[關(guān)鍵詞]
[摘要]
目的 建立甲型H1N1流感病毒(H1N1)繼發(fā)金黃色葡萄球菌Staphylococcus aureus感染模型,探討金銀花水提物(LJAE)對(duì)該模型藥效及機(jī)制。方法 小鼠按體質(zhì)量隨機(jī)分為對(duì)照組、模型組、磷酸奧司他韋(OT,抗病毒陽性藥,11 mg·kg-1,臨床等效劑量)組、頭孢克洛緩釋片(CF,抗菌陽性藥,106.38 mg·kg-1,臨床等效劑量)組和LJAE低、中、高劑量(6.5、13.0、26.0 mL·kg-1)組,除對(duì)照組外,建立致死型H1N1繼發(fā)S.aureus模型,連續(xù)給藥5 d,觀察死亡率;除對(duì)照組外,建立亞致死性H1N1繼發(fā)S.aureus模型,連續(xù)給藥5 d,測(cè)定肺指數(shù),通過實(shí)時(shí)熒光定量PCR(qRT-PCR)法檢測(cè)肺病毒量,平板法檢測(cè)肺菌量,蘇木精-伊紅(HE)染色觀察肺損傷;瑞氏-吉姆薩染色法測(cè)定肺泡灌洗液中巨噬細(xì)胞和中性粒細(xì)胞數(shù)目;通過qRT-PCR法檢測(cè)肺單核細(xì)胞趨化蛋白-1(MCP-1)、腫瘤壞死因子-α(TNF-α)、干擾素-γ(IFN-γ)、白介素-4(IL-4)、趨化因子(C-X-C基序)配體1(CXCL-1) mRNA表達(dá)量; ELISA法檢測(cè)肺組織MCP-1、TNF-α、IFN-γ水平;流式細(xì)胞術(shù)測(cè)定外周血CD4+IFN-γ+(Th1)和CD4+IL-4+(Th2)細(xì)胞比例。結(jié)果 與模型組比較,LJAE顯著降低致死型H1N1繼發(fā)S.aureus模型小鼠的死亡率(P<0.05、0.01、0.001),顯著降低亞致死型H1N1繼發(fā)S.aureus模型小鼠的肺指數(shù)(P<0.001)、肺病毒量(P<0.001)和肺菌量(P<0.001),減輕肺病理損傷; LJAE顯著減少肺部中性粒細(xì)胞和巨噬細(xì)胞的募集( P<0.001),顯著降低趨化因子MCP-1和促炎細(xì)胞因子TNF-α和IFN-γ mRNA和蛋白的表達(dá)(P<0.001),降低外周血單核細(xì)胞CD4+IFN-γ+/CD4+IL-4+(P<0.001)。結(jié)論 LJAE具有顯著的抗H1N1繼發(fā)S.aureus感染的活性,能夠通過調(diào)節(jié)過激的Th1型免疫反應(yīng),抑制Th1型細(xì)胞因子MCP-1、TNF-α和IFN-γ的釋放,減輕肺部炎癥細(xì)胞的過度浸潤,修復(fù)H1N1繼發(fā)S.aureus引發(fā)的肺損傷。
[Key word]
[Abstract]
Objective To establish a secondary Staphylococcus aureus (S. aureus) infection model of influenza A H1N1 virus (H1N1), and explore the efficacy evaluation and research of Lonicera japonica aqueous extract (LJAE) on this model. Methods Mice were randomly divided into the control group, model group, oseltamivir phosphate (OT, antiviral positive drug, 11 mg·kg-1, clinical equivalent dose) group, Cefaclor Sustained-Release Tablets (CF, antibacterial positive drug, 106.38 mg·kg-1, clinical equivalent dose) group and low, medium and high dose LJAE (6.5, 13.0, 26.0 mL·kg-1) groups according to body weight. Except for the control group, lethal H1N1 secondary S. aureus models were established, and continuous administration was given for five days to observe the mortality rate. Except for the control group, sublethal H1N1 secondary S. aureus models were established, and continuous administration was given for five days. The lung index was measured, the lung viral load was detected by real-time fluorescence quantitative PCR (qRT-PCR), the lung bacterial load was detected by plate method, and lung injury was observed by hematoxylineosin (HE) staining. The number of macrophages and neutrophils in the alveolar lavage fluid was determined by Wright-Giemsa staining. The mRNA expression levels of lung monocyte chemoattractant protein-1 (MCP-1), tumor necrosis factor-α (TNF-α), interferon-γ (IFN-γ), interleukin-4 (IL-4), and chemokine (C-X-C motif) ligand 1 (CXCL-1) were detected by qRT-PCR. The levels of MCP-1, TNF-α, and IFN-γ in lung tissue were detected by ELISA. The proportions of CD4+IFN-γ+ (Th1) and CD4+IL-4+ (Th2) cells in peripheral blood were determined by flow cytometry. Results Compared with the model group, LJAE significantly reduced the mortality rate of mice in the lethal H1N1 secondary S. aureus model (P < 0.05, 0.01, 0.001), significantly reduced the lung index (P < 0.001), lung viral load (P < 0.001), and lung bacterial load (P < 0.001) of mice in the sublethal H1N1 secondary S. aureus model, and alleviated lung injury. LJAE significantly reduced the recruitment of neutrophils and macrophages in the lungs (P < 0.001), significantly decreased the mRNA and protein expression of chemokine MCP-1 and pro-inflammatory cytokines TNF-α and IFN-γ (P < 0.001), and decreased the ratio of CD4+IFN-γ+/CD4+IL-4+ in peripheral blood mononuclear cells. Conclusion LJAE has significant anti-H1N1 secondary S. aureus activity, by inhibiting the excessive Th1 immune response, it inhibits the release of Th1 cytokines MCP-1, TNF- α, and IFN-γ, reduces excessive infiltration of lung inflammatory cells, and alleviates lung injury caused by H1N1 secondary S. aureus.
[中圖分類號(hào)]
R285.5
[基金項(xiàng)目]
山東省重點(diǎn)研發(fā)計(jì)劃項(xiàng)目(2021CXGC010511);校企合作項(xiàng)目-金銀花口服液技術(shù)開發(fā)項(xiàng)目(KYC2022035);2023年全國中藥特色技術(shù)傳承人才培訓(xùn)項(xiàng)目(T20234832005)