Herbal compound 861 regulates mRNA expression of collagen synthesis- and degradation-related genes in human hepatic stellate cells

AIM： To identify the role of herbal compound 861 （Cpd 861） in the regulation of mRNA expression of collagen synthesis- and degradation-related genes in human hepatic stellate cells （HSCs）. METHODS： mRNA levels of collagen types I and III, matrix metalloproteinase 1 （MMP-1）, matrix metalloproteinase 2 （MMP-2）, membrane type-1 matrix metalloproteinase （MT1-MMP）, tissue inhibitor of metalloproteinase 1 （TIMP-1）, and transforming growth factor β1 （TGF-β1） in cultured-activated HSCs treated with Cpd 861 or interferon-γ, （IFN-γ,） were determined by real-time PCR. RESULTS： Both Cpd 861 and IFN-γ reduced the mRNA levels of collagen type Ⅲ, MMP-2 and TGF-β1. Moreover, Cpd 861 significantly enhanced the MMP-1 mRNA levels while down-regulated the TIMP-1 mRNA expression, increasing the ratio of MMP-1 to TIMP-1 to （6.3 ＋ 0.3）- fold compared to the control group. CONCLUSION： The anti-fibrosis function of Cpd 861 may be mediated by both decreased interstitial collagen sythesis by inhibiting the transcription of collagen type Ⅲ and TGF-β1 and increased degradation of these collagens by up-regulating MMP-1 and down-regulating TIMP-1 mRNA levels.

Down-regulation of transforming growth factor β1/activin receptor-like kinase 1 pathway gene expression by herbal compound 861 is related to deactivation of LX-2 cells

AIM: To investigate the effect of herbal compound 861 (Cpd861) on the transforming growth factor-β1 (TGFβ1)/ activin receptor-like kinase 1 (ALK1, type Ⅰ receptor) signaling-pathway-related gene expression in the LX-2 cell line, and the inhibitory mechanism of Cpd861 on the activation of LX-2 cells. METHODS: LX-2 cells were treated with TGFβ1 (5 ng/mL) Cpd861 (0.1 mg/mL), TGFβ1 (5 ng/mL) plus Cpd861 (5 ng/mL) for 24 h to investigate the effect of Cpd861 on the TGFβ1/ALK1 pathway. Real-time PCR was performed to examine the expression of α-SMA (α-smooth muscle actin), ALK1, Id1 (inhibitor of differentiation 1). Western blotting was carried out to measure the levels of α-SMA and phosphorylated Smad1, and immunocytochemical analysis for the expression of α-SMA. RESULTS: In LX-2 cells, TGFβ1/ALK1-pathway-related gene expression could be stimulated by TGFβ1, which led to excessive activation of the cells. Cpd861 decreased the activation of LX-2 cells by reducing the expression of α-SMA mRNA and protein expression. This effect was related to inhibition of the above TGFβ1/ALK1-pathway- related expression of genes such as Id1 and ALK1, and phosphorylation of Smad1 in LX-2 cells, even with TGFβ1 co-treatment for 24 h. CONCLUSION: Cpd861 can restrain the activation of LX-2 cells by inhibiting the TGFβ1/ALK1/Smad1 pathway.

Effects of Herbal Compound 861 on Collagen Synthesis and Degradation in Rat Mesangial Cells Exposed to High Glucose

Objective： To investigate the effects of Herbal Compound 861 （Cpd 861） on collagen synthesis and degradation in rat mesangial cells exposed to high glucose. Methods： The third to fifth passage of rat mesangial cells were exposed to high glucose and Cpd 861 at a concentration of 0.25-4.00 g/L for 24, 48 and 72 h, respectively. Benazepril （107-10s mmol/L） was selected as positive control. The methyl thiazolyl tetrazolium colorimetric assay was used to evaluate the effect of Cpd 861 on cell proliferation. After incubation with Cpd 861 at a concentration of 2.00 g/L for 48 h, the protein secretions of collagen type IV, matrix metallopeptidase 9 （MMP-9）, tissue inhibitor of metalloproteinase-1 （TIMP-1）, transforming growth factor beta 1 （TGF-131）, and hepatocyte growth factor （HGF） were detected by enzyme-linked immunosorbent assay method. And rat mesangial cells were harvested to determine MMP-9, TIMP-1, TGF-131 and HGF mRNA expression by revers^transcription polymerase chain reaction. Results： Cpd 861 inhibited cell proliferation induced by high glucose in a dose- and time-dependent manner. Compared with high glucose, collagen type IV production was decreased significantly by Cpd 861 （P〈0.01）. Cpd 861 increased the protein secretions and mRNA expressions of MMP-9 and HGF, whereas the protein secretions and mRNA expressions of TIMP-1 and TGF-131 were reduced markedly （P〈0.05）. The ratio of MMP-9 to TIMP-1 was enhanced by Cpd 861 significantly. There was no significant difference in all above-mentioned effects between Cpd 861 （2.00 g/L） and benazepril （10-5 mmol/L）. Conclusion： The anti-glomerulosclerosis mechanisms of Cpd 861 were partly attributed to its effects of inhibiting mesangial cell proliferation, decreasing collagen synthesis and enhancing collagen degradation.

Suppressive effect of herbal compound 861 on chemical hepatocarcinogenesis in rats

Objective To investigate the effects of herbal compound 861 (Cpd861) on hepatocarcinogenesis induced by diethylntrosamine and 2-acetylaminofluorene (DEN-AAF) in female Sprague Dawley rats.Methods Liver preneoplastic foci were induced using the DEN-AAF method in female Sprague Dawley rats, which were then treated with Cpd861. For quantitative assessment of liver preneoplastic foci, the placental form of glutathione-S-transferase (GST-P) positive foci were measured using immunohistochemical staining and image analysis. GST-P protein expression was measured by Western blotting, mRNA expression was assessed by Northern blotting.Results Treatment using DEN-AAF caused a significant decrease in body weight and increase in liver weight compared to the control group. Oral Cpd861 administration essentially prevented DEN-AAF-induced body weight loss and liver weight increase. When 2-AAF was followed by treatment with Cpd861, there was a decrease in the number of large foci as compared to 2-AAF alone. However, there were still considerable numbers of small mixed clear/vacuolated cell foci, some of which were positive for GST-P. Significant increase in GST-P protein and mRNA expression were observed in the DEN-AAF group, while treatment with Cpd861 inhibited the increase. The effect of Cpd861 on hepatocarcinogenesis occurred in a concentration-dependent manner. Conclusion Chinese herbal compound Cpd861 prevents hepatocarcinogenesis in DEN-AAF-induced liver preneoplastic lesions in rats.

复方中药861对大鼠肝星状细胞系一氧化氮合酶表达及其活性的影响

目的 :研究复方中药 86 1对大鼠肝星状细胞系 (HSC T6 )一氧化氮合酶 (NOS)表达及其酶活性的影响 ,并探讨该药预防、治疗早期门脉高压的可行性。方法 :细胞密度为 1× 10 5/ml的HSC T6 接种于细胞培养皿中 ,95 %O2 ,5 %CO2 ,37℃ ,培养 2 4h进行以下分组实验 ,每组重复 6皿 ,继续培养 2 4h。 1组 :HSC T6 空白对照组 ;2组 :HSC T6 加复方中药 86 1(2mg/ml) ;3组 :HSC T6 加复方中药 86 1(4mg/ml) ;4组 :HSC T6 加复方中药 86 1(8mg/ml) ;5组 :HSC T6 加复方中药 86 1(4mg/ml)加L 硝基精氨酸甲酯 (L NAME ,4mg/ml)。采用化学比色法测定NOS活性 ,采用硝酸还原酶法测定培养液一氧化氮 (NO)水平。 4 %多聚甲醛固定细胞 2h ,采用免疫细胞化学法观察HSC T6 诱导型一氧化氮合酶 (iNOS)表达。结果 :复方中药 86 1能增加HSC T6 NOS活性 (P <0 0 1) ,上清液NO水平也平行增加 (P <0 0 1) ,L NAME不能抑制复方中药86 1刺激的HSC T6 合成、分泌NO增加 (P >0 0 5 )。免疫细胞化学研究发现 ,活化的HSC T6 细胞浆有iNOS表达 ,复方中药 86 1能增加HSC T6 iNOS表达。结论 :活化HSC T6 iNOS表达阳性 ,与自分泌NO有关 ;复方中药 86 1能明显增加HSC T6 iNOS表达及其酶活性 ,NO合成、分泌增加。复方中药 86

中药复方861合剂对大鼠急性心肌梗死后心肌纤维化影响的初步研究

目的探讨中药复方861合剂对大鼠急性心肌梗死(AMI)后心肌纤维化、心室重构的影响。方法雄性SD大鼠结扎左冠状动脉前降支制成心肌梗死模型,随机分为未治疗组(M组中药复方)、861合剂治疗组(T)组及假手术组(S组)。术后第14日及第56日时测定大鼠体重(BW)、心脏重量(HW)、左室重量(LVW)、心脏重量/体重比(HW/BW)、左室重量/体重比(LVW/BW)及心肌胶原含量。各组心肌组织MASSON染色,光镜下观察病理改变。结果T组在术后第14日HW、LVW、HW/BW、LVW/BW均明显低于M组及S组,术后第56日的上述指标显著低于M组但高于S组。T组术后14d及56d的胶原含量均显著低于M组,但仍高于S组。结论中药复方861合剂有明显的抑制和减轻大鼠AMI后心肌纤维化的作用,但并不能完全阻止其发生。

实验性肝癌前期病变的研究及中药复方861对其的影响

目的 研究肝癌前期病变有关指标的表达和中药复方861对癌前期病变形成过程的影响。方法 以二乙基亚硝胺诱发大鼠肝癌前期病变,以复方861对诱癌过程干预,利用免疫组化、蛋白质印迹(Westem—blotting)法分别检测胎盘型谷胱甘肽—S—转移酶(GST—P)、增殖细胞核抗原(PCNA)的表达。结果 免疫组化染色结果示第6周时见GST—P阳性细胞,第8周开始普遍见GST—P结节。PCNA免疫组化结果示,第6周开始有阳性细胞表达,部分GST—P阳性结节内PCNA阳性细胞较多。Westem—blotting结果示GST—P从诱癌第6周开始有阳性表达,造模组GST—P阳性表达比同期干预组表达量大。结论PCNA标识的GST—P阳性的细胞是早期癌前期病变的有效标志。复方861可抑制癌前期病变的进程。

中药861合剂对脓毒症大鼠心肌纤维化的干预

目的观察脓毒症状态下大鼠心肌纤维化的改变及其与心脏功能的关系,并探讨中药861合剂对心肌纤维化的影响。方法采用经阴茎静脉注射脂多糖的方法制作大鼠脓毒症模型。50只雄性Wistar大鼠随机分为模型组(20只)、中药861合剂干预组(20只)及对照组(10只)。模型组和中药861合剂干预组各分为低剂量组、高剂量组。各组动物处死前均行经右颈总动脉左心室内插管,监测血流动力学及左室功能变化。测定大鼠血清Ⅰ型前胶原羧基端肽(PICP)、转化生长因子β1(TGF-β1);心肌组织匀浆上清羟脯氨酸(HC)含量;及心脏组织Masson染色,光镜、电镜观察心肌病理改变,计算机图像分析系统测定心肌胶原容积分数(CVF)和心肌内血管周围胶原面积(PVCA)。结果 1各组大鼠血流动力学指标无显著差异(P>0.05);2各组大鼠左心功能指标比较无显著差异(P>0.05);3模型组大鼠的PICP、TGF-β1、HC浓度均有不同程度升高(P<0.05或P<0.01);且脓毒症越重,升高越显著。中药861合剂干预组大鼠的PICP、TGF-β1、HC升高程度和病理的损害均轻于模型组大鼠,差异有显著性(P<0.05或P<0.01);4模型组大鼠的CVF和PVCA有不同程度升高(P<0.05),中药861合剂干预组大鼠的CVF、PVCA较对照组大鼠无显著差异(P>0.05)。结论 1脓毒症早期存在心肌间质改变即心肌间质纤维化,脓毒症越严重,心肌纤维化改变越明显;2中药861合剂能抑制脓毒症心肌纤维化进展,其机制和抑制成纤维细胞的活化,减少TGF-β1的表达有关。

中药复方861治疗大鼠肺纤维化的实验研究

为寻找疗效满意、副作用小的中药治疗肺间质纤维化 ,本实验观察了气管内注入博莱霉素制成的大鼠肺纤维化模型及复方 86 1治疗该模型后第 7天和第 2 8天两个时间段的血、肺泡灌洗液 (BALF)中透明质酸 (HA)和层粘连蛋白 (LN)含量变化、病理变化及 2 8天血气变化。观察中药复方 86 1对大鼠肺纤维化的疗效。结果表明 :中药复方 86 1可减轻肺纤维化大鼠肺组织的早期肺泡炎和后期肺纤维化 (病理 ) ;中药复方 86 1可降低模型大鼠早期 (7天 )血清和BALF中的HA含量及血清LN含量 ,并可改善动脉血氧分压。因此 ,中药复方 86 1对大鼠实验性肺纤维化有一定治疗作用。

中药复方861合剂对小鼠急性肝损伤的保护作用研究

目的:研究中药复方861合剂对四氯化碳(CCl4)和D-半乳糖胺(D-GalN)所致小鼠急性肝损伤的保护作用,并观察小鼠的主要中毒症状,为临床合理用药提参考。方法:采用CCl4(0.02 ml/kg)和D-GalN(0.8 g/kg)制备小鼠急性肝损伤模型,以联苯双酯(0.029 g/kg)为阳性药,观察中药复方861合剂(1.5、3及6 g/kg)对小鼠血清丙氨酸转氨酶(ALT)、天冬氨酸转氨酶(AST)的影响和其对肝脏的保护作用;以最大给药容量(1.0 ml)和最大药物浓度(60 g/kg)给药,记录小鼠14 d内的中毒症状及死亡情况。结果:中药复方861合剂对一次性腹腔注射CCl4和D-GalN所致急性肝损伤小鼠血清ALT、AST水平的升高均有较强抑制作用。对于CCl4诱导肝损伤小鼠,中药复方861合剂高、中及低剂量组小鼠的ALT、AST水平与模型组相比,差异有统计学意义(P<0.05);对于D-GalN诱导肝损伤小鼠,中药复方861合剂高、中剂量组小鼠的ALT、AST水平与模型组相比,差异有统计学意义(P <0.05)。组织学检查结果显示,中药复方861合剂对肝损伤有一定的保护作用。中药复方861合剂在最大容量和浓度的给药条件下,小鼠无死亡,行为、活动如常。结论:中药复方861合剂对CCl4和D-GalN所致小鼠急性肝损伤具有较好的治疗作用,且在最大给药浓度条件下用药安全。

黄芪对免疫损伤性肝纤维化大鼠的治疗作用

目的:研究中药黄芪的抗肝纤维化作用,对比观察中药861合剂及其组分之一黄芪对肝纤维化的治疗作用。方法:采用大鼠白蛋白免疫损伤性肝纤维化动物模型,通过光镜、电镜观察、胶原免疫组化染色及胶原蛋白生化测定。结果,黄芪可使大鼠肝纤维化程度及超微结构的病理改变明显减轻,减少总胶原及Ⅰ、Ⅱ、Ⅴ型胶原在肝内的沉积,随着疗程的延长,作用更为显著。同时以肝总胶原蛋白含量为指标,黄芪的抗肝纤维化作用不如中药861合剂。结论:黄芪与其它成份组成中药复方可加强抗纤维化作用。

复方861对活化肝星状细胞超微结构及细胞骨架蛋白的影响

为探讨复方861抗肝纤维化的药理作用,研究其对活化的肝星状细胞系(HSC-T6)超微结构及细胞骨架蛋白的影响.

复方丹参合剂提高胶原酶活性逆转实验性肝纤维化的研究

目的了解复方861合剂对实验性肝纤维化的逆转作用及可能的机理。方法制备大鼠免疫损伤性肝纤维化模型,给予复方丹参861合剂治疗;观察肝组织、血清胶原酶活性及肝组织胶原蛋白含量的变化。结果治疗组大鼠在治疗过程中,肝组织胶原蛋白含量较模型组显著降低;而其肝组织活性胶原酶活性(A),肝组织潜在胶原酶活性(L),A/L 比值及血清胶原酶活性(S)均较模型组显著升高.结论复方丹参861合剂对已形成的肝纤维化有一定程度的逆转作用;其机理可能与胶原酶活性升高对沉积胶原的降解有关.