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[1]葉錦霞,付長龍,林潔,等.透骨消痛膠囊對毒胡蘿卜素誘導(dǎo)的內(nèi)質(zhì)網(wǎng)應(yīng)激PEKR信號通路介導(dǎo)的大鼠體外培養(yǎng)關(guān)節(jié)軟骨細胞凋亡的影響[J].中醫(yī)正骨,2017,29(06):1-7.
　YE Jinxia,FU Changlong,LIN Jie,et al.Effect of Tougu Xiaotong Jiaonang(透骨消痛膠囊)on apoptosis mediated by endoplasmic reticulum stress(PEKR signaling pathway)and induced by thapsigargin in rat's articular chondrocytes cultured in vitro[J].The Journal of Traditional Chinese Orthopedics and Traumatology,2017,29(06):1-7.
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透骨消痛膠囊對毒胡蘿卜素誘導(dǎo)的內(nèi)質(zhì)網(wǎng)應(yīng)激PEKR信號通路介導(dǎo)的大鼠體外培養(yǎng)關(guān)節(jié)軟骨細胞凋亡的影響()
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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06期
頁碼:
1-7
欄目:
基礎(chǔ)研究
出版日期:
2017-06-20

文章信息/Info

Title:
Effect of Tougu Xiaotong Jiaonang(透骨消痛膠囊)on apoptosis mediated by endoplasmic reticulum stress(PEKR signaling pathway)and induced by thapsigargin in rat's articular chondrocytes cultured in vitro
作者:
葉錦霞; 付長龍; 林潔; 吳廣文; 鄭春松; 陳俊; 葉蕻芝
福建中醫(yī)藥大學,福建 福州 350122
Author(s):
YE Jinxia; FU Changlong; LIN Jie; WU Guangwen; ZHENG Chunsong; CHEN Jun; YE Hongzhi
Fujian University of Traditional Chinese Medicine,Fuzhou 350112,Fujian,China
關(guān)鍵詞:
骨關(guān)節(jié)炎;  透骨消痛膠囊;  軟骨細胞;  軟骨; 關(guān)節(jié);  凋亡;  內(nèi)質(zhì)網(wǎng)應(yīng)激;  毒胡蘿卜素;  大鼠;  動物實驗
Keywords:
Key words osteoarthritis;  Tougu Xiaotong Jiaonang;  chondrocytes;  cartilage; articular;  apoptosis;  endoplasmic reticulum stress;  thapsigargin;  rats;  animal experimentation
摘要:
目的:觀察透骨消痛膠囊對毒胡蘿卜素(thapsigargin,TG)誘導(dǎo)的內(nèi)質(zhì)網(wǎng)應(yīng)激(PEKR信號通路)介導(dǎo)的大鼠體外培養(yǎng)關(guān)節(jié)軟骨細胞凋亡的影響。方法:采用4周齡SD大鼠膝關(guān)節(jié)軟骨建立軟骨細胞體外培養(yǎng)體系,將培養(yǎng)的第3代軟骨細胞分為空白組、模型組及透骨消痛膠囊高、中、低劑量組。空白組不進行干預(yù); 模型組及透骨消痛膠囊高、中、低劑量組先以2 μmol·L-1 TG干預(yù)4 h,然后模型組更換為正常培養(yǎng)基,透骨消痛膠囊高、中、低劑量組分別更換為濃度為100 μg·mL-1、50 μg·mL-1、25 μg·mL-1的含透骨消痛膠囊培養(yǎng)基干預(yù)24 h。干預(yù)后以MTT法檢測各組軟骨細胞活性、以AnnexinⅤ-FITC/PI雙染法流式細胞術(shù)檢測各組軟骨細胞凋亡率、以Western Blot法檢測各組軟骨細胞中激活轉(zhuǎn)錄因子4(activating transcription factor 4,ATF4)、結(jié)合免疫球蛋白(binding immunoglobulin protein,BIP)、天冬氨酸特異性半胱氨酸蛋白酶12(cysteine aspartate specific protease 12,Caspase12)和Caspase3的表達量。結(jié)果:①軟骨細胞活性。5組軟骨細胞吸光度比較,差異有統(tǒng)計學意義(0.428±0.009,0.226±0.028,0.345±0.025,0.301±0.035,0.269±0.033,F=39.462,P=0.000)。模型組吸光度低于空白組(P=0.000); 透骨消痛膠囊高、中、低劑量組吸光度均高于模型組(P=0.000,P=0.000,P=0.022); 透骨消痛膠囊中、低劑量組的吸光度均低于高劑量組(P=0.019,P=0.000); 透骨消痛膠囊低劑量組與中劑量組比較,差異無統(tǒng)計學意義(P=0.085)。②軟骨細胞凋亡率。5組軟骨細胞凋亡率比較,差異有統(tǒng)計學意義[(3.270±0.231)%,(30.003±4.128)%,(12.383±0.933)%,(15.387±2.961)%,(20.143±3.472)%,F=37.560,P=0.000]。模型組軟骨細胞凋亡率高于空白組(P=0.000); 透骨消痛膠囊高、中、低劑量組軟骨細胞凋亡率均低于模型組(P=0.000,P=0.000,P=0.001); 透骨消痛膠囊低劑量組軟骨細胞凋亡率低于高劑量組(P=0.007),透骨消痛膠囊高、低劑量組與中劑量組比較,差異均無統(tǒng)計學意義(P=0.216,P=0.063)。③軟骨細胞內(nèi)質(zhì)網(wǎng)應(yīng)激(PEKR信號通路)關(guān)鍵蛋白含量。5組軟骨細胞中ATF4含量比較,差異有統(tǒng)計學意義(0.257±0.028,0.435±0.033,0.315±0.023,0.276±0.038,0.351±0.043,F=13.057,P=0.001)。空白組及透骨消痛膠囊高、中、低劑量組的ATF4含量均低于模型組(P=0.000,P=0.001,P=0.000,P=0.012); 透骨消痛膠囊低劑量組的ATF4含量高于中劑量組(P=0.022); 透骨消痛膠囊高劑量組的ATF4含量與透骨消痛膠囊中、低劑量組比較,差異均無統(tǒng)計學意義(P=0.188,P=0.229)。5組軟骨細胞中BIP含量比較,差異有統(tǒng)計學意義(0.227±0.026,0.432±0.022,0.294±0.035,0.263±0.020,0.339±0.032,F=25.416,P=0.000)。空白組及透骨消痛膠囊高、中、低劑量組的BIP含量均低于模型組(P=0.000,P=0.000,P=0.000,P=0.002); 透骨消痛膠囊低劑量組的BIP含量高于中劑量組(P=0.006); 透骨消痛膠囊高劑量組的BIP含量與透骨消痛膠囊中、低劑量組比較,差異均無統(tǒng)計學意義(P=0.185,P=0.072)。5組軟骨細胞中Caspase12含量比較,差異有統(tǒng)計學意義(0.252±0.027,0.346±0.028,0.294±0.011,0.315±0.024,0.325±0.019,F=7.345,P=0.005)。模型組的Caspase12含量高于空白組(P=0.001); 透骨消痛膠囊高劑量組的Caspase12含量低于模型組(P=0.018); 透骨消痛膠囊中、低劑量組的Caspase12含量與模型組比較,差異均無統(tǒng)計學意義(P=0.126,P=0.277); 透骨消痛膠囊中、低劑量組的Caspase12含量與高劑量組比較,差異均無統(tǒng)計學意義(P=0.277,P=0.126); 透骨消痛膠囊中、低劑量組的Caspase12含量比較,差異無統(tǒng)計學意義(P=0.614)。5組軟骨細胞中Caspase3含量比較,差異有統(tǒng)計學意義(0.265±0.028,0.380±0.017,0.317±0.026,0.304±0.019,0.333±0.022,F=10.507,P=0.001)。模型組的Caspase3含量高于空白組(P=0.000); 透骨消痛膠囊高、中、低劑量組的Caspase3含量均低于模型組(P=0.006,P=0.002,P=0.029); 透骨消痛膠囊中、低劑量組的Caspase3含量與高劑量組比較,差異均無統(tǒng)計學意義(P=0.496,P=0.387),透骨消痛膠囊中、低劑量組的Caspase3含量比較,差異無統(tǒng)計學意義(P=0.138)。結(jié)論:透骨消痛膠囊能抑制TG誘導(dǎo)的大鼠體外培養(yǎng)關(guān)節(jié)軟骨細胞發(fā)生由內(nèi)質(zhì)網(wǎng)應(yīng)激(PEKR信號通路)介導(dǎo)的細胞凋亡,其作用效果與藥物劑量無明顯關(guān)系。
Abstract:
ABSTRACT Objective:To observe the effect of Tougu Xiaotong Jiaonang(透骨消痛膠囊,TGXTJN)on apoptosis mediated by endoplasmic reticulum stress(PEKR signaling pathway)and induced by thapsigargin(TG)in rat's articular chondrocytes cultured in vitro.Methods:The knee cartilage of 4-week-old SD rats were used to build the in-vitro cultivation system of chondrocytes,and the third-generation chondrocytes of SD rats cultured in vitro were divided into blank group,model group,TGXTJN high-dose group,TGXTJN middle-dose group and TGXTJN low-dose group.The cells in blank group were not be treated and the cells in other groups were treated with TG(2 μmol/L)for 4 hours.Then the cells in model group were cultured in normal nutritive medium,and the cells in TGXTJN high-,middle- and low-dose group were cultured in nutritive medium supplemented with TGXTJN with concentration of 100,50 and 25 μg/mL respectively for 24 hours.After the end of intervention,the chondrocyte activities were detected by using MTT method,the apoptosis rates of chondrocytes were detected by using AnnexinⅤ-FITC/PI double-staining flow cytometry(FCM),and the expressions of activating transcription factor 4(ATF4),binding immunoglobulin protein(BIP),cysteine aspartate specific protease 12(Caspase12)and Caspase3 in chondrocytes were detected by using Western Blot assays.Results:There was statistical difference in the absorbance of chondrocytes between the 5 groups(0.428+/-0.009,0.226+/-0.028,0.345+/-0.025,0.301+/-0.035,0.269+/-0.033,F=39.462,P=0.000).The absorbance of chondrocytes were lower in model group compared to blank group(P=0.000)and were higher in TGXTJN high-,middle- and low-dose group compared to model group(P=0.000,P=0.000,P=0.022)and were lower in TGXTJN middle- and low-dose group compared to TGXTJN high-dose group(P=0.019,P=0.000).There was no statistical difference in absorbance of chondrocytes between TGXTJN low-dose group and TGXTJN middle-dose group(P=0.085).There was statistical difference in apoptosis rate of chondrocytes between the 5 groups(3.270+/-0.231,30.003+/-4.128,12.383+/-0.933,15.387+/-2.961,20.143+/-3.472%,F=37.560,P=0.000).The apoptosis rate of chondrocytes was higher in model group compared to blank group(P=0.000)and were lower in TGXTJN high-,middle- and low-dose group compared to model group(P=0.000,P=0.000,P=0.001)and was lower in TGXTJN low-dose group compared to TGXTJN high-dose group(P=0.007).There was no statistical difference in apoptosis rate of chondrocytes between TGXTJN high-dose group and TGXTJN middle-dose group and between TGXTJN low-dose group and TGXTJN middle-dose group(P=0.216,P=0.063).There was statistical difference in contents of ATF4 in chondrocytes between the 5 groups(0.257+/-0.028,0.435+/-0.033,0.315+/-0.023,0.276+/-0.038,0.351+/-0.043,F=13.057,P=0.001).The ATF4 contents were lower in blank group and TGXTJN high-, middle- and low-dose group compared to model group(P=0.000,P=0.001,P=0.000,P=0.012)and were higher in TGXTJN low-dose group compared to TGXTJN middle-dose group(P=0.022).There was no statistical difference in ATF4 contents between TGXTJN high-dose group and TGXTJN middle-dose group and between TGXTJN high-dose group and TGXTJN low-dose group(P=0.188,P=0.229).There was statistical difference in contents of BIP in chondrocytes between the 5 groups(0.227+/-0.026,0.432+/-0.022,0.294+/-0.035,0.263+/-0.020,0.339+/-0.032,F=25.416,P=0.000).The BIP contents were lower in blank group,TGXTJN high-,middle- and low-dose group compared to model group(P=0.000,P=0.000,P=0.000,P=0.002).The BIP contents were higher in TGXTJN low-dose group compared to TGXTJN middle-dose group(P=0.006).There was no statistical difference in BIP contents between TGXTJN high-dose group and TGXTJN middle-dose group and between TGXTJN high-dose group and TGXTJN low-dose group(P=0.185,P=0.072).There was statistical difference in contents of Caspase 12 in chondrocytes between the 5 groups(0.252+/-0.027,0.346+/-0.028,0.294+/-0.011,0.315+/-0.024,0.325+/-0.019,F=7.345,P=0.005).The Caspase 12 contents were higher in model group compared to blank group(P=0.001)and TGXTJN high-dose group(P=0.018).There was no statistical difference in Caspase 12 contents between TGXTJN middle-dose group and model group and between TGXTJN low-dose group and model group(P=0.126,P=0.277).There was no statistical difference in Caspase 12 contents between TGXTJN middle-dose group and TGXTJN high-dose group and between TGXTJN low-dose group and TGXTJN high-dose group(P=0.277,P=0.126).There was no statistical difference in Caspase 12 contents between TGXTJN middle-dose group and TGXTJN low-dose group(P=0.614).There was statistical difference in contents of Caspase 3 in chondrocytes between the 5 groups(0.265+/-0.028,0.380+/-0.017,0.317+/-0.026,0.304+/-0.019,0.333+/-0.022,F=10.507,P=0.001).The Caspase 3 contents were higher in model group compared to blank group(P=0.000)and were lower in TGXTJN high-,middle- and low-dose group compared to model group(P=0.006,P=0.002,P=0.029).There was no statistical difference in Caspase 3 contents between TGXTJN middle-dose group and TGXTJN high-dose group and between TGXTJN low-dose group and TGXTJN high-dose group(P=0.496,P=0.387).There was no statistical difference in Caspase 3 contents between TGXTJN middle-dose group and TGXTJN low-dose group(P=0.138).Conclusion:TGXTJN can inhibit the apoptosis mediated by endoplasmic reticulum stress(PEKR signaling pathway)and induced by TG in rat's articular chondrocytes cultured in vitro,and its effect bears no obvious relation to its dosage.

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備注/Memo

備注/Memo:
基金項目:國家自然科學基金青年科學基金項目(81202836); 福建省自然科學基金項目(2016J01779) 通訊作者:葉蕻芝 E-mail:[email protected]
更新日期/Last Update: 2017-06-20