背景：
术后神经认知功能恢复延迟（delayed neurocognitive recovery，dNCR）是老年患者围术期常见的中枢神经系统（central nervous system，CNS）并发症，表现为记忆力、注意力以及思维能力障碍。dNCR发生于术后30天以内，通常表现为一过性的认知功能损害，但部分患者可发展为长期认知功能障碍，严重影响患者生活质量，也给患者家庭及社会带来巨大的负担。因此，探究dNCR发生机制，并制定防治策略是临床亟待解决的关键问题。
焦亡是依赖炎症小体的炎性、程序性细胞死亡，也是CNS清除衰老细胞的重要途径之一。焦亡（pyroptosis）主要通过半胱氨酸天冬氨酸蛋白酶（cysteine aspartic acid specific protease，Caspase）活化焦亡执行蛋白gasdermin D（GSDMD），使其活性N端（GSDMD-N）在质膜上聚集形成膜孔，细胞渗透性改变，最终导致细胞肿胀并裂解。过度激活的细胞焦亡引起局部炎症反应加重，从而导致组织损伤，是神经炎症性和神经退行性疾病重要的病理过程，在疾病的发生发展中发挥重要作用。
丝裂原活化蛋白激酶（mitogen-activated protein kinase，MAPK）是体内重要的信号转导途径，c-Jun氨基末端激酶（c-Jun N-terminal kinases，JNK）作为MAPK家族成员之一，在调控CNS增殖、分化及炎症反应中发挥重要作用。研究表明，JNK/MAPK信号途径在多种CNS疾病中调控细胞焦亡参与疾病的发生发展。特异性阻断JNK/MAPK信号途径可以有效缓解CNS疾病中细胞焦亡的过度激活，维持正常的神经功能。
外周伤害性刺激经神经和体液途径作用于CNS后导致小胶质细胞过度焦亡，炎症因子大量释放，是神经退行性疾病等炎性脑病的关键环节。研究表明，麻醉手术应激可致海马组织核苷酸结合寡聚化结构域样受体蛋白3（nucleotide-binding oligomerization domain leucine rich repeat and pyrin domain containing 3，NLRP3）炎症小体激活及细胞焦亡，伴随大量炎症因子释放，从而导致术后dNCR。然而，海马小胶质细胞焦亡在术后dNCR中的具体角色，以及JNK/MAPK信号途径是否参与dNCR中小胶质细胞焦亡的调控，目前尚未见报道。
本研究采用常用的术后dNCR动物及细胞模型，分别观察麻醉手术及脂多糖（lipopolysaccharide，LPS）对小胶质细胞焦亡及JNK/MAPK信号通路的影响；在此基础上，采用JNK特异性抑制剂SP600125观察阻断JNK/MAPK信号通路对dNCR小胶质细胞焦亡的影响，解析其对术后认知功能的改善作用，以期为dNCR的防治提供新思路。

目的：
1. 探究七氟烷麻醉下剖腹探查术对老龄小鼠术后认知功能及海马小胶质细胞焦亡的影响；
2. 探究七氟烷麻醉下剖腹探查术对老龄小鼠海马小胶质细胞JNK/MAPK信号通路的影响；
3. 探究JNK/MAPK信号通路在老龄小鼠海马小胶质细胞焦亡中的作用及分子机制。
 
方法：
第一部分：七氟烷麻醉下剖腹探查术对老龄小鼠术后认知功能及海马小胶质细胞焦亡的影响
（1）体内实验：18月龄雌性C57 BL/6小鼠44只，随机分为2组（n=22）：假手术组（Sham）和麻醉手术组（Sur）。Sham组在2.5%七氟烷麻醉下备皮及消毒，麻醉时间约25 min。Sur组在2.5%七氟烷麻醉下行剖腹探查术，手术时间约25 min。分别在术后1-6天采用Morris水迷宫（Morris water maze，MWM）（n=12）及术后第2、7天采用条件恐惧实验（fear conditioning test，FCT）（n=10）检测小鼠术后学习记忆功能。
（2）体内实验：18月龄雌性C57 BL/6小鼠24只，随机分为4组（n=6）：假手术组（Sham）、麻醉手术后1 h组（Sur 1 h）、6 h组（Sur 6 h）及24 h组（Sur 24 h）。Sham组与麻醉手术组实验方案与实验（1）相同。采用Western blot检测海马焦亡相关蛋白NLRP3、凋亡相关斑点样蛋白（apoptosis associated speck like protein containing Caspase recruitment domain，ASC）、Caspase-1、Cleaved-Caspase-1、GSDMD、GSDMD-N、白细胞介素（interleukin，IL）-1β、pro-IL-1β及IL-18表达情况，酶联免疫吸附试验（enzyme-linked immunosorbent assay，ELISA）检测海马IL-1β及IL-18表达情况。此外，采用免疫荧光（immunofluorescence，IF）检测焦亡最显著时间点（根据Western blot结果）海马小胶质细胞特异性表达蛋白血小板二磷酸腺苷受体亚基12（platelet adenosine diphosphate receptor subunit 12，P2Y12）与NLRP3或GSDMD-N共表达情况。
（3）体外实验：BV2小胶质细胞系，随机分为7组（n=3）：对照组（Control），脂多糖处理组（LPS），按药物浓度随机分为0.125 μg/ml组、0.25 μg/ml组、0.5 μg/ml组、1 μg/ml 组、2 μg/ml 组及5 μg/ml组。LPS组给予不同浓度LPS处理12 h，Control组给予等体积磷酸盐缓冲液（phosphate-buffered saline，PBS）。采用细胞活力试剂盒（cell counting kit-8，CCK-8）检测细胞活力情况，Western blot检测焦亡相关蛋白表达情况，碘化丙啶实验（propidium iodide，PI）检测细胞焦亡发生的比例，IF检测GSDMD-N表达情况。
（4）体外实验：BV2小胶质细胞系，随机分为6组（n=3）：对照组（Control），脂多糖处理组（LPS），并按药物处理时间随机分为1 h组、3 h组、6 h组、12 h组及24 h组。LPS组给予1 μg/ml LPS，Control组给予等体积PBS。具体检测方法及指标同实验（3）。

第二部分：七氟烷麻醉下剖腹探查术对老龄小鼠海马小胶质细胞JNK/MAPK信号通路的影响
（1）体内实验：18月龄雌性C57 BL/6小鼠24只，随机分为4组（n=6），分组及实验方案同第一部分实验（2）。采用Western blot检测海马MAPK信号通路相关蛋白JNK、p-JNK、P38、p-P38、细胞外信号调节激酶（extracellular signal-regulated kinase，ERK）及p-ERK表达情况。此外，采用IF检测p-JNK活性最显著时间点（根据Western blot结果）海马P2Y12与p-JNK共表达情况。
（2）体外实验：以不同浓度的LPS处理BV2小胶质细胞系12 h，具体分组及实验方案同第一部分实验（3）。采用Western blot检测MAPK信号通路相关蛋白表达情况。
（3）体外实验：以不同持续时间的1 μg/ml LPS处理BV2小胶质细胞系，具体分组及实验方案同第一部分实验（4）。具体检测方法及指标与实验（2）相同。

第三部分：JNK/MAPK信号通路调控小胶质细胞焦亡在老龄小鼠术后认知功能恢复延迟中的作用
（1）体外实验：BV2小胶质细胞系，随机分为4组（n=3）：对照组（Control）、SP600125组（SP600125）、LPS组（LPS）及LPS + SP600125组（LPS + SP600125）。给予SP600125（JNK抑制剂）25 μM预处理5 h，1 μg/ml LPS处理6 h。采用CCK-8检测细胞活力情况，乳酸脱氢酶实验（lactate dehydrogenase，LDH）检测LDH释放情况，Western blot检测p-JNK、JNK及焦亡相关蛋白表达情况，PI检测细胞焦亡发生的比例，IF检测NLRP3、GSDMD-N及IL-1β表达情况。
（2）体外实验：BV2小胶质细胞系，随机分为4组（n=3）：对照组（Control）、MCC950组（MCC950）、LPS组（LPS）及LPS + MCC950组（LPS + MCC950）。给予MCC950（NLRP3抑制剂）50 μM预处理5 h，1 μg/ml LPS处理6 h。采用Western blot检测p-JNK、JNK及焦亡相关蛋白表达情况，IF检测p-JNK表达情况。
（3）体内实验：18月龄雌性C57 BL/6小鼠112只，随机分为4组（n=28）：假手术组（Sham）、SP600125组（SP600125）、麻醉手术组（Sur）及麻醉手术+SP600125组（Sur + SP600125）。腹膜注射200 μl 10 mg/kg SP600125或对照溶剂预处理1周，Sham组与麻醉手术组实验方案与第一部分研究相同。采用MWM（n=12）及FCT（n=10）检测小鼠术后学习记忆功能；麻醉手术后24 h取材各组海马组织（n=6），采用Western blot检测海马p-JNK、JNK及焦亡相关蛋白表达情况，IF检测海马P2Y12与NLRP3或GSDMD-N共表达情况，ELISA检测海马IL-1β及IL-18表达情况。

结果：
第一部分：七氟烷麻醉下剖腹探查术对老龄小鼠术后认知功能及海马小胶质细胞焦亡的影响
（1）体内实验：MWM结果显示，与Sham组相比，Sur组小鼠术后第2、3、4天逃避潜伏期明显延长（P<0.05），术后第6天平台穿越次数及目标象限停留时间明显减少（P<0.05）。2组小鼠测试期间游泳速度无明显差异（P>0.05）。FCT结果显示，Sur组小鼠术后第2、7天场景条件恐惧实验中僵直时间较Sham组显著降低（P<0.05），但2组小鼠声音条件恐惧测试无明显差异（P>0.05）。
（2）体内实验：Western blot结果显示，与Sham组相比，Sur 6 h组和Sur 24 h组海马焦亡相关蛋白表达显著增加，且Sur 24 h组焦亡表达最显著（P<0.05）。ELISA结果显示，Sur 6 h组和Sur 24 h组炎症因子IL-1β和IL-18表达较Sham组显著升高（P<0.05）。IF结果显示，与Sham组相比，Sur 24 h组小鼠海马P2Y12和NLRP3或GSDMD-N表达显著增加（P<0.05），且P2Y12+细胞中NLRP3或GSDMD-N共表达的比例明显上升（P<0.05）。
（3）体外实验：与Control组相比，1 μg/ml、2 μg/ml及5 μg/ml LPS干预组细胞生存率显著降低（P<0.05）、PI阳性细胞百分率显著增加（P<0.05）、GSDMD-N蛋白表达显著升高（P<0.05）。Western blot结果显示，1 μg/ml LPS干预组焦亡相关蛋白表达较Control组显著增加（P<0.05）。
（4）体外实验：与Control组相比，LPS干预6 h、12 h及24 h组细胞生存率明显下降（P<0.05）、PI阳性细胞百分率显著增加（P<0.05）、GSDMD-N蛋白表达显著升高（P<0.05）。Western blot结果显示，LPS干预6 h组焦亡相关蛋白表达较Control组显著提高（P<0.05）。

第二部分：七氟烷麻醉下剖腹探查术对老龄小鼠海马小胶质细胞JNK/MAPK信号通路的影响
（1）体内实验：Western blot结果显示，与Sham组相比，Sur 1 h组、Sur 6 h组及Sur 24 h组小鼠海马p-JNK表达明显升高，且Sur 1 h组p-JNK表达最显著（P<0.05）,而各组小鼠海马p-P38和p-ERK表达无统计学差异（P>0.05）。IF结果显示，与Sham组相比，Sur 1 h组小鼠海马P2Y12和p-JNK表达明显升高（P<0.05），且P2Y12+细胞中p-JNK同时表达百分比也明显上升（P<0.05）。
（2）体外实验：Western blot结果观察到，与Control组相比，各浓度LPS干预组 p-JNK及p-ERK蛋白水平均明显上调（P<0.05）；0.25 μg/ml、0.5 μg/ml及1 μg/ml LPS干预组p-P38表达明显升高（P<0.05）。
（3）体外实验：Western blot结果显示，与Control组相比，各时间LPS干预组均可见p-JNK蛋白表达明显增高（P<0.05）；LPS干预6 h组p-P38和p-ERK表达显著提高（P<0.05）。

第三部分：JNK/MAPK信号通路调控小胶质细胞焦亡在老龄小鼠术后认知功能恢复延迟中的作用及机制研究
（1）体外实验：CCK-8结果显示，SP600125预处理明显缓解LPS诱导的BV2小胶质细胞活力下降（P<0.05）。LDH结果显示，SP600125预处理有效减轻LPS诱导的BV2小胶质细胞LDH释放（P<0.05）。Western blot结果显示，SP600125预处理有效抑制p-JNK后，显著抑制LPS诱导的焦亡相关分子表达（P<0.05）。IF结果显示，LPS + SP600125组NLRP3、GSDMD-N及IL-1β蛋白表达较LPS组明显下调（P<0.05）。PI结果表明，LPS + SP600125组显著降低LPS引起的PI阳性细胞百分率升高（P<0.05）。
（2）体外实验：Western blot结果显示，MCC950预处理抑制NLRP3表达后，可以明显减轻LPS诱导的ASC、GSDMD-N、IL-1β及IL-18蛋白表达（P<0.05）。MCC950预处理对LPS诱导的p-JNK、Cleaved-Caspase-1及pro-IL-1β表达上调无明显改善作用（P>0.05）。IF结果显示，MCC950预处理对LPS引起的p-JNK表达增加无明显缓解作用（P>0.05）。
（3）体内实验：MWM结果显示，与Sham组相比，Sur组小鼠术后第2、3、4天逃避潜伏期明显增加（P<0.05），术后第6天平台穿越次数及目标象限停留时间明显减少（P<0.05），SP600125预处理明显改善上述改变（P<0.05）。各组小鼠测试期间游泳速度无明显差异（P>0.05）。FCT结果显示， SP600125预处理明显逆转Sur组小鼠术后第2、7天场景条件恐惧实验中僵直时间降低（P<0.05），但4组小鼠声音条件恐惧测试无明显差异（P>0.05）。Western blot结果显示，SP600125预处理有效阻断p-JNK表达后，明显降低Sur组小鼠海马焦亡相关分子表达（P<0.05）。IF结果显示，SP600125预处理明显下调Sur组小鼠海马P2Y12和NLRP3或GSDMD-N表达（P<0.05），也明显减少P2Y12+细胞中 NLRP3或GSDMD-N同时表达百分比。ELISA结果显示，Sur + SP600125组海马炎症因子IL-1β及IL-18表达较Sur组明显减低（P<0.05）。

结论：
1. 七氟烷麻醉下剖腹探查术可以引起老龄小鼠术后2 - 7天海马依赖的学习和记忆功能下降，与术后24 h海马小胶质细胞焦亡及神经炎症反应加重有关；
2. 七氟烷麻醉下剖腹探查术可引起术后1 h老龄小鼠海马小胶质细胞JNK/MAPK信号通路的激活，并持续到术后24 h；
3. 抑制JNK/MAPK信号通路可减轻海马小胶质细胞焦亡及神经炎症反应，改善麻醉手术导致的老龄小鼠学习记忆功能损伤。

 

Background:
Postoperative delayed neurocognitive recovery (dNCR) is a common central nervous system (CNS) complication in elderly patients during the perioperative period, manifesting as dysfunction in memory, attention, thinking ability. dNCR occurs within 30 days after anesthesia and surgery, it usually performs as transient cognitive impairment, but some patients may develop long-term cognitive dysfunction, which seriously affects the quality of life of patients, and also brings a huge burden to patients' families and social medical resources. Therefore, exploring the pathogenesis of dNCR and formulating prevention and treatment strategies as early as possible are crucial clinical issues that need to be solved.
Pyroptosis is a form of inflammatory programmed cell death relying on inflammasome, and it is also one of the vital pathways for the CNS to eliminate senescent cells. Pyroptosis is mainly activated by cysteine aspartic acid specific protease (Caspase), which activates the pyroptotic executive protein gasdermin D (GSDMD), causing its active N-terminal (GSDMD-N) to accumulate on the membrane to form a membrane pore, which eventually causes cells swelling and lysis. Excessive activation of pyroptosis causes aggravation of local inflammation, which leads to tissue damage. Pyroptosis is an important pathological process of neuroinflammatory and neurodegenerative diseases, which plays a vital role in the occurrence and development of the diseases.
Mitogen-activated protein kinase (MAPK) is an important signal transduction pathway in the body, and c-Jun N-terminal kinases (JNK) are one of the members of the MAPK family. It plays a vital role in regulating the cell proliferation, differentiation and inflammation in the CNS. Studies have revealed that the JNK/MAPK signaling pathway regulates pyroptosis in an array of CNS diseases which participates in the occurrence and development of the disease. The special blockage of the JNK/MAPK signal pathway can effectively mitigate the excessive activation of pyroptosis in CNS diseases, thus reserving the normal function of the brain.
Upon acting on the CNS through the neural and humoral pathways, peripheral harmful stimuli results in excessive activation of microglia pyroptosis and the numerous release of inflammatory cytokines. It is a key pathological hub of the inflammatory encephalopathy such as neurodegenerative diseases. Studies have demonstrated that the stress of anesthesia and surgery can cause nucleotide-binding oligomerization domain leucine rich repeat and pyrin domain containing 3 (NLRP3) inflammasome activation and pyroptosis in the hippocampus, accompanied by the release of a large number of inflammatory cytokines, leading to the occurrence of postoperative dNCR. However, the specific role of hippocampal microglia pyroptosis in postoperative dNCR and whether the JNK/MAPK signal pathway is involved in the regulation of microglia pyroptosis in dNCR has not yet been reported.
In this study, the classical animal and cell models of postoperative dNCR were used to observe the effects of anesthesia surgery and lipopolysaccharide (LPS) on microglia pyroptosis and JNK/MAPK signaling pathways. On this basis, JNK specific inhibitor SP600125 was used to observe the effect of blockage of JNK/MAPK signaling pathway on microglia pyroptosis in dNCR, and analyze its effect on postoperative cognitive function, in order to provide new ideas for the prevention and treatment of dNCR.

Objective:
1. To explore the effects of sevoflurane anesthesia and exploratory laparotomy on postoperative cognitive function and microglia pyroptosis in the hippocampus of aged mice.
2. To explore the effects of sevoflurane anesthesia and exploratory laparotomy on microglia JNK/MAPK signal pathway in the hippocampus of aged mice.
3. To explore the effects and molecular mechanisms of JNK/MAPK signaling pathway on microglia pyroptosis in the hippocampus of aged mice.

Methods：
Part Ⅰ: The effects of exploratory laparotomy under sevoflurane anesthesia on postoperative cognitive function and microglia pyroptosis in the hippocampus of aged mice
(1) In vivo: Forty-four female C57 BL/6 mice, 18-month-old, were randomly divided into 2 groups (n=22): sham operation group (Sham) and anesthesia plus surgery group (Sur). Animals in the sham group were shaved and sterilized under 2.5% sevoflurane anesthesia, anesthesia was about 25 minutes. Sur group underwent exploratory laparotomy under 2.5% sevoflurane anesthesia, the operation was about 25 minutes. Morris water maze (MWM) (n=12) examined 1 - 6 days after surgery and fear conditioning test (FCT) (n=10) detected on the 2nd and 7th day after surgery were used to examine the learning and memory ability of the mice.
(2) In vivo: Twenty-four female C57 BL/6 mice, 18-month-old, were randomly assigned into 4 groups (n=6): sham operation group (Sham), 1 h after anesthesia and surgery group (Sur 1 h), 6 h after anesthesia and surgery group (Sur 6 h) and 24 h after anesthesia and surgery group (Sur 24 h). The experimental protocol of the Sham group and Sur group was the same as an experiment (1). Western blot was utilized to detect the expression of pyroptosis-associated proteins including NLRP3, apoptosis associated speck like protein containing Caspase recruitment domain (ASC), Caspase-1, Cleaved-Caspase-1, GSDMD, GSDMD-N, interleukin (IL)-1β、pro-IL-1β and IL-18 in the hippocampus. Enzyme-linked immunosorbent assay (ELISA) was applied to evaluate the expression of inflammatory factors IL-1β and IL-18 in the hippocampus. Moreover, immunofluorescence (IF) was used to detect the co-expression of microglial specific protein platelet adenosine diphosphate receptor subunit 12 (P2Y12) and NLRP3 or GSDMD-N in the hippocampus at the most significant time point of pyroptosis (according to Western blot results).
(3) In vitro: microglial cell line BV2 was randomly divided into 7 groups (n=3): control group (Control), and lipopolysaccharide (LPS) group which was randomly assigned to 0.125 μg/ml group, 0.25 μg/ml group, 0.5 μg/ml group, 1 μg/ml group, 2 μg/ml group and 5 μg/ml group based on LPS concentration. LPS group was administrated with different concentrations of LPS for 12 h, Control group was treated with the same volume of phosphate-buffered saline (PBS). Cell viability was detected by cell counting kit-8 (CCK-8), the expression of pyroptosis-related proteins was examined by Western blot, the percentage of pyroptotic cell death was assessed by propidium iodide (PI) test, and the expression of GSDMD-N was evaluated by IF.
(4) In vitro: microglial cell line BV2 was randomly allocated into 6 groups (n=3): control group (Control), and lipopolysaccharide (LPS) group which was randomly divided into 1 h group, 3 h group, 6 h group, 12 h group, and 24 h group according to LPS duration time. LPS group was treated with 1 μg/ml LPS, Control group was incubated with the same volume of PBS. The experimental methods and indicators are the same as the experiment (3).

Part ⅠI: The effects of exploratory laparotomy under sevoflurane anesthesia on microglia JNK/MAPK signal pathway in the hippocampus of aged mice
(1) In vivo: Twenty-four female C57 BL/6 mice, 18-month-old, were randomly assigned into 4 groups (n=6). The grouping and experimental protocol were the same as the first part of the experiment (2). Western blot was used to assess the expression of the MAPK signal pathway-related proteins JNK, p-JNK, P38, p-P38, extracellular signal-regulated kinase (ERK) and p-ERK in the hippocampus. Moreover, IF was subjected to detect the co-expression of P2Y12 and p-JNK in the hippocampus at the time point when the expression of p-JNK was highest (according to Western blot results).
(2) In vitro: microglial cell line BV2 was treated with different concentrations of LPS for 12 h. The grouping and experimental protocol were the same as the first part of the experiment (3). Western blot was performed to detect the expression of MAPK signal pathway-related proteins.
(3) In vitro: microglial cell line BV2 was treated with different duration of 1 μg/ml LPS. The grouping and experimental protocol were the same as the first part of the experiment (4). The experimental methods and indicators are the same as the experiment (2).

Part ⅠII: The effects and mechanisms of JNK/MAPK pathway-mediated microglia pyroptosis on delayed neurocognitive recovery in aged mice
(1) In vitro: microglial cell line BV2 was randomly allocated into 4 groups (n=3): control group (Control), SP600125 group (SP600125), LPS group (LPS) and LPS + SP600125 group (LPS + SP600125). BV2 cells were treated with SP600125 (JNK inhibitor) 25 μM for 5 h, or 1 μg/ml LPS for 6 h. The cell viability was evaluated by CCK-8, the release of lactate dehydrogenase (LDH) was detected by LDH test, the expression of p-JNK, JNK, and pyroptosis-related proteins were detected by Western blot, the pyroptotic cell death was examined by PI test, and the expression of NLRP3, GSDMD-N and IL-1β were investigated by IF.
(2) In vitro: microglial cell line BV2 was randomly allocated into 4 groups (n=3): control group (Control), MCC950 group (MCC950), LPS group (LPS) and LPS + MCC950 group (LPS + MCC950). BV2 cells were pretreated with 50 μ M MCC950 (NLRP3 inhibitor) for 5 h, or 1 μg/ml LPS for 6 h. Western blot was performed to evaluate the expression of p-JNK, JNK, and pyroptosis-related proteins. IF was executed to determine the expression of p-JNK.
(3) In vivo: One hundred and twelve 18-month-old female C57 BL/6 mice were randomly distributed into 4 groups (n=28): sham operation group (Sham), SP600125 group (SP600125), sevoflurane anesthesia and surgery group (Sur) and sevoflurane anesthesia surgery and SP600125 group (Sur + SP600125). Mice were intraperitoneally injected with 10 mg/kg SP600125 or vehicle at 200 μl daily for the consecutive 1 week, the experimental protocol of Sham and Surgery were the same as the first part. MWM (n=12) and FCT (n=10) were used to evaluate the learning and memory ability of the mice. The hippocampal tissues of each group were harvested 24 h after anesthesia and surgery (n=6). The expression of p-JNK, JNK, and pyroptosis-associated proteins in the hippocampus were examined by Western blot. The co-expression of P2Y12 and NLRP3 or GSDMD-N in the hippocampus was detected by IF. The expression of IL-1β and IL-18 in the hippocampus were evaluated by ELISA.

Results:
Part Ⅰ: The effects of exploratory laparotomy under sevoflurane anesthesia on postoperative cognitive function and microglia pyroptosis in the hippocampus of aged mice
(1) In vivo: The results of MWM displayed that, compared with the Sham group, the escape latency of the mice in the Sur group was significantly prolonged on the 2, 3, and 4 days after surgery (P<0.05), the times of platform crossings and the percentage of time spent in the target quadrant on the 6 days after surgery in the Sur group were significantly less than those in the Sham group (P<0.05). There was no significant difference in swimming speed between the two groups during the test (P>0.05). The results of FCT depicted that, compared with the Sham group, the mice in the Sur group had significantly reduced freezing time in the contextual fear conditioning test on the 2 and 7 days after surgery (P<0.05). No significant difference in the freezing time between the two groups was observed in the tone-related fear conditioning test on the 2 and 7 days after surgery (P>0.05).
(2) In vivo: The results of Western blot showed that the expression of pyroptosis-associated proteins in the hippocampus was significantly elevated in the Sur 6 h and Sur 24 h group compared with Sham group, and the highest expression of pyroptosis was in Sur 24 h group (P<0.05). The results of ELISA showed that in the hippocampus of mice, compared with the Sham group, the expression of IL-1β and IL-18 in the Sur 6 h and Sur 24 h group were significantly improved (P<0.05). The data of IF demonstrated that compared with the Sham group, the expression of P2Y12 and NLRP3 or GSDMD-N in the hippocampus of mice in the Sur 24 h group were remarkedly increased, and the percentage of co-expression of NLRP3 or GSDMD-N in P2Y12+ cells was also significantly elevated (P<0.05).
(3) In vitro: Compared with the Control group, the cell viability was decreased (P<0.05), the percentage of PI-positive cells was progressively elevated (P<0.05), the expression of GSDMD-N was markedly augmented (P<0.05) in 1 μg/ml, 2 μg/ml and 5 μg/ml LPS treatment group. The data of Western blot unveiled that the expression of pyroptosis-associated proteins in 1 μg/ml LPS group was markedly elevated compared with the Control group (P<0.05).
(4) In vitro: Compared with the Control group, the cell viability was significantly reduced (P<0.05), the percentage of PI-positive cells was markedly elevated (P<0.05), the expression of GSDMD-N was dramatically increased (P<0.05) in LPS 6 h group, 12 h group and 24 h group. The Western blot data indicated that the expression of pyroptosis-associated proteins was enhanced in LPS 6 h group compared with the Control group (P<0.05).

Part ⅠI: The effects of exploratory laparotomy under sevoflurane anesthesia on microglia JNK/MAPK signal pathway in the hippocampus of aged mice
(1) In vivo: The data of Western blot revealed that the expression of p-JNK was remarkedly up-regulated in the hippocampus of mice in Sur 1h, Sur 6h, and Sur 24h group, and the highest expression of p-JNK was in Sur 1 h group (P<0.05). While the expression of p-P38 and p-ERK were not significantly different in the hippocampus of mice among all the groups (P>0.05). The findings of IF indicated that compared with the Sham group, the expression of P2Y12 and p-JNK in the hippocampus of mice in the Sur 1 h group were remarkedly increased, and the percentage of co-expression of p-JNK in P2Y12+ cells was also obviously increased (P<0.05).
(2) In vitro: The results of Western blot unveiled that the expression of p-JNK and p-ERK in different concentrations of the LPS treatment group was significantly higher than that in the Control group (P<0.05). The levels of p-P38 expression in 0.25 μg/ml, 0.5 μg/ml and 1 μg/ml LPS treatment group were apparently higher than that in the Control group (P<0.05).
(3) In vitro: The Western blot results depicted that the level of p-JNK in different duration of the LPS treatment group was significantly higher than that in the Control group (P< 0.05). The expression of p-P38 and p-ERK in 6 h of the LPS treatment group was increased compared with the Control group (P< 0.05).

Part ⅠII: The effects and mechanisms of JNK/MAPK pathway-mediated microglia pyroptosis on delayed neurocognitive recovery in aged mice
(1) In vitro: The CCK-8 results revealed that SP600125 pretreatment significantly mitigated the decline of cell viability induced by LPS treatment (P<0.05). The data of LDH showed that the release of LDH stimulated by LPS incubation was dramatically decreased by SP600125 pretreatment (P<0.05). The results of Western blot showed that after p-JNK levels were significantly inhibited by SP600125 pretreatment, the increased expression of pyroptosis-associated proteins induced by LPS administration was markedly weakened (P<0.05). The results of IF showed that the expression of NLRP3, GSDMD-N and IL-1β protein in the LPS + SP600125 group was significantly lower than that in the LPS group (P<0.05). The results of PI revealed that LPS-induced increased percentage of PI-positive cells was significantly reduced in LPS + SP600125 group (P<0.05).
(2) In vitro: The results of Western blot displayed that MCC950 pretreatment could significantly reduce the expression of ASC, GSDMD-N, IL-1β, and IL-18 stimulated by LPS incubation after NLRP3 levels were effectively suppressed (P<0.05). While LPS-induced elevated expression of p-JNK, Cleaved-Caspase-1 and pro-IL-1β could not be abolished by MCC950 pretreatment (P>0.05). The graphs of IF showed that MCC950 pretreatment could not mitigate the augmented expression of p-JNK induced by LPS stimulation (P>0.05).
(3) In vivo: The results of MWM showed that compared with the Sham group, the escape latency in the Sur group was significantly increased on postoperative 2, 3, and 4 days (P<0.05), the number of platform crossing and the time spent in the target quadrant in Sur group were significantly decreased on 6 days after surgery, while SP600125 treatment significantly improved all these changes (P<0.05). There was no significant difference in swimming speed among the four groups during the test (P>0.05). The results of FCT revealed that in the context test, SP600125 pretreatment reversed the decreased freezing time caused by anesthesia and surgery on postoperative 2 and 7 days (P<0.05). No significant difference in the freezing time among the four groups in the tone test was observed on the 2 and 7 days after surgery (P>0.05). The data of Western blot exhibited that with an effective blockage of p-JNK expression, SP600125 pretreatment significantly decreased the enhanced expression of pyroptosis-associated proteins in the hippocampus of mice in the Sur group (P<0.05). The results of IF demonstrated that SP600125 pretreatment down-regulated the expression of P2Y12 and NLRP3 or GSDMD-N in the hippocampus of mice caused by anesthesia and surgery (P<0.05), and reduced the percentage of co-expression of NLRP3 or GSDMD-N in P2Y12+ cells (P<0.05). The ELISA results revealed that the expression of IL-1β and IL-18 in the hippocampus of the Sur + SP600125 group were notably attenuated than that in the Sur group (P<0.05).

Conclusion:
1. The exploratory laparotomy under sevoflurane anesthesia can cause hippocampal-dependent learning and memory decline in aged mice 2-7 days after surgery, which is related to microglia pyroptosis and increased neuroinflammation in the hippocampus at 24 h after surgery
2. The exploratory laparotomy under sevoflurane anesthesia can induce the activation of the microglia JNK/MAPK signal pathway in the hippocampus of aged mice 1 h after surgery, and lasts to 24 h after surgery
3. Inhibition of JNK/MAPK pathway can alleviate microglia pyroptosis and neuroinflammatory responses in the hippocampus, thus improving hippocampus-dependent learning and memory impairments caused by anesthesia and surgery in aged mice

 

 

第一章 文献综述 1
1.1 焦亡的概念 2
1.2 焦亡的形态学特征 2
1.3 焦亡的信号途径 3
1.3.1 经典途经细胞焦亡 3
1.3.2 非经典途经细胞焦亡 5
1.4 炎症小体的组分 6
1.5小胶质细胞焦亡与中枢神经系统相关疾病 7
1.5.1 小胶质细胞焦亡与阿尔茨海默病 8
1.5.2 小胶质细胞焦亡与帕金森病 9
1.5.3 小胶质细胞焦亡与脑卒中 10
1.5.4 小胶质细胞焦亡与围术期神经认知障碍 12
1.6 结语与展望 14
第二章 论文正文 15
2.1 引言 15
2.2 第一部分：七氟烷麻醉下剖腹探查术对老龄小鼠术后认知功能及海马小胶质细胞焦亡的影响 18
2.2.1 引言 18
2.2.2 对象与方法 19
2.2.3 结果 32
2.2.4 讨论 40
2.2.5 小结 44
2.3 第二部分：七氟烷麻醉下剖腹探查术对老龄小鼠海马小胶质细胞JNK/MAPK信号通路的影响 45
2.3.1 引言 45
2.3.2 对象与方法 46
2.3.3 结果 48
2.3.4 讨论 52
2.3.5 小结 54
2.4 第三部分：JNK/MAPK信号通路调控小胶质细胞焦亡在dNCR中的作用 55
2.4.1 引言 55
2.4.2 对象与方法 56
2.4.3 结果 60
2.4.4 讨论 73
2.4.5 小结 76
第三章 结论及展望 77
3.1 结论 77
3.2 展望 77
第四章 创新性与局限性 78
4.1 创新性 78
4.2 局限性 78
参考文献 79
致谢 95
北京大学学位论文原创性声明和使用授权说明 97
个人简历、在学期间发表的学术论文与研究成果 99

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