桑黄素通过抑制TXNIP/NLRP3/Caspase-1信号通路对脑缺血再灌注大鼠神经元凋亡的影响

doi:10.11958/20221258

摘要/Abstract

摘要：

目的探讨桑黄素（Morin）通过调控硫氧还蛋白互作蛋白（TXNIP）/核苷酸结合寡聚化结构域样受体蛋白3（NLRP3）/胱天蛋白酶1（Caspase-1）信号通路，对脑缺血再灌注大鼠神经元凋亡的影响。方法将60只大鼠随机均分为假手术组、模型组、Morin低剂量（Morin-L，10 mg/kg）组、Morin高剂量（Morin-H，40 mg/kg）组、Morin-H+pLVX-NC组（40 mg/kg Morin+ pLVX-NC）、Morin-H+pLVX-TXNIP组（40 mg/kg Morin+pLVX-TXNIP）。采用线栓法制作大鼠大脑中动脉栓塞模型（除外假手术组），给药1 h后再灌注。再灌注72 h后，进行神经功能缺损评分；取出全脑进行脑含水量测定；苏木精-伊红（HE）染色观察脑皮质半暗带病理损伤情况；2,3,5-氯化三苯基四氮唑（TTC）检测脑组织梗死面积；酶联免疫吸附试验检测血清中白细胞介素（IL）-1β、IL-18含量；Western blot检测脑皮质中TXNIP/NLRP3/Caspase-1信号通路蛋白表达水平。结果与假手术组比较，模型组神经功能缺损评分，IL-1β、IL-18水平，脑含水量，梗死面积，TXNIP、NLRP3、Caspase-1蛋白表达均增加（P<0.05）；与模型组比较，Morin-L组、Morin-H组神经功能缺损评分，IL-1β、IL-18水平，脑含水量，梗死面积，TXNIP、NLRP3、Caspase-1蛋白表达均降低（P<0.05）；与Morin-H+pLVX-NC组比较，Morin-H+pLVX-TXNIP组神经功能缺损评分，IL-1β、IL-18水平，脑含水量，梗死面积，TXNIP、NLRP3、Caspase-1蛋白表达增加（P<0.05）。结论 Morin可以减轻脑缺血再灌注大鼠脑损伤，抑制神经元凋亡，作用机制可能与抑制TXNIP/NLRP3/Caspase-1信号通路活化有关。

关键词: 再灌注损伤, 缺氧缺血,脑, 疾病模型,动物, 桑黄素, TXNIP/NLRP3/Caspase-1信号通路, 神经元凋亡

Abstract:

Objective To investigate the influence of Morin on neuronal apoptosis in cerebral ischemia-reperfusion rats by regulating TXNIP/NLRP3/Caspase-1 signaling pathway. Methods Sixty rats were randomly grouped into the sham operation group, the model group, the Morin low-dose group (Morin-L, 10 mg/kg), the Morin high-dose group (Morin-H, 40 mg/kg), the Morin-H+pLVX-NC group (40 mg/kg Morin+pLVX-NC) and Morin-H+pLVX-TXNIPgroup (pLVX-TXNIP, 40 mg/kg Morin+pLVX-TXNIP). The rat middle cerebral artery embolism model was established by the suture method (except for the sham group), and then perfused after 1 h of administration. After 72 hours of perfusion, neurological deficits were scored. The whole brain was removed for brain edema determination. Hematoxylin-Eosin (HE) staining was performed to observe the pathological damage in the penumbra area of brain tissue. TTC was performed to detect the infarct size of brain tissue. ELISA was performed to detect serum contents of IL-1β and IL-18. Western blot assay was used to detect expression levels of TXNIP/NLRP3/Caspase-1 signaling pathway-related proteins in brain tissue. Results Compared with the sham operation group, the neurological deficit score, contents of IL-1β and IL-18, brain water content, infarct size, expression levels of TXNIP, NLRP3 and Caspase-1 proteins were increased in the model group (P<0.05). Compared with the model group, the neurological deficit score, contents of IL-1β and IL-18, brain water content, infarct size, expression levels of TXNIP, NLRP3 and Caspase-1 proteins were decreased in the Morin-L group and the Morin-H group (P<0.05). Compared with Morin-H+pLVX-NC group, the neurological deficit score, contents of IL-1β and IL-18, brain water content, infarct size, expression levels of TXNIP, NLRP3 and Caspase-1 proteins were increased in the Morin-H+pLVX-TXNIP group (P<0.05). Conclusion Morin can reduce brain injury and inhibit neuronal apoptosis in rats with cerebral ischemia-reperfusion, which may be related to the inhibition of TXNIP/NLRP3/Caspase-1 signaling pathway.

Key words: reperfusion injury, hypoxia-ischemia, brain, disease models, animal, Samlutein, TXNIP/NLRP3/Caspase-1 signaling pathway, apoptosis of neuron

中图分类号:

R543.4

图/表 6

表1 各组血清中IL-1β、IL-18水平的比较（n=10，ng/L，$\bar{x}\pm s$）

Tab.1 Comparison of serum levels of IL-1β and IL-18 between the six groups

组别	IL-1β	IL-18
假手术组	10.27±1.03	75.15±7.52
模型组	29.12±2.92^a	138.52±13.86^a
Morin-L组	22.04±2.21^b	106.37±10.64^b
Morin-H组	15.34±1.54^bc	84.15±8.42^bc
Morin-H+pLVX-NC组	15.27±1.53	84.28±8.43
Morin-H+pLVX-TXNIP组	24.35±2.44^de	115.68±11.57^de
F	115.929^＊＊	54.256^＊＊

图1 各组大鼠大脑皮质半暗带组织病理学变化（HE，×400）

Fig.1 Histopathological changes of cerebral penumbra in each group of rats (HE, ×400)

图2 脑组织梗死面积变化（TTC染色，×100） A：假手术组；B：模型组；C：Morin-L组；D：Morin-H组；E：Morin-H+pLVX-NC组；F：Morin-H+pLVX-TXNIP组。

Fig.2 Changes in infarct size of brain tissue (TTC staining, ×100)

表2 各组脑组织梗死面积和脑含水量比较（n=3，$\bar{x}\pm s$）

Tab.2 Comparison of infarct size of brain tissue and brain water content between six groups of rats

组别	梗死面积（%）	脑含水量（%）
假手术组	0.00±0.00	65.12±3.01
模型组	21.05±2.11^a	85.34±3.55^a
Morin-L组	13.21±1.33^b	75.66±2.76^b
Morin-H组	8.51±0.86^bc	67.43±2.34^bc
Morin-H+pLVX-NC组	8.48±0.85	68.32±2.28
Morin-H+pLVX-TXNIP组	15.64±1.57^de	82.54±2.88^de
F	92.531^＊＊	26.751^＊＊

图3 各组TXNIP、NLRP3、Caspase-1蛋白表达（Western blot图） A：假手术组；B：模型组；C：Morin-L组；D：Morin-H组；E：Morin-H+pLVX-NC组；F：Morin-H+pLVX-TXNIP组。

Fig.3 Expression of TXNIP, NLRP3 and Caspase-1 proteins in each group (Western blot assay)

表3 各组TXNIP、NLRP3、Caspase-1蛋白表达水平比较（n=4，$\bar{x}\pm s$）

Tab.3 Comparison of TXNIP, NLRP3 and Caspase-1 protein expression between six groups of rats

组别	TXNIP	NLRP3	Caspase-1
假手术组	0.25±0.03	0.17±0.02	0.34±0.04
模型组	1.19±0.12^a	0.85±0.09^a	1.12±0.12^a
Morin-L组	0.63±0.07^b	0.54±0.06^b	0.77±0.08^b
Morin-H组	0.34±0.04^bc	0.24±0.03^bc	0.39±0.04^bc
Morin-H+pLVX-NC组	0.37±0.04	0.25±0.03	0.37±0.04
Morin-H+pLVX-TXNIP组	0.71±0.08^de	0.66±0.07^de	0.74±0.08^de
F	96.979^＊＊	95.663^＊＊	72.103^＊＊

参考文献 18

[1]	SHAO Z Q, DOU S S, ZHU J G, et al. Apelin-13 inhibits apoptosis and excessive autophagy in cerebral ischemia/reperfusion injury[J]. Neural Regen Res, 2021, 16(6):1044-1051. doi:10.4103/1673-5374.300725.
[2]	刘海颖, 冯子人, 孙辉, 等. 大鼠脑缺血再灌注早期过氧化物酶体增殖物激活受体γ活化与细胞焦亡的关系[J]. 天津医药, 2020, 48(1):34-37.
	LIU H Y, FENG Z R, SUN H, et al. Relationship between activation of peroxisome proliferator-activated receptor γ and cell apoptosis in the early stage of cerebral ischemia-reperfusion in rats[J]. Tianjin Med J, 2020, 48(1):34-37. doi:10.11958/20192153.
[3]	LIANG T Y, PENG S Y, MA M, et al. Protective effects of sevoflurane in cerebral ischemia reperfusion injury:a narrative review[J]. Med Gas Res, 2021, 11(4):152-154. doi:10.4103/2045-9912.318860.
[4]	HAN L, XI G, GUO N, et al. Expression and mechanism of TXNIP/NLRP3 inflammasome in sciatic nerve of type 2 diabetic rats[J]. Dis Markers, 2022, 2022:9696303. doi:10.1155/2022/9696303.
[5]	HEEBA G H, RABIE E M, ABUZEID M M, et al. Morin alleviates fructose-induced metabolic syndrome in rats viaameliorating oxidative stress,inflammatory and fibrotic markers[J]. Korean J Physiol Pharmacol, 2021, 25(3):177-187. doi:10.1002/jnr.24385.
[6]	XING F, LIU Y, DONG R, et al. miR-374 improves cerebral ischemia reperfusion injury by targeting Wnt5a[J]. Exp Anim, 2021, 70(1):126-136. doi:10.1538/expanim.20-0034.
[7]	张泽莲, 刘培俊, 陈娟, 等. 桑黄素通过抑制HMGB1/TLR4/NF-κB通路改善重症肺炎大鼠肺损伤[J]. 免疫学杂志, 2022, 38(6):478-486.
	ZHANG Z L, LIU P J, CHEN J, et al. Morgenin improves lung injury in severe pneumonia rats by inhibiting HMGB1/TLR4/NF-κB pathway[J]. Immunol J, 2022, 38(6):478-486.
[8]	潘琼, 郭科, 李亚倩, 等. TXNIP介导的氧化应激在雌激素延缓阿尔兹海默病中的作用[J]. 中南大学学报(医学版), 2019, 44(12):1360-1366.
	PAN Q, GUO K, LI Y Q, et al. The role of TXNIP-mediated oxidative stress in estrogen delaying Alzheimer's disease[J]. J Cent South Univ(Med Sci), 2019, 44(12):1360-1366.
[9]	GUO H, ZHU L, TANG P, et al. Carthamin yellow improves cerebral ischemia-reperfusion injury by attenuating inflammation and ferroptosis in rats[J]. Int J Mol Med, 2021, 47(4):52. doi:10.3892/ijmm.2021.4885.
[10]	LI J, PENG L, BAI W, et al. Biliverdin protects against cerebral ischemia/reperfusion injury by regulating the miR-27a-3p/rgs1 axis[J]. Neuropsychiatr Dis Treat, 2021, 17:1165-1181. doi:10.2147/NDT.S300773.
[11]	ZHANG C, ZHEN L, FANG Z, et al. Adiponectin treatment attenuates cerebral ischemia-reperfusion injury through HIF-1α-mediated antioxidation in mice[J]. Oxid Med Cell Longev, 2021, 2021:5531048. doi:10.1155/2021/5531048.
[12]	植天道, 黄齐慧. 桑色素的研究进展[J]. 中国中医药现代远程教育, 2009, 7(3):112-115.
	ZHI T D, HUANG Q H. Research progress of morin[J]. Chin Med Mod Distance Educat Chin, 2009, 7(3):112-115.
[13]	PARK H J, PARK J N, YOON S Y, et al. Morin disrupts cytoskeletonreorganization in osteoclasts through an ROS/SHP1/c-Src axis and grants protection from LPS-induced bone loss[J]. Antioxidants (Basel), 2022, 11(5):963. doi:10.3390/antiox11050963.
[14]	CHEN Y, LI Y, XU H, et al. Morin mitigates oxidative stress,apoptosis and inflammation in cerebral ischemic rats[J]. Afr J Tradit Complement Altern Med, 2017, 14(2):348-355. doi:10.21010/ajtcam.v14i2.36.
[15]	KHAMCHAI S, CHUMBOATONG W, HATA J, et al. Morin protects the blood-brain barrier integrity against cerebral ischemia reperfusion through anti-inflammatory actions in rats[J]. Sci Rep, 2020, 10(1):13379. doi:10.1038/s41598-020-70214-8.
[16]	ZHANG C R, ZHU W N, TAO W, et al. Moxibustion against cyclophosphamide-induced premature ovarian failure in rats through inhibiting NLRP3-/Caspase-1-/GSDMD-Dependent pyroptosis[J]. Evid Based Complement Alternat Med, 2021, 2021:8874757. doi:10.1155/2021/8874757.
[17]	CHEN D, DIXON B J, DOYCHEVA D M, et al. IRE1α inhibition decreased TXNIP/NLRP3 inflammasome activation through miR-17-5p after neonatal hypoxic-ischemic brain injury in rats[J]. J Neuroinflammation, 2018, 15(1):32. doi:10.1186/s12974-018-1077-9.
[18]	ZHOU Y, CHEN Z, YANG X, et al. Morin attenuates pyroptosis of nucleus pulposus cells and ameliorates intervertebral disc degeneration via inhibition of the TXNIP/NLRP3/Caspase-1/IL-1β signaling pathway[J]. Biochem Biophys Res Commu, 2021, 559:106-112. doi:10.1016/j.bbrc.2021.04.090.