青蒿琥酯对自发性干燥综合征NOD/Ltj小鼠的治疗作用

doi:10.3969/j.issn.1674-8115.2025.10.003

上海交通大学学报（医学版） ›› 2025, Vol. 45 ›› Issue (10): 1279-1287.doi: 10.3969/j.issn.1674-8115.2025.10.003

青蒿琥酯对自发性干燥综合征NOD/Ltj小鼠的治疗作用

李燕香, 石欢, 俞创奇()

上海交通大学医学院附属第九人民医院口腔外科，上海交通大学口腔医学院，国家口腔医学中心，国家口腔疾病临床医学研究中心，上海市口腔医学重点实验室，上海市口腔医学研究所，上海 200011

收稿日期:2024-11-27 接受日期:2025-04-27 出版日期:2025-10-28 发布日期:2025-10-28
通讯作者: 俞创奇，主任医师，博士；电子信箱：ycq616@hotmail.com。
基金资助:
国家自然科学基金(82174041);上海市自然科学基金(22ZR1437400);上海市扬帆计划(22YF1422300)

Therapeutic effect of artesunate on spontaneous mouse models of Sjögren syndrome (NOD/Ltj mouse)

LI Yanxiang, SHI Huan, YU Chuangqi()

Department of Oral Surgery, Shanghai Ninth People's Hospital, Shanghai Jiao Tong University School of Medicine; College of Stomatology, Shanghai Jiao Tong University; National Center for Stomatology; National Clinical Research Center for Oral Diseases; Shanghai Key Laboratory of Stomatology; Shanghai Research Institute of Stomatology, Shanghai 200011, China

Received:2024-11-27 Accepted:2025-04-27 Online:2025-10-28 Published:2025-10-28
Contact: YU Chuangqi, E-mail: ycq616@hotmail.com.
Supported by:
National Natural Science Foundation of China(82174041);Natural Science Foundation of Shanghai(22ZR1437400);Shanghai Young Science and Technology Talents Sailing Program(22YF1422300)

摘要/Abstract

摘要：

目的·探索青蒿琥酯（artesunate，ART）对干燥综合征模型非肥胖型糖尿病（non-obese diabetic，NOD）/Ltj小鼠的治疗效果，及其对小鼠外周B淋巴细胞亚群分布的影响。方法·以ICR小鼠为空白对照组，将NOD/Ltj小鼠随机分为疾病组和ART组。对ART组小鼠进行间隔1 d的ART（10 mg/kg）灌胃处理，空白对照组和疾病组同时进行等量的溶剂（0.5%羧甲基纤维素钠）灌胃处理，持续给药4周。测量小鼠体质量、唾液流率、下颌下腺指数、脾脏指数；采用高通量液相蛋白芯片（CBA）流式多因子检测血浆中B细胞活化因子（B-cell activating factor，BAFF）、白细胞介素-6（interleukin-6，IL-6）和γ干扰素（interferon-γ，IFN-γ）水平；苏木精-伊红（H-E）染色观察下颌下腺淋巴细胞浸润情况；流式细胞术分析脾脏B淋巴细胞各亚群的分布情况；RT-qPCR检测脾脏B淋巴细胞Prdm1、Il-6r、Il-6和Stat3的mRNA表达水平。通过CCK-8法和凋亡流式染色进一步检测ART对B细胞的作用。结果·与疾病组相比，ART组NOD/Ltj小鼠的干燥症状显著改善，外周血血浆中BAFF及促炎因子IL-6和IFN-γ的水平均显著降低（均P<0.05）；下颌下腺H-E染色结果显示ART组腺体导管周围的淋巴细胞浸润状态优于疾病组；流式分析结果显示，与疾病组比较，ART组脾脏中初始B细胞分布增加，双阴B细胞、转换记忆B细胞及浆母细胞的比例均显著降低（均P<0.05），脾脏B细胞中Prdm1、Il-6r和Stat3的mRNA水平均显著降低（均P<0.05）。CCK8法检测结果显示：作用6 h后，随着培养时间延长，ART组细胞的增殖受到显著抑制；作用24 h后，ART组凋亡细胞数量显著高于对照组（P<0.001）。结论·ART对NOD/Ltj小鼠的作用，可能是通过影响外周B淋巴细胞亚群的分布，同时抑制Prdm1表达，进而调节B淋巴细胞向浆细胞和浆母细胞分化。

关键词: 干燥综合征, 青蒿琥酯, NOD/Ltj小鼠, B淋巴细胞

Abstract:

Objective ·To elevate the therapeutic effect of artesunate (ART) on NOD/Ltj mice (non-obese diabetic mice, the spontaneous models of Sjögren syndrome), and explore its potential impact on the distribution of B lymphocyte subsets. Methods ·ICR mice were used as the blank control group, and NOD/Ltj mice were randomly divided into disease and ART groups. NOD/Ltj mice and ICR mice were treated with ART (10 mg/ kg) or its vehicle (0.5%CMC) by oral gavage every other day for 4 weeks. Body weight, salivary flow rate, submandibular gland index, and spleen index were measured. Cytokines in plasma, including interleukin-6 (IL-6), interferon-γ (IFN-γ), and B-cell activating factor (BAFF) in serum, were detected by cytometric bead array (CBA). Hematoxylin-Eosin (H-E) staining of submandibular glands was used to observe the infiltration of lymphocyte. Flow cytometry was applied to analyze the distribution of B lymphocyte subsets in the spleen. The mRNA expression of Prdm1, Il-6r, Il-6, and Stat3 in spleen B lymphocytes was detected by RT-qPCR. The effect of ART on B cells was further detected by CCK-8 and Annexin V-FITC/PI staining by flow cytometry. Results ·Compared to the disease group, ART significantly improved the symptoms of Sjögren syndrome in NOD/Ltj mice. ART treatment also resulted in a reduction in the levels of BAFF, IL-6, and IFN-γ in the plasma (all P<0.05). Moreover, lymphocyte infiltration around the glandular ducts in the submandibular glands was greatly improved in the ART group compared with the disease group. Flow cytometry analysis revealed that the proportion of Naïve B cells in the ART group was significantly increased compared with the disease group, along with a significant reduction in the proportions of double-negative B cells, switched memory B cells, and plasmablasts (all P<0.05). The relative mRNA expression levels of Prdm1, Il-6r,and Stat3 in the ART group were significantly lower than those in the disease group (all P<0.05). The CCK8 assay results showed that after 6 h of treatment, with the extension of the culture time, cell proliferation in the ART group was significantly inhibited; after 24 h of treatment, the number of apoptotic cells in the ART group was significantly higher than that in the control group (P<0.001). Conclusion ·ART demonstrates therapeutic effects in NOD/Ltj mice, potentially through modulating the distribution of peripheral B lymphocyte subsets. It can inhibit the expression of Prdm1, thereby regulating the differentiation of B lymphocytes into plasma cells and plasmablasts.

Key words: Sj?gren syndrome (SS), artesunate (ART), NOD/Ltj mouse, B lymphocyte

中图分类号:

R782.2

李燕香, 石欢, 俞创奇. 青蒿琥酯对自发性干燥综合征NOD/Ltj小鼠的治疗作用[J]. 上海交通大学学报（医学版）, 2025, 45(10): 1279-1287.

LI Yanxiang, SHI Huan, YU Chuangqi. Therapeutic effect of artesunate on spontaneous mouse models of Sjögren syndrome (NOD/Ltj mouse)[J]. Journal of Shanghai Jiao Tong University (Medical Science), 2025, 45(10): 1279-1287.

图/表 6

表1 qRT-PCR引物序列

Tab 1 Primer sequences for qRT-PCR

Primer	Sequence (5'→3')
Prdm1-Forward	CTTCTCTTGGAAAAACGTGTGGG
Prdm1-Reverse	TCATATCAGCGTCCTCCATGT
Stat3-Forward	GAGAGCAGAAGGGAGCAA
Stat3-Reverse	CTCACAGAGTGGGGCAA
Il-6r-Forward	TTGGGTTGCTTCTCTGTGT
Il-6r-Reverse	AAGGTCGGCTTCAGTGG
Il-6-Forward	GTATGAACAACGATGATGCAC
Il-6-Reverse	CTCCAGAAGACCAGAGGAAA

图1 小鼠干燥症状及血浆中促炎因子水平 (n=3)Note： A. Weight changes of the mice. B. Salivary flow rate of the mice. C/D. Submandibular gland index (C) and splenic index (D) of the mice. E‒G. Serum levels of IL-6 (E), IFN-γ (F) and BAFF(G) in the mice detected by CBA.

Fig 1 Sjögren syndrome-like symptoms and pro-inflammatory cytokine levels in the plasma of mice (n=3)

图2 ART对NOD/Ltj小鼠下颌下腺局部炎症浸润的影响 (n=3)Note： A. H-E staining of the submandibular glands. B.Number of lymphocyte infiltration foci in the submandibular glands. C. Chisholm and Mason (CM) histopathological grading of the submandibular glands.

Fig 2 Effect of ART on local lymphocytic infiltration in the submandibular glands of NOD/Ltj mice (n=3)

图3 ART对NOD/Ltj小鼠脾脏B淋巴细胞分布的影响Note： A. Representative flow cytometry analysis of B-cell subsets based on IgD and CD27 expression. B‒F. Distribution of Naïve B cells (B), double-negative B cells (C), unswitched memory B cells (D), switched memory B cells (E), and total memory B cells (F) in the three groups. G‒J. Representative flow cytometry analysis (G) and the distribution of autoimmunity-associated B cells (H), plasmablasts (I), and plasma cells (J) in the indicated groups (n=3).

Fig 3 Effect of ART on the distribution of splenic B-lymphocyte subsets in NOD/Ltj mice

图4 脾脏B淋巴细胞 Prdm1 (A)、Il-6r (B)、 Stat3 (C)和 Il-6 (D)的mRNA表达

Fig 4 mRNA expression levels of Prdm1 (A), Il-6r (B), Stat3 (C), and Il-6 (D) in splenic B lymphocytes

图5 ART对Raji B淋巴细胞凋亡的诱导作用Note： A. Inhibitory effect of Artesunate on the proliferation of Raji B lymphocytes detected by CCK-8 assay. B. Representative flow cytometry chart of Annexin V-FITC/PI apoptosis assay. C.Rescue effect of Z-VAD-FMK on artesunate detected by CCK-8 assay.

Fig 5 Inductive effect of ART on apoptosis in Raji B lymphocytes

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青蒿琥酯对自发性干燥综合征NOD/Ltj小鼠的治疗作用

Therapeutic effect of artesunate on spontaneous mouse models of Sjögren syndrome (NOD/Ltj mouse)

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