罗氟司特对大鼠创伤性脑损伤的作用及其机制研究

doi:10.11958/20221943

摘要/Abstract

摘要：

目的探讨罗氟司特（RF）对大鼠创伤性脑损伤（TBI）的作用及其可能的机制。方法按随机数字表法将36只大鼠分为3组：假手术（Sham）组、TBI组、TBI+RF组，每组12只。采用改良Feeney自由落体法建立大鼠TBI模型，TBI+RF组于建模后即刻、1 d、2 d时腹腔注射RF，Sham组和TBI组则予以等量溶剂。建模后1、2、3、7、14 d时对各组大鼠进行改良神经功能缺损严重程度评分（mNSS）。建模后3 d时，HE染色后观察病灶周围皮层神经元病理变化并分析死亡神经元数量，酶联免疫吸附试验（ELISA）检测大鼠损伤侧皮层白细胞介素（IL）-1β、IL-6、肿瘤坏死因子（TNF）-α含量，免疫组化检测大鼠病灶周围皮层NOD样受体热蛋白结构域相关蛋白3（NLRP3）含量。结果与TBI组比较，TBI+RF组的mNSS在建模后3 d内差异无统计学意义，但7 d、14 d时降低（P<0.05）。与Sham组比较，TBI组病灶周围脑组织结构疏松，细胞间质水肿明显，见较多死亡神经元（P<0.05）；TBI+RF组病灶周围仍可见细胞间质水肿，但死亡神经元百分比较TBI组减少（P<0.05）。与Sham组比较，TBI组的IL-1β、IL-6、TNF-α和NLRP3含量均升高（P<0.05）；而经罗氟司特治疗后，大鼠的IL-1β、IL-6、TNF-α和NLRP3含量均降低（P<0.05）。结论罗氟司特可能通过降低创伤性脑损伤早期的炎症介质水平，减轻大鼠创伤性脑损伤，从而起到神经保护作用。

关键词: 脑损伤,创伤性, NLR家族,热蛋白结构域包含蛋白3, 白细胞介素6, 白细胞介素1β, 肿瘤坏死因子α, 罗氟司特

Abstract:

Objective To investigate the effect of roflumilast (RF) on traumatic brain injury (TBI) in rats and its possible mechanism. Methods A total of 36 rats were divided into three groups by random number table method: the sham group, the TBI group and the TBI+RF group, with 12 rats in each group. The TBI model of rat was established by modified Feeney free fall method. Rats in the TBI+RF group were intraperitoneally injected with RF solution immediately, 1 day and 2 day after modeling, while rats in the Sham group and the TBI group were given equal volume solvent. The modified neurological severity score (mNSS) was performed on day 1, day 2, day 3, day 7 and day 14 after modeling. At 3 days after surgery, the pathological changes of neurons and the number of dead neurons in the perifocal cortex of rats were observed by HE staining. The contents of interleukin (IL)-1β, IL-6 and tumor necrosis factor (TNF)-α in the injured cortex of rats were detected by ELISA, and the contents of NOD-like receptor thermal protein domain associated protein 3 (NLRP3) in the perifocal cortex of rats were detected by immunohistochemistry. Results There was no significant difference in mNSS within 3 days after modeling between the TBI+RF group and the TBI group, but mNSS decreased at 7 and 14 days in the TBI+RF group (P<0.05). Compared with the Sham group, the TBI group had loose brain structure around the lesion, significant interstitial edema and more dead neurons (P<0.05). Interstitial edema was still observed around the lesion in the TBI+RF group, but the percentage of dead neurons was decreased compared with that in the TBI group (P<0.05). Compared with the Sham group, the contents of IL-1β, IL-6, TNF-α and NLRP3 were increased in the TBI group (P<0.05). The contents of IL-1β, IL-6, TNF-α and NLRP3 were decreased after RF treatment (P<0.05). Conclusion Roflumilast alleviates traumatic brain injury in rats by reducing the level of inflammatory mediators in the early stage of TBI, thus playing a neuroprotective role.

Key words: brain injuries, traumatic, NLR family, pyrin domain-containing 3 protein, interleukin-6, interleukin-1beta, tumor necrosis factor-alpha, Roflumilast

中图分类号:

R651.15

图/表 5

表1 各组大鼠建模后不同时间点mNSS比较（n=4，分，$\bar{x}\pm s$）

Tab.1 Comparison of mNSS at different time points after modeling between the three groups

组别	建模后时间（d）
组别	1	2	3	7	14
Sham组	1.50±0.58	1.00±0.82	1.25±0.50	1.00±0.82	1.00±0.82
TBI组	13.75±0.96^a	13.50±1.00^a	12.75±1.50^a	11.50±0.58^a	9.25±0.96^a
TBI+RF组	14.00±0.82^a	13.75±0.50^a	12.50±0.58^a	10.00±0.82^ab	7.25±0.96^ab
F	319.696^＊＊	332.739^＊＊	182.735^＊＊	232.200^＊＊	88.900^＊＊

表2 各组大鼠损伤侧皮层IL-1β、IL-6、TNF-α含量比较（n=4，ng/g，$\bar{x}\pm s$）

Tab.2 Comparison of contents of IL-1β, IL-6 and TNF-α in the injured cortex of rats between the three groups

组别	IL-1β	IL-6	TNF-α
Sham组	0.138±0.006	0.329±0.031	1.008±0.031
TBI组	0.174±0.013^a	0.506±0.037^a	1.444±0.127^a
TBI+RF组	0.158±0.004^ab	0.443±0.024^ab	1.286±0.044^ab
F	19.081^＊＊	33.698^＊＊	30.813^＊＊

图1 各组大鼠病灶周围神经元病理变化比较（HE染色）

Fig.1 Comparison of pathological changes of neurons in perifocal cortex of rats between the three groups (HE staining)

图2 各组大鼠病灶周围NLRP3含量比较（免疫组化染色）

Fig.2 Comparison of NLRP3 content around lesion between the three groups (Immunohistochemical staining)

表3 各组大鼠病灶周围皮层中死亡神经元数量和NLRP3含量的比较（n=4，$\bar{x}\pm s$）

Tab.3 Comparison of the number of dead neurons and NLRP3 content in the perifocal cortex of rats between the three groups

组别	死亡神经元（%）	NLRP3阳性表达AOD值
Sham组	23.430±0.933	0.035±0.002
TBI组	51.213±1.590^a	0.055±0.002^a
TBI+RF组	37.198±1.528^b	0.039±0.04^b
F	403.925^＊＊	57.907^＊＊

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