Bredehöft Janne, Dolga Amalia M, Honrath Birgit, Wache Sybille, Mazurek Sybille, Culmsee Carsten, Schoemaker Regien G, Gerstberger Rüdiger, Roth Joachim, Rummel Christoph
Institute of Veterinary Physiology and Biochemistry, Justus Liebig University Giessen, Giessen, Germany.
Department of Molecular Pharmacology, Groningen Research Institute of Pharmacy, University of Groningen, Groningen, Netherlands.
J Inflamm Res. 2022 Jan 23;15:509-531. doi: 10.2147/JIR.S338812. eCollection 2022.
Previously, we have shown that CyPPA (cyclohexyl-[2-(3,5-dimethyl-pyrazol-1-yl)-6-methyl-pyrimidin-4-yl]-amine), a pharmacological small-conductance calcium-activated potassium (SK)-channel positive modulator, antagonizes lipopolysaccharide (LPS)-induced cytokine expression in microglial cells. Here, we aimed to test its therapeutic potential for brain-controlled sickness symptoms, brain inflammatory response during LPS-induced systemic inflammation, and peripheral metabolic pathways in mice.
Mice were pretreated with CyPPA (15 mg/kg IP) 24 hours before and simultaneously with LPS stimulation (2.5 mg/kg IP), and the sickness response was recorded by a telemetric system for 24 hours. A second cohort of mice were euthanized 2 hours after CyPPA or solvent treatment to assess underlying CyPPA-induced mechanisms. Brain, blood, and liver samples were analyzed for inflammatory mediators or nucleotide concentrations using immunohistochemistry, real-time PCR and Western blot, or HPLC. Moreover, we investigated CyPPA-induced changes of UCP1 expression in brown adipose tissue (BAT)-explant cultures.
CyPPA treatment did not affect LPS-induced fever, anorexia, adipsia, or expression profiles of inflammatory mediators in the hypothalamus or plasma or microglial reactivity to LPS (CD11b staining and CD68 mRNA expression). However, CyPPA alone induced a rise in core body temperature linked to heat production via altered metabolic pathways like reduced levels of adenosine, increased protein content, and increased UCP1 expression in BAT-explant cultures, but no alteration in ATP/ADP concentrations in the liver. CyPPA treatment was accompanied by altered pathways, including NFκB signaling, in the hypothalamus and cortex, while circulating cytokines remained unaltered.
Overall, while CyPPA has promise as a treatment strategy, in particular according to results from in vitro experiments, we did not reveal anti-inflammatory effects during severe LPS-induced systemic inflammation. Interestingly, we found that CyPPA alters metabolic pathways inducing short hyperthermia, most likely due to increased energy turnover in the liver and heat production in BAT.
此前，我们已经证明，环己基-[2-(3,5-二甲基-吡唑-1-基)-6-甲基-嘧啶-4-基]-胺（CyPPA），一种药理学上的小电导钙激活钾（SK）通道正性调节剂，可拮抗脂多糖（LPS）诱导的小胶质细胞中细胞因子的表达。在此，我们旨在测试其对小鼠脑控疾病症状、LPS诱导的全身炎症期间的脑炎症反应以及外周代谢途径的治疗潜力。
在LPS刺激（腹腔注射2.5mg/kg）前24小时及同时，用CyPPA（腹腔注射15mg/kg）对小鼠进行预处理，并用遥测系统记录24小时的疾病反应。在CyPPA或溶剂处理2小时后，对另一组小鼠实施安乐死，以评估CyPPA诱导的潜在机制。使用免疫组织化学、实时PCR、蛋白质印迹或高效液相色谱法分析脑、血液和肝脏样本中的炎症介质或核苷酸浓度。此外，我们研究了CyPPA诱导的棕色脂肪组织（BAT）外植体培养物中解偶联蛋白1（UCP1）表达的变化。
CyPPA处理不影响LPS诱导的发热、厌食、拒食，或下丘脑、血浆中炎症介质的表达谱，也不影响小胶质细胞对LPS的反应性（CD11b染色和CD68 mRNA表达）。然而，单独使用CyPPA会导致核心体温升高，这与通过改变代谢途径产生热量有关，如腺苷水平降低、蛋白质含量增加以及BAT外植体培养物中UCP1表达增加，但肝脏中的ATP/ADP浓度没有改变。CyPPA处理伴随着下丘脑和皮质中包括核因子κB信号通路在内的途径改变，而循环细胞因子保持不变。
总体而言，虽然CyPPA有望成为一种治疗策略，特别是根据体外实验结果，但我们并未发现其在严重LPS诱导的全身炎症期间具有抗炎作用。有趣的是，我们发现CyPPA改变代谢途径，导致短暂体温过高，这很可能是由于肝脏能量转换增加和BAT产热增加所致。
