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[1]陳世宣,劉益杰,胡笑燊,等.基于瞬時(shí)受體電位香草素亞家族4信號(hào)通路探討牛蒡子苷元對(duì)體外培養(yǎng)軟骨細(xì)胞增殖及Ⅱ型膠原蛋白和軟骨蛋白聚糖表達(dá)的影響[J].中醫(yī)正骨,2017,29(10):13-19.
　CHEN Shixuan,LIU Yijie,HU Xiaoshen,et al.An experimental study of 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 signaling pathway[J].The Journal of Traditional Chinese Orthopedics and Traumatology,2017,29(10):13-19.
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基于瞬時(shí)受體電位香草素亞家族4信號(hào)通路探討牛蒡子苷元對(duì)體外培養(yǎng)軟骨細(xì)胞增殖及Ⅱ型膠原蛋白和軟骨蛋白聚糖表達(dá)的影響()
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《中醫(yī)正骨》[ISSN:1001-6015/CN:41-1162/R]

卷:
第29卷
期數(shù):
2017年10期
頁(yè)碼:
13-19
欄目:
基礎(chǔ)研究
出版日期:
2017-10-20

文章信息/Info

Title:
An experimental study of 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 signaling pathway
作者:
陳世宣1; 劉益杰1; 胡笑燊1; 李安琪1; 厲坤鵬1; 馮偉2
1.上海中醫(yī)藥大學(xué),上海 201203; 2.上海市第七人民醫(yī)院,上海 201137
Author(s):
CHEN Shixuan1; LIU Yijie1; HU Xiaoshen1; ZHANG Zengqiao1; LI Anqi1; LI Kunpeng1; FENG Wei2
1.Shanghai University of Traditional Chinese Medicine,Shanghai 201203,China 2.The Seventh People's Hospital of Shanghai,Shanghai 201137,China
關(guān)鍵詞:
骨關(guān)節(jié)炎;  牛蒡子甙;  軟骨細(xì)胞;  瞬時(shí)受體電位香草素亞家族4;  細(xì)胞增殖;  膠原Ⅱ型;  軟骨蛋白聚糖類(lèi)
Keywords:
Key words osteoarthritis;  arctiin;  chondrocytes;  TRPV4;  cell proliferation;  collagen typeⅡ;  aggrecans
摘要:
目的:基于瞬時(shí)受體電位香草素亞家族4(transient receptor potential vanilloid 4,TRPV4)信號(hào)通路探討牛蒡子苷元對(duì)體外培養(yǎng)軟骨細(xì)胞增殖及Ⅱ型膠原蛋白和軟骨蛋白聚糖表達(dá)的影響。方法:自新生SD大鼠膝關(guān)節(jié)軟骨組織提取軟骨細(xì)胞,進(jìn)行體外培養(yǎng),取第1代軟骨細(xì)胞分別加入不同培養(yǎng)液進(jìn)行干預(yù)。空白組僅加入常規(guī)培養(yǎng)液,牛蒡子苷元組加入含10 μmol牛蒡子苷元的培養(yǎng)液,牛蒡子苷元聯(lián)合阻斷劑組加入含10 μmol牛蒡子苷元和10 μmol GSK205的培養(yǎng)液,激動(dòng)劑組加入含1 μmol 4α-PPD的培養(yǎng)液,阻斷劑聯(lián)合激動(dòng)劑組加入含10 μmol GSK205和1 μmol 4α-PPD 的培養(yǎng)液,阻斷劑組加入含10 μmol GSK205的培養(yǎng)液。分別于干預(yù)24 h和72 h后以CCK-8法檢測(cè)軟骨細(xì)胞增殖情況,48 h后以Western blot法檢測(cè)軟骨蛋白聚糖和Ⅱ型膠原蛋白水平。結(jié)果:①軟骨細(xì)胞增殖測(cè)定結(jié)果。干預(yù)24 h后,6組的吸光度比較,差異有統(tǒng)計(jì)學(xué)意義(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)。牛蒡子苷元組、激動(dòng)劑組的吸光度均高于空白組(P=0.000,P=0.000); 牛蒡子苷元聯(lián)合阻斷劑組、阻斷劑聯(lián)合激動(dòng)劑組、阻斷劑組的吸光度與空白組比較,組間差異均無(wú)統(tǒng)計(jì)學(xué)意義(P=0.632,P=0.840,P=0.164); 牛蒡子苷元組的吸光度高于牛蒡子苷元聯(lián)合阻斷劑組、阻斷劑聯(lián)合激動(dòng)劑組(P=0.000,P=0.000); 牛蒡子苷元組與激動(dòng)劑組比較,差異無(wú)統(tǒng)計(jì)學(xué)意義(P=0.834); 牛蒡子苷元聯(lián)合阻斷劑組的吸光度低于激動(dòng)劑組(P=0.000),與阻斷劑聯(lián)合激動(dòng)劑組比較,差異無(wú)統(tǒng)計(jì)學(xué)意義(P=0.498); 激動(dòng)劑組的吸光度高于阻斷劑聯(lián)合激動(dòng)劑組(P=0.000)。干預(yù)72 h后,6組的吸光度比較,差異有統(tǒng)計(jì)學(xué)意義(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)。牛蒡子苷元組、牛蒡子苷元聯(lián)合阻斷劑組、激動(dòng)劑組的吸光度均高于空白組(P=0.000,P=0.001,P=0.000); 阻斷劑聯(lián)合激動(dòng)劑組、阻斷劑組與空白組比較,組間差異均無(wú)統(tǒng)計(jì)學(xué)意義(P=0.326,P=0.104); 牛蒡子苷元組的吸光度高于牛蒡子苷元聯(lián)合阻斷劑組、激動(dòng)劑組及阻斷劑聯(lián)合激動(dòng)劑組(P=0.000,P=0.000,P=0.000); 牛蒡子苷元聯(lián)合阻斷劑組的吸光度低于激動(dòng)劑組(P=0.010),與阻斷劑聯(lián)合激動(dòng)劑組比較,差異無(wú)統(tǒng)計(jì)學(xué)意義(P=0.098); 激動(dòng)劑組的吸光度高于阻斷劑聯(lián)合激動(dòng)劑組(P=0.000)。②軟骨蛋白聚糖和Ⅱ型膠原蛋白水平測(cè)定結(jié)果。5組軟骨細(xì)胞中軟骨蛋白聚糖水平比較,差異有統(tǒng)計(jì)學(xué)意義(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)。牛蒡子苷元組、激動(dòng)劑組的軟骨蛋白聚糖水平均高于空白組(P=0.000,P=0.000); 牛蒡子苷元聯(lián)合阻斷劑組、阻斷劑聯(lián)合激動(dòng)劑組與空白組比較,組間差異均無(wú)統(tǒng)計(jì)學(xué)意義(P=0.136,P=0.593); 牛蒡子苷元組的軟骨蛋白聚糖水平高于牛蒡子苷元聯(lián)合阻斷劑組、阻斷劑聯(lián)合激動(dòng)劑組(P=0.000,P=0.000); 牛蒡子苷元組與激動(dòng)劑組比較,差異無(wú)統(tǒng)計(jì)學(xué)意義(P=0.305); 牛蒡子苷元聯(lián)合阻斷劑組的軟骨蛋白聚糖水平低于激動(dòng)劑組(P=0.000),與阻斷劑聯(lián)合激動(dòng)劑組比較,差異無(wú)統(tǒng)計(jì)學(xué)意義(P=0.310); 激動(dòng)劑組的軟骨蛋白聚糖水平高于阻斷劑聯(lián)合激動(dòng)劑組(P=0.000)。5組軟骨細(xì)胞中Ⅱ型膠原蛋白水平比較,差異有統(tǒng)計(jì)學(xué)意義(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)。牛蒡子苷元組、牛蒡子苷元聯(lián)合阻斷劑組、激動(dòng)劑組的Ⅱ型膠原蛋白水平均高于空白組(P=0.000,P=0.000,P=0.000); 阻斷劑聯(lián)合激動(dòng)劑組的Ⅱ型膠原蛋白水平低于空白組(P=0.032); 牛蒡子苷元組的Ⅱ型膠原蛋白水平高于牛蒡子苷元聯(lián)合阻斷劑組、激動(dòng)劑組、阻斷劑聯(lián)合激動(dòng)劑組(P=0.000,P=0.000,P=0.000); 牛蒡子苷元聯(lián)合阻斷劑組的Ⅱ型膠原蛋白水平高于阻斷劑聯(lián)合激動(dòng)劑組(P=0.000),與激動(dòng)劑組比較,差異無(wú)統(tǒng)計(jì)學(xué)意義(P=0.193); 激動(dòng)劑組的Ⅱ型膠原蛋白水平高于阻斷劑聯(lián)合激動(dòng)劑組(P=0.000)。結(jié)論:牛蒡子苷元可通過(guò)TRPV4信號(hào)通路促進(jìn)體外培養(yǎng)軟骨細(xì)胞的增殖及軟骨細(xì)胞中Ⅱ型膠原蛋白和軟骨蛋白聚糖的表達(dá)。
Abstract:
ABSTRACT 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 blocker-agonist 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 the 5 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.

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備注/Memo:
基金項(xiàng)目:上海市自然科學(xué)基金資助項(xiàng)目(15ZR1441000) 通訊作者:馮偉 E-mail:[email protected]
更新日期/Last Update: 2018-03-10