Effects of endothelin-1 on hepatic stellate cell proliferation, collagen synthesis and secretion, intracellular free calcium concentration

AIM: To explore the effects of endothelin-1(ET-1) on hepatic stellate cells (HSCs) DNA uptake, DNA synthesis, collagen synthesis and secretion, inward whole-cell calcium concentration ([Ca2+]i) as well as the blocking effect of verapamil on ET-1-stimulated release of inward calcium (Ca2+) of HSC in vitro. METHODS: Rat hepatic stellate cells (HSCs) were isolated and cultivated. 3H-TdR and 3H-proline incorporation used for testing DNA uptake and synthesis, collagen synthesis and secretion of HSCs cultured in vitro; Fluorescent calcium indicator Fura-2/AM was used to measure [Ca2+]i inward HSCs. RESULTS: ET-1 at the concentration of 5X10(-8) mol/L, caused significant increase both in HSC DNA synthesis (2,247+/-344 cpm, P<0.05) and DNA uptake (P<0.05) when compared with the control group. ET-1 could also increase collagen synthesis (P<0.05 vs control group) and collagen secretion (P<0.05 vs control group). Besides, inward HSC [Ca2+]i reached a peak concentration (422+/-98 mol/L, P<0.001) at 2 min and then went down slowly to 165+/-51 mol/L (P<0.01) at 25 min from resting state (39+/-4 mol/L) after treated with ET-1. Verapamil (5 mol/L) blocked ET-1-activated [Ca2+]i inward HSCs compared with control group (P<0.05). Fura-2/AM loaded HSC was suspended in no Ca2+ buffer containing 1 mol/L EGTA, 5 min later, 10(-8) mol/L of ET-1 was added, [Ca2+]i inward HSCs rose from resting state to peak 399+/-123 mol/L, then began to come down by the time of 20 min. It could also raise [Ca2+]i inward HSCs even without Ca2+ in extracellular fluid, and had a remarkable dose-effect relationship(P<0.05). Meanwhile, verapamil could restrain the action of ET-1(P<0.05). CONCLUSION: Actions of ET-1 on collagen metabolism of HSCs may depend on the transportation of inward whole-cell calcium.     

Effect of rat serum containing Biejiajian oral liquid on proliferation of rat hepatic stellate cells

AIM: Liver fibrosis is a common pathological process of chronic liver diseases. Activation of hepatic stellate cells (HSCs) is the key issue in the occurrence of liver fibrosis. In this study, we observed the inhibitory action of rat serum containing Biejiajian oral liquid (BOL), a decoction of turtle shell, on proliferation of rat HSCs, and to explore the anti-hepatofibrotic mechanisms of BOL. METHODS: A rat model of hepatic fibrosis was induced by subcutaneous injection of CCl(4). Serum containing low, medium and high dosages of BOL was prepared respectively. Normal and fibrotic HSCs were isolated and cultured. The effect of sera containing BOL on proliferation of HSCs was determined by (3)H-TdR incorporation. RESULTS: The inhibitory rate of normal rat HSC proliferation caused by 100 mL/mL sera containing medium and high dosages of BOL showed a remarkable difference as compared with that caused by colchicine (medium dosage group: 34.56+/-4.21% vs 29.12+/-2.85%, P<0.01; high dosage group: 37.82+/-1.32% vs 29.12+/-2.85%, P<0.01). The inhibitory rate of fibrotic rat HSC proliferation caused by 100 mL/L serum containing medium and high dosages of BOL showed a remarkable difference as compared with that caused by colchicine (medium dosage group: 51.31+/-3.14% vs 38.32+/-2.65%, P<0.01; high dosage group: 60.15+/-5.36% vs 38.32+/-2.65%, P<0.01). The inhibitory rate of normal rat HSC proliferation caused by 100 mL/L and 200 mL/L sera containing a medium dosage of BOL showed a significant difference as compared with that caused by 50 mL/L (100 mL/L group: 69.02+/-9.96% vs 50.82+/-9.28%, P<0.05; 200 mL/L group: 81.78+/-8.92% vs 50.82+/-9.28%, P<0.01). The inhibitory rate of fibrotic rat HSC proliferation caused by 100 mL/L and 200 mL/L sera containing a medium dosage of BOL showed a significant difference as compared with that caused by 50 mL/L (100 mL/L group: 72.19+/-10.96% vs 61.38+/-7.16%, P<0.05; 200 mL/L group: 87.16+/-8.54% vs 61.38+/-7.16%, P<0.01). CONCLUSION: Rat serum containing BOL can inhibit proliferation of rat HSCs, and the inhibition depends on the dosage and concentration of BOL. The inhibitory effect on HSC proliferation is one of the main anti-hepatofibrotic mechanisms of BOL.     

木犀草素抑制肝星状细胞增殖及其胶原合成

目的 研究木犀草素对体外培养的肝星状细胞(hepatic stellate cells,HSC)增殖及其胶原表达、合成的影响。方法 从Wistar大鼠肝脏分离培养HSC,并用~3H-TdR和~3H-pro同位素掺入实验,基因探针原位杂交等技术研究了木犀草素对HSC增殖、胶原基因表达合成的影响。结果 当木犀草素的浓度分别达到10 μmol/L和20 μmol/L 时抑制HSC增殖(t=2.542,P＜0.05)和胶原合成(t=3.650,P＜0.01),其作用具有剂量依赖关系;25 μmol/L木犀草素使Ⅰ、Ⅲ型前胶原mRNA的表达降低,其中Ⅰ型前胶原基因表达降低具有统计学差异(x~2=6.850,P＜0.01)。结论 木犀草素在体外抑制HSC增殖和胶原表达合成,在体内可能会具有预防或冶疗肝纤维化的作用。     

血小板衍化生长因子对血管平滑肌细胞增殖及胶原蛋白合成的影响

目的 :观察血小板衍化生长因子 （PDGF）对培养的血管平滑肌细胞 （VSMC）增殖及胶原蛋白合成的影响。方法 :采用培养的兔动脉 VSMC,应用 3H- Td R的 3H-脯氨酸掺入方法 ,观察 PDGF- BB对兔 VSMC DNA合成以及胶原蛋白合成的影响。结果 :PDGF- BB可促进处于静止状态的兔 VSMC DNA及胶原蛋白的合成 ,并呈现明显的浓度依赖关系 ,在 40 μg/ L 的浓度时 DNA及胶原蛋白的合成达到高峰 ,DNA及胶原蛋白分别处于 36 h和 48h合成最为显著。结论 :PDGF- BB可明显促进培养的 VSMC增殖及胶原蛋白的合成。     

The regulatory role of AT 1 receptor on activated HSCs in hepatic fibrogenesis:effects of RAS inhibitors on hepatic fibrosis induced by CCl(4)

AIM:To assess the effect of ACE inhibitor and Ang II type 1 (AT1) receptor antagonist in preventing hepatic fibrosis caused by CCl(4) administration in rats;to investigate whether or not there are expression of AT 1 receptors on hepatic stellate cells; and to observe the effect of Ang II on proliferation and ECM synthesis of cultured HSCs.METHODS:Studies were conducted in male Sprague-Dawley rats. Except for the hepatofibrotic model group and the control group, in three treated groups, either enalapril (5mg/kg), or losartan (10mg/kg), or enalapril + losartan were given to the fibrotic rats by daily gavage, and saline vehicle was given to model and normal control rats. After 6 weeks, liver fibrosis was assessed directly by hepatic morphometric analysis, which has been considered the gold standard for the quantification of fibrosis. The expressions of AT 1 receptors and (alpha-mooth muscle actin,alpha-SMA) in liver tissue or isolated hepatic stellate cells (HSCs) were detected by immunohistochemical techniques. The effect of Ang II on HSC proliferation was determined by MTT method. Effect of Ang II on collagen synthesis of HSCs was determined by (3)H-proline incorporation.RESULTS:Contrasted to the fibrosis in rats of the model group, groups of rats treated with either enalapril or losartan, or a combination of two drugs showed a limited expansion of the interstitium (4.23 plus minus 3.70 vs 11.22 plus minus 4.79, P<0.05), but no difference was observed among three treated groups (5.38 plus minus3.43, 4.96 plus minus 2.96, 4.23 plus minus 2.70, P>0.05). Expression of AT 1 receptors was found in fibrotic interstitium of fibrotic rats, whereas in normal control rats they were limited to vasculature only to a very slight degree. AT 1 receptors were also expressed on activated HSCs in the culture. At concentrations from 10(-9) to 10(-5)mol/L, Ang II stimulated HSC proliferation in culture in a dose dependent manner. Increasing Ang II concentrations produced corresponding increases in (3)H-proline incorporation. Differences among groups were significant.CONCLUSION:Angiotensin converting enzyme inhibitors and AT 1 blocker may slow the progression of hepatic fibrosis;activated HSCs express AT 1 receptors, and Ang II can stimulate the proliferation and collagen synthesis of HSCs in a dose-dependent manner; and activation of RAS may be related to hepatic fibrogenesis induced by CCl(4).     

细胞外信号调节激酶信号通路对乙醛刺激的大鼠肝星状细胞周期的影响

目的 观察特异性丝裂原细胞外信号反应激酶1(MEK 1)阻断剂(PD98059)对乙醛刺激的大鼠肝星状细胞(HSC)增殖及细胞周期的影响,并探讨其作用机制。 方法 用不同浓度的PD98059对乙醛刺激的HSC进行处理;以四甲基偶氮唑蓝法检测细胞增殖,流式细胞仪检测细胞周期,逆转录聚合酶链反应方法检测HSC内细胞周期蛋白-D1(Cyclin D1)mRNA和细胞周期蛋白依赖性激酶(CDK4)mRNA的表达。 结果 20、50、100μmol/L的PD98059均能显著且剂量依赖性地抑制乙醛刺激的HSC增殖,3组A值分别为0.109±0.020、0.081±0.010、0.056±0.020,与乙醛组A值0.146±0.030相比较,F=31.385,P<0.05;20、50、100 μmol/L的PD98059可显著抑制乙醛刺激的HSC由G1期进入S期,G0/G1期细胞百分比逐渐升高,3组G0/G1期细胞百分比分别为(61.9±6.3)％、(64.1±3.3)％、(70.9±4.8)％,与乙醛组(55.2±4.4)％相比较,F=16.402,P<0.05;50、100μmol/L的PD98059能显著抑制乙醛刺激的HSC内Cylin D1 mRNA表达,2组平均光强度比值分别为0.56±0.04,0.46±0.03,与乙醛组0.65±0.07相比较,F=68.758,P<0.05;50、100μmol/L的PD98059能显著抑制乙醛刺激的HSC内CDK4 mRNA表达,2组平均光强度比值分别为0.39±0.07,0.33±0.05,与乙醛组0.50±0.06相比较,F=29.406,P<0.05。 结     

转化生长因子—β1单克隆抗体对大鼠肺纤维化的治疗观察

目的探讨转化生长因子－β（ＴＧＦ－β）单克隆抗体对博莱霉素所致肺间质纤维化的作用。方法结合体内和体外实验，利用３Ｈ－胸腺嘧啶核苷（３Ｈ－ＴｄＲ）掺入法和Ｎｏｒｔｈｅｒｎ杂交法。结果表明大鼠肺纤维化模型中肺泡巨噬细胞的条件培养基能促进成纤维细胞的增殖和胶原ｍＲＮＡ的表达。ＴＧＦ－β１单克隆抗体对其有抑制作用，同时亦能减轻肺组织的病变程度和胶原ｍＲＮＡ的表达。结论ＴＧＦ－β１单克隆抗体可部分地抑制成纤维细胞增殖和胶原合成。     

Bile acids induce hepatic stellate cell proliferation via activation of the epidermal growth factor receptor

BACKGROUND & AIMS: Hepatic stellate cell (HSC) proliferation is a key event in the development of liver fibrosis. In many liver diseases, HSCs are exposed to inflammatory cytokines, reactive oxygen species, and bile acids. Although inflammatory cytokines and reactive oxygen species are known to promote proliferation of HSCs, nothing is known about the effects of bile acids on HSC proliferation or apoptosis. The aim of this study was to investigate the effects of bile acids on HSC proliferation. METHODS: HSCs were exposed to bile acids with different hydrophobicity (5-200 micromol/L). HSC proliferation and cell cycle-related events were assessed by bromodeoxyuridine incorporation, cell counting and proliferating cell nuclear antigen and cyclin E expression, apoptosis by caspase-3 activity assay, immunocytochemistry for active caspase-3 and acridine orange staining, and activation of signal transduction pathways by Western blot using phospho-specific antibodies. Uptake of bile acids was investigated using fluorescent bile acids. RESULTS: All bile acids, at concentrations >25 micromol/L, induce a 2.5- to 3-fold increase in HSC proliferation via activation of the epidermal growth factor receptor. Bile acid-induced proliferation is mediated by activation of a protein kinase C/extracellular signal-regulated kinase/p70S6K-dependent pathway. Bile acids did not induce apoptosis in HSCs. HSCs do not take up fluorescent bile acids and do not express the bile acid importer ntcp. CONCLUSIONS: Bile acids at levels reached in cholestatic conditions are an independent profibrogenic factor. Bile acids induce HSC proliferation via the activation of the epidermal growth factor receptor, whereas HSCs are protected against bile acid-induced apoptosis by excluding bile acids.     

Effect of ligand of peroxisome proliferator-activated receptor γ on the biological characters of hepatic stellate cells

AIM: To study the effect of rosiglitazone, which is a ligand of peroxisome proliferator-activated receptor gamma (PPARgamma), on the expression of PPARgamma in hepatic stellate cells (HSCs) and on the biological characteristics of HSCs. METHODS: The activated HSCs were divided into three groups: control group, 3 micromol/L rosiglitazone group, and 10 micromol/L rosiglitazone group. The expression of PPARgamma, alpha-smooth muscle actin (alpha-SMA), and type I and III collagen was detected by RT-PCR, Western blot and immunocytochemical staining, respectively. Cell proliferation was determined with methylthiazolyltetrazolium (MTT) colorimetric assay. Cell apoptosis was demonstrated with flow cytometry. RESULTS: The expression of PPARgamma at mRNA and protein level markedly increased in HSCs of 10 micromol/L rosiglitazone group (t value was 10.870 and 4.627 respectively, P<0.01 in both). The proliferation of HSCs in 10 micromol/L rosiglitazone group decreased significantly (t = 5.542, P<0.01), alpha-SMA expression level and type I collagen synthesis ability were also reduced vs controls (t value = 10.256 and 14.627 respectively, P<0.01 in both). The apoptotic rate of HSCs significantly increased in 10 micromol/L rosiglitazone group vs control (chi(2) = 16.682, P<0.01). CONCLUSION: By increasing expression of PPARgamma in activated HSCs, rosiglitazone, an agonist of PPARgamma, decreases alpha-SMA expression and type I collagen synthesis, inhibits cell proliferation, and induces cell apoptosis.     

Effect of N-acetyl-seryl-aspartyl-lysyl-proline on DNA and collagen synthesis in rat cardiac fibroblasts

N:-Acetyl-seryl-aspartyl-lysyl-proline (Ac-SDKP) is a natural inhibitor of pluripotent hematopoietic stem cell entry into the S phase of the cell cycle and is normally present in human plasma. Ac-SDKP is exclusively hydrolyzed by ACE, and its plasma concentration is increased 5-fold after ACE inhibition in humans. We examined the effect of 0.05 to 100 nmol/L Ac-SDKP on 24-hour (3)H-thymidine incorporation (DNA synthesis) by cardiac fibroblasts both in the absence and presence of 5% FCS. Captopril (1 micromol/L) was added in all cases to prevent the degradation of Ac-SDKP. Treatment of cardiac fibroblasts with 5% FCS increased thymidine incorporation from a control value of 12 469+/-594 to 24 598+/-1051 cpm (P:<0.001). Cotreatment with 1 nmol/L Ac-SDKP reduced stimulation to control levels (10 373+/-200 cpm, P:<0.001). We measured hydroxyproline content and incorporation of (3)H-proline into collagenous fibroblast proteins and found that Ac-SDKP blocked endothelin-1 (10(-8) mol/L)-induced collagen synthesis in a biphasic and dose-dependent manner, causing inhibition at low doses, whereas high doses had little or no effect. It also blunted the activity of p44/p42 mitogen-activated protein kinase in a biphasic and dose-dependent manner in serum-stimulated fibroblasts, suggesting that the inhibitory effect of DNA and collagen synthesis may depend in part on blocking mitogen-activated protein kinase activity. Participation of p44/p42 in collagen synthesis was confirmed, because a specific inhibitor for p44/p42 activation (PD 98059, 25 micromol/L) was able to block endothelin-1-induced collagen synthesis, similar to the effect of Ac-SDKP. The fact that Ac-SDKP inhibits DNA and collagen synthesis in cardiac fibroblasts suggests that it may be an important endogenous regulator of fibroblast proliferation and collagen synthesis in the heart. Ac-SDKP may participate in the cardioprotective effect of ACE inhibitors by limiting fibroblast proliferation (and hence collagen production), and therefore it would reduce fibrosis in patients with hypertension.     

转化生长因子β1对不同活化状态肝星状细胞增殖与Ⅰ型胶原表达的影响

目的 观察不同活化状态肝星状细胞(HSC)对外源性转化生长因子-β_1(TGF-β_1)旁分泌刺激的生物学效应作用。方法 原代分离培养大鼠HSC,无包被塑料培养皿上分别培养1、4、7d,细胞处于静止、中间活化与完全活化状态,继以10～500 pmol/L TGF-β_1温育细胞24h,~3H—TdR掺入法测定细胞增殖,western blot法检测细胞α-平滑肌肌动蛋白(α-SMA)与Ⅰ型胶原蛋白表达沉积,~3H-脯氨酸掺入与胶原酶消化法测定细胞总胶原的分泌量。100pmol/L TGF-β_1温育细胞15～90min,northern blot法检测细胞Ⅰ型前胶原mRNA的表达水平。结果 TGF-β_1浓度依赖性抑制培养1d HSC的细胞增殖,10～500 pmol/L TGF-β_1浓度组细胞内~3H—TdR掺入率分别为对照组的52.8％～16.8％,与对照组比较,q值为5.44～10.37,P<0.01。但TGF-β_1对培养4d与7d的细胞增殖无影响。随细胞活化,HSC基础性α-SMA、Ⅰ型胶原蛋白与mRNA水平明显增加,而TGF-β_1刺激各培养时间HSC以上蛋白与基因的表达。培养1、4、7d HSC基础水平与TGF-β_1刺激的总胶原分泌量分别为(804±274)dpm/孔与(1 200±708)dpm/孔;(2 966±1 701)dpm/孔与(6 160±1 123)dpm/孔;(2 580±767)dpm/孔与(4 583±1 467)dpm/孔,后2组组内比较,t值分别为3.84与2.96,P<0.01或P<0.05。以培养4d HSC     

Progelatinase A is produced and activated by rat hepatic stellate cells and promotes their proliferation.

Activated hepatic stellate cells (HSCs) are a potential source of gelatinase A, which accumulates in fibrotic livers. Progelatinase A activation requires its binding to a complex of membrane-type matrix metalloproteinase (MT-MMP) and tissue inhibitor of metalloproteinases (TIMP)-2. These studies examine gelatinase A, MT1-MMP, and TIMP-2 synthesis by HSCs during activation in vitro and the potential role of gelatinase A in promoting HSC proliferation. Gelatinase A, MT1-MMP, and TIMP-2 messenger RNA (mRNA) were all upregulated in HSCs activated on plastic over 5 to 14 days. Gelatinase A expression was maximal at 7 days of culture, coinciding with the peak of HSC proliferation and the onset of procollagen I and alpha-smooth muscle actin (alpha-SMA) mRNA expression. Active forms of gelatinase A of 62 kd and 66 kd were secreted by activated HSCs and reached a maximum of 12.1% of total enzyme in 14-day culture supernatants. Treatment of HSCs with concanavalin A (con A) induced activation of MT1-MMP and enhanced secretion of activated gelatinase A, which reached a maximum of 44.4% of the total enzyme secreted into culture supernatants using 30 microgram/mL con A. [(14)C]-gelatin degradation assays confirmed the presence of gelatinolytic activity in activated HSC supernatants, which reached a maximum level at 7 days of culture. Antisense oligonucleotide inhibition of endogenous progelatinase A production, or the MMP inhibitor 1,10-phenanthroline inhibited (3)H-thymidine incorporation into HSC DNA by greater than 50%. We conclude that HSCs produce progelatinase A during activation in vitro and activate this enzyme coincident with MT1-MMP and TIMP-2 synthesis. Gelatinase A activity is required for maximal proliferation of HSCs in vitro suggesting this metalloproteinase is an autocrine proliferation factor for HSCs.     

Study on the mechanism of epidermal growth factor-induced proliferation of hepatoma cells

AIM: Many growth factors, such as epidermal growth factor(EGF), are associated with the carcinogenesis. EGF plays itsrole in the proliferation of hepatoma cells through bindingwith EGF receptor (EGFR) and a series of signal transduction.But the postreceptor pathway is still not clear. In the presentexperiment, we studied the effect of tyrosine kinase, proteinkinase C, Na+/H+ exchange, calmodulin and voltage-dependent Ca2+ channel on EGF-induced hepatoma cellproliferation.METHODS: Hepatoma cell line SMMC7721 was cultured inRPMI1640 serum-free medium. In order to study the effectof thyrosine kinase, protein kinase C, Na+/H+ exchange,calmodulin and voltage-dependent Ca2+ channel on humanheptoma cell proliferation induced by epidermal growth factor(EGF), DNA synthesis rate of hepatoma cells was measuredby the method of 3H-TdR incorporation.RESULTS: EGF (10-9 M) stimulated the proliferation of heptomacells significantly (3H-TdR incorporation was 1 880+281 cpm/well, P＜0.05), and this effect was significantly inhibited bytyrosine kinase inhibitor genistein (3H-TdR incorporation was808±209 cpm/well, P＜0.001). Calmedulin inhibitor W-7, proteinkinase C inhibitor H-7 and Na+/H+ exchange inhibitor amilorideindividually had significant inhibiting effect on EGF-inducedproliferation of hepatoma cells (3H-TdR incorporation was978±87.3 cpm/well, 1 241+147 cpm/well, 1 380+189 cpm/well, respectivly, P＜0.001, P＜0.01, P＜0.05), but they allhad no effect on the basal level proliferation of culturedhepatoma cells (3H-TdR incorporation was 1 284+260 cpm/well, 1 179+150 cpm/well, 1 392+152 cpm/well, respectivly,3H-TdR incorporation of the control was 1353+175 cpm/well, P＞0.05). Voltage-dependent Ca2+ channel inhibitorverapamil had no inhibition on EGF-induced proliferation ofhepatoma cells (3H-TdR incorporation was 1 637+133 cpm/well, P＞0.05), it also had no effect on the basal levelproliferation of cultured hepatoma cells (3H-TdR incorporationwas 1196+112 cpm/well,P＞0.05).CONCLUSION: Our data suggest that tyrosine kinase, Ca2+-calmodulin-dependent pathway, protein kinase C and Na+/H+ exchange play a critical role in EGF-induced proliferationof hepatoma cells and that the effect of EGF is independentof voltage-dependent Ca2+ channel.     

抗纤维化短肽N-乙酰基-丝氨酰-天门冬酰-赖氨酰-脯氨酸对大鼠心脏成纤维细胞胶原合成与降解的调节作用

目的探讨N-乙酰基-丝氨酰-天门冬酰-赖氨酰-脯氨酸（AcSDKP）对血小板源性生长因子（PDGF）诱导的大鼠心脏成纤维细胞增殖、胶原合成和降解代谢的调节作用。方法分离培养新生大鼠心脏成纤维细胞。采用^3H-TdR和。H-脯氨酸掺入法分别检测心脏成纤维细胞增殖与胶原蛋白合成。Western blot法检测心脏成纤维细胞Ⅰ、Ⅲ型胶原蛋白表达和基质金属蛋白酶（MMP）-1蛋白的表达。明胶酶谱法检测心脏成纤维细胞MMP-2和MMP-9活性的表达。结果PDGF促进心脏成纤维细胞增殖、胶原合成,Ⅰ、Ⅲ型胶原表达,以及MMP-2、MMP-9活性和MMP-1表达。AcSDKP对PDGF介导的心脏成纤维细胞增殖、胶原合成均有抑制作用。AcSDKP上调由PDGF介导的心脏成纤维细胞MMP-2、MMP-9活性和MMP-1的表达。结论AcSDKP抑制PDGF介导的心脏成纤维细胞增殖和胶原的合成,上调MMPs活性或表达,促进胶原的降解,这些可能与AcSDKP抗心脏纤维化作用相关。     

Catecholamines block 2-hydroxyestradiol-induced antimitogenesis in mesangial cells

Methylation of 2-hydroxyestradiol to 2-methoxyestradiol by catechol-O-methyl transferase (COMT) mediates the antimitogenic effects of 2-hydroxyestradiol on vascular smooth muscle cells. Moreover, 2-hydroxyestradiol inhibits growth of glomerular mesangial cells (GMCs). Because catecholamines are substrates for COMT, which is expressed in GMCs, we hypothesize that catecholamines may abrogate the antimitogenic effects of 2-hydroxyestradiol on GMCs by competing for COMT and inhibiting 2-methoxyestradiol formation. To test this hypothesis, we investigated the antimitogenic effects of 2-hydroxyestradiol on rat GMCs in the presence and absence of catecholamines. The capability of GMCs to methylate 2-hydroxyestradiol in the presence and absence of catecholamines was also evaluated. GMCs metabolized 2-hydoxyestradiol in a concentration-dependent manner with a V(max) of 12.03+/-0.32 pmol/10(6) cells/min and an apparent K(m) of 0.23+/-0.04 micromol/L. Norepinephrine (10 micromol/L) and epinephrine (10 micromol/L) significantly inhibited methylation of 0.25 micromol/L 2-hydroxyestradiol. Norepinephrine concentration-dependently abrogated the ability of 2-hydroxyestradiol to inhibit 3H-thymidine incorporation (index of DNA synthesis). In the presence of 5, 10, and 40 micromol/L norepinephrine, the inhibitory effect of 0.1 micromol/L 2-hydroxyestradiol on 3H-thymidine incorporation was reduced from 51+/-0.7% to 46+/-0.4%, 39+/-0.3%, and 25+/-0.7%, respectively. Similar to DNA synthesis, the inhibitory effects of 2-hydroxyestradiol on cell number and 3H-proline incorporation (index of collagen synthesis) on GMCs were abrogated by catecholamines. Our findings provide evidence that methylation of 2-hydroxyestradiol inhibits GMC proliferation and extracellular matrix synthesis and may in part protect against renal proliferative diseases. Moreover, catecholamines may abrogate the renoprotective effects of 2-hydroxyestradiol in the glomeruli by inhibiting COMT and 2-methoxyestradiol formation.     

大麻素受体2介导的N-花生四烯酸氨基乙醇对肝星状细胞增殖和活化的影响

目的 观察内源性大麻素N-花生四烯酸氨基乙醇(AEA)及大麻素受体(CBR)2对肝星状细胞(HSC)增殖活化的影响,以探讨内源性大麻素及其受体系统在肝纤维化发展中的作用.方法 采用免疫荧光观察血小板衍生生长因子(PDGF)刺激前后HSC中CBR1和CBR2的表达.Western blot、PCR法观察不同浓度AEA及CBR2拮抗剂AM630对PDGF刺激下HSC增殖及活化的影响,同时用四甲基偶氮唑盐、流式细胞仪分析AEA对HSC活力及凋亡的影响.结果 HSC中CBR2的表达较CBR1高(F=116.797,P<0.01),且PDGF刺激后CBR2的表达明显增强(F=7.878,P<0.05).AEA可剂量依赖地抑制HSC的增殖,在浓度为10,20、50μmol/L时抑制率分别为7.12%±0.34%、12.52%±0.78%、80.13%±1.57%,差异有统计学意义(F=533.41,P<0.01);但对HSC凋亡的影响不明显.同时AEA可抑制HSC的活化指标α-平滑肌肌动蛋白、转化生长因子β1、Ⅰ型胶原、Ⅲ型胶原及基质金属蛋白酶抑制因子等的表达,但这种抑制作用在给予CBR2拮抗剂AM630后明显减弱,差异有统计学意义(P<0.05).结论 CBR2在AEA引起的HSC增殖及活化抑制中起关键作用,AEA和CBR2可望成为肝纤维治疗的新靶点.     

Effects of the tyrosine protein kinase inhibitor genistein on the proliferation,activation of cultured rat hepatic stellate cells

AIM：Hepatic stellate cell(HSC)plays a pivotal role in liver fibrosis and is considered as the therapeutic target for the treatment of hepatic fibrosis,Tyrosine protein kinase plays an important role in the proliferation,activation of HSC.The purpose of the study is to investigate the effects of the tyosine protein kinase inhibitor genistein on the proliferation and activation of cultured rat HSC.METHODS:Rat HSC were isolated from Wistar rats by in situ perfusion of collagenase and pronase and single-step density Nycodenz gradient,Culture-activated HSC were serum-starved and incubated with10^-9to10^-5mol/L concentration of genistein for 24,48or 72h,In PDGF-induced HSC proliferation,HSC were stimulated with10μg&#183;L^-1PDGF-BBfo15min,and thentreated with genistein for the same time.Cell proliferation was measured by MПassay and based on flow cytometric analysis of cell cycle.The a-smooth muscle actin(α-SMA）expression in HSC was studied with confocallaser microscopy and flow cytometry.c-fos,c-jun and cyclinD1expression in HSCwas also detected by flow cytometry.RESULTS:Genistein inhibited basal and PDGF-induced proliferation of HSCat the concentration of 10^-8to10^-5mol/L,and treatment with10^-7mol/L concentration of genistein for 48h inhibited the HSCproliferation significantly(the inhibition rate was 70.3%,P&lt;0.05).Immunofluorescence detected by confocal laser microscopy and flow cytometry showed that treatment with10^-7mol/L genistein for48h suppressed the expression of α-SMA significantly in HSC(the specific fluorescence intensity were60.2&#177;21.5vs35.3&#177;11.6and12.8&#177;10.4vs9.54&#177;6.39,respectively,bothP&lt;0.05).The intensity of c-fos,c-jun and cyclinD1 expression of HSCs treated with 10^-7mol/L genistein for 48h was also significantly decreased compared with the controls.CONCLUSION;Genistein influences proliferation of HSC,suppresses the expression of α-SMA in HSC and tinhibits the intensity of c-fos,c-jun and cyclinD1 expression of HCSs,Genistein has therapeutic potential against liver fibrosis.     

Role of hepatic stellate cell/hepatocyte interaction and activation of hepatic stellate cells in the early phase of liver regeneration in the rat

BACKGROUND/AIMS: Hepatic stellate cells (HSCs) are known to play a role in hepatic regeneration. We investigated hepatocyte/HSC interaction and HSC activation at various times after 70% partial hepatectomy (PHx) in the rat. METHODS: The hepatic microcirculation was studied using intravital fluorescence microscopy (IVFM). Desmin and alpha-SMA within liver tissue were detected by immunohistochemistry. In isolated parenchymal liver cells (PLCs) and HSCs, double immunostaining was used to identify activated HSC. RESULTS: Using IVFM, hepatocyte-clusters were often seen in vivo at 3 days after PHx (PHx3). Distance between HSC fell from 61.7+/-2.1 microm in controls to 36.1+/-1.4 microm (P<0.001) while the HSC/hepatocyte ratio rose (0.71+/-0.01 to 1.08+/-0.03; P<0.001). In >80% of in vivo microscopic fields in the PHx3 group, clusters of HSCs were observed especially near hepatocyte-clusters. At PHx1 and PHx3, >20% of cells in the PLC-fraction were HSCs which adhered to hepatocytes. At PHx3, in addition to desmin staining, isolated HSCs were also positive for BrdU and alpha-SMA, and formed clusters. HSCs in the HSC-fraction were only positive for desmin which indicated that adherence to hepatocytes is required for HSC activation. CONCLUSIONS: Our data suggest that HSCs are activated by adhering to hepatocytes in the early phase of liver regeneration.     

三氟拉嗪对贮脂细胞增殖及胶原合成的影晌

目的:探讨三氟拉嗪抗肝纤维化作用的机制。方法:采用链酶蛋白酶、胶原酶及密度梯度离心,分离大鼠肝脏贮脂细胞,用MTT方法及~3H-脯氨酸掺入方法,观察了三氟拉嗪对贮脂细胞增殖的调控。结果:低浓度三氟拉嗪的抑制作用不明显(P>0.05),当药物浓度上升为10～20μmol/L~(-1)时,抑制作用增强,呈明显药物浓度依赖关系。结论:三氟拉嗪可通过抑制贮脂细胞的增殖及胶原合成,而达到抗肝纤维化的作用。     

骨髓间充质干细胞条件培养液对心脏成纤维细胞的影响

目的探讨体外2％氧条件下培养骨髓间充质干细胞（mesenchymal stem cells，MSCs）培养液对成年大鼠心肌成纤维细胞增殖及胶原合成情况的影响。方法分离Wistar大鼠下肢骨获取MSCs进行培养，收集培养3、6、9、24h培养液，用其刺激成年Wistar大鼠的心肌成纤维细胞30h，采用MTT法和^3H-脯氨酸掺入法检测心肌成纤维细胞的增殖及胶原合成情况。结果经3、6、9h的MSCs条件培养液刺激后的心肌成纤维细胞^3H-脯氨酸掺入量与无血清对照组比较显著增加，分别增加23％（P〈0．01）、44％（P〈0．01）和31％（P〈0．01），而24h组与无血清对照组比较差异无显著性：MSCs条件培养液刺激后的心肌成纤维细胞MTT结果与对照组比较差异无显著性。结论MSCs低氧条件培养液可以促进心肌成纤维细胞胶原合成，改善心功能。