肝豆灵片通过调节TLR4/NF-κB/NLRP3信号通路减轻肝豆状核变性神经炎症的机制

doi:10.3976/j.issn.1002-4026.2023.04.006

Abstract

Abstract:

This study aimed to investigate the effect of Gandouling tablet on neuroinflammation in hepatolenticular degeneration in TX mice and mechanism of generating CuCl₂-induced microglia inflammatory response based on TLR4/NF-κB/NLRP3 signaling pathway. TX mice were divided into control, model, Gandouling tablet low-dose, Gandouling tablet medium-dose, Gandouling tablet high-dose, and penicilamine groups. BV2 cells were divided into control, model, Gandouling tablet, TAK-242, and Gandouling tablet + TAK-242 groups. Hematoxylin and eosin staining was performed to detect histopathological changes in the hippocampus of mice in each group. Western blot was used to detect the expressions of TLR4, p65, NLRP3, and IL-1β in hippocampal tissue and BV2 cells of mice in each group. Enzyme-linked immunosorbent assay was performed to quantify TNF-α, IL-1β, and IL-6 levels in hippocampal tissue and BV2 cells of mice in each group. Real-time polymerase chain reaction was used to determine the expression of TLR4, p65, NLRP3, TNF-α, and IL-1β mRNA in all groups of BV2 cells. Compared with the control group, hippocampal tissue in the model group showed considerable inflammatory damage; increased protein expressions of TLR4, p65, NLRP3, and IL-1β; and significantly increased levels of TNF-α, IL-1β, and IL-6 (P<0.05). Compared with the model group, the pathological damage of hippocampal tissue improved in both Gandouling tablet and penicillamine groups, and the effect of Gandouling tablet in the Gandouling tablet high-dose group was more prominent than that in the other groups. The Gandouling tablet and TAK-242 groups inhibited the activation of BV2 cells. Additionally, the expression of TLR4, p65, NLRP3, and IL-1β protein and mRNA were significantly reduced in these two groups as compared with the model group, and TNF-α, IL-1β and IL-6 levels were significantly decreased (P < 0.05). Gandouling tablet can alleviate hippocampal inflammation and inhibit CuCl₂-induced hyperactivation of BV2 cells in TX mice probably by downregulating TLR4/NF-κB/NLRP3 signaling pathway.

Key words: Gandouling tablet, hepatolenticular degeneration, TLR4/NF-κB/NLRP3, neuroinflammation, action mechanism

CLC Number:

R285

WEN Yuya, DONG Ting, JIANG Zhangsheng, CHEN Jie, TIAN Liwei, ZHAO Chenling, TANG Lulu. The mechanism of Gandouling tablet in alleviating hepatolenticular degeneration neuroinflammation via the regulation of the TLR4/NF-κB/NLRP3 signaling pathway[J].Shandong Science, 2023, 36(4): 42-51.

Figures/Tables 8

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References 22

[1]	DONG T, QIU J, CHENGH D, et al. Impairment of time-based prospective memory in patients with Wilson's disease[J]. European Review for Medical and Pharmacological Sciences, 2016, 20(9): 1845-1851. doi: 10758 pmid: 27212179
[2]	DONG T, YANG W M, WU M C, et al. Microstructure changes in whiter matter relate to cognitive impairment in Wilson's disease[J]. Bioscience Reports, 2019, 39(3): BSR20181651. DOI: 10.1042/BSR20181651. doi: 10.1042/BSR20181651
[3]	DONG J J, WANG X, XU C C, et al. Inhibiting NLRP3 inflammasome activation prevents copper-induced neuropathology in a murine model of Wilson's disease[J]. Cell Death & Disease, 2021, 12(1): 1-12. DOI: 10.1038/s41419-021-03397-1. doi: 10.1038/s41419-021-03397-1
[4]	QIU W Q, AI W, ZHU F D, et al. Polygala saponins inhibit NLRP3 inflammasome-mediated neuroinflammation via SHP-2-mediated mitophagy[J]. Free Radical Biology and Medicine, 2022, 179: 76-94. DOI: 10.1016/j.freeradbiomed.2021.12.263. doi: 10.1016/j.freeradbiomed.2021.12.263
[5]	HU Z Q, YU F X, GONG P, et al. Subneurotoxic copper(II)-induced NF-κB-dependent microglial activation is associated with mitochondrial ROS[J]. Toxicology and Applied Pharmacology, 2014, 276(2): 95-103. DOI: 10.1016/j.taap.2014.01.020. doi: 10.1016/j.taap.2014.01.020 pmid: 24530511
[6]	JU M J, LIU B F, HE H Y, et al. microRNA-27a alleviates LPS-induced acute lung injury in mice via inhibiting inflammation and apoptosis through modulating TLR4/MyD88/NF-κB pathway[J]. Cell Cycle (Georgetown, Tex), 2018, 17(16): 2001-2018. DOI: 10.1080/15384101.2018.1509635. doi: 10.1080/15384101.2018.1509635
[7]	JIN X, LIU M Y, ZHANG D F, et al. Baicalin mitigates cognitive impairment and protects neurons from microglia-mediated neuroinflammation via suppressing NLRP3 inflammasomes and TLR4/NF-κB signaling pathway[J]. CNS Neuroscience & Therapeutics, 2019, 25(5): 575-590. DOI: 10.1111/cns.13086. doi: 10.1111/cns.13086
[8]	韩辉, 郑明翠, 吴丽敏, 等. 肝豆状核变性中医证型与ATP7B基因突变的相关性研究[J]. 中国中西医结合杂志, 2018, 38(7): 799-804. DOI: 10.7661/j.cjim.20180606.136. doi: 10.7661/j.cjim.20180606.136
[9]	许琛林. 双去甲氧基姜黄素激活AMPK/SIRT1通路改善Aβ_1-42对SK-N-SH细胞的毒性作用[D]. 衡阳: 南华大学, 2019.
[10]	赵红晔. 隐丹参酮对SD大鼠重要大脑细胞网络模型氧糖剥夺/复氧糖损伤的干预研究[D]. 大连: 大连医科大学, 2017.
[11]	董婷, 杨文明, 张娟, 等. 肝豆灵对Wilson病患者前瞻性记忆功能的影响[J]. 安徽中医药大学学报, 2018, 37(1): 13-16. DOI: 10.3969/j.issn.2095-7246.2018.01.005. doi: 10.3969/j.issn.2095-7246.2018.01.005
[12]	韩辉, 方向, 吴丽敏, 等. 化痰祛瘀法治疗肝豆状核变性认知功能障碍21例[J]. 安徽中医药大学学报, 2014, 33(1): 21-23. DOI: 10.3969/j.issn.2095-7246.2014.01.009. doi: 10.3969/j.issn.2095-7246.2014.01.009
[13]	DONG T, WU M C, TANG L L, et al. GanDouLing promotes proliferation and differentiation of neural stem cells in the mouse model of Wilson's disease[J]. Bioscience Reports, 2021, 41(1): BSR20202717. DOI: 10.1042/BSR20202717. doi: 10.1042/BSR20202717
[14]	林康, 王共强, 金平. 铜与认知障碍病理机制的研究[J]. 中医药临床杂志, 2017, 29(7): 977-979. DOI: 10.16448/j.cjtcm.2017.0325. doi: 10.16448/j.cjtcm.2017.0325
[15]	李祥, 汪瀚, 胡建鹏, 等. 120例肝豆状核变性合并抑郁症状患者的中医证型及神经递质与炎症因子变化特点研究[J]. 安徽中医药大学学报, 2021, 40(2): 14-18. DOI: 10.3969/j.issn.2095-7246.2021.02.005. doi: 10.3969/j.issn.2095-7246.2021.02.005
[16]	ZHOU Q, ZHANG Y, LU L, et al. Copper induces microglia-mediated neuroinflammation through ROS/NF-κB pathway and mitophagy disorder[J]. Food and Chemical Toxicology, 2022, 168: 113369. DOI: 10.1016/j.fct.2022.113369. doi: 10.1016/j.fct.2022.113369
[17]	HENN A, LUND S, HEDTJÄRN M, et al. The suitability of BV2 cells as alternative model system for primary microglia cultures or for animal experiments examining brain inflammation[J]. ALTEX, 2009, 26(2): 83-94. DOI: 10.14573/altex.2009.2.83. doi: 10.14573/altex.2009.2.83 pmid: 19565166
[18]	RAHIMIFARD M, MAQBOOL F, MOEINI-NODEH S, et al. Targeting the TLR4 signaling pathway by polyphenols: A novel therapeutic strategy for neuroinflammation[J]. Ageing Research Reviews, 2017, 36: 11-19. DOI: 10.1016/j.arr.2017.02.004. doi: S1568-1637(16)30286-0 pmid: 28235660
[19]	ZHU K Y, ZHU X, SUN S H, et al. Inhibition of TLR4 prevents hippocampal hypoxic-ischemic injury by regulating ferroptosis in neonatal rats[J]. Experimental Neurology, 2021, 345: 113828. DOI: 10.1016/j.expneurol.2021.113828. doi: 10.1016/j.expneurol.2021.113828
[20]	MIYAKE K. Innate recognition of lipopolysaccharide bytoll-like receptor 4-MD-2[J]. Trends in Microbiology, 2004, 12(4): 186-192. DOI: 10.1016/j.tim.2004.02.009. doi: 10.1016/j.tim.2004.02.009
[21]	XING Y, YAO X M, LI H, et al. Cutting edge: TRAF₆ mediates TLR/IL-1R signaling-induced nontranscriptional priming of the NLRP3 inflammasome[J]. Journal of Immunology (Baltimore, Md: 1950), 2017, 199(5): 1561-1566. DOI: 10.4049/jimmunol.1700175. doi: 10.4049/jimmunol.1700175
[22]	CUI W G, SUN C L, MA Y Q, et al. Inhibition of TLR4 induces M2 microglial polarization and provides neuroprotection via the NLRP3 inflammasome in alzheimer's disease[J]. Frontiers in Neuroscience, 2020, 14: 444. DOI: 10.3389/fnins.2020.00444. doi: 10.3389/fnins.2020.00444 pmid: 32508567

基因名称	引物序列	产物长度/bp
TLR4	上游5'- AATGAGGACTGGGTGAGAAATGAG-3' 下游5'-CTGTAGTGAAGGCAGAGGTGAAAG-3'	80
p65	上游5'-TCCTGTTCGAGTCTCCATGCAG-3' 下游5'-GGTCTCATAGGTCCTTTTGCGC-3'	133
NLRP3	上游5'-TCACAACTCGCCCAAGGAGGAA-3' 下游5'-AAGAGACCACGGCAGAAGCTAG-3'	147
TNF-α	上游5'- GGGTTGTACCTTGTCTACTCCCA-3' 下游5'-GAGATAGCAAATCGGCTGACG-3'	95
IL-1β	上游5'-TGGACCTTCCAGGATGAGGACA-3' 下游5'-GTTCATCTCGGAGCCTGTAGTG-3'	148
GAPDH	上游5'-GTGTTCCTACCCCCAATGTG-3' 下游5'-GTCATTGAGAGCAATGCCAG-3'	215

The mechanism of Gandouling tablet in alleviating hepatolenticular degeneration neuroinflammation via the regulation of the TLR4/NF-κB/NLRP3 signaling pathway

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