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Journal of Central South University Medical Sciences logoLink to Journal of Central South University Medical Sciences
. 2023 Dec 28;48(12):1793–1803. [Article in Chinese] doi: 10.11817/j.issn.1672-7347.2023.230208

盐诱导激酶抑制剂HG-9-91-01对小鼠脓毒症相关认知功能障碍的保护作用及其机制

Protective effects of the salt-induced kinase inhibitor HG-9-91-01 on sepsis-associated cognitive dysfunction in mice and the underlying mechanisms

WANG Xueqin 1,2,2, WANG Shuang 3, CUI Yanhui 1,✉
Editor: 郭 征
PMCID: PMC10930753  PMID: 38448372

Abstract

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.

Keywords: sepsis, cognitive dysfunction, salt-inducible kinase, cAMP response element-binding protein-regulated transcription coactivator 1, insulin-like growth factor 1, HG-9-91-01, neuroinflammation, synaptic plasticity

脓毒症是宿主对感染的反应失调所导致的危及生命的器官功能障碍，是一个重要的全球健康问题[1]。脓毒症相关认知功能障碍是脓毒症常见的后遗症，约有46%的脓毒症幸存患者出院后5年内出现认知功能损伤[2]，严重影响患者生活质量，给家庭和社会带来承重负担[3]。然而，脓毒症相关认知功能障碍的核心病理机制尚不完全清楚，亦缺乏有效的治疗靶点和方案。

盐诱导激酶(salt-induced kinase，SIK)属于单磷酸腺苷激活的蛋白激酶亚家族，包含3个亚型(SIK1、SIK2、SIK3)，在机体内广泛表达[4-5]。SIK是调节代谢、免疫、炎症反应等生理病理过程的重要分子[4-5]；而上述过程与脓毒症相关认知损伤的致病机制密切相关[6-7]。既往研究显示，SIK参与调节神经元的形态[8]和功能[9]，影响神经元的存活[10-11]，在多种神经系统疾病的进展中发挥作用[4-5, 11]。此外，SIK经典的作用机制之一是磷酸化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)，导致其滞留于细胞质，调控CREB下游的一系列基因表达[4-5]，从而参与突触可塑性、炎症、认知功能等生理病理过程[5, 12-13]。以上证据提示，SIK可能参与脓毒症相关认知功能障碍发生和发展，但目前尚未见研究报道。

研究显示，选择性SIK抑制剂HG-9-91-01可有效减轻炎症反应[14-15]，促进抗炎细胞因子的分泌[15-16]，在小鼠炎症性疾病中发挥保护作用[14, 16]。HG-9-91-01还可通过调节CRTC1-CREB-脑源性神经营养因子途径发挥抗抑郁效应[17]。然而，HG-9-91-01在脓毒症相关认知功能障碍这一免疫反应失调引发的脑损伤中的作用尚不清楚。

胰岛素样生长因子1(insulin-like growth factor 1，IGF-1)是CRTC1/CREB信号通路下游分子[18]，是重要的神经营养因子，在神经元分化和迁移、突触可塑性、神经胶质细胞发育和神经炎症等方面发挥调节作用[19-20]。脓毒症患者血清IGF-1水平明显降低[21]，低IGF-1水平预示患者可能病情严重、预后不良[22]。IGF-1治疗可提高脓毒症小鼠的存活率[21]；减少脓毒症大鼠海马组织细胞的凋亡，改善认知功能障碍[23]。基于此，本研究拟明确SIK在脓毒症小鼠海马的表达，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，并基于CRTC1/IGF-1通路探讨可能的分子机制。

1. 材料与方法

1.1. 主要试剂

脂多糖(lipopolysaccharide，LPS)(货号L2880)为美国Sigma公司产品；HG-9-91-01(货号T4599)购自上海陶术生物科技有限公司；TRIZol(货号15596026)为美国Thermo Fisher科技公司产品；反转录试剂盒(货号R223-01)和定量PCR检测试剂盒(货号Q711-02)购自南京诺唯赞生物科技股份有限公司；组织蛋白抽提试剂盒(货号CW0891M)和BCA蛋白质定量试剂盒(货号CW0014S)购自北京康为世纪生物科技有限公司。

蛋白质印迹法用SIK1一抗(货号51045-1-AP， 1꞉1 000)、诱导型一氧化氮合酶(inducible nitric oxide synthase，iNOS)一抗(货号18985-1-AP，1꞉500)、Tubulin一抗(货号66031-1-AP，1꞉5 000)为美国Proteintech公司产品；SIK2一抗(货号6919，1꞉1 000)为美国CST公司产品，SIK3一抗(货号AF9172，1꞉1 000)、CRTC1一抗(货号DF2415，1꞉500)、磷酸化的CRTC1(phosphorylated CRTC1，p-CRTC1)一抗(货号AF8518，1꞉500)为美国Affinity公司产品；CD68一抗(货号ab125212，1꞉500)、离子钙结合衔接分子1(ionized calcium binding adaptor molecule 1，Iba-1)一抗(货号ab5076，1꞉500)、N-甲基-D-天冬氨酸(N-methyl-D-aspartate，NMDA)受体(NMDA receptor，NR)1一抗(货号ab134308，1꞉800)、NR2A一抗(货号ab174636，1꞉800)、NR2B一抗(货号ab93610，1꞉1 000)为英国Abcam公司产品；IGF-1一抗(货号A11985，1꞉500)为美国Abclonal公司产品；山羊抗兔二抗(货号925-32211，1꞉20 000)、山羊抗鼠二抗(货号925-68070，1꞉20 000)和驴抗山羊二抗(货号925-32214，1꞉20 000)为美国LI-COR公司产品。

免疫组织化学(immunohistochemistry，IHC)用VECTASTAIN ABC-HRP试剂盒(货号PK-4001)为美国Vector Laboratories公司产品；Iba-1一抗(货号019-19741，1꞉1 000)购自日本富士胶片和光纯药株式会社。

1.2. 动物与分组

8~9周龄的SPF级雄性C57BL/6小鼠购于湖南斯莱克景达实验动物有限公司。实验开始前，小鼠适应性饲养1周。饲养条件为：温度(23±2) ℃，相对湿度(50±5)%，12 h明/暗周期。该研究获得长沙医学院医学伦理委员会批准。

首先，采用单次腹腔注射LPS建立脓毒症模型，检测小鼠海马组织中SIK的表达情况。将小鼠随机分为2组：对照组(Con组，n=7)、LPS组(n=21)。LPS组小鼠以8 mg/kg[24]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LPS后1、3、6 d取小鼠海马组织，分别通过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，研究SIK抑制剂HG-9-91-01对小鼠脓毒症相关认知损伤的影响及其机制。将小鼠随机分为3组：对照组(Con组，n=10)、LPS组(n=10)、HG组 (n=10)。HG组在注射LPS后的第3~6天(每天1次，共4次)以10 mg/kg的剂量腹腔注射HG-9-91-01，LPS组注射等体积的溶媒。末次药物注射后2 h开始旷场实验(open field test，OFT)(图1)。行为学检测后处死各组小鼠，取其海马组织，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iNOS、CD68、Iba-1、NR亚型、CRTC1、IGF-1的蛋白质表达水平，IHC检测海马CA1区、CA3区、齿状回(dentate gyrus，DG)Iba-1阳性细胞的表达。

图1.

图1

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给药和行为学检测的流程

Figure 1 Schedule of drug administration and behavioral tests

NS: Normal saline; LPS: Lipopolysaccharide; SIK: Salt-induced kinase; OFT: Open field test; MWM: Morris water maze.

1.3. 方法

1.3.1. OFT

将小鼠在旷场中央释放，让其自由探索5 min，采用Smart 3.0行为学视频记录与分析系统记录并分析小鼠的活动情况，将小鼠在旷场中的穿行总距离和运动速度作为评估自主活动度的指标。

1.3.2. Morris水迷宫实验

在注射LPS后的第7~11天进行Morris水迷宫实验(Morris water maze，MWM)以评估小鼠认知功能。学习期：将平台沉没于水下1 cm，把小鼠从任意象限面向池壁轻轻放入水中，让其自由寻找平台60 s，若小鼠成功找到水下平台，则让其在平台上停留20 s后取出；若60 s后小鼠仍未找到平台，则将其引导至平台，并停留20 s后取出。在注射LPS后的第7~10天，每天每只小鼠进行4次实验(即分别从4个象限入水)，每次实验间隔15~20 min。检测期：在注射LPS后的第11天撤去平台，将小鼠从距离原平台最远的位置放入水中。通过Smart 3.0行为学视频记录与分析系统追踪、记录小鼠60 s内到达平台区域(学习期)/原平台区域(检测期)的时间(逃避潜伏期)，以及检测期在目标象限即原平台所在象限停留时间百分比和运动速度。

1.3.3. qPCR

采用TRIzol法提取小鼠海马组织RNA，反转录试剂盒合成cDNA，定量PCR检测试剂盒检测SIK1、SIK2、SIK3、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)]和小胶质细胞标志分子[iNOS、CD68、CD206、精氨酸酶-1(arginase-1，Arg-1)]基因的表达水平。以GAPDH为内参，采用2-ΔΔCt方法计算目标基因的相对表达量。引物序列见表1。

表1.

定量聚合酶链反应引物序列

Table 1 Sequences of primers for quantitative PCR

Gene name Primer sequence (5'-3')
Forward Reverse
SIK1 GGCTTTTACGACGTGGAACG ATTGCAACCTGCGTTTTGGT
SIK2 ACGTCCCTTACACAAGGAATTG TTGGAGCTGAGGAGCAGTTG
SIK3 GTCCAAAAGGCACACACTGG TCCTTGTAGGTGGAGCGATG
TNF-α CCACCACGCTCTTCTGTCTA GAGGCCATTTGGGAACTTCTCATC
IL-1β GAAATGCCACCTTTTGACAGTG TGGATGCTCTCATCAGGACAG
IL-6 CTCTGGCTTTGTCTTTCTTGTTATCTTT AGTTGTGCAATGGCAATTCTGA
Arg-1 CTCCAAGCCAAAGTCCTTAGAG AGGAGCTGTCATTAGGGACATC
CD206 TTCAGCTATTGGACGCGAGG GAATCTGACACCCAGCGGAA
iNOS CAGATCGAGCCCTGGAAGAC CTGGTCCATGCAGACAACCT
CD68 TGTCTGATCTTGCTAGGACCG GAGAGTAACGGCCTTTTTGTGA
GAPDH CATGGCCTTCCGTGTTCCTA TACTTGGCAGGTTTCTCCAGG
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SIK: Salt-induced kinase; TNF-α: Tumor necrosis factor-α; IL-1β: Interleukin-1β; IL-6: Interleukin-6; Arg-1: Arginase-1; iNOS: Inducible nitric oxide synthase; GAPDH: Glyceraldehyde-3-phosphate dehydrogenase.

1.3.4. 蛋白质印迹法

采用组织蛋白抽提试剂盒提取小鼠海马组织总蛋白质，通过BCA蛋白质定量试剂盒测定蛋白质的浓度。蛋白变性后经SDS聚丙烯酰胺凝胶电泳(SDS polyacrylamide gel electrophoresis，SDS-PAGE)分离，转移至硝酸纤维素(nitrocellulose，NC)膜。用5%的脱脂牛奶在室温下封闭1 h后加入一抗，在4 ℃孵育过夜，次日用TBST洗涤后加入二抗，在室温下孵育1 h；洗涤后显影。用ImageJ软件分析蛋白质(SIK1、SIK2、SIK3、iNOS、CD68、Iba-1、NR1、NR2A、NR2B、CRTC1、p-CRTC1、IGF-1)的相对表达水平。

1.3.5. IHC

小鼠麻醉后自心尖进针灌注生理盐水，随后用4%的多聚甲醛灌注固定，获取脑组织。后固定过夜后经梯度蔗糖溶液脱水，OCT胶包埋。作冠状冰冻切片，收集海马切片进行IHC检测。用PBS漂洗切片后，加入3%的过氧化氢溶液处理10 min；用PBS漂洗切片后，加入5%的牛血清白蛋白在室温下封闭1 h，然后加入Iba-1一抗于4 ℃孵育过夜；次日用PBS漂洗后加入二抗(1꞉200)，在室温下孵育2 h；用PBS洗涤后加入ABC工作液(1꞉1꞉200)，在室温下孵育1 h；用PBS洗涤后，行DAB显色、贴片，随后经梯度酒精和二甲苯脱水透明，用中性树胶封片。用显微成像系统采图并分析Iba-1阳性细胞数量，通过Fiji软件将图像二值化，进行Sholl分析。

1.4. 统计学处理

采用Graphpad Prism(Version 8.3)软件进行数据分析，数据以均数±标准误表示，MWM学习期逃避潜伏期和小胶质细胞Sholl分析采用双因素方差分析合并事后Tukey检验，其他数据采用单因素方差分析合并事后Tukey检验。P<0.05为差异有统计学意义。

2. 结 果

2.1. 脓毒症小鼠海马组织中 SIK 的mRNA和蛋白质 表达均上调

与Con组比较，LPS组小鼠在注射LPS 3 d后，SIK1、SIK2、SIK3的mRNA表达均明显上调(均P<0.05，图2A)；在注射LPS 1、3、6 d后，SIK1的蛋白质表达均上调(均P<0.05，图1B)；在注射LPS 3、6 d后，SIK2和SIK3的蛋白质表达均上调(均P<0.05，图2B)。

图2.

图2

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小鼠海马组织中SIK的表达情况

Figure 2 Expression of SIK in the hippocampus of mice

A: Expression level of SIK mRNA (n=7); B: Expression level of SIK protein (n=4). Data are expressed as the mean±standard error of mean. *P<0.05, **P<0.01, ***P<0.001 vs the Con group. Con: Control; LPS: Lipopolysaccharide; SIK: Salt-induced kinase.

2.2. HG-9-91-01对小鼠脓毒症相关认知功能障碍的影响

3组小鼠的OFT代表性轨迹见图3A，各组小鼠在旷场中的穿行总距离(图3B)和运动速度(图3C)差异无统计学意义(均P>0.05)。MWM学习期(LPS注射后的第8~10天)LPS组小鼠的逃避潜伏期均明显长于Con组(均P<0.05，图3D)；学习期(LPS注射后的第9和10天)，HG组小鼠的逃避潜伏期相较于LPS组明显缩短(均P<0.05，图3D)。测试期(LPS注射后的第11天)，与Con组比较，LPS组小鼠的逃避潜伏期明显延长(P<0.05，图3E)，目标象限停留时间百分比降低 (P<0.05，图3F)，运动速度下降(P<0.05，图3G)；与LPS组相比，HG组小鼠的逃避潜伏期明显缩短(P<0.05，图3E)，目标象限停留时间百分比增加(P<0.05，图3F)。

图3.

图3

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HG-9-91-01对小鼠脓毒症相关认知功能障碍的影响

Figure 3 Effects of HG-9-91-01 on sepsis-associated cognitive dysfunction in mice

A: Representative diagrams of the motion trajectory in the OFT of the 3 groups; B: Total distance of the 3 groups in the OFT; C: Speed of the 3 groups in the OFT; D: Escape latency of the 3 groups during learning period of the MWM test; E: Escape latency of the 3 groups during detection period of the MWM test; F: Percentage of target quadrant dwell time of the 3 groups during detection period of the MWM test; G: Speed of the 3 groups during detection period of the MWM test. Data are expressed as the mean±standard error of mean (n=10). **P<0.01, ***P<0.001 vs the Con group; †P<0.05, ††P<0.01 vs the LPS group. OFT: Open field test; MWM: Morris water maze; Con: Control; LPS: Lipopolysaccharide; HG: HG-9-91-01.

2.3. HG-9-91-01对小鼠海马组织神经炎症的影响

qPCR结果显示：与Con组比较，LPS组海马组织炎症因子TNF-α、IL-1β、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Arg-1的mRNA表达均下调(均P<0.05，图4A)；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，图4A)。

图4.

图4

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HG-9-91-01对小鼠海马组织神经炎症的影响

Figure 4 Effects of HG-9-91-01 on neuroinflammation in the hippocampus of mice

A: mRNA levels of inflammatory factors and microglial markers in each group (n=7). *P<0.05, **P<0.01, ***P<0.001. B: Protein levels of microglial markers in each group (n=4). *P<0.05, **P<0.01, ***P<0.001. C: Number of hippocampal Iba-1 positive cells in each group of mice (n=3). Scale bar=50 μm. **P<0.01. D: Sholl analysis of microglia (n=3). *P<0.05, **P<0.01, ***P<0.001 vs the LPS group; †P<0.05, ††P<0.01 vs the HG group. Data are expressed as the mean±standard error of mean. Con: Control; LPS: Lipopolysaccharide; HG: HG-9-91-01; TNF-α: Tumor necrosis factor-α; IL-1β: Interleukin-1β; IL-6: Interleukin-6; iNOS: Inducible nitric oxide synthase; Arg-1: Arginase-1; Iba-1: Ionized calcium binding adaptor molecule 1; CA: Cornu ammonis; DG: Dentate gyrus.

蛋白质印迹结果显示：与Con组比较，LPS组海马组织iNOS、CD68、Iba-1的蛋白质表达均上调(均P<0.05，图4B)；与LPS组比较，HG组iNOS、CD68、Iba-1的蛋白质表达均下调(均P<0.05，图4B)。

IHC结果显示：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(均P<0.05，图4C)；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，图4C)。

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，图4D)；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，图4D)。

2.4. HG-9-91-01对小鼠海马组织突触相关蛋白的影响

蛋白质印迹结果显示：与Con组比较，LPS组小鼠海马突触相关蛋白NR1、NR2A、NR2B的蛋白质表达均下调(均P<0.05)；与LPS组比较，HG组小鼠海马突触相关蛋白NR1、NR2A、NR2B的蛋白质表达均下调(均P<0.05，图5)。

图5.

图5

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HG-9-91-01对小鼠海马组织突触相关蛋白NMDA受体表达的影响

Figure 5 Effects of HG-9-91-01 on synaptic-related protein NR in the hippocampus of mice

Data are expressed as the mean±standard error of mean (n=4). **P<0.01, ***P<0.001 vs the Con group; †P<0.05, ††P<0.01 vs the LPS group. Con: Control; LPS: Lipopolysaccharide; HG: HG-9-91-01; NMDA: N-methyl-D-aspartate; NR: NMDA receptor.

2.5. HG-9-91-01对CRTC1/IGF-1通路的影响

蛋白质印迹结果显示：与Con组比较，LPS组小鼠海马p-CRTC1的蛋白质表达上调，而IGF-1的蛋白质表达下调(均P<0.05)；与LPS组比较，HG组海马p-CRTC1的蛋白质表达下调，而IGF-1的蛋白质表达上调(均P<0.05，图6)。

图6.

图6

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HG-9-91-01对小鼠海马组织CRTC1/IGF-1通路的影响

Figure 6 Effects of HG-9-91-01 on the CRTC1/IGF-1 pathway in the hippocampus of mice Data are expressed as the mean±standard error of mean (n=4). **P<0.01, ***P<0.001 vs the Con group; †††P<0.001 vs the LPS group. Con: Control; LPS: Lipopolysaccharide; HG: HG-9-91-01; CRTC1: cAMP response element-binding protein-regulated transcription coactivator 1; p-CRTC1: Phosphorylated CRTC1; IGF-1: Insulin-like growth factor 1.

3. 讨 论

本研究采用腹腔注射LPS诱导小鼠脓毒症，这是评估脓毒症相关认知损伤最常用的模型之一，其优点在于方法简单，易于重现，且建模后运动受限较少，可在脓毒症早期评估认知功能障碍[25]。本研究结果显示，MWM中LPS组小鼠逃避潜伏期延长，目标象限停留时间百分比降低，表明小鼠认知功能受损。同时，LPS组小鼠SIK表达上调。本研究参照文献[16]并结合前期实验的结果，在建立脓毒症模型3 d后腹腔注射SIK抑制剂HG-9-91-01，观测其对小鼠认知功能的影响。结果显示，HG-9-91-01可明显缩短脓毒症小鼠的逃避潜伏期，增加小鼠在目标象限停留时间百分比，表明HG-9-91-01可改善小鼠脓毒症相关认知功能障碍。

神经炎症是脓毒症相关认知功能障碍的重要致病机制之一[6-7, 26]。在脓毒症中，神经炎症的主要特征是小胶质细胞的激活。小胶质细胞的激活通常导致细胞胞体变大、突起分支减少、突起变短等形态学改变[27-28]。一般来说，活化的小胶质细胞分为2种亚型，即促炎的M1型和抗炎的M2型[26]。既往研究[29-30]显示，减少M1型，增强M2型小胶质细胞极化有助于改善脓毒症相关认知损伤。内源性SIK2在神经元和小胶质细胞中表达，抑制SIK2可以减轻小鼠脑出血后神经炎症[31]。HG-9-91-01可促进结肠巨噬细胞IL-10的分泌，在小鼠结肠炎模型中显示出良好的抗结肠炎作用[16]。小胶质细胞是中枢神经系统中的巨噬细胞[32]，HG-9-91-01是否通过影响小胶质细胞活化来改善脓毒症相关认知功能障碍？本研究结果显示，LPS组小鼠海马组织小胶质细胞数量增加，M1型小胶质细胞标志分子和炎症因子表达增加，M2型标志分子表达减少；HG-9-91-01可减少小胶质细胞数量，下调M1型小胶质细胞标志分子和炎症因子的表达，上调M2型标志分子的表达。Sholl分析是一种可量化分析小胶质细胞形态变化的方法[28, 33]。Sholl分析结果显示，LPS组小胶质细胞突起与同心圆的交点减少，表明细胞形态复杂性降低，HG-9-91-01干预可增加交点数。以上结果表明，HG-9-91-01可能通过抑制M1型小胶质细胞活化，恢复小胶质细胞形态复杂性，减轻海马神经炎症，改善小鼠脓毒症相关认知功能障碍。

突触可塑性是认知功能的核心基础[34]，脓毒症相关认知损伤可能是由于突触丢失或功能变化引起的[35]。对因感染引发谵妄患者脑脊液的生物标志物进行研究，结果显示与突触形成和功能相关的蛋白质表达下调[36]。NR是谷氨酸门控离子通道，在突触传递和可塑性中起关键作用[37]。本课题组以前的研究[24]表明，NR激动剂可改善小鼠脓毒症相关认知损伤。本研究发现，HG-9-91-01可上调脓毒症小鼠海马组织中NR亚基NR1、NR2A、NR2B的表达，表明HG-9-91-01也可能通过调节突触可塑性发挥保护作用。

既往研究报道，CRTC1与突触可塑性、认知功能密切相关[12-13]，同时也参与神经炎症反应的调节[13]。IGF-1是受CRTC1/CREB下游信号通路调节的神经营养因子[18]，在中枢神经系统中具有多种作用[20]。研究[22]显示IGF-1水平与脓毒症患者病情和预后相关。本研究结果显示，HG-9-91-01可降低CRTC1的磷酸化水平，促进IGF-1表达上调。

综上所述，脓毒症小鼠海马SIK表达增加，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。

基金资助

国家自然科学基金(82200099)；湖南省自然科学基金(2020JJ5627)；湖南省教育厅科学研究项目(19C0197)。

This work was supported by the National Natural Science Foundation (82200099), the Natural Science Foundation of Hunan Province (2020JJ5627), and the Scientific Research Foundation of Hunan Provincial Education Department (19C0197), China.

利益冲突声明

作者声称无任何利益冲突。

作者贡献

王雪琴 实验操作，数据采集与分析，论文撰写与修改；王双 数据采集与分析，论文修改；崔艳慧 实验设计与指导，论文修改。所有作者阅读并同意最终的文本。

Footnotes

http://dx.chinadoi.cn/10.11817/j.issn.1672-7347.2023.230208

原文网址

http://xbyxb.csu.edu.cn/xbwk/fileup/PDF/2023121793.pdf

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