上海交通大学学报（医学版）

上海交通大学学报（医学版） ›› 2022, Vol. 42 ›› Issue (5): 570-577.doi: 10.3969/j.issn.1674-8115.2022.05.003

• 论著 · 基础研究 • 上一篇    下一篇

牙龈素提取物对小鼠脑神经炎症的影响

张桓瑜1(), 江旖婷1, 朱晓晨2, 何智妍2, 周薇2, 宋忠臣1()   

  1. 1.上海交通大学医学院附属第九人民医院牙周病科，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 200011
    2.上海交通大学医学院附属第九人民医院口腔微生态与系统性疾病实验室，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 200125
  • 收稿日期:2022-01-28 接受日期:2022-04-05 出版日期:2022-05-28 发布日期:2022-05-28
  • 通讯作者: 宋忠臣 E-mail:602895658@qq.com;szhongchen@sina.com
  • 作者简介:张桓瑜（1997—），男，硕士生；电子信箱：602895658@qq.com。
  • 基金资助:
    国家自然科学基金(82071112);上海交通大学医学院附属第九人民医院“交叉基金”(JYJC202005)

Effects of gingipain extracts on brain neuroinflammation in mice

ZHANG Huanyu1(), JIANG Yiting1, ZHU Xiaochen2, HE Zhiyan2, ZHOU Wei2, SONG Zhongchen1()   

  1. 1.Department of Periodontology, 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 200011, China
    2.Laboratory of Oral Microbiota and Systemic Disease, 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 200125, China
  • Received:2022-01-28 Accepted:2022-04-05 Online:2022-05-28 Published:2022-05-28
  • Contact: SONG Zhongchen E-mail:602895658@qq.com;szhongchen@sina.com
  • Supported by:
    National Natural Science Foundation of China(82071112);Cross-disciplinary Research Fund of Shanghai Ninth People's Hospital, Shanghai Jiao Tong University School of Medicine(JYJC202005)

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摘要：

目的·观察牙龈素提取物对小鼠脑神经炎症的影响。方法·采用超声破膜结合低温超速离心，并通过超滤浓缩的方法从牙龈卟啉单胞菌ATCC33277获得牙龈素提取物。对牙龈素提取物进行蛋白定量后行SDS聚丙烯酰胺凝胶电泳，经湿转法转移至聚偏二氟乙烯（polyvinylidene fluoride，PVDF）膜，封闭后分别使用赖氨酸特异性牙龈素（lysine-gingipain，Kgp）抗体和精氨酸特异性牙龈素（arginine-gingipain，Rgp）抗体对样本进行孵育，以检测提取物中的牙龈素免疫原性。通过向C57BL/6N小鼠腹腔注射牙龈素提取物建立小鼠牙龈素急性感染模型。在小鼠腹腔注射牙龈素提取物后的不同时间点，分别采用针对Kgp和Rgp的特异性荧光底物Z-His-Glu-Lys-MCA和Boc-Phe-Ser-Arg-MCA检测小鼠血浆中2种牙龈素的活性。将40只C57BL/6N小鼠随机分为4组，分别为对照组、牙龈素组、牙龈素＋Kgp抑制剂（COR388）组和COR388组。预先给予COR388处理1 h后，再腹腔注射牙龈素提取物，24 h后麻醉下取材，脑组织经固定、脱水、包埋、切片等步骤后，分别使用离子钙适配分子1（ionized calcium binding adapter molecule 1，Iba1）抗体和神经胶质纤维酸性蛋白（glial fibrillary acidic protein，GFAP）抗体标记小鼠大脑皮层小胶质细胞和星形胶质细胞，采用免疫组织化学技术观察上述4组小鼠大脑皮层小胶质细胞及星形胶质细胞的激活情况。采用酶联免疫吸附测定检测大脑皮层肿瘤坏死因子-α（tumor necrosis factor-α，TNF-α）和白介素-1β（interleukin-1β，IL-1β）的表达水平。结果·蛋白质印迹分析显示，该牙龈素提取物在截留分子量50 000和70 000附近有较清晰的条带，表明该提取物具有牙龈素免疫原性。此牙龈素提取物具有生物活性，腹腔注射牙龈素提取物后，小鼠血浆牙龈素活性呈现先升后降的趋势。腹腔注射牙龈素提取物后4~8 h，小鼠血浆牙龈素活性达到峰值，随后逐渐下降。免疫组织化学结果显示，小鼠大脑皮层的小胶质细胞和星形胶质细胞体积增大，且具有不规则突起，提示腹腔注射牙龈素提取物可激活小鼠脑内小胶质细胞和星形胶质细胞。酶联免疫吸附测定结果表明小鼠大脑皮层炎症因子TNF-α和IL-1β表达水平升高。结论·急性感染牙龈素提取物可促进小鼠脑神经炎症。

关键词: 牙龈卟啉单胞菌, 精氨酸特异性牙龈素, 赖氨酸特异性牙龈素, 神经炎症, 炎症因子

Abstract:

Objective·To observe the effects of gingipain extracts on brain inflammation in mice.

Methods·The gingipain extracts were obtained from Porphyromonas gingivalis ATCC33277 by sonication method combined with low temperature ultracentrifugation, followed by ultrafiltration and concentration. Gingipain extracts were identified by Western blotting after protein quantification. SDS polyacrylamide gel electrophoresis was performed and then transferred the proteins to the polyvinylidene fluoride (PVDF) membrane. After blockage, the proteins were probed with lysine-specific gingipain (Kgp) antibody and arginine-gingipain (Rgp) antibody, respectively, to detect the immunogenicity of gingipain in the extracts. The acute infection model was established by intraperitoneal gingipain extracts in C57BL/6N mice. The specific fluorescent substrates Z-His-Glu-Lys-MCA for Kgp and Boc-Phe-Ser-Arg-MCA for Rgp were used to detect the activities of two gingipains in plasma at different time points after intraperitoneal injection of gingipain extracts in mice. C57BL/6N mice were randomly divided into 4 groups, which were recorded as the control group, the gingipain group, the gingipain＋Kgp inhibitor (COR388) group and the COR388 group, respectively. Mice in the gingipain＋COR388 group were pre-treated with COR388 for 1 h, and then intraperitoneally injected with gingipain extracts. Twenty-four hours later, the samples were collected under anesthesia and the brains were processed sequentially by procedures such as fixing, dehydrating, embedding and dissecting. The microglia and astrocytes in the mouse cortex were labeled with ionized calcium binding adapter molecule 1 antibody (anti-Iba1) and glial fibrillary acidic protein antibody (anti-GFAP), respectively, and the activation of microglia and astrocytes in the cortex of the above four groups of mice were observed through immunohistochemistry. The expression of tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) were evaluated by enzyme linked immunosorbent assay (ELISA).

Results·There were relatively clear bands around 50 000 and 70 000 in the Western blotting analysis of the gingipain extracts, suggesting that the extracts contained gingipain immunogenicity. Meanwhile, the gingipain extracts were biologically active, and the activity of gingipain in the plasma of mice showed a trend of first increasing and then decreasing after intraperitoneal injection of the gingipain extracts. The activity of plasma gingipain in mice reached a peak 4?8 h after intraperitoneal injection of gingipain extracts, and then gradually decreased. Immunohistochemistry results showed that the microglia and astrocytes in the cortex had increased volume of cell bodies and irregular protrusions, suggesting that intraperitoneal injection of gingipain extracts could activate microglia and astrocytes in the mouse brain. ELISA results showed that the expression of inflammatory factors (TNF-α and IL-1β) in cortex increased.

Conclusion·Acute infection of gingipain extracts can induce neuroinflammation in mice.

Key words: Porphyromonas gingivalis, arginine-gingipain (Rgp), lysine-gingipain (Kgp), neuroinflammation, inflammatory factor

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