大黄素对骨关节炎模型小鼠痛觉行为的调节机制

doi:10.11958/20240056

摘要/Abstract

摘要：

目的基于线粒体关键基因探究大黄素缓解骨关节炎模型小鼠痛觉行为的作用机制。方法 30只C57BL/6J小鼠随机分为对照组、骨关节炎（OA）组和OA+大黄素组，每组10只。OA组和OA+大黄素组进行膝关节内注入完全弗氏佐剂（20 μL）建立OA模型，OA+大黄素组进行大黄素腹腔内给药（10 mg/kg）。行为学测试后，收集小鼠膝关节组织进行苏木素-伊红（HE）染色。蛋白免疫印迹分析膝关节组织炎性因子白细胞介素-1β（IL-1β）、肿瘤坏死因子-α（TNF-α）以及线粒体相关蛋白NADH脱氢酶（泛醌）黄素蛋白1（NDUFV1）、细胞色素C氧化酶亚基5B（COX5B）、细胞色素C氧化酶15（COX15）、NADH脱氢酶（泛醌）1α亚复合物亚基10（NDUFA10）的表达水平。结果与对照组相比，OA组小鼠机械痛阈值降低，转棒停留时间和运动距离下降（P<0.05）；与OA组相比，OA+大黄素组机械痛阈值升高，转棒停留时间和运动距离均增加（P<0.05）。对照组膝关节关节软骨和软骨下骨结构完整，OA组软骨层变薄，软骨下小梁退化，大黄素处理缓解了软骨变性。与对照组相比，OA组IL-1β、TNF-α、COX15和NDUFA10表达升高，NDUFV1和COX5B表达降低，大黄素处理可恢复上述蛋白表达水平（P<0.05）。结论大黄素可通过调控炎性因子和线粒体相关蛋白的表达来缓解OA小鼠痛觉行为。

关键词: 骨关节炎, 大黄素, 线粒体, 计算生物学, 炎症性疼痛

Abstract:

Objective To explore the regulatory mechanism of emodin on pain behavior in a mouse model of osteoarthritis based on mitochondrial key genes. Methods Thirty C57BL/6J mice were randomly divided into the control group, the osteoarthritis (OA) model group and the emodin-treated (OA+emodin) group, 10 mice per each group. The mice in the OA group and the OA+emodin group were intra-articular injection of complete Freund’s adjuvant (20 μL) in knee to establish the OA model, and mice in the OA+emodin group were treated by intraperitoneal emodin (10 mg/kg) injection. After behavioral testing, knee tissue of mice was collected for hematoxylin-eosin staining. Western blot analysis was used to detect expression levels of proinflammatory factors interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α) and mitochondria-related proteins NADH dehydrogenase (ubiquinone) flavoprotein 1 (NDUFV1), cytochrome C oxidase subunit 5B (COX5B), cytochrome C oxidase assembly protein COX15 homolog (COX15), NADH dehydrogenase (ubiquinone) 1 alpha subcomplex subunit 10 (NDUFA10) in knee tissue. Results Compared with the control group, mice in the OA group showed decreased mechanical nociceptive threshold (PWT), reduced latency and distance in rotarod test (P<0.05). Compared with the OA group, mice in the OA+emodin group showed increased PWT, latency, and distance (P<0.05). In the control group, the structures of cartilage and subchondral bone were intact, while in the OA group, the cartilage was thinner and the subchondral trabeculae was deteriorated. The treatment with emodin alleviated cartilage degeneration. The expression levels of IL-1β, TNF-α, COX15 and NDUFA10 were increased while expression levels of NDUFV1 and COX5B were decreased in the OA group compared with the control group. The emodin treatment restored the above-mentioned protein expression levels (P<0.05). Conclusion Emodin can alleviate pain behavior in OA mice by regulating the expressions of inflammatory factors and mitochondrial related proteins.

Key words: osteoarthritis, emodin, mitochondria, computational biology, inflammatory pain

中图分类号:

R965.1，R684.3

图/表 8

图1 大黄素调节OA模型小鼠痛觉行为的实验流程图

Fig.1 Schematic diagram of experimental procedures of emodin regulating pain behavior in a mouse model of osteoarthritis

表1 各组小鼠行为学测试结果比较（n=10，$\bar{x}±s$）

Tab.1 Comparison of behavioral tests between different groups of mice

组别	机械痛阈值/g	转棒停留时间/s	运动距离/m
对照组	1.48±0.25	543.92±46.50	23.89±2.17
OA组	0.21±0.13^a	215.18±50.45^a	8.42±2.16^a
OA+大黄素组	0.78±0.20^b	353.49±23.94^b	14.98±1.14^b
F	100.531^＊＊	154.783^＊＊	169.678^＊＊

图2 各组小鼠膝关节软骨组织HE染色 A：关节间隙；B：关节软骨；C软骨下骨；D：骨赘形成；▲：软骨下小梁；左侧标尺=100 μm，右侧标尺=50 μm。

Fig.2 HE staining of mouse knee cartilage tissue in each group

图3 OA差异性表达基因和线粒体相关分析 A：OA数据差异性表达基因火山图；B：OA差异性表达基因和线粒体相关重叠基因韦恩图；C：重叠差异性基因相互作用网络。

Fig.3 Correlation analysis of OA differentially expressed genes and mitochondria

图4 Western blot检测各组线粒体相关蛋白表达水平 A：对照组；B：OA组；C：OA+大黄素组。

Fig.4 Protein expression of mitochondria-associated proteins in each group detected by Western blot assay

表2 各组小鼠线粒体相关蛋白NDUFV1、COX5B、COX15和NDUFA10表达水平比较（n=3，$\bar{x}±s$）

Tab.2 Comparison of mitochondria-associated proteins NDUFV1, COX5B, COX15 and NDUFA10 expression between different groups of mice

组别	NDUFV1	COX5B	COX15	NDUFA10
对照组	1.00±0.04	1.00±0.04	1.00±0.09	1.00±0.03
OA组	0.53±0.03^a	0.55±0.05^a	1.77±0.08^a	2.40±0.09^a
OA+大黄素组	0.67±0.03^b	1.12±0.03^b	1.31±0.05^b	0.55±0.03^b
F	148.151^＊＊	133.223^＊＊	82.022^＊＊	767.383^＊＊

图5 Western blot检测各组膝关节组织IL-1β和TNF-α蛋白表达 A：对照组；B：OA组；C：OA+大黄素组。

Fig.5 Protein expressions of IL-1β and TNF-α in knee joint tissue of each group detected by Western blot assay

表3 各组小鼠膝关节组织IL-1β和TNF-α蛋白表达水平比较（$\bar{x}±s$）

Tab.3 Comparison of IL-1β and TNF-α protein expression between different groups of mice

组别	n	IL-1β	TNF-α
对照组	3	1.00±0.17	1.00±0.04
OA组	3	2.19±0.16^a	1.52±0.04^a
OA+大黄素组	3	1.14±0.16^b	1.15±0.05^b
F		47.405^＊＊	104.231^＊＊

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