Action mechanism of Coptidis Rhizoma Alkaloids against cerebral ischemia based on transcriptome sequencing

doi:10.12307/2025.063

Abstract

Abstract: BACKGROUND: Coptis chinensis can clear heat, dry dampness, relieve fire, and detoxify. Coptis chinensis and its components have a significant protective effect on cerebral ischemia. The action mechanism of anti-cerebral ischemia of Coptidis Rhizoma Alkaloids was explored based on network pharmacology and transcriptome sequencing.
OBJECTIVE: Based on the study of the protective effects of Coptidis Rhizoma Alkaloids on cerebral ischemia of rats, the action mechanism of Coptidis Rhizoma Alkaloids intervention in cerebral ischemia was investigated by using network pharmacology and transcriptome sequencing technology.
METHODS: The SD rats were randomly divided into sham operation group, ischemia/reperfusion group, positive drug group, and Coptidis Rhizoma Alkaloids group. The ischemia/reperfusion model of middle cerebral artery occlusion was prepared by modified thread method in the latter three groups. No thread was inserted and the other operations were the same in the sham operation group. TTC staining, Longa 5 neurological deficient score, hematoxylin and eosin staining, and Nissl staining were used to evaluate the protective effect of Coptidis Rhizoma Alkaloids on ischemia/reperfusion model rats. Transcriptome sequencing was performed on the brain tissues of rats in sham operation group, ischemia/reperfusion group, and Coptidis Rhizoma Alkaloids group. Differentially expressed genes, gene Ontology analysis, Kyoto encyclopedia of genes and genomes analysis, and Correlation Analysis of Transcriptomics and Network Pharmacology were used to elucidate the effect of Coptidis Rhizoma Alkaloids on cerebral ischemia. Finally, ELISA and immunofluorescence staining were used to verify the key targets of Coptidis Rhizoma Alkaloids in the intervention of cerebral ischemia.
RESULTS AND CONCLUSION: (1) Coptidis Rhizoma Alkaloids treatment decreased the Longa 5 neurological deficit scores and cerebral infarction area of ischemia/reperfusion model rats, increased the number of neurons and Nissl bodies. (2) Differentially expressed gene after Coptidis Rhizoma Alkaloids treatment analyzed by functional enrichment analysis of gene ontology includes biological processes such as inflammatory reaction and positive regulation of mitogen-activated protein kinase cascade. The enrichment analysis of Kyoto gene and genome encyclopedia analysis pathway mainly involves interleukin-17 signaling pathway, neuroactive ligand-receptor interaction, cyclic adenosine-3′,5′-mconophosphate signaling pathway and so on. (3) Analysis of transcriptomics showed that the main genes regulated by Coptidis Rhizoma Alkaloids were prostaglandin endoperoxide synthase 2, brain derived neurotrophic factor, and transient receptor potential A1. (4) Network pharmacology analysis revealed that nine components in Coptidis Rhizoma Alkaloids may exert their effects by associating with 87 targets related to prostaglandin endoperoxide synthase 2, brain derived neurotrophic factor, and transient receptor potential A1. (5) ELISA and immunofluorescence staining results further confirmed that Coptidis Rhizoma Alkaloids regulated the expression of prostaglandin endoperoxide synthase 2, brain derived neurotrophic factor, and transient receptor potential A1. (6) It is concluded that Coptidis Rhizoma Alkaloids treatment can significantly improve the injury in ischemia/reperfusion model rats, possibly by regulating prostaglandin endoperoxide synthase 2, brain derived neurotrophic factor, and transient receptor potential A1.

Key words: Coptidis Rhizoma Alkaloids, cerebral ischemia, transcriptome sequencing, prostaglandin endoperoxide synthase 2, brain derived neurotrophic factor, transient receptor potential A1

CLC Number:

Tian Liangliang, Zhou Rui, Cao Guangzhao, Zhang Jingjing. Action mechanism of Coptidis Rhizoma Alkaloids against cerebral ischemia based on transcriptome sequencing[J]. Chinese Journal of Tissue Engineering Research, 2025, 29(19): 4161-4171.

Figures/Tables 8

References

[1] ZHANG JJ,GUO FF, ZHOU R, et al. Proteomics and transcriptome reveal the key transcription factors mediating the protection of Panax notoginseng saponins (PNS) against cerebral ischemia/reperfusion injury. Phytomedicine. 2021;92:153613.
[2] BOEHME AK, ESENWA C, ELKIND MS. Stroke Risk Factors, Genetics, and Prevention. Circ Res. 2017;120(3):472-495.
[3] CAMPBELL BCV, DE SILVA DA, MACLEOD MR, et al. Ischaemic stroke. Nat Rev Dis Primers. 2019; 5(1):70.
[4] FU Y, LIU Q, ANRATHER J, et al. Immune interventions in stroke. Nat Rev Neurol. 2015; 11(9):524-535.
[5] SAINI V, GUADA L, YAVAGAL DR. Global Epidemiology of Stroke and Access to Acute Ischemic Stroke Interventions. Neurology. 2021;97(20 Supplement 2):S6-S16.
[6] 石晓花, 莽靖, 徐忠信. 脑缺血再灌注损伤细胞死亡模式的研究进展[J]. 吉林大学学报(医学版),2022,48(6):1635-1643.
[7] ZHENG P, ZHANG N, REN D, et al. Integrated spatial transcriptome and metabolism study reveals metabolic heterogeneity in human injured brain. Cell Rep Med. 2023;4(6): 101057.
[8] ZHENG K, LIN L, JIANG W, et al. Single-cell RNA-seq reveals the transcriptional landscape in ischemic stroke. J Cereb Blood Flow Metab. 2022;42(1):56-73.
[9] 丁实, 赵学荣, 赵亮, 等. 黄连素通过B细胞淋巴瘤-2/自噬相关基因复合体抑制自噬减轻脑缺血再灌注损伤的研究[J]. 中国临床药理学杂志,2021,37(9):1094-1097.
[10] HU J, CHAI Y, WANG Y, et al. PI3K p55γ promoter activity enhancement is involved in the anti-apoptotic effect of berberine against cerebral ischemia–reperfusion. Eur J Pharmacol. 2012;674(2):132-142.
[11] KIM M, KIM TW, KIM CJ, et al. Berberine Ameliorates Brain Inflammation in Poloxamer 407-Induced Hyperlipidemic Rats. Int Neurourol J. 2019;23(Suppl 2):S102-S110.
[12] CHENG F, WANG Y, LI J, et al. Berberine improves endothelial function by reducing endothelial microparticles-mediated oxidative stress in humans. Int J Cardiol. 2013;167(3): 936-942.
[13] SARNA LK, WU N, HWANG SY, et al. Berberine inhibits NADPH oxidase mediated superoxide anion production in macrophages. Can J Physiol Pharmacol. 2010;88(3):369-378.
[14] ZHOU R, GUO F, XIANG C, et al. Systematic Study of Crucial Transcription Factors of Coptidis rhizoma Alkaloids against Cerebral Ischemia-Reperfusion Injury. ACS Chem Neurosci. 2021;12(13):2308-2319.
[15] CHEN HY, YE XL, CUI XL, et al. Cytotoxicity and antihyperglycemic effect of minor constituents from Rhizoma Coptis in HepG2 cells. Fitoterapia. 2012;83(1):67-73.
[16] 李淑琴, 朱嘉宝, 武宇洲. 银杏叶提取物防治心脑血管疾病的研究进展[J]. 中国新药杂志,2016,25(1):76-81.
[17] ZHANG JJ, GUO FF, ZHOU R, et al. Proteomics and transcriptome reveal the key transcription factors mediating the protection of Panax notoginseng saponins (PNS) against cerebral ischemia/reperfusion injury. Phytomedicine. 2021:153613. doi: 10.1016/j.phymed.2021.153613.
[18] ZHANG JJ, ZHOU R, CAO GZ, et al. Guhong Injection Prevents Ischemic Stroke-Induced Neuro-Inflammation and Neuron Loss Through Regulation of C5ar1. Front Pharmacol. 2022; 13:818245.
[19] LIU R, DIAO J, HE S, et al. XQ-1H protects against ischemic stroke by regulating microglia polarization through PPARγ pathway in mice. Int Immunopharmacol. 2018;57:72-81.
[20] XU D, HOU K, LI F, et al. XQ-1H alleviates cerebral ischemia in mice through inhibition of apoptosis and promotion of neurogenesis in a Wnt/β-catenin signaling dependent way. Life Sci. 2019;235:116844.
[21] WANG Y, PEI DS, JI HX, et al. Protective effect of a standardized Ginkgo extract (ginaton) on renal ischemia/reperfusion injury via suppressing the activation of JNK signal pathway. Phytomedicine. 2008;15(11):923-931.
[22] LO EH, DALKARA T, MOSKOWITZ MA. Mechanisms, challenges and opportunities in stroke. Nat Rev Neurosci. 2003;4(5):399-415.
[23] MOSKOWITZ MA, LO EH, IADECOLA C. The Science of Stroke: Mechanisms in Search of Treatments. Neuron. 2010;67(2):181-198.
[24] 巩婷婷, 司凯, 刘会平, 等. MAPK级联调控细胞生长及其在免疫,炎症及癌症中作用的研究进展[J]. 中南大学学报(医学版), 2022, 47(12):1721-1728.
[25] 许川山, 余茜, 吴士明, 等. MAPK级联传导途径与脑缺血性损害[J]. 现代康复,2001 (13):62-63.
[26] ZHANG M, GONG JX, WANG JL, et al. p38 MAPK Participates in the Mediation of GLT-1 Up-regulation During the Induction of Brain Ischemic Tolerance by Cerebral Ischemic Preconditioning. Mol Neurobiol. 2016;54(1): 1-14.
[27] GELDERBLOM M, WEYMAR A, BERNREUTHER C, et al. Neutralization of the IL-17 axis diminishes neutrophil invasion and protects from ischemic stroke. Blood. 2012;120(18): 3793-3802.
[28] SUN L, ZHANG H, WANG W, et al. Astragaloside IV Exerts Cognitive Benefits and Promotes Hippocampal Neurogenesis in Stroke Mice by Downregulating Interleukin-17 Expression via Wnt Pathway. Front Pharmacol. 2020;11: 421.
[29] ZHANG Q, LUO T, YUAN D, et al. Retraction Note: Qilongtian ameliorate bleomycin-induced pulmonary fibrosis in mice via inhibiting IL-17 signal pathway. Sci Rep. 2023;13(1):11263.
[30] 潘玲珍,闫智勇,左长英,等.长期使用地西泮对神经活性配体受体相互作用信号通路的影响[J]. 中国药科大学学报,2011, 42(5):443-446.
[31] ZHANG S, WANG X, YANG Q, et al. Isopropyl 3-(3,4-dihydroxyphenyl)-2-hydroxypropanoate plays an anti-hypoxic role through regulating neuroactive ligand-receptor interaction signaling pathway in larval zebrafish. Biomed Pharmacother. 2023;161:114570.
[32] ZAN XY, LI L. Construction of lncRNA-mediated ceRNA network to reveal clinically relevant lncRNA biomarkers in glioblastomas. Oncol Lett. 2019;17(5):4369-4374.
[33] HU S, CAO Q, XU P, et al. Rolipram stimulates angiogenesis and attenuates neuronal apoptosis through the cAMP/cAMP-responsive element binding protein pathway following ischemic stroke in rats. Exp Ther Med. 2016;11(3):1005-1010.
[34] LIU C, DU L, ZHANG S, et al. Network pharmacology and experimental study of phenolic acids in salvia miltiorrhiza bung in preventing ischemic stroke. Front Pharmacol. 2023;14:1108518.
[35] WANG YL, ZHU XL, SUN MH, et al. Effects of astaxanthin onaxonal regeneration via cAMP/PKA signaling pathway in mice with focal cerebral infarction. Eur Rev Med Pharmacol Sci. 2019;23(3 Suppl):135-143.
[36] ZHOU XQ, ZENG XN, KONG H, et al. Neuroprotective effects of berberine on stroke models in vitro and in vivo. Neurosci Lett. 2008;447(1):31-36.
[37] ZHU JR, LU HD, GUO C, et al. Berberine attenuates ischemia–reperfusion injury through inhibiting HMGB1 release and NF-κB nuclear translocation. Acta Pharmacologica Sinica. 2018;39(11):1706-1715.
[38] JIN X, WANG RH, WANG H, et al. Brain protection against ischemic stroke using choline as a new molecular bypass treatment. Acta Pharmacologica Sinica. 2015;36(12):1416-1425.
[39] WANG J, TANG M, XIE X, et al. Efficacy of ferulic acid in the treatment of acute ischemic stroke injury in rats: a systematic review and meta-analysis. Front Pharmacol. 2023; 14: 1278036.
[40] ZHONG F, CHEN Y, CHEN J, et al. Jatrorrhizine: A Review of Sources, Pharmacology, Pharmacokinetics and Toxicity. Front Pharmacol. 2022;12:783127.
[41] LIANG H, CHANG X, XIA R, et al. Magnoflorine attenuates cerebral ischemia-induced neuronal injury via autophagy/sirt1/AMPK signaling pathway. Evid Based Complement Alternat Med. 2022;2022:2131561.
[42] TANG C, HONG J, HU C, et al. Palmatine protects against cerebral ischemia/reperfusion injury by activation of the AMPK/Nrf2 pathway. Oxid Med Cell Longev. 2021;2021:6660193.
[43] PEREIRA JF, DE SOUSA NEVES JC, FONTELES AA, et al. Palmatine, a natural alkaloid, attenuates memory deficits and neuroinflammation in mice submitted to permanent focal cerebral ischemia. J Neuroimmunol. 2023;381:578131.
[44] COLAIZZO D, FOFI L, TISCIA G, et al. The COX-2 G/C− 765 polymorphism may modulate the occurrence of cerebrovascular ischemia. Blood Coagul Fibrinolysis. 2006;17(2):93-96.
[45] LIN W, WANG Y, CHEN Y, et al. Role of calcium signaling pathway-related gene regulatory networks in ischemic stroke based on multiple WGCNA and single-cell analysis. Oxid Med Cell Longev. 2021;2021:8060477.
[46] ZHOU Z, LU C, MENG S, et al. Silencing of PTGS2 exerts promoting effects on angiogenesis endothelial progenitor cells in mice with ischemic stroke via repression of the NF-κB signaling pathway. J Cell Physiol. 2019;234(12):23448-23460.
[47] CUI Q, ZHANG YL, MA YH, et al. A network pharmacology approach to investigate the mechanism of Shuxuening injection in the treatment of ischemic stroke. J Ethnopharmacol. 2020;257:112891.
[48] 尚津锋, 焦家康, 李倩楠, 等. 白杨素通过抑制铁死亡减轻大鼠脑缺血再灌注损伤[J]. 中国中药杂志,2023,48(6): 597-1605.
[49] REX D, DAGAMAJALU S, GOUDA MM, et al. A comprehensive network map of IL-17A signaling pathway. J Cell Commun Signal. 2023; 17(1):209-215.
[50] LI JK, NIE L, ZHAO YP, et al. IL-17 mediates inflammatory reactions via p38/c-Fos and JNK/c-Jun activation in an AP-1-dependent manner in human nucleus pulposus cells. J Transl Med. 2016:14:77.
[51] BALKAYA M, CHO S. Genetics of stroke recovery: BDNF val66met polymorphism in stroke recovery and its interaction with aging. Neurobiol Dis. 2019;126:36-46.
[52] BÉJOT Y, MOSSIAT C, GIROUD M, et al. Circulating and Brain BDNF Levels in Stroke Rats. Relevance to Clinical Studies. PLOS ONE. 2011;6(12):e29405.
[53] DI LAZZARO V, PELLEGRINO G, DI PINO G, et al. Val66Met BDNF gene polymorphism influences human motor cortex plasticity in acute stroke. Brain Stimul. 2015;8(1):92-96.
[54] 何恩浩, 曾燕, 陈星星. Hap1在神经系统疾病中的研究进展[J]. 生命科学,2023,35(5): 592-600.
[55] PLOUGHMAN M, ATTWOOD Z, WHITE N, et al. Endurance exercise facilitates relearning of forelimb motor skill after focal ischemia. Eur J Neurosci. 2007;25(11):3453-3460.
[56] ZHAO D, ZHANG M, YANG L, et al. GPR68 improves nerve damage and myelination in an immature rat model induced by sevoflurane anesthesia by activating cAMP/CREB to mediate BDNF. ACS Chem Neurosci. 2022;13(3):423-431.
[57] EARLEY S, GONZALES AL, CRNICH R. Endothelium-dependent cerebral artery dilation mediated by TRPA1 and Ca2+-Activated K+ channels. Circ Res. 2009;104(8):987-994.
[58] PIRES PW, EARLEY S. Neuroprotective effects of TRPA1 channels in the cerebral endothelium following ischemic stroke. eLife. 2018;7:e35316.
[59] PIRES PW, SULLIVAN MN, EARLEY S. Endothelial TRPA1 Channel is Protective Against Ischemia and Hemorrhagic Strokes in Mice. Stroke. 2016;47(suppl_1):A141-A141.