人工冬虫夏草对博莱霉素所致大鼠肺纤维化的干预作用
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摘要
研究背景及目的:
特发性肺纤维化(idiopathic pulmonary fibrosis,IPF)是一种以普通型间质性肺炎(usual interstitial pneumonitis,UIP)为特征性病理改变的慢性、弥漫性肺间质病变的疾病,其临床表现主要为进行性呼吸困难和刺激性干咳,预后不良,诊断后的中位生存时间为3-5年。大部分患者因肺纤维化导致肺动脉高压、呼吸困难、肺源性心脏病和右心衰竭死亡。IPF目前还缺乏特异有效的治疗手段。糖皮质激素仍作为首选药物广泛用于临床,但其疗效不确切,且副作用大。因此研究IPF治疗具有非常重要的意义。
冬虫夏草是我国有两千多年历史的传统药用真菌,有显著的抗氧化、抗衰老、抗病毒、抗菌、抗炎、免疫调节等功效。冬虫夏草临床效果虽然明显,但由于产量有限,不能被广泛应用。蝙蝠蛾被毛孢、中华束丝孢、中国被毛孢为同物异名,且为冬虫夏草菌的无性型菌种。本研究采用的人工冬虫夏草(百令胶囊)是采用生物工程方法分离冬虫夏草后低温发酵精制而成的,是以中华束丝孢为生产菌种,与天然冬虫夏草有相似的化学成份。目前已初步证实人工冬虫夏草在肾间质纤维化、支气管哮喘、慢性阻塞性肺疾病动物模型有一定的干预作用。人工冬虫夏草对实验性肺纤维化动物模型的干预作用也已经有了初步的探讨,作用机制可能是通过抑制TGF-β1在肺组织中的表达,调整Ⅰ型/Ⅱ型细胞因子平衡达到的,但国内外尚未有人工冬虫夏草对肺纤维化早期肺泡炎阶段作用的研究。且从IPF的发病进程、临床症状发展迅速的特点及现有人工冬虫夏草的研究来看,人工冬虫夏草单独治疗肺纤维化仍缺乏有力的证据,仍需糖皮质激素控制IPF症状。目前国内外也尚未有研究报道人工冬虫夏草联合糖皮质激素对肺纤维化的治疗作用。故本实验分三部分研究,第一部分:人工冬虫夏草对肺纤维化的干预作用;第二部分:分别从肺泡炎阶段及肺纤维化阶段探讨人工冬虫夏草对大鼠肺纤维化的作用机制;第三部分:人工冬虫夏草联合糖皮质激素对肺纤维化的治疗作用。
第一部分人工冬虫夏草对博莱霉素所致大鼠肺纤维化的干预作用
目的
探讨人工冬虫夏草对博莱霉素诱导的大鼠肺纤维化的干预作用。
方法
40只SD大鼠,随机分为4组:①正常对照组(Ctr组);②模型组(Blm组);③虫草菌液组(Ccs组);④强的松组(Pred组)。采用气管内注射博莱霉素建立模型,次日起分别给予虫草菌液5 g·kg-1·d-1、强的松6mg·kg-1·d-1灌胃治疗,Ctr组和Blm组给予生理盐水灌胃。实验期间观察大鼠一般状况及称体重。第28d处死动物,肺组织标本行HE染色病理形态学观察及Ashcroft纤维化评分,肺组织MASSON染色测量胶原面积,碱水解法检测肺组织HYP含量。
结果
Ctr组大鼠一般状况佳,28d后体重较造模前增长(175.4±16.3)g,肺组织结构清晰,无明显纤维组织增生,纤维化评分为0.54±0.23,血管及气道壁少量绿色胶原沉积,胶原相对面积为(6.8±0.95)%,肺组织HYP含量为(2053.67±197)μg/g;与Ctr组比较,Blm组大鼠一般状况差,体重增长量(104.5±23.2g)明显减少(P<0.01),肺组织结构破坏,重度炎性细胞浸润,大块纤维组织增生实变,纤维化评分(5.83±1.21)明显增高(P<0.01),支气管、血管周边及肺间质大量绿色胶原沉积,胶原相对面积(67.83±16.23%)增加(P<0.01) ,肺组织HYP含量(4693.76±386μg/g)明显升高(P<0.01);与Blm组相比,Ccs组大鼠一般状况改善,体重增长量(173.6±12.6 g)增加(P<0.05),Ccs组可见肺组织损害减轻,纤维组织增生减少,纤维化评分(3.25±0.43)降低(P<0.01),胶原面积(35.22±11.34%)减少(P<0.01),肺组织HYP含量(3812.92±240μg/g)减少(P<0.05)。与Blm组相比,Pred组大鼠一般状况及体重增长量(125.9±16.7 g)无明显改善,Pred组可见肺组织损害减轻,纤维组织增生减少,纤维化评分(3.48±0.91)降低(P<0.01),胶原面积(31.48±8.65%)减少(P<0.01),肺组织HYP含量(3792.02±207μg/g)减少(P<0.05)。
结论
人工冬虫夏草能够减轻博莱霉素所致大鼠肺纤维化,改善大鼠一般状况。
第二部分人工冬虫夏草对大鼠肺纤维化作用机制的初步探讨
目的
探讨人工冬虫夏草对大鼠肺纤维化干预作用的机制。
方法
80只SD大鼠,随机分为14d组和28d组。14d组分为:①正常对照组(Ctrl4d组);②模型组(Blm14d组);③强的松(Pred14d组);④人工冬虫夏草组(Ccs14d组)。28d组分为:①正常对照组(Ctr28d组);②模型组(Blm28d组);③人工冬虫夏草组(Ccs28d组);④强的松(Pred28d组)。采用气管内注射博莱霉素建立模型,次日起Ccs28d组、Pred28d组分别给予虫草菌液5 g·kg-1·d-1、强的松6mg·kg-1·d-1灌胃治疗,Ctrl4d组、Ctr28d组、Blm14d组和Blm28d组分别给予生理盐水灌胃。14d组于14d处死动物,肺组织标本进行病理形态学观察及肺泡炎评分,免疫组化检测肺组织iNOS表达,ELISA法检测BALF TNF-α、GSH、MDA含量及SOD活性;28d组于28d处死动物,ELISA法检测BALF TGF-β1含量,免疫组化检测肺组织CTGF蛋白表达。
结果
Ctr14d组肺组织结构清晰,无炎性细胞浸润,肺泡炎评分为0.25±0.10;与Ctr14d组相比,Blm14d组可见肺组织结构破坏,重度炎性细胞浸润以及纤维组织增生实变,肺泡炎评分(2.5±0.42)明显升高(P<0.01),BALF中TNF-α、MDA含量明显升高,SOD活力、GSH含量降低,肺组织iNOS表达明显上调(P均<0.01);与Blm14d组相比,Ccs14d组、Pred14d组肺组织损害减轻,炎性细胞浸润以及纤维组织增生明显减少,肺泡炎评分分别为1.5±0.16、1.37±0.14,均明显降低(P均<0.01),BALF中TNF-α、MDA含量降低(P均<0.01),GSH含量及SOD活力升高(P均<0.01),肺组织iNOS表达下调(P均<0.01)。与Ctr28d组相比,Blm28d组BLAF中TGF-β1的含量明显升高,CTGF蛋白表达上调(P均<0.01);与Blm28d组相比,Ccs28d组、Pred28d组能够降低BALF中TGF-β1的含量(P<0.05),下调肺组织CTGF蛋白表达(P<0.05)。
结论
(1)人工冬虫夏草对博莱霉素所致肺纤维化早期肺泡炎阶段的干预作用,可能是通过下调BALF中TNF-α、肺组织iNOS等细胞因子的表达、减少脂质过氧化物的释放,达到减轻肺泡炎的作用。
(2)人工冬虫夏草对博莱霉素所致肺纤维化阶段的干预作用,可能是通过减少BALF含量,下调CTGF表达,达到减轻肺纤维化的作用。
(3)人工冬虫夏草对肺纤维化干预作用的机制,可能是通过肺泡炎阶段下调炎症介质及过氧化物表达,抑制炎症及氧化损伤对肺纤维阶段TGF-β1的活化,下调CTGF表达减少CTGF与TGF-β1结合,达到抑制ECM合成、减轻肺纤维化的作用。
第三部分人工冬虫夏草联合糖皮质激素对大鼠肺纤维化的干预作用
目的
人工冬虫夏草联合糖皮质激素对大鼠肺纤维化的干预作用。
方法
105只SD大鼠随机分7组:①正常对照组(Ctr组);②模型组(Blm组);③虫草菌液组(Ccs组);④常规剂量强的松组(C-Pred组);⑤小剂量强的松组(L-Pred组);⑥常规剂量强的松联合虫草菌液组(C-Com组);⑦小剂量强的松联合虫草菌液组(L-Com组)。采用气管内注射博莱霉素建立模型,次日起分别给药灌胃治疗,Ctr组和Blm组给予生理盐水灌胃。于第28d处死动物,肺组织标本行HE染色及Ashcroft纤维化评分,肺组织MASSON染色测量胶原面积,碱水解法检测肺组织HYP含量,免疫组化检测肺组织TGF-β1蛋白表达,蛋白免疫印迹法(Western blotting)检测肺组织PDGF-BB蛋白表达,ELISA法检测肺泡灌洗液上清bFGF含量。
结果
Ctr组大鼠一般状况佳,体重增长量为(184.4±19.4)g;与Ctr组比较,Blm组大鼠一般状况差,体重增长量(119.5±28.6g)明显减少(P<0.01);与Blm组相比,C-Pred组一般状况无明显改善,体重增长量(128.72±21.9g)无统计学差异(P>0.05);其余各治疗组大鼠一般状况改善,体重增长量增加(P<0.01),其中C-Com组(P<0.05)。Ctr组肺组织结构清晰,无明显纤维组织增生,纤维化评分为0.61±0.43,血管及气道壁少量绿色胶原纤维沉积,胶原面积为(9.41±1.86)%;与Ctr组相比,Blm组可见肺组织结构破坏,重度炎性细胞浸润,大块纤维组织增生实变,纤维化评分(4.63±1.21)升高(P<0.01),支气管、血管周边及肺间质大量绿色胶原沉积,胶原面积(69.63±7.09)增加(P<0.01),肺组织HYP含量升高(P<0.01),TGF-β1蛋白、PDGF-BB蛋白表达上调(P均<0.01),BLAF中bFGF含量升高(P<0.01);与Blm组相比,Ccs组、C-Pred组可见纤维组织增生减少,纤维化评分降低(P均<0.05),胶原面积减少(P均<0.05),HYP含量下降(P均<0.01),TGF-β1蛋白表达下调(P<0.01),PDGF-BB蛋白表达下调(P<0.05),Ccs组bFGF含量降低(P<0.01),C-Pred组bFGF含量无明显改变(P>0.05);C-Com组分别与Ccs组、C-Pred组比较,能够进一步降低肺纤维化评分,进一步减少胶原面积,降低HYP含量、bFGF含量,下调TGF-β1蛋白、PDGF-BB蛋白表达(P均<0.05),L-Com组分别与Ccs组、L-Pred组比较,能够进一步降低肺纤维化评分,进一步减少胶原面积,降低HYP含量、bFGF含量,下调TGF-β1蛋白(P均<0.05),其中PDGF-BB蛋白表达与Ccs组比较,无统计学差异(P>0.05)。
结论
(1)人工冬虫夏草联合强的松对博莱霉素所致肺纤维化有协同治疗作用,并能改善大鼠一般状况。
(2)人工冬虫夏草联合强的松对肺纤维化协同治疗作用的机制,可能是通过协同抑制TGF-β1、bFGF、PDGF-BB等促细胞外基质(ECM)合成因子,达到协同减轻肺纤维化的作用。
(3)小剂量强的松联合人工冬虫夏草与常规剂量强的松联合人工冬虫夏草对肺纤维化的治疗作用相同。
Background and Objective:
Idiopathic pulmonary fibrosis (IPF) is a chronic,diffuse interstitial pulmonary disease associated with a characteristic histologic appearance of usual interstitial pneumonia (UIP). The principal symptoms are described as dry cough and dyspnea. However, IPF generally progresses relentlessly and carries the poorest prognosis of the chronic IPF with a median survival of 3-5 years after diagnosis, there is no effective way to cure this disease yet. The current standard therapy recommended by ATS (American Thoracic society) for IPF involves treatment with corticosteroids and other immunosuppressive/cytotoxic agents. However, the beneficial effects of these agents are not well established, and adverse effects are often seen in the long treatment with glucocorticoid.
Cordyceps sinensis is a medicinal fungus of Traditional Chinese Medicine. There are a wide range of reported uses of Cordycep sinensis in the literature. However, the production of Cordyceps sinensis is so limited that can not be widely used.Cultured Cordyceps sinensis and natural Cordyceps sinensis have similar chemical composition. Cultured Cordyceps sinensis possesses anti-inflammatory,anti-hypoxia,anti-tumor effect function and regulating the endocrine system, enhanced immune function, which has a protective effect on the kidney,lung,liver and other organs. Recently, cultured Cordyceps sinensis has some beneficial effects on pulmonary fibrosis. So our experiments are to study the treatment effects and mechanism of cultured Cordyceps sinensis on pulmonary fibrosis,and the treatment effects of cultured Cordyceps sinensis combined with glucocorticosteroid on experimental pulmonary fibrosis in rats induced by Bleomycin.
The rats were respectively intratracheally instilled with Bleomycin to set up a pulmonary fibrosis model.
PartⅠThe effectiveness of cultured Cordyceps sinensis on pulmonary fibrosis induced by Bleomycin in rats
Aim: To study effectiveness of cultured Cordyceps sinensis on pulmonary fibrosis induced by Bleomycin in rats.
Methods: Fifty rats were randomly divided into five groups, including control group (Ctr group), model group(Blm group), cultured Cordyceps sinensis group(Ccs group),prednisone group(Pred group).On experimental day 0,the rats were respectively intratracheally instilled with Bleomycin, and rats in the Ctr group and Blm group with the same volume of normal saline. One day after the injection, Ccs group and Pred group was respectively given to rats daily by gastric gavage, while the same volume of normal saline was given to those in the Ctr group and Blm group.During the experimental,the rats were observed the general conditions and measured the body weight.On 28th d, bronchoalveolar lavage fluid(BALF) and lung tissue were collected. Histological changes of the lungs were evaluated by HE stain , Masson’s trichrome stain. Collagen content of the lung tissue was assessed by hydroxyprolin concentration.
Result: (1)Compared to Blm group, the Ccs group improved the body weight loss and general conditions;
(2) Compared to Blm group,pulmonary fibrosis and collagen deposition were alleviated in Ccs group and Pred group ;
(3) Compared to Blm group,hydroxyprolin concentrations were decreased in Ccs group and Pred group.
Conclusion:Ccs group can alleviate pulmonary fibrosis,improve the general conditions and the body weight loss induced by Bleomycin in rats.
PartⅡThe mechanism of cultured Cordyceps sinensis on pulmonary fibrosis induced by Bleomycin in rats
Aim: To study mechanism of cultured Cordyceps sinensis on experimental pulmonary fibrosis in rats induced by Bleomycin.
Methods: Eighty rats were randomly divided into14d group and 28 d group. 14 d group was divided into four groups, including control group(Ctrl4d group),model group(Blml4d group), prednisone group(Predl4d group) , and cultured Cordyceps sinensis group( Ccsl4d group). 28d group was divided into four groups, including control group(Ctr28d group),model group(Blm28d group), prednisone group(Pred28d group) , and cultured Cordyceps sinensis group( Ccs28d group). On experimental day 0,the rats were respectively intratracheally instilled with Bleomycin, and rats in the control group and model group with the same volume of normal saline. One day after the injection, cultured Cordyceps sinensis was given to rats daily by gastric gavage, and prednisone was given to the prednisone group, while the same volume of normal saline was given to those in the control group and model group.At 14d, bronchoalveolar lavage fluid(BALF) and lung tissue of l4 day group rats were collected.The contents of TNF-αin BALF were measured. The left lung was subjected to HE staining, iNOS immunohistochemical test, and homogenized for biochemical assays of MDA, GSH contents. At 28d, bronchoalveolar lavage fluid(BALF) and lung tissue of 28 day group rats were collected. The contents of TGF-β1 protein in BALF were measured. The lung were subjected to CTGF immunohistochemical test. Result: (1)Compared to Blml4d group, alveolitis were alleviated in Ccsl4d group,and the iNOS protein expression in lung tissue decreased.The MDA、TNF-αcontents decreased,and the contents of GSH,SOD increased in Ccsl4d group .
(2)Compared to Blm28d group, the TGF-β1 contents and the CTGF protein expression in lung tissue decreased in Ccs28d group.
Conclusion:
(1) Cultured Cordyceps sinensis alleviated alveolitis,which was through decreasing MDA、TNF-αcontents and iNOS protein expression, increasing the GSH,SOD contents.
(2) Cultured Cordyceps sinensis alleviated pulmonary fibrosis,which was through decreasing the TGF-β1 contents and the CTGF protein expression.
(3) The mechanism by which cultured Cordyceps sinensis resists pulmonary fibrosis is involved in alleviating alveolitis, enhancing the power of elimination oxygen radical,reducing ROS-mediated TGF-β1 activation and inhibiting the production CTGF to synthesis the extracellular matrix(ECM).
PartⅢThe effectiveness of cultured Cordyceps sinensis combined with glucocorticosteroid on pulmonary fibrosis induced by Bleomycin in rats
Aim: To study the treatment effects of cultured Cordyceps sinensis combined with glucocorticosteroid on experimental pulmonary fibrosis in rats induced by Bleomycin.
Methods: One hundred and five rats were randomly divided into seven groups, including control group(Ctr group), model group(Blm group), cultured Cordyceps sinensis group(Ccs group),conventional-dose prednisone group(C-Pred group), low-dose prednisone group(L-Pred group),cultured Cordyceps sinensis combined with conventional dose prednisone group(C-Com group),cultured Cordyceps sinensis combined with low dose prednisone group(L-Com group). On experimental day 0,the rats were respectively intratracheally instilled with Bleomycin, and rats in the Ctr group and Blm group with the same volume of normal saline. One day after the injection, cultured Cordyceps sinensis and glucocorticosteroid were respectively given to rats daily by gastric gavage, while the same volume of normal saline was given to those in the Ctr group and Blm group. During the experimental,the rats were observed the general conditions and measured the body weight.On 28th d, bronchoalveolar lavage fluid(BALF) and lung tissue were collected. Histological changes of the lungs were evaluated by HE stain , Masson’s trichrome stain. Collagen content of the lung tissue was assessed by hydroxyprolin concentration.Lung expression of TGF-β1 protein was assessed by immunohistochemistry.Lung expression of PDGF-BB protein was assessed by Western blotting.The level of bFGF protein was measured by enzyme-linked immunosorbent assay (ELISA).
Result:
(1)Compared to Blm group, the Ccs group,Pred group,C-Pred group L-Com group,C-Com group improved the body weight loss and general conditions;
(2)Compared to Blm group, pulmonary fibrosis were alleviated in Ccs group and C-Pred group,and PDGF TGF-β1 protein expression, hydroxyproline concentrations and protein bFGF were decreased.
(3)The combination effect of C-Com group or L-Com group was augmented compared with using Cordyceps sinensis or prednisone group alone.
Conclusion:
(1)The combination use of cultured Cordyceps sinensis and prednisone ,which may be through down-regulating the factors promoting extraceular matrix(ECM) synthesis TGF-B1,PDGF and bFGF,has synergia effects in anti - fibrous degeneration and improving the body weight loss ,general conditions.
(2) Cultured Cordyceps sinensis combined with low dose prednisone had the same anti-fibrosis effect as Cultured Cordyceps sinensis combined with conventional dose prednisone.
特发性肺纤维化(idiopathic pulmonary fibrosis,IPF)是一种以普通型间质性肺炎(usual interstitial pneumonitis,UIP)为特征性病理改变的慢性、弥漫性肺间质病变的疾病,其临床表现主要为进行性呼吸困难和刺激性干咳,预后不良,诊断后的中位生存时间为3-5年。大部分患者因肺纤维化导致肺动脉高压、呼吸困难、肺源性心脏病和右心衰竭死亡。IPF目前还缺乏特异有效的治疗手段。糖皮质激素仍作为首选药物广泛用于临床,但其疗效不确切,且副作用大。因此研究IPF治疗具有非常重要的意义。
冬虫夏草是我国有两千多年历史的传统药用真菌,有显著的抗氧化、抗衰老、抗病毒、抗菌、抗炎、免疫调节等功效。冬虫夏草临床效果虽然明显,但由于产量有限,不能被广泛应用。蝙蝠蛾被毛孢、中华束丝孢、中国被毛孢为同物异名,且为冬虫夏草菌的无性型菌种。本研究采用的人工冬虫夏草(百令胶囊)是采用生物工程方法分离冬虫夏草后低温发酵精制而成的,是以中华束丝孢为生产菌种,与天然冬虫夏草有相似的化学成份。目前已初步证实人工冬虫夏草在肾间质纤维化、支气管哮喘、慢性阻塞性肺疾病动物模型有一定的干预作用。人工冬虫夏草对实验性肺纤维化动物模型的干预作用也已经有了初步的探讨,作用机制可能是通过抑制TGF-β1在肺组织中的表达,调整Ⅰ型/Ⅱ型细胞因子平衡达到的,但国内外尚未有人工冬虫夏草对肺纤维化早期肺泡炎阶段作用的研究。且从IPF的发病进程、临床症状发展迅速的特点及现有人工冬虫夏草的研究来看,人工冬虫夏草单独治疗肺纤维化仍缺乏有力的证据,仍需糖皮质激素控制IPF症状。目前国内外也尚未有研究报道人工冬虫夏草联合糖皮质激素对肺纤维化的治疗作用。故本实验分三部分研究,第一部分:人工冬虫夏草对肺纤维化的干预作用;第二部分:分别从肺泡炎阶段及肺纤维化阶段探讨人工冬虫夏草对大鼠肺纤维化的作用机制;第三部分:人工冬虫夏草联合糖皮质激素对肺纤维化的治疗作用。
第一部分人工冬虫夏草对博莱霉素所致大鼠肺纤维化的干预作用
目的
探讨人工冬虫夏草对博莱霉素诱导的大鼠肺纤维化的干预作用。
方法
40只SD大鼠,随机分为4组:①正常对照组(Ctr组);②模型组(Blm组);③虫草菌液组(Ccs组);④强的松组(Pred组)。采用气管内注射博莱霉素建立模型,次日起分别给予虫草菌液5 g·kg-1·d-1、强的松6mg·kg-1·d-1灌胃治疗,Ctr组和Blm组给予生理盐水灌胃。实验期间观察大鼠一般状况及称体重。第28d处死动物,肺组织标本行HE染色病理形态学观察及Ashcroft纤维化评分,肺组织MASSON染色测量胶原面积,碱水解法检测肺组织HYP含量。
结果
Ctr组大鼠一般状况佳,28d后体重较造模前增长(175.4±16.3)g,肺组织结构清晰,无明显纤维组织增生,纤维化评分为0.54±0.23,血管及气道壁少量绿色胶原沉积,胶原相对面积为(6.8±0.95)%,肺组织HYP含量为(2053.67±197)μg/g;与Ctr组比较,Blm组大鼠一般状况差,体重增长量(104.5±23.2g)明显减少(P<0.01),肺组织结构破坏,重度炎性细胞浸润,大块纤维组织增生实变,纤维化评分(5.83±1.21)明显增高(P<0.01),支气管、血管周边及肺间质大量绿色胶原沉积,胶原相对面积(67.83±16.23%)增加(P<0.01) ,肺组织HYP含量(4693.76±386μg/g)明显升高(P<0.01);与Blm组相比,Ccs组大鼠一般状况改善,体重增长量(173.6±12.6 g)增加(P<0.05),Ccs组可见肺组织损害减轻,纤维组织增生减少,纤维化评分(3.25±0.43)降低(P<0.01),胶原面积(35.22±11.34%)减少(P<0.01),肺组织HYP含量(3812.92±240μg/g)减少(P<0.05)。与Blm组相比,Pred组大鼠一般状况及体重增长量(125.9±16.7 g)无明显改善,Pred组可见肺组织损害减轻,纤维组织增生减少,纤维化评分(3.48±0.91)降低(P<0.01),胶原面积(31.48±8.65%)减少(P<0.01),肺组织HYP含量(3792.02±207μg/g)减少(P<0.05)。
结论
人工冬虫夏草能够减轻博莱霉素所致大鼠肺纤维化,改善大鼠一般状况。
第二部分人工冬虫夏草对大鼠肺纤维化作用机制的初步探讨
目的
探讨人工冬虫夏草对大鼠肺纤维化干预作用的机制。
方法
80只SD大鼠,随机分为14d组和28d组。14d组分为:①正常对照组(Ctrl4d组);②模型组(Blm14d组);③强的松(Pred14d组);④人工冬虫夏草组(Ccs14d组)。28d组分为:①正常对照组(Ctr28d组);②模型组(Blm28d组);③人工冬虫夏草组(Ccs28d组);④强的松(Pred28d组)。采用气管内注射博莱霉素建立模型,次日起Ccs28d组、Pred28d组分别给予虫草菌液5 g·kg-1·d-1、强的松6mg·kg-1·d-1灌胃治疗,Ctrl4d组、Ctr28d组、Blm14d组和Blm28d组分别给予生理盐水灌胃。14d组于14d处死动物,肺组织标本进行病理形态学观察及肺泡炎评分,免疫组化检测肺组织iNOS表达,ELISA法检测BALF TNF-α、GSH、MDA含量及SOD活性;28d组于28d处死动物,ELISA法检测BALF TGF-β1含量,免疫组化检测肺组织CTGF蛋白表达。
结果
Ctr14d组肺组织结构清晰,无炎性细胞浸润,肺泡炎评分为0.25±0.10;与Ctr14d组相比,Blm14d组可见肺组织结构破坏,重度炎性细胞浸润以及纤维组织增生实变,肺泡炎评分(2.5±0.42)明显升高(P<0.01),BALF中TNF-α、MDA含量明显升高,SOD活力、GSH含量降低,肺组织iNOS表达明显上调(P均<0.01);与Blm14d组相比,Ccs14d组、Pred14d组肺组织损害减轻,炎性细胞浸润以及纤维组织增生明显减少,肺泡炎评分分别为1.5±0.16、1.37±0.14,均明显降低(P均<0.01),BALF中TNF-α、MDA含量降低(P均<0.01),GSH含量及SOD活力升高(P均<0.01),肺组织iNOS表达下调(P均<0.01)。与Ctr28d组相比,Blm28d组BLAF中TGF-β1的含量明显升高,CTGF蛋白表达上调(P均<0.01);与Blm28d组相比,Ccs28d组、Pred28d组能够降低BALF中TGF-β1的含量(P<0.05),下调肺组织CTGF蛋白表达(P<0.05)。
结论
(1)人工冬虫夏草对博莱霉素所致肺纤维化早期肺泡炎阶段的干预作用,可能是通过下调BALF中TNF-α、肺组织iNOS等细胞因子的表达、减少脂质过氧化物的释放,达到减轻肺泡炎的作用。
(2)人工冬虫夏草对博莱霉素所致肺纤维化阶段的干预作用,可能是通过减少BALF含量,下调CTGF表达,达到减轻肺纤维化的作用。
(3)人工冬虫夏草对肺纤维化干预作用的机制,可能是通过肺泡炎阶段下调炎症介质及过氧化物表达,抑制炎症及氧化损伤对肺纤维阶段TGF-β1的活化,下调CTGF表达减少CTGF与TGF-β1结合,达到抑制ECM合成、减轻肺纤维化的作用。
第三部分人工冬虫夏草联合糖皮质激素对大鼠肺纤维化的干预作用
目的
人工冬虫夏草联合糖皮质激素对大鼠肺纤维化的干预作用。
方法
105只SD大鼠随机分7组:①正常对照组(Ctr组);②模型组(Blm组);③虫草菌液组(Ccs组);④常规剂量强的松组(C-Pred组);⑤小剂量强的松组(L-Pred组);⑥常规剂量强的松联合虫草菌液组(C-Com组);⑦小剂量强的松联合虫草菌液组(L-Com组)。采用气管内注射博莱霉素建立模型,次日起分别给药灌胃治疗,Ctr组和Blm组给予生理盐水灌胃。于第28d处死动物,肺组织标本行HE染色及Ashcroft纤维化评分,肺组织MASSON染色测量胶原面积,碱水解法检测肺组织HYP含量,免疫组化检测肺组织TGF-β1蛋白表达,蛋白免疫印迹法(Western blotting)检测肺组织PDGF-BB蛋白表达,ELISA法检测肺泡灌洗液上清bFGF含量。
结果
Ctr组大鼠一般状况佳,体重增长量为(184.4±19.4)g;与Ctr组比较,Blm组大鼠一般状况差,体重增长量(119.5±28.6g)明显减少(P<0.01);与Blm组相比,C-Pred组一般状况无明显改善,体重增长量(128.72±21.9g)无统计学差异(P>0.05);其余各治疗组大鼠一般状况改善,体重增长量增加(P<0.01),其中C-Com组(P<0.05)。Ctr组肺组织结构清晰,无明显纤维组织增生,纤维化评分为0.61±0.43,血管及气道壁少量绿色胶原纤维沉积,胶原面积为(9.41±1.86)%;与Ctr组相比,Blm组可见肺组织结构破坏,重度炎性细胞浸润,大块纤维组织增生实变,纤维化评分(4.63±1.21)升高(P<0.01),支气管、血管周边及肺间质大量绿色胶原沉积,胶原面积(69.63±7.09)增加(P<0.01),肺组织HYP含量升高(P<0.01),TGF-β1蛋白、PDGF-BB蛋白表达上调(P均<0.01),BLAF中bFGF含量升高(P<0.01);与Blm组相比,Ccs组、C-Pred组可见纤维组织增生减少,纤维化评分降低(P均<0.05),胶原面积减少(P均<0.05),HYP含量下降(P均<0.01),TGF-β1蛋白表达下调(P<0.01),PDGF-BB蛋白表达下调(P<0.05),Ccs组bFGF含量降低(P<0.01),C-Pred组bFGF含量无明显改变(P>0.05);C-Com组分别与Ccs组、C-Pred组比较,能够进一步降低肺纤维化评分,进一步减少胶原面积,降低HYP含量、bFGF含量,下调TGF-β1蛋白、PDGF-BB蛋白表达(P均<0.05),L-Com组分别与Ccs组、L-Pred组比较,能够进一步降低肺纤维化评分,进一步减少胶原面积,降低HYP含量、bFGF含量,下调TGF-β1蛋白(P均<0.05),其中PDGF-BB蛋白表达与Ccs组比较,无统计学差异(P>0.05)。
结论
(1)人工冬虫夏草联合强的松对博莱霉素所致肺纤维化有协同治疗作用,并能改善大鼠一般状况。
(2)人工冬虫夏草联合强的松对肺纤维化协同治疗作用的机制,可能是通过协同抑制TGF-β1、bFGF、PDGF-BB等促细胞外基质(ECM)合成因子,达到协同减轻肺纤维化的作用。
(3)小剂量强的松联合人工冬虫夏草与常规剂量强的松联合人工冬虫夏草对肺纤维化的治疗作用相同。
Background and Objective:
Idiopathic pulmonary fibrosis (IPF) is a chronic,diffuse interstitial pulmonary disease associated with a characteristic histologic appearance of usual interstitial pneumonia (UIP). The principal symptoms are described as dry cough and dyspnea. However, IPF generally progresses relentlessly and carries the poorest prognosis of the chronic IPF with a median survival of 3-5 years after diagnosis, there is no effective way to cure this disease yet. The current standard therapy recommended by ATS (American Thoracic society) for IPF involves treatment with corticosteroids and other immunosuppressive/cytotoxic agents. However, the beneficial effects of these agents are not well established, and adverse effects are often seen in the long treatment with glucocorticoid.
Cordyceps sinensis is a medicinal fungus of Traditional Chinese Medicine. There are a wide range of reported uses of Cordycep sinensis in the literature. However, the production of Cordyceps sinensis is so limited that can not be widely used.Cultured Cordyceps sinensis and natural Cordyceps sinensis have similar chemical composition. Cultured Cordyceps sinensis possesses anti-inflammatory,anti-hypoxia,anti-tumor effect function and regulating the endocrine system, enhanced immune function, which has a protective effect on the kidney,lung,liver and other organs. Recently, cultured Cordyceps sinensis has some beneficial effects on pulmonary fibrosis. So our experiments are to study the treatment effects and mechanism of cultured Cordyceps sinensis on pulmonary fibrosis,and the treatment effects of cultured Cordyceps sinensis combined with glucocorticosteroid on experimental pulmonary fibrosis in rats induced by Bleomycin.
The rats were respectively intratracheally instilled with Bleomycin to set up a pulmonary fibrosis model.
PartⅠThe effectiveness of cultured Cordyceps sinensis on pulmonary fibrosis induced by Bleomycin in rats
Aim: To study effectiveness of cultured Cordyceps sinensis on pulmonary fibrosis induced by Bleomycin in rats.
Methods: Fifty rats were randomly divided into five groups, including control group (Ctr group), model group(Blm group), cultured Cordyceps sinensis group(Ccs group),prednisone group(Pred group).On experimental day 0,the rats were respectively intratracheally instilled with Bleomycin, and rats in the Ctr group and Blm group with the same volume of normal saline. One day after the injection, Ccs group and Pred group was respectively given to rats daily by gastric gavage, while the same volume of normal saline was given to those in the Ctr group and Blm group.During the experimental,the rats were observed the general conditions and measured the body weight.On 28th d, bronchoalveolar lavage fluid(BALF) and lung tissue were collected. Histological changes of the lungs were evaluated by HE stain , Masson’s trichrome stain. Collagen content of the lung tissue was assessed by hydroxyprolin concentration.
Result: (1)Compared to Blm group, the Ccs group improved the body weight loss and general conditions;
(2) Compared to Blm group,pulmonary fibrosis and collagen deposition were alleviated in Ccs group and Pred group ;
(3) Compared to Blm group,hydroxyprolin concentrations were decreased in Ccs group and Pred group.
Conclusion:Ccs group can alleviate pulmonary fibrosis,improve the general conditions and the body weight loss induced by Bleomycin in rats.
PartⅡThe mechanism of cultured Cordyceps sinensis on pulmonary fibrosis induced by Bleomycin in rats
Aim: To study mechanism of cultured Cordyceps sinensis on experimental pulmonary fibrosis in rats induced by Bleomycin.
Methods: Eighty rats were randomly divided into14d group and 28 d group. 14 d group was divided into four groups, including control group(Ctrl4d group),model group(Blml4d group), prednisone group(Predl4d group) , and cultured Cordyceps sinensis group( Ccsl4d group). 28d group was divided into four groups, including control group(Ctr28d group),model group(Blm28d group), prednisone group(Pred28d group) , and cultured Cordyceps sinensis group( Ccs28d group). On experimental day 0,the rats were respectively intratracheally instilled with Bleomycin, and rats in the control group and model group with the same volume of normal saline. One day after the injection, cultured Cordyceps sinensis was given to rats daily by gastric gavage, and prednisone was given to the prednisone group, while the same volume of normal saline was given to those in the control group and model group.At 14d, bronchoalveolar lavage fluid(BALF) and lung tissue of l4 day group rats were collected.The contents of TNF-αin BALF were measured. The left lung was subjected to HE staining, iNOS immunohistochemical test, and homogenized for biochemical assays of MDA, GSH contents. At 28d, bronchoalveolar lavage fluid(BALF) and lung tissue of 28 day group rats were collected. The contents of TGF-β1 protein in BALF were measured. The lung were subjected to CTGF immunohistochemical test. Result: (1)Compared to Blml4d group, alveolitis were alleviated in Ccsl4d group,and the iNOS protein expression in lung tissue decreased.The MDA、TNF-αcontents decreased,and the contents of GSH,SOD increased in Ccsl4d group .
(2)Compared to Blm28d group, the TGF-β1 contents and the CTGF protein expression in lung tissue decreased in Ccs28d group.
Conclusion:
(1) Cultured Cordyceps sinensis alleviated alveolitis,which was through decreasing MDA、TNF-αcontents and iNOS protein expression, increasing the GSH,SOD contents.
(2) Cultured Cordyceps sinensis alleviated pulmonary fibrosis,which was through decreasing the TGF-β1 contents and the CTGF protein expression.
(3) The mechanism by which cultured Cordyceps sinensis resists pulmonary fibrosis is involved in alleviating alveolitis, enhancing the power of elimination oxygen radical,reducing ROS-mediated TGF-β1 activation and inhibiting the production CTGF to synthesis the extracellular matrix(ECM).
PartⅢThe effectiveness of cultured Cordyceps sinensis combined with glucocorticosteroid on pulmonary fibrosis induced by Bleomycin in rats
Aim: To study the treatment effects of cultured Cordyceps sinensis combined with glucocorticosteroid on experimental pulmonary fibrosis in rats induced by Bleomycin.
Methods: One hundred and five rats were randomly divided into seven groups, including control group(Ctr group), model group(Blm group), cultured Cordyceps sinensis group(Ccs group),conventional-dose prednisone group(C-Pred group), low-dose prednisone group(L-Pred group),cultured Cordyceps sinensis combined with conventional dose prednisone group(C-Com group),cultured Cordyceps sinensis combined with low dose prednisone group(L-Com group). On experimental day 0,the rats were respectively intratracheally instilled with Bleomycin, and rats in the Ctr group and Blm group with the same volume of normal saline. One day after the injection, cultured Cordyceps sinensis and glucocorticosteroid were respectively given to rats daily by gastric gavage, while the same volume of normal saline was given to those in the Ctr group and Blm group. During the experimental,the rats were observed the general conditions and measured the body weight.On 28th d, bronchoalveolar lavage fluid(BALF) and lung tissue were collected. Histological changes of the lungs were evaluated by HE stain , Masson’s trichrome stain. Collagen content of the lung tissue was assessed by hydroxyprolin concentration.Lung expression of TGF-β1 protein was assessed by immunohistochemistry.Lung expression of PDGF-BB protein was assessed by Western blotting.The level of bFGF protein was measured by enzyme-linked immunosorbent assay (ELISA).
Result:
(1)Compared to Blm group, the Ccs group,Pred group,C-Pred group L-Com group,C-Com group improved the body weight loss and general conditions;
(2)Compared to Blm group, pulmonary fibrosis were alleviated in Ccs group and C-Pred group,and PDGF TGF-β1 protein expression, hydroxyproline concentrations and protein bFGF were decreased.
(3)The combination effect of C-Com group or L-Com group was augmented compared with using Cordyceps sinensis or prednisone group alone.
Conclusion:
(1)The combination use of cultured Cordyceps sinensis and prednisone ,which may be through down-regulating the factors promoting extraceular matrix(ECM) synthesis TGF-B1,PDGF and bFGF,has synergia effects in anti - fibrous degeneration and improving the body weight loss ,general conditions.
(2) Cultured Cordyceps sinensis combined with low dose prednisone had the same anti-fibrosis effect as Cultured Cordyceps sinensis combined with conventional dose prednisone.
引文
[1]中华医学会呼吸病学分会.特发性肺(间质)纤维化诊断和治疗指南(草案).中华结核和呼吸杂志. 2002. 25(7): 387-389.
[2] Demedts M, Costabel U. ATS/ERS international multidisciplinary consensus classification of the idiopathic interstitial pneumonias. Eur Respir J. 2002. 19(5): 794-6.
[3] Noth I, Martinez FJ. Recent advances in idiopathic pulmonary fibrosis. Chest. 2007. 132(2): 637-50.
[4] Flaherty KR. High-resolution computed tomography and the many faces of idiopathic pulmonary fibrosis. Am J Respir Crit Care Med. 2008. 177(4): 367-8.
[5] Raghu G, Weycker D, Edelsberg J, et al. Incidence and prevalence of idiopathic pulmonary fibrosis. Am J Respir Crit Care Med. 2006. 174(7): 810-6.
[6] Fernandez PER, Daniels CE, Schroeder DR, et al. Incidence, prevalence, and clinical course of idiopathic pulmonary fibrosis: a population-based study. Chest. 2010. 137(1): 129-37.
[7] Walter N, Collard HR, King TE Jr. Current perspectives on the treatment of idiopathic pulmonary fibrosis. Proc Am Thorac Soc. 2006. 3(4): 330-8.
[8] Bringardner BD, Baran CP, Eubank TD, et al. The role of inflammation in the pathogenesis of idiopathic pulmonary fibrosis. Antioxid Redox Signal. 2008. 10(2): 287-301.
[9] Inghilleri S, Morbini P, Oggionni T, et al.. In situ assessment of oxidant and nitrogenic stress in bleomycin pulmonary fibrosis. Histochem Cell Biol. 2006. 125(6): 661-9.
[10] Day BJ. Antioxidants as potential therapeutics for lung fibrosis. Antioxid Redox Signal. 2008. 10(2): 355-70.
[11] Daniil ZD, Papageorgiou E, Koutsokera A, et al. Serum levels of oxidative stress as a marker of disease severity in idiopathic pulmonary fibrosis. Pulm Pharmacol Ther. 2008. 21(1): 26-31.
[12] Phan SH. Biology of fibroblasts and myofibroblasts. Proc Am Thorac Soc. 2008. 5(3): 334-7.
[13] Camara J, Jarai G. Epithelial-mesenchymal transition in primary human bronchial epithelial cells is Smad-dependent and enhanced by fibronectin and TNF-alpha. Fibrogenesis Tissue Repair. 2010. 3(1): 2.
[14] Chen CM, Wang LF, Chou HC, et al.. Up-regulation of connective tissue growth factor in hyperoxia-induced lung fibrosis. Pediatr Res. 2007. 62(2): 128-33.
[15] Ask K, Martin GE, Kolb M, et al. Targeting genes for treatment in idiopathic pulmonary fibrosis: challenges and opportunities, promises and pitfalls. Proc Am Thorac Soc. 2006. 3(4): 389-93.
[16] Khalil N, Xu YD, O'Connor R, et al. Proliferation of pulmonary interstitial fibroblasts is mediated by transforming growth factor-beta1-induced release of extracellular fibroblast growth factor-2 and phosphorylation of p38 MAPK and JNK. J Biol Chem. 2005. 280(52): 43000-9.
[17] Drakopanagiotakis F, Xifteri A, Polychronopoulos V, Bouros D. Apoptosis in lung injury and fibrosis. Eur Respir J. 2008. 32(6): 1631-8.
[18] Suga M, Iyonaga K, Okamoto T, et al. Characteristic elevation of matrix metalloproteinase activity in idiopathic interstitial pneumonias. Am J Respir Crit Care Med. 2000. 162(5): 1949-56.
[19] Selman M, King TE, Pardo A. Idiopathic pulmonary fibrosis: prevailing and evolving hypotheses about its pathogenesis and implications for therapy. Ann Intern Med. 2001. 134(2): 136-51.
[20] Walter N, Collard HR, King TE Jr. Current perspectives on the treatment of idiopathic pulmonary fibrosis. Proc Am Thorac Soc. 2006. 3(4): 330-8.
[21] Klingsberg RC, Mutsaers SE, Lasky JA. Current clinical trials for the treatment of idiopathic pulmonary fibrosis. Respirology. 2010. 15(1): 19-31.
[22]周刚,王继峰,牛建昭,等.姜黄素抗肺纤维化大鼠细胞外基质过度形成的实验研究.中国中药杂志. 2006. (07): 570-573.
[23]杜钢军,张硕,林海红,等.灯盏花素对博来霉素诱导小鼠肺纤维化的保护作用.中国药理学通报. 2009. 25(2): 160-163.
[24]王昌明,何庆忠,张瑞祥.丹参酮对鼠肺纤维化过程中组织学变化的影响.中华结核和呼吸杂志. 1994. (05): 308-310+320.
[25] Paterson RR. Cordyceps: a traditional Chinese medicine and another fungal therapeutic biofactory. Phytochemistry. 2008. 69(7): 1469-95.
[26] Zhou X, Gong Z, Su Y, et al. Cordyceps fungi: natural products, pharmacological functions and developmental products. J Pharm Pharmacol. 2009. 61(3): 279-91.
[27]刘光全,陶水华,李辉,等.虫草胶囊菌粉的鉴定与分析.中国食品卫生杂志. 2009. (05): 418-421.
[28]魏鑫丽,印象初,郭英兰,等.冬虫夏草及其相关类群的分子系统学分析.菌物学报. 2006. (02): 192-202.
[29]蒋毅.冬虫夏草无性型研究概况.菌物系统. 2003. (01): 161-176.
[30]李风华,刘平,熊卫国,等.虫草多糖逆转DMN诱导大鼠肝纤维化的作用及机制研究.中国中药杂志. 2006. (23): 1968-1971.
[31] Liu YK, Shen W. Inhibitive effect of cordyceps sinensis on experimental hepatic fibrosis and its possible mechanism. World J Gastroenterol. 2003. 9(3): 529-33.
[32]朱运锋,谌贻璞,芮宏亮,等.虫草菌粉对慢性马兜铃酸肾病大鼠模型肾间质纤维化的保护作用.中华医学杂志. 2007. (38): 2667-2671.
[33]柴晶晶,谌贻璞,芮宏亮,等.虫草菌粉对慢性马兜铃酸肾病大鼠肾组织TGF-β_1及Snail表达和TEMT的影响.中国中西医结合杂志. 2009. (04): 325-329.
[34]王少杰,白文,王春玲,等.人工冬虫夏草菌液对博莱霉素致小鼠肺纤维化的保护作用.中国中药杂志. 2007. (24): 2623-2627.
[35]杨礼腾,程德云,聂莉,等.虫草菌粉对肺纤维化大鼠肺TGFβ_1及其信号通路分子mRNA表达的影响.四川中医. 2006. (02): 23-25.
[36]杨晶,刘忠英,郭家松,等.冬虫夏草预防肺纤维化的实验研究.实用医学杂志. 2008. (08): 1310-1312.
[37]曹志飞,蒋小岗,彭蕾,等.虫草提取物对肺纤维化小鼠的抗氧化作用研究.中国野生植物资源. 2009. (03): 52-57.
[38] Moeller A, Ask K, Warburton D, et al. The bleomycin animal model: a useful tool to investigate treatment options for idiopathic pulmonary fibrosis. Int J Biochem Cell Biol. 2008. 40(3): 362-82.
[39] Teixeira KC, Soares FS, Rocha LG, et al. Attenuation of bleomycin-induced lung injury and oxidative stress by N-acetylcysteine plus deferoxamine. Pulm Pharmacol Ther. 2008. 21(2): 309-16.
[40] Ashcroft T, Simpson JM, Timbrell V. Simple method of estimating severity of pulmonary fibrosis on a numerical scale. J Clin Pathol. 1988. 41(4): 467-70.
[41] American Thoracic Society/European Respiratory Society International Multidisciplinary Consensus Classification of the Idiopathic Interstitial Pneumonias. This joint statement of the American Thoracic Society (ATS), and the European Respiratory Society (ERS) was adopted by the ATS board of directors, June 2001 and by the ERS Executive Committee, June 2001. Am J Respir Crit Care Med. 2002. 165(2): 277-304.
[42]蒋捍东,刘豹.糖皮质激素在特发性肺纤维化中的应用.中华结核和呼吸杂志. 2007. 30(4): 249-250.
[43] Zhou XM, Zhang GC, Li JX, et al. Inhibitory effects of Hu-qi-yin on the bleomycin-induced pulmonary fibrosis in rats. J Ethnopharmacol. 2007. 111(2): 255-64.
[44] Gong LK, Li XH, Wang H, et al. Effect of Feitai on bleomycin-induced pulmonary fibrosis in rats. J Ethnopharmacol. 2005. 96(3): 537-44.
[45] Szapiel SV, Elson NA, Fulmer JD, et al. Bleomycin-induced interstitial pulmonary disease in the nude, athymic mouse. Am Rev Respir Dis. 1979. 120(4): 893-9.
[46] Maher TM, Wells AU, Laurent GJ. Idiopathic pulmonary fibrosis: multiplecauses and multiple mechanisms. Eur Respir J. 2007. 30(5): 835-9.
[47] Kim DS, Collard HR, King TE Jr. Classification and natural history of the idiopathic interstitial pneumonias. Proc Am Thorac Soc. 2006. 3(4): 285-92.
[48] Parambil JG, Myers JL, Ryu JH. Histopathologic features and outcome of patients with acute exacerbation of idiopathic pulmonary fibrosis undergoing surgical lung biopsy. Chest. 2005. 128(5): 3310-5.
[49] Day BJ. Antioxidants as potential therapeutics for lung fibrosis. Antioxid Redox Signal. 2008. 10(2): 355-70.
[50] Bringardner BD, Baran CP, Eubank TD, et al. The role of inflammation in the pathogenesis of idiopathic pulmonary fibrosis. Antioxid Redox Signal. 2008. 10(2): 287-301.
[51] Won SY, Park EH. Anti-inflammatory and related pharmacological activities of cultured mycelia and fruiting bodies of Cordyceps militaris. J Ethnopharmacol. 2005. 96(3): 555-61.
[52] Rao YK, Fang SH, Tzeng YM. Evaluation of the anti-inflammatory and anti-proliferation tumoral cells activities of Antrodia camphorata, Cordyceps sinensis, and Cinnamomum osmophloeum bark extracts. J Ethnopharmacol. 2007. 114(1): 78-85.
[53] Wang BJ, Won SJ, Yu ZR, et al. Free radical scavenging and apoptotic effects of Cordyceps sinensis fractionated by supercritical carbon dioxide. Food Chem Toxicol. 2005. 43(4): 543-52.
[54] Nan JX, Park EJ, Yang BK, et al. Antifibrotic effect of extracellular biopolymer from submerged mycelial cultures of Cordyceps militaris on liver fibrosis induced by bile duct ligation and scission in rats. Arch Pharm Res. 2001. 24(4): 327-32.
[55] Yamaguchi Y, Kagota S, Nakamura K, et al. Inhibitory effects of water extracts from fruiting bodies of cultured Cordyceps sinensis on raised serum lipid peroxide levels and aortic cholesterol deposition in atherosclerotic mice. Phytother Res. 2000. 14(8): 650-2.
[56]林晓霞,谢强敏,沈文会,等.虫草菌粉对致敏豚鼠肺功能和大鼠气道炎症反应的影响.中国中药杂志. 2001. 26(9): 622-625.
[57]刘进,童旭峰,管彩虹,等.冬虫夏草对慢性阻塞性肺疾病大鼠Th1/Th2类细胞因子平衡的干预作用.中华结核和呼吸杂志. 2003. 26(3): 191-192.
[58] Serrano-Mollar A, Closa D, Prats N, et al. In vivo antioxidant treatment protects against bleomycin-induced lung damage in rats. Br J Pharmacol. 2003. 138(6): 1037-48.
[59] Punithavathi D, Venkatesan N, Babu M. Curcumin inhibition of bleomycin-induced pulmonary fibrosis in rats. Br J Pharmacol. 2000. 131(2): 169-72.
[60] Kim KM, Kwon YG, Chung HT, et al. Methanol extract of Cordyceps pruinosa inhibits in vitro and in vivo inflammatory mediators by suppressing NF-kappaB activation. Toxicol Appl Pharmacol. 2003. 190(1): 1-8.
[61] Kikuchi N, Ishii Y, Morishima Y, et al. Nrf2 protects against pulmonary fibrosis by regulating the lung oxidant level and Th1/Th2 balance. Respir Res. 2010. 11: 31.
[62] Scotton CJ, Chambers RC. Molecular targets in pulmonary fibrosis: the myofibroblast in focus. Chest. 2007. 132(4): 1311-21.
[63] Pociask DA, Sime PJ, Brody AR. Asbestos-derived reactive oxygen species activate TGF-beta1. Lab Invest. 2004. 84(8): 1013-23.
[64] Sullivan DE, Ferris M, Pociask D, et al. The latent form of TGFbeta(1) is induced by TNFalpha through an ERK specific pathway and is activated by asbestos-derived reactive oxygen species in vitro and in vivo. J Immunotoxicol. 2008. 5(2): 145-9.
[65] Sime PJ, Marr RA, Gauldie D, et al. Transfer of tumor necrosis factor-alpha to rat lung induces severe pulmonary inflammation and patchy interstitial fibrogenesis with induction of transforming growth factor-beta1 and myofibroblasts. Am J Pathol. 1998. 153(3): 825-32.
[66] Chen CM, Wang LF, Chou HC, et al. Up-regulation of connective tissue growth factor in hyperoxia-induced lung fibrosis. Pediatr Res. 2007. 62(2):128-33.
[67] Rudd RM, Prescott RJ, Chalmers JC, et al. British Thoracic Society Study on cryptogenic fibrosing alveolitis: Response to treatment and survival. Thorax. 2007. 62(1): 62-6.
[68] Pereira CA, Malheiros T, Coletta EM, et al. Survival in idiopathic pulmonary fibrosis-cytotoxic agents compared to corticosteroids. Respir Med. 2006. 100(2): 340-7.
[69]薄守波,何汶婴.百令胶囊加泼尼松治疗慢性马兜铃酸肾病的临床观察.中国药师. 2003. (09): 570-571.
[70] Wen FQ, Skold CM, Liu XD, et al. Glucocorticoids and TGF-beta1 synergize in augmenting fibroblast mediated contraction of collagen gels. Inflammation. 2001. 25(2): 109-17.
[71] Cutroneo KR. Relationship between glucocorticoid-mediated early decrease of protein synthesis and the steady state decreases of glucocorticoid receptor and TGF-beta activator protein. Int J Biochem Cell Biol. 2002. 34(2): 194-203.
[72] Liu YK, Shen W. Inhibitive effect of cordyceps sinensis on experimental hepatic fibrosis and its possible mechanism. World J Gastroenterol. 2003. 9(3): 529-33.
[73]龚伟,黎磊石,陈丹,等.百令(冬虫夏草)对糖尿病大鼠转化生长因子β及其受体表达的影响.肾脏病与透析肾移植杂志. 2006. 15(4): 329-339.
[74] Chaudhary NI, Roth GJ, Hilberg F, et al. Inhibition of PDGF, VEGF and FGF signalling attenuates fibrosis. Eur Respir J. 2007. 29(5): 976-85.
[75] Yoo HS, Shin JW, Cho JH, et al. Effects of Cordyceps militaris extract on angiogenesis and tumor growth. Acta Pharmacol Sin. 2004. 25(5): 657-65.
[76] Ogata S, Yorioka N, Kohno N. Glucose and prednisolone alter basic fibroblast growth factor expression in peritoneal mesothelial cells and fibroblasts. J Am Soc Nephrol. 2001. 12(12): 2787-96.
[77] Oku H, Shimizu T, Kawabata T, et al. Antifibrotic action of pirfenidone and prednisolone: different effects on pulmonary cytokines and growth factors in bleomycin-induced murine pulmonary fibrosis. Eur J Pharmacol. 2008.590(1-3): 400-8.
[78]方向群,朱元珏,胡晓玲,等.科氯沙坦对大鼠肺纤维化模型的干预作用及对MCP-1和bFGF表达的影响.中华结核和呼吸杂志. 2002. 25(5): 268-272.
[79] Levitzki A. PDGF receptor kinase inhibitors for the treatment of PDGF driven diseases. Cytokine Growth Factor Rev. 2004. 15(4): 229-35.
[2] Demedts M, Costabel U. ATS/ERS international multidisciplinary consensus classification of the idiopathic interstitial pneumonias. Eur Respir J. 2002. 19(5): 794-6.
[3] Noth I, Martinez FJ. Recent advances in idiopathic pulmonary fibrosis. Chest. 2007. 132(2): 637-50.
[4] Flaherty KR. High-resolution computed tomography and the many faces of idiopathic pulmonary fibrosis. Am J Respir Crit Care Med. 2008. 177(4): 367-8.
[5] Raghu G, Weycker D, Edelsberg J, et al. Incidence and prevalence of idiopathic pulmonary fibrosis. Am J Respir Crit Care Med. 2006. 174(7): 810-6.
[6] Fernandez PER, Daniels CE, Schroeder DR, et al. Incidence, prevalence, and clinical course of idiopathic pulmonary fibrosis: a population-based study. Chest. 2010. 137(1): 129-37.
[7] Walter N, Collard HR, King TE Jr. Current perspectives on the treatment of idiopathic pulmonary fibrosis. Proc Am Thorac Soc. 2006. 3(4): 330-8.
[8] Bringardner BD, Baran CP, Eubank TD, et al. The role of inflammation in the pathogenesis of idiopathic pulmonary fibrosis. Antioxid Redox Signal. 2008. 10(2): 287-301.
[9] Inghilleri S, Morbini P, Oggionni T, et al.. In situ assessment of oxidant and nitrogenic stress in bleomycin pulmonary fibrosis. Histochem Cell Biol. 2006. 125(6): 661-9.
[10] Day BJ. Antioxidants as potential therapeutics for lung fibrosis. Antioxid Redox Signal. 2008. 10(2): 355-70.
[11] Daniil ZD, Papageorgiou E, Koutsokera A, et al. Serum levels of oxidative stress as a marker of disease severity in idiopathic pulmonary fibrosis. Pulm Pharmacol Ther. 2008. 21(1): 26-31.
[12] Phan SH. Biology of fibroblasts and myofibroblasts. Proc Am Thorac Soc. 2008. 5(3): 334-7.
[13] Camara J, Jarai G. Epithelial-mesenchymal transition in primary human bronchial epithelial cells is Smad-dependent and enhanced by fibronectin and TNF-alpha. Fibrogenesis Tissue Repair. 2010. 3(1): 2.
[14] Chen CM, Wang LF, Chou HC, et al.. Up-regulation of connective tissue growth factor in hyperoxia-induced lung fibrosis. Pediatr Res. 2007. 62(2): 128-33.
[15] Ask K, Martin GE, Kolb M, et al. Targeting genes for treatment in idiopathic pulmonary fibrosis: challenges and opportunities, promises and pitfalls. Proc Am Thorac Soc. 2006. 3(4): 389-93.
[16] Khalil N, Xu YD, O'Connor R, et al. Proliferation of pulmonary interstitial fibroblasts is mediated by transforming growth factor-beta1-induced release of extracellular fibroblast growth factor-2 and phosphorylation of p38 MAPK and JNK. J Biol Chem. 2005. 280(52): 43000-9.
[17] Drakopanagiotakis F, Xifteri A, Polychronopoulos V, Bouros D. Apoptosis in lung injury and fibrosis. Eur Respir J. 2008. 32(6): 1631-8.
[18] Suga M, Iyonaga K, Okamoto T, et al. Characteristic elevation of matrix metalloproteinase activity in idiopathic interstitial pneumonias. Am J Respir Crit Care Med. 2000. 162(5): 1949-56.
[19] Selman M, King TE, Pardo A. Idiopathic pulmonary fibrosis: prevailing and evolving hypotheses about its pathogenesis and implications for therapy. Ann Intern Med. 2001. 134(2): 136-51.
[20] Walter N, Collard HR, King TE Jr. Current perspectives on the treatment of idiopathic pulmonary fibrosis. Proc Am Thorac Soc. 2006. 3(4): 330-8.
[21] Klingsberg RC, Mutsaers SE, Lasky JA. Current clinical trials for the treatment of idiopathic pulmonary fibrosis. Respirology. 2010. 15(1): 19-31.
[22]周刚,王继峰,牛建昭,等.姜黄素抗肺纤维化大鼠细胞外基质过度形成的实验研究.中国中药杂志. 2006. (07): 570-573.
[23]杜钢军,张硕,林海红,等.灯盏花素对博来霉素诱导小鼠肺纤维化的保护作用.中国药理学通报. 2009. 25(2): 160-163.
[24]王昌明,何庆忠,张瑞祥.丹参酮对鼠肺纤维化过程中组织学变化的影响.中华结核和呼吸杂志. 1994. (05): 308-310+320.
[25] Paterson RR. Cordyceps: a traditional Chinese medicine and another fungal therapeutic biofactory. Phytochemistry. 2008. 69(7): 1469-95.
[26] Zhou X, Gong Z, Su Y, et al. Cordyceps fungi: natural products, pharmacological functions and developmental products. J Pharm Pharmacol. 2009. 61(3): 279-91.
[27]刘光全,陶水华,李辉,等.虫草胶囊菌粉的鉴定与分析.中国食品卫生杂志. 2009. (05): 418-421.
[28]魏鑫丽,印象初,郭英兰,等.冬虫夏草及其相关类群的分子系统学分析.菌物学报. 2006. (02): 192-202.
[29]蒋毅.冬虫夏草无性型研究概况.菌物系统. 2003. (01): 161-176.
[30]李风华,刘平,熊卫国,等.虫草多糖逆转DMN诱导大鼠肝纤维化的作用及机制研究.中国中药杂志. 2006. (23): 1968-1971.
[31] Liu YK, Shen W. Inhibitive effect of cordyceps sinensis on experimental hepatic fibrosis and its possible mechanism. World J Gastroenterol. 2003. 9(3): 529-33.
[32]朱运锋,谌贻璞,芮宏亮,等.虫草菌粉对慢性马兜铃酸肾病大鼠模型肾间质纤维化的保护作用.中华医学杂志. 2007. (38): 2667-2671.
[33]柴晶晶,谌贻璞,芮宏亮,等.虫草菌粉对慢性马兜铃酸肾病大鼠肾组织TGF-β_1及Snail表达和TEMT的影响.中国中西医结合杂志. 2009. (04): 325-329.
[34]王少杰,白文,王春玲,等.人工冬虫夏草菌液对博莱霉素致小鼠肺纤维化的保护作用.中国中药杂志. 2007. (24): 2623-2627.
[35]杨礼腾,程德云,聂莉,等.虫草菌粉对肺纤维化大鼠肺TGFβ_1及其信号通路分子mRNA表达的影响.四川中医. 2006. (02): 23-25.
[36]杨晶,刘忠英,郭家松,等.冬虫夏草预防肺纤维化的实验研究.实用医学杂志. 2008. (08): 1310-1312.
[37]曹志飞,蒋小岗,彭蕾,等.虫草提取物对肺纤维化小鼠的抗氧化作用研究.中国野生植物资源. 2009. (03): 52-57.
[38] Moeller A, Ask K, Warburton D, et al. The bleomycin animal model: a useful tool to investigate treatment options for idiopathic pulmonary fibrosis. Int J Biochem Cell Biol. 2008. 40(3): 362-82.
[39] Teixeira KC, Soares FS, Rocha LG, et al. Attenuation of bleomycin-induced lung injury and oxidative stress by N-acetylcysteine plus deferoxamine. Pulm Pharmacol Ther. 2008. 21(2): 309-16.
[40] Ashcroft T, Simpson JM, Timbrell V. Simple method of estimating severity of pulmonary fibrosis on a numerical scale. J Clin Pathol. 1988. 41(4): 467-70.
[41] American Thoracic Society/European Respiratory Society International Multidisciplinary Consensus Classification of the Idiopathic Interstitial Pneumonias. This joint statement of the American Thoracic Society (ATS), and the European Respiratory Society (ERS) was adopted by the ATS board of directors, June 2001 and by the ERS Executive Committee, June 2001. Am J Respir Crit Care Med. 2002. 165(2): 277-304.
[42]蒋捍东,刘豹.糖皮质激素在特发性肺纤维化中的应用.中华结核和呼吸杂志. 2007. 30(4): 249-250.
[43] Zhou XM, Zhang GC, Li JX, et al. Inhibitory effects of Hu-qi-yin on the bleomycin-induced pulmonary fibrosis in rats. J Ethnopharmacol. 2007. 111(2): 255-64.
[44] Gong LK, Li XH, Wang H, et al. Effect of Feitai on bleomycin-induced pulmonary fibrosis in rats. J Ethnopharmacol. 2005. 96(3): 537-44.
[45] Szapiel SV, Elson NA, Fulmer JD, et al. Bleomycin-induced interstitial pulmonary disease in the nude, athymic mouse. Am Rev Respir Dis. 1979. 120(4): 893-9.
[46] Maher TM, Wells AU, Laurent GJ. Idiopathic pulmonary fibrosis: multiplecauses and multiple mechanisms. Eur Respir J. 2007. 30(5): 835-9.
[47] Kim DS, Collard HR, King TE Jr. Classification and natural history of the idiopathic interstitial pneumonias. Proc Am Thorac Soc. 2006. 3(4): 285-92.
[48] Parambil JG, Myers JL, Ryu JH. Histopathologic features and outcome of patients with acute exacerbation of idiopathic pulmonary fibrosis undergoing surgical lung biopsy. Chest. 2005. 128(5): 3310-5.
[49] Day BJ. Antioxidants as potential therapeutics for lung fibrosis. Antioxid Redox Signal. 2008. 10(2): 355-70.
[50] Bringardner BD, Baran CP, Eubank TD, et al. The role of inflammation in the pathogenesis of idiopathic pulmonary fibrosis. Antioxid Redox Signal. 2008. 10(2): 287-301.
[51] Won SY, Park EH. Anti-inflammatory and related pharmacological activities of cultured mycelia and fruiting bodies of Cordyceps militaris. J Ethnopharmacol. 2005. 96(3): 555-61.
[52] Rao YK, Fang SH, Tzeng YM. Evaluation of the anti-inflammatory and anti-proliferation tumoral cells activities of Antrodia camphorata, Cordyceps sinensis, and Cinnamomum osmophloeum bark extracts. J Ethnopharmacol. 2007. 114(1): 78-85.
[53] Wang BJ, Won SJ, Yu ZR, et al. Free radical scavenging and apoptotic effects of Cordyceps sinensis fractionated by supercritical carbon dioxide. Food Chem Toxicol. 2005. 43(4): 543-52.
[54] Nan JX, Park EJ, Yang BK, et al. Antifibrotic effect of extracellular biopolymer from submerged mycelial cultures of Cordyceps militaris on liver fibrosis induced by bile duct ligation and scission in rats. Arch Pharm Res. 2001. 24(4): 327-32.
[55] Yamaguchi Y, Kagota S, Nakamura K, et al. Inhibitory effects of water extracts from fruiting bodies of cultured Cordyceps sinensis on raised serum lipid peroxide levels and aortic cholesterol deposition in atherosclerotic mice. Phytother Res. 2000. 14(8): 650-2.
[56]林晓霞,谢强敏,沈文会,等.虫草菌粉对致敏豚鼠肺功能和大鼠气道炎症反应的影响.中国中药杂志. 2001. 26(9): 622-625.
[57]刘进,童旭峰,管彩虹,等.冬虫夏草对慢性阻塞性肺疾病大鼠Th1/Th2类细胞因子平衡的干预作用.中华结核和呼吸杂志. 2003. 26(3): 191-192.
[58] Serrano-Mollar A, Closa D, Prats N, et al. In vivo antioxidant treatment protects against bleomycin-induced lung damage in rats. Br J Pharmacol. 2003. 138(6): 1037-48.
[59] Punithavathi D, Venkatesan N, Babu M. Curcumin inhibition of bleomycin-induced pulmonary fibrosis in rats. Br J Pharmacol. 2000. 131(2): 169-72.
[60] Kim KM, Kwon YG, Chung HT, et al. Methanol extract of Cordyceps pruinosa inhibits in vitro and in vivo inflammatory mediators by suppressing NF-kappaB activation. Toxicol Appl Pharmacol. 2003. 190(1): 1-8.
[61] Kikuchi N, Ishii Y, Morishima Y, et al. Nrf2 protects against pulmonary fibrosis by regulating the lung oxidant level and Th1/Th2 balance. Respir Res. 2010. 11: 31.
[62] Scotton CJ, Chambers RC. Molecular targets in pulmonary fibrosis: the myofibroblast in focus. Chest. 2007. 132(4): 1311-21.
[63] Pociask DA, Sime PJ, Brody AR. Asbestos-derived reactive oxygen species activate TGF-beta1. Lab Invest. 2004. 84(8): 1013-23.
[64] Sullivan DE, Ferris M, Pociask D, et al. The latent form of TGFbeta(1) is induced by TNFalpha through an ERK specific pathway and is activated by asbestos-derived reactive oxygen species in vitro and in vivo. J Immunotoxicol. 2008. 5(2): 145-9.
[65] Sime PJ, Marr RA, Gauldie D, et al. Transfer of tumor necrosis factor-alpha to rat lung induces severe pulmonary inflammation and patchy interstitial fibrogenesis with induction of transforming growth factor-beta1 and myofibroblasts. Am J Pathol. 1998. 153(3): 825-32.
[66] Chen CM, Wang LF, Chou HC, et al. Up-regulation of connective tissue growth factor in hyperoxia-induced lung fibrosis. Pediatr Res. 2007. 62(2):128-33.
[67] Rudd RM, Prescott RJ, Chalmers JC, et al. British Thoracic Society Study on cryptogenic fibrosing alveolitis: Response to treatment and survival. Thorax. 2007. 62(1): 62-6.
[68] Pereira CA, Malheiros T, Coletta EM, et al. Survival in idiopathic pulmonary fibrosis-cytotoxic agents compared to corticosteroids. Respir Med. 2006. 100(2): 340-7.
[69]薄守波,何汶婴.百令胶囊加泼尼松治疗慢性马兜铃酸肾病的临床观察.中国药师. 2003. (09): 570-571.
[70] Wen FQ, Skold CM, Liu XD, et al. Glucocorticoids and TGF-beta1 synergize in augmenting fibroblast mediated contraction of collagen gels. Inflammation. 2001. 25(2): 109-17.
[71] Cutroneo KR. Relationship between glucocorticoid-mediated early decrease of protein synthesis and the steady state decreases of glucocorticoid receptor and TGF-beta activator protein. Int J Biochem Cell Biol. 2002. 34(2): 194-203.
[72] Liu YK, Shen W. Inhibitive effect of cordyceps sinensis on experimental hepatic fibrosis and its possible mechanism. World J Gastroenterol. 2003. 9(3): 529-33.
[73]龚伟,黎磊石,陈丹,等.百令(冬虫夏草)对糖尿病大鼠转化生长因子β及其受体表达的影响.肾脏病与透析肾移植杂志. 2006. 15(4): 329-339.
[74] Chaudhary NI, Roth GJ, Hilberg F, et al. Inhibition of PDGF, VEGF and FGF signalling attenuates fibrosis. Eur Respir J. 2007. 29(5): 976-85.
[75] Yoo HS, Shin JW, Cho JH, et al. Effects of Cordyceps militaris extract on angiogenesis and tumor growth. Acta Pharmacol Sin. 2004. 25(5): 657-65.
[76] Ogata S, Yorioka N, Kohno N. Glucose and prednisolone alter basic fibroblast growth factor expression in peritoneal mesothelial cells and fibroblasts. J Am Soc Nephrol. 2001. 12(12): 2787-96.
[77] Oku H, Shimizu T, Kawabata T, et al. Antifibrotic action of pirfenidone and prednisolone: different effects on pulmonary cytokines and growth factors in bleomycin-induced murine pulmonary fibrosis. Eur J Pharmacol. 2008.590(1-3): 400-8.
[78]方向群,朱元珏,胡晓玲,等.科氯沙坦对大鼠肺纤维化模型的干预作用及对MCP-1和bFGF表达的影响.中华结核和呼吸杂志. 2002. 25(5): 268-272.
[79] Levitzki A. PDGF receptor kinase inhibitors for the treatment of PDGF driven diseases. Cytokine Growth Factor Rev. 2004. 15(4): 229-35.