利多卡因抑制糖尿病小鼠Kupffer细胞炎症反应及对肝脓肿形成的影响

王睿斌; 卢雨征; 朱静林; 王为; 贾光

doi:10.3969/j.issn.1001-5256.2022.06.023

利多卡因抑制糖尿病小鼠Kupffer细胞炎症反应及对肝脓肿形成的影响

DOI: 10.3969/j.issn.1001-5256.2022.06.023

首都医科大学附属北京世纪坛医院急诊科，北京 100038

基金项目:

北京世纪坛医院科研发展基金 (2016-Q18);

北京市重点实验室开放课题 (2019-KF04)

伦理学声明：本研究方案于2016年10月经由北京世纪坛医院实验动物伦理委员会审批，批号：sjtkyll-lx-2016(103)，符合实验室动物管理与使用准则。

利益冲突声明：本研究不存在研究者、伦理委员会成员以及与公开研究成果有关的利益冲突。

作者贡献声明：王睿斌负责课题设计，资料分析，撰写论文; 卢雨征、朱静林参与整理数据及数据分析; 王为、贾光参与实验操作及论文撰写。

详细信息

通信作者:
王睿斌，wangruibin@bjsjth.cn

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出版历程
- 收稿日期: 2021-10-18
- 录用日期: 2022-01-04
- 出版日期: 2022-06-20

Inhibitory effect of lidocaine on Kupffer cell inflammatory response and its effect on liver abscess formation in diabetic mice

Department of Emergency, Beijing Shijitan Hospital, Capital Medical University, Beijing 100038, China

Research funding:

Scientific Research and Development Fund of Beijing Shijitan Hospital (2016-Q18);

Open Project of Beijing Key Laboratory (2019-KF04)

More Information

Corresponding author: WANG Ruibin, wangruibin@bjsjth.cn(ORCID: 0000-0001-9592-1539)

摘要

摘要: 目的观察利多卡因是否能逆转糖尿病小鼠Kupffer细胞功能障碍，阐明利多卡因通过改善糖尿病小鼠Kupffer细胞吞噬功能并影响肝脓肿形成的机制。方法将C57BLKS/J db/db小鼠分为糖尿病对照组和糖尿病+利多卡因组，将C57BLKS/J db/m小鼠分为非糖尿病对照组和非糖尿病+利多卡因组，每组5只，均喂食肺炎克雷伯杆菌悬液。分别取Kupffer细胞体外培养，电镜观察超微结构改变，测定Kupffer细胞的炎症介质分泌水平，细胞间黏附分子1(ICAM-1)表达水平，中性粒细胞趋化功能，吞噬功能，以及肝脓肿的形成。计量资料多组间比较采用Kruskal-Wallis H秩和检验，进一步两两比较采用Mann-Whitney U检验；计数资料组间比较采用χ²检验。结果糖尿病小鼠Kupffer细胞中线粒体和粗面内质网的数量减少, 内质网扩张，线粒体肿胀及脂滴增多。与非糖尿病对照组比较，糖尿病对照组Kupffer细胞表达NO[(4.95±0.06) μmol/L vs (1.34±0.13)μmol/L]、IL-6[(740.04±8.58) pg/mL vs (515.77±4.62)pg/mL]、TNFα[(774.23±7.98) pg/mL vs (461.51±1.76)pg/mL]、IFNγ[(842.33±14.79) pg/mL vs (542.47±6.75)pg/mL]、ICAM-1(2.40±0.02 vs 1.33±0.01)水平明显升高(P值均＜0.05)，中性粒细胞趋化增强(100.80±10.18 vs 13.80±3.70，P＜0.05)，吞噬能力减弱(9.86±1.82 vs 60.00±3.54，P＜0.05)，肝脓肿形成无影响(40% vs 0，P＞0.05)。与糖尿病对照组比较，糖尿病+利多卡因组Kupffer细胞表达NO[(3.35±0.28) μmol/L vs (4.95±0.06)μmol/L]、IL-6[(688.42±36.34) pg/mL vs (740.04±8.58) pg/mL]、TNFα[(631.15±4.30) pg/mL vs (774.23±7.98) pg/mL]、IFNγ[(704.56±3.64) pg/mL vs (842.33±14.79)pg/mL]、ICAM-1(1.50±0.02 vs 2.40±0.02) 水平明显降低(P值均＜0.05)，中性粒细胞趋化减弱(33.40±5.60 vs 100.80±10.18，P＜0.05)，吞噬能力增强(49.20±2.59 vs 9.86±1.82，P＜0.05)、肝脓肿形成无影响(0 vs 40%，P＞0.05)。结论利多卡因可以抑制糖尿病小鼠Kupffer细胞炎症反应并改善其吞噬功能，对Kupffer细胞起到保护作用，但对肝脓肿的形成无影响。
- 糖尿病 /
- 肝脓肿 /
- 枯否细胞 /
- 利多卡因
Abstract: Objective To investigate whether lidocaine can reverse Kupffer cell dysfunction in diabetic mice, as well as the mechanism of lidocaine in affecting liver abscess formation by improving the phagocytic function of Kupffer cells. Methods C57BLKS/J db/db mice were divided into diabetes control group and diabetes+lidocaine group, and C57BLKS/J db/m mice were divided into non-diabetes control group and non-diabetes+lidocaine group, with 5 mice in each group. All mice were fed with the suspension of Klebsiella pneumoniae. Kupffer cells were collected from each group and were cultured in vitro; an electron microscope was used to measure the change in ultrastructure, and Kupffer c ells were measured in terms of the levels of inflammatory mediators, the expression level of intercellular adhesion molecule-1 (ICAM-1), the chemotactic function of neutrophils, and phagocytic function; liver abscess formation was also observed. The Kruskal-Wallis H test was used for comparison of continuous data between multiple groups, and the Mann-Whitney U test was used for further comparison between two groups; the chi-square test was used for comparison of categorical data between groups. Results Compared with the non-diabetic mice, the diabetic mice had significant reductions in mitochondria and rough endoplasmic reticulum, endoplasmic reticulum dilation, mitochondrial swelling, and an increase in lipid droplets in Kupffer cells. Compared with the non-diabetes control group, the diabetes control group had significant increases in the levels of nitric oxide (NO) (4.95±0.06 μmol/L vs 1.34±0.13 μmol/L, P < 0.05), interleukin-6 (IL-6) (740.04±8.58 pg/mL vs 515.77±4.62 pg/mL, P < 0.05), tumor necrosis factor-α (TNFα) (774.23±7.98 pg/mL vs 461.51±1.76 pg/mL, P < 0.05), interferon gamma (IFNγ) (842.33±14.79 pg/mL vs 542.47±6.75 pg/mL, P < 0.05), and ICAM-1 (2.40±0.02 vs 1.33±0.01, P < 0.05) in Kupffer cells, a significant increase in neutrophil chemotaxis (100.80±10.18 vs 13.80±3.70, P < 0.05), and a significant reduction in phagocytic capacity (9.86±1.82 vs 60.00±3.54, P < 0.05), with no effect on liver abscess formation (40% vs 0, P > 0.05). Compared with the diabetes control group, the diabetes+lidocaine group had significant reductions in the levels of NO (3.35±0.28 μmol/L vs 4.95±0.06 μmol/L, P < 0.05), IL-6 (688.42±36.34 pg/mL vs 740.04±8.58 pg/mL, P < 0.05), TNFα (631.15±4.30 pg/mL vs 774.23±7.98 pg/mL, P < 0.05), IFNγ (704.56±3.64 pg/mL vs 842.33±14.79 pg/mL, P < 0.05), and ICAM-1 (1.50±0.02 vs 2.40±0.02, P < 0.05) in Kupffer cells, a significant reduction in neutrophil chemotaxis (33.40±5.60 vs 100.80±10.18, P < 0.05), and a significant increase in phagocytic capacity (49.20±2.59 vs 9.86±1.82, P < 0.05), with no effect on liver abscess formation (0 vs 40%, P > 0.05). Conclusion Lidocaine can inhibit Kupffer cell inflammatory response and improve the phagocytic function of Kupffer cells in diabetic mice, thereby exerting a protective effect on Kupffer cells, but it had no effect on liver abscess formation.
- Diabetes Mellitus /
- Liver Abscess /
- Kupffer Cells /
- Lidocaine

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图 1 非糖尿病与糖尿病小鼠Kupffer细胞超微结构观察(透射电镜，×5000)

注：a，糖尿病小鼠; b，非糖尿病小鼠。红色箭头为线粒体; 绿色箭头为粗面内质网。

Figure 1. Ultrastructural observation of mitochondria and endoplasmic reticulum in Kupffer cells in nondiabetic vs diabetic mice (transmission electron microscopy, ×5000)

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图 2 Western Blot检测各组小鼠Kupffer细胞ICAM-1表达

Figure 2. Expression of ICAM-1 in Kupffer cells detected by Western Blot

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图 3 各组小鼠中性粒细胞趋化(0.1%结晶紫染色，×100)

注：a，非糖尿病对照组; b，糖尿病对照组; c，非糖尿病+利多卡因组; d，糖尿病+利多卡因组。

Figure 3. Neutrophil chemotaxis of mice in each group (0.1% crystal violet staining, ×100)

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图 4 各组小鼠Kupffer细胞吞噬功能(Giemsa染色，×100)

注：a，非糖尿病对照组; b，糖尿病对照组; c，非糖尿病+利多卡因组; d，糖尿病+利多卡因组。

Figure 4. Phagocytosis of Kupffer cells in each group (Giemsa staining, ×100)

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图 5 小鼠肝脓肿形成

注：a，肝脓肿形成; b，正常肝脏。

Figure 5. Formation of liver abscess in mice

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表 1 各组小鼠Kupffer细胞NO、IL-6、TNFα、IFNγ、ICAM-1水平

Table 1. Levels of NO, IL-6, TNFα, IFNγ and ICAM-1 in Kupffer cells of mice in each group

指标	非糖尿病对照组 (n=5)	糖尿病对照组 (n=5)	非糖尿病+利多卡因组(n=5)	糖尿病+利多卡因组(n=5)	H值	P值
NO(μmol/L)	1.34±0.13	4.95±0.06¹⁾	1.47±0.16	3.35±0.28²⁾	16.50	＜0.001
IL-6(pg/mL)	515.77±4.62	740.04±8.58¹⁾	512.79±2.24	688.42±36.34²⁾	15.79	0.001
TNFα(pg/mL)	461.51±1.76	774.23±7.98¹⁾	468.48±7.42	631.15±4.30²⁾	16.71	＜0.001
IFNγ(pg/mL)	542.47±6.75	842.33±14.79¹⁾	543.49±8.38	704.56±3.64²⁾	16.07	0.001
ICAM-1	1.33±0.01	2.40 ±0.02¹⁾	1.31±0.03	1.50±0.02²⁾	16.52	0.001
注：与非糖尿病对照组相比，1)P＜0.05;与糖尿病对照组相比，2)P＜0.05。

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表 2 各组小鼠中性粒细胞招募、吞噬功能水平

Table 2. Neutrophil recruitment and phagocytic function levels in each group

项目	非糖尿病对照组 (n=5)	糖尿病对照组 (n=5)	非糖尿病+利多卡因组(n=5)	糖尿病+利多卡因组(n=5)	H值	P值
中性粒细胞趋化	13.80±3.70	100.80±10.18¹⁾	14.40±4.67	33.40±5.60²⁾	16.12	0.001
Kupffer细胞吞噬功能	60.00±3.54	9.86 ±1.82¹⁾	62.60±1.95	49.20±2.59²⁾	16.87	0.001
注：与非糖尿病对照组相比，1)P＜0.05;与糖尿病对照组相比，2)P＜0.05。

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