基于lncRNA DLEU2/miR-655/SMAD7信号通路探讨山茱萸新苷对镉暴露大鼠骨损伤骨关节炎的影响

摘要/Abstract

摘要：

目的基于长链非编码 RNA 淋巴细胞白血病 2 型缺失因子（long non coding RNA deleted in lymphocytic leukemia 2，lncRNA DLEU2）/微小RNA-655（microRNA-655，miR-655）/SMAD同源物7（Mothers against decapentaplegic homolog 7，SMAD7）信号通路探讨山茱萸新苷对镉暴露大鼠骨损伤骨关节炎的影响。方法 2023年6—12月于秦皇岛市第一医院动物房开展动物实验，40只大鼠分为正常组、镉暴露模型组、山茱萸新苷低剂量组、山茱萸新苷高剂量组。除对照组外，其余各组予以二氯化镉（cadmium dichloride，CdCl₂）持续灌胃暴露建立镉暴露大鼠股骨损伤骨关节炎模型，在CdCl₂暴露期间，山茱萸新苷低、高剂量组（2 000、4 000 mg/kg）大鼠每日予以相应剂量的山茱萸新苷灌胃，正常组、镉暴露模型组给予等体积的生理盐水灌胃。实验结束后，用SkyScan 1076 Bruker-Micro CT扫描仪测量大鼠股骨骨密度（bone density，BMD）、骨小梁数量、骨小梁厚度、骨小梁分散度；测定大鼠热刺激潜伏期、股骨关节炎症积、足趾容积；实时荧光逆转录法及蛋白印迹法测定大鼠股骨lncRNA DLEU2、miR-655、SMAD7水平；酶联免疫吸附法测定大鼠股骨白介素-1β（interleukin-1β，IL-1β）、肿瘤坏死因子（tumor necrosis factor-α，TNF-α）、半胱氨酸的天冬氨酸蛋白水解酶-3（cysteinyl aspartate specific proteinase-3，caspase-3）蛋白水平。结果镉暴露模型组大鼠股骨骨小梁分散度水平、股骨关节炎症积、足趾容积，股骨miR-655 mRNA水平，股骨IL-1β、TNF-α、caspase-3蛋白水平高于正常组，BMD、骨小梁数量、骨小梁厚度水平、PEL、lncRNA DLEU2 mRNA（0.95±0.17 vs 4.32±0.16）、SMAD7 mRNA（0.42±0.29 vs 4.82±0.10）和蛋白（0.32±0.06 vs 1.52±0.16）水平低于正常组，差异均有统计学意义（均P<0.05）；山茱萸新苷低、高剂量组大鼠骨小梁分散度水平、关节炎症积、足趾容积，股骨miR-655 mRNA水平，股骨IL-1β、TNF-α、caspase-3蛋白水平低于镉暴露模型组，BMD、骨小梁数量、骨小梁厚度水平、热刺激潜伏期（paw withdrawal thermal latency，PEL）、lncRNA DLEU2 mRNA（1.72±0.11、2.87±0.07 vs 0.95±0.17）、SMAD7 mRNA（1.11±0.20、2.89±0.17 vs 0.42±0.29）和蛋白（0.74±0.12、0.95±0.11 vs 0.32±0.06）水平高于镉暴露模型组，差异均有统计学意义（均P<0.05）；山茱萸新苷高剂量组骨小梁分散度水平、股骨关节炎症积、足趾容积，股骨miR-655 mRNA水平，股骨IL-1β、TNF-α、caspase-3蛋白水平低于山茱萸新苷低剂量组，BMD、骨小梁数量、骨小梁厚度水平、PEL、lncRNA DLEU2 mRNA（2.87±0.07 vs 1.72±0.11）、SMAD7 mRNA（2.89±0.17 vs 1.11±0.20）和蛋白（0.95±0.11 vs 0.74±0.12）水平高于山茱萸新苷低剂量组，差异均有统计学意义（均P<0.05）。结论山茱萸新苷对镉暴露大鼠骨损伤骨关节炎具有明显治疗作用；其机制与山茱萸新苷激活镉暴露大鼠股骨lncRNA DLEU2/miR-655/SMAD7信号通路进而抑制了股骨细胞炎症与凋亡有关。

关键词: 长链非编码RNA淋巴细胞白血病2型缺失因子, 微小RNA-655, SMAD同源物7, 山茱萸新苷, 镉, 骨损伤, 骨关节炎

Abstract:

Objective To investigate the effect of cornus officinalis glycosides on bone injury and osteoarthritis in rats exposed to cadmium based on the long non coding RNA deleted in lymphocytic leukemia 2（lncRNA DLEU2）/microRNA-655（miR-655）/Mothers against decapentaplegic homolog 7（SMAD7） signaling pathway. Methods Animal experiments were conducted from June to December 2023 in the animal room of Qinhuangdao First Hospital. Forty rats were divided into a normal group，a cadmium exposure model group，a low-dose cornus officinalis glycosides group，and a high-dose cornus officinalis glycosides group. Except for the control group，the femoral osteoarthritis model of cadmium-exposed rats was established by continuous intragastric exposure to cadmium dichloride（CdCl₂） in all other groups. During the CdCl₂ exposure period，rats in the low-dose and high-dose cornus officinalis glycosides groups（2 000 and 4 000 mg/kg） were intragastrically administered with the corresponding dose of cornus officinalis glycosides daily，and the normal group and the cadmium exposure model group were intragastricly administered with an equal volume of normal saline. After the experiment，SkyScan 1076 Bruker-Micro CT scanner was used to measure the femoral bone density（BMD），trabecular number，trabecular thickness，and trabecular dispersion of the rats. The thermal stimulation latency，femoral osteoarthritis score，and toe volume of the rats were measured. Real-time fluorescence reverse transcription and Western blotting were used to measure the levels of lncRNA DLEU2，miR-655，and SMAD7 in the femur of the rats. And enzyme-linked immunosorbent assay was used to measure the levels of interleukin-1β（IL-1β），tumor necrosis factor-α（TNF-α），and cysteinyl aspartate specific proteinase-3（caspase-3） proteins in the femur of the rats. Results The cadmium exposure model group had higher levels of femoral trabecular dispersion，femoral joint inflammation area，toe volume，femoral miR-655 mRNA，and femoral IL-1β，TNF-α，and caspase-3 protein than those of the normal group，while BMD，trabecular number，trabecular thickness，paw withdrawal thermal latency（PEL），lncRNA DLEU2 mRNA（0.95±0.17 vs 4.32±0.16），SMAD7 mRNA（0.42±0.29 vs 4.82±0.10） and protein（0.32±0.06 vs 1.52±0.16） levels were lower than those of the normal group，and the differences were statistically significant（all P<0.05）. The trabecular dispersion，joint inflammation area，toe volume，femoral miR-655 mRNA，femoral IL-1β，TNF-α，and caspase-3 protein levels in the low-dose cornus officinalis glycosides groups and high-dose cornus officinalis glycosides groups were lower than those in the cadmium exposure model group，while BMD，trabecular number，trabecular thickness，PEL，lncRNA DLEU2 mRNA（1.72±0.11，2.87±0.07 vs 0.95±0.17），SMAD7 mRNA（1.11±0.20，2.89±0.17 vs 0.42±0.29） and protein levels（0.74±0.12，0.95±0.11 vs 0.32±0.06） were higher than those in the cadmium exposure model group，and the differences were statistically significant（all P<0.05）. The trabecular dispersion，femoral osteoarthritis area，toe volume，femoral miR-655 mRNA， femoral IL-1β，TNF-α，and caspase-3 protein levels in the high-dose cornus officinalis glycosides group were lower than those in the low-dose cornus officinalis glycosides group，while BMD，trabecular number，trabecular thickness，PEL，lncRNA DLEU2 mRNA（2.87±0.07 vs 1.72±0.11），SMAD7 mRNA（2.89±0.17 vs 1.11±0.20） and protein levels（0.95±0.11 vs 0.74±0.12） were higher than those in the low-dose cornus officinalis glycosides group（all P<0.05）. Conclusion Cornus officinalis glycosides have a significant therapeutic effect on bone injury and osteoarthritis in cadmium exposed rats. The mechanism is related to the activation of lncRNA DLEU2/miR-655/SMAD7 signaling pathway in the femur of cadmium exposed rats，thereby inhibiting inflammation and apoptosis of femoral cells.

Key words: lncRNA DLEU2, miR-655, SMAD7, Cornus officinalis glycosides, Cadmium, Bone injury, Osteoarthritis

中图分类号:

R-332

白波, 马佳良, 刘永权, 张玉舰, 陈国栋, 龚珊. 基于lncRNA DLEU2/miR-655/SMAD7信号通路探讨山茱萸新苷对镉暴露大鼠骨损伤骨关节炎的影响. [J]职业与健康, 2026, 42(1): 65-72.

BAI Bo, MA Jialiang, LIU Yongquan, ZHANG Yujian, CHEN Guodong, GONG Shan. Exploring the effect of cornus officinalis glycosides on bone injury and osteoarthritis in cadmium exposed rats based on lncRNA DLEU2/miR-655/SMAD7 signaling pathway. [J]OCCUPATION AND HEALTH, 2026, 42(1): 65-72.

图/表 7

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组别	只数	骨小梁分散度（mm）	BMD（g/cm³）	骨小梁数量（1/mm²）	骨小梁厚度（mm）
正常组	10	0.26±0.03	1.98±0.12	7.78±0.28	0.27±0.04
镉暴露模型组	10	0.75±0.03^a	1.11±0.11^a	2.12±0.26^a	0.05±0.02^a
山茱萸新苷低剂量组	10	0.54±0.04^b	1.36±0.13^b	3.24±0.38^b	0.12±0.03^b
山茱萸新苷高剂量组	10	0.40±0.03^bc	1.58±0.12^bc	4.75±0.37^bc	0.19±0.03^bc

组别	只数	骨小梁分散度（mm）	BMD（g/cm³）	骨小梁数量（1/mm²）	骨小梁厚度（mm）
正常组	10	0.26±0.03	1.98±0.12	7.78±0.28	0.27±0.04
镉暴露模型组	10	0.75±0.03^a	1.11±0.11^a	2.12±0.26^a	0.05±0.02^a
山茱萸新苷低剂量组	10	0.54±0.04^b	1.36±0.13^b	3.24±0.38^b	0.12±0.03^b
山茱萸新苷高剂量组	10	0.40±0.03^bc	1.58±0.12^bc	4.75±0.37^bc	0.19±0.03^bc

组别	只数	PEL （s）	关节炎症积分	足趾容积（μL）
对照组	10	21.85±1.41	0.00±0.00	1.38±0.50
镉暴露模型组	10	7.96±0.85^a	13.59±0.87^a	13.74±1.10^a
山茱萸新苷组低剂量组	10	10.54±1.11^b	11.50±0.79^b	10.41±0.69^b
山茱萸新苷组高剂量组	10	17.39±1.26^bc	5.62±0.49^bc	3.73±0.67^bc

组别	只数	PEL （s）	关节炎症积分	足趾容积（μL）
对照组	10	21.85±1.41	0.00±0.00	1.38±0.50
镉暴露模型组	10	7.96±0.85^a	13.59±0.87^a	13.74±1.10^a
山茱萸新苷组低剂量组	10	10.54±1.11^b	11.50±0.79^b	10.41±0.69^b
山茱萸新苷组高剂量组	10	17.39±1.26^bc	5.62±0.49^bc	3.73±0.67^bc

组别	只数	lncRNA DLEU2 mRNA	miR-655 mRNA	SMAD7 mRNA	SMAD7蛋白（/GAPDH）
对照组	10	4.32±0.16	0.93±0.17	4.82±0.10	1.52±0.16
镉暴露模型组	10	0.95±0.17^a	5.14±0.30^a	0.42±0.29^a	0.32±0.06^a
山茱萸新苷低剂量组	10	1.72±0.11^b	3.81±0.22^b	1.11±0.20^b	0.74±0.12^b
山茱萸新苷高剂量组	10	2.87±0.07^bc	2.14±0.11^bc	2.89±0.17^bc	0.95±0.11^bc