清金益气方对金黄色葡萄球菌诱导大鼠急性咽炎的作用研究

Study on the effects of Qingjin Yiqi Decoction on Staphylococcus aureus-induced acute pharyngitis in rats

关键词:  急性咽炎  金黄色葡萄球菌  黏蛋白5AC  清金益气方  前列腺素E2

Key words:  acute pharyngitis  Staphylococcus aureus  mucin 5AC  Qingjin Yiqi Decoction  prostaglandin E2

摘要:

[目的] 探讨清金益气方对金黄色葡萄球菌（S. aureus）诱导的大鼠急性咽炎（AP）的作用研究。[方法] 选取雄性SD大鼠24只，随机分为对照组、模型组、清金益气方组（3.4 g/kg）和阿莫西林组（0.36 g/kg），每组6只。清金益气方组预给药3 天后，对照组注射生理盐水，其余大鼠连续2 天在咽部注射S. aureus（2×10⁷ CFU/天），造模后对照组和模型组继续灌胃生理盐水，清金益气方组和阿莫西林组分别灌胃给药，并持续7 d。末次给药1 h后对大鼠行为学和咽部病变打分。通过埃文斯蓝染色（EB）和苏木精-伊红染色（HE）检测咽部EB染料渗出量及观察咽、气管组织病理学变化。通过血常规分析外周血和气管灌洗液（TLF）中白细胞和中性粒细胞变化。通过流式细胞术统计外周血中T淋巴细胞变化。通过免疫荧光法考察咽组织中E-cadherin表达。通过酶联免疫吸附测定法（ELISA）检测血清中肿瘤坏死因子-α（TNF-α）、白细胞介素-1β（IL-1β）、IL-6、前列腺素E2（PGE2）及免疫球蛋白A（IgA）水平，TLF中分泌型免疫球蛋白A（SIgA）水平，及咽、气管组织和TLF中黏蛋白5AC（MUC5AC）水平。通过阿利新蓝-过碘酸-雪夫染色（AB-PAS）评估咽组织黏液蛋白表达。采用脂磷壁酸（LTA）刺激人淋巴结转移的肺癌细胞NCI-H292细胞建立上皮损伤模型，检测QJYQ对E-cadherin，IL-1β和MUC5AC表达的影响。[结果] QJYQ可以显著上调S. aureus诱导的AP大鼠体质量（P<0.001）和胸腺指数（P<0.05），改善大鼠活动能力（P<0.05）和皮毛色泽（P<0.001），降低咽部EB染料渗出量（P<0.01）。咽和气管组织上皮血管增多、腺体壁增厚及炎性细胞浸润亦得到明显改善。QJYQ显著降低外周血及TLF中白细胞数目（P<0.05，P<0.001）、中性粒细胞比例（P<0.01），增加CD4⁺ T细胞，降低CD8⁺ T细胞比例，降低IgA水平（P<0.01）以及上调SIgA水平（P<0.001）。QJYQ上调咽组织E-cadherin表达及显著降低IL-1β（P＜0.001）、PGE₂（P＜0.05）、TNF-α（P＜0.001）和IL-6（P＜0.001）含量。QJYQ抑制咽组织黏液蛋白表达，明显抑制咽、气管和TLF中MUC5AC含量（P＜0.001，P＜0.001，P＜0.001）。给药QJYQ可以增加NCI-H292细胞中E-cadherin表达，降低IL-1β（P＜0.01）和MUC5AC的表达。[结论] QJYQ可以通过抑制炎症因子水平和MUC5AC表达抑制S. aureus诱导的大鼠AP。

Abstract:

[Objective] To investigate the effect of Qingjin Yiqi Decoction(QJYQ) on acute pharyngitis(AP) induced by Staphylococcus aureus(S. aureus) in rats.[Methods] SD rats were randomly divided into four groups:Control group,Model group,QJYQ group(3.4 g/kg),and Amoxicillin group(0.36 g/kg). After 3 days of pre-administration with QJYQ,rats of Control group were injected with saline,the other rats were injected with 2×10⁷ CFU of S. aureus in the pharynx for 2 consecutive days. After modeling,rats of QJYQ group and Amoxicillin group were respectively given gavage for 7 days. 1 h after the last administration,the behavioral and pharyngeal lesions were scored. The exudation of Evans blue(EB) dye in pharynx and the pathological changes in the pharynx and trachea were performed by EB staining and hematoxylin-eosin(HE) staining. The change of white blood cells(WBC) and neutrophils in peripheral blood and tracheal lavage fluid(TLF) was analyzed by blood routine. The changes of T lymphocytes in peripheral blood were measured by flow cytometry. The expression of E-cadherin in pharynx was investigated by immunofluorescence(IF). Serum levels of tumor necrosis factor-α(TNF-α),interleukin-1β(IL-1β),IL-6,prostaglandin E2(PGE2) and immunoglobulin A(IgA),secreted immunoglobulin A(SIgA) in TLF and mucin 5AC(MUC5AC) levels in pharynx,tracheal tissue and TLF were detected by enzyme-linked immunosorbent assay(ELISA). The expression of mucin of the pharynx was performed by alcian blue-periodic acid schiff(AB-PAS) staining. Human lymph node metastatic lung cancer cells NCI-H292 were stimulated by lipoteichoic acid(LTA) to establish an epithelial injury model,and the expression of E-cadherin,IL-1β and MUC5AC was detected by IF and reverse transcription-quantity PCR(RT-qPCR).[Results] QJYQ significantly upregulated the body weight and thymus index,improved the mobility and fur coloration and reduced exudation of EB dye in pharynx of S. aureus-induced AP rats. Histopathological data showed the blood vessels,congestion with gland walls in lamina propria and thickening of submucosa extensive infiltration of inflammatory cells was much increased,which were all inhibited after QJYQ administration. QJYQ significantly decreased WBC number and the percentage of neutrophils were significantly inhibited after QJYQ administration. Consistently,the decreased CD4⁺ T cells and SIgA levels,and increased CD8⁺ T cells and IgA levels were all reversed by QJYQ. QJYQ also up-regulated E-cadherin expression in pharynx and decreased the levels of IL-1β,PGE2,TNF-α and IL-6 in serum. Moreover,the increased mucin expression in pharynx and MUC5AC levels in pharynx,trachea and TLF were significantly inhibited after GPS administration. QJYQ increased the expression of E-cadherin, significantly reduced the mRNA expression of IL-1β and MUC5AC in NCI-H292 cells.[Conclusion] QJYQ inhibited S. aureus-induced AP in rats by inhibiting the levels of inflammatory cytokines and the expression of MUC5AC.