化浊解毒中药血清及补骨脂素对肝星状细胞生物活性的影响
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摘要
肝纤维化是以结节形成和肝脏收缩为特征,是肝脏疾病末期的所有并发症包括门脉高压、腹水、肝性脑病、合成及代谢功能障碍的基础,因此肝纤维化既损伤肝细胞的功能又能增加门静脉压力。几乎在所有的慢性肝损伤的病人中都会产生纤维化,最终形成肝硬化甚至肝癌,严重影响人的生命。目前一般认为肝纤维化发生的原理为:肝纤维化是肝脏发生慢性非自限性损伤之后的一个创伤修复反应;激活的肝星状细胞是肝纤维化形成最主要的细胞。
肝纤维化最主要的事件是肝星状细胞的活化,肝星状细胞是正常和纤维化肝脏细胞外基质的基本来源,是肝细胞和窦内皮细胞之间的内皮下间隙非游走的窦旁细胞,是体内储存视黄醛衍生物的首要部位,在星状细胞被激活的过程中,静止的富含维生素A的细胞转化为高度致纤维化的细胞,其形态特征为粗面内质网增大、维生素A颗粒减少、核膜皱缩,出现可收缩性的纤维丝及细胞增殖。被激活的肝星状细胞造成肝纤维化的最直接的方式就是增加细胞外基质的形成。细胞外基质是指一系列参与构成正常肝和纤维化肝间质框架的大分子,包括几个结构和支撑分子家族:胶原、非胶原糖蛋白、基质结合生长因子、黏多糖、蛋白多糖和基质细胞蛋白。细胞外基质是细胞功能的动态调节器,纤维化肝脏的基质成分在性质和含量上均有别于正常肝脏,其胶原总含量增加了3~10倍,在肝损伤区域内首先出现的基质改变是细胞纤连蛋白的增加,这些变化导致肝星状细胞的激活和纤维化速度的增加。而激活后的肝星状细胞表达针对胶原和层粘连蛋白的整合素受体则又有助于基质成分的沉积。最终导致细胞外基质合成增加,降解减少,造成细胞外基质的过度沉积。
研究表明纤维化在多种疾病中可以被逆转,如酒精性肝病随着戒酒,其肝纤维化可被显著逆转;乙肝患者在应用抗病毒药拉米夫定之后肝纤维化程度也显著改善;丙肝患者应用干扰素和利巴韦林之后肝纤维化有短期的退化或肝纤维化进展减缓。
目前肝纤维化的治疗主要集中在(1)治愈原发疾病如清除慢性病毒性感染;戒酒和停用有毒性的药物;去除过剩的铁和铜;解除胆道梗阻等。(2)减轻炎症和免疫反应。(3)抑制星状细胞的活化包括能够抑制星状细胞增殖、促进星状细胞凋亡、抑制胶原等细胞外基质的合成;增加细胞外基质的降解,起到抗纤维形成的作用。
现在研究多将肝星状细胞作为治疗肝纤维化的靶点。治疗上多用抗氧化剂如生育酚、水飞蓟素等,可以经保护肝细胞和抑制库普弗细胞功能的双重作用减少肝损伤。另一类为抑制细胞外基质产生的物质,主要是通过直接阻断基质合成和处理或间接抑制转化生子因子(TGF-β)的活性而实现,由于中和转化生长因子能够取得加速基质降解和抑制基质产生的双重效果,所以转化生长因子拮抗剂也是目前的热点,如松弛肽等。PPAR-γ在星状细胞内有表达,其配体可以下调星状细胞的活性,在活化的肝星状细胞中PPAR-γ表达显著减少。上调PPAR-γ表达能够使肝星状细胞从激活态转为静止态,从而抑制肝星状细胞活性。所以本实验将肝星状细胞及TGF-β和PPAR-γ作为靶点观察化浊解毒方的作用机制。
中医多将肝纤维化、肝硬化归为“癥积”“臌胀”“积聚”等范畴。历代医家多认为是由气血瘀滞造成,多用活血化瘀、软肝散结之品治疗,虽能取效,但是也仅能治标而不治本。经过多年的临床实践,导师李佃贵教授认为浊毒在肝纤维化形成中起着关键作用。浊毒内蕴可以影响肝的疏泄功能,导致气机郁滞,血不归藏,变生诸症,日久正虚,浊毒与虚共同致病。故治疗上固本与驱邪不可偏废,以化浊解毒为大法,兼补益肝肾,经多年临床验证化浊解毒方在临床应用中取得良好的效果,但是对其作用机制尚不明确,本实验从细胞水平观察化浊解毒中药血清对肝星状细胞的作用,从而探讨其作用机制。
在治疗肝纤维化的中药复方中除了大剂量的应用化浊解毒、软肝散结、活血化瘀之品外,根据中医的辨证论治的原则我们会选择性的应用一些补益肝肾的药物其中较常用的是补骨脂,其主要成分是补骨脂素,结合现代研究,在治疗肝纤维化中的天然黄酮类药物的应用如水飞蓟素,姜黄素等能够通过抑制肝星状细胞的增殖和分化及细胞外基质的合成而阻止肝纤维化的发生和肝硬化的形成。在以往的研究中发现补骨脂素也有很多与姜黄素相似的作用,我们推测属于香豆素类化合物补骨脂素有可能也具有类似的防治肝纤维化作用,所以本实验观察补骨脂素对肝星状细胞增殖及氧化应激的作用,从而观察其在肝纤维化治疗中的作用机理。进而研究化浊毒与抗氧化应激之间是否存在一定联系。本研究将分五部分来探讨化浊解毒中药血清及补骨脂素对肝星状细胞生物活性的影响。
第一部分化浊解毒中药血清对体外活化肝星状细胞增殖及凋亡的影响
目的:化浊解毒方是临床上治疗肝纤维化的验方,但对其作用机理缺乏研究。本研究通过观察化浊解毒中药血清对体外培养的活化肝星状细胞增殖与凋亡的影响,探讨化浊解毒方治疗肝纤维化的作用机制。
方法:选择健康的SD大鼠,制备含药血清,分为干扰素组(阳性对照组),化浊解毒方大、中、小剂量组及空白对照组。体外培养活化的肝星状细胞,加入各含药血清进行培养24小时、48小时、72小时,利用MTT法观察化浊解毒中药血清对肝星状细胞增殖的影响;培养48小时后运用流式细胞仪分析各组肝星状细胞的凋亡情况。
结果:肝星状细胞在不同浓度化浊解毒中药血清培养基中培养24h,与空白对照组比较,大剂量组(0.216±0.051)和阳性对照组(0.232±0.029)有抑制肝星状细胞增殖的作用(P<0.05);培养48 h后,化浊解毒中药血清各剂量组均有抑制肝星状细胞增殖的作用,其中大剂量组(0.160±0.044)效果更为明显(P<0.05),且与阳性对照组(0.20±0.039)比较无差别,其效果优于中(0.266±0.046)、小剂量组(0.315±0.043)(P<0.05);培养72h后,阳性对照组和化浊解毒中药血清各剂量组均有明显的抑制HSC-T6增殖的作用(P<0.05),且化浊解毒中药血清大剂量组效果优于阳性对照组(P<0.05)。
经过流式细胞术分析后显示:肝星状细胞经过含药血清干预48h后,空白对照组的凋亡率约2.68%,即说明正常的肝星状细胞也存在着凋亡,阳性对照组15.78 %和化浊解毒中药血清各组的凋亡率均较空白对照组高(P<0.05);与阳性对照组比较,化浊解毒中药血清的大剂量组的细胞凋亡率更高为20.02 %(P<0.05);中剂量组细胞凋亡率13.38%与阳性对照组没有明显差别(P>0.05),小剂量组的凋亡率12.88%要低于阳性对照组(P<0.05)。
结论:化浊解毒中药血清各浓度组在体外均能够抑制肝星状细胞的增殖并诱导其凋亡,同时存在一定的量效关系,即浓度越大其抑制增殖和促进凋亡的作用越强。与干扰素组比较,化浊解毒中药血清尤其是大剂量组能有效的抑制肝星状细胞的增殖和诱导其凋亡。
第二部分化浊解毒中药血清对肝星状细胞细胞外基质的影响
目的:本部分实验主要观察化浊解毒中药血清对肝星状细胞细胞外基质尤其是对I型胶原、Ⅲ型胶原、层粘连蛋白、透明质酸的影响。
方法:肝星状细胞加入含药血清后培养48小时后,运用酶联免疫吸附法检测化浊解毒中药血清对肝星状细胞细胞外基质I型胶原、Ⅲ型胶原、层粘连蛋白、透明质酸的影响。
结果:肝星状细胞在不同浓度化浊解毒中药血清培养基中培养48 h后,与空白对照组比较,各组均能抑制肝星状细胞分泌I型胶原、Ⅲ型胶原、层粘连蛋白、透明质酸。其中I型胶原、Ⅲ型胶原的含量在大剂量组(分别为117.9 ng·mL~(-1),56.3 ng·mL~(-1))最低,其作用效果明显优于中(158.0 ng·mL~(-1),70.9 ng·mL~(-1))、小剂量组(162.6 ng·mL~(-1),79.6 ng·mL~(-1))及阳性对照组(148.3 ng·mL~(-1),64.3 ng·mL~(-1))(P<0.05);层粘连蛋白、透明质酸的含量在大剂量组(60.1 ng·mL~(-1),31.2 ng·mL~(-1))与阳性对照组(60.0±5.1 ng·mL~(-1),32.8±4.1 ng·mL~(-1))均较低,两组之间无明显差异(P>0.05),均优于中药的中、小剂量组(P<0.05)。
结论:化浊解毒中药血清能够减少体外培养的肝星状细胞分泌的细胞外基质,包括降低I型胶原、Ⅲ型胶原、透明质酸和层粘连蛋白的含量。
第三部分化浊解毒中药血清对肝星状细胞TGF-β/Smad4信号通路的影响
目的:观察化浊解毒中药血清对肝星状细胞内TGF-β1 mRNA和Smad4 mRNA的影响。
方法:运用RT-PCR方法观察化浊解毒中药血清各浓度组对TGF-β1 mRNA和Smad4 mRNA基因表达的影响。
结果:经化浊解毒中药血清干预48小时后,化浊解毒方各剂量组的TGF-β1 mRNA表达均较正常血清对照组显著减少(P<0.05)。各组的Smad4 mRNA表达量均显著减少(P<0.05)。
结论:化浊解毒中药血清抑制肝星状细胞增殖、促进其凋亡及减少合成细胞外基质可能是通过下调TGF-β1 mRNA与Smad4 mRNA表达,影响TGF-β/Smad4通路起作用。
第四部分化浊解毒中药血清对肝星状细胞PPARγmRNA的影响
目的:本研究将观察化浊解毒中药血清对肝星状细胞内PPARγ的影响,并最终发现对PPARγ及TGF-β/Smad通路的作用,进一步明确化浊解毒中药血清抗肝纤维化的作用机制。
方法:运用RT-PCR方法观察化浊解毒中药血清各浓度组对PPARγmRNA基因表达的影响。
结果:与空白对照组比较,各组的PPARγmRNA表达量均显著增加(P<0.05)。其中化浊解毒中药血清大剂量组的PPARγmRNA表达量明显优于阳性对照组和中、小剂量组(P<0.05)。
结论:化浊解毒中药血清能够增加肝星状细胞内PPARγmRNA的表达量,以化浊解毒中药血清大剂量组最为明显,这也表明了化浊解毒方之所以能干预TGF-β/Smad信号通路可能是通过上调PPARγmRNA来起作用的。
第五部分补骨脂素对肝星状细胞的增殖及活性的影响
目的:观察化浊解毒方中补骨脂所含有的主要成分补骨脂素对肝星状细胞增殖及活性的影响。
方法:常规培养肝星状细胞,并用0.1 mmo·L~(-1)的H2O2造成HSC-T6氧化应激的模型。加入补骨脂素,并用MTT法、放射免疫法和ELISA法分别检测肝星状细胞增殖、细胞上清液中超氧化物歧化酶(SOD),丙二醛(MDA),还原性谷胱甘肽(GSH),谷胱甘肽过氧化物酶(GSH-PX)及I型胶原的含量。
结果:与正常对照组比较,补骨脂素在浓度为10μmol·L~(-1),1μmol·L~(-1),0.1μmol·L~(-1),均呈现出抑制HSC增殖的作用(P<0.05),且效果最好的是在作用48h(P<0.05);与模型组比较,补骨脂素各个浓度组能够提高SOD和GSH-PX的活性(P<0.05),并降低细胞上清液中MDA和GSH的含量(P<0.05);与模型组比较,补骨脂素各个浓度组在作用48 h后,细胞上清液中的I型胶原的表达量均降低(P<0.05)。
结论:补骨脂素能有效的抑制肝星状细胞的增殖及氧化应激,说明补骨脂素是化浊解毒中药血清治疗肝纤维化的物质基础之一,从另一层面上也说明了浊毒导致肝纤维化的机制可能和氧化应激有关,化浊毒的方法之所以能在治疗肝纤维化中起作用,很可能与其中的抗氧化成分有关,即化浊毒与抗氧化应激有关。
总论
1化浊解毒中药血清各浓度组均能够抑制肝星状细胞的增殖。
2化浊解毒中药血清各浓度组均促进肝星状细胞的凋亡。
3化浊解毒中药血清各浓度组均能抑制HSCs细胞分泌I型胶原、Ⅲ型胶原、透明质酸和层粘连蛋白。
4经化浊解毒中药血清干预后,化浊解毒中药血清大剂量组的HSC内TGF-β1mRNA表达量明显降低。
5化浊解毒中药血清大剂量组的Smad4 mRNA表达量明显低于中、小剂量组(P<0.05)。
6化浊解毒中药血清大剂量组的PPARγmRNA表达量明显优于阳性对照组和中、小剂量组。
7化浊解毒方防治肝纤维化的机制可能是通过上调PPARγmRNA表达而干预肝星状细胞内TGF-β/Smad4信号通路,从而促进肝星状细胞凋亡和抑制肝星状细胞的增殖及减少其分泌细胞外基质含量而实现的。
8不同浓度补骨脂素均有明显抑制肝星状细胞增殖的作用,其中10μmol/L组效果更为明显,在培养48h、72 h后作用更为明显,并呈现出量效关系和时效关系。
补骨脂素能有效的抑制肝星状细胞的增殖及氧化应激,说明补骨脂素是化浊解毒方治疗肝纤维化的物质基础之一,化浊毒的方法之所以能在治疗肝纤维化中起作用,很可能与其中的植物雌激素成分有关。
Liver fibrosis is formed in almost all the chronic liver injury patients with the characterized of nodules and liver contraction and finally ended withcirrhosis or liver cancer.Liver fibrosis is the basis of all complications in the telophase of liver disease, including portal hypertension, ascites, hepatic encephalopathy, synthesis and metabolic disorders. Liver fibrosis can damage
the function of liver cells and increase the portal vein pressure. At present, the general principle of the formation of liver fibrosis was considered that: Liver fibrosis is the repair response after a chronic, self-limited damage of liver. Activated hepatic stellate cells were the main cell in the formation of liver fibrosis. The activation of hepatic stellate cells is the key point of the fomation of liver fibrosis.
Hepatic stellate cells, lie between liver cells and sinus endothelial cells, the main cells which produce extracellular matrix in the normal and fibrosis liver, and the main cells of storing retinaldehyde derivatives. In the process of the activation of stellate cells, the quiescent hepatic stellate cells with copious vitamin A were transtormed to the cells which could induce fibrosis and its morphological characteristics were the augumentation of rough endoplasmic reticulum, the decrease vitamin A particle, corrugation aryotheca, the appearance of contractility cellosilk and cell proliferation. The most direct way of the formation of Activated hepatic stellate cells to liver fibrosis was to increase the content of extracellular matrix. Extracellular matrix was a series of macromolecules which pariticitated the constraction of normal liver and fibrosis liver interstitial framework, including the family of several structures and support molecular: the collagen, the collagen glycoprotein and matrix combining growth factor, mucopolysaccharide, proteoglycan and matrix cells protein. The nature and content of matrix components were different from normal liver in liver fibrosis, its collagen total content increased 3~10 times. In liver damage area, these changes accelerated the activation of hepatic stellate cells and the formation speed of fibrosis. Further more, the activated hepatic stellate cells expressed integrin receptor also help matrix components of deposits, which eventually lead to extracellular matrix synthesis increase, degradation decline, and resulted in the extracellular matrix of excessive deposition.
Researches showed that fibrosis can be reversed in many diseases such as alcohol withdrawal; the liver fibrosis can be significant reversed in alcoholic liver disease. Patients with chronic hepatitis b patients in application disease-resistant poison lamivudine after liver fibrosis degree is improved markedly. After application of ribavirin, the fibrosis progression was slowdown in patients with hepatitics.
At present, hepatic fibrosis treatment mainly focus on: (1) To cure primary diseases such as chronic viral infection; alcohol withdrawal and poisonous drug discontinuation, to remove excess iron and copper, to remove biliopancreatic obstruction, etc. (2) To ease inflammation and immune response. (3)To restrain stellate cell activation including the antiproliferative of stellate cells; To promote stellate cell apoptosis, resist fibrogenesis inhibit collagen synthesis of extracellular matrix, and increase the degradation of extracellular matrix.
Hepatic stellate cells were the target of treatment of hepatic fibrosis. Firstly, antioxidants such as tocopherol, silymarin etc, could reduce liver damage through protective liver cells and inhibit kupffer cells. Secondly, the material was to restrain the production of matrix, which mainly through blocking matrix synthesis and processing or indirectly inhibiting activity of TGF-β. Neutralize transformation growth factor can accelerat matrix biodegradation and inhibit matrix production. The focus was transformation growth factor (beta) antagonists, such as flabby peptide, etc. Ppar-gamma was expressed in stellate cells, whose ligand can degrade stellate cell activity. In the activated hepatic stellate cells, and the expressed ppar-gamma was declined significantly. Up-regulated Ppar-gamma expression could make hepatic stellate cells from activted state to rested state, so that inhibit hepatic stellate cell activity. In the study, the mechanism of action of HuaZhuojiedu Formula was observed by its action on TGF - beta and Ppar-gamma.
TCM regarded hepatic fibrosis and cirrhosis as "ZhengJia" "GuZhang" "JiJu". It is used to be considered as the results of blood stasis sluggish, treated by promoting blood circulation to remove blood stasis. In the clinical practice, Mr Li (Diangui) considered turbidity poison playing a key role in the formation of liver fibrosis. Turbidity Poison could affect the liver function and lead to qiji yuzhi. The cause of liver fibrosis was Turbidity Poison and asthenia. Guben and QuXie were important in treatment of liver fibrosis. The effectiveness of HuaZhuojiedu formula was satisfactory after clinical verification, but the mechanism of its action pathway is unclear. In this study, the mechanism of action of HuaZhuojiedu formula on hepatic stellate cells would be observated in the cell level.
In the treatment of liver fibrosis, according to TCM treatment principle, those tonify hepatorenal drugs would be applicated besides Huazhuojiedu, soft liver improving quality, promoting blood circulation to remove blood stasis, and psoralea corylifolia main applicated in the treatment of liver fibrosis. In the treatment of liver fibrosis, medicine such as natural flavonoids silymarin and curcumin could inhibit hepatic stellate cell proliferation, differentiation and extracellular matrix synthesis and prevent the formation of hepatic fibrosis and cirrhosis. In the former research psoralea was found also had much similar effect with curcumin, we assume that psoralen may also have similar function in preventing liver fibrosis, so in this study the function of psoralen on hepatic stellate cells will be observed. This study would be divided into five parts to explore the mechanism of action of HuaZhuojiedu formula and the effect of its anti-oxidant psoralen on the prevention of liver fibrosis, and to find the correlation of ZhuoDu and oxidative stress.
Objective: HuaZhuojiedu Formula is a popular folk medicine which has been used for treatment of liver fibrosis in clinic. The mechanism was unclear.
In the study, the effect of HuaZhuojiedu Formula medicated serum on the proliferation and apoptosis of hepatic stellate cells in vitro was analyzed, in order to explore its mechanism of action.
Methods: SD rats, choosed to produce the medicated serum, were divided into five groups: interferon group (positive group), HuaZhuojiedu Formula large, medium and small dose groups and control group. Hepatic stellate cells were cultivated in vitro, hepatic stellate cell proliferation was observed after addition of each medicated serum 24 hours, 48 hours, 72 hours, using the MTT method, and hepatic stellate cell apoptosis was analysed after cultivating 48 hours using flow cytometric analysis.
Results: After cultivated 24h, compared with control group, HSC-T6 proliferation have been inhibited in the HuaZhuojiedu high-dose group (0.216±0.051) and positive group (0.232±0.029) (P<0.05). After cultivated 48h, HSC-T6 proliferation have been inhibited in HuaZhuojiedu groups, especially in high-dose group (0.160±0.044) (P<0.05), but the effect of inhibition on HSC was indiscriminately between HuaZhuojiedu high-dose group and positive group. After cultivated 72h, the inhibition action of HuaZhuojiedu high-dose group was better than positive group (P<0.05).
Flow cytometric analysis showed that: The apoptosis rate of HSC was about 2.68% in control group after 48h,the apoptosis rate was higher in positive control 15.78% and HuaZhuojiedu high-dose group than that in control group (P<0.05). Comared with positive group, the apoptosis rate of HuaZhuojiedu high-dose group was 20.02% higher (P<0.05) than positive group. The apoptosis rate of HuaZhuojiedu middle-dose group 13.38% was indiscriminately with positive control group (P>0.05), and in the small-dose group, the apoptpsis rate was 12.88% lower than positive control group (P <0.05).
Conclusions: HuaZhuojiedu Formula medicated serum can inhibit hepatic stellate cells proliferation and induce apoptosis in a dose-response Part one Effect of HuaZhuojiedu Formula medicated serum on the proliferation and apoptosis of Hepatic Stellate Cells in vitro relation, namely the inhibiting proliferation and the concentration of apoptosis role to promote the stronger. Compared with positive group, HuaZhuojiedu Formula medicated serum restrains hepatic stellate cell proliferation and induced the apoptosis more effectively.
Part two Effect of HuaZhuojiedu Formula medicated serum on Extr-acellular Matrix Secretion of Hepatic Stellate cells in Vitro
Objective: To observe the effect of HuaZhuojiedu Formula medicated serum on extracellular matrix secretion of hepatic stellate cells in vitro.
Methods: HSC-T6 was incubated with different medicated serum for 48 hours. Collagen type I, collagen typeⅢ, layer adhesion protein, hyaluronic acid was determined by enzyme-linked immunosorbent adsorption method (ELISA).
Results: Addition of HuaZhuojiedu Formula medicated serum into culture medium inhibited Type I collage, hyaluronic acid and laminin secretion. Type I collagen, the content ofⅢcollagen type in high-dose group (117.9±17.0 ng·mL~(-1), 56.3±6.2 ng·mL~(-1))were lowest, and its function is obviously superior (plus or minus 158.0±15.2 ng·mL~(-1) 170.9±6.3 ng·mL~(-1)) than small dose group (162.6±1.98 ng·mL~(-1), 79.6±5.9 ng·mL~(-1)) and control group (148.3±17.8 ng·mL~(-1), 164.3±8.8 ng·mL~(-1)) (P < 0.05). Layer adhesion protein, hyaluronic acid content in high-dose group (60.1±4.5 ng·mL~(-1), 31.2±4.2 ng·mL~(-1)) and positive control my monitor section (60.0±5.1 ng·mL~(-1), 32.8±4.1ng·mL~(-1)) were lower, there was no obvious difference between the two groups (P > 0.05).
Conclusion: HuaZhuojiedu Formula medicated serum can reduce the secretion of the extracellular matrix of hepatic stellate cells in vitro.
Part three Effect of HuaZhuojiedu Formula medicated serum on expression of Transforming Growth Factor- beta1 and Protein Smads in Hepatic Stellate Cells
Objective: To investigate the effect HuaZhuojiedu Formula medicated serum on the expresion of TGF-β1 mRNA and Smad4 mRNA.
Methods: Effect of different volume HuaZhuojiedu Formula medicated serum on TGF-β1 mRNA and Smad4 mRNA were detected through RT-PCR method.
Results: After treated with HuaZhuojiedu Formula medicated serum for 48h, Contrasted to the black control group, the expression of TGF-βmRNA of HSC were significantly decreased in high-dose HuaZhuojiedu Formula (P<0.05), and the expressions of Smad4 mRNA was down-regulated (P<0.05). Conclusion: HuaZhuojiedu Formula medicated serums inhibited hepatic stellate cell proliferation and promoted its apoptosis and reduced synthesis of extracellular matrix, which action mechanism possibly was via down-regulated TGF -β1 mRNA and Smad4 mRNA
Part four Effect of HuaZhuojiedu Formula medicated serum on expression of Ppar-gamma mRNA influence
Objective: Aim to study the effect of HuaZhuojiedu Formula medicated serum on peroxisome proliferator-activated receptorγ(PPARγ) of hepatic stellate cells (HSC) and the correlation between PPARγand TGF-β.
Methods: The effect of HuaZhuojiedu Formula medicated serum on the express of PPARγmRNA in HSC was observed by applying RT-PCR.
Results: At the transcription and translation level, HuaZhuojiedu Formula medicated serum groups upregulated the express of PPARγmRNA (P<0.05), the express of PPARγmRNA was significiently higher in high-dose group than that in other groups (P<0.05).
Conclusion: HuaZhuojiedu Formula medicated serum can upregulate the expression of PPAR gamma mRNA in hepatic stellate cells especialy in HuaZhuojiedu Formula medicated serum high-dose group, which showed that the interferen of HuaZhuojiedu Formula medicated serum in the signal pathway of TGF-β/Smad is funcional through upregulating the express of PPARγmRNA.
Part five Effect of Psoralen on the proliferation and activation of Hepatic Stellate Cell
Objective: To investigate the effect of antioxidant psoralen on the proliferation and lipid peroxidation of oxidative stress in rat hepatic stellate cells in vitro.
Methods: HSC-T6 was incubated with H2O2, which were treated with psoralen.MTT colorimetry was used for assaying proliferation of HSC. Intracellular MDA, SOD, GSH and GSH-PX levels in the culture media were measured by ELISA reagent boxes, collagen I was determined by ELISA.
Results: Comapred with model group, Psoralen significantly decreased the number of HSC in dose of 10μmol·L~(-1), 1μmol·L~(-1), 0.1μmol·L~(-1)(P<0.05) especially at 48h and psoralen in the concentration of 10mM, 1mM, 0.1 mM groups could decrease the content of collagen type I and intracellular MDA and GSH (P<0.05), and increased the activity of SOD and GSH-PX (P<0.05). psoralen in the concentration of 10 mM, 1mM, 0.1 mM groups can improve SOD and GSH - PX activity (P < 0.05), reduce the content of MDA and GSH (P < 0.05), and decrease the content of collagen type I(P < 0.05).
Conclusion: Psoralen could inhibit the proliferation and oxidative stress of HSC. Psoralen was one of material basis of HuaZhuojiedu Formula which prevents liver fibrosis. The mechanism of ZhuoDu induce the liver fibrosis may be correlate with oxidative stress.HuaZhuodu was related to anti-oxidant stress.
General Introduction
1 HuaZhuojiedu Formula medicated serum can inhibit hepatic stellate cell proliferation.
2 HuaZhuojiedu Formula medicated serums can promote hepatic stellate cell apoptosis.
3 HuaZhuojiedu Formula medicated serums can restrain the secretion of collagen type I,Ⅲcollagen type, hyaluronic acid and layer adhesion protein.
4 In HuaZhuojiedu Formula high-dose medicated serum group, expression of TGF -β1mRNA was decreased obviously in HSC.
5 In HuaZhuojiedu Formula high-dose medicated serum group, expression of Smad4 mRNA was lower obviously than that in medium and small dose group (P < 0.05).
6 In HuaZhuojiedu Formula high-dose medicated serum group, expression of PPAR gamma mRNA upgrated obviously more than that in the control group and positive.
7 The preventive effect of HuaZhuojiedu Formula on hepatic fibrosis could be implement through upregulating PPARγmRNA and interventing TGF - beta/Smad4 signaling pathways in stellate cells, promote the stellate cell apoptosis and inhibit stellate cell proliferation and reduce secrete extracellular matrix content.
8 In different concentrations, psoralen significantly inhibits the proliferation of HSC-T6, especially in 10mM at 48h, 72 h, in the concentration-response relation.
Psoralen could inhibit the proliferation and oxidative stress of HSC. Psoralen was one of material basis of HuaZhuojiedu Formula which prevents liver fibrosis.
肝纤维化最主要的事件是肝星状细胞的活化,肝星状细胞是正常和纤维化肝脏细胞外基质的基本来源,是肝细胞和窦内皮细胞之间的内皮下间隙非游走的窦旁细胞,是体内储存视黄醛衍生物的首要部位,在星状细胞被激活的过程中,静止的富含维生素A的细胞转化为高度致纤维化的细胞,其形态特征为粗面内质网增大、维生素A颗粒减少、核膜皱缩,出现可收缩性的纤维丝及细胞增殖。被激活的肝星状细胞造成肝纤维化的最直接的方式就是增加细胞外基质的形成。细胞外基质是指一系列参与构成正常肝和纤维化肝间质框架的大分子,包括几个结构和支撑分子家族:胶原、非胶原糖蛋白、基质结合生长因子、黏多糖、蛋白多糖和基质细胞蛋白。细胞外基质是细胞功能的动态调节器,纤维化肝脏的基质成分在性质和含量上均有别于正常肝脏,其胶原总含量增加了3~10倍,在肝损伤区域内首先出现的基质改变是细胞纤连蛋白的增加,这些变化导致肝星状细胞的激活和纤维化速度的增加。而激活后的肝星状细胞表达针对胶原和层粘连蛋白的整合素受体则又有助于基质成分的沉积。最终导致细胞外基质合成增加,降解减少,造成细胞外基质的过度沉积。
研究表明纤维化在多种疾病中可以被逆转,如酒精性肝病随着戒酒,其肝纤维化可被显著逆转;乙肝患者在应用抗病毒药拉米夫定之后肝纤维化程度也显著改善;丙肝患者应用干扰素和利巴韦林之后肝纤维化有短期的退化或肝纤维化进展减缓。
目前肝纤维化的治疗主要集中在(1)治愈原发疾病如清除慢性病毒性感染;戒酒和停用有毒性的药物;去除过剩的铁和铜;解除胆道梗阻等。(2)减轻炎症和免疫反应。(3)抑制星状细胞的活化包括能够抑制星状细胞增殖、促进星状细胞凋亡、抑制胶原等细胞外基质的合成;增加细胞外基质的降解,起到抗纤维形成的作用。
现在研究多将肝星状细胞作为治疗肝纤维化的靶点。治疗上多用抗氧化剂如生育酚、水飞蓟素等,可以经保护肝细胞和抑制库普弗细胞功能的双重作用减少肝损伤。另一类为抑制细胞外基质产生的物质,主要是通过直接阻断基质合成和处理或间接抑制转化生子因子(TGF-β)的活性而实现,由于中和转化生长因子能够取得加速基质降解和抑制基质产生的双重效果,所以转化生长因子拮抗剂也是目前的热点,如松弛肽等。PPAR-γ在星状细胞内有表达,其配体可以下调星状细胞的活性,在活化的肝星状细胞中PPAR-γ表达显著减少。上调PPAR-γ表达能够使肝星状细胞从激活态转为静止态,从而抑制肝星状细胞活性。所以本实验将肝星状细胞及TGF-β和PPAR-γ作为靶点观察化浊解毒方的作用机制。
中医多将肝纤维化、肝硬化归为“癥积”“臌胀”“积聚”等范畴。历代医家多认为是由气血瘀滞造成,多用活血化瘀、软肝散结之品治疗,虽能取效,但是也仅能治标而不治本。经过多年的临床实践,导师李佃贵教授认为浊毒在肝纤维化形成中起着关键作用。浊毒内蕴可以影响肝的疏泄功能,导致气机郁滞,血不归藏,变生诸症,日久正虚,浊毒与虚共同致病。故治疗上固本与驱邪不可偏废,以化浊解毒为大法,兼补益肝肾,经多年临床验证化浊解毒方在临床应用中取得良好的效果,但是对其作用机制尚不明确,本实验从细胞水平观察化浊解毒中药血清对肝星状细胞的作用,从而探讨其作用机制。
在治疗肝纤维化的中药复方中除了大剂量的应用化浊解毒、软肝散结、活血化瘀之品外,根据中医的辨证论治的原则我们会选择性的应用一些补益肝肾的药物其中较常用的是补骨脂,其主要成分是补骨脂素,结合现代研究,在治疗肝纤维化中的天然黄酮类药物的应用如水飞蓟素,姜黄素等能够通过抑制肝星状细胞的增殖和分化及细胞外基质的合成而阻止肝纤维化的发生和肝硬化的形成。在以往的研究中发现补骨脂素也有很多与姜黄素相似的作用,我们推测属于香豆素类化合物补骨脂素有可能也具有类似的防治肝纤维化作用,所以本实验观察补骨脂素对肝星状细胞增殖及氧化应激的作用,从而观察其在肝纤维化治疗中的作用机理。进而研究化浊毒与抗氧化应激之间是否存在一定联系。本研究将分五部分来探讨化浊解毒中药血清及补骨脂素对肝星状细胞生物活性的影响。
第一部分化浊解毒中药血清对体外活化肝星状细胞增殖及凋亡的影响
目的:化浊解毒方是临床上治疗肝纤维化的验方,但对其作用机理缺乏研究。本研究通过观察化浊解毒中药血清对体外培养的活化肝星状细胞增殖与凋亡的影响,探讨化浊解毒方治疗肝纤维化的作用机制。
方法:选择健康的SD大鼠,制备含药血清,分为干扰素组(阳性对照组),化浊解毒方大、中、小剂量组及空白对照组。体外培养活化的肝星状细胞,加入各含药血清进行培养24小时、48小时、72小时,利用MTT法观察化浊解毒中药血清对肝星状细胞增殖的影响;培养48小时后运用流式细胞仪分析各组肝星状细胞的凋亡情况。
结果:肝星状细胞在不同浓度化浊解毒中药血清培养基中培养24h,与空白对照组比较,大剂量组(0.216±0.051)和阳性对照组(0.232±0.029)有抑制肝星状细胞增殖的作用(P<0.05);培养48 h后,化浊解毒中药血清各剂量组均有抑制肝星状细胞增殖的作用,其中大剂量组(0.160±0.044)效果更为明显(P<0.05),且与阳性对照组(0.20±0.039)比较无差别,其效果优于中(0.266±0.046)、小剂量组(0.315±0.043)(P<0.05);培养72h后,阳性对照组和化浊解毒中药血清各剂量组均有明显的抑制HSC-T6增殖的作用(P<0.05),且化浊解毒中药血清大剂量组效果优于阳性对照组(P<0.05)。
经过流式细胞术分析后显示:肝星状细胞经过含药血清干预48h后,空白对照组的凋亡率约2.68%,即说明正常的肝星状细胞也存在着凋亡,阳性对照组15.78 %和化浊解毒中药血清各组的凋亡率均较空白对照组高(P<0.05);与阳性对照组比较,化浊解毒中药血清的大剂量组的细胞凋亡率更高为20.02 %(P<0.05);中剂量组细胞凋亡率13.38%与阳性对照组没有明显差别(P>0.05),小剂量组的凋亡率12.88%要低于阳性对照组(P<0.05)。
结论:化浊解毒中药血清各浓度组在体外均能够抑制肝星状细胞的增殖并诱导其凋亡,同时存在一定的量效关系,即浓度越大其抑制增殖和促进凋亡的作用越强。与干扰素组比较,化浊解毒中药血清尤其是大剂量组能有效的抑制肝星状细胞的增殖和诱导其凋亡。
第二部分化浊解毒中药血清对肝星状细胞细胞外基质的影响
目的:本部分实验主要观察化浊解毒中药血清对肝星状细胞细胞外基质尤其是对I型胶原、Ⅲ型胶原、层粘连蛋白、透明质酸的影响。
方法:肝星状细胞加入含药血清后培养48小时后,运用酶联免疫吸附法检测化浊解毒中药血清对肝星状细胞细胞外基质I型胶原、Ⅲ型胶原、层粘连蛋白、透明质酸的影响。
结果:肝星状细胞在不同浓度化浊解毒中药血清培养基中培养48 h后,与空白对照组比较,各组均能抑制肝星状细胞分泌I型胶原、Ⅲ型胶原、层粘连蛋白、透明质酸。其中I型胶原、Ⅲ型胶原的含量在大剂量组(分别为117.9 ng·mL~(-1),56.3 ng·mL~(-1))最低,其作用效果明显优于中(158.0 ng·mL~(-1),70.9 ng·mL~(-1))、小剂量组(162.6 ng·mL~(-1),79.6 ng·mL~(-1))及阳性对照组(148.3 ng·mL~(-1),64.3 ng·mL~(-1))(P<0.05);层粘连蛋白、透明质酸的含量在大剂量组(60.1 ng·mL~(-1),31.2 ng·mL~(-1))与阳性对照组(60.0±5.1 ng·mL~(-1),32.8±4.1 ng·mL~(-1))均较低,两组之间无明显差异(P>0.05),均优于中药的中、小剂量组(P<0.05)。
结论:化浊解毒中药血清能够减少体外培养的肝星状细胞分泌的细胞外基质,包括降低I型胶原、Ⅲ型胶原、透明质酸和层粘连蛋白的含量。
第三部分化浊解毒中药血清对肝星状细胞TGF-β/Smad4信号通路的影响
目的:观察化浊解毒中药血清对肝星状细胞内TGF-β1 mRNA和Smad4 mRNA的影响。
方法:运用RT-PCR方法观察化浊解毒中药血清各浓度组对TGF-β1 mRNA和Smad4 mRNA基因表达的影响。
结果:经化浊解毒中药血清干预48小时后,化浊解毒方各剂量组的TGF-β1 mRNA表达均较正常血清对照组显著减少(P<0.05)。各组的Smad4 mRNA表达量均显著减少(P<0.05)。
结论:化浊解毒中药血清抑制肝星状细胞增殖、促进其凋亡及减少合成细胞外基质可能是通过下调TGF-β1 mRNA与Smad4 mRNA表达,影响TGF-β/Smad4通路起作用。
第四部分化浊解毒中药血清对肝星状细胞PPARγmRNA的影响
目的:本研究将观察化浊解毒中药血清对肝星状细胞内PPARγ的影响,并最终发现对PPARγ及TGF-β/Smad通路的作用,进一步明确化浊解毒中药血清抗肝纤维化的作用机制。
方法:运用RT-PCR方法观察化浊解毒中药血清各浓度组对PPARγmRNA基因表达的影响。
结果:与空白对照组比较,各组的PPARγmRNA表达量均显著增加(P<0.05)。其中化浊解毒中药血清大剂量组的PPARγmRNA表达量明显优于阳性对照组和中、小剂量组(P<0.05)。
结论:化浊解毒中药血清能够增加肝星状细胞内PPARγmRNA的表达量,以化浊解毒中药血清大剂量组最为明显,这也表明了化浊解毒方之所以能干预TGF-β/Smad信号通路可能是通过上调PPARγmRNA来起作用的。
第五部分补骨脂素对肝星状细胞的增殖及活性的影响
目的:观察化浊解毒方中补骨脂所含有的主要成分补骨脂素对肝星状细胞增殖及活性的影响。
方法:常规培养肝星状细胞,并用0.1 mmo·L~(-1)的H2O2造成HSC-T6氧化应激的模型。加入补骨脂素,并用MTT法、放射免疫法和ELISA法分别检测肝星状细胞增殖、细胞上清液中超氧化物歧化酶(SOD),丙二醛(MDA),还原性谷胱甘肽(GSH),谷胱甘肽过氧化物酶(GSH-PX)及I型胶原的含量。
结果:与正常对照组比较,补骨脂素在浓度为10μmol·L~(-1),1μmol·L~(-1),0.1μmol·L~(-1),均呈现出抑制HSC增殖的作用(P<0.05),且效果最好的是在作用48h(P<0.05);与模型组比较,补骨脂素各个浓度组能够提高SOD和GSH-PX的活性(P<0.05),并降低细胞上清液中MDA和GSH的含量(P<0.05);与模型组比较,补骨脂素各个浓度组在作用48 h后,细胞上清液中的I型胶原的表达量均降低(P<0.05)。
结论:补骨脂素能有效的抑制肝星状细胞的增殖及氧化应激,说明补骨脂素是化浊解毒中药血清治疗肝纤维化的物质基础之一,从另一层面上也说明了浊毒导致肝纤维化的机制可能和氧化应激有关,化浊毒的方法之所以能在治疗肝纤维化中起作用,很可能与其中的抗氧化成分有关,即化浊毒与抗氧化应激有关。
总论
1化浊解毒中药血清各浓度组均能够抑制肝星状细胞的增殖。
2化浊解毒中药血清各浓度组均促进肝星状细胞的凋亡。
3化浊解毒中药血清各浓度组均能抑制HSCs细胞分泌I型胶原、Ⅲ型胶原、透明质酸和层粘连蛋白。
4经化浊解毒中药血清干预后,化浊解毒中药血清大剂量组的HSC内TGF-β1mRNA表达量明显降低。
5化浊解毒中药血清大剂量组的Smad4 mRNA表达量明显低于中、小剂量组(P<0.05)。
6化浊解毒中药血清大剂量组的PPARγmRNA表达量明显优于阳性对照组和中、小剂量组。
7化浊解毒方防治肝纤维化的机制可能是通过上调PPARγmRNA表达而干预肝星状细胞内TGF-β/Smad4信号通路,从而促进肝星状细胞凋亡和抑制肝星状细胞的增殖及减少其分泌细胞外基质含量而实现的。
8不同浓度补骨脂素均有明显抑制肝星状细胞增殖的作用,其中10μmol/L组效果更为明显,在培养48h、72 h后作用更为明显,并呈现出量效关系和时效关系。
补骨脂素能有效的抑制肝星状细胞的增殖及氧化应激,说明补骨脂素是化浊解毒方治疗肝纤维化的物质基础之一,化浊毒的方法之所以能在治疗肝纤维化中起作用,很可能与其中的植物雌激素成分有关。
Liver fibrosis is formed in almost all the chronic liver injury patients with the characterized of nodules and liver contraction and finally ended withcirrhosis or liver cancer.Liver fibrosis is the basis of all complications in the telophase of liver disease, including portal hypertension, ascites, hepatic encephalopathy, synthesis and metabolic disorders. Liver fibrosis can damage
the function of liver cells and increase the portal vein pressure. At present, the general principle of the formation of liver fibrosis was considered that: Liver fibrosis is the repair response after a chronic, self-limited damage of liver. Activated hepatic stellate cells were the main cell in the formation of liver fibrosis. The activation of hepatic stellate cells is the key point of the fomation of liver fibrosis.
Hepatic stellate cells, lie between liver cells and sinus endothelial cells, the main cells which produce extracellular matrix in the normal and fibrosis liver, and the main cells of storing retinaldehyde derivatives. In the process of the activation of stellate cells, the quiescent hepatic stellate cells with copious vitamin A were transtormed to the cells which could induce fibrosis and its morphological characteristics were the augumentation of rough endoplasmic reticulum, the decrease vitamin A particle, corrugation aryotheca, the appearance of contractility cellosilk and cell proliferation. The most direct way of the formation of Activated hepatic stellate cells to liver fibrosis was to increase the content of extracellular matrix. Extracellular matrix was a series of macromolecules which pariticitated the constraction of normal liver and fibrosis liver interstitial framework, including the family of several structures and support molecular: the collagen, the collagen glycoprotein and matrix combining growth factor, mucopolysaccharide, proteoglycan and matrix cells protein. The nature and content of matrix components were different from normal liver in liver fibrosis, its collagen total content increased 3~10 times. In liver damage area, these changes accelerated the activation of hepatic stellate cells and the formation speed of fibrosis. Further more, the activated hepatic stellate cells expressed integrin receptor also help matrix components of deposits, which eventually lead to extracellular matrix synthesis increase, degradation decline, and resulted in the extracellular matrix of excessive deposition.
Researches showed that fibrosis can be reversed in many diseases such as alcohol withdrawal; the liver fibrosis can be significant reversed in alcoholic liver disease. Patients with chronic hepatitis b patients in application disease-resistant poison lamivudine after liver fibrosis degree is improved markedly. After application of ribavirin, the fibrosis progression was slowdown in patients with hepatitics.
At present, hepatic fibrosis treatment mainly focus on: (1) To cure primary diseases such as chronic viral infection; alcohol withdrawal and poisonous drug discontinuation, to remove excess iron and copper, to remove biliopancreatic obstruction, etc. (2) To ease inflammation and immune response. (3)To restrain stellate cell activation including the antiproliferative of stellate cells; To promote stellate cell apoptosis, resist fibrogenesis inhibit collagen synthesis of extracellular matrix, and increase the degradation of extracellular matrix.
Hepatic stellate cells were the target of treatment of hepatic fibrosis. Firstly, antioxidants such as tocopherol, silymarin etc, could reduce liver damage through protective liver cells and inhibit kupffer cells. Secondly, the material was to restrain the production of matrix, which mainly through blocking matrix synthesis and processing or indirectly inhibiting activity of TGF-β. Neutralize transformation growth factor can accelerat matrix biodegradation and inhibit matrix production. The focus was transformation growth factor (beta) antagonists, such as flabby peptide, etc. Ppar-gamma was expressed in stellate cells, whose ligand can degrade stellate cell activity. In the activated hepatic stellate cells, and the expressed ppar-gamma was declined significantly. Up-regulated Ppar-gamma expression could make hepatic stellate cells from activted state to rested state, so that inhibit hepatic stellate cell activity. In the study, the mechanism of action of HuaZhuojiedu Formula was observed by its action on TGF - beta and Ppar-gamma.
TCM regarded hepatic fibrosis and cirrhosis as "ZhengJia" "GuZhang" "JiJu". It is used to be considered as the results of blood stasis sluggish, treated by promoting blood circulation to remove blood stasis. In the clinical practice, Mr Li (Diangui) considered turbidity poison playing a key role in the formation of liver fibrosis. Turbidity Poison could affect the liver function and lead to qiji yuzhi. The cause of liver fibrosis was Turbidity Poison and asthenia. Guben and QuXie were important in treatment of liver fibrosis. The effectiveness of HuaZhuojiedu formula was satisfactory after clinical verification, but the mechanism of its action pathway is unclear. In this study, the mechanism of action of HuaZhuojiedu formula on hepatic stellate cells would be observated in the cell level.
In the treatment of liver fibrosis, according to TCM treatment principle, those tonify hepatorenal drugs would be applicated besides Huazhuojiedu, soft liver improving quality, promoting blood circulation to remove blood stasis, and psoralea corylifolia main applicated in the treatment of liver fibrosis. In the treatment of liver fibrosis, medicine such as natural flavonoids silymarin and curcumin could inhibit hepatic stellate cell proliferation, differentiation and extracellular matrix synthesis and prevent the formation of hepatic fibrosis and cirrhosis. In the former research psoralea was found also had much similar effect with curcumin, we assume that psoralen may also have similar function in preventing liver fibrosis, so in this study the function of psoralen on hepatic stellate cells will be observed. This study would be divided into five parts to explore the mechanism of action of HuaZhuojiedu formula and the effect of its anti-oxidant psoralen on the prevention of liver fibrosis, and to find the correlation of ZhuoDu and oxidative stress.
Objective: HuaZhuojiedu Formula is a popular folk medicine which has been used for treatment of liver fibrosis in clinic. The mechanism was unclear.
In the study, the effect of HuaZhuojiedu Formula medicated serum on the proliferation and apoptosis of hepatic stellate cells in vitro was analyzed, in order to explore its mechanism of action.
Methods: SD rats, choosed to produce the medicated serum, were divided into five groups: interferon group (positive group), HuaZhuojiedu Formula large, medium and small dose groups and control group. Hepatic stellate cells were cultivated in vitro, hepatic stellate cell proliferation was observed after addition of each medicated serum 24 hours, 48 hours, 72 hours, using the MTT method, and hepatic stellate cell apoptosis was analysed after cultivating 48 hours using flow cytometric analysis.
Results: After cultivated 24h, compared with control group, HSC-T6 proliferation have been inhibited in the HuaZhuojiedu high-dose group (0.216±0.051) and positive group (0.232±0.029) (P<0.05). After cultivated 48h, HSC-T6 proliferation have been inhibited in HuaZhuojiedu groups, especially in high-dose group (0.160±0.044) (P<0.05), but the effect of inhibition on HSC was indiscriminately between HuaZhuojiedu high-dose group and positive group. After cultivated 72h, the inhibition action of HuaZhuojiedu high-dose group was better than positive group (P<0.05).
Flow cytometric analysis showed that: The apoptosis rate of HSC was about 2.68% in control group after 48h,the apoptosis rate was higher in positive control 15.78% and HuaZhuojiedu high-dose group than that in control group (P<0.05). Comared with positive group, the apoptosis rate of HuaZhuojiedu high-dose group was 20.02% higher (P<0.05) than positive group. The apoptosis rate of HuaZhuojiedu middle-dose group 13.38% was indiscriminately with positive control group (P>0.05), and in the small-dose group, the apoptpsis rate was 12.88% lower than positive control group (P <0.05).
Conclusions: HuaZhuojiedu Formula medicated serum can inhibit hepatic stellate cells proliferation and induce apoptosis in a dose-response Part one Effect of HuaZhuojiedu Formula medicated serum on the proliferation and apoptosis of Hepatic Stellate Cells in vitro relation, namely the inhibiting proliferation and the concentration of apoptosis role to promote the stronger. Compared with positive group, HuaZhuojiedu Formula medicated serum restrains hepatic stellate cell proliferation and induced the apoptosis more effectively.
Part two Effect of HuaZhuojiedu Formula medicated serum on Extr-acellular Matrix Secretion of Hepatic Stellate cells in Vitro
Objective: To observe the effect of HuaZhuojiedu Formula medicated serum on extracellular matrix secretion of hepatic stellate cells in vitro.
Methods: HSC-T6 was incubated with different medicated serum for 48 hours. Collagen type I, collagen typeⅢ, layer adhesion protein, hyaluronic acid was determined by enzyme-linked immunosorbent adsorption method (ELISA).
Results: Addition of HuaZhuojiedu Formula medicated serum into culture medium inhibited Type I collage, hyaluronic acid and laminin secretion. Type I collagen, the content ofⅢcollagen type in high-dose group (117.9±17.0 ng·mL~(-1), 56.3±6.2 ng·mL~(-1))were lowest, and its function is obviously superior (plus or minus 158.0±15.2 ng·mL~(-1) 170.9±6.3 ng·mL~(-1)) than small dose group (162.6±1.98 ng·mL~(-1), 79.6±5.9 ng·mL~(-1)) and control group (148.3±17.8 ng·mL~(-1), 164.3±8.8 ng·mL~(-1)) (P < 0.05). Layer adhesion protein, hyaluronic acid content in high-dose group (60.1±4.5 ng·mL~(-1), 31.2±4.2 ng·mL~(-1)) and positive control my monitor section (60.0±5.1 ng·mL~(-1), 32.8±4.1ng·mL~(-1)) were lower, there was no obvious difference between the two groups (P > 0.05).
Conclusion: HuaZhuojiedu Formula medicated serum can reduce the secretion of the extracellular matrix of hepatic stellate cells in vitro.
Part three Effect of HuaZhuojiedu Formula medicated serum on expression of Transforming Growth Factor- beta1 and Protein Smads in Hepatic Stellate Cells
Objective: To investigate the effect HuaZhuojiedu Formula medicated serum on the expresion of TGF-β1 mRNA and Smad4 mRNA.
Methods: Effect of different volume HuaZhuojiedu Formula medicated serum on TGF-β1 mRNA and Smad4 mRNA were detected through RT-PCR method.
Results: After treated with HuaZhuojiedu Formula medicated serum for 48h, Contrasted to the black control group, the expression of TGF-βmRNA of HSC were significantly decreased in high-dose HuaZhuojiedu Formula (P<0.05), and the expressions of Smad4 mRNA was down-regulated (P<0.05). Conclusion: HuaZhuojiedu Formula medicated serums inhibited hepatic stellate cell proliferation and promoted its apoptosis and reduced synthesis of extracellular matrix, which action mechanism possibly was via down-regulated TGF -β1 mRNA and Smad4 mRNA
Part four Effect of HuaZhuojiedu Formula medicated serum on expression of Ppar-gamma mRNA influence
Objective: Aim to study the effect of HuaZhuojiedu Formula medicated serum on peroxisome proliferator-activated receptorγ(PPARγ) of hepatic stellate cells (HSC) and the correlation between PPARγand TGF-β.
Methods: The effect of HuaZhuojiedu Formula medicated serum on the express of PPARγmRNA in HSC was observed by applying RT-PCR.
Results: At the transcription and translation level, HuaZhuojiedu Formula medicated serum groups upregulated the express of PPARγmRNA (P<0.05), the express of PPARγmRNA was significiently higher in high-dose group than that in other groups (P<0.05).
Conclusion: HuaZhuojiedu Formula medicated serum can upregulate the expression of PPAR gamma mRNA in hepatic stellate cells especialy in HuaZhuojiedu Formula medicated serum high-dose group, which showed that the interferen of HuaZhuojiedu Formula medicated serum in the signal pathway of TGF-β/Smad is funcional through upregulating the express of PPARγmRNA.
Part five Effect of Psoralen on the proliferation and activation of Hepatic Stellate Cell
Objective: To investigate the effect of antioxidant psoralen on the proliferation and lipid peroxidation of oxidative stress in rat hepatic stellate cells in vitro.
Methods: HSC-T6 was incubated with H2O2, which were treated with psoralen.MTT colorimetry was used for assaying proliferation of HSC. Intracellular MDA, SOD, GSH and GSH-PX levels in the culture media were measured by ELISA reagent boxes, collagen I was determined by ELISA.
Results: Comapred with model group, Psoralen significantly decreased the number of HSC in dose of 10μmol·L~(-1), 1μmol·L~(-1), 0.1μmol·L~(-1)(P<0.05) especially at 48h and psoralen in the concentration of 10mM, 1mM, 0.1 mM groups could decrease the content of collagen type I and intracellular MDA and GSH (P<0.05), and increased the activity of SOD and GSH-PX (P<0.05). psoralen in the concentration of 10 mM, 1mM, 0.1 mM groups can improve SOD and GSH - PX activity (P < 0.05), reduce the content of MDA and GSH (P < 0.05), and decrease the content of collagen type I(P < 0.05).
Conclusion: Psoralen could inhibit the proliferation and oxidative stress of HSC. Psoralen was one of material basis of HuaZhuojiedu Formula which prevents liver fibrosis. The mechanism of ZhuoDu induce the liver fibrosis may be correlate with oxidative stress.HuaZhuodu was related to anti-oxidant stress.
General Introduction
1 HuaZhuojiedu Formula medicated serum can inhibit hepatic stellate cell proliferation.
2 HuaZhuojiedu Formula medicated serums can promote hepatic stellate cell apoptosis.
3 HuaZhuojiedu Formula medicated serums can restrain the secretion of collagen type I,Ⅲcollagen type, hyaluronic acid and layer adhesion protein.
4 In HuaZhuojiedu Formula high-dose medicated serum group, expression of TGF -β1mRNA was decreased obviously in HSC.
5 In HuaZhuojiedu Formula high-dose medicated serum group, expression of Smad4 mRNA was lower obviously than that in medium and small dose group (P < 0.05).
6 In HuaZhuojiedu Formula high-dose medicated serum group, expression of PPAR gamma mRNA upgrated obviously more than that in the control group and positive.
7 The preventive effect of HuaZhuojiedu Formula on hepatic fibrosis could be implement through upregulating PPARγmRNA and interventing TGF - beta/Smad4 signaling pathways in stellate cells, promote the stellate cell apoptosis and inhibit stellate cell proliferation and reduce secrete extracellular matrix content.
8 In different concentrations, psoralen significantly inhibits the proliferation of HSC-T6, especially in 10mM at 48h, 72 h, in the concentration-response relation.
Psoralen could inhibit the proliferation and oxidative stress of HSC. Psoralen was one of material basis of HuaZhuojiedu Formula which prevents liver fibrosis.
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