目的： 研究白细胞介素-22（IL-22）在牙周炎症环境下对牙龈上皮屏障的调控作用及其可能机制。 方法： 构建IL-22敲除小鼠，采用混合菌液口腔灌洗法构建牙周炎模型。收集同窝生野生型牙周炎组及IL-22敲除牙周炎组小鼠（每组7只）口腔菌斑并提取DNA，建立牙周炎相关风险微生物数据库“PD-RiskMicroDB”并结合16S rRNA测序结果，测定两组小鼠口腔菌群变化和微生物功能的关系。通过改良胰酶消化法培养牙龈上皮细胞（GEC），以100 μg/L IL-22、感染复数为100的牙龈卟啉单胞菌（Pg）分别或联合刺激GEC，实验分组为：对照组（细胞未加刺激）、IL-22组、Pg组及Pg+IL-22组，处理时间为3和12 h；采用细胞免疫荧光、实时荧光定量PCR（RT-qPCR）及蛋白质印迹法检测各组细胞3 h后上皮屏障蛋白E-钙黏蛋白（E-cadherin）的表达；通过异硫氰酸荧光素-葡聚糖（FITC-D）介导的上皮细胞通透性实验明确各组GEC 3和12 h细胞通透性的变化。RT-qPCR检测同窝生野生型牙周炎组及IL-22敲除牙周炎组小鼠牙龈上皮E-cadherin表达水平。将15只C57BL/6野生型小鼠按随机数字表法随机分为对照组、牙周炎组和牙周炎+IL-22处理组，每组5只。RT-qPCR及免疫组织化学（IHC）染色法检测各组小鼠牙龈上皮E-cadherin的表达水平。 结果： 16S rRNA测序结果显示，与同窝生野生型牙周炎组小鼠相比，IL-22敲除牙周炎组小鼠口腔菌群组成发生改变，其中与牙周组织侵袭相关的菌属丰度显著增高（线性判别分析得分为2.22，P=0.009）。体外细胞实验显示，Pg感染GEC 3 h后，Pg组GEC的细胞连接中断，Pg组E-cadherin荧光强度较对照组减弱，E-cadherin的mRNA（0.69±0.12）和蛋白（0.60±0.12）表达水平均显著低于对照组（分别为1.00±0.00、1.04±0.08）（P=0.043，P=0.003）；而Pg+IL-22组的E-cadherin荧光强度较Pg组稍增强，Pg+IL-22组E-cadherin的mRNA（1.16±0.10）和蛋白（0.98±0.07）表达水平均显著高于Pg组（分别为0.69±0.12、0.60±0.12）（P=0.005，P=0.007）；上皮通透性实验结果显示，Pg处理GEC 3 h后，对照组、IL-22组、Pg组及Pg+IL-22组各组间总体比较上皮屏障通透性差异无统计学意义（F=0.20，P=0.893）；处理12 h后，相较于对照组（1.00±0.00），Pg组GEC上皮屏障通透性（1.39±0.15）显著增加（P=0.027），而Pg+IL-22组上皮屏障通透性（1.02±0.18）显著低于Pg组（P=0.034）；体内RT-qPCR结果显示，IL-22敲除牙周炎组小鼠牙龈上皮E-cadherin的mRNA表达水平（0.32±0.21）显著低于同窝生野生型牙周炎组（1.01±0.01）（t=5.70，P=0.005）。RT-qPCR及IHC染色结果显示，牙周炎组小鼠牙龈上皮组织E-cadherin的mRNA表达水平（0.40±0.07）和E-cadherin阳性表达吸光度值（0.02±0.00）均显著低于对照组（分别为1.00±0.00、0.04±0.01）（P=0.005，P=0.006）；牙周炎+ IL-22处理组E-cadherin的mRNA表达水平（1.06±0.24）和E-cadherin阳性表达吸光度值（0.03±0.01）均显著高于牙周炎组（P=0.003，P=0.039）。 结论： IL-22可能通过调节口腔菌群的侵袭性以及宿主屏障蛋白表达，发挥其对炎症环境下牙龈上皮屏障的保护作用。.

2024-01-01·BIO-PROTOCOL

Fluorescent Labeling and Imaging of IL-22 mRNA-Loaded Lipid Nanoparticles

Article

作者: Bolay, Eunice ; Mow, Rabeya Jafrin ; Srinivasan, Anand ; Merlin, Didier ; Yang, Chunhua

Lipid nanoparticle (LNP)-based drug delivery systems (DDSs) are widely recognized for their ability to enhance efficient and precise delivery of therapeutic agents, including nucleic acids like DNA and mRNA. Despite this acknowledgment, there is a notable knowledge gap regarding the systemic biodistribution and organ accumulation of these nanoparticles. The ability to track LNPs in vivo is crucial for understanding their fate within biological systems. Fluorescent labeling of LNPs facilitates real-time tracking, quantification, and visualization of their behavior within biological systems, providing valuable insights into biodistribution, cellular uptake, and the optimization of drug delivery strategies. Our prior research established reversely engineered LNPs as an exceptional mRNA delivery platform for treating ulcerative colitis. This study presents a detailed protocol for labeling interleukin-22 (IL-22) mRNA-loaded LNPs, their oral administration to mice, and visualization of DiR-labeled LNPs biodistribution in the gastrointestinal tract using IVIS spectrum. This fluorescence-based approach will assist researchers in gaining a dynamic understanding of nanoparticle fate in other models of interest. Key features • This protocol is developed to assess the delivery of IL-22 mRNA to ulcerative colitis sites using lipid nanoparticles. • This protocol uses fluorescent DiR dye for imaging of IL-22 mRNA-loaded lipid nanoparticles in the gastrointestinal tract of mice. • This protocol employs the IVIS spectrum for imaging.

2019-02-06·ACS Applied Materials & Interfaces

Targeted Interleukin-22 Gene Delivery in the Liver by Polymetformin and Penetratin-Based Hybrid Nanoparticles to Treat Nonalcoholic Fatty Liver Disease

Article

作者: Liu, Hongrui ; Wu, Zimei ; Hao, Qiang ; Tang, Shijie ; Fan, Jiajun ; Jin, Xin ; Mei, Xiaobin ; Zhang, Xuyao ; Luan, Jingyun ; Zai, Wenjing ; Ju, Dianwen ; Chen, Wei

Nonalcoholic fatty liver disease (NAFLD) is now a leading cause of chronic liver disease, and there is currently no available treatment strategy. Interleukin-22 (IL-22) has been recognized as a promising agent for alleviating NAFLD, but the efficacy of IL-22 is far from satisfactory because safe dose of IL-22 elicited limited improvement, whereas higher concentration might induce serious side effects and off-target toxicities. Thus, targeted and sustained expression of IL-22 in the liver is necessary. To meet the challenge, we elaborately developed a novel polymetformin carrier by conjugating biguanide to chitosan, termed chitosan-metformin (CM), which could exert advanced gene delivery efficiency and possess intrinsic therapeutic efficacy from metformin for NAFLD. CM accompanied with penetratin and DSPE-PEG2000 could self-assemble to form stable nanocomplexes with IL-22 gene via electrostatic interaction. This nanoparticle (CDPIA) exerted desirable particle size at ∼100 nm, fine morphology, and efficient cellular internalization. Furthermore, CDPIA also demonstrated a unique superiority in endosomal escape capacity and satisfactory biocompatibility as well as predominant liver accumulation. Most importantly, CDPIA distinctly alleviated hepatic steatosis, restored insulin sensitivity, and improved metabolic syndrome in high-fat-diet-fed mice model. This liver-targeted delivery of IL-22 activated STAT3/Erk1/2 and Nrf2/SOD1 signaling transductions as well as modulated lipid-metabolism-related gene expression. These findings altogether demonstrated that the polymetformin and penetratin-based hybrid nanoparticles could be exploited as a novel safe and efficient strategy for the improvement of NAFLD.

100 项与 IL-22(Synthekine) 相关的药物交易

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