[1]刘 阳,石 芸,钟林平,等.25-OH 维生素D 通过调控TGF-β 介导的YAP/TAZ 核转位机制对新生儿感染性肺炎的抗炎作用研究[J].现代检验医学杂志,2024,39(04):28-33+39.[doi:10.3969/j.issn.1671-7414.2024.04.006]
　LIU Yang,SHI Yun,ZHONG Linping,et al.Anti-inflammatory Effect of 25-OH Vitamin D in Neonatal Infectious Pneumonia by Modulating the TGF-β-mediated YAP/TAZ Nuclear Translocation[J].Journal of Modern Laboratory Medicine,2024,39(04):28-33+39.[doi:10.3969/j.issn.1671-7414.2024.04.006]
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25-OH 维生素D 通过调控TGF-β 介导的YAP/TAZ 核转位机制对新生儿感染性肺炎的抗炎作用研究()
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《现代检验医学杂志》[ISSN:/CN:]

卷:
第39卷
期数:
2024年04期
页码:
28-33+39
栏目:
论著
出版日期:
2024-07-15

文章信息/Info

Title:
Anti-inflammatory Effect of 25-OH Vitamin D in Neonatal Infectious Pneumonia by Modulating the TGF-β-mediated YAP/TAZ Nuclear Translocation
文章编号:
1671-7414（2024）04-028-07
作者:
刘 阳; 石 芸; 钟林平; 秦膏阳
（电子科技大学医学院附属妇女儿童医院/ 成都市妇女儿童中心医院新生儿科，成都 611731）
Author(s):
LIU Yang;  SHI Yun;  ZHONG Linping;  QIN Gaoyang
（Department of Neonatology, School of Medicine, University of Electronic Science and Technology of China/Chengdu Women and Children’s Center Hospital, Chengdu 611731, China）
关键词:
新生儿感染性肺炎; 25-OH 维生素D; 转化生长因子β; Yes 相关蛋白/ 细胞核转录共激活因子PDZ 结合基序核转位
分类号:
R722.135；R392.11
DOI:
10.3969/j.issn.1671-7414.2024.04.006
文献标志码:
A
摘要:
目的 探讨25-OH 维生素D（25-OH vitamin D，25-OH-VD） 对新生儿感染性肺炎（neonatal infectiouspneumonia，NIP）的抗炎作用及其作用机制。方法 选取2022 年1 月～ 2023 年1 月成都市妇女儿童中心医院收治的65 例NIP 患儿，根据病情严重程度，分为轻症组（n=34）和重症组（n=31），另选取同期60 例健康新生儿为对照组。通过酶联免疫吸附试验（ELISA）检测血清25-OH-VD，白细胞介素-2（inteleukin-2，IL-2）、γ 干扰素（interferon-γ，IFN-γ）、肿瘤坏死因子α（tumor necrosis factor-α，TNF-α）和IL-1β 水平；蛋白免疫印迹法检测外周血单个核细胞中维生素D 受体（vitamin D receptor，VDR）、细胞核转化生长因子β（transforming growth factor β，TGF-β）/Yes相关蛋白（nucleus-Yes-associated protein，n-YAP）和细胞核转录共激活因子PDZ 结合基序（nucleus-transcriptional coactivatorwith PDZ-binding motif，n-TAZ）通路相关蛋白表达。脂多糖（lipopolysaccharide，LPS）刺激人肺上皮细胞构建NIP 体外模型，分为Control 组、LPS 组和LPS+VD 组；CCK-8 检测细胞活力；qRT-PCR 检测各组细胞中细胞色素P450家族成员28B1（recombinant cytochrome P450 27B1，CYP27B1）和VDR 的mRNA 表达；免疫细胞化学法检测各组细胞YAP 阳性细胞核数；免疫荧光检测各组细胞YAP/TAZ 复合物核转位；蛋白免疫印迹法检测各组细胞炎性因子相关蛋白表达。结果 与对照组相比，轻症组和重症组患儿血清中IL-2（1.91±0.18 μg/L，2.63±0.27 μg/L vs 1.05±0.12 μg/L），IFN-γ（1.73±0.13 μg/L，2.18±0.19 μg/L vs 1.03±0.07 μg/L），TNF-α（1.79±0.08 μg/L，2.38±0.13 μg/L vs 0.97±0.04μg/L）和IL-1β（2.18±0.07 μg/L，2.59±0.11 μg/L vs 0.96±0.02 μg/L）含量以及TGF-β（1.67±0.21，2.43±0.42vs 1.02±0.04），n-YAP（2.08±0.11，4.23±0.37 vs 0.99±0.02） 和n-TAZ（2.47±0.42，4.21±0.58 vs 1.03±0.05） 蛋白表达逐渐升高，25-OH-VD（12.57±2.21 μg/L，7.85±2.03 μg/L vs 16.76±1.02 μg/L）含量和VDR（0.73±0.09，0.51±0.06 vs 1.03±0.08）蛋白表达逐渐降低，差异具有统计学意义（F=18.983 ～ 56.782，均P<0.001）。细胞实验结果显示，与Control 组相比，LPS 组细胞存活率在12h（76.23%±0.73% vs 116.72%±2.14%），24 h（57.23%±0.94% vs 125.76%±1.67%）和48 h（41.23%±0.56% vs 138.56%±1.35%）时显著降低（t=10.342，26.562，37.821）；YAP 阳性细胞数（47.35±3.47 个 vs 12.46±1.34 个）和YAP/TAZ 复合物核转位率（2.56%±0.32% vs 1.01%±0.06%）升高（t=46.362，26.921）；IL-2（2.03±0.09 vs 1.03±0.08），IFN-γ（2.07±0.21 vs 1.02±0.04），TNF-α（2.18±0.11vs 0.99±0.02）和IL-1β（3.17±0.42 vs 1.03±0.05）的蛋白表达水平升高（t=28.341，26.713，31.235，47.823），差异具有统计学意义（均P<0.001），而两组间CYP27B1 和VDR mRNA 表达差异无统计学意义（t=0.872，0.786，均P>0.05）。与LPS 组比较，LPS+VD 组在12 h（85.23%±0.36%），24 h（79.82%±0.63%），48 h（76.28%±0.72%）的细胞存活率和CYP27B1（4.42±0.14），VDR mRNA（5.13±0.56）表达升高，YAP 阳性细胞核数（24.41±3.23 个）和YAP/TAZ 复合物核转位率（1.47%±0.26%）降低，IL-2（1.21±0.06），IFN-γ（1.13±0.42），TNF-α（1.03±0.37）和IL-1β（1.61±0.58）蛋白表达降低，差异具有统计学意义（t=7.263，19.892，23.145，27.872，26.982，14.762，13.623，18.273，25.314，27.873，22.134，均P<0.01）。结论 血清25-OH-VD 水平与NIP 严重程度相关，外源性给予VD 补充可通过调控TGF-β 介导的YAP/TAZ 核转位机制发挥抗炎作用，减轻NIP 损伤。
Abstract:
Objective To investigate the anti-inflammatory effect of 25-OH vitamin D (25-OH-VD) on neonatal infectious pneumonia (NIP) and its mechanism. Methods A total of 65 children with NIP admitted to Chengdu Women and Children’s Central Hospital from January 2022 to January 2023 were selected. According to the severity of the disease, they were divided into mild group (n=34) and severe group (n=31), and 60 healthy neonates in the same period were selected as the control group. Serum 25-OH-VD, interleukin-2 (IL-2), interferon-γ (IFN-γ), tumor necrosis factor-α (TNF-α) and IL-1β levels were measured by ELISA. The expressions of vitamin D receptor (VDR), transforming growth factor-β (TGF-β)/nuolea Yesassociated protein (n-YAP) and nuclear transcriptional coactivator PDZ-binding motif (n-TAZ) pathway related proteins in peripheral blood mononuclear cells were detected by Western blot. NIP in vitro models were established by lipopolysaccharide (LPS) stimulation of human lung epithelial cells, which were divided into control group, LPS group and LPS+VD group. Cell viability was detected by CCK-8 assay. The mRNA expressions of recombinant cytochrome P450 27B1 (CYP27B1) and VDR in each group were detected by qRT-PCR. The number of YAP positive nuclei in each group was detected by immunocytochemistry. The nuclear translocation of YAP/TAZ complex in each group was detected by immunofluorescence. The expression of inflammatory factor-related proteins in each group was detected by Western blot. Results Compared with the control group, the serum levels of IL-2 (1.91±0.18 μg/L, 2.63±0.27 μg/L vs 1.05±0.12 μg/L), IFN-γ (1.73±0.13 μg/L, 2.18±0.19 μg/L vs 1.03±0.07 μg/L), TNF-α (1.79±0.08 μg/L, 2.38±0.13 μg/L vs 0.97±0.04 μg/L), IL-1β (2.18±0.07 μg/L, 2.59±0.11 μg/L vs 0.96±0.02 μg/L), TGF-β (1.67±0.21, 2.43±0.42 vs 1.02±0.04), n-YAP (2.08±0.11, 4.23±0.37 vs 0.99±0.02) and n-TAZ (2.47±0.42, 4.21±0.58 vs 1.03±0.05) proteins of the children in the mild and severe groups were gradually increased, but the content of 25-OH-VD (12.57±2.21 μg/L, 7.85±2.03 μg/L vs 16.76±1.02 μg/L) and the expression of VDR (0.73±0.09, 0.51±0.06 vs 1.03±0.08) proteins were gradually decreased, and the differences were significant (F=18.983 ～ 56.782, all P<0.001). Cell experiment results showed that cellular survival rate was lower at 12 h (76.23%±0.73% vs 116.72%±2.14%), 24 h (57.23%±0.94% vs 125.76%±1.67%) and 48 h (41.23%±0.56% vs 138.56%±1.35%) in the LPS group compared with the control group(t=10.342, 26.562, 37.821), but the rate of cytosolic n-YAP positivity (47.35±3.47 vs 12.46±1.34), the rate of nuclear translocation of the YAP/TAZ complex (2.56%±0.32% vs 1.01%±0.06%)(t=46.362, 26.921), the protein expression levels of IL-2 (2.03±0.09 vs 1.03±0.08), IFN-γ (2.07±0.21 vs 1.02±0.04), TNF-α (2.18±0.11 vs 0.99±0.02) and IL-1β (3.17±0.42 vs 1.03±0.05) were up-regulated(t=28.341, 26.713, 31.235, 47.823), with significant differences (all P<0.001), while there was no significant difference in the mRNA expression of CYP27B1 and VDR (t=0.872, 0.786, all P>0.05). Compared with the LPS group, the cell survival rate at 12h (85.23%±0.36%), 24h (79.82%±0.63%) and 48h (76.28%±0.72%) and the mRNA expression of CYP27B1 (4.42±0.14) and VDR (5.13±0.56) were elevated in the LPS+VD group, while the nucleus n-YAP positivity (24.41±3.23), nuclear translocation of the YAP/TAZ complex (1.47%±0.26%), IL-2 (1.21±0.06), IFN-γ (1.13±0.42), TNF-α (1.03±0.37) and IL-1β (1.61±0.58) protein levels were down-regulated, and the differences were significant (t=7.263, 19.892, 23.145, 27.872, 26.982, 14.762, 13.623, 18.273, 25.314, 27.873, 22.134, all P<0.01). Conclusion The serum 25-OH-VD level is related to the severity of NIP. Exogenous VD supplementation may play an anti-inflammatory role in reducing NIP injury by regulating the TGF-β-mediated YAP/TAZ nuclear translocation mechanism.

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备注/Memo

备注/Memo:
基金项目：四川省科技计划项目（编号：19RKX0835）。
作者简介： 刘阳（1978-），女，本科，副主任医师，研究方向：新生儿循环相关，E-mail:liuyang5748@126.com。
更新日期/Last Update: 2024-07-15