不同外感致病因素对病证结合CIA模型建立的比较研究
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摘要
目的:该研究通过复制改良病证结合CIA模型,观察比较不同外感致病因素对大鼠滑膜细胞凋亡的影响。方法:将50只SPF级雌性Wistar大鼠随机分为5组:空白对照组、CIA对照组、风寒湿痹证CIA组、风湿热痹证CIA组、寒热错杂痹证CIA组。分别采用风寒湿、风湿热等外感致病因素刺激,结合牛II型胶原乳化剂诱导的方法制备类风湿关节炎大鼠模型。通过对大鼠一般状态观察,关节炎指数评分和关节肿胀度测量,对比三种RA病证结合动物模型的发病情况;采用ELISA法检测血清RF、ACPA、IL-1β、IL-10、TNF-α含量;HE染色观察踝关节病理学改变;TUNEL法检测滑膜细胞凋亡情况。结果:与空白对照组相比,CIA模型各组大鼠关节炎指数和关节肿胀度明显升高,血清RF、ACPA、IL-1β、TNF-α含量上升,血清IL-10含量下降。踝关节HE染色结果显示,CIA模型各组大鼠滑膜组织均有不同程度的增生和血管翳的形成,关节软骨可见不同程度的退行性变及骨质的破坏。TUNEL法检测结果显示,与空白对照组相比,CIA模型各组大鼠滑膜组织细胞凋亡率显著下降,以寒热错杂痹证CIA组下降最为明显,其次为风湿热痹证CIA组,风寒湿痹证CIA组次之,CIA对照组下降程度最低。结论:风、寒、湿、热等外感致病因素对类风湿关节炎发病是有影响的,在CIA模型基础上,联合风寒湿、风湿热刺激,可以诱导出稳定可靠、符合中医证候特点的类风湿关节炎病证结合动物模型。其中,以寒热错杂痹证CIA组最为严重,滑膜细胞凋亡率最低。
Objective: To observe and compare the effect of different exogenous pathogenic factors on the apoptosis of synoviocytes by replicating and improving collagen-induced arthritis(CIA) models of combined disease and syndrome. Methods: Fifty SPF female Wistar rats were randomly divided into five groups: the blank control group,the CIA control group,the wind-cold-dampness Bi syndrome CIA group,the wind-dampness-heat Bi syndrome CIA group,the cold-heat complex Bi syndrome CIA group. The rat models of rheumatoid arthritis(RA) were prepared by using the wind-cold-dampness,wind-dampness-heat stimulation and combing with bovine type II collagen. General state,arthritis index(AI) and joint swelling degree of the rats were observed to evaluate the onset of RA models of combined disease and syndrome. Serum rheumatoid factors(RF),anti-cyclic citrullinated peptide antibodies(ACPA),interleukin-1β(IL-1β),interleukin-10(IL-10) and tumour necrosis factor-α(TNF-α) were detected by enzyme linked immunosorbent assay(ELISA). Histopathologic changes of ankle joint were observed by hematoxylin-eosin(HE) staining. The method of terminal-deoxynucleoitidyl transferase mediated nick end labeling(TUNEL) was used to detect the apoptosis of synoviocytes. Results: Compared with the blank control group,AI and joint swelling degree of all CIA model groups were increased significantly. The contents of serum RF,ACPA,IL-1β and TNF-α increased while serum IL-10 decreased. The result of ankle HE staining showed that there were various degrees of synovial tissue hyperplasia and pannus formation in the CIA model groups. Articular cartilage can be seen with varying degrees of degenerative degeneration and bone destruction. The result of TUNEL showed that the apoptosis rate of synovial tissues were markly decreased than that of the blank control group. The decline of the cold-heat complex Bi syndrome CIA group was the most obvious,followed by the wind-dampness-heat Bi syndrome CIA group and the wind-cold-dampness Bi syndrome CIA group. The decreasing degree of the CIA control group was the minimum. Conclusion: The invasion of wind,cold,dampness and heat has an effect on the happening of RA. The CIA models combined with the wind-cold-dampness and wind-dampness-heat stimulation could induce stable and reliable RA models of combined disease and syndrome which conformed to the characteristics of traditional Chinese medicine(TCM) syndrome. The cold-heat complex Bi syndrome CIA group was the most severe and the apoptosis rate of synovial cells was the lowest among them.
Objective: To observe and compare the effect of different exogenous pathogenic factors on the apoptosis of synoviocytes by replicating and improving collagen-induced arthritis(CIA) models of combined disease and syndrome. Methods: Fifty SPF female Wistar rats were randomly divided into five groups: the blank control group,the CIA control group,the wind-cold-dampness Bi syndrome CIA group,the wind-dampness-heat Bi syndrome CIA group,the cold-heat complex Bi syndrome CIA group. The rat models of rheumatoid arthritis(RA) were prepared by using the wind-cold-dampness,wind-dampness-heat stimulation and combing with bovine type II collagen. General state,arthritis index(AI) and joint swelling degree of the rats were observed to evaluate the onset of RA models of combined disease and syndrome. Serum rheumatoid factors(RF),anti-cyclic citrullinated peptide antibodies(ACPA),interleukin-1β(IL-1β),interleukin-10(IL-10) and tumour necrosis factor-α(TNF-α) were detected by enzyme linked immunosorbent assay(ELISA). Histopathologic changes of ankle joint were observed by hematoxylin-eosin(HE) staining. The method of terminal-deoxynucleoitidyl transferase mediated nick end labeling(TUNEL) was used to detect the apoptosis of synoviocytes. Results: Compared with the blank control group,AI and joint swelling degree of all CIA model groups were increased significantly. The contents of serum RF,ACPA,IL-1β and TNF-α increased while serum IL-10 decreased. The result of ankle HE staining showed that there were various degrees of synovial tissue hyperplasia and pannus formation in the CIA model groups. Articular cartilage can be seen with varying degrees of degenerative degeneration and bone destruction. The result of TUNEL showed that the apoptosis rate of synovial tissues were markly decreased than that of the blank control group. The decline of the cold-heat complex Bi syndrome CIA group was the most obvious,followed by the wind-dampness-heat Bi syndrome CIA group and the wind-cold-dampness Bi syndrome CIA group. The decreasing degree of the CIA control group was the minimum. Conclusion: The invasion of wind,cold,dampness and heat has an effect on the happening of RA. The CIA models combined with the wind-cold-dampness and wind-dampness-heat stimulation could induce stable and reliable RA models of combined disease and syndrome which conformed to the characteristics of traditional Chinese medicine(TCM) syndrome. The cold-heat complex Bi syndrome CIA group was the most severe and the apoptosis rate of synovial cells was the lowest among them.
引文
[1]沈丕安.类风湿关节炎中医临床诊疗[M].北京:人民军医出版社,2015.
[2]曾小峰,朱松林,谭爱春,等.我国类风湿关节炎疾病负担和生存质量研究的系统评价[J].中国循证医学杂志,2013,13(3):300-307.
[3]陈程,黄光辉,黄豆豆,等.类风湿关节炎啮齿类实验动物模型的研究进展[J].现代药物与临床,2013,28(3):428-434.
[4]李艳玲,祁芳,许明,等.常用类风湿关节炎动物模型研究概况及展望[J].湖南中医药大学学报,2015,35(4):63-66.
[5]刘佳维,王永辉,李艳彦,等.黄芪桂枝五物汤对CIA模型大鼠关节滑膜细胞凋亡的影响[J].中国实验方剂学杂志,2017,23(14):171-176.
[6]镇兰芳,宋玉,张六通.外感风寒湿热对胶原诱导关节炎大鼠血清CD44水平的影响[J].辽宁中医药大学学报,2013,15(10):159-161.
[7]张晓园,王永辉,李艳彦,等.风湿宁胶囊对寒湿闭阻型类风湿关节炎模型大鼠治疗作用机制研究[J].山西中医,2016,32(5):54-56.
[8]秦川.实验动物学[M].北京:人民卫生出版社,2010:234-240.
[9]Yi JK,Kim HJ,Yu DH,et al.Regulation of inflammatory responses and fibroblast-like synoviocyte apoptosis by calcineurin-binding protein 1 in mice with collagen-induced arthritis[J].Arthritis Rheum,2012,64(7):2191-2200.
[10]马艳苗.风湿宁胶囊对胶原诱导性关节炎大鼠的治疗作用及机制研究[D].武汉:湖北中医药大学,2013.
[11]Trentham DE,Townes AS,Kang AH.Autoimmunity to typeⅡcollagen:an experimental model of arthritis[J].Exp Med,1977,146:857-868.
[12]Jung SM,Lee J,Baek SY,et al.Ethyl pyruvate ameliorates inflammatory arthritis in mice[J].International Immunopharmacology,2017,52:333-341.
[13]周仲英,周学平.从瘀热论治内科难治病[M].北京:人民卫生出版社,2010:416-420.
[14]马俊福,侯秀娟,刘小平,等.类风湿关节炎寒证动物模型的建立与评价[J].中华中医药杂志,2016,31(5):1967-1970.
[15]王惠萱.临床疾病检验项目选择与应用[M].北京:人民军医出版社,2010:226.
[16]Svensson B,Andersson M,Forslind K,et al.Persistently active disease is common in patients with rheumatoid arthritis,particularly in women:a long-term inception cohort study[J].Scand J Rheumatol,2016,4(20):1-8.
[17]Ma Z,Wang B,Wang M,et al.TL1A increased IL-6 production on fibroblast-like synoviocytes by preferentially activating TNF receptor2 in rheumatoid arthritis[J].Cytokine,2016,12(83):92-98.
[2]曾小峰,朱松林,谭爱春,等.我国类风湿关节炎疾病负担和生存质量研究的系统评价[J].中国循证医学杂志,2013,13(3):300-307.
[3]陈程,黄光辉,黄豆豆,等.类风湿关节炎啮齿类实验动物模型的研究进展[J].现代药物与临床,2013,28(3):428-434.
[4]李艳玲,祁芳,许明,等.常用类风湿关节炎动物模型研究概况及展望[J].湖南中医药大学学报,2015,35(4):63-66.
[5]刘佳维,王永辉,李艳彦,等.黄芪桂枝五物汤对CIA模型大鼠关节滑膜细胞凋亡的影响[J].中国实验方剂学杂志,2017,23(14):171-176.
[6]镇兰芳,宋玉,张六通.外感风寒湿热对胶原诱导关节炎大鼠血清CD44水平的影响[J].辽宁中医药大学学报,2013,15(10):159-161.
[7]张晓园,王永辉,李艳彦,等.风湿宁胶囊对寒湿闭阻型类风湿关节炎模型大鼠治疗作用机制研究[J].山西中医,2016,32(5):54-56.
[8]秦川.实验动物学[M].北京:人民卫生出版社,2010:234-240.
[9]Yi JK,Kim HJ,Yu DH,et al.Regulation of inflammatory responses and fibroblast-like synoviocyte apoptosis by calcineurin-binding protein 1 in mice with collagen-induced arthritis[J].Arthritis Rheum,2012,64(7):2191-2200.
[10]马艳苗.风湿宁胶囊对胶原诱导性关节炎大鼠的治疗作用及机制研究[D].武汉:湖北中医药大学,2013.
[11]Trentham DE,Townes AS,Kang AH.Autoimmunity to typeⅡcollagen:an experimental model of arthritis[J].Exp Med,1977,146:857-868.
[12]Jung SM,Lee J,Baek SY,et al.Ethyl pyruvate ameliorates inflammatory arthritis in mice[J].International Immunopharmacology,2017,52:333-341.
[13]周仲英,周学平.从瘀热论治内科难治病[M].北京:人民卫生出版社,2010:416-420.
[14]马俊福,侯秀娟,刘小平,等.类风湿关节炎寒证动物模型的建立与评价[J].中华中医药杂志,2016,31(5):1967-1970.
[15]王惠萱.临床疾病检验项目选择与应用[M].北京:人民军医出版社,2010:226.
[16]Svensson B,Andersson M,Forslind K,et al.Persistently active disease is common in patients with rheumatoid arthritis,particularly in women:a long-term inception cohort study[J].Scand J Rheumatol,2016,4(20):1-8.
[17]Ma Z,Wang B,Wang M,et al.TL1A increased IL-6 production on fibroblast-like synoviocytes by preferentially activating TNF receptor2 in rheumatoid arthritis[J].Cytokine,2016,12(83):92-98.