利奈唑胺对MRSA感染致慢性骨髓炎大鼠细菌负荷和骨修复的影响

doi:10.11958/20221569

摘要/Abstract

摘要：

目的探究利奈唑胺对耐甲氧西林金黄色葡萄球菌（MRSA）感染致慢性骨髓炎大鼠细菌负荷及骨修复的影响。方法将45只Wistar大鼠随机均分为假手术组、模型组、利奈唑胺组，每组15只。模型组、利奈唑胺组大鼠双侧胫骨近端骨缺损后接种10 μL 1.0×10⁸ CFU/mL MRSA，构建慢性骨髓炎模型。造模后7 d，利奈唑胺组腹腔注射利奈唑胺54 mg/kg，假手术组及模型组给予等体积葡萄糖注射液，连续14 d。观察创口愈合情况、Rissing评分、胫骨X线Norden评分和细菌负荷情况，HE染色观察胫骨组织病理改变，酶联免疫吸附试验检测病灶周围骨骼肌组织转化生长因子-β1（TGF-β1）、白细胞介素（IL）-1β、IL-6水平；蛋白免疫印迹法检测胫骨组织TGF-β1、激活素受体样激酶1（ALK-1）、Smad1/5、p-Smad1/5、骨形态发生蛋白2（BMP-2）蛋白表达。结果假手术组全部甲级愈合，模型组乙级愈合3只、丙级愈合12只，利奈唑胺组乙级愈合9只、丙级愈合6只，差异有统计学意义（χ²=35.111，P<0.01）；利奈唑胺组大鼠Rissing、Norden评分及细菌负荷低于模型组（P<0.05）。模型组大鼠局部骨质破坏、髓腔内及骨膜下脓肿且有炎细胞浸润和局部纤维化，利奈唑胺组大鼠骨质破坏、炎细胞浸润、纤维化明显减轻。与假手术组相比，模型组骨骼肌TGF-β1、IL-1β、IL-6水平和胫骨组织TGF-β1、ALK-1、p-Smad1/5蛋白表达升高，BMP-2蛋白表达降低（P<0.05）；与模型组比较，利奈唑胺干预后可以逆转上述指标的改变（P<0.05）。结论利奈唑胺可通过抑制TGF-β/BMP通路活化减轻MRSA感染所致慢性骨髓炎大鼠细菌负荷，促进骨修复。

关键词: 骨髓炎, 耐甲氧西林金黄色葡萄球菌, 利奈唑酮, 转化生长因子β1, 骨形态发生蛋白质2, 激活素受体，Ⅰ型, Smad蛋白质类

Abstract:

Objective To explore the effects of linezolid on bacterial load and bone repair in rats with chronic osteomyelitis induced by methicillin-resistant Staphylococcus aureus (MRSA) infection. Methods A total of 45 Wistar rats were randomly and averagely divided into the sham operation group, the model group and the linezolid group, 15 rats in each group. Rats in the model group and the linezolid group were inoculated with 10 μL of MRSA (1×10⁸ CFU/mL) after bilateral proximal tibia bone defects to construct rat models of chronic osteomyelitis. At 7 d after modeling, rats in the linezolid group was intraperitoneally injected with linezolid (54 mg/kg), while the sham operation group and the model group were given the same volume of glucose injection for 14 d. The wound healing, scores of Rissing and tibia X-ray Norden, and bacterial load were observed. The pathological changes of tibia tissue were observed by HE staining. The levels of transforming growth factor-β1 (TGF-β1), interleukin (IL)-1β and IL-6 in skeletal muscle tissue around lesions were detected by enzyme-linked immunosorbent assay. The expression levels of TGF-β1, activin receptor-like kinase 1 (ALK-1), Smad1/5, p-Smad1/5 and bone morphogenetic protein 2 (BMP-2) in tibial tissue were detected by Western blot assay. Results All the sham operation group healed in class-A. In the model group, there were 3 rats with class-B healing and 12 rats with class-C healing. In the linezolid group, there were 9 rats with class-B healing and 6 rats with class-C healing, there was a significant difference between the two groups (χ²=35.111, P<0.01). Rissing score, Norden score and bacterial load were lower in the linezolid group than those in the model group (P<0.05). In the model group, there were local bone destruction, intramedullary and subperiosteal abscesses, inflammatory cell infiltration and local fibrosis. In the linezolid group, bone destruction, inflammatory cell infiltration and fibrosis were significantly relieved. Compared with the sham operation group, levels of TGF-β1, IL-1β and IL-6 in skeletal muscle tissue, and expression levels of TGF-β1, ALK-1 and p-Smad1/5 in tibia tissue were increased, while BMP-2 was decreased in the model group (P<0.05). Compared with the model group, linezolid intervention could reverse changes of the above indexes (P<0.05). Conclusion Linezolid can relieve bacterial load and promote bone repair in rats with chronic osteomyelitis induced by MRSA infection by inhibiting the activation of TGF-β/BMP pathways.

Key words: osteomyelitis, methicillin-resistant staphylococcus aureus, linezolid, transforming growth factor beta1, bone morphogenetic protein 2, activin receptors, type Ⅰ, Smad proteins

中图分类号:

R681.21

图/表 5

表1 模型组与利奈唑胺组大鼠Rissing、Norden评分及细菌负荷比较 (n=15, $\bar{x}±s$)

Tab.1 Comparison of Rissing, Norden scores and bacterial load between the model group and the linezolid group

组别	Rissing评分/分	Norden评分/分	细菌负荷/（CFU/mL）
模型组	2.85±0.32	2.75±0.43	2.13±0.42
利奈唑胺组	1.62±0.38	1.35±0.31	0.51±0.04
t	9.589^＊＊	10.229^＊＊	14.871^＊＊

图1 各组大鼠胫骨组织病理变化（HE染色，×100）

Fig.1 Pathological changes of tibia tissue in each group（HE staining, ×100）

表2 各组大鼠骨骼肌组织TGF-β1、IL-1β、IL-6水平比较 (n=15,ng/L, $\bar{x}±s$)

Tab.2 Comparison on levels of TGF-β1, IL-1β and IL-6 in skeletal muscle tissue between different groups

组别	TGF-β1	IL-1β	IL-6
假手术组	21.24±5.17	29.51±6.12	11.35±2.42
模型组	185.18±16.51^a	115.32±25.73^a	69.51±17.64^a
利奈唑胺组	75.35±23.48^ab	58.25±14.38^ab	28.61±7.25^ab
F	369.144^＊＊	94.726^＊＊	108.634^＊＊

表3 各组大鼠胫骨组织中TGF-β1、ALK-1、p-Smad1/5、BMP-2蛋白表达水平比较 (n=15, $\bar{x}±s$)

Tab.3 Comparison on expression levels of TGF-β1, ALK-1, p-Smad1/5 and BMP-2 in tibia tissue between different groups

组别	TGF-β1	ALK-1	p-Smad1/5	BMP-2
假手术组	0.42±0.07	0.31±0.05	0.35±0.06	0.53±0.09
模型组	1.18±0.25^a	1.32±0.27^a	0.95±0.24^a	0.35±0.06^a
利奈唑胺组	0.78±0.14^ab	0.65±0.18^ab	0.67±0.17^ab	0.42±0.07^ab
F	74.759^＊＊	110.246^＊＊	45.017^＊＊	22.319^＊＊

图2 Western blot检测TGF-β/BMP通路相关蛋白的表达

Fig.2 Expressions of TGF-β/BMP pathway-related proteins detected by Western blot assay

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