目的 探讨连翘提取物对肺炎链球菌感染的肺泡上皮细胞A549损伤的影响及其可能作用机制。方法 采用肺炎链球菌感染肺泡上皮细胞A549建立细胞损伤模型，不同剂量的连翘提取物处理细胞；微小RNA-223-3p（micro RNA-223-3p，miR-223-3p）特异性寡核苷酸抑制剂（anti-miR-223-3p）及其阴性对照序列（anti-miR-NC）分别转染至A549细胞后用肺炎链球菌培养细胞；miR-223-3p寡核苷酸模拟物（miR-223-3p mimics）转染至A549细胞后用连翘提取物处理细胞，然后用肺炎链球菌培养细胞。MTT、流式细胞术分别检测细胞增殖与凋亡能力；ELISA法检测白细胞介素-6（interleukin-6，IL-6）、γ干扰素（interferon γ，IFN-γ）、肿瘤坏死因子-α（tumor necrosis factor-α，TNF-α）、白细胞介素-10（interleukin-10，IL-10）的水平；qRT-PCR法检测miR-223-3p的表达量；Western blotting法检测活化的含半胱氨酸的天冬氨酸蛋白水解酶3（cleaved cysteinyl aspartate-specific protease-3，cleaved Caspase-3）蛋白表达量。结果 连翘提取物能够以剂量相关方式增强肺炎链球菌感染的A549细胞活力（P<0.05），升高IL-10水平（P<0.05），降低细胞凋亡率和cleaved Caspase-3蛋白表达水平（P<0.05），还可降低miR-223-3p的表达量和IL-6、INF-γ、TNF-α的水平（P<0.05）；转染anti-miR-223-3p后感染肺炎链球菌的A549细胞活力和IL-10的水平升高（P<0.05），细胞凋亡率和cleaved Caspase-3蛋白水平降低（P<0.05），IL-6、INF-γ、TNF-α的水平降低（P<0.05）；转染miR-223-3p mimics可逆转连翘提取物对肺炎链球菌感染的A549细胞增殖、凋亡及炎症反应的作用。结论 连翘提取物可能通过抑制miR-223-3p的表达而促进细胞增殖及抑制细胞凋亡、炎症反应从而减轻肺炎链球菌感染的肺泡上皮细胞损伤。

Objective To explore the effect of Lianqiao (Forsythiae Fructus) extract on injury of alveolar epithelial cells A549 infected by Streptococcus pneumoniae and its possible mechanism. Methods Streptococcus pneumoniae was used to infect alveolar epithelial cells A549 to establish a cell injury model, and different doses of Forsythiae Fructus extract were used to treat the cells. Micro RNA-223-3p (miR-223-3p) specific oligonucleotide inhibitor (anti-miR-223-3p) and its negative control sequence (anti-miR-NC) were respectively transfected into A549 cells and cultured with Streptococcus pneumoniae. After miR-223-3p oligonucleotide mimics (miR-223-3p mimics) was transfected into A549 cells, the cells were treated with Forsythiae Fructus extract, and then the cells were cultured with Streptococcus pneumoniae. MTT and flow cytometry were used to detect cell proliferation and apoptosis. ELISA method was used to detect the levels of interleukin-6 (IL-6), interferon γ (INF-γ), tumor necrosis factor-α (TNF-α) and IL-10. qRT-PCR method was used to detect the expression of miR-223-3p. Western blotting method was used to detect the expression of cleaved Caspase-3 protein. Results Forsythiae Fructus extract could enhance the viability of A549 cells infected by Streptococcus pneumoniae in a dose-dependent manner (P < 0.05), and could increase the level of IL-10 (P < 0.05), but could reduce the apoptosis rate and the protein level of cleaved Caspase3 (P < 0.05), it could also reduce the expression of miR-223-3p and the levels of IL-6, INF-γ, TNF-α (P < 0.05). After transfection with anti-miR-223-3p, the viability and IL-10 levels of A549 cells infected by Streptococcus pneumoniae were increased (P < 0.05), while the apoptosis rate and the protein level of cleaved Caspase-3 were decreased (P < 0.05), and the levels of IL-6, INF-γ, TNF-α were decreased (P < 0.05). Transfection of miR-223-3p mimics could reverse the effects of Forsythiae Fructus extract on the proliferation, apoptosis and inflammation of A549 cells infected with Streptococcus pneumoniae. Conclusion Forsythiae Fructus extract may promote cell proliferation and inhibit cell apoptosis and inflammation by inhibiting the expression of miR-223-3p, thereby reducing the damage of alveolar epithelial cells infected by Streptococcus pneumoniae.

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