Let-7e-5p调控Fas/FasL影响小鼠变应性鼻炎发病的机制研究

doi:10.11958/20221110

天津医药 ›› 2023, Vol. 51 ›› Issue (4): 360-365.doi: 10.11958/20221110

Let-7e-5p调控Fas/FasL影响小鼠变应性鼻炎发病的机制研究

唐桥斐¹(), 曹建秋², 张爽¹^,^△()

1 沈阳医学院附属第二医院耳鼻喉科（邮编110002）
2 沈阳医学院附属第二医院干诊科（邮编110002）

收稿日期:2022-07-14 修回日期:2022-09-22 出版日期:2023-04-15 发布日期:2023-04-20
通讯作者: 张爽 E-mail:tanghuashengmi@163.com;zs_5257@163.com
作者简介:唐桥斐（1977），女，副主任医师，主要从事变应性鼻炎的发病机制及治疗方面研究。E-mail：tanghuashengmi@163.com
基金资助:
辽宁省科学技术计划项目(2018225020);沈阳市科学技术计划资助项目(21-173-9-17)

Study on the mechanism of let-7e-5p regulating Fas/FasL to affect the pathogenesis of allergic rhinitis in mice

TANG Qiaofei¹(), CAO Jianqiu², ZHANG Shuang¹^,^△()

1 Department of Otolaryngology, the Second Hospital of Shenyang Medical College, Shenyang 110002, China
2 Department of Cadre, the Second Hospital of Shenyang Medical College, Shenyang 110002, China

Received:2022-07-14 Revised:2022-09-22 Published:2023-04-15 Online:2023-04-20
Contact: ZHANG Shuang E-mail:tanghuashengmi@163.com;zs_5257@163.com

唐桥斐, 曹建秋, 张爽. Let-7e-5p调控Fas/FasL影响小鼠变应性鼻炎发病的机制研究[J]. 天津医药, 2023, 51(4): 360-365.

TANG Qiaofei, CAO Jianqiu, ZHANG Shuang. Study on the mechanism of let-7e-5p regulating Fas/FasL to affect the pathogenesis of allergic rhinitis in mice[J]. Tianjin Medical Journal, 2023, 51(4): 360-365.

摘要/Abstract

摘要：

目的探讨let-7e-5p对小鼠变应性鼻炎的影响及机制。方法将18只BALB/c小鼠按随机数字表法均分为对照（Control）组、变应性鼻炎（AR）组、let-7e-5p激动剂阴性对照（AR+agomir-NC）组、let-7e-5p激动剂（AR+agomir）组、let-7e-5p抑制剂阴性对照（AR+antagomir-NC）组和let-7e-5p抑制剂（AR+antagomir）组。除Control组外，其余组应用卵白蛋白（OVA）致敏建立AR模型，AR+agomir组和AR+antagomir组同时给予let-7e-5p agomir或let-7e-5p antagomir进行干预，末次激发致敏后对小鼠变应性鼻炎进行评分，HE染色观察鼻黏膜组织，统计嗜酸性粒细胞数量，real-time PCR检测let-7e-5p、Fas、FasL mRNA表达，Western blot检测Fas、FasL蛋白表达，酶联免疫吸附试验检测血清免疫球蛋白E（IgE）、特异性IgE（sIgE）、干扰素γ（IFN-γ）、白细胞介素（IL）-12、IL-4、IL-13水平，双荧光素酶实验分析let-7e-5p和Fas、FasL的靶向关系。结果与Control组比较，AR组小鼠变应性鼻炎评分升高，鼻黏膜增生明显，嗜酸性粒细胞数量增加，let-7e-5p mRNA表达及IFN-γ、IL-12水平下降，Fas、FasL表达及IgE、sIgE、IL-4、IL-13水平升高（均P<0.05）。与AR+agomir-NC组比较，AR+agomir组小鼠变应性鼻炎评分降低，鼻黏膜结构清晰，嗜酸性粒细胞数量减少，let-7e-5p mRNA表达及IFN-γ、IL-12水平升高，Fas、FasL表达及IgE、sIgE、IL-4、IL-13水平下降（均P<0.05）。与AR+antagomir-NC组比较，AR+antagomir组小鼠变应性鼻炎评分升高，鼻黏膜增厚，嗜酸性粒细胞数量增加，let-7e-5p mRNA表达减少，Fas、FasL表达及IgE、sIgE、IL-4、IL-13水平升高（均P<0.05），IL-12、IFN-γ水平差异无统计学意义。与NC+3'UTR-WT组比较，let-7e-5p agomir+3'UTR-WT组荧光素酶活性下降（P<0.05）。结论 Let-7e-5p通过靶向调节Fas/FasL影响1型辅助性T细胞（Th1）/2型辅助性T细胞（Th2）平衡，从而参与AR小鼠变应性鼻炎发病的进展。

关键词: 鼻炎，变应性，常年性, 疾病模型，动物, let-7e-5p, Fas, FasL, 1型辅助性T细胞, 2型辅助性T细胞

Abstract:

Objective To investigate the effect and mechanism of let-7e-5p on allergic rhinitis in mice. Methods Eighteen BALB/c mice were randomly divided into the control group, the allergic rhinitis (AR) group, the let-7e-5p agonist negative control (AR+agomir-NC) group, the let-7e-5p agonist (AR+agomir) group, the let-7e-5p inhibitor negative control (AR+antagomir-NC) group and the let-7e-5p inhibitor (AR+antagomir) group. Except the control group, the other groups were sensitized with ovalbumin (OVA) to establish AR model. The AR+agomir group and the AR+antagomir group were treated with let-7e-5p agomir or let-7e-5p antagomir at the same time for intervention. After the last sensitization, the allergic rhinitis of mice were scored. HE staining was used to observe nasal mucosa. The number of eosinophils was counted. Real-time PCR was used to detect mRNA expression levels of let-7e-5p, Fas and FasL, and Western blot assay was used to detect protein expression levels of Fas and FasL. Enzyme-linked immunosorbent assay (ELISA) was used to detect serum levels of immunoglobulin E (IgE), specific immunoglobulin E (sIgE), interferon gamma (IFN-γ), interleukin (IL)-12, IL-4 and IL-13. The targeting relationship between let-7e-5p and Fas and FasL were analyzed by dual luciferase assay. Results Compared with the control group, the allergic rhinitis score was increased in the AR group, nasal mucosa hyperplasia was obvious and the number of eosinophils was increased. The expression levels of let-7e-5p and IFN-γ and IL-12 were decreased, while the expression of Fas and FasL and levels of IgE, sIgE, IL-4 and IL-13 were increased (all P<0.05). Compared with the AR+agomir-NC group, the allergic rhinitis score was decreased in the AR+agomir group, the nasal mucosa structure was clear and the number of eosinophils was reduced. The expression of let-7e-5p and levels of IFN-γ and IL-12 were increased, while the expression of Fas and FasL and levels of IgE, sIgE, IL-4 and IL-13 were decreased (all P<0.05). Compared with the AR+antagomir-NC group, the allergic rhinitis score was increased in the AR+antagomir group, the nasal mucosa was significantly thickened, and the number of eosinophils was increased. The expression of let-7e-5p was decreased, and the expression of Fas, FasL and levels of IgE, sIgE, IL-4 and IL-13 were increased (all P<0.05). There were no significant differences in IL-12 and IFN-γ between the AR+antagomir-NC group and the AR+antagomir group (P>0.05). Compared with the NC+3'UTR-WT group, luciferase activity was decreased in the let-7e-5p agomir+3'UTR-WT group (P<0.05). Conclusion Let-7e-5p may affect the balance of T helper type 1 (Th1)/ T helper type 2 (Th2) cells by targeting Fas/FasL, and thus participate in the progression of AR in mice.

Key words: rhinitis, allergic, perennial, disease models, animal, let-7e-5p, Fas, FasL, T helper type 1, T helper type 2

中图分类号:

R765.21

图/表 11

参考文献 21

[1]	ZHANG Y, LAN F, ZHANG L. Advances and highlights in allergic rhinitis[J]. Allergy, 2021, 76(11):3383-3389. doi:10.1111/all.15044.
[2]	SIDDIQUI Z A, WALKER A, PIRWANI M M, et al. Allergic rhinitis: Diagnosis and management[J]. Br J Hosp Med (Lond), 2022, 83(2):1-9. doi:10.12968/hmed.2021.0570.
[3]	JAIN A, IRIZARRY-CARO R A, MCDANIEL M M, et al. T cells instruct myeloid cells to produce inflammasome-independent IL-1β and cause autoimmunity[J]. Nat Immunol, 2020, 21(1):65-74. doi:10.1038/s41590-019-0559-y.
[4]	KINIKLI G, ATES A, TURGAY M, et al. Serum-soluble Fas antigen level in patients with allergic rhinitis: Its relation to specific immunotherapy[J]. Allergy Asthma Proc, 2006, 27(2):145-147.
[5]	PIRAYESH A, SHAHSAVAN S, ZARGARI SAMANI O Z, et al. Differential expression of Fas in moderate/severe and mild persistent allergic rhinitis and its correlation with pathological parameters[J]. Am J Rhinol Allergy, 2019, 33(3):286-293. doi:10.1177/1945892418824246.
[6]	LI W Q, ZHANG W T, LIU J, et al. Down-regulation of miR-let-7e attenuates LPS-induced acute lung injury in mice via inhibiting pulmonary inflammation by targeting SCOS1/NF-κB pathway[J]. Biosci Rep, 2021, 41(1): BSR20201089. doi:10.1042/BSR20201089.
[7]	SUOJALEHTO H, LINDSTROM I, MAJURI M L, et al. Altered microRNA expression of nasal mucosa in long-term asthma and allergic rhinitis[J]. Int Arch Allergy Immunol, 2014, 163(3):168-178. doi:10.1159/000358486.
[8]	LI L H, ZHANG S R, JIANG X S, et al. MicroRNA-let-7e regulates the progression and development of allergic rhinitis by targeting suppressor of cytokine signaling 4 and activating Janus kinase 1/signal transducer and activator of transcription 3 pathway[J]. Exp Ther Med, 2018, 15(4):3523-3529. doi:10.3892/etm.2018.5827.
[9]	姜玉秋, 唐桥斐, 闫智永, 等. LncRNA PVT1通过竞争miR-149-5p上调CHI3L1并影响变应性鼻炎进展的研究[J]. 天津医药, 2022, 50(3):259-264.
	JIANG Y Q, TANG Q F, YAN Z Y, et al. LncRNA PVT1 upregulates CHI3L1 and affects the progression of allergic rhinitis by competition with miR-149-5p[J]. Tianjin Med J, 2022, 50(3):259-264. doi:10.11958/20211109.
[10]	RONG J P, XU L, HU Y Y, et al. Inhibition of let-7b-5p contributes to an anti-tumorigenic macrophage phenotype through the SOCS1/STAT pathway in prostate cancer[J]. Cancer Cell Int, 2020, 20:470. doi:10.1186/s12935-020-01563-7.
[11]	SUOJALEHTO H, TOSKALA E, KILPELÄINEN M, et al. MicroRNA profiles in nasal mucosa of patients with allergic and nonallergic rhinitis and asthma[J]. Int Forum Allergy Rhinol, 2013, 3(8):612-620. doi:10.1002/alr.21179.
[12]	DONG J H, XU O, WANG J X, et al. Luteolin ameliorates inflammation and Th1/Th2 imbalance via regulating the TLR4/NF-κB pathway in allergic rhinitis rats[J]. Immunopharmacol Immunotoxicol, 2021, 43(3):319-327. doi:10.1080/08923973.2021.1905659.
[13]	LI J J, RAGHAV P, HU C Y. Ajwain oil attenuates allergic response of ovalbumin-induced allergic rhinitis via alteration of inflammatory,oxidative stress,and Th1/Th2 responses[J]. J Food Biochem, 2021, 45(12):e13963. doi:10.1111/jfbc.13963.
[14]	GUAN H B, FAN D P, MRELASHVILI D, et al. MicroRNA let-7e is associated with the pathogenesis of experimental autoimmune encephalomyelitis[J]. Eur J Immunol, 2013, 43(1):104-114. doi:10.1002/eji.201242702.
[15]	KAGAWA T, WATANABE M, INOUE N, et al. Increases of microRNA let-7e in peripheral blood mononuclear cells in Hashimoto's disease[J]. Endocr J, 2016, 63(4):375-380. doi:10.1507/endocrj.EJ15-0577.
[16]	RASQUINHA M T, SUR M, LASRADO N, et al. IL-10 as a Th2 cytokine:Differences between mice and humans[J]. J Immunol, 2021, 207(9):2205-2215. doi:10.4049/jimmunol.2100565.
[17]	KODA Y, TERATANI T, CHU P S, et al. CD8(+) tissue-resident memory T cells promote liver fibrosis resolution by inducing apoptosis of hepatic stellate cells[J]. Nat Commun, 2021, 12(1):4474. doi:10.1038/s41467-021-24734-0.
[18]	JIMBO H, NAGAI H, FUJIWARA S, et al. Fas-FasL interaction in cytotoxic T cell-mediated vitiligo:The role of lesional expression of tumor necrosis factor-alpha and interferon-gamma in Fas-mediated melanocyte apoptosis[J]. Exp Dermatol, 2020, 29(1):61-70. doi:10.1111/exd.14053.
[19]	UPADHYAY R, BOIARSKY J A, PANTSULAIA G, et al. A critical role for fas-mediated off-target tumor killing in T-cell immunotherapy[J]. Cancer Discov, 2021, 11(3):599-613. doi:10.1158/2159-8290.CD-20-0756.
[20]	ZHOU L L, XUE C J, CHEN Z Y, et al. c-Fos is a mechanosensor that regulates inflammatory responses and lung barrier dysfunction during ventilator-induced acute lung injury[J]. BMC Pulm Med, 2022, 22(1):9. doi:10.1186/s12890-021-01801-2.
[21]	侯穗波, 戴勇, 刘明, 等. 变应性鼻炎患者血清可溶性Fas与Fas配体的变化[J]. 中华微生物学和免疫学杂志, 2001, 21(S):111-113.
	HOU S B, DAI Y, LIU M, et al. Changes of serum soluble Fas and Fas ligand in patients with allergic rhinitis[J]. Chinese Journal of Microbiology and Immunology, 2001, 21(S):111-113.

分级评分（分）	喷嚏（个/30 min）	清涕	搔鼻（次）
0	0	无	无
1	1~3	流涕至鼻前孔	轻微抓鼻几次
2	4~10	流涕超过鼻前孔	双爪反复挠鼻
3	>11	涕流满面	抓挠鼻面不止、四处摩擦

分级评分（分）	喷嚏（个/30 min）	清涕	搔鼻（次）
0	0	无	无
1	1~3	流涕至鼻前孔	轻微抓鼻几次
2	4~10	流涕超过鼻前孔	双爪反复挠鼻
3	>11	涕流满面	抓挠鼻面不止、四处摩擦

基因名称	引物序列（5'→3'）	产物大小（bp）
let-7e-5p	上游：TGAGGTAGGAGGTTGTATAGTT	54
	下游：GCAGGGTCCGAGGTATTC	54
U6	上游：CGCAAGGATGACACGCAAAT	74
	下游：GCAGGGTCCGAGGTATTC	74
Fas	上游：AACCAGACTTCTACTGCGATTC	144
	下游：TCAACAACCATAGGCGATTT	144
FasL	上游：GCCATCACAACCACTCCC	107
	下游：CCAGAGCCACCAGAACCA	107
β-actin	上游：CTGTGCCCATCTACGAGGGCTAT	155
	下游：TTTGATGTCACGCACGATTTCC	155

基因名称	引物序列（5'→3'）	产物大小（bp）
let-7e-5p	上游：TGAGGTAGGAGGTTGTATAGTT	54
	下游：GCAGGGTCCGAGGTATTC	54
U6	上游：CGCAAGGATGACACGCAAAT	74
	下游：GCAGGGTCCGAGGTATTC	74
Fas	上游：AACCAGACTTCTACTGCGATTC	144
	下游：TCAACAACCATAGGCGATTT	144
FasL	上游：GCCATCACAACCACTCCC	107
	下游：CCAGAGCCACCAGAACCA	107
β-actin	上游：CTGTGCCCATCTACGAGGGCTAT	155
	下游：TTTGATGTCACGCACGATTTCC	155

组别	评分（分）	嗜酸性粒细胞（个/视野）
Control组	1.33±0.58	21.50±2.65
AR组	7.67±0.58^a	147.75±6.75^a
AR+agomir-NC组	7.33±0.58^a	136.25±6.13^a
AR+agomir组	4.00±1.00^b	75.00±10.42^b
AR+antagomir-NC组	7.33±0.58^a	149.75±1.71^a
AR+antagomir组	9.00±0.00^c	226.25±8.18^c
F	63.310^**	441.500^**

Let-7e-5p调控Fas/FasL影响小鼠变应性鼻炎发病的机制研究

Study on the mechanism of let-7e-5p regulating Fas/FasL to affect the pathogenesis of allergic rhinitis in mice

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参考文献 21

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组别	let-7e-5p	Fas	FasL
Control组	1.00±0.07	1.00±0.07	1.00±0.06
AR组	0.30±0.00^a	3.53±0.16^a	2.80±0.05^a
AR+agomir-NC组	0.34±0.02^a	3.34±0.25^a	2.41±0.04^a
AR+agomir组	0.70±0.04^b	1.98±0.05^b	1.33±0.13^b
AR+antagomir-NC组	0.31±0.02^a	3.48±0.36^a	2.59±0.16^a
AR+antagomir组	0.20±0.00^c	4.59±0.30^c	3.59±0.22^c
F	226.200^**	92.260^**	173.300^**

组别	Fas	FasL
Control组	1.00±0.02	1.01±0.03
AR组	4.91±0.12^a	3.15±0.06^a
AR+agomir-NC组	4.85±0.07^a	3.12±0.09^a
AR+agomir组	1.97±0.09^b	1.82±0.15^b
AR+antagomir-NC组	4.64±0.14^a	3.14±0.10^a
AR+antagomir组	5.57±0.37^c	4.50±0.16^c
F	334.900^**	382.100^**

组别	IgE （μg/L）	sIgE （μg/L）	1型辅助性T细胞（Th1）		2型辅助性T细胞（Th2）
组别	IgE （μg/L）	sIgE （μg/L）	IL-12（ng/L）	IFN-γ（ng/L）	IL-4（ng/L）	IL-13（ng/L）
Control组	28.77±3.15	2.18±0.24	186.44±20.53	70.77±8.24	29.14±2.93	18.56±1.97
AR组	179.68±16.82^a	39.37±3.98^a	93.57±10.48^a	32.62±3.73^a	72.93±7.49^a	146.29±16.68^a
AR+agomir-NC组	192.71±21.11^a	35.67±3.59^a	89.58±9.79^a	34.47±4.19^a	73.87±8.21^a	151.06±17.66^a
AR+agomir组	48.16±4.14^ab	26.56±3.11^ab	167.82±18.15^b	55.21±6.78^ab	49.96±5.67^ab	106.80±10.84^ab
AR+antagomir-NC组	187.66±21.23^a	39.63±4.00^a	91.68±10.14^a	32.71±3.83^a	74.71±8.20^a	150.27±16.83^a
AR+antagomir组	272.61±28.86^abc	55.23±6.35^abc	75.08±8.56^ab	20.77±2.39^ab	116.89±14.16^abc	195.96±20.68^abc
F	78.040^**	59.920^**	35.280^**	36.440^**	35.960^**	46.590^**

组别	Fas	FasL
NC+3'UTR-WT组	0.92±0.12	0.98±0.15
let-7e-5p agomir+3'UTR-WT组	0.49±0.06^a	0.50±0.06^a
NC+3'UTR-MT组	1.00±0.11	1.00±0.13
let-7e-5p agomir+3'UTR-MT组	0.91±0.13^b	1.02±0.11^b
F	13.230^**	13.740^**

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[15]	张凤涛 , 李晓红 , 王景景 , 孙洪涛 , 陈翀 , 陈锋△. miR-322对大鼠高原性肺动脉高压的作用及机制研究[J]. 天津医药, 2020, 48(7): 621-624.