梓醇对IL-1β诱导软骨细胞损伤的保护机制研究

doi:10.11958/20221441

摘要/Abstract

摘要：

目的探讨梓醇对白细胞介素-1β（IL-1β）诱导软骨细胞损伤的影响及其可能作用机制。方法分离培养人膝关节软骨细胞，分为对照（Con）组、IL-1β组（用10 μg/L IL-1β处理48 h），IL-1β+梓醇-低组、IL-1β+梓醇-中组、IL-1β+梓醇-高组（分别用10、20及50 ng/L的梓醇处理软骨细胞后，用10 μg/L IL-1β处理24 h），IL-1β+miR-NC组、IL-1β+miR-140-5p组（分别转染miR-NC和miR-140-5p mimics后，用10 μg/L IL-1β处理24 h），IL-1β+梓醇+anti-miR-NC组、IL-1β+梓醇+anti-miR-140-5p组（分别转染anti-miR-NC和anti-miR-140-5p后，用50 ng/L梓醇和10 μg/L IL-1β处理24 h）。细胞计数试剂盒-8（CCK-8）法检测细胞活力；酶联免疫吸附试验（ELISA）检测白细胞介素-6（IL-6）、肿瘤坏死因子α（TNF-α）、γ干扰素（IFN-γ）的水平；流式细胞术检测细胞凋亡；采用实时荧光定量PCR检测miR-140-5p的表达水平；Western blot检测活化的胱天蛋白酶3（Cleaved-caspase 3）、Cleaved-caspase 9蛋白表达。结果与IL-1β组比较，IL-1β+梓醇-低组、IL-1β+梓醇-中组、IL-1β+梓醇-高组IL-6、TNF-α、IFN-γ水平，细胞凋亡率和Cleaved-caspase 3、Cleaved-caspase 9蛋白表达水平降低（P<0.05），细胞活力和miR-140-5p表达水平升高（P<0.05）；与IL-1β+miR-NC组比较，IL-1β+miR-140-5p组IL-6、TNF-α、IFN-γ水平，细胞凋亡率和Cleaved-caspase 3、Cleaved-caspase 9蛋白表达水平降低（P<0.05），细胞活力和miR-140-5p表达水平升高（P<0.05）；与IL-1β+梓醇+anti-miR-NC组比较，IL-1β+梓醇+anti-miR-140-5p组IL-6、TNF-α、IFN-γ水平，细胞凋亡率和Cleaved-caspase 3、Cleaved-caspase 9蛋白表达水平升高（P<0.05），细胞活力和miR-140-5p表达水平降低（P<0.05）。结论梓醇可能通过上调miR-140-5p表达，抑制细胞炎性因子释放及细胞凋亡，减轻IL-1β诱导的软骨细胞损伤。

关键词: 膝关节, 软骨，关节, 梓醇, 细胞凋亡, miR-140-5p, 炎性因子

Abstract:

Objective To investigate the effect of catalpol on interleukin-1β (IL-1β)-induced chondrocyte injury and its possible mechanism. Methods Human knee joint chondrocytes were isolated, cultured and divided into the control (Con) group, the IL-1β group (treated with 10 μg/L IL-1β for 48 h), the IL-1β+catalpol-low group, the IL-1β+catalpol -medium group, the IL-1β+catalpol -high group (treated with 10, 20 and 50 ng/L catalpol and then treated with 10 μg/L IL-1β for 24 h), the IL-1β+miR-NC group (transfected with miR-NC, treated with 10 μg/L IL-1β for 24 h), the IL-1β+miR-140-5p group (transfected with miR-140-5p mimics, treated with 10 μg/L IL-1β for 24 h), the IL-1β+catalpol+anti-miR-NC group (after transfection with anti-miR-NC, treated with 50 ng/L catalpol and 10 μg/L IL-1β for 24 h) and the IL-1β+catalpol+anti-miR-140-5p group (transfected with anti-miR-140-5p, treated with 50 ng/L catalpol and 10 μg/L IL-1β for 24 h). Cell viability was detected by cell count Kit 8 (CCK-8 method). Levels of interleukin-6 (IL-6), tumor necrosis factor α (TNF-α) and interferon γ (IFN-γ) were detected by enzyme-linked immunosorbent assay (ELISA). The apoptosis rate was detected by flow cytometry. The expression of miR-140-5p was detected by real-time quantitative fluorescence PCR (qPCR). Expression levels of cleaved aspartate specific cysteine proteinase (Cleaved-caspase) 3 and Cleaved-caspase 9 were detected by Western blot assay. Results Compared with the IL-1β group, levels of IL-6, TNF-α, IFN-γ, cell apoptosis rate, Cleaved-caspase 3 and Cleaved-caspase 9 protein expression levels were decreased in the IL-1β+catalpol-low group, the IL-1β+catalpol-medium group and the IL-1β+catalpol-high group, but cell viability and miR-140-5p expression were increased (P<0.05). Compared with the IL-1β+miR-NC group, levels of IL-6, TNF-α, IFN-γ, cell apoptosis rate, Cleaved-caspase 3 and Cleaved-caspase 9 protein expression levels were decreased in the IL-1β+miR-140-5p group (P<0.05), and cell viability and miR-140-5p were increased (P<0.05). Compared with the IL-1β+catalpol +anti-miR-NC group, levels of IL-6, TNF-α, IFN-γ, cell apoptosis rate, Cleaved-caspase 3 and Cleaved-caspase 9 protein expression levels were increased in the IL-1β+ catalpol+anti-miR-140-5p group (P<0.05), and cell viability was decreased (P<0.05). Conclusion Catalpol may reduce IL-1β-induced chondrocyte injury by up-regulating the expression of miR-140-5p, inhibiting the release of cellular inflammatory factors and apoptosis.

Key words: knee joint, cartilage, articular, catalpol, apoptosis, miR-140-5p, inflammatory factor

中图分类号:

R684.76

图/表 10

参考文献 17

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组别	细胞活力/A₄₅₀			IL-6/（ng/L）
Con组	0.78±0.08			9.97±0.93
IL-1β组	0.31±0.04^a			86.55±7.18^a
IL-1β+梓醇-低组	0.42±0.05^ab			64.19±5.47^ab
IL-1β+梓醇-中组	0.52±0.06^abc			43.40±4.87^abc
IL-1β+梓醇-高组	0.64±0.07^abcd			16.67±1.39^abcd
F	80.005^＊＊			429.566^＊＊
组别		TNF-α/（ng/L）	IFN-γ/（ng/L）
Con组		5.78±0.56	31.71±3.11
IL-1β组		74.05±7.01^a	163.14±11.33^a
IL-1β+梓醇-低组		55.88±4.56^ab	115.22±10.34^ab
IL-1β+梓醇-中组		30.88±3.44^abc	79.56±7.38^abc
IL-1β+梓醇-高组		9.89±0.85^bcd	44.61±4.17^abcd
F		471.325^＊＊	408.467^＊＊

组别	细胞活力/A₄₅₀			IL-6/（ng/L）
Con组	0.78±0.08			9.97±0.93
IL-1β组	0.31±0.04^a			86.55±7.18^a
IL-1β+梓醇-低组	0.42±0.05^ab			64.19±5.47^ab
IL-1β+梓醇-中组	0.52±0.06^abc			43.40±4.87^abc
IL-1β+梓醇-高组	0.64±0.07^abcd			16.67±1.39^abcd
F	80.005^＊＊			429.566^＊＊
组别		TNF-α/（ng/L）	IFN-γ/（ng/L）
Con组		5.78±0.56	31.71±3.11
IL-1β组		74.05±7.01^a	163.14±11.33^a
IL-1β+梓醇-低组		55.88±4.56^ab	115.22±10.34^ab
IL-1β+梓醇-中组		30.88±3.44^abc	79.56±7.38^abc
IL-1β+梓醇-高组		9.89±0.85^bcd	44.61±4.17^abcd
F		471.325^＊＊	408.467^＊＊

组别	凋亡率/%		Cleaved-caspase 3
Con组	6.31±0.52		0.13±0.02
IL-1β组	32.63±3.16^a		0.58±0.04^a
IL-1β+梓醇-低组	22.90±2.13^ab		0.45±0.04^ab
IL-1β+梓醇-中组	15.71±1.41^abc		0.31±0.03^abc
IL-1β+梓醇-高组	9.04±0.73^abcd		0.19±0.02^abcd
F	297.530^＊＊		314.265^＊＊
组别		Cleaved-caspase 9		miR-140-5p
Con组		0.28±0.03		1.00±0.00
IL-1β组		0.77±0.06^a		0.26±0.03^a
IL-1β+梓醇-低组		0.64±0.05^ab		0.46±0.03^ab
IL-1β+梓醇-中组		0.51±0.04^abc		0.69±0.06^abc
IL-1β+梓醇-高组		0.35±0.03^abcd		0.96±0.07^bcd
F		193.026^＊＊		442.922^＊＊

组别	凋亡率/%		Cleaved-caspase 3
Con组	6.31±0.52		0.13±0.02
IL-1β组	32.63±3.16^a		0.58±0.04^a
IL-1β+梓醇-低组	22.90±2.13^ab		0.45±0.04^ab
IL-1β+梓醇-中组	15.71±1.41^abc		0.31±0.03^abc
IL-1β+梓醇-高组	9.04±0.73^abcd		0.19±0.02^abcd
F	297.530^＊＊		314.265^＊＊
组别		Cleaved-caspase 9		miR-140-5p
Con组		0.28±0.03		1.00±0.00
IL-1β组		0.77±0.06^a		0.26±0.03^a
IL-1β+梓醇-低组		0.64±0.05^ab		0.46±0.03^ab
IL-1β+梓醇-中组		0.51±0.04^abc		0.69±0.06^abc
IL-1β+梓醇-高组		0.35±0.03^abcd		0.96±0.07^bcd
F		193.026^＊＊		442.922^＊＊

组别	细胞活力/A₄₅₀		IL-6/（ng/L）
IL-1β+miR-NC组	0.33±0.04		87.72±7.93
IL-1β+miR-140-5p组	0.56±0.06		23.21±2.18
t	9.569^＊＊		23.532^＊＊
组别	TNF-α/（ng/L）	IFN-γ/（ng/L）
IL-1β+miR-NC组	77.31±6.24	165.82±14.54
IL-1β+miR-140-5p组	13.61±1.19	55.09±4.99
t	30.083^＊＊	21.830^＊＊