七氟烷调控Wnt/β-catenin信号通路对脓毒症急性肺损伤的保护机制

doi:10.3969/j.issn.1006-5725.2025.19.006

摘要/Abstract

摘要：

目的探究七氟烷（SEV）在脓毒症诱导的急性肺损伤（ALI）中的作用及对Wnt/β-catenin信号通路的影响。方法将40只C57小鼠随机分为Sham组、CLP组、SEV组、SEV + XAV组（β-catenin抑制剂），每组10只。采用盲肠结扎穿孔术建立脓毒症模型，通过HE染色、肺湿/干重比、TUNEL染色评估肺损伤；ELISA检测炎症因子［肿瘤坏死因子-α（TNF-α）、白细胞介素-1β（IL-1β）、白细胞介素-6（IL-6）］水平；比色法及流式细胞术检测氧化应激指标［超氧化物歧化酶（SOD）、丙二醛（MDA）、活性氧（ROS）］；激光散斑血流仪检测下肢血流灌注及氧合；RT-qPCR和Western blot检测Wnt通路关键分子及下游靶基因（c-Myc、Cyclin D1）的表达；免疫荧光染色检测肺组织中β-catenin与肺泡Ⅱ型上皮细胞标记SP-C的共定位情况。结果与Sham组相比，CLP组小鼠的脓毒症严重程度、肺病理损伤（包括肺泡结构破坏、炎症浸润及细胞凋亡）、促炎因子水平均显著升高，氧化应激指标明显恶化（表现为SOD活性降低，MDA和ROS水平升高），下肢血流及氧合水平显著下降，β-catenin及其下游靶基因的表达、β-catenin与SP-C的共定位信号及荧光强度均显著下调（P < 0.05）。与CLP组相比，SEV组上述各项指标均显著改善。然而，与SEV组相比，SEV + XAV组的保护效应出现逆转，各项指标接近CLP组水平（P < 0.05）。结论七氟烷通过激活Wnt/β-catenin信号通路发挥抗炎、抗氧化作用，增强肺泡Ⅱ型上皮细胞中β-catenin的表达与定位，从而缓解脓毒症诱导的急性肺损伤。

关键词: 七氟烷, Wnt/β-catenin, 脓毒症急性肺损伤, 氧化应激, 肺泡Ⅱ型上皮细胞

Abstract:

Objective To explore the role of sevoflurane （SEV） in sepsis-induced acute lung injury （ALI） and observe its impact on the Wnt/β-catenin signaling pathway. Methods Forty C57 mice were randomly divided into 4 groups （n = 10 each）： Sham， CLP， SEV， and SEV + XAV （β-catenin inhibitor）. A sepsis model was established via cecal ligation and puncture. Lung injury was evaluated using HE staining， lung wet/dry weight ratio， and TUNEL staining. Levels of inflammatory factors （TNF-α， IL-1β， IL-6） were detected by ELISA. Oxidative stress indices （SOD， MDA， ROS） were measured by colorimetry and flow cytometry. Hindlimb blood perfusion and oxygenation were assessed with laser speckle flowmetry. Expressions of key Wnt pathway molecules and downstream target genes （c-Myc， Cyclin D1） were detected by RT-qPCR and Western blot. Co-localization of β-catenin and SP-C （a marker of type Ⅱ alveolar epithelial cells） in lung tissues was determined by immunofluorescence staining. Results Compared with the Sham group， the CLP group exhibited significant increases in sepsis severity， lung pathological damage including alveolar structure destruction， inflammatory infiltration， and apoptosis， elevation in pro-inflammatory cytokine levels， and significant decrease in SOD and increase in MDA and ROS. Additionally， lower limb blood flow and oxygenation levels were significantly reduced， while the expression of β-catenin and its downstream target genes， as well as the co-localization signal and fluorescence intensity of β-catenin with SP-C， were significantly downregulated （all P < 0.05）. Compared with the CLP group， the SEV group showed significant improvements in all these indicators. However， compared with the SEV group， the SEV + XAV group demonstrated a reversed protective effect， with all indicators approaching the levels observed in the CLP group （all P < 0.05）. Conclusion Sevoflurane alleviates sepsis-induced ALI by activating Wnt/β-catenin signaling pathway， exerting anti-inflammatory and antioxidant effects， and enhancing the expression and localization of β-catenin in type Ⅱ alveolar epithelial cells.

Key words: sevoflurane, Wnt/β-catenin, sepsis-induced acute lung injury, oxidative stress, type Ⅱ alveolar epithelial cells

中图分类号:

R563.1

郭晋言,尤雨晴,陈可,潘芬,赖嘉慧,陈素芳,姚伟锋. 七氟烷调控Wnt/β-catenin信号通路对脓毒症急性肺损伤的保护机制[J]. 实用医学杂志, 2025, 41(19): 2991-2999.

Jinyan GUO,Yuqing YOU,Ke CHEN,Fen PAN,Jiahui LAI,Sufang CHEN,Weifeng. YAO. Protective mechanism of sevoflurane on acute lung injury in sepsis by regulating the Wnt/β-catenin signaling pathway[J]. The Journal of Practical Medicine, 2025, 41(19): 2991-2999.

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基因名称	基因长度/bp	上游序列	下游序列	基因符号
β?catenin	150	ATGGAACCAGACAGAAAAGC	CTAGTCATTGCAATTTTGC	Ctnnb1
c?Myc	120	GGCTCCTGGCAAAAGGTCA	CTGCGTAGTTGTGCTGATGT	Myc
Cyclin D1	180	GCTGCGAAGTGGAAACCATC	CCTCCTTCTGCACACATTTGAA	Ccnd1
GSK?3β	150	TTGGAGCCACTGATTACACG	CCAACTGATCCACACCACTG	Gsk3b
GAPDH	100	GAAGGTGAAGGTCGGAGTCA	CATGGGTGGAATCATATTGGAA	GAPDH

组别	例数	肺组织评分/分	每视野细胞凋亡	肺湿/干比
Sham组	10	1.90 ± 0.74	32.80 ± 6.36	2.79 ± 0.47
CLP组	10	9.50 ± 1.84^??	264.29 ± 39.04^?	6.62 ± 0.85
SEV组	10	7.30 ± 0.95^ΔΔ	214.61 ± 36.21^ΔΔΔ	5.18 ± 0.92^Δ
SEV + XAV组	10	10.10 ± 2.42^###	265.72 ± 35.61^#	6.87 ± 0.84^##
F值		52.13	117.42	56.13
P值		< 0.001	< 0.001	< 0.001

组别	例数	SOD/（U/mgprot）	MDA/（mmol/mgprot）	ROS/（AU/mgprot）
Sham组	10	213.59 ± 20.72	0.64 ± 0.16	94.85 ± 26.80
CLP组	10	80.63 ± 25.08^?	2.92 ± 0.50^?	423.23 ± 77.83^?
SEV组	10	109.12 ± 28.05^Δ	2.22 ± 0.54^Δ	290.67 ± 82.34^ΔΔ
SEV + XAV组	10	72.19 ± 17.16^##	3.19 ± 0.52^#	453.03 ± 75.53^##
F值		79.2	62.5	55.3
P值		< 0.001	< 0.001	< 0.001

组别	例数	IL?1β/（pg/mL）	TNF?α/（pg/mL）	IL?6/（pg/mL）
Sham组	10	22.38 ± 10.08	27.84 ± 13.6	129.21 ± 72.60
CLP组	10	144.45 ± 25.24^?	146.80 ± 23.78^?	953.68 ± 199.98^?
SEV组	10	112.88 ± 20.24^Δ	111.27 ± 18.00^Δ	700.13 ± 149.31^ΔΔ
SEV + XAV组	10	147.55 ± 26.04^##	142.70 ± 23.76^##	973.18 ± 201.09^###
F值		74.84	74.38	57.31
P值		< 0.001	< 0.001	< 0.001

组别	例数	β-catenin mRNA	c-Myc mRNA	Cyclin D1 mRNA	GSK-3β mRNA
Sham组	10	1.83 ± 0.44	1.75 ± 0.52	1.23 ± 0.40	0.79 ± 0.18
CLP组	10	1.14 ± 0.28^??	0.84 ± 0.40^??	0.61 ± 0.32^??	1.25 ± 0.38^?
SEV组	10	1.59 ± 0.42^Δ	1.44 ± 0.58^Δ	0.83 ± 0.53^Δ	0.74 ± 0.20^ΔΔ
SEV + XAV组	10	0.98 ± 0.30^#	0.68 ± 0.22^#	0.54 ± 0.29^#	0.99 ± 0.32^#
F值		11.92	7.25	3.39	7.00
P值		< 0.001	< 0.001	<0.05	<0.001