加减三甲散及其虫类药对肝纤维化大鼠TGF-β/Smads信号通路及MMPs/TIMPs体系影响的研究
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摘要
目的：观察加减三甲散及其虫类药对实验性肝纤维化肝组织TGF-β/Smad转导通路及MMPs/TIMPs的影响,探讨其抗肝纤维化的效果和作用机理。
方法：选用SD雄性大鼠,设正常对照组,模型组,阳性组,加减三甲散组,虫药组,去虫药组等共6组。除正常对照组外,其余五组采用40%四氯化碳(CCL4)植物油溶液腹腔注射制备肝纤维化模型,造模的同时予药物灌胃治疗。8周后,处死各组大鼠,腹主动脉取血,放射免疫法检测层粘蛋白(LN)、Ⅳ型胶原(Ⅳ-C)等肝纤维化血清学指标；光镜和电镜观察肝组织病理变化；免疫组化法检测肝组织TGF-β1、TNF-α、Smad2/3、Smad7,原位杂交法检测肝组织MMP-2、TIMP-2、TIMP-1并作图像分析。
结果：
1. TGF-β/Smad转导通路：
TGF-β1的表达：正常对照组几乎不表达TGF-β1,模型组表达增高(P<0.01),与模型组相比,加减三甲散组、阳性组、虫药组、去虫药组表达降低(P<0.01),与阳性组相比,加减三甲散组有差异(P<0.05),虫药组、去虫药组无差异性。
Smad2/3的表达：正常对照组几乎不表达Smad2/3,模型组表达增高(P<0.01),与模型组比较,加减三甲散组、阳性组、虫药组、去虫药组表达降低(P<0.01),与阳性组比较,加减三甲散组、虫药组表达降低(P<0.01,P<0.05)。
Smad7的表达：与正常对照组比较,模型组明显降低(P<0.01),与模型组比较,加减三甲散组、虫药组、去虫药组Smad7的表达明显升高(P<0.01),与阳性组比较,加减三甲散组(P<0.01)、虫药组、去虫药组(P<0.05)均升高。
TNF-α的表达：与正常对照组比较,模型组TNF-α的表达明显升高(P<0.01),与模型组相比,阳性组、加减三甲散组、虫药组、去虫药组表达明显降低(P<0.01),与阳性组相比,加减三甲散组有差异(P<0.05)。
2.MMPs/TIMPs体系：
MMP-2mRNA的表达：与正常对照组相比,模型组无差异,虫药组表达升高(P<0.05),与模型组相比,虫药组表达升高(P<0.05),加减三甲散、阳性组、去虫药组无差异(P>0.05)。
TIMP-2mRNA的表达：与正常对照组相比,模型组表达明显升高(P<0.01),与模型组相比,各治疗组表达降低(P<0.01),且与阳性组比较,加减三甲散与虫药组亦有降低且加减三甲散降低明显(P<0.01),虫药组(P<0.05),去虫药组无差异(P>0.05)。
TIMP-1mRNA的表达：与正常对照组相比,模型组TIMP-1mRNA的表达明显升高P<0.01),与模型组相比,各治疗组的表达均有降低(P<0.01),且加减三甲散与虫药组低于阳性组(P<0.05),去虫药组无差异(P>0.05)。
结论：
1.加减三甲散可显著改善肝纤维化大鼠肝组织肝纤维化程度。
2.加减三甲散能降低肝纤维化大鼠肝组织TGF-β1、Smad2/3的表达,升高Smad7的表达,且效果优于秋水仙碱。通过抑制TGF-β/Smad转导通路,达到抗纤维化的效果。
3.在降低TNF-α的表达方面,加减三甲散优于秋水仙碱。
4.加减三甲散可降低肝纤维化大鼠肝组织TIMP-1mRNA、TIMP-2mRNA的表达,从而抑制MMP-2降解ECM,达到抗纤维化的效果。
5.虫药组在降低纤维化大鼠肝组织TIMP-1mRNA的表达方面效同加减三甲散(均为P<0.05)。在降低纤维化大鼠肝组织TIMP-2mRNA的表达方面,加减三甲散效果明显,优于秋水仙碱组、虫药组、去虫药组。虫药组效果也优于秋水仙碱组。去虫药组效果与秋水仙碱组无差异。
Objective:To obsreve the effect of SJS and its parts on transforming growth factor beta/smad pathway(TGF-β/Smad),tumor necrosis factor alpha(TNF-α) and matrix metalloproteinases/tissue inhibitor of metalioproteinases(MMP/TIMP) of hepatic tissue of hepatic fibrosis rats and to discuss the curative and mechanism of jiajiansanjiasanfang on the treatment of hepatic fibrosis.
Methods:Male SD rats were randomized into 6 groups:normal control group, model group, positive control group, JJSJS group, insect drugs group and expel insect drugs group. Except the rats in the normal group, others were inflicted with hepatic fibrosis by intraperitoneal injection of 40% carbon tetrachloride, and were given medication at the same time. After eight weeks, all the rats were excuted and the hepatic tissues were kept for pathologic examination to observe hepatic fibrosis and immunohistochemical to detect TGF-β1, Smad2/3, Smad7 and TNF-α, and in situ hybridization to detect MMP-2, TIMP-2, TIMP-1.
Results:
1.TGF-β/Smad pathway:Expression of TGF-β1 in hepatic tissue:TGF-β1 expression scarcely appeared in the hepatic tissues of rats in the normal group, but increased visibly in rat liver of the model group(P<0.01).Compared with the model group, their expression decreased in rat liver of the positive control group(P<0.05) and the JJSJS group(P<0.01),but the expression of insect drugs and expel insect drugs groups increased.
Expression of Smad2/3 in hepatic tissue:Smad2/3 expression scarcely appeared in the hepatic tissues of rats in the normal group, but increased visibly in rat liver of the model group(P< 0.01).Compared with the model group,their expression decreased in rat liver of the positive control group and the JJSJS group(P<0.01).Compared with the positive control group, the JJSJS group is more lower(P<0.05). but the expression of insect drugs and expel insect drugs groups increased.
Expression of Smad7 in hepatic tissue:Compared with the normal group, Smad7 expression decreased visibly in the hepatic tissues of rats in the model group. Compared with the model group, the expression increased visibly in the JJSJS one(P<0.01),but didn't have difference with the positive control one. Compared with the JJSJS group, their expression increased visibly in rat liver of the insect drugs and expel insect drugs groups(P<0.01).
Expression of TNF-αin hepatic tissue:TNF-αexpression scarcely appeared in the hepatic tissues of rats in the normal group, but increased visibly in rat liver of the model group(P< 0.01).Compared with the model group,their expression decreased in rat liver of the positive control group and the JJSJS group(P<0.01).But the expression of insect drugs and expel insect drugs groups increased.
2.MMP/TIMP System:Expression of MMP-2mRNA in hepatic tissue; Compared with the normal group, expression of MMP-2mRNA in hepatic tissue didn't have difference with it in the model group, and the expression increased in the insect drugs group(P<0.05).Compared with the model group, the expression increased in the insect drugs group (P<0.05)while there was not different in the others.
Expression of TIMP-2mRNA in hepatic tissue:Compared with the normal group, expression of MMP-2mRNA in hepatic tissue increased visibly in the model group(P<0.01).Compared with the model group, the expression decreased in the other groups(P<0.01).Compared with the positive control group, the expression decreased in the JJSJS group(P<0.01) and the insect drugs groups(P<0.05),and lower in the JJSJS group.
Expression of TIMP-1 mRNA in hepatic tissue:Compared with the normal group, the expression of TIMP-1 mRNA in hepatic tissue increased visibly in the model group(P< 0.01).Compared with the model group the expression all decreased in the other groups(P< 0.01),and compared with the positive control group, the expression decreased in the JJSJS and the insect drugs group(P<0.05).
Conclusions:
1. JJSJS reduces the fibrotic degree of rats hepatic tissue significantly which means that this formular has powerful anti-fibrosis effect.
2. JJSJS inhibits expression of TGF-β1 and Smad2/3 in hepatic tissues and increases the expression of Smad7.It is better than the colchicines.It has powerful anti-fibrotic effect through inhibiting the TGF-β/Smad pathway.
3. There isn't different between the JJSJS and the colchicines in decressing the expression of TNF-a in hepatic tissue in the fibrosis rats.
4. JJSJS decresses the expression of TIMP-lmRNA,TIMP-2mRNA and MMP-2mRNA in hepatic tissue in the fibrosis rats, so it suppresses ECM's degradation process by decresses the expression of MMP-2mRNA.
5. It has not difference between the JJSJS and the insect drugs group in decressing the expression of TIMP-lmRNA.
方法：选用SD雄性大鼠,设正常对照组,模型组,阳性组,加减三甲散组,虫药组,去虫药组等共6组。除正常对照组外,其余五组采用40%四氯化碳(CCL4)植物油溶液腹腔注射制备肝纤维化模型,造模的同时予药物灌胃治疗。8周后,处死各组大鼠,腹主动脉取血,放射免疫法检测层粘蛋白(LN)、Ⅳ型胶原(Ⅳ-C)等肝纤维化血清学指标；光镜和电镜观察肝组织病理变化；免疫组化法检测肝组织TGF-β1、TNF-α、Smad2/3、Smad7,原位杂交法检测肝组织MMP-2、TIMP-2、TIMP-1并作图像分析。
结果：
1. TGF-β/Smad转导通路：
TGF-β1的表达：正常对照组几乎不表达TGF-β1,模型组表达增高(P<0.01),与模型组相比,加减三甲散组、阳性组、虫药组、去虫药组表达降低(P<0.01),与阳性组相比,加减三甲散组有差异(P<0.05),虫药组、去虫药组无差异性。
Smad2/3的表达：正常对照组几乎不表达Smad2/3,模型组表达增高(P<0.01),与模型组比较,加减三甲散组、阳性组、虫药组、去虫药组表达降低(P<0.01),与阳性组比较,加减三甲散组、虫药组表达降低(P<0.01,P<0.05)。
Smad7的表达：与正常对照组比较,模型组明显降低(P<0.01),与模型组比较,加减三甲散组、虫药组、去虫药组Smad7的表达明显升高(P<0.01),与阳性组比较,加减三甲散组(P<0.01)、虫药组、去虫药组(P<0.05)均升高。
TNF-α的表达：与正常对照组比较,模型组TNF-α的表达明显升高(P<0.01),与模型组相比,阳性组、加减三甲散组、虫药组、去虫药组表达明显降低(P<0.01),与阳性组相比,加减三甲散组有差异(P<0.05)。
2.MMPs/TIMPs体系：
MMP-2mRNA的表达：与正常对照组相比,模型组无差异,虫药组表达升高(P<0.05),与模型组相比,虫药组表达升高(P<0.05),加减三甲散、阳性组、去虫药组无差异(P>0.05)。
TIMP-2mRNA的表达：与正常对照组相比,模型组表达明显升高(P<0.01),与模型组相比,各治疗组表达降低(P<0.01),且与阳性组比较,加减三甲散与虫药组亦有降低且加减三甲散降低明显(P<0.01),虫药组(P<0.05),去虫药组无差异(P>0.05)。
TIMP-1mRNA的表达：与正常对照组相比,模型组TIMP-1mRNA的表达明显升高P<0.01),与模型组相比,各治疗组的表达均有降低(P<0.01),且加减三甲散与虫药组低于阳性组(P<0.05),去虫药组无差异(P>0.05)。
结论：
1.加减三甲散可显著改善肝纤维化大鼠肝组织肝纤维化程度。
2.加减三甲散能降低肝纤维化大鼠肝组织TGF-β1、Smad2/3的表达,升高Smad7的表达,且效果优于秋水仙碱。通过抑制TGF-β/Smad转导通路,达到抗纤维化的效果。
3.在降低TNF-α的表达方面,加减三甲散优于秋水仙碱。
4.加减三甲散可降低肝纤维化大鼠肝组织TIMP-1mRNA、TIMP-2mRNA的表达,从而抑制MMP-2降解ECM,达到抗纤维化的效果。
5.虫药组在降低纤维化大鼠肝组织TIMP-1mRNA的表达方面效同加减三甲散(均为P<0.05)。在降低纤维化大鼠肝组织TIMP-2mRNA的表达方面,加减三甲散效果明显,优于秋水仙碱组、虫药组、去虫药组。虫药组效果也优于秋水仙碱组。去虫药组效果与秋水仙碱组无差异。
Objective:To obsreve the effect of SJS and its parts on transforming growth factor beta/smad pathway(TGF-β/Smad),tumor necrosis factor alpha(TNF-α) and matrix metalloproteinases/tissue inhibitor of metalioproteinases(MMP/TIMP) of hepatic tissue of hepatic fibrosis rats and to discuss the curative and mechanism of jiajiansanjiasanfang on the treatment of hepatic fibrosis.
Methods:Male SD rats were randomized into 6 groups:normal control group, model group, positive control group, JJSJS group, insect drugs group and expel insect drugs group. Except the rats in the normal group, others were inflicted with hepatic fibrosis by intraperitoneal injection of 40% carbon tetrachloride, and were given medication at the same time. After eight weeks, all the rats were excuted and the hepatic tissues were kept for pathologic examination to observe hepatic fibrosis and immunohistochemical to detect TGF-β1, Smad2/3, Smad7 and TNF-α, and in situ hybridization to detect MMP-2, TIMP-2, TIMP-1.
Results:
1.TGF-β/Smad pathway:Expression of TGF-β1 in hepatic tissue:TGF-β1 expression scarcely appeared in the hepatic tissues of rats in the normal group, but increased visibly in rat liver of the model group(P<0.01).Compared with the model group, their expression decreased in rat liver of the positive control group(P<0.05) and the JJSJS group(P<0.01),but the expression of insect drugs and expel insect drugs groups increased.
Expression of Smad2/3 in hepatic tissue:Smad2/3 expression scarcely appeared in the hepatic tissues of rats in the normal group, but increased visibly in rat liver of the model group(P< 0.01).Compared with the model group,their expression decreased in rat liver of the positive control group and the JJSJS group(P<0.01).Compared with the positive control group, the JJSJS group is more lower(P<0.05). but the expression of insect drugs and expel insect drugs groups increased.
Expression of Smad7 in hepatic tissue:Compared with the normal group, Smad7 expression decreased visibly in the hepatic tissues of rats in the model group. Compared with the model group, the expression increased visibly in the JJSJS one(P<0.01),but didn't have difference with the positive control one. Compared with the JJSJS group, their expression increased visibly in rat liver of the insect drugs and expel insect drugs groups(P<0.01).
Expression of TNF-αin hepatic tissue:TNF-αexpression scarcely appeared in the hepatic tissues of rats in the normal group, but increased visibly in rat liver of the model group(P< 0.01).Compared with the model group,their expression decreased in rat liver of the positive control group and the JJSJS group(P<0.01).But the expression of insect drugs and expel insect drugs groups increased.
2.MMP/TIMP System:Expression of MMP-2mRNA in hepatic tissue; Compared with the normal group, expression of MMP-2mRNA in hepatic tissue didn't have difference with it in the model group, and the expression increased in the insect drugs group(P<0.05).Compared with the model group, the expression increased in the insect drugs group (P<0.05)while there was not different in the others.
Expression of TIMP-2mRNA in hepatic tissue:Compared with the normal group, expression of MMP-2mRNA in hepatic tissue increased visibly in the model group(P<0.01).Compared with the model group, the expression decreased in the other groups(P<0.01).Compared with the positive control group, the expression decreased in the JJSJS group(P<0.01) and the insect drugs groups(P<0.05),and lower in the JJSJS group.
Expression of TIMP-1 mRNA in hepatic tissue:Compared with the normal group, the expression of TIMP-1 mRNA in hepatic tissue increased visibly in the model group(P< 0.01).Compared with the model group the expression all decreased in the other groups(P< 0.01),and compared with the positive control group, the expression decreased in the JJSJS and the insect drugs group(P<0.05).
Conclusions:
1. JJSJS reduces the fibrotic degree of rats hepatic tissue significantly which means that this formular has powerful anti-fibrosis effect.
2. JJSJS inhibits expression of TGF-β1 and Smad2/3 in hepatic tissues and increases the expression of Smad7.It is better than the colchicines.It has powerful anti-fibrotic effect through inhibiting the TGF-β/Smad pathway.
3. There isn't different between the JJSJS and the colchicines in decressing the expression of TNF-a in hepatic tissue in the fibrosis rats.
4. JJSJS decresses the expression of TIMP-lmRNA,TIMP-2mRNA and MMP-2mRNA in hepatic tissue in the fibrosis rats, so it suppresses ECM's degradation process by decresses the expression of MMP-2mRNA.
5. It has not difference between the JJSJS and the insect drugs group in decressing the expression of TIMP-lmRNA.
引文
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