小儿健脾益肺方改善哮喘小鼠气道高反应的实验研究
Experimental study of
Xiao ’er Jianpi Yifei Formula improving airway hyperresponsiveness in asthmatic mice- 北京中医药大学学报 2023年46卷第9期 页码：1267-1279
- 作者机构：
中国中医科学院西苑医院 北京 100091
- 作者简介：
陈恂，男，博士，主治医师
# 冀晓华，教授，主任医师，主要研究方向：中医药防治儿童呼吸系统过敏性疾病，E-mail：13611199408@163.com
- 基金信息：国家自然科学基金青年项目(82104934);中国中医科学院科技创新工程项目(CI2021A02502;CI2021A02508);中国中医科学院优秀青年科技人才培养专项(ZZ15-YQ-007)
DOI：10.3969/j.issn.1006-2157.2023.09.012
中图分类号： R285.5纸质出版日期：2023-09-30，
网络出版日期：2023-07-27，
收稿日期：2023-03-29，
- 稿件说明：
移动端阅览
陈恂, 燕晓茹, 李敏, 等. 小儿健脾益肺方改善哮喘小鼠气道高反应的实验研究[J]. 北京中医药大学学报, 2023,46(9):1267-1279.
CHEN Xun, YAN Xiaoru, LI Min, et al. Experimental study of
Xiao ’er Jianpi Yifei Formula improving airway hyperresponsiveness in asthmatic mice[J]. Journal of Beijing University of Traditional Chinese Medicine, 2023,46(9):1267-1279.陈恂, 燕晓茹, 李敏, 等. 小儿健脾益肺方改善哮喘小鼠气道高反应的实验研究[J]. 北京中医药大学学报, 2023,46(9):1267-1279. DOI： 10.3969/j.issn.1006-2157.2023.09.012.
CHEN Xun, YAN Xiaoru, LI Min, et al. Experimental study of
Xiao ’er Jianpi Yifei Formula improving airway hyperresponsiveness in asthmatic mice[J]. Journal of Beijing University of Traditional Chinese Medicine, 2023,46(9):1267-1279. DOI： 10.3969/j.issn.1006-2157.2023.09.012.
摘要
目的
2
探讨小儿健脾益肺方通过高迁移率组蛋白B1(HMGB1)/Toll样受体4(TLR4)/核转录因子-κB(NF-κB)炎症信号通路改善哮喘小鼠气道高反应的作用机制。
方法
2
60只SPF级Balb/c小鼠随机数字表法分为空白组、模型组、布地奈德组、小儿健脾益肺方组、布地奈德＋小儿健脾益肺方组，用卵清白蛋白(OVA)致敏、激发建立小鼠急性哮喘模型，分别予布地奈德(0.2 g/L)、小儿健脾益肺方(1.875 g/kg)干预。肺功能检测气道阻力，支气管肺泡灌洗液吉姆萨染色检测小鼠各种细胞占比情况，肺组织切片HE及PAS染色观察，ELISA检测血清及支气管肺泡灌洗液中OVA-IgE、白细胞介素(IL)-6、IL-1β、γ干扰素(IFN-γ)、肿瘤坏死因子-α(TNF-α)含量，蛋白质印迹法及免疫荧光检测HMGB1、髓样分化因子88(MyD88)、TLR4、p-NF-κB/NF-κB等通路蛋白表达情况。
结果
2
与空白组比较，模型组气道阻力明显升高(
P
<
0.01)，支气管肺泡灌洗液中嗜酸性粒细胞比例明显升高(
P
<
0.01)，组织病理学染色较空白组出现明显炎性细胞浸润、杯状细胞增生、气道上皮水肿、管腔狭窄，血清及支气管肺泡灌洗液中OVA-IgE、IL-6、IL-1β、IFN-γ、TNF-α表达升高(
P
<
0.01)，HMGB1、MyD88、TLR4、NF-κB等通路蛋白表达升高(
P
<
0.05)，NF-κB磷酸化程度升高(
P
<
0.05)。与模型组比较，布地奈德组、小儿健脾益肺方组、布地奈德＋小儿健脾益肺方组气道阻力降低，支气管肺泡灌洗液中巨噬细胞占比升高、嗜酸性粒细胞降低(
P
<
0.05)，组织病理学染色结果显示炎症细胞浸润改善、平滑肌层增厚缓解、上皮完整性增加，杯状细胞化生减少，HE评分、PAS评分与模型组比较差异具有统计学意义(
P
<
0.05)。血清和支气管肺泡灌洗液中，小儿健脾益肺方组、布地奈德组、布地奈德＋小儿健脾益肺方组与模型组比较均能明显降低各项指标的含量(
P
<
0.01)；与布地奈德组比较，小儿健脾益肺方组血清OVA-IgE含量明显升高(
P
<
0.05)，小儿健脾益肺方组和布地奈德＋小儿健脾益肺方组HMGB1含量明显降低(
P
<
0.05)。蛋白质印迹法检测结果显示，与模型组比较，小儿健脾益肺方组、布地奈德组、布地奈德＋小儿健脾益肺方组均能有效降低HMGB1、MyD88和TLR4蛋白的表达和NF-κB的磷酸化程度，差异具有统计学意义(
P
<
0.01)；与布地奈德组相比，小儿健脾益肺方组HMGB1的表达明显下降，而TLR4、MyD88的表达升高，布地奈德＋小儿健脾益肺方组HMGB1、TLR4、MyD88的表达均明显下降，差异均具有统计学意义(
P
<
0.05)。免疫荧光检测结果显示，小儿健脾益肺方组、布地奈德组、布地奈德＋小儿健脾益肺方组HMGB1与模型组比较明显降低(
P
<
0.01)，布地奈德＋小儿健脾益肺方组的HMGB1和TLR4较布地奈德组均明显降低(
P
<
0.05)。
结论
2
小儿健脾益肺方和布地奈德均能有效缓解哮喘相关病理改变，降低炎症因子表达，抑制HMGB1/TLR4/NF-κB炎症信号通路，改善气道高反应。小儿健脾益肺方和布地奈德联合应用优于布地奈德单独应用。HMGB1/TLR4/NF-κB炎症信号通路是健脾益肺方改善儿童哮喘气道高反应性的潜在作用机制。
Abstract
Objective
2
We sought to explore the mechanism of
Xiao
’
er Jianpi Yifei
Formula in improving airway hyperresponsiveness in asthmatic mice signaling pathway through the HMGB1/TLR4/NF-κB inflammatory signaling pathway.
Methods
2
Sixty SPF Balb/c mice were randomly divided into the control group
the model group
the budesonide group
the
Xiao
’
er Jianpi Yifei
Formula group
and the budesonide +
Xiao
’
er Jianpi Yifei
Formula group. The acute asthma model was established by sensitization and stimulation with ovalbumin(OVA). Budesonide(0.2 g/L) and
Xiao
’
er Jianpi Yifei
Formula(1.875 g/kg) were used for intervention. The degree of airway hyperresponsiveness was evaluated by measuring airway resistance. The proportion of different types of cells in bronchoalveolar lavage fluid(BALF) was detected by Giemsa staining. Pathological changes were observed by HE staining and PAS staining in lung tissue sections. The expression levels of OVA-IgE
IL-6
IL-1β
IFN-γ
and TNF-α in serum and BALF were evaluated by ELISA
and the expression of HMGB1
MyD88
TLR4
and p-NF-κB/NF-κB were detected by Western blotting and immunofluorescence.
Results
2
Compared to the control group
the model group showed a significant increase in airway resistance(
P
<
0.01)
an obvious increase in the proportion of eosinophils in bronchoalveolar lavage fluid(
P
<
0.01)
significant inflammatory cell infiltration
goblet cell hyperplasia
epithelial swelling
luminal stenosis in tissue pathology staining
and a significant increase in the expression levels of OVA-IgE
IL-6
IL-1β
IFN-γ
and TNF-α in serum and BALF(
P
<
0.01). The expression levels of HMGB1
MyD88
TLR4
and NF-κB were also elevated(
P
<
0.05). The phosphorylation of NF-κB was increased(
P
<
0.05). Compared to the model group
the budesonide group
the
Xiao
’
er Jianpi Yifei
Formula group
and the budesonide+
Xiao
’
er Jianpi Yifei
Formula group exhibited reduced airway resistance
an increased proportion of macrophages in BALF
and reduced eosinophil counts(
P
<
0.05). The histopathological staining result showed that the infiltration of inflammatory cells was improved
the smooth muscle layer thickening reduced
the epithelial integrity was increased
and the metaplasia of Goblet cell was reduced. HE scores and PAS scores compared to the model group were statistical significant(
P
<
0.05). In the serum and BALF
the contents of various indexes in the
Xiao
’
er Jianpi Yifei
Formula group
the budesonide group
and the budesonide+
Xiao
’
er Jianpi Yifei
Formula group were lower than those in the model group(
P
<
0.01); Compared to the budesonide group
the serum OVA-IgE content in the
Xiao
’
er Jianpi Yifei
Formula group was greater (
P
<
0.05)
while the HMGB1 content in the
Xiao
’
er Jianpi Yifei
Formula group and the budesonide+
Xiao
’
er Jianpi Yifei
Formula group was lower(
P
<
0.05). Western blotting result showed that
compared to the model group
the expression of HMGB1
MyD88
and TLR4 proteins
and the phosphorylation degree of NF-κB could be effectively reduced (
P
<
0.01) in the
Xiao
’
er Jianpi Yifei
Formula group
the budesonide group
and the budesonide+
Xiao
’
er Jianpi Yifei
Formula group . Compared to the budesonide group
the expression of HMGB1 in the
Xiao
’
er Jianpi Yifei
Formula group was decreased significantly
while the expression of both TLR4 and MyD88 was increased
and the expressions of HMGB1
TLR4
and MyD88 in the budesonide+
Xiao
’
er Jianpi Yifei
Formula group were decreased significantly
with statistically significant differences(
P
<
0.05). The IF result showed that HMGB1 expression levels in the
Xiao
’
er Jianpi Yifei
Formula group
budesonide group
and budesonide+
Xiao
’
er Jianpi Yifei
Formula group were significantly lower than that in the model group(
P
<
0.01)
and the HMGB1 and TLR4 expression levels in the budesonide+
Xiao
’
er Jianpi Yifei
Formula group were significantly lower than those in the budesonide group(
P
<
0.05).
Conclusion
2
Xiao
’
er Jianpi Yifei
Formula and budesonide could effectively alleviate asthma-related pathological changes
reduce the expression of inflammatory cytokines
inhibit the HMGB1/TLR4/NF-κB inflammatory signaling pathway
and improve airway hyperresponsiveness. The combination of
Xiao
’
er Jianpi Yifei
Formula and budesonide was superior to budesonide alone. The HMGB1/TLR4/NF-κB inflammatory signaling pathway may be a potential mechanism for
Xiao
’
er Jianpi
Yifei Formula to improve airway hyperresponsiveness in children with asthma.
关键词
儿童哮喘小儿健脾益肺方HMGB1/TLR4/NF-κB炎症信号通路气道高反应小鼠
Keywords
childhood asthmaXiao’er Jianpi Yifei FormulaHMGB1/TLR4/NF-κB inflammatory signaling pathwayairway hyperresponsivenessmice
references
中华儿科杂志编辑委员会，中华医学会儿科学分会呼吸学组，中国医师协会儿科医师分会儿童呼吸专业委员会．儿童支气管哮喘规范化诊治建议(2020年版) [J]. 中华儿科杂志， 2020, 58(9): 708－717.
Global Initiative for Asthma. Global Strategy for Asthma Management and Prevention [EB/OL].(2023－05－02)[2023－06－26].https：//ginasthma.org/wp-content/uploads/2023/05/GINA－2023－Full-Report－2023－WMS.pdf.
肖惠迪， 书文， 李梦龙， 等. 中国2011—2018年儿童哮喘患病率Meta分析 [J]. 中国学校卫生， 2020, 41(8): 1208－1211.
ELISEEVA TI, GEPPE NA, TUSH EV, et al. Body Height of Children with Bronchial Asthma of Various Severities [J/OL]. Can Respir J, 2017, 2017：8761404[2022－11－08].https：//www.hindawi.com/journals/crj/2017/8761404/https：//www.hindawi.com/journals/crj/2017/8761404/.
ULLAH MA, LOH Z, GAN WJ, et al. Receptor for advanced glycation end products and its ligand high-mobility group box－1 mediate allergic airway sensitization and airway inflammation [J].J Allergy Clin Immunol, 2014, 134(2): 440－450.
ZHOU Y, JIANG YQ, WANG WX, et al. HMGB1 and RAGE levels in induced sputum correlate with asthma severity and neutrophil percentage [J]. Hum Immunol, 2012, 73(11): 1171－1174.
SHALABY KH, HEIALY SA, TSUCHIYA K, et al. The TLR4－TRIF pathway can protect against the development of experimental allergic asthma [J]. Immunology, 2017, 152(1): 138－149.
ZHANG H, YANG N, WANG TY, et al. Vitamin D reduces inflammatory response in asthmatic mice through HMGB1/TLR4/NF－κB signaling pathway [J]. Mol Med Rep, 2018, 17(2): 2915－2920.
刘礼荣， 符诒慧， 周黎明， 等. 抑制HMGB1对支气管哮喘小鼠气道炎症的影响及机制 [J]. 中华医院感染学杂志， 2022, 32(11): 1632－1637.
姚如婕， 刘沉涛， 黄榕， 等. 布地奈德雾化治疗对哮喘小鼠糖皮质激素受体及核因子－κB表达的影响[J]. 中国当代儿科杂志， 2015, 17(1): 86－89.
KANG JY, LEE SY, RHEE CK, et al. Effect of aging on airway remodeling and muscarinic receptors in a murine acute asthma model [J]. Clin Interv Aging, 2013, 8：1393－1403.
PADRID P, SNOOK S, FINUCANE T, et al. Persistent airway hyperresponsiveness and histologic alterations after chronic antigen challenge in cats [J]. Am J Respir Crit Care Med, 1995, 151(1): 184－193.
高桂玲. 参苓白术散辅助丙酸氟替卡松气雾剂治疗肺脾气虚型哮喘缓解期患儿的效果 [J]. 临床医学， 2022, 42(8): 115－157.
邬素珍， 莫珊， 陆柳丹， 等. 健脾补肺膏对咳嗽变异性哮喘缓解期儿童的影响 [J]. 中国中西医结合杂志， 2023，43(1)：113－116.
杨宏志， 郭光辉， 杨俊姝， 等. 益肺Ⅱ号方对支气管哮喘缓解期免疫功能和血清炎症因子的影响 [J]. 中华中医药学刊， 2020, 38(4): 94－97.
吴振奎， 钟丽花， 刘延霞， 等. 女贞子多糖联合布地奈德在哮喘幼鼠模型中的免疫调节及抗氧化作用研究 [J]. 中国免疫学杂志， 2022, 38(9): 1069－1074.
HOU CC, KONG JC, LIANG Y, et al. HMGB1 contributes to allergen-induced airway remodeling in a murine model of chronic asthma by modulating airway inflammation and activating lung fibroblasts [J]. Cell Mol Immunol, 2015, 12(4): 409－423.
LV YH, LI YL, ZHANG DD, et al. HMGB1－induced asthmatic airway inflammation through GRP75－mediated enhancement of ER-mitochondrial Ca2＋ transfer and ROS increased [J]. J Cell Biochem, 2018, 119(5): 4205－4215.
DI CANDIA L, GOMEZ E, VENEREAU E, et al. HMGB1 is upregulated in the airways in asthma and potentiates airway smooth muscle contraction via TLR4 [J]. J Allergy Clin Immunol, 2017, 140(2): 584－587.
HOU CC, ZHAO HJ, LIU LY, et al. High mobility group protein B1(HMGB1) in Asthma: comparison of patients with chronic obstructive pulmonary disease and healthy controls [J]. Mol Med, 2011, 17(7/8): 807－815.
OTTOW MK, KLAVER EJ, VAN DER POUW KRAAN TC, et al. The helminth Trichuris suis suppresses TLR4－induced inflammatory responses in human macrophages [J]. Genes Immun, 2014, 15(7): 477－486.
KIM HS, CHUNG DH. TLR4－mediated IL－12 production enhances IFN－γ and IL－1β production, which inhibits TGF－β production and promotes antibody-induced joint inflammation [J/OL]. Arthritis Res Ther, 2012, 14(5): R210[2022－11－08]. https：//arthritis-research.biomedcentral.com/articles/10.1186/ar4048https：//arthritis-research.biomedcentral.com/articles/10.1186/ar4048.
ZHU HT, BIAN C, YUAN JC, et al. Curcumin attenuates acute inflammatory injury by inhibiting the TLR4/MyD88/NF－κB signaling pathway in experimental traumatic brain injury [J/OL]. J Neuroinflammation, 2014, 11：59[2022－11－08]. https：//jneuroinflammation.biomedcentral.com/articles/10.1186/1742－2094－11－59https：//jneuroinflammation.biomedcentral.com/articles/10.1186/1742－2094－11－59.
乔俊英， 宋丽， 张艳丽， 等. 哮喘小鼠HMGB1/TLR4/NF－κB信号通路及维生素D的作用 [J]. 中国当代儿科杂志， 2017, 19(1): 95－103.
LEE CC, LAI YT, CHANG HT, et al. Inhibition of high-mobility group box 1 in lung reduced airway inflammation and remodeling in a mouse model of chronic asthma [J]. Biochem Pharmacol, 2013, 86(7): 940－949.
SHANG LQ, WANG L, SHI XL, et al. HMGB1 was negatively regulated by HSF1 and mediated the TLR4/MyD88/NF－κB signal pathway in asthma [J/OL]. Life Sci, 2020, 241：117120[2022－11－08].http：// www.sciencedirect.com/science/article/abs/pii/S0024320519310-471？via%3Dihubhttp：// www.sciencedirect.com/science/article/abs/pii/S0024320519310-471？via%3Dihub.
何苗. 布地奈德对高氧致新生小鼠支气管肺发育不良及HMGB1/TLR4/NF－κB通路的影响 [J]. 中国医师杂志， 2019, 21(8): 1199－1204.
刘虹， 王洁英， 刘娜， 等. 布地奈德混悬液对哮喘模型小鼠肺组织TLR4/MyD88/NF－κB通路的影响 [J]. 现代生物医学进展， 2021, 21(23): 4432－4435.
李星， 李竹英， 王珏. 平喘颗粒对哮喘气道相关炎症因子及肺组织HMGB1、TLR4表达的影响 [J]. 中国中医急症， 2021, 30(6): 941－944.
程哲， 代灵灵， 曹德飞， 等．支气管哮喘患者诱导痰中HMGB1和RAGE水平的变化及临床意义[J] . 中华医学杂志，2011，91(22): 1538－1542.
DONG YM, YAN H, ZHAO X, et al. Gu-Ben-Fang-Xiao Decoction Ameliorated Murine Asthma in Remission Stage by Modulating Microbiota-Acetate-Tregs Axis [J/OL]. Front Pharmacol, 2020, 11：549[2022－11－08]. https：//www.frontiersin.org/articles/10.3389/fphar.2020.00549/fullhttps：//www.frontiersin.org/articles/10.3389/fphar.2020.00549/full.
陈国榕， 吴东. 肠道菌群代谢产物丁酸减轻重症急性胰腺炎相关肺损伤的机制[J].中华临床营养杂志，2020，28(6)：363－367.
吴玉苗， 吴要伟， 朱万青， 等. 短链脂肪酸与儿童哮喘的关系及治疗研究进展[J].中国现代医学杂志，2022，32(16)：37－42.
丁泽君， 孙明洁， 刘佳， 等．丁酸钠对脂多糖诱导的急性肺损伤大鼠的肺保护研究[J]. 国际麻醉学与复苏杂志，2013，34(9)：779－782.
LI N, LIU XX, HONG M, et al. Sodium butyrate alleviates LPS-induced acute lung injury in mice via inhibiting HMGB1 release [J]. Int Immunopharmacol, 2018, 56：242－248.
0
浏览量
25
下载量
0
CSCD
- 网络PDF下载
- BibTeX下载
- Endnote下载
- RefMan 下载
- NoteExpress下载
- NoteFirst下载
关联资源
相关文章
相关作者
相关机构
- 地址：北京市朝阳区北三环东路11号 邮编：100029
- 联系电话：(010)53911857 Email：jbutcm@bucm.edu.cn
- 技术支持由北京北大方正电子有限公司提供 京ICP备09064830号-19
京公网安备11010802024621 - 本系统建议在Chrome、 IE9+ 以上版本浏览器阅读本站内容，360浏览器请切换至极速模式
- Cookies帮助我们提供服务并提供个性化体验。使用本网站，即表示您同意我们使用Cookies