甘草酸能减轻小鼠肺炎病毒引起的小鼠肺脏损伤

doi:10.12300/j.issn.1674-5817.2024.014

摘要/Abstract

摘要：

目的通过小鼠肺炎病毒（pneumonia virus of mice，PVM）感染近交系BALB/c小鼠建立病毒感染肺炎动物模型，观察PVM感染过程中促炎症警报素分子高迁移率族蛋白B1（high mobility group box 1，HMGB1）的变化，以及HMGB1抑制剂甘草酸（glycyrrhizic acid，GA）对小鼠肺脏感染损伤的在体干预作用。方法将3周龄的雌性BALB/c小鼠随机分为3组，每组6只。一组未经PVM感染，作为对照组（Control）；另外两组先以1×10⁴半数组织培养感染剂量（50% tissue culture infective dose，TCID₅₀）/25 μL剂量滴鼻接种PVM，然后分别给予GA生理盐水溶液灌胃（GA组）或单纯生理盐水灌胃（normal saline，NS组）处理，连续15 d。其间，观察并记录各组小鼠体重、外观等改变。在实验终点采集各组小鼠的肺脏组织样本，通过苏木精-伊红染色和免疫组织化学法检测小鼠肺脏组织内PVM和HMGB1蛋白的分布情况；通过实时荧光定量PCR法检测小鼠肺脏组织中HMGB1、Toll样受体4（Toll-like receptor 4，TLR-4）、晚期糖基化终产物特异性受体（advanced glycosylation end-product-specific receptor，AGER），以及白细胞介素（interleukin，IL）-1β、IL-2和肿瘤坏死因子α（tumor necrosis factor-α，TNF-α）等炎症因子的表达水平。结果与Control组相比，NS组小鼠6 d后体重显著下降（P<0.05），组织病理学结果显示其肺脏有明显的炎症病变，免疫组织化学结果显示HMGB1从细胞核释放至细胞质，实时荧光定量PCR结果显示HMGB1、IL-1β和IL-2的表达水平均显著上调（P<0.05）；GA干预组的临床症状和体重无明显变化。而与NS组相比，GA干预组肺组织的病理损伤明显减轻，且肺组织中HMGB1、IL-1β、IL-2和干扰素γ（interferon-γ，IFN-γ）的表达水平显著下降（P<0.05），但AGER的表达水平显著增高（P<0.05）。结论 PVM感染能引起明显的小鼠肺部炎症性病理损伤，而GA能有效减轻其损伤，其作用机制可能与激活HMGB1信号通路相关。

关键词: 甘草酸, 高迁移率族蛋白B1, 小鼠肺炎病毒, 肺损伤, BALB/c小鼠

Abstract:

Objective In this study, inbred BALB/c mice infected with the pneumonia virus of mice (PVM) were used to establish an animal model of viral pneumonia, and the changes in the pro-inflammatory alarmin molecule, high mobility group box 1 protein (HMGB1), during PVM infection were observed, as well as the in vivo intervention effects of the HMGB1 inhibitor, glycyrrhizic acid (GA), on PVM-induced lung injury. Methods Three-week-old female BALB/c mice were randomly divided into three groups, each consisting of 6 mice. One group, uninfected by PVM, served as the control group (Control). The other two groups were inoculated intranasally with PVM at a dose of 1×10⁴ 50% tissue culture infective dose (TCID₅₀)/25 μL, and subsequently treated with GA saline solution (GA group) or plain saline solution (normal saline, NS group) via gavage for 15 consecutive days. During this period, changes in body weight and appearance were monitored in each group. At the end of the experiment, lung tissue samples were collected from all groups. The distribution of PVM and HMGB1 proteins in the lung tissues was analyzed using hematoxylin-eosin staining and immunohistochemistry. The expression levels of HMGB1 and its Toll-like receptor 4 (TLR-4), advanced glycosylation end-product-specific receptor (AGER), and inflammatory cytokines such as interleukin (IL)-1β, IL-2, and tumor necrosis factor-α (TNF-α) in lung tissues of mice were measured using real time fluorescence quantitative PCR. Results Compared with the Control group, the NS group showed a significant weight loss after 6 days (P<0.05). Histopathological tests revealed pronounced inflammatory lesions in their lungs. Immunohistochemistry results showed that HMGB1 was released from the nucleus to the cytoplasm, and real time fluorescence quantitative PCR results indicated that the expression levels of HMGB1, IL-1β, and IL-2 were significantly upregulated (P<0.05). In the GA group, there was no significant change in the clinical symptoms or body weight. However, compared with the NS group, the pathological damages of lung tissues in the GA group were significantly reduced, and the expression levels of HMGB1, IL-1β, IL-2, and interferon-γ (IFN-γ) in lung tissues were also significantly decreased (P<0.05), although the expression level of AGER was significantly increased (P<0.05). Conclusion PVM infection can cause significant inflammatory pathological lung damages in mice, and GA can effectively alleviate the damages. Its therapeutic effect may be related to the activation of HMGB1 signaling pathway.

Key words: Glycyrrhizic acid, High mobility group box 1, Pneumonia virus of mice, Lung damage, BALB/c mice

中图分类号:

Q95-33

刘芸,冯婷婷,佟巍,等. 甘草酸能减轻小鼠肺炎病毒引起的小鼠肺脏损伤[J]. 实验动物与比较医学, 2024, 44(3): 251-258. DOI: 10.12300/j.issn.1674-5817.2024.014.

Yun LIU,Tingting FENG,Wei TONG,et al. Glycyrrhizic Acid Showed Therapeutic Effects on Severe Pulmonary Damages in Mice Induced by Pneumonia Virus of Mice Infection[J]. Laboratory Animal and Comparative Medicine, 2024, 44(3): 251-258. DOI: 10.12300/j.issn.1674-5817.2024.014.

图/表 5

注：在小鼠肺炎病毒（PVM）感染前3 d，将所有小鼠引入动物生物安全二级（ABSL-2）设施。第0天，测量小鼠体重，进行PVM滴鼻接种，同时分别灌胃给药，甘草酸（GA）或生理盐水（NS）。此后，每日给予GA或NS治疗，每3 d称重一次。每组6只小鼠。实验终点设为病毒感染后第15天。
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图1 动物实验时间表
注：在小鼠肺炎病毒（PVM）感染前3 d，将所有小鼠引入动物生物安全二级（ABSL-2）设施。第0天，测量小鼠体重，进行PVM滴鼻接种，同时分别灌胃给药，甘草酸（GA）或生理盐水（NS）。此后，每日给予GA或NS治疗，每3 d称重一次。每组6只小鼠。实验终点设为病毒感染后第15天。

Figure 1 Animal experiment timeline
Note： Three days before pneumonia virus of mice (PVM) infection, all mice were transferred to the ABSL-2 facility. On Day 0, the body weights of mice were measured, PVM intranasal inoculation was performed, and glycyrrhizic acid (GA) or normal saline (NS) was administered via gavage. Subsequently, GA or NS treatment was given daily and body weights were measured every 3 days. Each group consisted of 6 mice. The endpoint of the experiment was set at 15 dpi (day post infection).

表1 引物序列

Table 1 Primer Sequence

目的基因 Target gene	上游引物（5’-3’） Forward primer	下游引物（5’-3’） Reverse primer
PVM	GCCTGCATCAACACAGTGTGT	GCCTGATGTGGCAGTGCTT
TLR-4	ATGGCATGGCTTACACCACC	GAGGCCAATTTTGTCTCCACA
AGER	ACATGTGTGTCTGAGGGAAGC	AGCTCTGACCGCAGTGTAAAG
HMGB1	CTTCGGCCTTCTTCTTGTTCT	GGCAGCTTTCTTCTCATAGGG
IL-2	CCCAAGCAGGCCACAGAATTGAAA	AGTCAAATCCAGAACATGCCGCAG
IFN-γ	AGAGGATGGTTTGCATCTGGGTCA	ACAACGCTATGCAGCTTGTTCGTG
CCL2	GGCTCAGCCAGATGCAGTTAA	CCTACTCATTGGGATCATCTTGCT
TNF-α	GACAAGGCTGCCCCGACTA	TTTCTCCTGGTATGAGATAGCAAATC
IL-1β	ATGAGGACATGAGCACCT TC	CATTGAGTTGGAGAGCTTTC
IL-6	GACTTCCATCCAGTTGCCTTC TT	TCCACGATTTCCCAGAGAACA

注：Control，未感染对照组；NS，小鼠肺炎病毒（PVM）感染后生理盐水（NS）处理组；GA，感染后（GA）甘草酸处理组。3组小鼠体重以平均值±SD表示（n=6）。与对照组相比，NS治疗组小鼠在感染后第3、6 天时体重下降，9天后体重增加，与对照组相比仍有显著差异（P<0.05）；GA组小鼠与对照组相比无显著差异（P>0.05）。
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图2 PVM感染后小鼠生长曲线
注：Control，未感染对照组；NS，小鼠肺炎病毒（PVM）感染后生理盐水（NS）处理组；GA，感染后（GA）甘草酸处理组。3组小鼠体重以平均值±SD表示（n=6）。与对照组相比，NS治疗组小鼠在感染后第3、6 天时体重下降，9天后体重增加，与对照组相比仍有显著差异（P<0.05）；GA组小鼠与对照组相比无显著差异（P>0.05）。

Figure 2 Body weight curves of mice after PVM infection
Note：The body weights of mice in the three groups were expressed as mean value±SD (n=6). Compared with the Control group, the NS group experienced weight loss at 3 and 6 dpi. Although weight gain in the NS group could be detected after 9 dpi, there were significant differences compared to the Control group (P<0.05). There were no significant weight differences in GA group compared to the Control group (P>0.05). Control: uninfected control group; NS: group treated with normal saline (NS) after infection; GA: group treated with glycyrrhizic acid (GA) after infection.

注：A～C，组织病理学分析显示PVM感染后的肺组织病理变化（苏木精-伊红染色，×10）。与Control组相比，NS组小鼠肺泡间隔增厚，肺泡腔出血，血管充血；GA组无明显的病理症状。D～F，免疫组织化学检测结果显示GA组肺组织中PVM病毒载量低于NS组（TMB染色，×40）。G～I，免疫组织化学分析显示HMGB1在肺组织中的表达（TMB染色，×100）。Control组中箭头指示HMGB1在细胞核位置；NS组可见HMGB1过表达，且分布于细胞质。Control：未感染对照组；NS：PVM感染后生理盐水处理组；GA：PVM感染后甘草酸处理组。
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图3 PVM感染后小鼠肺部病理变化
注：A～C，组织病理学分析显示PVM感染后的肺组织病理变化（苏木精-伊红染色，×10）。与Control组相比，NS组小鼠肺泡间隔增厚，肺泡腔出血，血管充血；GA组无明显的病理症状。D～F，免疫组织化学检测结果显示GA组肺组织中PVM病毒载量低于NS组（TMB染色，×40）。G～I，免疫组织化学分析显示HMGB1在肺组织中的表达（TMB染色，×100）。Control组中箭头指示HMGB1在细胞核位置；NS组可见HMGB1过表达，且分布于细胞质。Control：未感染对照组；NS：PVM感染后生理盐水处理组；GA：PVM感染后甘草酸处理组。

Figure 3 Pathological changes in the lungs of mice after PVM infection in three groups
Note：A-C, Histopathological analysis showed the pulmonary tissue pathological changes after PVM infection (HE staining, ×10). Compared with the Control group, thickened alveolar septa, hemorrhage in alveolar cavities, and vascular hyperemia were detected in the NS group; No significant pathological symptom was observed in the GA group. D-F, Immunohistochemical analysis demonstrated lower PVM viral load in pulmonary tissues in the GA group compared to the NS group (TMB staining, ×40). G-I， Immunohistochemical analysis showed the HMGB1 expression in pulmonary tissues (TMB staining, ×100). In the Control group, the cell nucleus locations of HMGB1 were indicated by arrows; in the NS group, HMGB1 was overexpressed and distributed in the cytoplasm. Control: uninfected control group; NS: group treated with normal saline（0.9%NaCl） after PVM infection; GA: group treated with glycyrrhizic acid after PVM infection. PVM: pneumonia virus of mice.

注：实时荧光定量PCR检测3组小鼠肺组织HMGB1、AGER、TLR-4、IL-1β、TNF-α、CCL2、IL-6、IFN-γ和IL-2相对于内参基因GAPDH的表达。n=6，^*P<0.05。Control：未感染对照组；NS：PVM感染后生理盐水处理组；GA：PVM感染后甘草酸处理组。
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图4 PVM感染后小鼠肺组织中炎症因子与警报素HMGB1及其受体AGER的表达
注：实时荧光定量PCR检测3组小鼠肺组织HMGB1、AGER、TLR-4、IL-1β、TNF-α、CCL2、IL-6、IFN-γ和IL-2相对于内参基因GAPDH的表达。n=6，^*P<0.05。Control：未感染对照组；NS：PVM感染后生理盐水处理组；GA：PVM感染后甘草酸处理组。

Figure 4 Expression of inflammatory factors and the alarmin HMGB1 and its receptor AGER after PVM infection in pulmonary tissue
Note：Real-time fluorescence quantitative PCR was performed on the pulmonary tissues from mice in three groups. The expression of high mobility group box 1 (HMGB1), advanced glycosylation end-product-specific receptor (AGER), Toll-like receptor 4 (TLR4), interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), C-C motif chemokine ligand 2 (CCL2), IL-6, interferon-γ (IFN-γ), and IL-2 were shown as ratios to GAPDH. n=6, ^*P<0.05. Control: uninfected control group; NS: group treated with normal saline（0.9%NaCl） after PVM infection; GA: group treated with glycyrrhizic acid after PVM infection. PVM: pneumonia virus of mice.

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