丙酮酸乙酯对脓毒症大鼠认知功能的改善作用及机制研究

doi:10.3877/cma.j.issn.2096-1537.2018.02.015

目的

探讨丙酮酸乙酯对脓毒症大鼠认知功能的影响及其机制。

方法

实验一：采用盲肠结扎穿孔法（CLP）制备脓毒症大鼠模型，50只健康雄性SD大鼠随机分为5组（n=10），分别为假手术组（C组）、脓毒症组（S组），脓毒症＋高迁移率运动蛋白族1（HMGB1）处理组（SH组），假手术＋HMGB1处理组（H组），脓毒症＋丙酮酸乙酯处理组（SE组）。SH组、H组则行侧脑室注射0.5 μg/μl重组HMGB1 5 μl，余3组侧脑室给予等体积0.9%NaCl；SE组皮下注射含80 mg/kg丙酮酸乙酯，余4组给予等量0.9%NaCl；造模后观察不同组别大鼠的7 d死亡率。实验二：分组处理同前（n=20），造模处理48 h后行6 d Morris水迷宫定位航行训练及随后1次空间探索实验，检测大鼠学习记忆能力。实验三：分组同前（n=30），造模处理48 h后，取脑组织测脑水含量，尼氏染色观察海马区神经元的形态数目，实时定量基因扩增荧光检测系统（RT-qPCR）检测海马HMGB1、离子钙接头分子（Iba-1）、肿瘤坏死因子α（TNF-α）、白介素1β（IL-1β）及β样淀粉前体蛋白（APP）的mRNA表达。

结果

（1）与C组相比，S组、SH组生存率明显减低（P<0.05），H组、SE组改变差异无统计学意义（P>0.05）。（2）Morris水迷宫行为学实验，5组间游泳速度差异无统计学意义（P>0.05）；与C组比较，S组、SH组、H组、SE组游泳距离、逃避潜伏期延长，穿越虚拟平台次数及目标象限停留时间百分比减少（P<0.05）；与S组比较，SH组游泳距离、逃避潜伏期增加，SE组缩短。（3）与C组比较，其余4组脑水含量增高（P<0.05）；与S组比较，SH组脑水含量增高，SE组脑水含量减低（P<0.05），H组脑水含量低于S组但高于C组（P<0.05）。尼氏染色显示，与C组比较，S组、SH组、H组、SE组导致海马CA1区正常神经元数量减少（P<0.05），与S组比较，SH组神经元数量较少，SE增高（P<0.05）。与C组比较，S组、SH组HMGB1、TNF-α、IL-1β、Iba-1、APP mRNA表达均上调（P<0.05），SH组更为明显，SE组HMGB1、TNF-α、IL-1β、Iba-1 mRNA表达上调（P<0.05），APP mRNA表达增加但差异无统计学意义（P>0.05）；与S组比较，SE组HMGB1、TNF-α、IL-1β、Iba-1 mRNA表达下调，APP mRNA表达差异无统计学意义（P>0.05）。

结论

丙酮酸乙酯可改善脓毒症所致的认知功能障碍，其机制与减轻炎症反应，改善血脑屏障功能有关。

关键词: 丙酮酸乙酯, 脓毒症相关性脑病, 高迁移率运动蛋白族1

Objective

To determine the effect of ethyl pyruvate on cognitive function in rats with sepsis and its underling mechanism.

Methods

Part Ⅰ: The sepsis model was conducted by cecal ligation and puncture (CLP). Fifty healthy male SD rats were randomly divided into 5 groups (n=10): sham operation group (group C), sepsis group (group S), sepsis + High mobility group box-1 (HMGB1) treatment group (group SH), sham + HMGB1 treatment group (group H), sepsis + ethyl pyruvate treatment group (group SE). In the group SH and H, 5 μl of 0.5 μg/μl recombinant HMGB1 was injected into the lateral ventricle and 5 μl of saline was administered to the other 3 groups. In the SE group, ethyl pyruvate (80 mg/kg) was injected subcutaneously, and the remaining 4 groups were given the same volume of saline. Severn day survival curve analysis was performed among different groups. Part Ⅱ: Group dividing and experiment process are the same as before (n=20). Forty-eight hours after CLP or CLP sham a 6-days positioning navigation training of Morris water maze and the subsequent space exploration experiment was used to test cognitive ability. Part Ⅲ: Forty-eight hours post CLP or CLP sham, the brain tissue was harvest forbrain water content. Nissl′s staining was used to observe the number of neurons in the hippocampus and RT-qPCR was used to detect mRNA expression of hippocampal HMGB1, Ionized calcium bindingadaptor molecule-1 (Iba-1), Tumor necrosis factor-α (TNF-α), interleukin 1β (IL-1β), and amyloid precursor protein (APP).

Results

(1) Compared to group C, the survival rate of group S and SH was significantly decreased (P<0.05) and there was no significant difference between group H and SE (P>0.05). (2) There was no difference in swimming speed among the five groups in Morris water maze experiment. Compared to group C, swimming distance and escape latency were longer and the percentage of staying time passing through virtual platform and target quadrant decreased significantly (P<0.05) in group S, SH, Hgroup SE. The swimming distance and escape latency increased in SH group and decreased in S group relative group S. (3) Compared to group C, brain water increased in the other four groups (P<0.05). In addition, brain water increased in group SH and decreased in group SE and H relative group S (all P<0.05). Nissl staining showed that compared to group C, group S, SH, H and SE showed a decreased number of normal neurons in hippocampal CA1 region (P<0.05). The number of neurons decreased in group SH and increased in group SE compared to group S (both P<0.05). Compared to group C, the expressions of HMGB1, TNF-α, IL-1β, Iba-1 and APP mRNA in group S and SH were up-regulated (P<0.05). The expression of HMGB1, TNF-α, IL-1β and Iba-1 mRNA was also up-regulated in SE group (P<0.05) but there was no difference of APP mRNA expression between group SE and C (P>0.05). Compared with S group, the expression of HMGB1, TNF-α, IL-1β and Iba-1 other than APP mRNA was down-regulated in SE group.

Conclusion

Ethyl pyruvate can relieve cognitive dysfunction that induced by sepsis, which may be associated with inflammation reduction and blood-brain barrier function improvement.

Key words: Ethyl pyruvate, Sepsis-associated encephalopathy, High-mobility protein group 1

图1 脓毒症大鼠不同处理组间生存率的比较

表1 不同组别脓毒症大鼠6 d定位航行距离的比较（cm，±s）

组别	第1天	第2天	第3天	第4天	第5天	第6天
C组（20只）	727.62±122.14	519.25±149.47	433.00±177.10	323.31±117.69	233.08±71.99	152.91±82.78
S组（11只）	769.58±248.25	676.96±304.60^a	564.99±232.47^a	546.71±308.04^a	431.59±308.31^a	319.00±206.98^a
SH组（8只）	720.17±255.52	487.61±255.93^ab	510.18±196.21^a	410.91±189.13^ab	474.11±285.38^a	362.03±210.40^ab
H组（18只）	752.52±186.92	682.84±227.27^a	546.76±290.58^a	407.88±210.46^ab	288.69±158.65^a	259.78±111.69^a
SE组（13只）	689.53±221.49	464.61±315.72^ab	471.38±260.15^ab	450.62±319.49^ab	340.81±191.78^ab	197.39±98.73^ab

表1 不同组别脓毒症大鼠6 d定位航行距离的比较（cm，±s）

组别	第1天	第2天	第3天	第4天	第5天	第6天
C组（20只）	727.62±122.14	519.25±149.47	433.00±177.10	323.31±117.69	233.08±71.99	152.91±82.78
S组（11只）	769.58±248.25	676.96±304.60^a	564.99±232.47^a	546.71±308.04^a	431.59±308.31^a	319.00±206.98^a
SH组（8只）	720.17±255.52	487.61±255.93^ab	510.18±196.21^a	410.91±189.13^ab	474.11±285.38^a	362.03±210.40^ab
H组（18只）	752.52±186.92	682.84±227.27^a	546.76±290.58^a	407.88±210.46^ab	288.69±158.65^a	259.78±111.69^a
SE组（13只）	689.53±221.49	464.61±315.72^ab	471.38±260.15^ab	450.62±319.49^ab	340.81±191.78^ab	197.39±98.73^ab

表2 不同组别脓毒症大鼠6 d定位航行逃避潜伏期的比较（s，±s）

组别	第1天	第2天	第3天	第4天	第5天	第6天
C组（20只）	31.99±4.37	25.51±6.42	20.11±5.31	13.67±4.05	11.78±4.49	13.37±5.74
S组（11只）	31.83±9.90	29.69±7.44	22.61±6.71	26.46±9.81^a	19.48±7.57^a	19.89±11.45^a
SH组（8只）	35.31±11.70	30.07±14.90	32.65±13.09^ab	31.37±14.82^ab	31.64±15.61^ab	26.64±12.39^ab
H组（18只）	37.70±9.72	35.41±13.64	29.40±12.03^ab	24.54±9.35^a	17.67±8.33^a	16.41±8.57^a
SE组（13只）	29.49±9.81	25.61±16.13	23.86±18.96	25.33±13.75^a	19.32±10.24^a	14.24±11.87^ab

表2 不同组别脓毒症大鼠6 d定位航行逃避潜伏期的比较（s，±s）

组别	第1天	第2天	第3天	第4天	第5天	第6天
C组（20只）	31.99±4.37	25.51±6.42	20.11±5.31	13.67±4.05	11.78±4.49	13.37±5.74
S组（11只）	31.83±9.90	29.69±7.44	22.61±6.71	26.46±9.81^a	19.48±7.57^a	19.89±11.45^a
SH组（8只）	35.31±11.70	30.07±14.90	32.65±13.09^ab	31.37±14.82^ab	31.64±15.61^ab	26.64±12.39^ab
H组（18只）	37.70±9.72	35.41±13.64	29.40±12.03^ab	24.54±9.35^a	17.67±8.33^a	16.41±8.57^a
SE组（13只）	29.49±9.81	25.61±16.13	23.86±18.96	25.33±13.75^a	19.32±10.24^a	14.24±11.87^ab

表3 不同组别脓毒症大鼠6 d定位航行游泳速度的比较（cm/s，±s）

组别	第1天	第2天	第3天	第4天	第5天	第6天
C组（20只）	19.88±3.49	22.15±4.71	19.73±4.67	20.90±5.30	24.30±5.93	20.99±4.21
S组（11只）	23.23±3.20	23.44±3.73	21.70±5.78	22.73±4.60	23.88±4.45	22.48±4.21
SH组（8只）	20.35±2.88	16.44±3.14	16.65±5.46	14.77±4.59	15.29±5.43	14.32±4.79
H组（18只）	20.43±4.12	20.05±4.08	18.02±3.68	16.03±3.62	16.11±4.15	16.64±3.28
SE组（13只）	17.82±9.50	18.10±2.66	19.24±4.99	17.76±5.25	17.76±4.61	15.39±5.85

表3 不同组别脓毒症大鼠6 d定位航行游泳速度的比较（cm/s，±s）

组别	第1天	第2天	第3天	第4天	第5天	第6天
C组（20只）	19.88±3.49	22.15±4.71	19.73±4.67	20.90±5.30	24.30±5.93	20.99±4.21
S组（11只）	23.23±3.20	23.44±3.73	21.70±5.78	22.73±4.60	23.88±4.45	22.48±4.21
SH组（8只）	20.35±2.88	16.44±3.14	16.65±5.46	14.77±4.59	15.29±5.43	14.32±4.79
H组（18只）	20.43±4.12	20.05±4.08	18.02±3.68	16.03±3.62	16.11±4.15	16.64±3.28
SE组（13只）	17.82±9.50	18.10±2.66	19.24±4.99	17.76±5.25	17.76±4.61	15.39±5.85

图2 不同组别脓毒症大鼠Morris水迷宫空间探索实验的比较

图3 不同组别脓毒症大鼠海马CA1区尼氏染色（×400）

图4 各组实验大鼠海马CA1区神经元细胞计数

图5 不同组别脓毒症大鼠脑水含量的比较

图6 不同组别脓毒症大鼠海马区HMGB1、TNF-α、IL-1β、Iba-1、APP mRNA表达情况的比较

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Effect of ethyl pyruvate on cognitive function in sepsis rats

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