芍芪多苷的研制及其对大鼠化学性肝纤维化的作用和促进胶原代谢的分子机制
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摘要
肝纤维化(hepatic fibrosis,HF)是大多数慢性肝病所共有的病理特征,是肝脏细胞外基质(extracellular matrix,ECM)合成和降解失衡、导致其过度沉积的结果,也是慢性肝炎进一步向肝硬化、肝癌发展的重要环节。近年来的研究证实HF在未进入肝硬化之前,尚有逆转可能,因此预防和逆转HF对慢性肝病、肝硬化等治疗有积极意义。但HF的形成是一个复杂的过程,目前用于治疗或逆转HF的方法大多效果不理想或长期应用不良反应较大。因此,探究HF的发病机制,并在中医药领域寻找高效低毒、能控制HF病情的新药是当前研究的重要方向。白芍和黄芪是中医临床常用的保肝药物,尤其是益气活血、养血柔肝方剂的主要组份。本实验采用均匀设计法优选出白芍和黄芪的最佳剂量配比后,运用现代提取分离技术制得白芍和黄芪复方的芍芪多苷有效部位群(shaoqiduogan,SQDG);在大鼠化学性HF模型上,探讨SQDG对大鼠HF的治疗作用及其特点;并从细胞因子、胶原代谢相关酶的蛋白表达水平等方面研究SQDG治疗大鼠HF的可能机制,从而为抗HF中药新药——SQDG制剂的进一步开发奠定基础。
目的:研究SQDG有效部位群的制备工艺,并建立相应的质量控制方法;从大鼠肝功能的改变、反映胶原水平的血清和肝脏指标及肝脏病理形态学等的改变,明确SQDG对大鼠HF的治疗作用;从细胞因子、胶原代谢相关酶等方面研究SQDG
Hepatic fibrosis (HF) is the hallmark of most chronic liver diseases. Its essense is excessive deposition of extracellular matrix (ECM) components in liver, which occurs due to an imbalance between the production and degradation of matrix. Collagen is the most important part of ECM. Its final stage is cirrhosis. HF is generally considered reversible conditions. Thus, to prevent or reverse HF has positive significance for therapeutics on chronic liver diseases and cirrhosis. But HF is a complex process, and current therapies targeting at arresting or revering HF are largely ineffective and some has unacceptable side effects in longterm therapy. Therefore, one of important trends focuses on investigation the mechanisms of HF and exploration new effective drugs without ill effects. Radix Paeonia Pall and Radix Astragali are two common traditional herbs in treatment for liver diseases. The present study was designed to optimize the proportion of the two herbs by uniform design and produce the effective compound------shaoqiduogan (SQDG) by modern techniques of extract, purity and separation, andto investigate the effects of SQDG on chemical HF of rat and its possible mechanisms. AIM To study on preparation process of SQDG and establish its quality standards. From the changes of liver function indices, liver index, spleen index, ECM levels on
serum and liver, and histological morphological of HF rats induced by CCI4, we observed the effects of SQDG on HF of rat. We explored its possible mechanisms on HF from cytokines and collagen metabolism. At last, we observed the effect on promotion HSC-T6 collagen degradation of SQDG and the changes of the expressions of G a i and G a s of HSC-T6 stimulated with TGF-pM by Western - blot analysis. METHODS The CCI4 -treated mice acute liver injury model was used to observe the protective effects of total glucosides of paeony (TGP) and astragalosides (ASTs). Uniform design was used to optimize the best proportion of Radix Paeonia Pall and Radix Astragali. The optimum extraction was investigated by orthogonal design. Macroporous resin was used to separate SQDG. High performance liquid chromatography(HPLC), high performance liquid chromatography-evaporative light scattering detector ( HPLC-ELSD) and colormetric method were used to determine paeoniflorin(Pae), astragaloside IV and ASTs, respectively. Experimental rat models of chemical HF induced by CCI4 was used to investigated intervention effects of SQDG. SQDG (42.5, 85, 170 mg-kg-l-d-1, 6d-w-l) were given by intragastric administration. Colchicine (Col), TPG, ASTs were given as positive control.The alanine aminotransferase (ALT), aspartate aminotransferase (AST) levels, A/G in plasma and hydroxyproline (Hyp) contents in liver tissue were assayed by spectrophotometry. The body weight, liver and spleen weight were assayed. The hyaluronic acid (HA), procollagen III (PCIII) and collagen I (C- I ) were assessed by radioimmunoassay. The transforming growth factor betal (TGF-pi) level in plasma was determined by enzyme-linked immunosorbent assay (ELISA).TGF-pi, C- I , matrix metalloproteinasel3 (MMP-13) and tissue inhibitors of metalloproteinasel (TIMP-1) levels in liver were assayed by immunohistochemistry. Hepatic tissue sections were stained with hematoxylin-eosin (HE), Masson, and Gomori examined under a light microscope, and scored.In vitro, the collagen production of HSC-T6 cells stimulated with TGF-pi was
measured with 3H-Proline incorporation. MMP-13 and TIMP-1 levels in culture supernatants of HSC-T6 cells stimulated with TGF-pl were determined by ELISA.Western-blot analysis was used to detect the changes of the expression of G-protein on HSC-T6 cell membrane stimulated by TGF-0 Qug-L"1) and effects on its of SQDG.RESULTS1. Preparation process and quality standards of SQDG were established.According to the extraction ratio of TGP and ASTs, the optimum proportion of Radix Paeonia Pall and Radix Astragali was decided to be 4 I 1 by uniform design. The optimum preparation process of SQDG was investrgated by orthogonal design and macroporous resin column separation as the paeoniflorin content index by HPLC. The optimum alcohol-extracting factors was as follows: 6 times of 70% alcohol, refluxing and extracting for 2 times, 1.5h per times. The extracts water solution was separated by D101 macroporous resin. The optimal separation conditions were as follows: The ratio of herbs and resin was 1 '. 2. The extracts water solution was absorbed by resin with a flow rate of 4 BV/h. Water, 20% alcohol, 40% alcohol and 70% alcohol were used to elute the resin column, respectively. Portions of 20% ethanol and 70 % ethanol were collected, and evaporated to dryness under reduced pressure, which yielded yellow dry powder......SQDG.Scientific method of quality control was established. TLC indentification of Radix Paeonia Pall and Radix Astragali were carried out. HPLC, HPLC-ELSD and colormetric method were used to determine Pae, AST IV and ASTs, respectively. The purity of Pae, ASTs and AST IVin SQDG were 51.23%, 5.03% and 0.686%, respectively.All these results showed that the process of extraction and purity was feasible, and the methods of quality control were scientific and reliable.2, SQDG could intervene in chemical hepatic fibrosis of ratsThe HF models in rats were induced by CCU. The histological morphological
examination of rat livers showed that SQDG (85, 170 mg-kg"1, ig) significantly reduced the scores of liver fibrosis of HF rats. SQDG (85, 170 mg-kg"1, ig) treatment markedly alleviated the degree of liver fibrogenesis and formation of pseudolobulus.Administration with SQDG (85, 170 mg-kg"1, ig) significantly reduced the elevated serum ALT, AST activities and elevated the reduced A/G of HF rats. Administration with SQDG (85, 170 mg-kg"1, ig) significantly reduced the elevated liver index and administration with SQDG (170 mg-kg"1, ig) significantly reduced the elevated spleen index. The results illustrated that SQDG has protective effects on liver of HF, and suggested that its effects on HF was related to improving liver functions.Administration with SQDG (85, 170 mg-kg"1, ig) significantly reduced the elevated serum HA, PCIII, C- I levels and Hyp content in liver homogenate. It suggested that SQDG could prevent HF via reducing collagen deposition.3. Inhibiting secretion of TGF-pi was one of important characteristics of SQDG preventing HFTGF-J31 is one of the most powerful profibrofenic activity cytokines. The results of ELISA and immunohistochemistry demonstrated that SQDG significantly reduced the elevated serum TGF-pi level, and significantly decreased TGF-pl expression in liver. The results showed that SQDG could inhibit HSC activation and decrease collagen production by reducing secretion of TGF-pi.4. Controlling the levels of MMP-13 and TIMP-1 to promote collagen degradation SQDG is one of important mechanisms of SQDG preventing HFThe results immunohistochemistry demonstrated that SQDG significantly increased MMP-13 expression and significantly decreased TIMP-1 expression in liver. In vitro, SQDG (20~160mg-L'1) significantly raised MMP-13 level and reduced the elevated TIMP-1 level in culture supernatants of HSC-T6 cells stimulated by TGF-pl. Therefore, SQDG could facilitate MMP-13 expression and inhibit TIMP-1 expression. The results suggested that SQDG intervene in rat HF via elevating the decreased
目的:研究SQDG有效部位群的制备工艺,并建立相应的质量控制方法;从大鼠肝功能的改变、反映胶原水平的血清和肝脏指标及肝脏病理形态学等的改变,明确SQDG对大鼠HF的治疗作用;从细胞因子、胶原代谢相关酶等方面研究SQDG
Hepatic fibrosis (HF) is the hallmark of most chronic liver diseases. Its essense is excessive deposition of extracellular matrix (ECM) components in liver, which occurs due to an imbalance between the production and degradation of matrix. Collagen is the most important part of ECM. Its final stage is cirrhosis. HF is generally considered reversible conditions. Thus, to prevent or reverse HF has positive significance for therapeutics on chronic liver diseases and cirrhosis. But HF is a complex process, and current therapies targeting at arresting or revering HF are largely ineffective and some has unacceptable side effects in longterm therapy. Therefore, one of important trends focuses on investigation the mechanisms of HF and exploration new effective drugs without ill effects. Radix Paeonia Pall and Radix Astragali are two common traditional herbs in treatment for liver diseases. The present study was designed to optimize the proportion of the two herbs by uniform design and produce the effective compound------shaoqiduogan (SQDG) by modern techniques of extract, purity and separation, andto investigate the effects of SQDG on chemical HF of rat and its possible mechanisms. AIM To study on preparation process of SQDG and establish its quality standards. From the changes of liver function indices, liver index, spleen index, ECM levels on
serum and liver, and histological morphological of HF rats induced by CCI4, we observed the effects of SQDG on HF of rat. We explored its possible mechanisms on HF from cytokines and collagen metabolism. At last, we observed the effect on promotion HSC-T6 collagen degradation of SQDG and the changes of the expressions of G a i and G a s of HSC-T6 stimulated with TGF-pM by Western - blot analysis. METHODS The CCI4 -treated mice acute liver injury model was used to observe the protective effects of total glucosides of paeony (TGP) and astragalosides (ASTs). Uniform design was used to optimize the best proportion of Radix Paeonia Pall and Radix Astragali. The optimum extraction was investigated by orthogonal design. Macroporous resin was used to separate SQDG. High performance liquid chromatography(HPLC), high performance liquid chromatography-evaporative light scattering detector ( HPLC-ELSD) and colormetric method were used to determine paeoniflorin(Pae), astragaloside IV and ASTs, respectively. Experimental rat models of chemical HF induced by CCI4 was used to investigated intervention effects of SQDG. SQDG (42.5, 85, 170 mg-kg-l-d-1, 6d-w-l) were given by intragastric administration. Colchicine (Col), TPG, ASTs were given as positive control.The alanine aminotransferase (ALT), aspartate aminotransferase (AST) levels, A/G in plasma and hydroxyproline (Hyp) contents in liver tissue were assayed by spectrophotometry. The body weight, liver and spleen weight were assayed. The hyaluronic acid (HA), procollagen III (PCIII) and collagen I (C- I ) were assessed by radioimmunoassay. The transforming growth factor betal (TGF-pi) level in plasma was determined by enzyme-linked immunosorbent assay (ELISA).TGF-pi, C- I , matrix metalloproteinasel3 (MMP-13) and tissue inhibitors of metalloproteinasel (TIMP-1) levels in liver were assayed by immunohistochemistry. Hepatic tissue sections were stained with hematoxylin-eosin (HE), Masson, and Gomori examined under a light microscope, and scored.In vitro, the collagen production of HSC-T6 cells stimulated with TGF-pi was
measured with 3H-Proline incorporation. MMP-13 and TIMP-1 levels in culture supernatants of HSC-T6 cells stimulated with TGF-pl were determined by ELISA.Western-blot analysis was used to detect the changes of the expression of G-protein on HSC-T6 cell membrane stimulated by TGF-0 Qug-L"1) and effects on its of SQDG.RESULTS1. Preparation process and quality standards of SQDG were established.According to the extraction ratio of TGP and ASTs, the optimum proportion of Radix Paeonia Pall and Radix Astragali was decided to be 4 I 1 by uniform design. The optimum preparation process of SQDG was investrgated by orthogonal design and macroporous resin column separation as the paeoniflorin content index by HPLC. The optimum alcohol-extracting factors was as follows: 6 times of 70% alcohol, refluxing and extracting for 2 times, 1.5h per times. The extracts water solution was separated by D101 macroporous resin. The optimal separation conditions were as follows: The ratio of herbs and resin was 1 '. 2. The extracts water solution was absorbed by resin with a flow rate of 4 BV/h. Water, 20% alcohol, 40% alcohol and 70% alcohol were used to elute the resin column, respectively. Portions of 20% ethanol and 70 % ethanol were collected, and evaporated to dryness under reduced pressure, which yielded yellow dry powder......SQDG.Scientific method of quality control was established. TLC indentification of Radix Paeonia Pall and Radix Astragali were carried out. HPLC, HPLC-ELSD and colormetric method were used to determine Pae, AST IV and ASTs, respectively. The purity of Pae, ASTs and AST IVin SQDG were 51.23%, 5.03% and 0.686%, respectively.All these results showed that the process of extraction and purity was feasible, and the methods of quality control were scientific and reliable.2, SQDG could intervene in chemical hepatic fibrosis of ratsThe HF models in rats were induced by CCU. The histological morphological
examination of rat livers showed that SQDG (85, 170 mg-kg"1, ig) significantly reduced the scores of liver fibrosis of HF rats. SQDG (85, 170 mg-kg"1, ig) treatment markedly alleviated the degree of liver fibrogenesis and formation of pseudolobulus.Administration with SQDG (85, 170 mg-kg"1, ig) significantly reduced the elevated serum ALT, AST activities and elevated the reduced A/G of HF rats. Administration with SQDG (85, 170 mg-kg"1, ig) significantly reduced the elevated liver index and administration with SQDG (170 mg-kg"1, ig) significantly reduced the elevated spleen index. The results illustrated that SQDG has protective effects on liver of HF, and suggested that its effects on HF was related to improving liver functions.Administration with SQDG (85, 170 mg-kg"1, ig) significantly reduced the elevated serum HA, PCIII, C- I levels and Hyp content in liver homogenate. It suggested that SQDG could prevent HF via reducing collagen deposition.3. Inhibiting secretion of TGF-pi was one of important characteristics of SQDG preventing HFTGF-J31 is one of the most powerful profibrofenic activity cytokines. The results of ELISA and immunohistochemistry demonstrated that SQDG significantly reduced the elevated serum TGF-pi level, and significantly decreased TGF-pl expression in liver. The results showed that SQDG could inhibit HSC activation and decrease collagen production by reducing secretion of TGF-pi.4. Controlling the levels of MMP-13 and TIMP-1 to promote collagen degradation SQDG is one of important mechanisms of SQDG preventing HFThe results immunohistochemistry demonstrated that SQDG significantly increased MMP-13 expression and significantly decreased TIMP-1 expression in liver. In vitro, SQDG (20~160mg-L'1) significantly raised MMP-13 level and reduced the elevated TIMP-1 level in culture supernatants of HSC-T6 cells stimulated by TGF-pl. Therefore, SQDG could facilitate MMP-13 expression and inhibit TIMP-1 expression. The results suggested that SQDG intervene in rat HF via elevating the decreased
引文
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81. Isbrucker RA, Peterson TC. Platelet-derived growth factor and pentoxifylline modulate of collagen synthesis in myofibroblasts. Toxicol and Appl Pharmacol, 1998; 149(1): 120—6
82. Schuppan D, Porov Y. Hepatic fibrosis: from bench to bedside. J Gastroenterol Hepatol, 2002; 17(suppl 3): S300~5
83. Safadi R, Friedman SL. Hepatic fibrosis-role of hepatic stellate cell activation. Med