三黄膏对耐甲氧西林金黄色葡萄球菌感染大鼠皮下软组织炎症及TLR2/NF-κB信号通路的影响

Effects of Sanhuang Ointment on Subcutaneous Soft Tissue Inflammation and TLR2/NF-κB Signaling Pathway in MRSA Infection Model Rats

目的观察三黄膏对耐甲氧西林金黄色葡萄球菌(MRSA)感染大鼠皮下软组织的影响,从TLR2/NF-κB信号通路探讨其作用机制。方法将96只SPF级Wistar大鼠随机分为空白组、模型组、百多邦组和三黄膏高、中、低剂量组,每组16只。除空白组外,其余各组以MRSA菌悬液皮下注射制备软组织感染模型,百多邦组和三黄膏高、中、低剂量组分别于患处涂抹相应药物,空白组和模型组涂抹等量凡士林,每日2次,共7 d。HE染色观察感染组织病理变化,ELISA检测血清及感染组织肿瘤坏死因子(TNF)-α、白细胞介素(IL)-1β、IL-7含量,RT-qPCR检测感染组织TNF-α、IL-1β、IL-7、Toll样受体2(TLR2)、转化生长因子-β活化激酶结合蛋白1(TAB1)、核因子(NF)-κB p65 mRNA表达,Western blot检测TLR2、TAB1、NF-κB p65蛋白表达。结果与空白组比较,模型组大鼠感染组织表皮层鳞状上皮普遍增厚,胶原纤维聚集成片状、团块状,各层有大量炎性细胞浸润;血清及感染组织TNF-α、IL-1β、IL-7含量显著增加(P<0.01),感染组织TNF-α、IL-1β、IL-7、TLR2、TAB1、NF-κB p65 mRNA和TLR2、TAB1、NF-κB p65蛋白表达均显著升高(P<0.01)。与模型组比较,三黄膏高剂量组大鼠感染组织胶原纤维聚集明显减少,周围肉芽组织填充,有新生的薄壁毛细血管;血清及感染组织TNF-α、IL-1β、IL-7含量显著减少,感染组织TNF-α、IL-1β、IL-7、TLR2、TAB1、NF-κB p65 mRNA和TLR2、TAB1、NF-κB p65蛋白表达显著降低,差异均有统计学意义(P<0.05,P<0.01)。结论三黄膏可能通过降低感染组织TLR2、TAB1、NF-κB p65 mRNA和蛋白表达,抑制TLR2/NF-κB信号通路下游炎症因子TNF-α、IL-1β、IL-7分泌,进而发挥促进感染创面愈合及抑制炎症反应的作用。

Objective To observe the effects of Sanhuang Ointment in treating subcutaneous soft tissues of rats infected with methicillin-resistant staphylococcus aureus(MRSA);To explore its mechanism from the TLR2/NF-κB signaling pathway.Methods Totally 96 SPF Wistar rats were randomly divided into blank group,model group,Mupirocin ointment group and Sanhuang Ointment high-,medium-,and high-dosage groups,with 16 rats in each group.Except for the blank group,the other groups were subcutaneously injected with MRSA bacterial suspension to make soft tissue infection models.The Mupirocin ointment group and Sanhuang Ointment high-,medium-,and lowdosage groups were applied externally with corresponding drugs on the affected areas,respectively,and the blank group and the model group were applied with the same amount of vaseline,twice a day,for a total of 7 days.HE staining was used to observe the pathological changes of skin tissue.ELISA was used to detect the contents of TNF-α,IL-1β,and IL-7 in serum and infected tissue.The expressions of TNF-α,IL-1β,IL-7,TLR2,TAB1,NF-κB p65mRNA in infected tissue were detected by RT-qPCR,and the proteins expression of TLR2,TAB1,NF-κB p65 were detected by Western blot.Results Compared with the blank group,the squamous epithelium of the epidermis of the skin tissue of the rats in model group were generally thickened,the collagen fibers aggregated into sheets and clumps,and a large number of inflammatory cells were infiltrated in each layer.The contents of TNF-α,IL-1βand IL-7 in serum and infected tissue significantly increased(P<0.01),the expressions of TNF-α,IL-1β,IL-7,TLR2,TAB1,NF-κB p65 mRNA and TLR2,TAB1,NF-κB p65 protein in infected tissue significantly increased(P<0.01).Compared with the model group,the aggregation of collagen fibers in the infected tissue of the rats in Sanhuang Ointment high-dosage group was significantly reduced,and the surrounding granulation tissue was filled with new thin-walled capillaries.The contents of TNF-α,IL-1β,IL-7 in serum and infected tissue were significantly reduced,the expressions of TNF-α,IL-1β,IL-7,TLR2,TAB1,NF-κB p65 m RNA and TLR2,TAB1,NF-κB p65 protein in infected tissue were significantly decreased,with statistical significance(P<0.05,P<0.01).Conclusion Sanhuang Ointment may decrease the expressions of TLR2,TAB1,NF-κB p65 mRNA and protein in infected tissue and inhibit the secretion of inflammatory factors TNF-α,IL-1βand IL-7 downstream of the TLR2/NF-κB signaling pathway,thereby promoting wound healing and inhibiting inflammatory response.