[1]叶锦霞,付长龙,林洁,等.透骨消痛胶囊对毒胡萝卜素诱导的内质网应激PEKR信号通路介导的大鼠体外培养关节软骨细胞凋亡的影响[J].中医正骨,2017,29(06):1-7.
　YE Jinxia,FU Changlong,LIN Jie,et al.Effect of Tougu Xiaotong Jiaonang(透骨消痛胶囊)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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透骨消痛胶囊对毒胡萝卜素诱导的内质网应激PEKR信号通路介导的大鼠体外培养关节软骨细胞凋亡的影响()
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《中医正骨》[ISSN:1001-6015/CN:41-1162/R]

卷:
第29卷
期数:
2017年06期
页码:
1-7
栏目:
基础研究
出版日期:
2017-06-20

文章信息/Info

Title:
Effect of Tougu Xiaotong Jiaonang(透骨消痛胶囊)on apoptosis mediated by endoplasmic reticulum stress(PEKR signaling pathway)and induced by thapsigargin in rat's articular chondrocytes cultured in vitro
作者:
叶锦霞; 付长龙; 林洁; 吴广文; 郑春松; 陈俊; 叶蕻芝
福建中医药大学,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
关键词:
骨关节炎;  透骨消痛胶囊;  软骨细胞;  软骨; 关节;  凋亡;  内质网应激;  毒胡萝卜素;  大鼠;  动物实验
Keywords:
Key words osteoarthritis;  Tougu Xiaotong Jiaonang;  chondrocytes;  cartilage; articular;  apoptosis;  endoplasmic reticulum stress;  thapsigargin;  rats;  animal experimentation
摘要:
目的:观察透骨消痛胶囊对毒胡萝卜素(thapsigargin,TG)诱导的内质网应激(PEKR信号通路)介导的大鼠体外培养关节软骨细胞凋亡的影响。方法:采用4周龄SD大鼠膝关节软骨建立软骨细胞体外培养体系,将培养的第3代软骨细胞分为空白组、模型组及透骨消痛胶囊高、中、低剂量组。空白组不进行干预; 模型组及透骨消痛胶囊高、中、低剂量组先以2 μmol·L-1 TG干预4 h,然后模型组更换为正常培养基,透骨消痛胶囊高、中、低剂量组分别更换为浓度为100 μg·mL-1、50 μg·mL-1、25 μg·mL-1的含透骨消痛胶囊培养基干预24 h。干预后以MTT法检测各组软骨细胞活性、以AnnexinⅤ-FITC/PI双染法流式细胞术检测各组软骨细胞凋亡率、以Western Blot法检测各组软骨细胞中激活转录因子4(activating transcription factor 4,ATF4)、结合免疫球蛋白(binding immunoglobulin protein,BIP)、天冬氨酸特异性半胱氨酸蛋白酶12(cysteine aspartate specific protease 12,Caspase12)和Caspase3的表达量。结果:①软骨细胞活性。5组软骨细胞吸光度比较,差异有统计学意义(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)。模型组吸光度低于空白组(P=0.000); 透骨消痛胶囊高、中、低剂量组吸光度均高于模型组(P=0.000,P=0.000,P=0.022); 透骨消痛胶囊中、低剂量组的吸光度均低于高剂量组(P=0.019,P=0.000); 透骨消痛胶囊低剂量组与中剂量组比较,差异无统计学意义(P=0.085)。②软骨细胞凋亡率。5组软骨细胞凋亡率比较,差异有统计学意义[(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]。模型组软骨细胞凋亡率高于空白组(P=0.000); 透骨消痛胶囊高、中、低剂量组软骨细胞凋亡率均低于模型组(P=0.000,P=0.000,P=0.001); 透骨消痛胶囊低剂量组软骨细胞凋亡率低于高剂量组(P=0.007),透骨消痛胶囊高、低剂量组与中剂量组比较,差异均无统计学意义(P=0.216,P=0.063)。③软骨细胞内质网应激(PEKR信号通路)关键蛋白含量。5组软骨细胞中ATF4含量比较,差异有统计学意义(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)。空白组及透骨消痛胶囊高、中、低剂量组的ATF4含量均低于模型组(P=0.000,P=0.001,P=0.000,P=0.012); 透骨消痛胶囊低剂量组的ATF4含量高于中剂量组(P=0.022); 透骨消痛胶囊高剂量组的ATF4含量与透骨消痛胶囊中、低剂量组比较,差异均无统计学意义(P=0.188,P=0.229)。5组软骨细胞中BIP含量比较,差异有统计学意义(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)。空白组及透骨消痛胶囊高、中、低剂量组的BIP含量均低于模型组(P=0.000,P=0.000,P=0.000,P=0.002); 透骨消痛胶囊低剂量组的BIP含量高于中剂量组(P=0.006); 透骨消痛胶囊高剂量组的BIP含量与透骨消痛胶囊中、低剂量组比较,差异均无统计学意义(P=0.185,P=0.072)。5组软骨细胞中Caspase12含量比较,差异有统计学意义(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)。模型组的Caspase12含量高于空白组(P=0.001); 透骨消痛胶囊高剂量组的Caspase12含量低于模型组(P=0.018); 透骨消痛胶囊中、低剂量组的Caspase12含量与模型组比较,差异均无统计学意义(P=0.126,P=0.277); 透骨消痛胶囊中、低剂量组的Caspase12含量与高剂量组比较,差异均无统计学意义(P=0.277,P=0.126); 透骨消痛胶囊中、低剂量组的Caspase12含量比较,差异无统计学意义(P=0.614)。5组软骨细胞中Caspase3含量比较,差异有统计学意义(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)。模型组的Caspase3含量高于空白组(P=0.000); 透骨消痛胶囊高、中、低剂量组的Caspase3含量均低于模型组(P=0.006,P=0.002,P=0.029); 透骨消痛胶囊中、低剂量组的Caspase3含量与高剂量组比较,差异均无统计学意义(P=0.496,P=0.387),透骨消痛胶囊中、低剂量组的Caspase3含量比较,差异无统计学意义(P=0.138)。结论:透骨消痛胶囊能抑制TG诱导的大鼠体外培养关节软骨细胞发生由内质网应激(PEKR信号通路)介导的细胞凋亡,其作用效果与药物剂量无明显关系。
Abstract:
ABSTRACT Objective:To observe the effect of Tougu Xiaotong Jiaonang(透骨消痛胶囊,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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备注/Memo

备注/Memo:
基金项目:国家自然科学基金青年科学基金项目(81202836); 福建省自然科学基金项目(2016J01779) 通讯作者:叶蕻芝 E-mail:yelin0930@163.com
更新日期/Last Update: 2017-06-20