[1]于兰,解冬千,王霞,等.蒺藜皂苷对白细胞介素-1β诱导的软骨细胞凋亡和炎症因子分泌的影响[J].中医正骨,2024,36(01):23-32.
　YU Lan,XIE Dongqian,WANG Xia,et al.Effects of tribulus terrestris saponins on interleukin-1β-induced chondrocyte apoptosis and inflammatory factor secretion[J].The Journal of Traditional Chinese Orthopedics and Traumatology,2024,36(01):23-32.
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蒺藜皂苷对白细胞介素-1β诱导的软骨细胞凋亡和炎症因子分泌的影响()
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《中医正骨》[ISSN:1001-6015/CN:41-1162/R]

卷:
第36卷
期数:
2024年01期
页码:
23-32
栏目:
基础研究
出版日期:
2024-01-20

文章信息/Info

Title:
Effects of tribulus terrestris saponins on interleukin-1β-induced chondrocyte apoptosis and inflammatory factor secretion
作者:
于兰; 解冬千; 王霞; 及超
保定市第二医院,河北 保定 071001
Author(s):
YU Lan; XIE Dongqian; WANG Xia; JI Chao
The No.2 Hospital of Baoding,Baoding 071001,Hebei,China
关键词:
骨关节炎;  软骨细胞;  细胞凋亡;  蒺藜;  皂苷类;  白细胞介素-1β;  RNA; 环状;  炎症介导素类
Keywords:
osteoarthritis;  chondrocytes;  apoptosis;  fructus tribuli;  saponins;  interleukin-1 beta;  RNA; circular;  inflammation mediators
摘要:
目的:观察蒺藜皂苷对白细胞介素-1β(interleukin-1β,IL-1β)诱导的软骨细胞凋亡和炎症因子分泌的影响,并探讨其作用机制。方法:①软骨细胞培养和转染。采用DMEM培养液培养小鼠软骨细胞(ATDC5细胞),培养后采用转染试剂分别转染pcDNA-circ_0045714、pcDNA、si-circ_0045714、si-NC。②蒺藜皂苷浓度筛选。将ATDC5细胞接种于96孔板中,一组正常培养(正常组),一组加入10 ng·mL-1的IL-1β(IL-1β组),其他组在加入10 ng·mL-1 IL-1β的基础上分别加入浓度为25 μg·mL-1、50 μg·mL-1、100 μg·mL-1、200 μg·mL-1、400 μg·mL-1的蒺藜皂苷。培养后计算软骨细胞存活率,并确定后续实验蒺藜皂苷的浓度。③软骨细胞增殖抑制率检测。将ATDC5细胞分为对照组、IL-1β组、IL-1β+蒺藜皂苷低剂量组、IL-1β+蒺藜皂苷中剂量组、IL-1β+蒺藜皂苷高剂量组,其中对照组采用常规培养液培养,IL-1β组采用含10 ng·mL-1的IL-1β培养液培养,IL-1β+蒺藜皂苷低、中、高剂量组分别采用含50 μg·mL-1、100 μg·mL-1、200 μg·mL-1的蒺藜皂苷和10 ng·mL-1的IL-1β培养液培养。转染pcDNA-circ_0045714、pcDNA的ATDC5细胞,培养方法同IL-1β组,并分别标记为IL-1β+pcDNA-circ_0045714组、IL-1β+pcDNA组。转染si-circ_0045714、si-NC的ATDC5细胞,培养方法同IL-1β+蒺藜皂苷高剂量组,并分别标记为IL-1β+蒺藜皂苷高剂量+si-circ_0045714组、IL-1β+蒺藜皂苷高剂量+si-NC组。培养后计算软骨细胞增殖抑制率。④软骨细胞凋亡率检测。于6孔板中接种ATDC5细胞及转染pcDNA-circ_0045714、pcDNA、si-circ_0045714、si-NC的ATDC5细胞,培养后采用流式细胞仪检测各组软骨细胞凋亡率。⑤软骨细胞中肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)和白细胞介素6(interleukin 6,IL-6)含量检测。收集各组软骨细胞的培养液,检测上清液中TNF-α和IL-6含量。⑥软骨细胞中circ_0045714表达量检测。提取各组软骨细胞中总RNA,逆转录合成cDNA,然后扩增,最后计算circ_0045714的表达量。结果:①蒺藜皂苷浓度筛选结果。IL-1β组的软骨细胞存活率低于正常组(LSD-t=15.396,P=0.000)。IL-1β组与IL-1β+25 μg·mL-1蒺藜皂苷组的软骨细胞存活率比较,差异无统计学意义(LSD-t=1.555,P=0.918)。IL-1β+50 μg·mL-1蒺藜皂苷组、IL-1β+100 μg·mL-1蒺藜皂苷组、IL-1β+200 μg·mL-1蒺藜皂苷组、IL-1β+400 μg·mL-1蒺藜皂苷组的软骨细胞存活率均高于IL-1β组(LSD-t=4.879,P=0.047; LSD-t=7.686,P=0.001; LSD-t=10.657,P=0.000; LSD-t=10.073,P=0.000)。IL-1β+400 μg·mL-1蒺藜皂苷组与IL-1β+200 μg·mL-1蒺藜皂苷组的软骨细胞存活率比较,差异无统计学意义(LSD-t=0.584,P=0.999)。因此,选择浓度为50 μg·mL-1、100 μg·mL-1、200 μg·mL-1的蒺藜皂苷进行实验,并分别标记为蒺藜皂苷低剂量组、中剂量组、高剂量组。②软骨细胞增殖抑制率、软骨细胞凋亡率、软骨细胞中TNF-α和IL-6含量检测结果。IL-1β组的软骨细胞增殖抑制率、软骨细胞凋亡率、软骨细胞中TNF-α和IL-6含量均高于对照组、IL-1β+蒺藜皂苷低剂量组、IL-1β+蒺藜皂苷中剂量组、IL-1β+蒺藜皂苷高剂量组(软骨细胞增殖抑制率:LSD-t=55.829,P=0.000; LSD-t=8.879,P=0.001; LSD-t=20.507,P=0.000; LSD-t=30.315,P=0.000; 软骨细胞凋亡率:LSD-t=27.508,P=0.000; LSD-t=5.076,P=0.032; LSD-t=11.689,P=0.000; LSD-t=21.284,P=0.000; 软骨细胞中TNF-α含量:LSD-t=29.990,P=0.000; LSD-t=7.720,P=0.002; LSD-t=17.182,P=0.000; LSD-t=24.615,P=0.000; 软骨细胞中IL-6含量:LSD-t=33.441,P=0.000; LSD-t=6.324,P=0.008; LSD-t=15.440,P=0.000; LSD-t=25.096,P=0.000)。IL-1β+蒺藜皂苷低剂量组的软骨细胞增殖抑制率、软骨细胞凋亡率、软骨细胞中TNF-α和IL-6含量均高于IL-1β+蒺藜皂苷中剂量组、IL-1β+蒺藜皂苷高剂量组(软骨细胞增殖抑制率:(LSD-t=11.627,P=0.000; LSD-t=21.436,P=0.000; 软骨细胞凋亡率:LSD-t=6.613,P=0.006; LSD-t=16.209,P=0.000; 软骨细胞中TNF-α含量:LSD-t=9.463,P=0.000; LSD-t=16.895,P=0.000; 软骨细胞中IL-6含量:LSD-t=9.117,P=0.001; LSD-t=18.773,P=0.000)。IL-1β+蒺藜皂苷中剂量组的软骨细胞增殖抑制率、软骨细胞凋亡率、软骨细胞中TNF-α和IL-6含量均高于IL-1β+蒺藜皂苷高剂量组(LSD-t=9.808,P=0.000; LSD-t=9.595,P=0.000; LSD-t=7.432,P=0.003; LSD-t=9.656,P=0.000)。③软骨细胞中circ_0045714表达量检测结果。IL-1β组软骨细胞中circ_0045714表达量低于对照组、IL-1β+蒺藜皂苷低剂量组、IL-1β+蒺藜皂苷中剂量组、IL-1β+蒺藜皂苷高剂量组(LSD-t=43.218,P=0.000; LSD-t=9.487,P=0.000; LSD-t=22.136,P=0.000; LSD-t=34.785,P=0.000)。IL-1β+蒺藜皂苷低剂量组软骨细胞中circ_0045714表达量低于IL-1β+蒺藜皂苷中剂量组、IL-1β+蒺藜皂苷高剂量组(LSD-t=12.649,P=0.000; LSD-t=25.298,P=0.000)。IL-1β+蒺藜皂苷中剂量组软骨细胞中circ_0045714表达量低于IL-1β+蒺藜皂苷高剂量组(LSD-t=12.649,P=0.000)。④过表达和干扰circ_0045714的软骨细胞构建结果。转染pcDNA、pcDNA-circ_0045714的软骨细胞中circ_0045714表达量比较,差异有统计学意义(1.00±0.00,4.18±0.14,t=39.342,P=0.000),说明过表达circ_0045714的软骨细胞构建成功。转染si-NC、si-circ_0045714的软骨细胞中circ_0045714表达量比较,差异有统计学意义(1.00±0.00,0.28±0.04,t=31.177,P=0.000),说明干扰circ_0045714的软骨细胞构建成功。⑤过表达circ_0045714对IL-1β诱导的软骨细胞影响结果。IL-1β+pcDNA-circ_0045714组的软骨细胞增殖抑制率、软骨细胞凋亡率、软骨细胞中TNF-α和IL-6含量均低于IL-1β+pcDNA组(t=16.290,P=0.000; t=11.359,P=0.000; t=11.988,P=0.000; t=12.266,P=0.000)。⑥干扰circ_0045714对IL-1β诱导的软骨细胞影响结果。IL-1β+蒺藜皂苷高剂量+si-circ_0045714组软骨细胞增殖抑制率、软骨细胞凋亡率、软骨细胞中TNF-α和IL-6含量均高于IL-1β+蒺藜皂苷高剂量+si-NC组(t=9.586,P=0.001; t=9.120,P=0.001; t=7.069,P=0.002; t=10.548,P=0.001)。结论:蒺藜皂苷能抑制IL-1β诱导的软骨细胞凋亡及炎症因子表达,具有治疗骨关节炎的潜在价值,其作用机制可能与上调软骨细胞中circ_0045714表达有关,且200 μg·mL-1蒺藜皂苷的效果更佳。
Abstract:
Objective:To observe the effects of tribulus terrestris saponins(TTS)on interleukin-1β(IL-1β)-induced chondrocyte apoptosis and inflammatory cytokine secretion,and to explore its underlying mechanism.Methods:①The mouse chondrocytes(ATDC5 cells)were harvested and cultured in the Dulbecco's Modified Eagle's Medium(DMEM).After 24-hour culture,the ATDC5 cells were transfected with pcDNA-circ_0045714,pcDNA,si-circ_0045714,and si-NC,respectively,by using transfection reagents.②The ATDC5 cells were inoculated onto the 96-well plates,with one group cultured in normal DMEM(normal group),one group in DMEM containing 10 ng/mL IL-1β(IL-1β group),and the rest in DMEM adding with 10 ng/mL IL-1β and TTS with concentration of 25,50,100,200,and 400 μg/mL,respectively.After 24-hour culture,the survival rate of chondrocytes in each group was calculated,based on which the concentration of TTS used in subsequent experiments was determined.③The ATDC5 cells were divided into control group,IL-1β group,IL-1β+low-dose TTS(L-TTS)group,IL-1β+medium-dose TTS(M-TTS)group,and IL-1β+high-dose TTS(H-TTS)group.The ATDC5 cells in control group were cultured in conventional DMEM,the ones in IL-1β group in DMEM with 10 ng/mL IL-1β,and the ones in the other 3 groups in DMEM adding with 10 ng/mL IL-1β and TTS with concentration of 50,100,and 200 μg/mL,respectively.The pcDNA-circ_0045714- and pcDNA-transfected ATDC5 cells were cultured with the same method as IL-1β group,and were labeled as IL-1β+pcDNA-circ_0045714 group and IL-1β+pcDNA group,respectively.The si-circ_0045714- and si-NC-transfected ATDC5 cells were cultured with the same method as IL-1β+H-TTS group,and were labeled as IL-1β+H-TTS+si-circ_0045714 group and IL-1β+H-TTS+si-NC group,respectively.After 24-hour culture,the proliferation inhibition rate of the chondrocytes in each group was calculated.④The ATDC5 cells and the pcDNA-circ_0045714-,pcDNA-,si-circ_0045714- and si-NC-transfected ATDC5 cells were inoculated onto the 6-well plates.After 12-hour culture,the apoptosis rate of chondrocytes in each group was detected by using flow cytometry.⑤The culture medium of chondrocytes was collected from each group,and the levels of tumor necrosis factor-α(TNF-α)and interleukin 6(IL-6)in the supernatant were detected.⑥The total RNA was extracted from chondrocytes in each group for synthesizing the cDNA by reverse transcription,followed by amplification for calculating the expression level of circ_0045714 in chondrocytes.Results:①The survival rate of chondrocytes was lower in IL-1β group compared to normal group(LSD-t=15.396,P=0.000),and lower compared with that of IL-1β+50 μg/mL TTS group,IL-1β+100 μg/mL TTS group,IL-1β+200 μg/mL TTS group and IL-1β+400 μg/mL TTS group(LSD-t=4.879,P=0.047; LSD-t=7.686,P=0.001; LSD-t=10.657,P=0.000; LSD-t=10.073,P=0.000); while the differences between IL-1β group and IL-1β+25 μg/mL TTS group,and between IL-1β+200 μg/mL TTS group and IL-1β+400 μg/mL TTS group were not statistically significant(LSD-t=1.555,P=0.918; LSD-t=0.584,P=0.999).Therefore,the TTS with concentrations of 50,100 and 200 μg/mL were selected for the further experiments and were labeled as L-TTS group,M-TTS group,and H-TTS group,respectively,based on the concentrations.②The proliferation inhibition rate,the apoptosis rate,and the levels of TNF-α and IL-6 in the chondrocytes were higher in IL-1β group compared with those of control group,IL-1β+L-TTS group,IL-1β+M-TTS group,and IL-1β+H-TTS group(the proliferation inhibition rate:LSD-t=55.829,P=0.000; LSD-t=8.879,P=0.001; LSD-t=20.507,P=0.000; LSD-t=30.315,P=0.000; the apoptosis rate:LSD-t=27.508,P=0.000; LSD-t=5.076,P=0.032; LSD-t=11.689,P=0.000; LSD-t=21.284,P=0.000; the level of TNF-α in the chondrocytes:LSD-t=29.990,P=0.000; LSD-t=7.720,P=0.002; LSD-t=17.182,P=0.000; LSD-t=24.615,P=0.000; the level of IL-6 in the chondrocytes:LSD-t=33.441,P=0.000; LSD-t=6.324,P=0.008; LSD-t=15.440,P=0.000; LSD-t=25.096,P=0.000),and they were higher in IL-1β+L-TTS group compared to IL-1β+M-TTS group and IL-1β+H-TTS group(the proliferation inhibition rate:LSD-t=11.627,P=0.000; LSD-t=21.436,P=0.000; the apoptosis rate:LSD-t=6.613,P=0.006; LSD-t=16.209,P=0.000; the level of TNF-α in the chondrocytes:LSD-t=9.463,P=0.000; LSD-t=16.895,P=0.000; the level of IL-6 in the chondrocytes:LSD-t=9.117,P=0.001; LSD-t=18.773,P=0.000),furthermore,they were higher in IL-1β+M-TTS group compared to IL-1β+H-TTS group(LSD-t=9.808,P=0.000; LSD-t=9.595,P=0.000; LSD-t=7.432,P=0.003; LSD-t=9.656,P=0.000).③The expression level of circ_0045714 in the chondrocytes was lower in IL-1β group compared to control group,IL-1β+L-TTS group,IL-1β+M-TTS group,and IL-1β+H-TTS group(LSD-t=43.218,P=0.000; LSD-t=9.487,P=0.000; LSD-t=22.136,P=0.000; LSD-t=34.785,P=0.000),and was lower in IL-1β+L-TTS group compared to IL-1β+M-TTS group and IL-1β+H-TTS group(LSD-t=12.649,P=0.000; LSD-t=25.298,P=0.000),moreover,it was lowly expressed in IL-1β+M-TTS group compared to IL-1β+H-TTS group(LSD-t=12.649,P=0.000).④The expression level of circ_0045714 showed a significant difference in the chondrocytes transfected with pcDNA and pcDNA-circ_0045714(1.00±0.00,4.18±0.14,t=39.342,P=0.000),which indicated the chondrocytes with overexpression of circ_0045714 were successfully constructed.The difference in the expression level of circ_0045714 among the chondrocytes transfected with si-NC and si-circ_0045714 indicated the successful silencing of circ-0045714 in chondrocytes(1.00±0.00,0.28±0.04,t=31.177,P=0.000).⑤The IL-1β+pcDNA-circ_0045714 group showed lower proliferation inhibition rate,apoptosis rate,and TNF-α and IL-6 levels in the chondrocytes compared to IL-1β+pcDNA group(t=16.290,P=0.000; t=11.359,P=0.000; t=11.988,P=0.000; t=12.266,P=0.000).⑥The IL-1β+H-TTS+si-circ_0045714 group exhibited higher proliferation inhibition rate,apoptosis rate,and TNF-α and IL-6 levels in the chondrocytes compared to IL-1β+H-TTS+si-NC group(t=9.586,P=0.001; t=9.120,P=0.001; t=7.069,P=0.002; t=10.548,P=0.001).Conclusion:The TTS can inhibit IL-1β-induced chondrocyte apoptosis and the expression of inflammatory cytokine,which has potential value in treatment of osteoarthritis.It may exert the effects by upregulating the expression of circ_0045714 in the chondrocytes,and the effects are stronger at the concentration of 200 μg/mL.

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备注/Memo

备注/Memo:
基金项目:保定市科技计划项目(2041ZF019)
更新日期/Last Update: 1900-01-01