基于瞬时受体电位香草素亚家族4信号通路探讨牛蒡子苷元对体外培养软骨细胞增殖及Ⅱ型胶原蛋白和软骨蛋白聚糖表达的影响

详细信息    查看全文 | 推荐本文 |

英文篇名：An experimental study of effect of arctigenin on proliferation of chondrocyte cultured in vitro and expression of type Ⅱ collagen protein and aggrecan based on the transient receptor potential vanilloid 4 signaling pathway 作者：陈世宣 ; 刘益杰 ; 胡笑燊 ; 张增乔 ; 李安琪 ; 厉坤鹏 ; 冯伟 英文作者：CHEN Shixuan;LIU Yijie;HU Xiaoshen;ZHANG Zengqiao;LI Anqi;LI Kunpeng;FENG Wei;Shanghai University of Traditional Chinese Medicine;The Seventh People's Hospital of Shanghai; 关键词：骨关节炎 ; 牛蒡子甙 ; 软骨细胞 ; 瞬时受体电位香草素亚家族4 ; 细胞增殖 ; 胶原Ⅱ型 ; 软骨蛋白聚糖类 英文关键词：osteoarthritis;;arctiin;;chondrocytes;;TRPV4;;cell proliferation;;collagen typeⅡ;;aggrecans 中文刊名：ZYZG 英文刊名：The Journal of Traditional Chinese Orthopedics and Traumatology 机构：上海中医药大学;上海市第七人民医院; 出版日期：2017-10-20 出版单位：中医正骨 年：2017 期：v.29;No.271 基金：上海市自然科学基金资助项目(15ZR1441000) 语种：中文; 页：ZYZG201710002 页数：7 CN：10 ISSN：41-1162/R 分类号：19-25

摘要

目的:基于瞬时受体电位香草素亚家族4(transient receptor potential vanilloid 4,TRPV4)信号通路探讨牛蒡子苷元对体外培养软骨细胞增殖及Ⅱ型胶原蛋白和软骨蛋白聚糖表达的影响。方法:自新生SD大鼠膝关节软骨组织提取软骨细胞,进行体外培养,取第1代软骨细胞分别加入不同培养液进行干预。空白组仅加入常规培养液,牛蒡子苷元组加入含10μmol牛蒡子苷元的培养液,牛蒡子苷元联合阻断剂组加入含10μmol牛蒡子苷元和10μmol GSK205的培养液,激动剂组加入含1μmol 4α-PPD的培养液,阻断剂联合激动剂组加入含10μmol GSK205和1μmol 4α-PPD的培养液,阻断剂组加入含10μmol GSK205的培养液。分别于干预24 h和72 h后以CCK-8法检测软骨细胞增殖情况,48 h后以Western blot法检测软骨蛋白聚糖和Ⅱ型胶原蛋白水平。结果:(1)软骨细胞增殖测定结果。干预24 h后,6组的吸光度比较,差异有统计学意义(1.03±0.72,1.32±0.11,1.01±0.05,1.33±0.34,1.04±0.50,1.10±0.06;F=18.309,P=0.000)。牛蒡子苷元组、激动剂组的吸光度均高于空白组(P=0.000,P=0.000);牛蒡子苷元联合阻断剂组、阻断剂联合激动剂组、阻断剂组的吸光度与空白组比较,组间差异均无统计学意义(P=0.632,P=0.840,P=0.164);牛蒡子苷元组的吸光度高于牛蒡子苷元联合阻断剂组、阻断剂联合激动剂组(P=0.000,P=0.000);牛蒡子苷元组与激动剂组比较,差异无统计学意义(P=0.834);牛蒡子苷元联合阻断剂组的吸光度低于激动剂组(P=0.000),与阻断剂联合激动剂组比较,差异无统计学意义(P=0.498);激动剂组的吸光度高于阻断剂联合激动剂组(P=0.000)。干预72 h后,6组的吸光度比较,差异有统计学意义(1.66±0.02,2.21±0.05,1.84±0.04,1.92±0.07,1.71±0.10,1.74±0.08;F=37.629,P=0.000)。牛蒡子苷元组、牛蒡子苷元联合阻断剂组、激动剂组的吸光度均高于空白组(P=0.000,P=0.001,P=0.000);阻断剂联合激动剂组、阻断剂组与空白组比较,组间差异均无统计学意义(P=0.326,P=0.104);牛蒡子苷元组的吸光度高于牛蒡子苷元联合阻断剂组、激动剂组及阻断剂联合激动剂组(P=0.000,P=0.000,P=0.000);牛蒡子苷元联合阻断剂组的吸光度低于激动剂组(P=0.010),与阻断剂联合激动剂组比较,差异无统计学意义(P=0.098);激动剂组的吸光度高于阻断剂联合激动剂组(P=0.000)。(2)软骨蛋白聚糖和Ⅱ型胶原蛋白水平测定结果。5组软骨细胞中软骨蛋白聚糖水平比较,差异有统计学意义(1.00±0.00,20.74±5.01,4.20±0.66,22.87±1.82,2.09±0.63;F=194.544,P=0.000)。牛蒡子苷元组、激动剂组的软骨蛋白聚糖水平均高于空白组(P=0.000,P=0.000);牛蒡子苷元联合阻断剂组、阻断剂联合激动剂组与空白组比较,组间差异均无统计学意义(P=0.136,P=0.593);牛蒡子苷元组的软骨蛋白聚糖水平高于牛蒡子苷元联合阻断剂组、阻断剂联合激动剂组(P=0.000,P=0.000);牛蒡子苷元组与激动剂组比较,差异无统计学意义(P=0.305);牛蒡子苷元联合阻断剂组的软骨蛋白聚糖水平低于激动剂组(P=0.000),与阻断剂联合激动剂组比较,差异无统计学意义(P=0.310);激动剂组的软骨蛋白聚糖水平高于阻断剂联合激动剂组(P=0.000)。5组软骨细胞中Ⅱ型胶原蛋白水平比较,差异有统计学意义(1.00±0.00,2.94±0.11,1.92±0.17,2.04±0.12,0.78±0.09;F=58.701,P=0.000)。牛蒡子苷元组、牛蒡子苷元联合阻断剂组、激动剂组的Ⅱ型胶原蛋白水平均高于空白组(P=0.000,P=0.000,P=0.000);阻断剂联合激动剂组的Ⅱ型胶原蛋白水平低于空白组(P=0.032);牛蒡子苷元组的Ⅱ型胶原蛋白水平高于牛蒡子苷元联合阻断剂组、激动剂组、阻断剂联合激动剂组(P=0.000,P=0.000,P=0.000);牛蒡子苷元联合阻断剂组的Ⅱ型胶原蛋白水平高于阻断剂联合激动剂组(P=0.000),与激动剂组比较,差异无统计学意义(P=0.193);激动剂组的Ⅱ型胶原蛋白水平高于阻断剂联合激动剂组(P=0.000)。结论:牛蒡子苷元可通过TRPV4信号通路促进体外培养软骨细胞的增殖及软骨细胞中Ⅱ型胶原蛋白和软骨蛋白聚糖的表达。
        Objective: To explore the effect of arctigenin on proliferation of chondrocyte cultured in vitro and expression of typeⅡcollagen protein and aggrecan based on the transient receptor potential vanilloid 4( TRPV4) signaling pathway. Methods: The chondrocytes were extracted from the knee cartilage tissues of newborn SD rats and were cultured in vitro,then the first-generation chondrocytes in blank group,arctigenin group,arctigenin-blocker group,agonist group,blocker-agonist group and blocker group were cultured in conventional culture medium,arctigenin( 10 μmol) culture medium,arctigenin( 10 μmol)-GSK205( 10 μmol) culture medium,4α-PPD( 1 μmol) culture medium,GSK205( 10 μmol)-4α-PPD( 1 μmol) culture medium and GSK205( 10 μmol) culture medium respectively. The chondrocyte proliferation was detected by using CCK-8 method after 24-and 72-hour intervention respectively,and the expression of aggrecan and typeⅡcollagen protein were detected by using Western blot method after 48-hour intervention. Results: After 24-hour intervention,there was statistical difference in the absorbance between the 6 groups( 1. 03 +/-0. 72,1. 32 +/-0. 11,1. 01 +/-0. 05,1. 33 +/-0. 34,1. 04 +/-0. 50,1. 10 +/-0. 06; F = 18. 309,P = 0. 000). The absorbance was higher in arctigenin group and agonist group compared to blank group( P = 0. 000,P = 0. 000). There was no statistical difference in the absorbance between arctigenin-blocker group and blank group and between blocker-agonist group and blank group and between blocker group and blank group( P = 0. 632,P = 0. 840,P =0. 164). The absorbance was higher in arctigenin group compared to arctigenin-blocker group and blocker-agonist group( P = 0. 000,P =0. 000). There was no statistical difference in the absorbance between arctigenin group and agonist group( P = 0. 834). The absorbance was lower in arctigenin-blocker group compared to agonist group( P = 0. 000). There was no statistical difference in the absorbance between arctigenin-blocker group and blocker-agonist group( P = 0. 498). The absorbance was higher in agonist group compared to blocker-agonist group( P = 0. 000). After 72-hour intervention,there was statistical difference in the absorbance between the 6 groups( 1. 66 +/-0. 02,2. 21 +/-0. 05,1. 84 +/-0. 04,1. 92 +/-0. 07,1. 71 +/-0. 10,1. 74 +/-0. 08; F = 37. 629,P = 0. 000). The absorbance were higher in arctigenin group,arctigenin-blocker group and agonist group compared to blank group( P = 0. 000,P = 0. 001,P = 0. 000). There was no statistical difference in the absorbance between blocker-agonist group and blank group and between blocker group and blank group( P = 0. 326,P = 0. 104). The absorbance was higher in arctigenin group compared to arctigenin-blocker group,agonist group and blockeragonist group( P = 0. 000,P = 0. 000,P = 0. 000),and was lower in arctigenin-blocker group compared to agonist group( P = 0. 010).There was no statistical difference in the absorbance between arctigenin-blocker group and blocker-agonist group( P = 0. 098),and the absorbance was higher in agonist group compared to blocker-agonist group( P = 0. 000). There was statistical difference in aggrecan level in chondrocytes between the 5 groups( 1. 00 +/-0. 00,20. 74 +/-5. 01,4. 20 +/-0. 66,22. 87 +/-1. 82,2. 09 +/-0. 63; F = 194. 544,P =0. 000). The aggrecan level was higher in arctigenin group and agonist group compared to blank group( P = 0. 000,P = 0. 000). There was no statistical difference in aggrecan level between arctigenin-blocker group and blank group and between blocker-agonist group and blank group( P = 0. 136,P = 0. 593). The aggrecan level was higher in arctigenin group compared to arctigenin-blocker group and blocker-agonist group( P = 0. 000,P = 0. 000). There was no statistical difference in aggrecan level between arctigenin group and agonist group( P =0. 305). The aggrecan level was lower in arctigenin-blocker group compared to agonist group( P = 0. 000),and there was no statistical difference in aggrecan level between arctigenin-blocker group and blocker-agonist group( P = 0. 310). The aggrecan level was higher in agonist group compared to blocker-agonist group( P = 0. 000). There was statistical difference in typeⅡcollagen protein level between the5 groups( 1. 00 +/-0. 00,2. 94 +/-0. 11,1. 92 +/-0. 17,2. 04 +/-0. 12,0. 78 +/-0. 09; F = 58. 701,P = 0. 000). The typeⅡcollagen protein levels were higher in arctigenin group,arctigenin-blocker group and agonist group compared to blank group( P = 0. 000,P =0. 000,P = 0. 000),and was lower in blocker-agonist group compared to blank group( P = 0. 032),and was higher in arctigenin group compared to arctigenin-blocker group,agonist group and blocker-agonist group( P = 0. 000,P = 0. 000,P = 0. 000),and was higher in arctigenin-blocker group compared to blocker-agonist group( P = 0. 000). There was no statistical difference in typeⅡcollagen protein level between arctigenin-blocker group and agonist group( P = 0. 193),and the typeⅡcollagen protein level was higher in agonist group compared to blocker-agonist group( P = 0. 000). Conclusion: Arctigenin can promote the proliferation of chondrocyte cultured in vitro and the expression of typeⅡcollagen protein and aggrecan through the TRPV4 signaling pathway.

引文

[1]VINCENT TL.Targeting mechanotransduction pathways in osteoarthritis:a focus on the pericellular matrix[J].Curr Opin Pharmacol,2013,13(3):449-454.
    [2]原晓强,金王东,周云婧,等.纯化血小板对大鼠软骨细胞增殖及膝骨关节炎大鼠软骨修复的作用研究[J].中医正骨,2016,28(12):6-12.
    [3]MCNULTY AL,LEDDY HA,LIEDTKE W,et al.TRPV4 as a therapeutic target for joint diseases[J].Naunyn Schmiedebergs Arch Pharmacol,2015,388(4):437-450.
    [4]NISHIMURA G,LAUSCH E,SAVARIRAYAN R,et al.TRPV4-associated skeletal dysplasias[J].Am J Med Genet C Semin Med Genet,2012,160C(3):190-204.
    [5]KANG SS,SHIN SH,AUH CK,et al.Human skeletal dysplasia caused by a constitutive activated transient receptor potential vanilloid 4(TRPV4)cation Channel mutation[J].Exp Mol Med,2012,44(12):707-722.
    [6]CLARK AL,VOTTA BJ,KUMAR S,et al.Chondroprotective role of the osmotically sensitive ion Channel transient receptor potential vanilloid 4:age-and sex-dependent progression of osteoarthritis in Trpv4-deficient mice[J].Arthritis Rheum,2010,62(10):2973-2983.
    [7]LIEDTKE W,CHOE Y,MARTí-RENOM MA,et al.Vanilloid receptor-related osmotically activated Channel(VROAC),a candidate vertebrate osmoreceptor[J].Cell,2000,103(3):525-535.
    [8]O’CONOR CJ,GRIFFIN TM,LIEDTKE W,et al.Increased susceptibility of Trpv4-deficient mice to obesity and obesity-induced osteoarthritis with very high-fat diet[J].Ann Rheum Dis,2013,72(2):300-304.
    [9]石琤,屠安琪,吴军豪.石氏经验方牛蒡子汤在伤科的临床应用[J].中医文献杂志,2015,33(2):44-46.
    [10]陈世宣,冯伟,周一心,等.牛蒡子苷元对关节软骨细胞增殖及Ⅱ型胶原表达的影响[J].上海中医药杂志,2015,49(7):69-71.