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Title: Protective effects of the salt-induced kinase inhibitor HG-9-91-01 on sepsis-associated cognitive dysfunction in mice and the underlying mechanisms.
Author: Wang X, Wang S, Cui Y.
Journal: Zhong Nan Da Xue Xue Bao Yi Xue Ban; 2023 Dec 28; 48(12):1793-1803. PubMed ID: 38448372.
Abstract:
OBJECTIVES: Sepsis-associated cognitive dysfunction is a common complication in patients with sepsis and lack of effective treatment. Its pathological mechanisms remain unclear. Salt-induced kinase (SIK) is an important molecule in the regulation of metabolism, immunity, and inflammatory response. It is associated with the development of many neurological diseases. This study aims to investigate the expression of SIK in the hippocampus of septic mice, and to evaluate the role and mechanism of the SIK inhibitor HG-9-91-01 in sepsis-associated cognitive dysfunction. METHODS: Firstly, C57BL/6 mice were randomly divided into a control group (Con group) and a sepsis model group [lipopolysaccharide (LPS) group]. The model group was injected intraperitoneally with LPS at a dose of 8 mg/kg and the Con group was injected with an equal volume of normal saline. Hippocampal tissues were harvested at 1, 3, and 6 days after injection and the expressions of SIK1, SIK2, and SIK3 were detected by real-time fluorescence quantitative PCR (qPCR) and Western blotting. Secondly, C57BL/6 mice were randomly divided into a Con group, a LPS group, and a SIK inhibitor group (HG group). The LPS and HG groups were injected with LPS to establish a sepsis model; in the HG group, HG-9-91-01 (10 mg/kg) was injected intraperitoneally at 3-6 days after LPS injection, and the LPS group was injected with the same volume of vehicle. Cognitive function was assessed at 7-11 days after LPS injection using the Morris water maze (MWM). Hippocampal tissues were harvested after the behavioral tests, and the mRNA levels of inflammatory factors and microglial markers were assessed by qPCR. The protein levels of inducible nitric oxide synthase (iNOS), CD68, ionized calcium binding adaptor molecule 1 (Iba-1), N-methyl-D-aspartate (NMDA) receptor (NR) subunit, cAMP response element-binding protein (CREB)-regulated transcription coactivator 1 (CRTC1), and insulin-like growth factor 1 (IGF-1) were detected by Western blotting. Immunohistochemistry (IHC) was used to detect the expression of Iba-1 positive cells in the CA1, CA3 and dentate gyrus (DG) of the hippocampus, followed by Sholl analysis. RESULTS: Compared with the Con group, the mRNA and protein levels of SIK1, SIK2, and SIK3 in the hippocampus were increased in the LPS group (all P<0.05). Compared with the Con group, mice in the LPS group had a significantly longer escape latency, a lower percentage of target quadrant dwell time and a reduced locomotor speed (all P<0.05); the HG group had a decreased escape latency and an increased percentage of time spent in the target quadrant in comparison with the LPS group (both P<0.05). The mRNA levels of inflammatory factors [tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), interleukin-6 (IL-6)], and the M1-type microglial markers iNOS and CD68 in the hippocampus of the LPS group were increased in comparison with the Con group, while the M2-type microglial markers CD206 and arginase-1 (Arg-1) were decreased. Compared with the LPS group, the mRNA levels of TNF-α, IL-1β, IL-6, and iNOS were downregulated, while the levels of CD206 and Arg-1 were upregulated in the HG group (all P<0.05). The protein levels of iNOS, CD68, and Iba-1 in the hippocampus of the LPS group were increased in comparison with the Con group, but they were downregulated in the HG group in comparison with the LPS group (all P<0.05). The number of Iba-1 positive cells in CA1, CA3, and DG of the hippocampus was increased in the LPS group in comparison with the Con group, but they were decreased in the HG group in comparison with the LPS group (all P<0.05). Sholl analysis showed that the number of intersections at all radii between 8-38 µm from the microglial soma was decreased in the LPS group in comparison with the Con group (all P<0.05). Compared with the LPS group, the number of intersections at all radii between 14-20 µm was significantly increased in the HG group (all P<0.05). The protein levels of NR subunit NR1, NR2A, NR2B, and IGF-1 were downregulated in the hippocampus of the LPS group in comparison with the Con group, while the expression of phosphorylated CRTC1 (p-CRTC1) was increased. Compared with the LPS group, the levels of NR1, NR2A, NR2B, and IGF-1 were upregulated, while p-CRTC1 was downregulated in the HG group (all P<0.05). CONCLUSIONS: SIK expression is upregulated in the hippocampus of septic mice. The SIK inhibitor HG-9-91-01 ameliorates sepsis-associated cognitive dysfunction in mice, and the mechanism may involve in the activation of the CRTC1/IGF-1 pathway, inhibition of neuroinflammation, and enhancement of synaptic plasticity. 目的: 脓毒症相关认知功能障碍是脓毒症患者常见的并发症，目前病理机制不明，缺乏有效的防治手段。盐诱导激酶(salt-induced kinase，SIK)是调节代谢、免疫、炎症反应等的重要分子，与多种神经系统疾病的发生和发展相关。本研究旨在探讨SIK在脓毒症小鼠海马中的表达，以及SIK抑制剂HG-9-91-01在脓毒症相关认知功能障碍中的作用及其机制。方法: 首先，将C57BL/6小鼠随机分为对照组(Con组)和脓毒症模型组[脂多糖(lipopolysaccharide，LPS)组]。LPS组小鼠以8 mg/kg的剂量腹腔注射LPS，Con组小鼠予注射等体积生理盐水。在注射后1、3、6 d取小鼠海马组织，分别采用实时荧光定量聚合酶链反应(quantitative PCR，qPCR)和蛋白质印迹法检测SIK1、SIK2、SIK3的mRNA和蛋白质的表达水平。然后，将小鼠随机分为Con组、LPS组、SIK抑制剂组(HG组)。LPS组和HG组注射LPS构建脓毒症模型，HG组在注射LPS后的第3~6天以10 mg/kg的剂量腹腔注射HG-9-91-01，LPS组注射等体积溶媒。在注射LPS后的第7~11天进行Morris水迷宫实验(Morris water maze，MWM)以评估3组小鼠的认知功能。行为学检测后取3组小鼠的海马组织，采用qPCR检测炎症因子和小胶质细胞标志分子的mRNA表达水平，蛋白质印迹法检测诱导型一氧化氮合酶(inducible nitric oxide synthase，iNOS)、CD68、离子钙结合衔接分子1(ionized calcium binding adaptor molecule 1，Iba-1)、N-甲基-D-天冬氨酸(N-methyl-D-aspartate，NMDA)受体(NMDA receptor，NR)亚型、cAMP反应元件结合蛋白(cAMP response element-binding protein，CREB)调控的转录共激活因子1(CREB-regulated transcription coactivator 1，CRTC1)、胰岛素样生长因子1(insulin-like growth factor 1，IGF-1)的蛋白质表达水平，免疫组织化学(immunohistochemistry，IHC)检测海马CA1区、CA3区、齿状回(dentate gyrus，DG)Iba-1阳性细胞的表达，并进行Sholl分析。结果: 与Con组比较，LPS组小鼠海马组织SIK1、SIK2、SIK3的mRNA和蛋白质表达水平均上调(均P<0.05)。与Con组比较，LPS组小鼠的逃避潜伏期明显延长，目标象限停留时间百分比降低，运动速度下降(均P<0.05)；与LPS组相比，HG组小鼠的逃避潜伏期明显缩短，目标象限停留时间百分比增加(均P<0.05)。与Con组比较，LPS组海马组织炎症因子[肿瘤坏死因子α(tumor necrosis factor-α，TNF-α)、白细胞介素1β(interleukin-1β，IL-1β)、白细胞介素6(interleukin-6，IL-6)]和I型小胶质细胞标志分子iNOS、CD68的mRNA表达均上调，而II型小胶质细胞标志分子CD206和精氨酸酶-1(arginase-1，Arg-1)的mRNA表达均下调；与LPS组比较，HG组TNF-α、IL-1β、IL-6、iNOS的mRNA表达均下调，而CD206和Arg-1的mRNA表达均上调(均P<0.05)。与Con组比较，LPS组海马组织iNOS、CD68、Iba-1的蛋白质表达均上调；与LPS组比较，HG组iNOS、CD68、Iba-1的蛋白质表达均下调(均P<0.05)。与Con组比较，LPS组海马组织CA1、CA3、DG区Iba-1阳性细胞数量均增加；与LPS组比较，HG组CA1、CA3、DG区Iba-1阳性细胞数量均减少(均P<0.05)。Sholl分析结果显示：距离小胶质细胞胞体8~38 μm半径范围内，LPS组小胶质细胞突起与同心圆的交点较Con组明显减少(均P<0.05)；在距离小胶质细胞胞体14~20 μm半径范围内，HG组小胶质细胞突起与同心圆的交点较LPS组明显增多(均P<0.05)。与Con组比较，LPS组小鼠海马突触相关蛋白NR亚型NR1、NR2A、NR2B及IGF-1的蛋白质表达下调，磷酸化的CRTC1(phosphorylated CRTC1，p-CRTC1)的蛋白质表达上调；与LPS组比较，HG组小鼠海马突触相关蛋白NR1、NR2A、NR2B及IGF-1的蛋白质表达上调，p-CRTC1的蛋白质表达下调(均P<0.05)。结论: 脓毒症小鼠海马组织SIK表达上调，SIK抑制剂HG-9-91-01可改善小鼠脓毒症相关认知功能障碍，其机制可能与激活CRTC1/IGF-1通路，抑制神经炎症，增强突触可塑性有关。. OBJECTIVE: Sepsis-associated cognitive dysfunction is a common complication in patients with sepsis and lack of effective treatment. Its pathological mechanisms remain unclear. Salt-induced kinase (SIK) is an important molecule in the regulation of metabolism, immunity, and inflammatory response. It is associated with the development of many neurological diseases. This study aims to investigate the expression of SIK in the hippocampus of septic mice, and to evaluate the role and mechanism of the SIK inhibitor HG-9-91-01 in sepsis-associated cognitive dysfunction. METHODS: Firstly, C57BL/6 mice were randomly divided into a control group (Con group) and a sepsis model group [lipopolysaccharide (LPS) group]. The model group was injected intraperitoneally with LPS at a dose of 8 mg/kg and the Con group was injected with an equal volume of normal saline. Hippocampal tissues were harvested at 1, 3, and 6 days after injection and the expressions of SIK1, SIK2, and SIK3 were detected by real-time fluorescence quantitative PCR (qPCR) and Western blotting. Secondly, C57BL/6 mice were randomly divided into a Con group, a LPS group, and a SIK inhibitor group (HG group). The LPS and HG groups were injected with LPS to establish a sepsis model; in the HG group, HG-9-91-01 (10 mg/kg) was injected intraperitoneally at 3-6 days after LPS injection, and the LPS group was injected with the same volume of vehicle. Cognitive function was assessed at 7-11 days after LPS injection using the Morris water maze (MWM). Hippocampal tissues were harvested after the behavioral tests, and the mRNA levels of inflammatory factors and microglial markers were assessed by qPCR. The protein levels of inducible nitric oxide synthase (iNOS), CD68, ionized calcium binding adaptor molecule 1 (Iba-1), N-methyl-D-aspartate (NMDA) receptor (NR) subunit, cAMP response element-binding protein (CREB)-regulated transcription coactivator 1 (CRTC1), and insulin-like growth factor 1 (IGF-1) were detected by Western blotting. Immunohistochemistry (IHC) was used to detect the expression of Iba-1 positive cells in the CA1, CA3 and dentate gyrus (DG) of the hippocampus, followed by Sholl analysis. RESULTS: Compared with the Con group, the mRNA and protein levels of SIK1, SIK2, and SIK3 in the hippocampus were increased in the LPS group (all P<0.05). Compared with the Con group, mice in the LPS group had a significantly longer escape latency, a lower percentage of target quadrant dwell time and a reduced locomotor speed (all P<0.05); the HG group had a decreased escape latency and an increased percentage of time spent in the target quadrant in comparison with the LPS group (both P<0.05). The mRNA levels of inflammatory factors [tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), interleukin-6 (IL-6)], and the M1-type microglial markers iNOS and CD68 in the hippocampus of the LPS group were increased in comparison with the Con group, while the M2-type microglial markers CD206 and arginase-1 (Arg-1) were decreased. Compared with the LPS group, the mRNA levels of TNF-α, IL-1β, IL-6, and iNOS were downregulated, while the levels of CD206 and Arg-1 were upregulated in the HG group (all P<0.05). The protein levels of iNOS, CD68, and Iba-1 in the hippocampus of the LPS group were increased in comparison with the Con group, but they were downregulated in the HG group in comparison with the LPS group (all P<0.05). The number of Iba-1 positive cells in CA1, CA3, and DG of the hippocampus was increased in the LPS group in comparison with the Con group, but they were decreased in the HG group in comparison with the LPS group (all P<0.05). Sholl analysis showed that the number of intersections at all radii between 8-38 µm from the microglial soma was decreased in the LPS group in comparison with the Con group (all P<0.05). Compared with the LPS group, the number of intersections at all radii between 14-20 µm was significantly increased in the HG group (all P<0.05). The protein levels of NR subunit NR1, NR2A, NR2B, and IGF-1 were downregulated in the hippocampus of the LPS group in comparison with the Con group, while the expression of phosphorylated CRTC1 (p-CRTC1) was increased. Compared with the LPS group, the levels of NR1, NR2A, NR2B, and IGF-1 were upregulated, while p-CRTC1 was downregulated in the HG group (all P<0.05). CONCLUSION: SIK expression is upregulated in the hippocampus of septic mice. The SIK inhibitor HG-9-91-01 ameliorates sepsis-associated cognitive dysfunction in mice, and the mechanism may involve in the activation of the CRTC1/IGF-1 pathway, inhibition of neuroinflammation, and enhancement of synaptic plasticity.

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