Abstract：OBJECTIVE To investigate the effects and mechanism of Buyang Huanwu Decoction and the like on primary cultured neonate hippocampal neurons induced by oxygen deprivation and reoxygenation. METHODS The primary cultured hippocampal neurons cell model was established, oxygen sugar deprivation/reoxygenation (OGD/R) model was established in vitro. CCK-8 was used to determine the concentration of Buyang Huanwu Decoction and the like drug-containing serum to protect hippocampal neurons cell. The experimental groups were divided into control group, model group, Buyang Huanwu Decoction drug-containing serum group(10%), Naoxintong Capsule drug-containing serum group(10%), Yangyin Tongnao Granules drug-containing serum group(10%), Buyang Huanwu Decoction drug-containing serum+TAK-242 group(10%,1 μmol·L-1), Naoxintong Capsule drug-containing serum+TAK-242 group(10%,1 μmol·L-1), Yangyin Tongnao Granules drug-containing serum+TAK-242 group(10%,1 μmol·L-1). The morphology of hippocampal neuron was observed under inverted microscope. The expression of tumor necrosis factor-α(TNF-α) and interleukin 1(IL-1β) in the cell supernatant was detected by ELISA kit. The neuronal cell apoptosis was observed by Hoechst 33358 staining fluorescence microscope and the expression of TLR4/NF-κB signaling pathway proteins was detected by Western blot. RESULTS The results showed that hgher purity neuronal cells can be obtained by primary culture. Compared with model group, Buyang Huanwu Decoction and the like all can reduce the release of TNF-α and IL-1β in hippocampal neurons, significantly reduced the number of apoptosis. At the same time, the three traditional Chinese medicine compounds all can inhibit the expression of TLR4 and NF-κB protein in TLR4/NF-κB signaling pathway, and protect the inflammatory response of oxygenated sugar deprivation and reoxygenated hippocampal neurons. The addition of TLR4-specific inhibitor TAK-242 blocked the conduction of this signaling pathway and reduced the protective effect of Buyang Huanwu Decoction and the like. CONCLUSION Buyang Huanwu Decoction and the like have protective effects on OGD/R-induced inflammatory response in hippocampal neurons, its mechanism may be related to TLR4/NF-κB signaling pathway, and the anti-inflammatory protective effect of Naoxintong Capsule is relatively obvious.
赵梦迪, 杨洁红, 周惠芬, 虞立, 李畅, 万海同. 补阳还五汤类方对氧糖剥夺再复糖复氧海马神经元细胞的体外保护作用及其机制研究[J]. 中国药学杂志, 2020, 55(9): 728-736.
ZHAO Meng-di, YANG Jie-hong, ZHOU Hui-fen, YU Li, LI Chang, WAN Hai-tong. Effects and Mechanism of Buyang Huanwu Decoction and the like on Oxygen Deprivation and Reoxygenation of Hippocampal Neurons in Vitro. Chinese Pharmaceutical Journal, 2020, 55(9): 728-736.
LUO X. Protective effect of ENT1 on brain tissue after cerebral ischemia-reperfusion in rats [D]. Zunyi:Zunyi Medical College, 2017.
[2]
WEN Y L, TUNG H T, TIN Y H, et al. Neuroprotective effect of paeonol mediates anti-inflammation via suppressing toll-like receptor 2 and toll-like receptor 4 signaling pathways in cerebral ischemia-reperfusion injured rats [J]. Evid Based Complement Alternat Med, 2016,2016:3704647.
[3]
ZHENG L, HAO R L, WEN L L, et al. Complement component C3 promotes cerebral ischemia/reperfusion injury mediated by TLR2/NFκB activation in diabetic mice [J]. Neurochem Res, 2018, 43(8):1599-1607.
[4]
YIN D, ZHOU S, XU X, et al. Dexmedetomidine attenuated early brain injury in rats with subarachnoid haemorrhage by suppressing the inflammatory response: the tlr4/nf-κb pathway and the nlrp3 inflammasome may be involved in the mechanism [J]. Brain Res, 2018, 1698:1-10.
[5]
WANG J, XU Z, CHEN X, et al. Microrna-182-5p attenuates cerebral ischemia-reperfusion injury by targeting toll-like receptor 4 [J]. Biochem Biophys Res Commun, 2018, 505(3):677-684.
[6]
WANG C Y, CHEN L J, HUANG S. Research progress of Toll-like receptor ligands and mechanisms of cerebral ischemic injury [J]. Chin Clin Neuroscie(中国临床神经科学), 2016, 24(4):446-452.
[7]
MEI X Y, WU Y X, ZHOU L. Effect and mechanism of buyang huanwu decoction on denervated muscle atrophy in rats [J]. Chin Pharm J (中国药学杂志), 2014, 49(9):726-730.
[8]
SUN Z, ZHOU L, LI Z L, et al. Chemical constituents of Naoxintong Capsules based on ultra performance liquid chromatography-quadrupole/electrostatic field orbitrap high resolution mass spectrometry [J]. Chin Pharm J (中国药学杂志), 2018, 53(19):1687-1695.
[9]
CHEN K. Clinical observation of Buchang Naoxintong in treating cerebral infarction [J]. World Latest Med Inform Abstracts(世界最新医学信息文摘), 2018, 18(A3):192-193.
[10]
WU Z, GAO W J. Research progress of Buyang Huanwu Decoction in repairing ischemic brain injury through Notch signaling pathway [J]. Hebei J Tradit Chin Med (河北中医药学报), 2018, 33(2):55-58.
[11]
WANG H Y, ZHOU H F, HE Y, et al. Protective effect and mechanism of Naoxintong Capsule on brain microvascular endothelial cells injured by hypoxia and hypoxia [J]. Chin Herb Med (中草药), 2018, 49(14):3318-3325.
[12]
WANG Y, HE Y, YANG J H, et al. Effect of Yangyin Tongnao granule on expression of caspase-3, Bcl-2 and Bax mRNA after cerebral ischemia/reperfusion injury in rats [J]. Chin J Tradit Chin Med (中华中医药杂志), 2018, 33(9):3875-3878.
[13]
LIU J. Effect of dexmedetomidine on hypoxia-reoxygenation injury in rat hippocampal neurons and its mechanism [D]. Qingdao:Qingdao University, 2017.
[14]
DU C H, WAN H T, ZHOU H F, et al. Protective effect of Guhong Injection on hypoxia and hypoglycemia of hippocampal neurons in neonatal rats [J]. Chin J Tradit Chin Med (中华中医药杂志), 2017, 32(1):289-293.
[15]
XIAO Q. Neuroprotective effect and mechanism of ginkgolide B on fetal rat hippocampal neurons [D]. Xian:Fourth Military Medical University, 2011.
[16]
LI Z. Effects of resveratrol on hippocampal neuronal injury in rats with focal cerebral ischemia/reperfusion and its molecular mechanism [D]. Hefei:Anhui Medical University, 2017.
[17]
LING C, LIANG J, ZHANG C, et al. Synergistic effects of salvianolic acid B and puerarin on cerebral ischemia reperfusion injury [J]. Molecules, 2018, 23(3):564.
[18]
ZYZAK J, MATUSZYK J, SIEDNIENKO J. Multilevel maturation of Toll-like receptor 9[J]. Postepy Hig Med Dosw (Online), 2013, 67:1034-1046.
[19]
ANDRADE W A, SOUZA M D, RAMOS M E, et al. Combined action of nucleic acid-sensing toll-like receptors and TLR11/TLR12 heterodimers imparts resistance to toxoplasma gondii in mice[J]. Cell Host Microbe, 2013, 13(1):42-53.
[20]
BRENCICOVA E, DIEBOLD S S. Nucleic acids and endosomal pattern recognition:how to tell friend from foe?[J]. Front Cell Infect Microbiol, 2013, 30:3-37.
[21]
CUI H X, CHEN J H, LI J W, et al. Protection of anthocyanin from myrica rubra against cerebral ischemia-reperfusion injury via modulation of the tlr4/nf-κb and nlrp3 pathways [J]. Molecules, 2018, 23(7):1788.
[22]
CHE H, RUAN Y Y, LUO T Y, et al. Regulation of the NHL protein family by Toll-like receptors in inflammatory response [J]. Acta Physiol Sin(生理学报), 2018, 70(5):521-530.
[23]
YUE S Q, SUN Z Y, LI L, et al. Advances in TLR4-mediated Chinese medicine intervention for cerebral ischemic inflammatory injury [J]. J Tianjin Univ Tradit Chin Med (天津中医药大学学报), 2018, 37(2):173-176.
[24]
WANG X, STRIDH L, LI W, et al. Lipopolysaccharide sensitizes neonatal hypoxic-ischemic brain injury in a MyD88dependent manner [J]. J Immunol, 2009, 183(11):7471-7477.
[25]
DING Y, YANG H, XIANG W, et al. CD200R1 agonist attenuates LPS-induced inflammatory response in human renal proximal tubular epithelial cells by regulating TLR4MyD88-TAK1-mediated NF-κB and MAPK pathway [J]. Biochem Biophys Res Commun, 2015, 460(2):287-294.
[26]
YANG M, AN R D, LI M H, et al. B-caryophyllene alleviates focal cerebral ischemia-reperfusion injury in mice by acting on HMGB1/TLR4/NF-κB pathway [J]. Chin J Immunol (中国免疫学杂志), 2017, 33(7):1009-1013.
[27]
LUO S Y, LI R, LE Z Y, et al. Anfatide protects against rat cerebral ischemia/reperfusion injury via tlr4/jnk/caspase-3 pathway [J]. Eur J Pharmacol, 2017, 807:127-137.
[28]
LU M R, ZHU Z F, ZHANG H P, et al. Correlation between cerebral hemorrhage and TLR4/NF-κB-mediated autophagy in microglia [J]. Stroke Neurol Dis (卒中与神经疾病), 2018, 25(4):373-376.
2020, Vol. 55 
