氢分子对咪喹莫特诱导的小鼠银屑病样皮损的抑制作用

doi:10.19586/j.2095-2341.2023.0112

生物技术进展 ›› 2023, Vol. 13 ›› Issue (6): 945-953.DOI: 10.19586/j.2095-2341.2023.0112

• 研究论文 • 上一篇

氢分子对咪喹莫特诱导的小鼠银屑病样皮损的抑制作用

朱钧锴¹(), 葛玲智², 张超², 曹璨², 吴嘉惠², 穆震²()

^1.泰安八十八医院皮肤科，山东泰安 271000
^2.山东第一医科大学第二附属医院皮肤科，山东泰安 271000

收稿日期:2023-09-16 接受日期:2023-11-01 出版日期:2023-11-25 发布日期:2023-12-12
通讯作者: 穆震
作者简介:朱钧锴 E-mail: zhujunkai_lucky@163.com;
基金资助:
泰安市科技创新发展项目(2020NS100)

Inhibitory Effect of Hydrogen Molecule on Imiquimod-induced Psoriasis-like Dermatitis in Mice

Junkai ZHU¹(), Lingzhi GE², Chao ZHANG², Can CAO², Jiahui WU², Zhen MU²()

^1.Dermatology Department，Tai’an 88 Hospital，Shandong Tai’an 271000，China
^2.Dermatology Department，the Second Affiliated Hospital of Shandong First Medical University，Shandong Tai’an 271000，China

Received:2023-09-16 Accepted:2023-11-01 Online:2023-11-25 Published:2023-12-12
Contact: Zhen MU

摘要/Abstract

摘要：

氢分子是一种新型的气体信号分子，对多种炎症疾病有一定的作用效果。银屑病是一种慢性炎症性疾病，氢分子有望作为银屑病辅助治疗的新手段。连续7 d使用5%咪喹莫特（imiquimod, IMQ）乳膏诱导小鼠银屑病模型，期间每天2次、每次2 h将小鼠置于35%的氢分子环境中进行干预。每天观察小鼠皮损变化并进行银屑病面积和严重程度指数（psoriasis area and severity index, PASI）评分；第8天，处死小鼠取皮损，苏木精-伊红（hematoxylin-eosin，HE）染色并使用Baker评分评估组织病理学损害程度；免疫组化检测皮损中炎症因子IL-6、TNF-α和IFN-γ的表达；实时荧光定量PCR（real-time quantitative fluorescent polymerase chain reaction, RT-qPCR）和蛋白免疫印迹检测皮损中炎症因子IL-6、TNF-α和IFN-γ的相对mRNA水平和蛋白水平的表达；用试剂盒检测皮损组织中氧化指标丙二醛（malondialdehyde, MDA）含量和抗氧化相关的过氧化氢酶（catalase, CAT）活力。结果显示，氢分子干预降低了IMQ诱导小鼠的PASI评分（P<0.05）和Baker评分（P<0.001），改善了组织的病理学变化；氢分子干预组小鼠皮损组织中IL-6、TNF-α和IFN-γ的mRNA及蛋白表达水平均下降，MDA的含量下降，CAT的活力增加（P<0.05）。结果表明，氢分子可抑制银屑病样皮损的形成、减轻炎症反应、减轻氧化应激损伤，有望成为治疗和缓解银屑病的新方法。

关键词: 氢分子, 银屑病样皮损, 氧化应激, 炎症反应

Abstract:

Hydrogen molecule is a new type of gas signaling molecule， which has a certain effect on a variety of inflammation diseases. Psoriasis is a chronic， inflammatory disease， and molecular hydrogen is expected to be a new adjuvant therapy for psoriasis. The psoriasis model of mice was induced by 5% imquimod （IMQ） cream for 7 consecutive days， during which the mice were exposed to 35% hydrogen molecular environment twice a day for 2 h each time for intervention. The changes of skin lesions were observed daily and the psoriasis area and severity index （PASI） score was performed. On the 8th day， the skin lesions were taken by death， hematoxylin-eosin （HE） staining， and the histopathological lesion was evaluated using Baker score. The expressions of inflammatory cytokines IL-6， TNF-α and IFN-γ were detected by immunohistochemistry. Real-time quantitative fluorescent PCR （RT-qPCR） and Western blot were used to detect the relative mRNA and protein levels of inflammatory cytokines IL-6， TNF-α and IFN-γ in skin lesions. The content of oxidation index malondialdehyde （MDA） and the activity of antioxidation-related catalase （CAT） were detected by the kit. Results showed that， molecular hydrogen intervention decreased PASI score （P<0.05） and Baker score （P<0.001）， and improved histopathological changes of IMQ-induced mice. The mRNA and protein expression levels of IL-6， TNF-α and IFN-γ in the skin tissue of mice in the hydrogen molecular intervention group were decreased， the content of MDA was decreased， and the activity of CAT was increased （all P<0.05）. The results indicated that， molecular hydrogen can inhibit the formation of psoriasis skin lesions， reduce inflammation and oxidative stress damage， which has the potential to treat psoriasis.

Key words: hydrogen molecular, psoriasis-like dermatitis, oxidative stress, inflammatory response

中图分类号:

Q819

朱钧锴, 葛玲智, 张超, 曹璨, 吴嘉惠, 穆震. 氢分子对咪喹莫特诱导的小鼠银屑病样皮损的抑制作用[J]. 生物技术进展, 2023, 13(6): 945-953.

Junkai ZHU, Lingzhi GE, Chao ZHANG, Can CAO, Jiahui WU, Zhen MU. Inhibitory Effect of Hydrogen Molecule on Imiquimod-induced Psoriasis-like Dermatitis in Mice[J]. Current Biotechnology, 2023, 13(6): 945-953.

图/表 7

表1 RT-qPCR目标基因的引物序列

Table 1 Primers sequence of RT-qPCR target gene

基因名称	引物序列（5'→3'）
Actin	F：5'-TGCTGTCCCTGTATGCCTCTG-3'
Actin	R：5'-TGATGTCACGCACGATTTCC-3'
IL-6	F：5'-TTCACAAGTCGGAGGCTTA-3'
IL-6	R：5'-CAAGTGCATCATCGTTGTTC-3'
TNF-α	F：5'-CGCTGAGGTCAATCTGC-3'
TNF-α	R：5'-GGCTGGGTAGAGAATGGA-3'
IFN-γ	F：5'-CTGCTGATGGGAGGAGAT-3'
IFN-γ	R：5'-CTGCTTTCTTTCAGGGACA-3'

图1 氢分子对小鼠银屑病样皮损的形态学影响A：第8天各组小鼠背部皮损形态学表现；B：各组小鼠PASI评分比较。*，**，***分别表示与空白组相比差异在P<0.05， P<0.01， P<0.001水平上有统计学意义；#，##，###分别表示与IMQ组相比差异在 P<0.05， P<0.01，P<0.001水平上有统计学意义。

Fig. 1 Morphological effects of molecular hydrogen on psoriasis-like mice

图2 氢分子对小鼠银屑病样皮损的组织病理影响A：各组小鼠7 d后皮损HE染色；B：两组小鼠皮损组织学Baker评分；C：各组小鼠皮损HE染色下表皮垂直厚度比较。 ***表示两组数据间在 P<0.001水平差异具有统计学意义。

Fig. 2 Histopathological effects of hydrogen molecular on psoriasis skin lesions in mice

图3 氢分子对小鼠银屑病样皮损中炎性细胞因子的影响A：免疫组化法检测各组小鼠炎性细胞表达； B：各组小鼠炎性细胞IL-6表达的OD值；C：各组小鼠炎性细胞TNF-α表达的OD值；D：各组小鼠炎性细胞IFN-γ表达的OD值。 *，**，*** 分别表示两组数据间在P<0.05、P<0.01、P<0.001水平差异具有统计学意义。

Fig. 3 Effect of hydrogen molecular on inflammatory cytokines in psoriatic skin lesions in mice

图4 氢分子对小鼠皮损中IL-6、TNF-α和IFN-γ的蛋白水平的影响A：Western Blot法检测各组小鼠炎性细胞因子的蛋白表达；B：各组小鼠皮损中炎性细胞因子IL-6蛋白表达水平；C：各组小鼠皮损中炎性细胞因子TNF-α蛋白表达水平；D：各组小鼠皮损中炎性细胞因子IFN-γ蛋白表达水平。*，**分别表示两组数据间在P<0.05，P<0.01水平差异具有统计学意义。

Fig. 4 Effect of hydrogen molecular on the protein levels of IL-6, TNF-α and IFN-γ in mouse skin lesions

图5 氢分子对小鼠皮损中炎症因子的mRNA水平的影响A：氢分子对小鼠皮损IL-6 mRNA水平的影响；B：氢分子对小鼠皮损TNF-α mRNA水平的影响；C：氢分子对小鼠皮损IFN-γ mRNA水平的影响。*， *** 分别表示两组数据间在P<0.05， P<0.001水平差异具有统计学意义。

Fig. 5 Effect of hydrogen molecular on mRNA levels of inflammatory cytokines in mouse skin lesions

图6 氢分子对小鼠银屑病样皮损氧化指标的影响A：氢分子对小鼠银屑病样皮损CAT表达的影响；B：氢分子对小鼠银屑病样皮损MDA表达的影响。*，**分别表示两组数据间在P<0.05，P<0.01水平差异具有统计学意义。

Fig. 6 Effect of hydrogen molecular on oxidation index of psoriatic skin lesions in mice

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氢分子对咪喹莫特诱导的小鼠银屑病样皮损的抑制作用

Inhibitory Effect of Hydrogen Molecule on Imiquimod-induced Psoriasis-like Dermatitis in Mice

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