cytoplasm and nuclei of L-02cells. TrkANGFR highly and p75^NTRweakly were expressed in cell membrane and cytoplasm. Exogenousp75^NTRprotein promoted L-02cells proliferation, and had an inhibition apoptosis tendencyby promoting cell mitosis from G1to S phase. Exogenous NGF-β and p75^NTRproteinpromoted L-02proliferation synergistically. Specific neutralizing antibody of TrkANGFRdecreased the proliferation of L-02cells. However, blocking the binding of NGF to p75^NTRby anti-p75^NTRdid not affect the proliferation of L-02cells.CONCSLUSION: Ectogenesis recombinant human p75^NTRprotein promotes L-02cellexpression of NGF, TrkANGFRand p75^NTR, and facilitates L-02cells proliferation in a dose-dependent manner via TrkANGFR/p75^NTRheterodimer signal pathway possibly.