组织蛋白酶 C 通过激活 Ca2+ 依赖性 PKC/p38MAPK/NF-κB 通路促进小胶质细胞 M1 极化并加重神经炎症。
Cathepsin C promotes microglia M1 polarization and aggravates neuroinflammation via activation of Ca-dependent PKC/p38MAPK/NF-κB pathway.
机构信息
Department of Anatomy, Dalian Medical University, West Section No.9, South Road, Lvshun, Dalian, 116044, Liaoning, China.
Liaoning Provincial Key Laboratory of Brain Diseases, Dalian Medical University, Dalian, 116044, Liaoning, China.
出版信息
J Neuroinflammation. 2019 Jan 16;16(1):10. doi: 10.1186/s12974-019-1398-3.
BACKGROUND
Microglia-derived lysosomal cathepsins are important inflammatory mediators to trigger signaling pathways in inflammation-related cascades. Our previous study showed that the expression of cathepsin C (CatC) in the brain is induced predominantly in activated microglia in neuroinflammation. Moreover, CatC can induce chemokine production in brain inflammatory processes. In vitro studies further confirmed that CatC is secreted extracellularly from LPS-treated microglia. However, the mechanisms of CatC affecting neuroinflammatory responses are not known yet.
METHODS
CatC over-expression (CatCOE) and knock-down (CatCKD) mice were treated with intraperitoneal and intracerebroventricular LPS injection. Morris water maze (MWM) test was used to assess the ability of learning and memory. Cytokine expression in vivo was detected by in situ hybridization, quantitative PCR, and ELISA. In vitro, microglia M1 polarization was determined by quantitative PCR. Intracellular Ca concentration was determined by flow cytometry, and the expression of NR2B, PKC, p38, IkBα, and p65 was determined by western blotting.
RESULTS
The LPS-treated CatCOE mice exhibited significantly increased escape latency compared with similarly treated wild-type or CatCKD mice. The highest levels of TNF-α, IL-1β, and other M1 markers (IL-6, CD86, CD16, and CD32) were found in the brain or serum of LPS-treated CatCOE mice, and the lowest levels were detected in CatCKD mice. Similar results were found in LPS-treated microglia derived from CatC differentially expressing mice or in CatC-treated microglia from wild-type mice. Furthermore, the expression of NR2B mRNA, phosphorylation of NR2B, Ca concentration, phosphorylation of PKC, p38, IκBα, and p65 were all increased in CatC-treated microglia, while addition of E-64 and MK-801 reversed the phosphorylation of above molecules.
CONCLUSION
The data suggest that CatC promotes microglia M1 polarization and aggravates neuroinflammation via activation of Ca-dependent PKC/p38MAPK/NF-κB pathway. CatC may be one of key molecular targets for alleviating and controlling neuroinflammation in neurological diseases.
背景
小胶质细胞来源的溶酶体组织蛋白酶是触发炎症相关级联中信号通路的重要炎症介质。我们之前的研究表明，在神经炎症中，大脑中组织蛋白酶 C（CatC）的表达主要在激活的小胶质细胞中诱导。此外，CatC 可以诱导脑炎症过程中的趋化因子产生。体外研究进一步证实，CatC 从 LPS 处理的小胶质细胞中外泌释放。然而，CatC 影响神经炎症反应的机制尚不清楚。
方法
CatC 过表达（CatCOE）和敲低（CatCKD）小鼠接受腹腔和侧脑室 LPS 注射。通过 Morris 水迷宫（MWM）测试评估学习和记忆能力。通过原位杂交、定量 PCR 和 ELISA 检测体内细胞因子表达。体外通过定量 PCR 测定小胶质细胞 M1 极化。通过流式细胞术测定细胞内 Ca 浓度，通过 Western blot 测定 NR2B、PKC、p38、IkBα 和 p65 的表达。
结果
与同样接受 LPS 处理的野生型或 CatCKD 小鼠相比，LPS 处理的 CatCOE 小鼠的逃避潜伏期明显延长。在 LPS 处理的 CatCOE 小鼠的大脑或血清中发现 TNF-α、IL-1β 和其他 M1 标志物（IL-6、CD86、CD16 和 CD32）水平最高，在 CatCKD 小鼠中发现水平最低。在 CatC 表达不同的小鼠来源的 LPS 处理的小胶质细胞或来自野生型小鼠的 CatC 处理的小胶质细胞中也发现了类似的结果。此外，CatC 处理的小胶质细胞中 NR2B mRNA 的表达、NR2B 的磷酸化、Ca 浓度、PKC、p38、IκBα 和 p65 的磷酸化均增加，而添加 E-64 和 MK-801 则逆转了上述分子的磷酸化。
结论
数据表明，CatC 通过激活 Ca 依赖性 PKC/p38MAPK/NF-κB 通路促进小胶质细胞 M1 极化并加重神经炎症。CatC 可能是缓解和控制神经退行性疾病中神经炎症的关键分子靶标之一。
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