白花丹醌对人肝星状细胞作用的分子机制研究
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摘要
肝纤维化是指肝细胞发生坏死及炎症刺激时,肝脏中胶原蛋白等细胞外基质的增生与降解失去平衡,导致肝脏内纤维结缔组织异常沉积的病理过程,它是一种可逆性病变。目前认为,肝星状细胞激活、增殖并大量生成(extracellular matrix, ECM)的过程是肝纤维化病理机制的中心环节,肝星状细胞(hepatic Stellate cell,HSC)的激活和增殖受多种细胞因子的调控。因此,阻断细胞因子的促增殖作用,有望成为肝纤维化治疗的有效手段。近几年来中医药抗肝纤维化治疗研究取得较大进展。前期在国家自然科学基金的资助下(N0:30760321)证实白花丹具有抗肝纤维化作用,白花丹的主要抗肝纤维化药物活性成分是白花丹醌,白花丹醌具有很强的活血化瘀作用。瘦素(leptin)是新发现的促肝纤维化的细胞因子,最近研究显示,leptin具有诱导HSC活化和增殖作用。
目的:本课题将以人HSC为对象,从细胞和分子水平深入开展白花丹醌对外源性leptin刺激后HSC之生物学特性和细胞内信号分子活化方面的研究,主要从抑制HSC激活、增殖、分泌、凋亡及胶原合成等几方面集中探究白花丹醌减少(抑制)HSC-LX2分泌胶原蛋白的靶位点和可能的作用分子机制,为开发广西特色民族药材抗肝纤维化提供理论和实验依据。
方法:
1. MTT法检测白花丹醌对HSC-LX2和人胚肝细胞(L-O2)毒性的影响以及leptin与HSC-LX2作用的时间和浓度的选择。
2. ELISA法检测HSC-LX2细胞培养上清液中leptin的分泌;检测白花丹醌对HSC-LX2细胞培养上清液TGF-β1,α-SMA, TNF-α, PDGF-BB, I型胶原表达的影响。
3.流式细胞术检测肝星状细胞细胞周期分布及细胞凋亡的变化。
4透射电镜观察肝星状细胞凋亡超微结构。
5.免疫细胞化学法检测HSC-LX2凋亡相关蛋白p53, Bax, Bcl-2的表达。
6.实时荧光定量PCR法检测白花丹醌对HSC-LX2 TGF-β1、α-SMA等细胞因子表达。
7. Western blot法检测白花丹醌对HSC-LX2细胞周期相关蛋白p53、Bax、Bcl-2;细胞因子TGF-β1,α-SMA, TNF-α, PDGF-BB;leptin刺激HSC-LX2信号转导通路相关蛋白OB-Rb, JAK2, STAT3, ERK1/2等蛋白表达的影响。
结果:
1.白花丹醌对HSC-LX2 IC50值平均为5.016μg/ml,对人正常胚肝细胞L-O2 IC50值为18.852μg/ml。白花丹醌对HSC-LX2毒性选择指数(SI)为3.76,说明白花丹醌对HSC-LX2具有一定的选择性毒性作用。
2.给药12 h、24 h后,与空白对照组比较,白花丹醌低、中剂量组细胞上清液SOD、MDA、LDH含量均无显著性差异(P>0.05),白花丹醌高剂量组与空白对照组比较有显著性差异(P<0.05),给药48 h后白花丹醌高剂量组与空白对照组比较有显著性差异(P<0.05或P<0.01)。实验结果表明白花丹醌对人肝细胞L-O2有一定的毒性作用,且药物浓度越高,作用的时间越长,毒性越大。考虑到白花丹醌的毒性作用,在后续实验选择白花丹醌2μmol/L、8μmol/L作为实验浓度。
1. HSC自分泌leptin的浓度随着培养时间的延长而递减,24 h达到最低,48 h又上升到原来水平,每个时间点分泌的leptin浓度比较无显著性差异(P>0.05),说明HSC自分泌leptin的量变化仍不明确。
2.流式细胞术PI染色法检测各周期细胞的DNA含量,结果显示,白花丹醌作用HSC-LX2 24 h后,HSC-LX2细胞G0/G1期细胞的百分比均明显增高,而S期+G2/M期细胞总数的百分比明显降低;并且随着药物剂量的增加,G0/G1期细胞的百分比增高,而S期+G2/M期细胞总数的百分比降低,呈剂量依赖性。白花丹醌阻断HSC细胞由G0/G1期向S期、G2/M期转变,从而抑制细胞DNA合成,抑制细胞分裂,使细胞周期进程减缓,阻止其发生增殖。
3.与空白对照组比较,leptin刺激显著增加了HSC-LX2细胞周期蛋白Cyclin DI、Cyclin E的含量,阻断了抑制蛋白p21的表达;白花丹醌则明显降低了Cyclin Dl、Cyclin E蛋白水平,增加了p21的蛋白表达。
4.细胞流式术结果显示,白花丹醌作用HSC-LX2 24 h,HSC-LX2凋亡率明显增加,白花丹醌2, 8μmol/L组和秋水仙碱组HSC凋亡率分别为(3.07±0.30)%,(5.21±0.41)%,(10.1±1.08)%,均显著高于空白对照组(1.40±0.13)%(P<0.01)。表明白花丹醌对HSC-LX2有一定的促凋亡和致坏死的作用。
5.电镜结果显示,空白对照组细胞形态清晰,细胞膜、核膜完整,细胞表面有大量微绒毛。白花丹醌组可见少量坏死细胞和许多不同程度的凋亡细胞。阳性对照组可见少量坏死细胞和许多不同程度的凋亡细胞,其细胞体积变小,核膜消失,胞质浓缩,胞质内出现大量空泡,染色质边集,核固缩。
6.受leptin刺激后HSC-LX2胞浆内表达少量的bax, Bcl-2, p53,可见少量的棕黄色颗粒,经白花丹醌2.8μmol/L干预后, HSC-LX2促凋亡基因Bax, p53蛋白表达明显升高,抗凋亡基因Bcl-2蛋白表达显著降低,与leptin对照组相比,差异均有统计学意义(P<0.01)。
1. ELISA法检测细胞上清液中细胞因子分泌情况,结果显示,与空白对照组比较,leptin组HSC培养上清液中TGF-β1、α-SMA、TNF-α、PDGF-BB含量显著增加(P<0.01)。与leptin组比较,白花丹醌各药物浓度组上清液中TGF-β1、α-SMA、TNF-α、PDGF-BB的含量明显降低(P<0.05或P<0.01),且随着白花丹醌作用浓度增加,对TGF-β1、α-SMA、TNF-α、PDGF-BB的活性抑制越显著。
2.用2-△△CT方法的公式进行计算,得到相对于leptin对照组的TGF-β1、α-SMA、TNF-α、PDGF-BB mRNA相对表达量。荧光实时定量PCR反应结果显示:空白对照组HSC有一定量TGF-β1、α-SMA、TNF-α、PDGF-BB mRNA表达,白花丹醌和秋水仙碱作用24 h后,活化的HSC-LX2内TGF-β1、α-SMA、TNF-α、PDGF-BBmRNA含量较leptin对照组均明显下降(P<0.05或P<0.01)。
3. Western blot结果显示,leptin与HSC-LX2孵育24 h,可刺激的HSC-LX2 TGF-β1、α-SMA、PDGF-BB蛋白表达明显升高,轻微增加TNF-α蛋白的表达,白花丹醌与HSC-LX2孵育24 h后,明显减少HSC-LX2TGF-β1、α-SMA、PDGF-BB蛋白表达,对TNF-α表达无明显影响。
1.以最佳刺激浓度(100 ng/ml)的leptin作用于HSC-LX2,检测不同时间点OB-Rb的表达水平。结果显示,leptin(100 ng/ml)可诱导HSCOB-Rb的表达,并呈现一定的时相变化。leptin刺激HSC 6 h后,即见OB-Rb表达增强,但无统计学意义;24 h时达到高峰(P<0.01); 36 h时仍保持较高的活化状态P<0.01);48 h后则基本接近初始水平。
2. Western blot法研究白花丹醌对leptin刺激HSC-LX2后蛋白酪氨酸激酶(JAK)-信号转导子和转录激活子(STAT)信号转导通路中相关信号因子:OB-Rb、JAK2、STAT3、ERK1/2及其磷酸化蛋白、MMP-1、MMP-13的影响。
①leptin刺激显著地增加了OB-Rb受体蛋白的表达,白花丹醌处理则明显地降低了OB-Rb受体蛋白的表达。
②与空白对照组比较,leptin刺激显著地增加了JAK2、pJAK2蛋白的表达。与leptin对照组比较,白花丹醌处理则明显地降低了pJAK2蛋白的表达水平(P<0.01),但对JAK2蛋白表达无明显影响(P>0.05)。
③与空白对照组相比较,leptin刺激更加显著地增加了STAT3、pSTAT3受体蛋白表达水平(P<0.01)。白花丹醌处理则明显地降低了STAT3、pSTAT3受体蛋白的表达(P<0.05)。
④与空白对照组相比较,leptin刺激更加显著地增加了ERK1/2、pERK1/2蛋白的丰度(P<0.01或P<0.05)。白花丹醌处理则明显地降低了ERK1/2、pERK1/2蛋白的表达(P<0.01或P<0.05)。
⑤与空白对照组比较,leptin刺激可明显降低HSC-LX2 MMP-1的表达,MMP-13表达水平无显著变化(P>0.05)。白花丹醌作用HSC-LX224h后MMP-l蛋白表达水平升高,MMP-13无显著变化(P>0.05)。
⑥.与空白对照组比较,leptin(100 ng/ml)可刺激HSC-LX2 I型胶原的增生和表达显著增加,白花丹醌处理组可明显抑制HSC胞内I胶原的表达和分泌。
3. ELISA检测白花丹醌对leptin刺激HSC-LX2I型胶原表达。结果显示:与空白对照组比较,leptin对照组HSC培养上清液中I型胶原的含量显著增加(P<0.01),与leptin对照组比较,白花丹醌各药物浓度组上清液中I型胶原的含量明显降低,差异有显著性(P<0.05)。
结论:
1.白花丹醌对正常人肝细胞L-O2具有一定的毒性作用,且药物浓度越高,作用时间越长,毒性越大。该药对HSC-LX2的毒性作用具有一定的选择性。
2.外源性leptin可促进HSC增殖和活化,并呈现剂量、时间依赖性。
3.白花丹醌可通过阻止HSC-LX2由G0/G1期进入S期,而抑制leptin所致的细胞增殖,其作用机制可能是与其降低cyclin D1、cyclin E1蛋白表达,提高P21蛋白表达有关。
4.白花丹醌对HSC-LX2有一定的促凋亡和致坏死的作用。白花丹醌诱导HSC凋亡具体机制在于上调活化的HSC中p53、bax蛋白表达,下调Bcl-2蛋白表达。
5.白花丹醌从基因和蛋白水平抑制外源性leptin刺激HSC-LX2的TGF-β1、α-SMA、PDGF-BB分泌,从基因水平抑制TNF-α的表达。
6.白花丹醌从细胞膜受体水平抑制外源性leptin刺激HSC的增殖,降低HSC-LX2OB-Rb蛋白的表达。
7.白花丹醌可通过阻断HSC-LX2细胞内JAK2-STAT3信号通路,降低JAK2、STAT3蛋白磷酸化水平,从而抑制外源性leptin刺激的HSC-LX2增殖。
8.白花丹醌阻断leptin诱导MAPK信号转导途径相关因子ERK蛋白磷酸化,在HSC的MAPK信号转导途径上显著抑制HSC的增殖。
9.白花丹醌抑制leptin诱导I型胶原、MMP-1的表达。
Hepatic fibrosis is a pathological process of abnormal deposition of fibrous connective tissue in liver resulted from the loss of balance between the proliferation and degradation of extracellular matrix(ECM), like collagen protein, when hepatocytes necrosis and inflammation stimulation occur in liver; it is a reversible lesion. At present, it is considered that the activation and proliferation of hepatic satellite cells and the great production of ECM play key roles in the pathological mechanism of hepatic fibrosis. The activation and proliferation of HSC are subject to the regulation of many cytokines. Therefore, to block the pro-proliferation effect of cytokine shows promise as an effective therapeutic measure for hepatic fibrosis. In recent years, anti-hepatic fibrosis treatment with TCM has achieved greater progress.In the previous work funded by National Natural Science Foundation of China, this topic team has confirmed the anti-hepatic fibrosis effect of Baihuadan (Ceylon Leadword Root or Leaf) and the main active ingredient of Baihuadan (Ceylon Leadword Root or Leaf) for resisting hepatic fibrosis is plumbagin.Plumbagin, as the main active ingredient of Baihuadan, had robust effect on activating blood circulation to dissipate blood stasis. Leptin is a new cytokine with the effect of pro-hepatic fibrosis and recent studies show that leptin can induce the activation and proliferation of HSC.
Objective This topic is to thoroughly explore the effects of plumbagin on biological features and the activation of signaling molecule in HSC after stimulation by external leptin at cellular and molecular level, which is mainly focused on its inhibition effect on activation, proliferation, secretion, apoptosis and collagen synthesis with the aim to determine the target sites and potential molecular mechanism of plumbagin in reducing the secretion of collagen by HSC-LX2.The actually mechanism of plumbagin in resisting hepatic fibrosis will be explored at cellular and molecular level, and thus to provide theoretical and experimental evidences for developing ethnic drugs in resisting hepatic fibrosis with characteristics of GuangXi region.
Methods 1.To test the effects of plumbagin on the toxicities of HSC-LX2 and human embryonic hepatocyte (L-O2) by MTT method; to determine the content of SOD, MDA and LDH in supernate of HSC-LX2 by ELIASA after action of plumbagin.
2. Test the secretion of leptin by ELISA method in culture supernate of HSC-LX2 cells; test the acting timing and concentration choice of leptin when acting with HSC-LX2; test changes of cell cycle by flow cytometry; test the effect of plumbagin on the expression of proteins related to HSC-LX2 cell cycle by Western blot ;test the effects of plumbagin on apoptosis of HSC-LX2 cells by V-FITC and PI double staining; observe the ultrastructure of apoptotic HSC cells by transmission electron microscope; test the expressions of apoptosis-associated proteins p53, Bax and Bcl-2 by cell immunohistochemistry.
3. Test the effect of plumbagin on the expression of HSC-LX2TGF-β1 and other cytokines by ELISA, Real-time quantitative PCR and Western blot. 4. Test the secretion of type-1 collagen in supernate of HSC culture by western blot; observe the activation phase of OB-Rb, JAK2, STAT3, ERK1/2 after stimulation by leptin by Western blot; test the effects of ; test the effect of plumbagin on the expression of proteins related to HSC-LX2 signal transduction after stimulation by leptin by Western blot.
Results
1. The average IC50 of plumbagin to HSC-LX2 is 5.016μg/ml and 18.852μg/ml to human normal embryonic hepatocyte. The toxic selective index (SI) of Plumbagin to plumbagin is 3.76, which indicates a certain selective toxic effect of plumbagin towards HSC-LX2.
2. There are no significant differences of the contents of SOD, MDA and LDH in supernate of low-, and middle-dose groups from blank control at 12h and 24h after addition of drug (p>0.05); there are significant differences in supernate of high-dose group from blank control (p<0.05); and there are significant differences in supernate of high-dose group from blank control at 48h after addition of drug (P<0.05 or P<0.01).The results show certain toxicity of plumbagin to human heptocytes L-O2 and the toxicity will be greater with higher concentration of plumbagin and longer acting duration. In light of the toxicity of plumbagin, 2umol/L and 8μmol/L of plumbagin are selected for subsequent experiments.
1. The concentration of leptin auto-secreted by HSC is to decrease with time and to the minimum at 24h and recovery of original level at 48h (P>0.05). There is no significant difference between the concentrations at each point, which shows the ambiguity of the change of auto-secretion amount of leptin by HSC.
2. Test the DNA content at each phase of cell cycle by PI staining for Flow cytometry: at 24h after addition of plumbagin, the percentage of HSC-LX2 cells at G0/G1 phase is apparent increased and the total percentage of HSC-LX2 cells at S phase+G2/M phase is apparent decreased; and with the increase of dose, the percentage of HSC-LX2 cells at G0/G1 phase becomes higher and the total percentage of HSC-LX2 cells at S phase+G2/M phase becomes lower, indicating dose-dependency. Plumbagin may block the transformation of HSCs from G0/G1 to S, G2/M phase and further inhibit the synthesis of DNA and cell division, which finally slow the cellular cycle progress and block cell proliferation.
3. Compared to blank control, the stimulation by leptin may significantly increase the content of HSC Cyclin DI and Cyclin E, and block the expression of inhibiting protein P21; however, plumbagin may significantly decrease the level of Cyclin DI and Cyclin E and increase the expression of p21 protein.
4. The results of flow cytometry show significant increased apoptotic rates of HSCs at 24h after addition of plumbagin to HSC-LX2; the apoptotic rates in 2 , 8μmol/L and colchicine groups are(3.07±0.30)%,(5.21±0.41)%,(10.1±1.08)% respectively, which are both significantly higher than blank control (1.40±0.13) %, (P<0.01).The result indicates that plumbagin can promote the apoptosis and necrosis of HSC-LX2.
5. The results from electron microscope show that HSCs in blank control group have clear forms, intact nuclear membrane and cell membrane. There is lots of microvillus on cell surface. After addition of plumbagin, a few of necrotic cells and many apoptotic cells of various degrees can be observed in HSC-LX2.
6. Small quantities of bax, Bcl-2 and p53 are expressed and a few of brownish particles may be seen after stimulation by leptin; after intervention by 2 and 8μmol/l of plumbagin, the expression of pro-apoptotic genes , Bax and P53, are significantly increased and the expression of anti-apoptotic gene, Bcl-2, is significantly decreased. All these differences are statistically significant compared to leptin-stimulation group (P<0.01).
1. Test the secretion of cytokins in supernate by ELISA method. Compared to blank control group, the contents of TGF-β1,α-SMA, TNF-αand PDGF-BB are significantly increased (P<0.01). Compared to leptin group, the contents of TGF-β1,α-SMA, TNF-αand PDGF-BB in each plumbagin groups are significantly decreased (P<0.05 or P<0.01). Additionally, the activity inhibition effect of plumbagin to TGF-β1,α-SMA, TNF-αand PDGF-BB becomes stronger with the increasing concentration of plumbagin.
2. Acquire the expression amount of TGF-β1,α-SMA, TNF-αand PDGF-BB relative to leptin group through 2-△△CT formula. The result of real-time PCR shows: certain quantity of expressions of TGF-β1,α-SMA, TNF-α, PDGF-BB mRNAa are observed in HSC of blank control group; at 24h after addition of plumbagin and colchicin, the quantities of TGF-β1,α-SMA, TNF-αand PDGF-BB mRNA in activated HSC-LX2 are all significantly decreased from leptin group(P < 0.05 or P <0.01).
3. Result of Western blot shows that, after incubation of HSC-LX2 with leptin for 24h, the expressions of HSC-LX2TGF-β1,α-SMA and PDGF-BB are all apparently increased and the expression of TNF-αis slightly increased. After incubation of HSC-LX2 with plumbagin for 24h, the expressions of HSC-LX2TGF-β1,α-SMA and PDGF-BB are all decreased and the expression of TNF-αis not affected apparently.
1. Test the expression level of OB-Rb at different point after addition of leptin of optimal stimulation concentration (100 ng/ml), and the result shows that leptin (100 ng/ml) can induce the expression of HSCOB-Rb with the manner of phase changing. At 6h after leptin stimulation, the expression of OB-Rb is increased without statistical significance; at 24, it reaches to the peak concentration (P<0.01); at 36h, it remains relative high activity (P<0.01); and after 48h, it returns to the initial level.
2. The study on the regulation effect of plumbagin on the expression of OB-Rb after stimulation by leptin through Western blot shows that leptin can significantly stimulate the expression of OB-Rb receptor protein and the treatment by plumbagin can significantly decrease the expression of OB-Rb receptor protein.
3. The study on the effects of plumbagin on the expression of JAK2 and PJAK2 through western blot, leptin can significantly increase the expression of JAK2 and pJAK2 compared to blank control group, however, the treatment by plumbagin can apparently decrease the expression level of pJAK2 (P<0.01) and has no significant effect on the expression of JAK2 (P<0.05).
4. The study on the regulation effect of plumbagin on the expression of STAT3 and pSTAT3 after stimulation of HSC-LX2 by leptin through Western blot, leptin can significantly increase the expression of STAT3 and pSTAT3 compared to blank control group (P<0.01), however, the treatment by plumbagin can apparently decrease the expression level of STAT3 and pSTAT3 receptor proteins (P<0.05).
5. The study on the regulation effect of plumbagin on the expression of ERK1/2 and pERK1/2 after stimulation of HSC-LX2 by leptin through Western blot, the result of western blot shows that leptin can significantly increase the abundances of ERK1/2 and pERK1/2 compared to blank control group(P<0.01 or P<0.05). However, the treatment by plumbagin can apparently decrease the expression level of ERK1/2 and pERK1/2 proteins (P<0.01 or P<0.05).
6. The study on the effect of plumbagin on the expressions of HSC-LX2 MMP-1 and MMP-13 after stimulation by leptin through Western blot, the analysis result shows that leptin can significantly decrease the expression of HSC-LX2MMP-1 and has no effect on the expression of MMP-13 compared to blank control group(P<0.05). However, after addition of plumbagin for 24h, the expression level of MMP-1 protein is increased and the expression of MMP-13 has no significantly alteration (P>0.05).
7. The study on the effect of plumbagin on the expression of HSC-LX2 type-1 collagen after stimulation by leptin through ELISA, the result shows that the content of type-1 collagen in HSC culture supernate is significantly increased compared to blank control group (P<0.01). However, the content of type-1 collagen in HSC culture supernate in each concentration group of plumbagin is apparently decreased compared to leptin group and the difference is of statistical significance(P<0.05).
8. In the test of the effect of plumbagin on the expression of HSC-LX2 type-1 collagen after stimulation by leptin through western blot, the result shows that leptin can increase the proliferation and expression of type-I collagen compared to control group (100 ng/ml) and the proliferation and expression of type-I collagen in HSCs are significantly inhibited in plumbagin treatment group.
Conclusions
1. Plumbagin shows some toxicity to human normal hepatocytes L-O2 and the toxicity will be greater with higher concentration of plumbagin and longer acting duration.
2. External leptin can promote the proliferation and activation of HSC with dose- and time-dependent manner. Plumbagin can inhibit the stimulatory effect of external lepton on the proliferation of HSC-LX2, which indicates that plumbagin can decrease the inflammation amplification effect and such effect may inhibit the development of hepatic fibrosis.
3. Plumbagin can block HSC-LX2 entering S phase from HSC-LX2 and in this way to inhibit the proliferation induced by leptin. Its mechanism may be related to the decrease of the expressions of cyclin D1 and cyclin E1 and the increase of P21 protein.
4. Plumbagin shows certain functions in promotions of apoptosis and necrosis of HSC-LX2. The actual mechanism of plumbagin in induction of HSC apoptosis relies on up-regulating the expressions of p53 and bax and down-regulating the expression of Bcl-2.
5. Plumbagin can inhibit the stimulation of external leptin on the secretions of HSC-LX2 TGF-β1,α-SMA, PDGF-BB at the level of gene and protein; it also can inhibit the expression of TNF-αat the level of gene.
6. Plumbagin can inhibit the proliferation of HSC induced by external leptin at the level of membrane receptor and decrease the expression of HSC-LX2 OB-Rb protein.
7. Plumbagin can decrease the protein phosphorylation level of JAK2 and STAT3, and inhibit the proliferation of HSC-LX2 induced by external leptin through blocking the signal transduction of JAK2-STAT3 in HSC-LX2.
8. Plumbagin also can block the protein phosphorylation of the protein factor, ERK, of the leptin-dependent MAPK signal transduction, and by this way, it can significantly inhibit the proliferation of HSC induced by HSC MAPK signal transduction.
9. Plumbagin can inhibit the expressions of type-I collagen and MMP-1.
目的:本课题将以人HSC为对象,从细胞和分子水平深入开展白花丹醌对外源性leptin刺激后HSC之生物学特性和细胞内信号分子活化方面的研究,主要从抑制HSC激活、增殖、分泌、凋亡及胶原合成等几方面集中探究白花丹醌减少(抑制)HSC-LX2分泌胶原蛋白的靶位点和可能的作用分子机制,为开发广西特色民族药材抗肝纤维化提供理论和实验依据。
方法:
1. MTT法检测白花丹醌对HSC-LX2和人胚肝细胞(L-O2)毒性的影响以及leptin与HSC-LX2作用的时间和浓度的选择。
2. ELISA法检测HSC-LX2细胞培养上清液中leptin的分泌;检测白花丹醌对HSC-LX2细胞培养上清液TGF-β1,α-SMA, TNF-α, PDGF-BB, I型胶原表达的影响。
3.流式细胞术检测肝星状细胞细胞周期分布及细胞凋亡的变化。
4透射电镜观察肝星状细胞凋亡超微结构。
5.免疫细胞化学法检测HSC-LX2凋亡相关蛋白p53, Bax, Bcl-2的表达。
6.实时荧光定量PCR法检测白花丹醌对HSC-LX2 TGF-β1、α-SMA等细胞因子表达。
7. Western blot法检测白花丹醌对HSC-LX2细胞周期相关蛋白p53、Bax、Bcl-2;细胞因子TGF-β1,α-SMA, TNF-α, PDGF-BB;leptin刺激HSC-LX2信号转导通路相关蛋白OB-Rb, JAK2, STAT3, ERK1/2等蛋白表达的影响。
结果:
1.白花丹醌对HSC-LX2 IC50值平均为5.016μg/ml,对人正常胚肝细胞L-O2 IC50值为18.852μg/ml。白花丹醌对HSC-LX2毒性选择指数(SI)为3.76,说明白花丹醌对HSC-LX2具有一定的选择性毒性作用。
2.给药12 h、24 h后,与空白对照组比较,白花丹醌低、中剂量组细胞上清液SOD、MDA、LDH含量均无显著性差异(P>0.05),白花丹醌高剂量组与空白对照组比较有显著性差异(P<0.05),给药48 h后白花丹醌高剂量组与空白对照组比较有显著性差异(P<0.05或P<0.01)。实验结果表明白花丹醌对人肝细胞L-O2有一定的毒性作用,且药物浓度越高,作用的时间越长,毒性越大。考虑到白花丹醌的毒性作用,在后续实验选择白花丹醌2μmol/L、8μmol/L作为实验浓度。
1. HSC自分泌leptin的浓度随着培养时间的延长而递减,24 h达到最低,48 h又上升到原来水平,每个时间点分泌的leptin浓度比较无显著性差异(P>0.05),说明HSC自分泌leptin的量变化仍不明确。
2.流式细胞术PI染色法检测各周期细胞的DNA含量,结果显示,白花丹醌作用HSC-LX2 24 h后,HSC-LX2细胞G0/G1期细胞的百分比均明显增高,而S期+G2/M期细胞总数的百分比明显降低;并且随着药物剂量的增加,G0/G1期细胞的百分比增高,而S期+G2/M期细胞总数的百分比降低,呈剂量依赖性。白花丹醌阻断HSC细胞由G0/G1期向S期、G2/M期转变,从而抑制细胞DNA合成,抑制细胞分裂,使细胞周期进程减缓,阻止其发生增殖。
3.与空白对照组比较,leptin刺激显著增加了HSC-LX2细胞周期蛋白Cyclin DI、Cyclin E的含量,阻断了抑制蛋白p21的表达;白花丹醌则明显降低了Cyclin Dl、Cyclin E蛋白水平,增加了p21的蛋白表达。
4.细胞流式术结果显示,白花丹醌作用HSC-LX2 24 h,HSC-LX2凋亡率明显增加,白花丹醌2, 8μmol/L组和秋水仙碱组HSC凋亡率分别为(3.07±0.30)%,(5.21±0.41)%,(10.1±1.08)%,均显著高于空白对照组(1.40±0.13)%(P<0.01)。表明白花丹醌对HSC-LX2有一定的促凋亡和致坏死的作用。
5.电镜结果显示,空白对照组细胞形态清晰,细胞膜、核膜完整,细胞表面有大量微绒毛。白花丹醌组可见少量坏死细胞和许多不同程度的凋亡细胞。阳性对照组可见少量坏死细胞和许多不同程度的凋亡细胞,其细胞体积变小,核膜消失,胞质浓缩,胞质内出现大量空泡,染色质边集,核固缩。
6.受leptin刺激后HSC-LX2胞浆内表达少量的bax, Bcl-2, p53,可见少量的棕黄色颗粒,经白花丹醌2.8μmol/L干预后, HSC-LX2促凋亡基因Bax, p53蛋白表达明显升高,抗凋亡基因Bcl-2蛋白表达显著降低,与leptin对照组相比,差异均有统计学意义(P<0.01)。
1. ELISA法检测细胞上清液中细胞因子分泌情况,结果显示,与空白对照组比较,leptin组HSC培养上清液中TGF-β1、α-SMA、TNF-α、PDGF-BB含量显著增加(P<0.01)。与leptin组比较,白花丹醌各药物浓度组上清液中TGF-β1、α-SMA、TNF-α、PDGF-BB的含量明显降低(P<0.05或P<0.01),且随着白花丹醌作用浓度增加,对TGF-β1、α-SMA、TNF-α、PDGF-BB的活性抑制越显著。
2.用2-△△CT方法的公式进行计算,得到相对于leptin对照组的TGF-β1、α-SMA、TNF-α、PDGF-BB mRNA相对表达量。荧光实时定量PCR反应结果显示:空白对照组HSC有一定量TGF-β1、α-SMA、TNF-α、PDGF-BB mRNA表达,白花丹醌和秋水仙碱作用24 h后,活化的HSC-LX2内TGF-β1、α-SMA、TNF-α、PDGF-BBmRNA含量较leptin对照组均明显下降(P<0.05或P<0.01)。
3. Western blot结果显示,leptin与HSC-LX2孵育24 h,可刺激的HSC-LX2 TGF-β1、α-SMA、PDGF-BB蛋白表达明显升高,轻微增加TNF-α蛋白的表达,白花丹醌与HSC-LX2孵育24 h后,明显减少HSC-LX2TGF-β1、α-SMA、PDGF-BB蛋白表达,对TNF-α表达无明显影响。
1.以最佳刺激浓度(100 ng/ml)的leptin作用于HSC-LX2,检测不同时间点OB-Rb的表达水平。结果显示,leptin(100 ng/ml)可诱导HSCOB-Rb的表达,并呈现一定的时相变化。leptin刺激HSC 6 h后,即见OB-Rb表达增强,但无统计学意义;24 h时达到高峰(P<0.01); 36 h时仍保持较高的活化状态P<0.01);48 h后则基本接近初始水平。
2. Western blot法研究白花丹醌对leptin刺激HSC-LX2后蛋白酪氨酸激酶(JAK)-信号转导子和转录激活子(STAT)信号转导通路中相关信号因子:OB-Rb、JAK2、STAT3、ERK1/2及其磷酸化蛋白、MMP-1、MMP-13的影响。
①leptin刺激显著地增加了OB-Rb受体蛋白的表达,白花丹醌处理则明显地降低了OB-Rb受体蛋白的表达。
②与空白对照组比较,leptin刺激显著地增加了JAK2、pJAK2蛋白的表达。与leptin对照组比较,白花丹醌处理则明显地降低了pJAK2蛋白的表达水平(P<0.01),但对JAK2蛋白表达无明显影响(P>0.05)。
③与空白对照组相比较,leptin刺激更加显著地增加了STAT3、pSTAT3受体蛋白表达水平(P<0.01)。白花丹醌处理则明显地降低了STAT3、pSTAT3受体蛋白的表达(P<0.05)。
④与空白对照组相比较,leptin刺激更加显著地增加了ERK1/2、pERK1/2蛋白的丰度(P<0.01或P<0.05)。白花丹醌处理则明显地降低了ERK1/2、pERK1/2蛋白的表达(P<0.01或P<0.05)。
⑤与空白对照组比较,leptin刺激可明显降低HSC-LX2 MMP-1的表达,MMP-13表达水平无显著变化(P>0.05)。白花丹醌作用HSC-LX224h后MMP-l蛋白表达水平升高,MMP-13无显著变化(P>0.05)。
⑥.与空白对照组比较,leptin(100 ng/ml)可刺激HSC-LX2 I型胶原的增生和表达显著增加,白花丹醌处理组可明显抑制HSC胞内I胶原的表达和分泌。
3. ELISA检测白花丹醌对leptin刺激HSC-LX2I型胶原表达。结果显示:与空白对照组比较,leptin对照组HSC培养上清液中I型胶原的含量显著增加(P<0.01),与leptin对照组比较,白花丹醌各药物浓度组上清液中I型胶原的含量明显降低,差异有显著性(P<0.05)。
结论:
1.白花丹醌对正常人肝细胞L-O2具有一定的毒性作用,且药物浓度越高,作用时间越长,毒性越大。该药对HSC-LX2的毒性作用具有一定的选择性。
2.外源性leptin可促进HSC增殖和活化,并呈现剂量、时间依赖性。
3.白花丹醌可通过阻止HSC-LX2由G0/G1期进入S期,而抑制leptin所致的细胞增殖,其作用机制可能是与其降低cyclin D1、cyclin E1蛋白表达,提高P21蛋白表达有关。
4.白花丹醌对HSC-LX2有一定的促凋亡和致坏死的作用。白花丹醌诱导HSC凋亡具体机制在于上调活化的HSC中p53、bax蛋白表达,下调Bcl-2蛋白表达。
5.白花丹醌从基因和蛋白水平抑制外源性leptin刺激HSC-LX2的TGF-β1、α-SMA、PDGF-BB分泌,从基因水平抑制TNF-α的表达。
6.白花丹醌从细胞膜受体水平抑制外源性leptin刺激HSC的增殖,降低HSC-LX2OB-Rb蛋白的表达。
7.白花丹醌可通过阻断HSC-LX2细胞内JAK2-STAT3信号通路,降低JAK2、STAT3蛋白磷酸化水平,从而抑制外源性leptin刺激的HSC-LX2增殖。
8.白花丹醌阻断leptin诱导MAPK信号转导途径相关因子ERK蛋白磷酸化,在HSC的MAPK信号转导途径上显著抑制HSC的增殖。
9.白花丹醌抑制leptin诱导I型胶原、MMP-1的表达。
Hepatic fibrosis is a pathological process of abnormal deposition of fibrous connective tissue in liver resulted from the loss of balance between the proliferation and degradation of extracellular matrix(ECM), like collagen protein, when hepatocytes necrosis and inflammation stimulation occur in liver; it is a reversible lesion. At present, it is considered that the activation and proliferation of hepatic satellite cells and the great production of ECM play key roles in the pathological mechanism of hepatic fibrosis. The activation and proliferation of HSC are subject to the regulation of many cytokines. Therefore, to block the pro-proliferation effect of cytokine shows promise as an effective therapeutic measure for hepatic fibrosis. In recent years, anti-hepatic fibrosis treatment with TCM has achieved greater progress.In the previous work funded by National Natural Science Foundation of China, this topic team has confirmed the anti-hepatic fibrosis effect of Baihuadan (Ceylon Leadword Root or Leaf) and the main active ingredient of Baihuadan (Ceylon Leadword Root or Leaf) for resisting hepatic fibrosis is plumbagin.Plumbagin, as the main active ingredient of Baihuadan, had robust effect on activating blood circulation to dissipate blood stasis. Leptin is a new cytokine with the effect of pro-hepatic fibrosis and recent studies show that leptin can induce the activation and proliferation of HSC.
Objective This topic is to thoroughly explore the effects of plumbagin on biological features and the activation of signaling molecule in HSC after stimulation by external leptin at cellular and molecular level, which is mainly focused on its inhibition effect on activation, proliferation, secretion, apoptosis and collagen synthesis with the aim to determine the target sites and potential molecular mechanism of plumbagin in reducing the secretion of collagen by HSC-LX2.The actually mechanism of plumbagin in resisting hepatic fibrosis will be explored at cellular and molecular level, and thus to provide theoretical and experimental evidences for developing ethnic drugs in resisting hepatic fibrosis with characteristics of GuangXi region.
Methods 1.To test the effects of plumbagin on the toxicities of HSC-LX2 and human embryonic hepatocyte (L-O2) by MTT method; to determine the content of SOD, MDA and LDH in supernate of HSC-LX2 by ELIASA after action of plumbagin.
2. Test the secretion of leptin by ELISA method in culture supernate of HSC-LX2 cells; test the acting timing and concentration choice of leptin when acting with HSC-LX2; test changes of cell cycle by flow cytometry; test the effect of plumbagin on the expression of proteins related to HSC-LX2 cell cycle by Western blot ;test the effects of plumbagin on apoptosis of HSC-LX2 cells by V-FITC and PI double staining; observe the ultrastructure of apoptotic HSC cells by transmission electron microscope; test the expressions of apoptosis-associated proteins p53, Bax and Bcl-2 by cell immunohistochemistry.
3. Test the effect of plumbagin on the expression of HSC-LX2TGF-β1 and other cytokines by ELISA, Real-time quantitative PCR and Western blot. 4. Test the secretion of type-1 collagen in supernate of HSC culture by western blot; observe the activation phase of OB-Rb, JAK2, STAT3, ERK1/2 after stimulation by leptin by Western blot; test the effects of ; test the effect of plumbagin on the expression of proteins related to HSC-LX2 signal transduction after stimulation by leptin by Western blot.
Results
1. The average IC50 of plumbagin to HSC-LX2 is 5.016μg/ml and 18.852μg/ml to human normal embryonic hepatocyte. The toxic selective index (SI) of Plumbagin to plumbagin is 3.76, which indicates a certain selective toxic effect of plumbagin towards HSC-LX2.
2. There are no significant differences of the contents of SOD, MDA and LDH in supernate of low-, and middle-dose groups from blank control at 12h and 24h after addition of drug (p>0.05); there are significant differences in supernate of high-dose group from blank control (p<0.05); and there are significant differences in supernate of high-dose group from blank control at 48h after addition of drug (P<0.05 or P<0.01).The results show certain toxicity of plumbagin to human heptocytes L-O2 and the toxicity will be greater with higher concentration of plumbagin and longer acting duration. In light of the toxicity of plumbagin, 2umol/L and 8μmol/L of plumbagin are selected for subsequent experiments.
1. The concentration of leptin auto-secreted by HSC is to decrease with time and to the minimum at 24h and recovery of original level at 48h (P>0.05). There is no significant difference between the concentrations at each point, which shows the ambiguity of the change of auto-secretion amount of leptin by HSC.
2. Test the DNA content at each phase of cell cycle by PI staining for Flow cytometry: at 24h after addition of plumbagin, the percentage of HSC-LX2 cells at G0/G1 phase is apparent increased and the total percentage of HSC-LX2 cells at S phase+G2/M phase is apparent decreased; and with the increase of dose, the percentage of HSC-LX2 cells at G0/G1 phase becomes higher and the total percentage of HSC-LX2 cells at S phase+G2/M phase becomes lower, indicating dose-dependency. Plumbagin may block the transformation of HSCs from G0/G1 to S, G2/M phase and further inhibit the synthesis of DNA and cell division, which finally slow the cellular cycle progress and block cell proliferation.
3. Compared to blank control, the stimulation by leptin may significantly increase the content of HSC Cyclin DI and Cyclin E, and block the expression of inhibiting protein P21; however, plumbagin may significantly decrease the level of Cyclin DI and Cyclin E and increase the expression of p21 protein.
4. The results of flow cytometry show significant increased apoptotic rates of HSCs at 24h after addition of plumbagin to HSC-LX2; the apoptotic rates in 2 , 8μmol/L and colchicine groups are(3.07±0.30)%,(5.21±0.41)%,(10.1±1.08)% respectively, which are both significantly higher than blank control (1.40±0.13) %, (P<0.01).The result indicates that plumbagin can promote the apoptosis and necrosis of HSC-LX2.
5. The results from electron microscope show that HSCs in blank control group have clear forms, intact nuclear membrane and cell membrane. There is lots of microvillus on cell surface. After addition of plumbagin, a few of necrotic cells and many apoptotic cells of various degrees can be observed in HSC-LX2.
6. Small quantities of bax, Bcl-2 and p53 are expressed and a few of brownish particles may be seen after stimulation by leptin; after intervention by 2 and 8μmol/l of plumbagin, the expression of pro-apoptotic genes , Bax and P53, are significantly increased and the expression of anti-apoptotic gene, Bcl-2, is significantly decreased. All these differences are statistically significant compared to leptin-stimulation group (P<0.01).
1. Test the secretion of cytokins in supernate by ELISA method. Compared to blank control group, the contents of TGF-β1,α-SMA, TNF-αand PDGF-BB are significantly increased (P<0.01). Compared to leptin group, the contents of TGF-β1,α-SMA, TNF-αand PDGF-BB in each plumbagin groups are significantly decreased (P<0.05 or P<0.01). Additionally, the activity inhibition effect of plumbagin to TGF-β1,α-SMA, TNF-αand PDGF-BB becomes stronger with the increasing concentration of plumbagin.
2. Acquire the expression amount of TGF-β1,α-SMA, TNF-αand PDGF-BB relative to leptin group through 2-△△CT formula. The result of real-time PCR shows: certain quantity of expressions of TGF-β1,α-SMA, TNF-α, PDGF-BB mRNAa are observed in HSC of blank control group; at 24h after addition of plumbagin and colchicin, the quantities of TGF-β1,α-SMA, TNF-αand PDGF-BB mRNA in activated HSC-LX2 are all significantly decreased from leptin group(P < 0.05 or P <0.01).
3. Result of Western blot shows that, after incubation of HSC-LX2 with leptin for 24h, the expressions of HSC-LX2TGF-β1,α-SMA and PDGF-BB are all apparently increased and the expression of TNF-αis slightly increased. After incubation of HSC-LX2 with plumbagin for 24h, the expressions of HSC-LX2TGF-β1,α-SMA and PDGF-BB are all decreased and the expression of TNF-αis not affected apparently.
1. Test the expression level of OB-Rb at different point after addition of leptin of optimal stimulation concentration (100 ng/ml), and the result shows that leptin (100 ng/ml) can induce the expression of HSCOB-Rb with the manner of phase changing. At 6h after leptin stimulation, the expression of OB-Rb is increased without statistical significance; at 24, it reaches to the peak concentration (P<0.01); at 36h, it remains relative high activity (P<0.01); and after 48h, it returns to the initial level.
2. The study on the regulation effect of plumbagin on the expression of OB-Rb after stimulation by leptin through Western blot shows that leptin can significantly stimulate the expression of OB-Rb receptor protein and the treatment by plumbagin can significantly decrease the expression of OB-Rb receptor protein.
3. The study on the effects of plumbagin on the expression of JAK2 and PJAK2 through western blot, leptin can significantly increase the expression of JAK2 and pJAK2 compared to blank control group, however, the treatment by plumbagin can apparently decrease the expression level of pJAK2 (P<0.01) and has no significant effect on the expression of JAK2 (P<0.05).
4. The study on the regulation effect of plumbagin on the expression of STAT3 and pSTAT3 after stimulation of HSC-LX2 by leptin through Western blot, leptin can significantly increase the expression of STAT3 and pSTAT3 compared to blank control group (P<0.01), however, the treatment by plumbagin can apparently decrease the expression level of STAT3 and pSTAT3 receptor proteins (P<0.05).
5. The study on the regulation effect of plumbagin on the expression of ERK1/2 and pERK1/2 after stimulation of HSC-LX2 by leptin through Western blot, the result of western blot shows that leptin can significantly increase the abundances of ERK1/2 and pERK1/2 compared to blank control group(P<0.01 or P<0.05). However, the treatment by plumbagin can apparently decrease the expression level of ERK1/2 and pERK1/2 proteins (P<0.01 or P<0.05).
6. The study on the effect of plumbagin on the expressions of HSC-LX2 MMP-1 and MMP-13 after stimulation by leptin through Western blot, the analysis result shows that leptin can significantly decrease the expression of HSC-LX2MMP-1 and has no effect on the expression of MMP-13 compared to blank control group(P<0.05). However, after addition of plumbagin for 24h, the expression level of MMP-1 protein is increased and the expression of MMP-13 has no significantly alteration (P>0.05).
7. The study on the effect of plumbagin on the expression of HSC-LX2 type-1 collagen after stimulation by leptin through ELISA, the result shows that the content of type-1 collagen in HSC culture supernate is significantly increased compared to blank control group (P<0.01). However, the content of type-1 collagen in HSC culture supernate in each concentration group of plumbagin is apparently decreased compared to leptin group and the difference is of statistical significance(P<0.05).
8. In the test of the effect of plumbagin on the expression of HSC-LX2 type-1 collagen after stimulation by leptin through western blot, the result shows that leptin can increase the proliferation and expression of type-I collagen compared to control group (100 ng/ml) and the proliferation and expression of type-I collagen in HSCs are significantly inhibited in plumbagin treatment group.
Conclusions
1. Plumbagin shows some toxicity to human normal hepatocytes L-O2 and the toxicity will be greater with higher concentration of plumbagin and longer acting duration.
2. External leptin can promote the proliferation and activation of HSC with dose- and time-dependent manner. Plumbagin can inhibit the stimulatory effect of external lepton on the proliferation of HSC-LX2, which indicates that plumbagin can decrease the inflammation amplification effect and such effect may inhibit the development of hepatic fibrosis.
3. Plumbagin can block HSC-LX2 entering S phase from HSC-LX2 and in this way to inhibit the proliferation induced by leptin. Its mechanism may be related to the decrease of the expressions of cyclin D1 and cyclin E1 and the increase of P21 protein.
4. Plumbagin shows certain functions in promotions of apoptosis and necrosis of HSC-LX2. The actual mechanism of plumbagin in induction of HSC apoptosis relies on up-regulating the expressions of p53 and bax and down-regulating the expression of Bcl-2.
5. Plumbagin can inhibit the stimulation of external leptin on the secretions of HSC-LX2 TGF-β1,α-SMA, PDGF-BB at the level of gene and protein; it also can inhibit the expression of TNF-αat the level of gene.
6. Plumbagin can inhibit the proliferation of HSC induced by external leptin at the level of membrane receptor and decrease the expression of HSC-LX2 OB-Rb protein.
7. Plumbagin can decrease the protein phosphorylation level of JAK2 and STAT3, and inhibit the proliferation of HSC-LX2 induced by external leptin through blocking the signal transduction of JAK2-STAT3 in HSC-LX2.
8. Plumbagin also can block the protein phosphorylation of the protein factor, ERK, of the leptin-dependent MAPK signal transduction, and by this way, it can significantly inhibit the proliferation of HSC induced by HSC MAPK signal transduction.
9. Plumbagin can inhibit the expressions of type-I collagen and MMP-1.
引文
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