田七注射液对大鼠肾纤维化的作用及TGF-β1/Smads信号转导机制的研究
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摘要
目的：
1.研究田七注射液对阿霉素肾纤维化大鼠模型肾脏结构和功能、细胞外基质成分等的影响,明确田七注射液抗肾纤维化的作用。
2.从TGF-β1/Smads信号转导通路研究田七注射液抗大鼠阿霉素性肾纤维化的作用机制。
方法：
1.采用切除右肾、尾静脉注射阿霉素(ADR)的方法建立阿霉素性肾纤维化大鼠模型。
2.将健康雄性SD大鼠48只随机分为正常组、假手术组、模型组和田七注射液组。正常组大鼠正常条件饲养,不造模,予以生理盐水(10mL/kg·d)腹腔注射；假手术组大鼠切除右肾后,不注射ADR,予以生理盐水(10mL/kg·d)腹腔注射；模型组大鼠按上法造模后,予以生理盐水(10mL/kg·d)腹腔注射；田七注射液组大鼠按上法造模后,予以田七注射液(2g生药/kg.d)腹腔注射给药。4组大鼠均连续给药8周后处死,收集各组大鼠的尿液、血液和肾组织。检测各组大鼠24h尿蛋白定量和肾功能(BUN和Scr),苏木素伊红(HE)和马松(Masson)染色检测大鼠肾组织病理情况并半定量评分。RT-PCR、免疫组织化学法和Western Blot法检测各组大鼠肾组织Ⅳ型胶原(CL-Ⅳ)、a-平滑肌肌动蛋白(α-SMA)、转化生长因子-β1(TGF-β1)、转化生长因子βⅠ型受体(TGF-βR-Ⅰ)、转化生长因子βⅡ型受体(TGF-βR-Ⅱ)、磷酸化Smad2(p-Smad2)、磷酸化Smad7(p-Smad7)mRNA和／或蛋白的表达。
结果：
1.切除右肾静注ADR后,大鼠尿蛋白排出增加(P<0.01),肾功能BUN和Scr水平升高(P<0.01),肾组织出现肾小球硬化、肾小管萎缩、间质纤维化、炎症细胞浸润等病理改变,提示肾纤维化大鼠模型建立成功。
2.与假手术组比较,模型组大鼠尿蛋白排出增加,BUN和Scr水平升高(P<0.01),肾小管CL-Ⅳ和α-SMA表达增多(P<0.01),提示肾纤维化大鼠存在小管上皮-间质细胞转分化(EMT)过程。与模型组比较,经田七注射液处理后,大鼠尿蛋白排出减少(P<0.01),肾功能BUN和Scr水平下降P<0.01),肾小球硬化、肾小管萎缩、间质纤维化、炎症细胞浸润等病理改变减轻,肾小球硬化面积及肾小管间质纤维化半定量评分均显著下降(P<0.01);肾小管CL-Ⅳ和α-SMA表达下调(P<0.01),提示田七注射液具有抗肾纤维化的作用,其机制可能与影响EMT过程有关。
3.与假手术组比较,阿霉素肾纤维化大鼠肾组织TGF-β1、TGF-βR-Ⅰ、TGF-βR-Ⅱ、p-Smad2、p-Smad7 mRNA和/或蛋白表达明著显上调(P<0.01),提示阿霉素肾纤维化大鼠肾组织TGF-β1/Smads信号通路活化。经田七注射液处理后,阿霉素肾纤维化大鼠肾组织TGF-β1、TGF-βR-Ⅰ、TGF-βR-Ⅱ、p-Smad2 mRNA和/或蛋白表达显著下调(P<0.01),但p-Smad7蛋白表达显著上调(P<0.01),提示田七注射液的抗肾纤维化作用可能与抑制肾组织TGF-β1/Smads信号通路活化有关。
结论：
1.阿霉素肾纤维化大鼠肾组织TGF-β1/Smads信号通路活化,参与肾小管上皮-间质细胞转分化过程。
2.田七注射液具有抗肾纤维化和改善肾功能的作用,其机制可能与抑制肾组织TGF-β1/Smads信号通路活化介导的肾小管上皮-间质细胞转分化过程有关。
Objective:
1. To observe the effect of TianQi injection on the structure, fuction and Extracellular matrix of kidney, confirming the anti-fibrosis role of TianQi injection.
2. To investigate the anti-fibrosis mechanism of TianQi injection on the TGF-β1/Smads signaling conduction.
Methods:
1. The adriamycin-induced kidney fibrosis models were established by ways of excising right kidney and injecting Adriamycin into vena caudalis.
2. The 48 healthy male Sprague-Dawley (SD) rats were randomly divided into four groups:normal group, sham group, model group, TianQi injection treatment group. The rats in the normal group feed under normal condition without experienceing the operation were dealed with normal sodium(NS) (10 ml/kg·d) by intraperitoneal injection; The rats in the sham group after remove right kidney were dealed with normal sodium(NS) (10 ml/kg·d) by intraperitoneal injection without receiving adriamycin;The rats in the model group received NS (10 ml/kg·d) by intraperitoneal injection after operation; The rats in the TianQi injection treatment group received TianQi injection (10 ml/kg·d) by intraperitoneal injection after operation; All the rats were sacrificed after receiving treatments for 4 weeks, respectively, and the urine, blood and kidneys were collected.24h quantity of urinary protein and kidney function were detected; Kidney histological examinations was performed by HE and Masson staining, and the degree of tubular-interstitial damage was valuated in terms of semi-quantitative score. The mRNA and/or protein expression of TGF-β1, TGF-βR-Ⅰ, TGF-βR-Ⅱ, p-Smad2 anc p-Smad7 were detected by means of reverse transcription-Polymerase chair reaction (RT-PCR), inununohistochemistry and Western blot.
Results:
1. After excising right kidney and injecting Adriamycin into vena caudalis 24h quantity of urinary protein in rats was increased (P<0.01), and the BUN and Scr level in serum sample was enhanced (P<0.01), and the kideny pathology such as glomcrulus sclerosis, tubular atrophy, interstitial fibrosis and inflammation cells infiltration were found in rats, it suggests a successful kidney fibrosis model.
2. When compared with sham group, the urinary protein in model group rats was increased (P<0.01), and the BUN and Scr level in serum sample was enhanced (P<0.01). The protein expression of CL-IV and a-SMA in rats model witl Adriamycin-induced kidney fibrosis were increased when compared with sham group (P<0.01), which suggests an EMT course occuring in kidney fibrosis rats TianQi injection treatment could reduce the quantity of urinary protein (P<0.01) lower the BUN and Scr level(P<0.01), ameliorate the pathology in kidney such a glomcrulus sclerosis, tubular atrophy, interstitial fibrosis and inflammation cells infiltration, reduce the scores of glomcrulus sclerosis area and interstitia fibrosis(P<0.01),down-regulate the protein expression of collagen type IV(P<0.01 when compared with model group, which indicates that TianQi injection plays anti-kidney fibrosis role, and the mechanism is involved in influencing the course o EMT.
3. The mRNA and/or protein expression of TGF-β1, TGF-βtypeⅠreceptor, TGF-βtypeⅡreceptor, p-Smad2 and p-Smad7 in Adriamycin-induced kidney fibrosis models were up-regulated when compared with sham group (P<0.01),which suggests that the TGF-β1/Smads signaling is activated. When compared with model group, TianQi injection treatment could significantly down-regulate the mRNA and/or protein expression of TGF-β1, TGF-βtypeⅠreceptor, TGF-P typeⅡreceptor, p-Smad2 (P<0.01), and up-regulate protein expression of p-Smad7 (P<0.01), which indicates that anti-kidney fibarosis role of TianQi injection is associated with repressing the activation of TGF-β1/Smads signaling.
Conclusion:
1. The activation of TGF-β1/Smads signaling in the adriamycin-induced kidney fibrosis models is involved in the course of EMT in the tubular.
2. TianQi injection plays a anti-fibrosis role that is perhaps associated with repressing TGF-β1/Smads signaling mediated tubular EMT.
1.研究田七注射液对阿霉素肾纤维化大鼠模型肾脏结构和功能、细胞外基质成分等的影响,明确田七注射液抗肾纤维化的作用。
2.从TGF-β1/Smads信号转导通路研究田七注射液抗大鼠阿霉素性肾纤维化的作用机制。
方法：
1.采用切除右肾、尾静脉注射阿霉素(ADR)的方法建立阿霉素性肾纤维化大鼠模型。
2.将健康雄性SD大鼠48只随机分为正常组、假手术组、模型组和田七注射液组。正常组大鼠正常条件饲养,不造模,予以生理盐水(10mL/kg·d)腹腔注射；假手术组大鼠切除右肾后,不注射ADR,予以生理盐水(10mL/kg·d)腹腔注射；模型组大鼠按上法造模后,予以生理盐水(10mL/kg·d)腹腔注射；田七注射液组大鼠按上法造模后,予以田七注射液(2g生药/kg.d)腹腔注射给药。4组大鼠均连续给药8周后处死,收集各组大鼠的尿液、血液和肾组织。检测各组大鼠24h尿蛋白定量和肾功能(BUN和Scr),苏木素伊红(HE)和马松(Masson)染色检测大鼠肾组织病理情况并半定量评分。RT-PCR、免疫组织化学法和Western Blot法检测各组大鼠肾组织Ⅳ型胶原(CL-Ⅳ)、a-平滑肌肌动蛋白(α-SMA)、转化生长因子-β1(TGF-β1)、转化生长因子βⅠ型受体(TGF-βR-Ⅰ)、转化生长因子βⅡ型受体(TGF-βR-Ⅱ)、磷酸化Smad2(p-Smad2)、磷酸化Smad7(p-Smad7)mRNA和／或蛋白的表达。
结果：
1.切除右肾静注ADR后,大鼠尿蛋白排出增加(P<0.01),肾功能BUN和Scr水平升高(P<0.01),肾组织出现肾小球硬化、肾小管萎缩、间质纤维化、炎症细胞浸润等病理改变,提示肾纤维化大鼠模型建立成功。
2.与假手术组比较,模型组大鼠尿蛋白排出增加,BUN和Scr水平升高(P<0.01),肾小管CL-Ⅳ和α-SMA表达增多(P<0.01),提示肾纤维化大鼠存在小管上皮-间质细胞转分化(EMT)过程。与模型组比较,经田七注射液处理后,大鼠尿蛋白排出减少(P<0.01),肾功能BUN和Scr水平下降P<0.01),肾小球硬化、肾小管萎缩、间质纤维化、炎症细胞浸润等病理改变减轻,肾小球硬化面积及肾小管间质纤维化半定量评分均显著下降(P<0.01);肾小管CL-Ⅳ和α-SMA表达下调(P<0.01),提示田七注射液具有抗肾纤维化的作用,其机制可能与影响EMT过程有关。
3.与假手术组比较,阿霉素肾纤维化大鼠肾组织TGF-β1、TGF-βR-Ⅰ、TGF-βR-Ⅱ、p-Smad2、p-Smad7 mRNA和/或蛋白表达明著显上调(P<0.01),提示阿霉素肾纤维化大鼠肾组织TGF-β1/Smads信号通路活化。经田七注射液处理后,阿霉素肾纤维化大鼠肾组织TGF-β1、TGF-βR-Ⅰ、TGF-βR-Ⅱ、p-Smad2 mRNA和/或蛋白表达显著下调(P<0.01),但p-Smad7蛋白表达显著上调(P<0.01),提示田七注射液的抗肾纤维化作用可能与抑制肾组织TGF-β1/Smads信号通路活化有关。
结论：
1.阿霉素肾纤维化大鼠肾组织TGF-β1/Smads信号通路活化,参与肾小管上皮-间质细胞转分化过程。
2.田七注射液具有抗肾纤维化和改善肾功能的作用,其机制可能与抑制肾组织TGF-β1/Smads信号通路活化介导的肾小管上皮-间质细胞转分化过程有关。
Objective:
1. To observe the effect of TianQi injection on the structure, fuction and Extracellular matrix of kidney, confirming the anti-fibrosis role of TianQi injection.
2. To investigate the anti-fibrosis mechanism of TianQi injection on the TGF-β1/Smads signaling conduction.
Methods:
1. The adriamycin-induced kidney fibrosis models were established by ways of excising right kidney and injecting Adriamycin into vena caudalis.
2. The 48 healthy male Sprague-Dawley (SD) rats were randomly divided into four groups:normal group, sham group, model group, TianQi injection treatment group. The rats in the normal group feed under normal condition without experienceing the operation were dealed with normal sodium(NS) (10 ml/kg·d) by intraperitoneal injection; The rats in the sham group after remove right kidney were dealed with normal sodium(NS) (10 ml/kg·d) by intraperitoneal injection without receiving adriamycin;The rats in the model group received NS (10 ml/kg·d) by intraperitoneal injection after operation; The rats in the TianQi injection treatment group received TianQi injection (10 ml/kg·d) by intraperitoneal injection after operation; All the rats were sacrificed after receiving treatments for 4 weeks, respectively, and the urine, blood and kidneys were collected.24h quantity of urinary protein and kidney function were detected; Kidney histological examinations was performed by HE and Masson staining, and the degree of tubular-interstitial damage was valuated in terms of semi-quantitative score. The mRNA and/or protein expression of TGF-β1, TGF-βR-Ⅰ, TGF-βR-Ⅱ, p-Smad2 anc p-Smad7 were detected by means of reverse transcription-Polymerase chair reaction (RT-PCR), inununohistochemistry and Western blot.
Results:
1. After excising right kidney and injecting Adriamycin into vena caudalis 24h quantity of urinary protein in rats was increased (P<0.01), and the BUN and Scr level in serum sample was enhanced (P<0.01), and the kideny pathology such as glomcrulus sclerosis, tubular atrophy, interstitial fibrosis and inflammation cells infiltration were found in rats, it suggests a successful kidney fibrosis model.
2. When compared with sham group, the urinary protein in model group rats was increased (P<0.01), and the BUN and Scr level in serum sample was enhanced (P<0.01). The protein expression of CL-IV and a-SMA in rats model witl Adriamycin-induced kidney fibrosis were increased when compared with sham group (P<0.01), which suggests an EMT course occuring in kidney fibrosis rats TianQi injection treatment could reduce the quantity of urinary protein (P<0.01) lower the BUN and Scr level(P<0.01), ameliorate the pathology in kidney such a glomcrulus sclerosis, tubular atrophy, interstitial fibrosis and inflammation cells infiltration, reduce the scores of glomcrulus sclerosis area and interstitia fibrosis(P<0.01),down-regulate the protein expression of collagen type IV(P<0.01 when compared with model group, which indicates that TianQi injection plays anti-kidney fibrosis role, and the mechanism is involved in influencing the course o EMT.
3. The mRNA and/or protein expression of TGF-β1, TGF-βtypeⅠreceptor, TGF-βtypeⅡreceptor, p-Smad2 and p-Smad7 in Adriamycin-induced kidney fibrosis models were up-regulated when compared with sham group (P<0.01),which suggests that the TGF-β1/Smads signaling is activated. When compared with model group, TianQi injection treatment could significantly down-regulate the mRNA and/or protein expression of TGF-β1, TGF-βtypeⅠreceptor, TGF-P typeⅡreceptor, p-Smad2 (P<0.01), and up-regulate protein expression of p-Smad7 (P<0.01), which indicates that anti-kidney fibarosis role of TianQi injection is associated with repressing the activation of TGF-β1/Smads signaling.
Conclusion:
1. The activation of TGF-β1/Smads signaling in the adriamycin-induced kidney fibrosis models is involved in the course of EMT in the tubular.
2. TianQi injection plays a anti-fibrosis role that is perhaps associated with repressing TGF-β1/Smads signaling mediated tubular EMT.
引文
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