益气逐瘀解毒颗粒调控MMPs/TIMPs平衡抗大鼠肝纤维化作用研究
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摘要
目的:探讨益气逐瘀解毒颗粒对四氯化碳(CCl4)诱导的大鼠肝纤维化的治疗作用及机制。方法:将SD雄性大鼠给予CCl4橄榄油混合液进行皮下注射7周,制备肝纤维化大鼠模型,随机分为益气逐瘀解毒颗粒高、中、低剂量组、阳性对照组、模型组、空白组,给予相应治疗,5周后处死大鼠,检测外周血生化指标,逆转录-定量PCR法和蛋白印迹法分别检测大鼠肝组织中基质蛋白酶1 (Matrix metalloproteinase1, MMP-1)、MMP-2、MMP-9、MMP-13及基质蛋白酶抑制因子1 (tissue Inhibitor of metalloproteinases1,TIMP-1)表达水平,肝组织苏木精-伊红(Hematoxylin-Eosin staining, HE染色)、MASSON染色检测肝纤维化程度。结果:治疗后与空白组比较,模型组的ALT、AST无显著性差异(P> 0.05),MMP-1、MMP-2、MMP-9、MMP-13基因表达显著降低(P <0.05),TIMP-1基因表达显著升高(P <0.05),肝纤维化半定量计分(SSS)评分显著升高(P <0.01);与模型组比较,益气逐瘀解毒颗粒各剂量组ALT、AST无显著性差异(P> 0.05),益气逐瘀解毒颗粒高剂量组MMP-1、MMP-2、MMP-9、MMP-13基因表达明显升高(P <0.05),中剂量组MMP-9、MMP-13基因表达明显增高(P <0.05),低剂量组MMP-13基因表达明显增高(P <0.05),高、中剂量组TIMP-1基因表达明显降低(P <0.05),益气逐瘀解毒颗粒各剂量组MMP-2、MMP-9蛋白表达量无显著性差异(P> 0.05),高、中剂量组MMP-13蛋白表达量显著升高(P <0.05),各剂量组TIMP-1蛋白表达量均显著下降(P <0.05),以低剂量组下降尤为显著,优于高、中剂量组(P <0.05),各药物干预组SSS评分显著降低(P <0.01);与阳性对照组比较,益气逐瘀解毒颗粒各剂量组ALT、AST无显著性差异(P> 0.05),益气逐瘀解毒颗粒高剂量组MMP-2基因表达明显升高(P <0.05),高、中剂量组TIMP-1基因表达明显降低(P <0.05),益气逐瘀解毒颗粒高、中剂量组MMP-13蛋白表达显著升高(P <0.05),益气逐瘀解毒颗粒各剂量组TIMP-1蛋白表达显著降低(P <0.05),低剂量组下降尤为显著,优于高、中剂量组(P <0.05),益气逐瘀解毒颗粒高、中、低剂量组SSS评分显著降低(P <0.01)。结论:调控MMPs/TIMPs平衡可能是益气逐瘀解毒颗粒抗肝维化的作用机制之一,其通过升高MMP-13、降低TIMP-1表达水平,调控MMPs/TIMPs平衡,促进过度沉积的细胞外基质(Extracelluar matrix,ECM)降解,改善CCl4诱导肝纤维化大鼠模型的肝脏功能,从而改善其肝纤维化程度。
Objective: To observe the clinical effect of Yiqi Zhuyu Jiedu granule for rats with liver fibrosis induced by carbon tetrachloride(CCl4), and its functional mechanism. Methods: The model of SD male rats with liver fibrosis was established by subcutaneous injection of the mixture of CCl4 and olive oil. The rats were divided into the groups of Yiqi Zhuyu Jiedu granule in high,middle and low doses, the positive control group, the model group and the blank group randomly, which received treatment accordingly. After 5 weeks, the rats were sacrificed. Detected biochemical indexes in the peripheral blood, measured the expression levels of matrix metalloproteinase(MMP)-1,MMP-2,MMP-9,MMP-13 and tissue inhibitor of metalloproteinases 1(TIMP-1)in the liver tissue of rats respectively via reverse transcription PCR and Western blot,and detected the degree of liver fibrosis in the liver tissue via hematoxylin-eosin staining(HE)and MASSON staining. Results: After treatment, comparing with the blank group, there was nosignificant difference being found in ALT and AST in the model group(P > 0.05), the gene expression levels of MMP-1, MMP-2,MMP-9 and MMP-13 were significantly decreased(P < 0.05),the gene expression level of TIMP-1 was significantly increased(P < 0.05),and the scores of Semiquantative scorning system(SSS)for liver fibrosis were significantly increased(P < 0.01). Comparing with the model group,there was no significant difference being found in ALT and AST in the groups of Yiqi Zhuyu Jiedu granule in each dose(P > 0.05),the gene expression levels of MMP-1,MMP-2, MMP-9 and MMP-13 in the high-dose Yiqi Zhuyu Jiedu granule group were significantly increased(P < 0.05),the gene expression levels of MMP-9 and MMP-13 in the middle-dose Yiqi Zhuyu Jiedu granule group were significantly increased(P < 0.05),the gene expression level of MMP-13 in the low-dose Yiqi Zhuyu Jiedu granule group was significantly increased(P < 0.05),and the gene expression level of TIMP-1 in the groups of Yiqi Zhuyu Jiedu granule in high and middle doses was significantly decreased(P < 0.05);there was no significant difference being found in the protein expression levels of MMP-2 and MMP-9 in the groups of Yiqi Zhuyu Jiedu granule in each dose(P > 0.05),the protein expression level of MMP-13 in the groups of Yiqi Zhuyu Jiedu granule in high and middle doses was significantly increased(P < 0.05),the protein expression level of TIMP-1 in the groups of Yiqi Zhuyu Jiedu granule in each dose was significantly decreased(P < 0.05),and the protein expression level of TIMP-1 in the low-dose Yiqi Zhuyu Jiedu granule group was decreased particularly significantly, being better than that in the groups of Yiqi Zhuyu Jiedu granule in high and middle doses(P < 0.05);the scores of SSS in each medicine intervention group were significantly decreased(P<0.01). Comparing with the positive control group,there was no significant difference being found in ALT and AST in the groups of Yiqi Zhuyu Jiedu granule in each dose(P > 0.05),the gene expression level of MMP-2 in the high-dose Yiqi Zhuyu Jiedu granule group was significantly increased(P < 0.05),the gene expression level of MMP-1 in the groups of Yiqi Zhuyu Jiedu granule in high and middle doses was significantly decreased(P < 0.05);the protein expression level of MMP-13 in the groups of Yiqi Zhuyu Jiedu granule in high and middle doses was significantly increased(P < 0.05),the gene expression level of TIMP-1 in the groups of Yiqi Zhuyu Jiedu granule in each dose was significantly decreased(P < 0.05),and the protein expression level of TIMP-1 in the low-dose Yiqi Zhuyu Jiedu granule group was decreased particularly significantly,being better than that in the groups of Yiqi Zhuyu Jiedu granule in high and middle doses(P < 0.05); the scores of SSS in the groups of Yiqi Zhuyu Jiedu granule in each dose were significantly decreased(P < 0.01).Conclusion: One of the functional mechanism of Yiqi Zhuyu Jiedu granule may be regulating the balance of MMPs/TIMPs by increasing the level of MMP-13 and decreasing the expression level of TIMP-1. It promotes the degradation of excessively deposited extracelluar matrix(ECM)for rats with liver fibrosis induced by carbon tetrachloride(CCl4),and promote liver functions of the model of rats with liver fibrosis induced by CCl4,thus to improve the degree of liver fibrosis.
Objective: To observe the clinical effect of Yiqi Zhuyu Jiedu granule for rats with liver fibrosis induced by carbon tetrachloride(CCl4), and its functional mechanism. Methods: The model of SD male rats with liver fibrosis was established by subcutaneous injection of the mixture of CCl4 and olive oil. The rats were divided into the groups of Yiqi Zhuyu Jiedu granule in high,middle and low doses, the positive control group, the model group and the blank group randomly, which received treatment accordingly. After 5 weeks, the rats were sacrificed. Detected biochemical indexes in the peripheral blood, measured the expression levels of matrix metalloproteinase(MMP)-1,MMP-2,MMP-9,MMP-13 and tissue inhibitor of metalloproteinases 1(TIMP-1)in the liver tissue of rats respectively via reverse transcription PCR and Western blot,and detected the degree of liver fibrosis in the liver tissue via hematoxylin-eosin staining(HE)and MASSON staining. Results: After treatment, comparing with the blank group, there was nosignificant difference being found in ALT and AST in the model group(P > 0.05), the gene expression levels of MMP-1, MMP-2,MMP-9 and MMP-13 were significantly decreased(P < 0.05),the gene expression level of TIMP-1 was significantly increased(P < 0.05),and the scores of Semiquantative scorning system(SSS)for liver fibrosis were significantly increased(P < 0.01). Comparing with the model group,there was no significant difference being found in ALT and AST in the groups of Yiqi Zhuyu Jiedu granule in each dose(P > 0.05),the gene expression levels of MMP-1,MMP-2, MMP-9 and MMP-13 in the high-dose Yiqi Zhuyu Jiedu granule group were significantly increased(P < 0.05),the gene expression levels of MMP-9 and MMP-13 in the middle-dose Yiqi Zhuyu Jiedu granule group were significantly increased(P < 0.05),the gene expression level of MMP-13 in the low-dose Yiqi Zhuyu Jiedu granule group was significantly increased(P < 0.05),and the gene expression level of TIMP-1 in the groups of Yiqi Zhuyu Jiedu granule in high and middle doses was significantly decreased(P < 0.05);there was no significant difference being found in the protein expression levels of MMP-2 and MMP-9 in the groups of Yiqi Zhuyu Jiedu granule in each dose(P > 0.05),the protein expression level of MMP-13 in the groups of Yiqi Zhuyu Jiedu granule in high and middle doses was significantly increased(P < 0.05),the protein expression level of TIMP-1 in the groups of Yiqi Zhuyu Jiedu granule in each dose was significantly decreased(P < 0.05),and the protein expression level of TIMP-1 in the low-dose Yiqi Zhuyu Jiedu granule group was decreased particularly significantly, being better than that in the groups of Yiqi Zhuyu Jiedu granule in high and middle doses(P < 0.05);the scores of SSS in each medicine intervention group were significantly decreased(P<0.01). Comparing with the positive control group,there was no significant difference being found in ALT and AST in the groups of Yiqi Zhuyu Jiedu granule in each dose(P > 0.05),the gene expression level of MMP-2 in the high-dose Yiqi Zhuyu Jiedu granule group was significantly increased(P < 0.05),the gene expression level of MMP-1 in the groups of Yiqi Zhuyu Jiedu granule in high and middle doses was significantly decreased(P < 0.05);the protein expression level of MMP-13 in the groups of Yiqi Zhuyu Jiedu granule in high and middle doses was significantly increased(P < 0.05),the gene expression level of TIMP-1 in the groups of Yiqi Zhuyu Jiedu granule in each dose was significantly decreased(P < 0.05),and the protein expression level of TIMP-1 in the low-dose Yiqi Zhuyu Jiedu granule group was decreased particularly significantly,being better than that in the groups of Yiqi Zhuyu Jiedu granule in high and middle doses(P < 0.05); the scores of SSS in the groups of Yiqi Zhuyu Jiedu granule in each dose were significantly decreased(P < 0.01).Conclusion: One of the functional mechanism of Yiqi Zhuyu Jiedu granule may be regulating the balance of MMPs/TIMPs by increasing the level of MMP-13 and decreasing the expression level of TIMP-1. It promotes the degradation of excessively deposited extracelluar matrix(ECM)for rats with liver fibrosis induced by carbon tetrachloride(CCl4),and promote liver functions of the model of rats with liver fibrosis induced by CCl4,thus to improve the degree of liver fibrosis.
引文
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[5]叶放,薛博瑜,吴勉华,等.重视对慢性肝炎肝纤维化进程中湿热瘀毒证治研究[J].中华中医药学刊,2007,25(12):2477-2480.
[6]ROEB E.Matrix metalloproteinases and liver fibrosis(translational aspects)[J].Matrix Biol,2018,68-69:463-473.
[7]谭勤锐,李晖,杨琪,等.基质金属蛋白酶及其特定抑制剂在肝纤维化形成中的作用研究进展[J].山东医药,2017,57(34):109-111.
[8]ROBERT S,GICQUEL T,VICTONI T,et al.Involvement of matrix metalloproteinases(MMPs)and inflammasome pathway in molecular mechanisms of fibrosis[J].Biosci Rep,2016,36(4):e00360.
[9]DUARTE S,BABER J,FUJII T,et al.Matrix metalloproteinases in liver injury,repair and fibrosis[J].Matrix Biol,2015,44-46:147-156.
[10]GIANNANDREA M,Parks W C.Diverse functions of matrix metalloproteinases during fibrosis[J].Dis Model Mech,2014,7(2):193-203.
[11]HAN Y P.Matrix metalloproteinases,the pros and cons,in liver fibrosis[J].J Gastroenterol Hepatol,2006,21(Suppl.3):S88-S91.
[2]叶放,薛博瑜,周珉,等.论湿热瘀毒与肝纤维化[J].南京中医药大学学报,2005,21(6):346-349.
[3]吕嫒嫒,薛博瑜.关幼波治疗慢性肝病经验[J].河南中医,2013,33(4):521-522.
[4]戴克敏.姜春华治疗肝病验方[J].山西中医,2007,23(6):10-11.
[5]叶放,薛博瑜,吴勉华,等.重视对慢性肝炎肝纤维化进程中湿热瘀毒证治研究[J].中华中医药学刊,2007,25(12):2477-2480.
[6]ROEB E.Matrix metalloproteinases and liver fibrosis(translational aspects)[J].Matrix Biol,2018,68-69:463-473.
[7]谭勤锐,李晖,杨琪,等.基质金属蛋白酶及其特定抑制剂在肝纤维化形成中的作用研究进展[J].山东医药,2017,57(34):109-111.
[8]ROBERT S,GICQUEL T,VICTONI T,et al.Involvement of matrix metalloproteinases(MMPs)and inflammasome pathway in molecular mechanisms of fibrosis[J].Biosci Rep,2016,36(4):e00360.
[9]DUARTE S,BABER J,FUJII T,et al.Matrix metalloproteinases in liver injury,repair and fibrosis[J].Matrix Biol,2015,44-46:147-156.
[10]GIANNANDREA M,Parks W C.Diverse functions of matrix metalloproteinases during fibrosis[J].Dis Model Mech,2014,7(2):193-203.
[11]HAN Y P.Matrix metalloproteinases,the pros and cons,in liver fibrosis[J].J Gastroenterol Hepatol,2006,21(Suppl.3):S88-S91.