千草方熏洗对膝骨关节炎大鼠软骨YAP/Smad3表达的影响
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摘要
目的:观察千草方熏洗对膝骨关节炎(KOA)大鼠软骨Yes相关蛋白(YAP)和Smad3表达的影响,研究千草方熏洗对膝骨关节炎大鼠软骨的保护作用及机制。方法:32只SD大鼠随机分成4组:对照组、模型组、中药组和透明质酸(HA)组,模型组、中药组和HA组均采用改良Hulth法构建大鼠KOA模型,中药组大鼠进行千草方熏洗,HA组大鼠予以关节腔内注射HA。处理后,取各组大鼠膝关节软骨组织和血清,软骨组织进行苏木精-伊红(HE)染色和Mankin’s评分,ELISA法检测血清中软骨寡聚基质蛋白(COMP)和Ⅱ型胶原C端肽(CTX-II)的表达量,TUNEL染色观察细胞凋亡情况,免疫组化检测YAP和高迁移率族蛋白B2(HMGB2)的表达,并采用Western blot检测YAP、Smad3、骨成型蛋白2(BMP2)、Bax和Bcl-2的表达水平。结果:HE染色结果表明,模型组软骨细胞排列紊乱,结构不清晰;与模型组相比,中药组软骨表面更加光滑,结构较清晰,软骨基质着色较均匀。与模型组相比,中药组和HA组的Mankin’s评分显著下降,血清CTX-II含量显著升高,COMP含量显著下降(P<0.05),软骨细胞的凋亡率显著下降(P<0.05)。免疫组化和Western blot的结果表明,与模型组相比,中药组和HA组YAP、HMGB2、Smad3和Bcl-2的表达量增加,BMP2和Bax的表达量下降(P<0.05)。结论:千草方熏洗能够通过YAP/Smad3信号通路缓解KOA。
AIM: To observe the effects of Qiancaofang fumigation on the expression of Yes-associated protein(YAP) and Smad3 in the cartilage of the rats with knee osteoarthritis(KOA), and to investigate the protective effects and mechanisms of Qiancaofang fumigation on the cartilage of the rats with KOA. METHODS: SD rats(n=32) were randomly divided into 4 groups: control group, model group, Chinese medicine group and hyaluronic acid(HA) group. The improved Hulth method was used to construct the rat model of KOA in model group, Chinese medicine group and HA group. The rats in Chinese medicine group were treated with Qiancaofang fumigation. The rats in HA group were treated with intra-articular injection of HA. After treatments, the cartilage tissues and serum of the rats in each group were taken. Hematoxylin-eosin(HE) staining and Mankin's scores were measured. The expression levels of cartilage oligomefic matrix protein(COMP) and C-telopeptide of type Ⅱ collagen(CTX-II) in the serum were detected. The method of TdT-mediated dUTP nick-end labeling(TUNEL) was used to observe the apoptosis of the cartilage cells. Immunohistochemistry was performed to detect the expression of YAP and high mobility group protein B2(HMGB2). Western blot was used to detect the expression of YAP, Smad3, bone morphogenetic protein 2(BMP2), Bax and Bcl-2. RESULTS: The results of HE staining showed that the cartilage cells in model group were disordered and the structure was not clear. Compared with model group, the cartilage surface in Chinese medicine group was more smooth, the structure was clearer, and the staining of the cartilage matrix was more uniform. Compared with model group, the Mankin's scores in Chinese medicine group and HA group were significantly decreased(P<0.05), the serum concentration of CTX-Ⅱ was significantly increased, the serum concentration of COMP was significantly decreased, and the apoptosis rate of the cells was decreased(P<0.05). The results of immunohistochemistry and Western blot showed that the expression levels of YAP, HMGB2, Smad3 and Bcl-2 were increased in Chinese medicine group and HA group, and the expression levels of BMP2 and Bax were decreased as compared with model group(P<0.05). CONCLUSION: Qiancaofang fumigation relieves KOA partially through the YAP/Smad3 signaling pathway.
AIM: To observe the effects of Qiancaofang fumigation on the expression of Yes-associated protein(YAP) and Smad3 in the cartilage of the rats with knee osteoarthritis(KOA), and to investigate the protective effects and mechanisms of Qiancaofang fumigation on the cartilage of the rats with KOA. METHODS: SD rats(n=32) were randomly divided into 4 groups: control group, model group, Chinese medicine group and hyaluronic acid(HA) group. The improved Hulth method was used to construct the rat model of KOA in model group, Chinese medicine group and HA group. The rats in Chinese medicine group were treated with Qiancaofang fumigation. The rats in HA group were treated with intra-articular injection of HA. After treatments, the cartilage tissues and serum of the rats in each group were taken. Hematoxylin-eosin(HE) staining and Mankin's scores were measured. The expression levels of cartilage oligomefic matrix protein(COMP) and C-telopeptide of type Ⅱ collagen(CTX-II) in the serum were detected. The method of TdT-mediated dUTP nick-end labeling(TUNEL) was used to observe the apoptosis of the cartilage cells. Immunohistochemistry was performed to detect the expression of YAP and high mobility group protein B2(HMGB2). Western blot was used to detect the expression of YAP, Smad3, bone morphogenetic protein 2(BMP2), Bax and Bcl-2. RESULTS: The results of HE staining showed that the cartilage cells in model group were disordered and the structure was not clear. Compared with model group, the cartilage surface in Chinese medicine group was more smooth, the structure was clearer, and the staining of the cartilage matrix was more uniform. Compared with model group, the Mankin's scores in Chinese medicine group and HA group were significantly decreased(P<0.05), the serum concentration of CTX-Ⅱ was significantly increased, the serum concentration of COMP was significantly decreased, and the apoptosis rate of the cells was decreased(P<0.05). The results of immunohistochemistry and Western blot showed that the expression levels of YAP, HMGB2, Smad3 and Bcl-2 were increased in Chinese medicine group and HA group, and the expression levels of BMP2 and Bax were decreased as compared with model group(P<0.05). CONCLUSION: Qiancaofang fumigation relieves KOA partially through the YAP/Smad3 signaling pathway.
引文
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[17] 王晶,张洹.雌激素对人骨髓间质细胞BMP-2 mRNA表达的影响[J].中国病理生理杂志,2007,23(3):584-586.
[18] 杨俊龙,王艳娉,张源,等.透明质酸治疗膝骨关节炎相关机制的研究进展[J].中国疼痛医学杂志,2017,23(6):451-454.
[2] Litwic A,Edwards MH,Dennison EM,et al.Epidemiology and burden of osteoarthritis[J].Br Med Bull,2013,105(1):185-199.
[3] 曾花,蔡道章,陈郁鲜,等.人膝骨关节炎血清生物学标志物的筛选[J].中国病理生理杂志,2012,28(5):884-888.
[4] 刘强,洪加源,胡维界.膝骨性关节炎治疗进展[J].医学综述,2015,21(3):474-476.
[5] 姜伟华,潘立群,陈荣明.中药外治膝骨关节炎研究进展[J].江苏中医药,2012,44(1):76-77.
[6] 李宏军.透明质酸钠注射配合千草方薰洗治疗膝骨关节炎的临床及实验研究[D].武汉:湖北中医学院,2008.
[7] 齐梦迭,王学敏.YAP调节细胞增殖和凋亡作用机制的研究进展[J].医学综述,2015,21(6):994-996.
[8] Qin Z,Xia W,Fisher GJ,et al.YAP/TAZ regulates TGF-β/Smad3 signaling by induction of Smad7 via AP-1 in human skin dermal fibroblasts[J].Cell Commun Signal,2018,16(1):18.
[9] 牛维,孙志涛,曹学伟,等.单味药鹿茸调控大鼠骨关节炎软骨组织Smad2、3表达的研究[J].中国中西医结合杂志,2014,34(2):209-213.
[10] Mankin HJ,Dorfman H,Lippiello L,et al.Biochemical and metabolic abnormalities in articular cartilage from osteo-arthritic human hips.II.Correlation of morphology with biochemical and metabolic data[J].J Bone Joint Surg Am,1971,53(3):523-537.
[11] Michael JW,Schlüter-Brust KU,Eysel P.The epidemiology,etiology,diagnosis,and treatment of osteoarthritis of the knee[J].Dtsch Arztebl Int,2010,107(9):152-162.
[12] Sarukawa J,Takahashi M,Doi M,et al.A longitudinal analysis of urinary biochemical markers and bone mineral density in STR/Ort mice as a model of spontaneous osteoarthritis[J].Arthritis Rheum,2010,62(2):463-471.
[13] King KB,Lindsey CT,Dunn TC,et al.A study of the relationship between molecular biomarkers of joint degeneration and the magnetic resonance-measured characteristics of cartilage in 16 symptomatic knees[J].Mag Reson Imaging,2004,22(8):1117-1123.
[14] 熊勇,彭锐,王华.“双固一通”温针灸对实验性兔膝骨性关节炎软骨细胞凋亡的影响[J].中国中医骨伤科杂志,2011,19(11):4-6.
[15] 唐国栋,杨国夫.HMGB2与骨性关节炎相关性的研究进展[J].中国矫形外科杂志,2015,23(13):1199-1201.
[16] Taniguchi N,Caramés B,Ronfani L,et al.Aging-related loss of the chromatin protein HMGB2 in articular cartilage is linked to reduced cellularity and osteoarthritis[J].Proc Natl Acad Sci U S A,2009,106(4):1181-1186.
[17] 王晶,张洹.雌激素对人骨髓间质细胞BMP-2 mRNA表达的影响[J].中国病理生理杂志,2007,23(3):584-586.
[18] 杨俊龙,王艳娉,张源,等.透明质酸治疗膝骨关节炎相关机制的研究进展[J].中国疼痛医学杂志,2017,23(6):451-454.