转化生长因子β1在大鼠心肌细胞肥大中的作用

目的探讨转化生长因子β1(transforminggrowth factorβ1，TGF-β1)在诱导大鼠心肌细胞肥大中的作用。方法培养新生大鼠的心肌细胞，检测[^3H]-Leu的掺入量和胚胎抗原心房利钠因子(atrial natriuretic factor，ANF)的表达。结果不同浓度的转化生长因子β1均能明显增加心肌细胞[^2H]-Leu的掺入量和ANF的表达。结论转化生长因子β1能诱导大鼠的心肌细胞肥大。     

葛根素对糖尿病大鼠肾功能及肾组织MMP-2与TIMP-2表达的影响

目的探讨葛根素对糖尿病大鼠肾功能及肾组织基质金属蛋白酶2（MMP-2）及其组织抑制剂2（TIMP-2）表达的影响。方法单侧肾切除大鼠ip链脲佐菌素诱发糖尿病模型。用原位杂交法检测肾小球MMP-2及TIMP-2 mRNA表达，流式细胞术和免疫组织化学检测肾皮质TGFβ1，MMP-2，TIMP-2,IV型胶原及层粘连蛋白表达。结果 糖尿病组较对照组肾小球MMP-2 mRNA及蛋白表达降低而TIMP-2 mRNA及蛋白表达升高，TGFβ1，IV型胶原及层粘连蛋白表达亦增加，肾功能恶化；葛根素用药组较糖尿病组肾小球MMP-2 mRNA及蛋白表达升高，而TGFβ1，TIMP-2，IV型胶原及层粘连蛋白表达减少，肾功能改善。结论葛根素对糖尿病大鼠肾功能具有保护作用，除降低血糖外，调节肾小球MMP-2及TIMP-2表达，从而减轻肾小球细胞外基质沉积也可能是其作用途径之一。     

地塞米松抑制肿瘤坏死因子α介导的肾小球系膜细胞

目的 探讨肿瘤坏死因子α(TNFα)对人肾小球系膜细胞(MC)促增殖效应及地塞米松(DXM)对其的抑制作用。方法 培养人肾MC，采用^3H—胸腺嘧啶掺入法(^3H—TdR掺入法)测定MC增殖：ELISA法测定细胞因子(IL1)及细胞外基质ECM(FN、LN)。结果 TNFa明显刺激MC^3H—TdR掺入，与对照组比较P＜0.01，同时，TNFa促进MC分泌IL1、和LN、FN，与对照组比较P＜0.01。DXM抑制TNFa刺激MC^3H—TdR掺入及分泌ILl、LN、FN，呈剂量依赖关系(P＜0.01)。结论 细胞因子TNFa能明显促进MC增殖及细胞外基质合成，而DXM具有抑制TNFa的作用，为临床上使用DXM治疗肾小球疾病提供了新的理论依据。     

缬沙坦对高糖培养的肾小球系膜细胞p38MAPK传导通路的影响

目的探讨高糖状态下肾小球系膜细胞中p38丝裂原活化蛋白激酶(p38 MAPK)、其上游因子MAPK激酶3/6(MKK3/6)和下游因子cAMP反应元件结合蛋白1(CREB1)的表达以及血管紧张素受体1拮抗剂(AT1Ra)缬沙坦的影响。方法体外培养大鼠肾小球系膜细胞,分别给予高糖和缬沙坦干预,采用Western bolt检测MKK3/6、p38 MAPK和CREB1及其磷酸化蛋白的表达,逆转录-聚合酶链反应(RT-PCR)检测系膜细胞内TGF-β1和FN mRNA的表达。放免法测定细胞上清液中纤维连接蛋白(FN)和IV型胶原的含量。MTT法检测缬沙坦在不同时间、不同药物浓度对细胞增殖状态的影响。结果①与低糖对照组相比,高糖组系膜细胞p-p38 MAPK、p-MKK3/6和p-CREB1表达明显上调,TGF-β1和FN mRNA的表达增加,FN和IV型胶原含量增加。②缬沙坦组p-p38 MAPK、p-MKK3/6和p-CREB1的表达明显下调,TGF-β1和FN mRNA的表达降低,同时FN和IV型胶原的含量减少。③MTT法检测显示不同浓度的缬沙坦对细胞增殖状态都有所抑制,并随药物浓度的增加而作用增强。结论缬沙坦抑制肾小球系膜细胞TGF-β1的表达和细胞外基质的分泌可能部分是通过影响p38 MAPK传导通路的激活来实现的。     

苦参碱抑制小鼠腹膜巨噬细胞纤维化细胞因子的产生和作用

目的：研究苦参碱对小鼠腹腔巨噬细胞释放纤维化细胞因子以及对巨噬细胞条件培养基（MCM）促HSC-T6大鼠储脂细胞和NIH3T3成纤维细胞增殖和胶原合成的影响。方法：巨噬细胞先后用卡西霉素1μmol/L和脂多糖100μg/L刺激诱导产生纤维化因子,细胞上清中促细胞增殖活性和促胶原合成活性分别用结晶紫染色法和[^3H]-脯氨酸掺入法测定,转化生长因子β活性采用貂肺上皮Mv-l-Lu细胞增殖抑制法测定。结果：苦参碱（0.5-2mmol/L）显著抑制LPS诱导的促胶原合成活性和TGFβ的产生,但不能抑制巨噬细胞产生促细胞增殖活性;苦参碱还能剂量依赖地抑制MCM诱导的HSC-T6细胞以及NIH3T3细胞增殖和胶原合成。结论：苦参碱抗肝纤维化作用与抑制巨噬细胞纤维化因子的产生和阻断其作用有关。     

血小板源生生长因子刺激血管平滑肌细胞增殖及其分子机制

目的：探讨血小板源生生长因子（PDGF－BB）刺激血管平滑肌细胞（VSMC）增殖及其分子机制。方法：用Western Blot法测定p44/p42 CCDPK活性。[^3H]脱氧胸腺嘧啶核苷酸掺入测定VSMC DNA合成。原位杂交检测c-myc mRNA的表达。结果：PDGFBB诱导的磷酸化CCDPK蛋白表达和[^3H]脱氧胸腺嘧啶核苷酸掺入呈浓度依赖性,此作用可被PTK抑制剂Genistein,外钙络合剂依他酸和MEK抑制剂PD 98059抑制。PDGF－BB刺激可引起c-myc mRNA的明显表达,此作用可被PD 98059抑制。结论：PDGF－BB通过激活p44/p42 CCDPK,上调c-myc mRNA的表达从而促进VSMC增殖,其作用是由PTK和Ca^2 介导的。     

缬沙坦对高糖培养系膜细胞信号转导和转录活化因子1、3表达的影响

目的探讨高糖状态下肾小球系膜细胞中信号转导和转录活化因子1、3的改变以及血管紧张素受体1拮抗剂(AT1Ra)缬沙坦的影响。方法体外培养大鼠肾小球系膜细胞,分别给予高糖和缬沙坦干预,采用W estern印迹检测信号转导和转录活化因子1、3(STAT1、STAT3)及其磷酸化蛋白(p-STAT1、p-STAT3)的表达,酶联免疫吸附实验(ELISA)和放免法测定细胞上清液中TGF-β1、纤维连接蛋白(F ibronectin,FN)和IV型胶原的含量,逆转录-聚合酶链反应(RT-PCR)检测TGF-β1mRNA的表达。结果与低糖对照组相比,高糖组系膜细胞p-STAT1和p-STAT3表达明显上调,TGF-β1、FN和IV型胶原含量增加,TGF-β1mRNA的表达增加。缬沙坦组p-STAT1和p-STAT3的表达明显下调,TGF-β1、FN和IV型胶原的含量减少,同时TGF-β1mRNA的表达降低。结论高糖状态下p-STAT1和p-STAT3表达明显升高,缬沙坦抑制肾小球系膜细胞TGF-β1和细胞外基质的分泌可能部分是通过影响STAT1和STAT3的激活而实现。     

卡托普利对缺氧诱导的肺动脉平滑肌细胞增殖和胶原合成的抑制作用(英文)

目的:观察卡托普利(Cap)抑制缺氧诱导的肺动脉平滑肌细胞(VSMC)增殖和胶原合成的作用,方法:采用细胞计数,[~3H]脱氧胸苷,[~3H]脯氨酸掺入和细胞内游离钙测定的方法。结果:卡托普利(Cap,1μmol·L~(-1))抑制缺氧诱导的VSMC中细胞数目,[~3H]脱氧胸苷和[~3H]脯氨酸掺入及细胞内游离钙的增高,较缺氧组分别降低了25％,36％,21％和16％,硝苯吡啶也具有上述抑制作用,Bay-K-8644促进VSMC中细胞数目,[~3H]脱氧胸苷和[~3H]脯氨酸掺入及细胞内游离钙的增高,分别增加35％,55％,36％,34％,这种作用可被Cap阻断。结论:Cap抑制缺氧诱导的肺动脉平滑肌细胞增殖和胶原合成,这可能与阻断L型钙通道有关。     

BMP-7对TGF-β_1诱导人肾小管上皮细胞外基质表达的影响

目的观察骨形态发生蛋白-7(BMP-7)对TGF-β1诱导人近端肾小管上皮细胞(HK-2)细胞外基质(ECM)成分表达的影响,以探讨其延缓肾间质纤维化病变的作用机制。方法将体外培养的HK-2细胞分为空白对照组,3μg.L-1TGF-β1组,BMP-7组,TGF-β1加不同浓度BMP-7组。免疫荧光检测FN的表达;RT-PCR和Western blot分别检测FN、ColⅠα1mRNA及蛋白的表达。结果免疫荧光检测结果显示,3μg.L-1TGF-β1刺激HK-2细胞48h后,FN表达明显增强;加入200μg.L-1BMP-7干预后荧光明显减弱。RT-PCR和Western blot结果显示,TGF-β1刺激HK-2细胞48h后,FN、ColⅠα1mRNA及蛋白表达均明显高于对照组(P<0.01);BMP-7(100～400μg.L-1)可在mRNA及蛋白水平呈剂量依赖性下调TGF-β1诱导的FN、ColⅠα1的表达。结论BMP-7能抑制TGF-β1诱导的HK-2细胞FN、ColⅠα1的合成。表明BMP-7抑制肾小管间质纤维化的进展、改善肾功能的作用,部分是通过抑制肾小管上皮细胞分泌细胞外基质实现的。     

Rhein reverses the diabetic phenotype of mesangial cells over-expressing the glucose transporter (GLUT1) by inhibiting the hexosamine pathway

BACKGROUND AND PURPOSE: Rhein, an anthraquinone compound isolated from rhubarb, has been proved effective in treatment of experimental diabetic nephropathy (DN). To explore the mechanism of its therapeutic effect on DN, rhein was tested for its effect on the hexosamine pathway. EXPERIMENTAL APPROACH: The influence of rhein on cellular hypertrophy, fibronectin synthesis, glucose uptake, glutamine: fructose 6-phosphate aminotransferase (GFAT) activity, UDP-N-acetylglucosamine (UDP-GlcNAc) level and TGF-beta1 and p21 expression was evaluated in MCGT1 cells, a GLUT1 transgenic rat mesangial cell line. GFAT activity in normal rat mesangial cells in high glucose concentrations and in vitro was also measured. KEY RESULTS: Significantly increased fibronectin synthesis, cellular hypertrophy, much higher GFAT activity and UDP-GlcNAc level and increased TGF-beta1 and p21 expression were found in MCGT1 cells cultured in normal glucose concentration. Rhein treatment decreased all these features of MCGT1 cells but did not exert a direct effect on GFAT enzymatic activity. CONCLUSIONS AND IMPLICATIONS: There was over-activity of the hexosamine pathway in MCGT1 cells, which may explain the higher expression of TGF-beta1 and p21, the cellular hypertrophy and the increased expression of extracellular matrix (ECM) components in the cells. By inhibiting the increased activity the hexosamine pathway, rhein decreased TGF-beta1 and p21 expression and thus contributed to the decreased cellular hypertrophy and ECM synthesis. Inhibition of the hexosamine pathway may be one of the mechanism through which rhein exerts its therapeutic role in diabetic nephropathy.     

大黄酸对人肾小球系膜细胞功能的影响

目的探讨大黄酸防治糖尿病肾病的作用机制。方法用细胞培养、ELISA、明胶酶谱及免疫沉淀、Western blot等技术,研究了大黄酸对肾小球系膜细胞转化生长因子(TGFβ₁)、基质金属蛋白酶-2,-9(MMP-2和MMP-9)及p38活化丝裂原活化蛋白激酶(p38MAPK)活性的作用。结果大黄酸可显著抑制肾小球系膜细胞的增殖,对抗高糖诱导肾小球系膜细胞TGFβ₁活性升高的作用;对高糖诱导肾小球系膜细胞MMP-2,MMP-9,pro-MMP-2及pro-MMP-9的活性增加无明显影响,但可明显对抗高糖引起的肾小球系膜细胞p38MAPK活性增加。结论大黄酸减少FN的分泌可能与其降低p38MAPK及TGFβ₁活性有关。     

卡托普利对缺氧诱导的肺动脉平滑肌细胞增殖和胶的合成的抑制作用

AIM: To study the effect of captopril (Cap) on hypoxia-induced proliferation and collagen synthesis in vascular smooth muscle cells (VSMC). METHODS: VSMC were isolated from rabbit pulmonary artery. Cultured VSMC were evaluated by incorporation of [3H]thymidine and [3H]proline, cell number, and intracellular calcium concentration ([Ca2+]i). RESULTS: Pretreatment of pulmonary VSMC with Cap 1 mumol.L-1 blocked hypoxia-induced increase in cell number and incorporation of [3H]proline and [3H]thymidine, which were decreased 25%, 21%, and 36%, respectively, as compared with hypoxic control. It also inhibited the increase of intracellular Ca2+ concentration under hypoxic condition. Addition of nifedipine inhibited hypoxia-stimulated increase in the collagen, DNA synthesis, and [Ca2+]i. Bay-K-8644 increased cell number (35%), DNA (55%), collagen synthesis (36%), and [Ca2+]i (33%) in pulmonary VSMC, that was completely abolished by Cap 1 mumol.L-1. CONCLUSION: Cap inhibited hypoxia-induced proliferation and collagen synthesis in VSMC.     

Rhodamine B inhibits collagen synthesis by human lip fibroblasts in culture.

To investigate the effect of the cosmetic dye, rhodamine B, on the metabolism of collagen in fibroblasts, confluent KD cells, an established cell line of fibroblasts from human lip, were cultured for 6 h in a serum-free medium in the presence of the dye at 100 micrograms/ml and below. It was found that rhodamine B significantly decreased the content of procollagen type I C-terminal peptide antigen in both the cell layer and the medium with an only slight decrease in the cell number. Rhodamine B significantly decreased the incorporation of [3H]proline into either the collagen-digestible protein or the non-collagen protein in the cell layer. The incorporation of both [3H]thymidine and [14C]leucine into the acid-insoluble fraction of the cell layer was significantly decreased by rhodamine B; the activity of lactate dehydrogenase that leaked into the medium was not changed by the dye. From these results, it was suggested that rhodamine B has the capacity of decreasing the collagen content of the fibroblast cell layer of the human lip, which may result from a non-specific inhibition of protein synthesis without non-specific cell damage. Rhodamine B may impair the formation of extracellular matrix which is important for the maintenance of the lip tissue.     

Effect of a single injection of betamethasone disodium phosphate on the synthesis of collagen and noncollagen protein of carrageenin granuloma in rats

The effect of a single injection of betamethasone disodium phosphate on the incorporation of [³H]proline into collagen and noncollagen protein of rat carrageenin granuloma was studied. In all series of experiments, both [³H]proline and the steroid were given intravenously and the animals were killed at 30 min after the labeled proline injection in order to investigate precisely the time course of the drug action. The incorporation was not influenced at all by the steroid when it was given 30 min before the injection of [³H]proline. The inhibitory effect of the steroid, for both the collagen and the noncollagen protein, became significant at 1 hr after its intravenous administration, increasing progressively with the passage of time. Inhibition of the labeled proline incorporation into collagen hydroxyproline was significantly greater than that into noncollagen protein. Neither accumulation of protocollagen nor any change of protocollagen proline hydroxylase activity was demonstrated in the granuloma obtained from the rats treated with the steroid. The results show that the steroid does not inhibit the process of hydroxylation of protocollagen, suggesting indirectly the inhibition of synthesis of protocollagen or of [³H]proline transport through cell membrane, or of both.     

Urotensin Ⅱ accelerates cardiac fibrosis and hypertrophy of rats induced by isoproterenol1

AIM: To study whether urotensin II (UII), a potent vasoconstrictive peptide, is involved in the development of cardiac hypertrophy and fibrogenesis of rats induced by isoproterenol (ISO). METHODS: Thirty male Wistar rats were randomly divided into 3 groups. Group 1 was the healthy control group, group 2 was the ISO group, and group 3 was the ISO+UII group. In groups 2 and 3, ISO (5 mg x kg(-1) x d(-1)) was given (sc) once daily for 7 d. Group 3 was also given UII in the first day [3 nmol/kg (5 microg/kg), iv], followed by sc (1.5 microg/kg) twice daily. Group 1 received 0.9% saline. UII receptor (UT) mRNA expression was determined by RT-PCR. The contents of UII and angiotensin II (Ang II) were determined by radioimmunoassay. In vitro, the effects of UII on DNA/collagen synthesis of cardiac fibroblasts were determined by [3H]thymidine/[3H]proline incorporation. RESULTS: The ratio of heart weight/body weight, plasma lactate dehydrogenase activity, myocardial malondialdehyde and hydroxyproline concentration increased significantly in the ISO group, as well as UT mRNA expression, plasma and cardiac UII and ventricular Ang II, compared with the control group (P< 0.01). ISO induced significant myocardial fibrogenesis. Moreover, UII+ISO co-treatment significantly increased the changes of biochemical markers of injury and the degree of cardiac hypertrophy and fibrosis. In vitro, 5 x 10(-9 )-5 x 10(-7 ) mol/L UII stimulated [3H]thymidine/[3H] proline incorporation into cardiac fibroblasts in a dose-dependent manner (P< 0.01). CONCLUSION: These results suggest that UII was involved in the development of cardiac fibrosis and hypertrophy by synergistic effects with ISO.     

金雀异黄素和槲皮素对大鼠肝星状细胞增殖、胶原合成及Ⅰ型原胶原nRNA水平的影响(英文)

目的:研究金雀异黄素和槲皮素对HSC-T6大鼠肝星状细胞增殖和胶原合成及Ⅰ型原胶原mRNA表达的影响。方法:细胞增殖和胶原合成分别采用结晶紫染色法和[~3H]-脯氨酸掺入法。原胶原mRNA水平用RT-PCR法测定。结果:金雀异黄素(25-70μmol/L)和槲皮素(6.25-50μmol/L)浓度依赖抑制HSC-T6细胞增殖和胶原合成,对Ⅰ型原胶原mRNA表达也有抑制作用。结论:金雀异黄素和槲皮素抑制肝星状细胞增殖和胶原合成可能对肝纤维化有保护作用。     

Strong antiproliferative effects of baicalein in cultured rat hepatic stellate cells.

Recently, antifibrogenetic effects of Sho-saiko-to, a traditional herbal medicine in Japan, have been shown in experimental hepatic fibrosis, and flavonoids in Sho-saiko-to are suspected as active ingredients. Thus, we evaluated the effects of baicalein, a major flavonoid in Sho-saiko-to, on proliferation and protein synthesis in cultured rat hepatic stellate cells. Baicalein decreased [3H]thymidine incorporation in cells stimulated with platelet-derived growth factor-B subunit homodimer (PDGF-BB) in a concentration-dependent manner (approximate ED50<10 microM, P<0.0001), and the decrease observed with 10 microM baicalein was greater than those observed with 5 microM retinol or 500 microM 3-isobutyl-1-methylxanthine (IBMX). Baicalein consistently decreased [3H]thymidine incorporation and cell number in cells stimulated with fetal calf serum (ED50<10 microM, P<0.0001), and moderately suppressed [3H]leucine and [3H]proline incorporation (P<0.0001). These results demonstrate the strong antiproliferative effect of baicalein in hepatic stellate cells, showing the possibility of baicalein as an antifibrogenetic drug for hepatic fibrosis.