浙江中西医结合杂志

王香英,魏金凤,田小辉,盛美玲,许如菊.灯盏花素调节lncRNA NEAT1/miR-9-5p/SLC26A2轴抑制哮喘小鼠的气道炎症[J].浙江中西医结合杂志,2024,34(2):

灯盏花素调节lncRNA NEAT1/miR-9-5p/SLC26A2轴抑制哮喘小鼠的气道炎症

Breviscapine alleviates airway inflammation via regulating lncRNA NEAT1/miR-9-5p/SLC26A2 axis in mice with asthma

投稿时间：2023-04-21 修订日期：2024-01-05

DOI：

中文关键词: 小鼠哮喘气道炎症灯盏花素 lncRNA NEAT1 miR-9-5p SLC26A2

英文关键词:Mouse Breviscapine Asthma Airway inflammation lncRNA NEAT1 miR-9-5p SLC26A2

基金项目:浙江省医药卫生科技计划项目(2021KY928, 2021KY932)作者单位: 杭州市儿童医院儿内科(王香英,盛美玲,许如菊),呼吸科(魏金凤),健康管理中心(田小辉)(杭州 310014)通讯作者王香英；Tel：13750821390；E-mail：13750821390@163.com

作者	单位	E-mail
王香英^*	杭州市儿童医院	13750821390@163.com
魏金凤
田小辉
盛美玲
许如菊

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中文摘要:

摘要：目的：探究灯盏花素通过长链非编码RNA（lncRNA）NEAT1/microRNA（miR）-9-5p/SLC26A2轴对哮喘模型小鼠气道炎症的抑制作用及分子机制。方法 15只6~8周雄性C57BL/6J小鼠，按照随机数字表法分为三组，即对照组、哮喘模型组和灯盏花素组。灯盏花素组小鼠在哮喘模型构建完成后每天1次灌胃10 mg/kg灯盏花素，连续7 d；对照组及哮喘模型组则使用等体积0.9%生理盐水进行灌胃处理。采用苏木素-伊红（HE）染色和过碘酸雪夫（PAS）染色对小鼠肺组织进行组织病理学观察。采用Wright-Giemsa对支气管肺泡灌洗液（BALF）进行染色及细胞学检测。酶联免疫吸附实验检测BALF及血清中炎性因子的水平。荧光原位杂交检测NEAT1的表达。实时荧光定量PCR检测lncRNA-NEAT1、miR-9-5p和SLC26A2 mRNA的表达。蛋白免疫印迹检测SLC26A2的蛋白表达。结果与对照组比较，哮喘模型组小鼠的BALF炎性细胞总数[（68.32±7.25）×104/mL比（17.71±3.14）×104/mL，P<0.01]、中性粒细胞数[（5.50±0.58）×104/mL比（0.72±0.15）×104/mL，P<0.01]、巨噬细胞数[（13.02±1.04）×104/mL比（2.70±0.61）×104/mL，P<0.01]、淋巴细胞数[（23.07±2.90）×104/mL比（4.13±0.53）×104/mL，P<0.01]、嗜酸性粒细胞数[（22.13±2.21）×104/mL比（1.63±0.22）×104/mL，P<0.01]增加；肺组织炎性细胞浸润水平增加[（2.49±0.25）分比（0.26±0.04）分，P<0.01]，PAS评分升高[（2.24±0.16）分比（0.26±0.08）分，P<0.01]；血清IgE[（3.52±0.32） IU/mL比（1.02±0.08） IU/mL，P<0.01]及BALF中白细胞介素-5（IL-5）[（154.48±9.27） pg/mL比（54.56±3.35） pg/mL，P<0.01]、白细胞介素-13（IL-13）[（96.86±6.45） pg/mL比（79.18±3.34） pg/mL，P<0.05]、白细胞介素-17（IL-17）[（185.24±7.24） pg/mL比（73.32±4.15） pg/mL，P<0.01]表达上升，干扰素-γ（INF-γ）表达下降[（48.46±3.81） pg/mL比（84.76±2.91） pg/mL，P<0.01]。与哮喘模型组比较，灯盏花素组小鼠BALF炎性细胞总数[（52.10±7.59）×104/mL比（68.32±7.25）×104/mL，P<0.05]、中性粒细胞数[（4.21±0.54）×104/mL比（5.50±0.58）×104/mL，P<0.05]、巨噬细胞数[（3.61±0.28）×104/mL比（13.02±1.04）×104/mL，P<0.01]、淋巴细胞数[（9.52±1.23）×104/mL比（23.07±2.90）×104/mL，P<0.01]、嗜酸性粒细胞数[（8.87±1.33）×104/mL比（22.13±2.21）×104/mL，P<0.01]降低；肺组织炎性细胞浸润水平[（1.46±0.15）分比（2.49±0.25）分，P<0.01]及PAS评分降低[（0.58±0.07）分比（2.24±0.16）分，P<0.01]；血清IgE[（1.83±0.14） IU/mL比（3.52±0.32） IU/mL，P<0.01]及BALF中IL-5[（78.25±4.44） pg/mL比（154.48±9.27） pg/mL，P<0.01]、IL-13[（59.34±5.32） pg/mL比（96.86±6.45） pg/mL，P<0.01]、IL-17[（125.60±5.88） pg/mL比（185.24±7.24） pg/mL，P<0.01]表达下降，INF-γ[（65.48±4.49） pg/mL比（48.46±3.81） pg/mL，P<0.05]表达上升。与对照组比较，哮喘模型组小鼠肺组织中NEAT1[（3.96±0.32）比（1.00±0.15），P<0.01]、SLC26A2相对表达上升[（3.91±0.32）比（1.00±0.08），P<0.01]，miR-9-5p相对表达量显著下降[（0.48±0.07）比（1.00±0.09），P<0.01]。与哮喘模型组比较，灯盏花素组小鼠NEAT1[（1.82±0.28）比（3.96±0.32），P<0.01]、SLC26A2相对表达降低[（2.00±0.23）比（3.91±0.32），P<0.01]，miR-9-5p相对表达增加[（0.67±0.06）比（0.48±0.07），P<0.05]。结论灯盏花素可能通过调节lncRNA NEAT1/miR-9-5p/SLC26A2轴抑制哮喘小鼠的气道炎症。

英文摘要:

Abstract Aim: To investigate the inhibitory effect and molecular mechanisms of breviscapine on airway inflammation of mice with asthma via long noncoding RNA (lncRNA) NEAT1/ microRNA (miR)-9-5p/SLC26A2. Methods 15 male C57BL/6J mice aged 6-8 weeks were divided into 3 groups according to random number table method: control group, asthmatic model group and breviscapine group. The mice in the breviscapine group were given 10 mg/kg breviscapine once a day for 7 consecutive days after the establishment of the asthma model. The control group and asthmatic mice were treated with 0.9%NaCl at the same volume. Hematoxylin-eosin (HE) staining and periodic acid-Schiff (PAS) staining were used for histopathological observation of lung tissue of mice. Wright-Giemsa staining and cytological examination of bronchoalveolar lavage fluid (BALF) were performed. The levels of inflammatory factors in BALF and serum were detected by enzyme-linked immunosorbent assay. The expression of NEAT1 was detected by fluorescence in situ hybridization. Quantitative real-time polymerase chain reaction (qRT-PCR) was used to detect the expression of lncRNA-NEAT1, miR-9-5p and SLC26A2 mRNA. The protein expression level of SLC26A2 was assessed by western blot. Results In inflammatory index, compared with the control group, the total number inflammatory cells of asthmatic mice [(68.32±7.25)×104/mL vs. (17.71±3.14)×104/mL, P<0.01], the number of neutrophil [(5.50±0.58)×104/mL vs. (0.72±0.15)×104/mL, P<0.01], the number of macrophage[(13.02±1.04)×104/mL vs. (2.70±0.61)×104/mL, P<0.01], the number of lymphocyte[(23.07±2.90)×104/mL vs. (4.13±0.53)×104/mL, P<0.01], the number of Eosinophils increased [(22.13±2.21)×104/mL vs. (17.71±3.14)×104/mL, P<0.01]. The level of inflammatory cell infiltration in lung tissue increased [(2.49±0.25) vs. (0.26±0.04), P<0.01], PAS score increased [(2.24±0.16) vs. (0.26±0.08), P<0.01]. Serum IgE [(3.52±0.32)IU/mL vs. (1.02±0.08)IU/mL, P<0.01] ,IL-5 [(154.48±9.27)pg/mL vs. (54.56±3.35)pg/mL, P<0.01], IL-13[(96.86±6.45)pg/mL vs. (79.18±3.34)pg/mL, P<0.05], IL-17[(185.24±7.24)pg/mL vs. (73.32±4.15)pg/mL, P<0.01]decreased, INF-γ[(48.46±3.81)pg/mL vs. (84.76±2.91)pg/mL, P<0.01]increased in BALF. While Breviscapine inhibits these inflammatory markers in asthma mice, compared with the asthma group, the number total inflammatory cell of the mice in breviscapine group decreased [(52.10±7.59)×104/mL vs. (68.32±7.25)×104/mL, P<0.05], the number of neutrophil decreased [(4.21±0.54)×104/mL vs. (5.50±0.58)×104/mL, P<0.05], the number of macrophage decreased [(3.61±0.28)×104/mL vs. (13.02±1.04)×104/mL, P<0.01],the number of lymphocyte decreased [(9.52±1.23)×104/mL vs. (23.07±2.90)×104/mL, P<0.01], the number of Eosinophils decreased [(8.87±1.33)×104/mL vs. (22.13±2.21)×104/mL, P<0.01]. The level of inflammatory cell infiltration in lung tissue decreased [(1.46±0.15) vs. (2.49±0.25), P<0.01], PAS score decreased [(0.58±0.07) vs. (2.24±0.16), P<0.01]. Serum IgE decreased [(1.83±0.14)IU/mL vs. (3.52±0.32)IU/mL, P 0.01], IL-5 [(78.25±4.44)pg/mL vs. (154.48±9.27)pg/mL, P<0.01], IL-13[(59.34±5.32)pg/mL vs. (96.86±6.45)pg/mL, P<0.01], IL-17[(125.60±5.88)pg/mL vs. (185.24±7.24)pg/mL, P<0.01], INF-γ decreased [(65.48±4.49)pg/mL vs. (48.46±3.81)pg/mL, P＜0.05] in BALF. In molecular mechanisms, compared with the control group, the relative expression of NEAT1[(3.96±0.32) vs. (1.00±0.15), P<0.01] and SLC26A2 increased [(3.91±0.32) vs. (1.00±0.08), P<0.01] , the relative expression of miR-9-5p decreased [(0.48±0.07) vs. (1.00±0.09), P<0.01] in asthmatic mice. Compared with the asthma group, the relative expression of NEAT1 in the breviscapine group mice decreased [(1.82±0.28) vs. (3.96±0.32), P<0.01], the relative expression of SLC26A2 decreased [(2.00±0.23) vs. (3.91±0.32), P<0.01], the relative expression of miR-9-5p increased [(0.67±0.06) vs. (0.48±0.07), P<0.05]. Conclusion Breviscapine could alleviate airway inflammation via regulating lncRNA NEAT1/miR-9-5p/SLC26A2 axis in mice with asthma.

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