The Regulatory Effects Of Sympathetic Neurotransmitter On HSCs Biological Behaviours And Its Mechanism

Hepatic fibrosis, the final stage of which is cirrhosis, can result from several chronic liver diseases caused by many pathogenic factors. The main pathological characteristic of hepatic fibrosis is the increased irregular deposition of extracellular matrix (ECM). Hepatic stellate cells (HSCs) play a pivotal role in this process. The activation of HSCs may result in their spontaneous proliferation with a strong fibrogenic activity. In recovery period, the apoptosis of HSCs increases significantly. So the proliferation and apoptosis of HSCs play a key role in the process of formation and resolution of hepatic fibrosis.It has been confirmed that HSCs can express many nerve markers and accept the innervation of sympathetic nerves and parasympathetic nerves. HSCs can also synthesize and release many neurotransmitters, such as norepinephrine (NE), dopamine (DA), hydroxytryptamine (5-HT), and expressα1B,α1D,β1,β2 adrenoceptor. This indicated that HSCs might come from nerve crist and act as neuroendocrine cells in liver. When stimulated, HSCs experience phenotypic alternation and regulate the function of the liver.Sympathetic nervous system (SNS) distributed extensively and involved in the functional regulation of almost every system and organ of the body. It has been reported that liver cirrosis patients had a high SNS activity with increased catecholamine in blood circulation. And sympathetic neurotransmitter NE could induce the proliferation of HSCs in vitro. So we can say sympathetic nervous system is contributed to the development of hepatic fibrosis by the regulation of HSC biological behaviors. However, to the best of our knowledge, few researches have been reported about the influences of SNS classical neurotransmitter (NE) and specific adrenoceptor on the development of hepatic fibrosis. The mechanisms of NE on HSCs proliferation and apoptosis as well as the intercellular signal transduction pathways remain unclear. Hence, we detected the expression of adrenoceptor subtypes in the hepatic fibrosis process of the rat models established by bile duct ligation. Through experiments in vitro we aim to investigate the effects of different adrenoceptor subtypes on HSC proliferation and apoptosis as well as collagen metabolism. The roles of signaling pathways in hepatic fibrosis and HSCs proliferation and apoptosis were studied to illuminate the effects and mechanisms of SNS on HSCs biological behaviors, in order to provide new viewpoint and defined strategy for preventing and treating hepatic fibrosis. The project contain four parts as below:Part 1:Dynamic changes ofα-AR,β1-AR andβ2-AR expression during hepatic fibrogenesisObjective: To investigate the dynamic changes ofα-AR,β1-AR,β2-AR expression in hepatic fibrosis rats.Methods: The present research was designed to establish rat hepatic fibrosis models by bile duct ligation (BDL). HE and Masson staining were used to determine hepatic fibrosis levels. Immunohistochemistry was applied to detect HSC activation indicator,α-SMA; Western blot and Real-time Q-PCR were used to measure the dynamic changes ofα-AR,β1-AR,β2-AR expression on protein and mRNA levels respectively during the development of hepatic fibrosis.Results: (1) General condition in rat hepatic fibrosis models: 1~2 h after BDL, rats recovered from operation and begin daily activities again. At about 48 h, urine became yellow; 3~4 d later, their skin and hairs began to turn yellow and they gradually became weak, ate less than their peers, with insignificant increasing or slight decreasing of body weights. 8~9 d later, the general state of model rats became poorer, with more sleep, slow response, less activities, significant jaundice and grey feces. 20 d later, their food-intake and body weights decreased significantly compared with that of control group, and some rats expressed abdominal bulge gradually. (2) Histopathological changes: The liver of rat after BDL appears to be brownish green or brown, with fine granule and hard texture on surface. HE and Masson trichrome staining showed that the liver tissue of sham operation group rats had complete hepatic lobule, well arranged hepatic plate, clear nucleous, and small quantity of connective tissue limited in portal area, without swollen hepatocytes, bile duct proliferation, silt chole and lymphocytes infiltration. 1 week after BDL, there was sparkling degeneration, necrosis and inflammation cell infiltration, fragmental necrosis in partial areas, small bile duct epithelial proliferation in portal area in the liver tissue of model rats. At 2 weeks after modeling, the liver tissue of model group rats showed abnormal arrangement of hepatic plates, disordered lobule structure, extensive proliferation small bile duct in portal areas extending to lobule, chaplet like hepatic lobule, fibrous tissues around portal areas, enlarging portal areas, fibrous tissues proliferation in hepatic lobule. At 3~4 weeks after modeling, the liver tissue demonstrated extensive fibrous connective tissue proliferation, and the proliferated fibers connected, enclosed and divided with each other to change the original hepatic lobule, even forming pseudo lobule. Masson trichrome stain collagen area density measurement demonstrated that 1 wk, 2 wk, 3 wk and 4 wk after BDL, collagen area and density in the model group(10.11%±1.14%, 21.14%±1.22%, 28.87%±2.05%, 37.44%±3.17%) were significantly higher than that of the sham operation group(3.22%±0.77%), P<0.01; (3) Changes ofα-SMA expression in hepatic fibrosis rats:α-SMA immunohistochemistry showed that it only expressed weakly in smooth muscle cells of vessel wall in normal rats hepatic tissues; with the development of hepatic fibrosis,α-SMA positive cells obviously increased in liver of modeo rats, distributing mainly in header area, fiber compartment, hepatic sinusoid surroundings, and proliferated bile duct surrounding cells. At 1 wk, 2 wk, 3 wk, 4 wk after BDL,α-SMA positive area density in the model group (10.58%±1.75%, 24.14%±2.02%, 29.74%±2.59%, 34.28%±2.01%) was significantly higher than that of the sham operation group(4.12%±1.51%), P<0.01; that is,α-SMA positive area density increased with the development of the hepatic fibrosis. (4) Western blot to detect the protein expression ofα-AR,β1-AR andβ2-AR. The results demonstratedα-AR specific bands were present at about 57 kD,β1-AR at about 50 kD,β2-AR at about 47 kD andβ-actin at 43 kD. At 1 wk, 2 wk, 3 wk, 4 wk after BDL, Western blot showed that the expression ofα-AR,β1-AR,β2-AR protein increased with the development of the hepatic fibrosis and the protein expression in the 4 groups were significantly higher than that of the sham operation group(1.54±0.08, 1.87±0.15, 2.72±0.09, 2.84±0.18 vs 0.85±0.12, P<0.05; 1.57±0.18, 1.92±0.11, 2.51±0.17, 2.89±0.19 vs 0.98±0.15, P<0.05; 1.84±0.20, 1.97±0.09, 2.85±0.14, 3.87±0.18 vs 1.24±0.18, P<0.05). (5)α-AR,β1-AR andβ2-AR mRNA expression variations in hepatic fibrosis rats detected by Real-time Q-PCR: At 1 wk, 2 wk, 3 wk, 4 wk after BDL,α-AR,β1-AR andβ2-AR mRNA relative expression content was calculated according to△c(t)intervention=c(t)purpose gene-c(t)GAPDH,△c(t)control=c(t)purpose gene - c(t)GAPDH,△△c (t)mRNA=△c(t) intervention -△c(t)control. relative expression content =2-△△c(t),α-AR,β1-AR andβ2-AR mRNA expression increased with the development of the hepatic fibrosis and the mRNA content in the 4 wk group were highest(1.54±0.08, 2.98±0.09, 3.23±0.11, 3.94±0.13 vs 1.00±0.07, P<0.01; 1.47±0.10, 2.13±0.09, 2.54±0.12, 2.81±0.10 vs 1.00±0.10, P<0.01; 1.24±0.07, 2.78±0.10, 3.54±0.14, 4.24±0.15 vs 1.00±0.08, P<0.01), respectively. (6) Multielement correlation analysis ofα-SMA andα-AR,β1-AR,β2-AR. The relation betweenα-SMA andα-AR,β1-AR,β2-AR were positive correlation, r value were 0.564, 0.753 and 0.606, respectively.Conclusions: Rat hepatic fibrosis models with bile duct ligation (BDL) were established succesfully. HSCs activated and proliferated greatly during the fibrosis. The expression ofα-SMA, the marker of HSCs activation, increased dramaticaly.α-AR,β1-AR,β2-AR increased on both protein and mRNA levels during the development of hepatic fibrosis, and were positive correlated withα-SMA.Part 2:Effects of norepinephrine (NE) on hepatic stellate cell biological behaviorsObjective: To investigate the effects of NE on hepatic stellate cell (HSC) proliferation and apoptosis as well as collagen metabolism.Methods: Using cell culture techniques in vitro, after NE of different concentrations were administered to HSCs, MTT method was used to evaluate cell proliferation. Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay was applied to detect cell apoptosis. And flow cytometry (FCM) was used to detect cell apoptosis and the protein expression of bcl-2 and bax. Inverted microscope was used to observe the morphological changes of HSCs. Real-time Q-PCR was used to measure the expression of collagenⅠon mRNA level. And Western blot and Real-time Q-PCR were used to measure the expressions of MMP-13, TIMP-1 on protein and mRNA levels.Results: (1) Compared with control group, NE at concentrations of 1, 10 and 100μmol·L-1 induced HSCs proliferation in a time-dependent manner detected by MMT; The action of NE at concentrations of 10μmol·L-1 reached the peak (0.262±0.085 vs 0.084±0.053, P<0.05). (2) After exposure of HSCs to NE at concentration of 10μmol·L-1 for 24 h, apoptosis rates decreased significantly compared with control group(6.60%±3.05% vs 12.60%±4.76% by TUNEL, P<0.01; 2.29%±0.22% vs 3.06%±0.57% by FCM, P<0.05). (3)The expression of apoptosis regulating gene bax decreased when NE was administrated to the HSCs for 24 h (6.60%±2.75% vs 9.60%±1.76%, P<0.05); while the expression of bcl-2 increased when NE was administrated to the HSCs (14.29%±0.41% vs 7.06%±0.54%, P<0.05). (4) No obvious morphological changes of HSCs were found after exposure of HSCs to NE. HSCs grow adhered on the wall of the culture flask and most cells appear to be polygon. After 24 h, the growth of HSCs came into exponential phase and form compact cell layer. After NE was added for 72 h, the cells proliferated dramatically, no obvious morphological changes of HSCs were found by the inverted microscope. (5) CollagenⅠmRNA expression in HSCs when NE of different concentrations were added: The relative expression content of collagenⅠmRNA of the NE groups were significantly higher than that of the control group (1.47±0.09, 1.91±0.11, 2.46±0.15, P<0.05). (6) Western blot was used to detect the protein expression of MMP-13 and TIMP-1. The results demonstrated MMP-13 specific bands were present at about 60 kD, TIMP-1 specific bands at about 28 kD andβ-actin bands at 43 kD. Western blot showed that the expression of MMP-13 of the NE groups were significantly lower than that of the control group (0.75±0.24,0.57±0.11,0.37±0.28 vs 0.98±0.21), P<0.05; The expressions of TIMP-1 of the NE groups were significantly higher than that of control group (0.57±0.09, 1.01±0.14, 1.15±0.17 vs 0.55±0.05), P<0.01; The ratio of MMP-13/TIMP-1 were dicreased significantly(1.32±0.05, 0.56±0.10, 0.32±0.09 vs 1.78±0.10, P<0.01). (7) MMP-13 and TIMP-1 mRNA expression in HSCs when NE of different concentrations were added: MMP-13 and TIMP-1 mRNA relative expression content was calculated according to△c(t)intervention=c(t)purpose gene - c(t)GAPDH,△c(t)control= c(t)purpose gene - c(t)GAPDH,△△c(t)mRNA=△c(t)intervention -△c(t) control. relative expression content =2-△△c(t), The expression of MMP-13 of the NE groups were significantly lower than that of the control group (0.81±0.13, 0.59±0.07, 0.48±0.08), P<0.05; The expression of TIMP-1 of the NE groups were significantly higher than that of the control group (1.47±0.12, 1.91±0.14, 2.06±0.11), P<0.05; The ratio of MMP-13/TIMP-1 were dicreased significantly (0.55±0.09, 0.31±0.10, 0.23±0.07, P<0.05).Conclusions: Sympathetic neurotransmitter, NE, could induce HSCs proliferation, and inhibit HSCs apoptosis in vitro. NE could induce the expression of bcl-2 and inhibit bax expression. MMP-13 expression decreased and TIMP-1 increased significantly on protein and mRNA levels when NE was added to the HSCs, and the banlance of MMP-13/TIMP-1 was broken. Thus the collagen degradation reduced and the expression of collagenⅠincreased which might represent a mechanism promoting the development of hepatic fibrosis.Part 3:Effects of adrenoceptor subtypes on hepatic stellate cell biological behaviorsObjective: To investigate the effects of adrenoceptor subtypes on the biological behaviors of hepatic stellate cells (HSCs).Methods: Using cell culture techniques in vitro, immunohistochemistry was applied to detect the expression ofα-AR,β1-AR andβ2-AR in HSCs; Western blot and Real-time Q-PCR were used to measure the expression ofα-AR,β1-AR andβ2-AR on both protein and mRNA levels. HSCs were devided in 6 groups as below: (1) control group; (2) NE group; (3) phentolamine (α-AR antagonist) group; (4) CGP20712A (β1-AR antagonist) group; (5) ICI118551 (β2-AR antagonist) group; (6) phentolamine plus propranolol group. After the antagonists of adrenoceptor subtypes were administered to the cultured HSCs, MTT assay was used to evaluate the cell proliferation. Terminal deoxyribonucleotidyltransferase-mediated dUTP nick end labelling (TUNEL) assay was used to detect cell apoptosis. And flow cytometry (FCM) was used to detect cell apoptosis and bcl-2 and bax protein expression. Real-time Q-PCR was used to measure the expression of collagenⅠon mRNA level. Western blot and Real-time Q-PCR were used to detect the expressions of MMP-13, TIMP-1 on both protein and mRNA levels.Results: (1) Western blot analysis demonstratedα-AR specific bands were present at about 57 kD,β1-AR specific bands at about 50 kD,β2-AR specific bands at about 47 kD andβ-actin bands at 43 kD.Immunohistochemistry affirmed the expression ofα-AR,β1-AR andβ2-AR in HSCs, mainly located at the cytoplasm and membrene; Real time Q-PCR revealed mRNA expression ofα-AR,β1-AR andβ2-AR in HSCs. (2) Compared with control group, NE induced HSCs proliferation detected by MTT. When the antagonists ofα-AR,β1-AR,β2-AR were administrated into the HSCs, the proliferation was inhibited. The inhibition was most severe whenα-AR antagonist andβ-AR antagonist were administrated into the HSCs together. (3) When the antagonists were added to the HSCs, apoptosis rates increased greatly detected by TUNEL. The effects ofα-AR andβ2-AR antagonist were significant (17.40%±4.51% and 21.40%±3.51% vs 12.60%±4.76%, P<0.01); whileβ1-AR antagonist CGP20712A can induce the apoptosis, too (13.60%±3.29% vs 12.60%±4.76%, P>0.05). (4) HSCs apoptosis rates increased greatly when the antagonists were added detected by FCM. The effects ofα-AR andβ2-AR antagonist were significant (5.04%±1.44% and 5.99%±2.14% vs 3.06%±0.57%, P<0.05); whileβ1-AR antagonist CGP20712A can induce apoptosis too, though P>0.05. (5) The expression of apoptosis regulating gene bax increased while bcl-2 decreased when the antagonists were added to the HSCs. The effects ofα-AR andβ2-AR antagonist were significant, (11.40±2.51, 10.40±1.41 vs 9.60±1.76, P<0.05; 5.04±1.44,4.99±1.41 vs 7.06±0.54, P<0.05); while the effect ofβ1-AR antagonist CGP20712A was weak, P>0.05. (6) When the antagonists ofα-AR,β1-AR,β2-AR were added to the HSCs, the expression of collagenⅠmRNA in HSCs decreased (0.40±0.11, 0.74±0.10 and 0.31±0.09, P<0.05). The effects were most significant when the antagonists ofα-AR andβ-AR were added together. (7) Western blot was used to detect the protein expression of MMP-13 and TIMP-1. The results demonstrated MMP-13 specific bands were present at about 60 kD, TIMP-1 specific bands at about 28 kD andβ-actin bands at 43 kD. Western blot showed that the expression of MMP-13 increased of HSCs when the antagonists ofα-AR,β1-AR,β2-AR were added to the HSCs (0.99±0.08, 1.26±0.13 and 1.27±0.07 vs 0.98±0.21, P<0.05) while TIMP-1 decreased (0.37±0.02, 0.54±0.21, 0.44±0.09 vs 0.55±0.05); The ratio of MMP-13/TIMP-1 increased significantly (2.68±0.07, 2.33±0.08, 2.89±0.12 vs 1.78±0.10, P<0.01). The effects were most significant when the antagonists ofα-AR andβ-AR were added together. (8) MMP-13 and TIMP-1 mRNA expression in HSCs when NE was added: MMP-13 and TIMP-1 mRNA relative expression content were calculated according to△c(t)intervention=c(t)purpose gene-c(t)GAPDH,△c(t)control=c(t)purpose gene - c(t)GAPDH,△△c(t)mRNA=△c(t) intervention -△c(t)control. Relative expression content =2-△△c (t). The expression of MMP-13 increased when the antagonists were added to the HSCs (1.98±0.18, 1.08±0.15 and 1.83±0.14, P<0.05) while TIMP-1 decreased (0.54±0.08, 0.89±0.10, 0.49±0.07, P<0.05); The ratio of MMP-13/TIMP-1 increased significantly (3.67±0.08, 1.21±0.11, 3.73±0.12, P<0.01). The effects were most significant when the antagonists ofα-AR andβ-AR were added together.Conclusions: HSCs can expressα-AR,β1-AR,β2-AR both on protein and mRNA level, mainly located on the membrene and cytoplasm. The antagonists ofα-AR,β1-AR,β2-AR can inhibit the proliferation of HSCs, and induce the apoptosis of HSCs. They can inhibit the expression of bax and induce the expression of bcl-2. The effects of phentolamine and ICI118551 were most significant. The antagonists can increase the expression of MMP-13 and decrease the expression of TIMP-1 thus decrease the ratio of MMP-13/TIMP-1. The expression of collagenⅠd ecreased significantly, too.Part 4: The signaling transduction mechanisms of norepinephrine (NE) on HSC biological behaviorsObjective: To investigate the signaling transduction pathway of NE on hepatic stellate cell (HSC) biological behaviors.Methods: Using cell culture techniques in vitro, after NE and the antagonist of PI-3K signaling transduction pathway were administered to HSCs, MTT method was used to evaluate cell proliferation. Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay and flow cytometry (FCM) were used to detect cell apoptosis. And Western blot was used to detect the protein expression of PI-3K.Results: (1) Compared with control group, NE induced HSC proliferation in a time-dependent manner; PI-3K signaling transduction pathway antagonist LY294002 inhibited the proliferation of HSCs. (2) After exposure of HSCs to NE at concentration of 10μmol/L for 24 h, apoptosis rates decreased significantly compared with control group. When the PI-3K signaling transduction pathway antagonist LY294002 were added to the HSCs, the apoptosis rates increased (14.40%±4.51% vs 6.60%±3.05% by TUNEL, P<0.05; 5.04%±1.44% vs 2.29%±0.22% by FCM, P<0.05). (3) When the antagonist LY294002 were administrated, the protein expression of PI-3K decreased significantly by Western blot.Conclusions: Sympathetic neurotransmitter, NE, could induce HSCs proliferation, and inhibit HSCs apoptosis. This is correlated with PI-3K signaling transduction pathway.