高脂饲喂小鼠结肠ILC3s变化对肠屏障应激能力的影响

doi:10.3877/cma.j.issn.2095-9133.2024.02.008

目的

探讨高脂饲喂小鼠结肠三型固有淋巴细胞（ILC3s）的变化对肠黏膜屏障应激能力影响的潜在机制。

方法

将24只SPF级雄性C57BL/6J小鼠适应性饲养1周后，随机分为空白对照组（N-CON组）、重症急性胰腺炎（SAP）对照组（N-SAP组）、空白高脂组（H-CON组）、SAP高脂组（H-SAP组），每组6只。N-CON、N-SAP给予普通饮食，H-CON、H-SAP给予高脂饮食，持续21周；N-SAP组、H-SAP组采用腹腔注射20%左旋精氨酸（L-Arg）作为应激刺激制备小鼠SAP模型，N-CON组、H-CON组予以腹腔注射等体积等渗盐水。造模48 h后，酶联免疫吸附试验（ELISA）检测甘油三酯（TG）、总胆固醇（TC）、血清淀粉酶（AMS）、脂多糖（LPS）、肿瘤坏死因子-α（TNF-α）、白细胞介素-6（IL-6）水平；HE染色观察胰腺、肺、结肠组织病理学形态；流式细胞术分析结肠ILC3s占淋巴细胞比例和IL-22⁺ ILC3s占结肠ILC3s比例；免疫荧光法检测结肠荆豆凝集素（UEA-1）、白细胞介素-22受体A1（IL-22RA1）表达情况；Western blot检测岩藻糖基转移酶2（Fut2）、IL-22RA1蛋白的表达水平。

结果

与N-CON组相比，H-CON组小鼠体重、TG、TC以及血清AMS、LPS、IL-6、TNF-α水平显著升高（P<0.05），结肠ILC3s占淋巴细胞比例显著下降（P<0.05），IL-22⁺ILC3s占结肠ILC3s比例升高（P>0.05），Fut2、IL-22RA1蛋白表达下降（P>0.05），UEA-1、IL-22RA1平均荧光强度略有下降（P>0.05）；给予20%Arg应激刺激后，与N-CON组相比，N-SAP组血清AMS、LPS、IL-6、TNF-α水平显著升高（P<0.05），结肠ILC3s占淋巴细胞比例显著下降（P<0.05），IL-22⁺ILC3s占结肠ILC3s比例变化显著升高（P<0.05），Fut2、IL-22RA1蛋白表达升高（P>0.05），UEA-1平均荧光强度显著下降（P<0.05），IL-22RA1平均荧光强度升高（P>0.05）；与N-SAP组相比，H-SAP组结肠ILC3s占淋巴细胞比例降低（P>0.05），IL-22⁺ILC3s占结肠ILC3s比例升高（P>0.05），UEA-1平均荧光强度下降（P>0.05），IL-22RA1平均荧光强度显著升高（P<0.05），Fut2、IL-22RA1蛋白表达增加（P>0.05）。

结论

高脂饲喂小鼠的肠黏膜屏障应激能力削弱，可能与结肠ILC3s调节岩藻糖基化过程有关。

关键词: ILC3s, 高脂饮食, Fut2, 肠黏膜屏障, 应激能力

Objective

To investigate the impact of colonic group 3 innate lymphoid cells (ILC3s) on intestinal barrier stress capability in mice fed with high-fat diet.

Methods

Twenty-four SPF male C57BL/6J mice were adaptively fed for 1 week and then randomly divided into 4 groups (n=6): blank control group (N-CON), SAP control group (N-SAP), high-fat diet control group (H-CON), and SAP high-fat diet group (H-SAP). N-CON and N-SAP received a normal diet, while H-CON and H-SAP were fed a high-fat diet for 21 weeks. In the SAP group, 20% Arg was injected intraperitoneally as stress stimulation to prepare the mouse SAP model, while an equal volume of saline was injected into the CON groups. After 48 hours of modeling, serum levels of TC, TG, AMY, LPS, TNF-α, IL-6 were measured by ELISA; pancreatic histopathology as well as lung and colon tissue morphology were observed using HE staining; flow cytometry was employed to analyze changes in the proportion of colonic ILC3s among lymphocytes and changes in the proportion of IL-22⁺ ILC3s among colonic ILC3s; immunofluorescence was conducted to detect UEA-Ⅰ and IL-22RA1 expression; Western blot was performed to measure Fut2 and IL-22RA1 protein expression levels.

Results

Compared with the N-CON group, the H-CON group exhibited significantly increased levels of body weight, TG, TC, serum AMS, LPS, IL-6 and TNF-α (P<0.05), along with a significant decrease in the proportion of colon ILC3s in lymphocytes (P<0.05). The proportion of IL-22⁺ILC3s in the colon showed an increase (P>0.05), while the expressions of Fut2 and IL-22RA1 were decreased (P>0.05). The average fluorescence intensity of UEA-1 and IL-22RA1 slightly decreased (P>0.05). After stimulation with a 20% Arg stress in the N-SAP group compared to the N-CON group, there was a significant increase in serum AMS, LPS, IL-6 and TNF-a levels (P<0.05) and a significant decrease in the proportion of colon ILC3s in lymphocytes (P<0.05). The proportion of IL-22+ILC3s in the colon significantly increased (P<0.05), while protein expression of Fut2 and IL-22RA1 was increased as well (P>0.05). The average fluorescence intensity of UEA-I significantly decreased (P<0.05), but that for IL-22 RA1was increased (P>0.05). In comparison to the N-SAP group, the H-SAP group showed no significant changes regarding the proportions between colonic ILC3s and lymphocytes (P>0.05); however, the percentage of IL-22 ⁺ILC3s in colonic ILC3s was significantly increased (P>0.05), while the average fluorescence intensity of UEA-I was decreased significantly (P<0.05), and also the protein expression of Fut2 and IL-22 RA1 was increased (P>0.05).

Conclusion

The intestinal barrier stress capability of mice fed with high-fat diet is impaired, which may be related to the regulation of fucosylation by ILC3s in the colon.

Key words: High-fat diet, ILC3s, Fut2, Intestinal mucosal barrier, Intestinal stress capability

表1 结肠组织损伤评分标准

评分	评分标准
0分	无炎症证据
2分	低度水平炎症，散在浸润的单核细胞（1~2个病灶）
4分	中度水平炎症，有多发病灶
6分	高度水平炎症，血管密度增加，管壁明显增厚
8分	炎症最严重，伴有透壁白细胞浸润

表1 结肠组织损伤评分标准

评分	评分标准
0分	无炎症证据
2分	低度水平炎症，散在浸润的单核细胞（1~2个病灶）
4分	中度水平炎症，有多发病灶
6分	高度水平炎症，血管密度增加，管壁明显增厚
8分	炎症最严重，伴有透壁白细胞浸润

图1 高脂饲喂21周后小鼠体重及血清TG、TC变化情况注：TG为甘油三酯，TC为总胆固醇；a为体重含量分析，b为血清甘油三酯含量分析，c为血清总胆固醇含量分析

表2 小鼠体重及血清TG、TC变化情况（

±s）

组别	初始体重（g）	21周喂养后体重（g）	21周喂养后TG水平（mg/dl）	21周喂养后TC水平（mg/dl）
对照组	23.2±0.83	29.61±1.32	13.66±4.90	79.03±15.75
高脂组	23.5±0.60	36.12±2.21^#	19.44±8.55^#	134.20±27.54^#

表2 小鼠体重及血清TG、TC变化情况（

±s）

组别	初始体重（g）	21周喂养后体重（g）	21周喂养后TG水平（mg/dl）	21周喂养后TC水平（mg/dl）
对照组	23.2±0.83	29.61±1.32	13.66±4.90	79.03±15.75
高脂组	23.5±0.60	36.12±2.21^#	19.44±8.55^#	134.20±27.54^#

图2 SAP刺激后小鼠的血清AMS、LPS、IL-6、TNF-α表达水平注：AMS为血清淀粉酶，LPS为脂多糖，IL-6为白细胞介素-6，TNF-α为肿瘤坏死因子-α；a为AMS含量分析，b为LPS含量分析，c为IL-6含量分析，d为TNF-α含量分析

图3 小鼠胰腺组织HE染色（×500）

图4 小鼠肺组织HE染色（×200）

图5 小鼠结肠组织HE染色（×100）

表3 胰腺、肺、结肠组织病理学评分（

±s）

组别	胰腺组织病理学评分	肺组织病理学评分	结肠组织病理学评分
N-CON组	0.27±0.12	1.20±0.35	0.53±0.46
N-SAP组	3.53±0.23^a	5.47±0.12^a	4.93±0.23^a
H-CON组	1.53±0.12	2.67±0.31^a	2.53±1.29^a
H-SAP组	6.63±1.82^bc	6.53±1.29^b	6.40±0.40^bc

表3 胰腺、肺、结肠组织病理学评分（

±s）

组别	胰腺组织病理学评分	肺组织病理学评分	结肠组织病理学评分
N-CON组	0.27±0.12	1.20±0.35	0.53±0.46
N-SAP组	3.53±0.23^a	5.47±0.12^a	4.93±0.23^a
H-CON组	1.53±0.12	2.67±0.31^a	2.53±1.29^a
H-SAP组	6.63±1.82^bc	6.53±1.29^b	6.40±0.40^bc

图6 小鼠结肠ILC3s所占结肠固有层淋巴细胞比例及IL-22⁺ILC3s占结肠ILC3s比例注:a为Lin^-、CD45⁺的ILC3s细胞群，b为CD127⁺、RORγt⁺的ILC3s细胞群，c为IL-22⁺的ILC3细胞群

图7 小鼠结肠ILC3s所占结肠固有层淋巴细胞比例及IL-22⁺ILC3s占结肠ILC3s比例变化注：a为各组小鼠结肠ILC3s占结肠固有层淋巴细胞比例统计分析，b为各组小鼠结肠IL-22+ILC3s占结肠ILC3s的比例统计分析

图8 小鼠结肠UEA-1、IL-22RA1免疫荧光染色注：绿色为结肠UEA-1，红色为IL-22RA1免疫荧光染色图，标尺为50 μm

图9 小鼠结肠UEA-1、IL-22RA1平均荧光强度值分析注：UEA-1为荆豆凝集素，IL-22RA1为白细胞介素-22受体A1；a为各组小鼠结肠UEA-1平均荧光强度，b为各组小鼠结肠IL-22RA1平均荧光强度

图10 小鼠结肠Fut2、IL-22RA1蛋白表达分析注：a为Fut2、IL-22RA1蛋白的Western Blot结果，b为各组小鼠Fut2蛋白表达结果分析，c为各组小鼠IL-22RA1蛋白表达结果分析

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Effects of colonic ILC3s alterations on intestinal barrier stress capability in mice fed with high-fat diet

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Effects of colonic ILC3s alterations on intestinal barrier stress capability in mice fed with high-fat diet