“Baihui” (DU20)-penetrating “Qubin” (GB7) acupuncture on blood–brain barrier integrity in rat intracerebral hemorrhage models via the RhoA/ROCK II/MLC 2 signaling pathway

Abstract

Background: Blocking the RhoA/ROCK II/MLC 2 (Ras homolog gene family member A/Rho kinase II/myosin light chain 2) signaling pathway can initiate neuroprotective mechanisms against neurological diseases such as stroke, cerebral ischemia, and subarachnoid hemorrhage. Nevertheless, it is not clear whether and how disrupting the RhoA/ROCK II/MLC 2 signaling pathway changes the pathogenic processes of the blood–brain barrier (BBB) after intracerebral hemorrhage (ICH). The present investigation included the injection of rat caudal vein blood into the basal ganglia area to replicate the pathophysiological conditions caused by ICH.

Methods: Scalp acupuncture (SA) therapy was performed on rats with ICH at the acupuncture point “Baihui”-penetrating “Qubin,” and the ROCK selective inhibitor fasudil was used as a positive control to evaluate the inhibitory effect of acupuncture on the RhoA/ROCK II/MLC 2 signaling pathway. Post-assessments included neurological deficits, brain edema, Evans blue extravasation, Western blot, quantitative polymerase chain reaction, and transmission electron microscope imaging.

Results: We found that ROCK II acts as a promoter of the RhoA/ROCK II/MLC 2 signaling pathway, and its expression increased at 6 h after ICH, peaked at 3 days, and then decreased at 7 days after ICH, but was still higher than the pre-intervention level. According to some experimental results, although 3 days is the peak, 7 days is the best time point for acupuncture treatment. Starting from 6 h after ICH, the neurovascular structure and endothelial cell morphology around the hematoma began to change. Based on the changes in the promoter ROCK II, a 7-day time point was selected as the breakthrough point for treating ICH model rats in the main experiment. The results of this experiment showed that both SA at “Baihui”-penetrating “Qubin” and treatment with fasudil could improve the expression of endothelial-related proteins by inhibiting the RhoA/ROCK II/MLC 2 signaling pathway and reduce neurological dysfunction, brain edema, and BBB permeability in rats.

Conclusion: This study found that these experimental data indicated that SA at “Baihui”-penetrating “Qubin” could preserve BBB integrity and neurological function recovery after ICH by inhibiting RhoA/ROCK II/MLC 2 signaling pathway activation and by regulating endothelial cell–related proteins.

Keywords

blood–brain barrier / caveolae / intracerebral hemorrhage / RhoA/ROCK II/MLC 2 signaling pathway / scalp acupuncture

References

[1] Cordonnier C, Demchuk A, Ziai W, Anderson CS. Intracerebral haemorrhage: current approaches to acute management [published correction appears in lancet. 2019 Feb 2;393(10170):406]. Lancet. 2018;392(10154):1257-1268.

[2] Chen Y, Chen S, Chang J, Wei J, Feng M, Wang R. Perihematomal edema after intracerebral hemorrhage: an update on pathogenesis, risk factors, and therapeutic advances. Front Immunol. 2021;12:740632.

[3] Hostettler IC, Seiffge DJ, Werring DJ. Intracerebral hemorrhage: an update on diagnosis and treatment. Expert Rev Neurother. 2019;19(7):679-694.

[4] Zhou Y, Wang Y, Wang J, Anne Stetler R, Yang QW. Inflammation in intracerebral hemorrhage: from mechanisms to clinical translation. Prog Neurobiol. 2014;115:25-44.

[5] Kushwaha R, Li Y, Makarava N, et al. Reactive astrocytes associated with prion disease impair the blood brain barrier. Preprint. bioRxiv. 2023;2023.03.21.533684.

[6] Gautam J, Xu L, Nirwane A, Nguyen B, Yao Y. Loss of mural cell-derived laminin aggravates hemorrhagic brain injury. J Neuroinflammation. 2020;17(1):103.

[7] Pimentel E, Sivalingam K, Doke M, Samikkannu T. Effects of drugs of abuse on the blood-brain barrier: a brief overview. Front Neurosci. 2020;14:513.

[8] Sun Q, Xu X, Wang T, et al. Neurovascular units and neural-glia networks in intracerebral hemorrhage: from mechanisms to translation. Transl Stroke Res. 2021;12(3):447-460.

[9] Lee MJ, Zhu J, An JH, et al. A transcriptomic analysis of cerebral microvessels reveals the involvement of Notch1 signaling in endothelial mitochondrial-dysfunction-dependent BBB disruption. Fluids Barriers CNS. 2022;19(1):64.

[10] Schmidt SI, Blaabjerg M, Freude K, Meyer M. RhoA signaling in neurodegenerative diseases. Cells. 2022;11(9):1520.

[11] Cai R, Wang Y, Huang Z, et al. Role of RhoA/ROCK signaling in Alzheimer’s disease. Behav Brain Res. 2021;414:113481.

[12] Luo Z, Deng H, Fang Z, et al. Ligustilide inhibited rat vascular smooth muscle cells migration via c-Myc/MMP2 and ROCK/JNK signaling pathway. J Food Sci. 2019;84(12):3573-3583.

[13] Hong L, Chen W, He L, et al. Effect of Naoluoxintong on the NogoA/RhoA/ROCK pathway by down-regulating DNA methylation in MCAO rats. J Ethnopharmacol. 2021;281:114559.

[14] Li MY, Dai XH, Yu XP, et al. Scalp acupuncture protects against neuronal Ferroptosis by activating the p62-Keap1-Nrf2 pathway in rat models of intracranial Haemorrhage. J Mol Neurosci. 2022;72(1):82-96.

[15] Guan R, Li Z, Dai X, et al. Electroacupuncture at GV20-GB7 regulates mitophagy to protect against neurological deficits following intracerebral hemorrhage via inhibition of apoptosis. Mol Med Rep. 2021;24(1):492.

[16] Liu P, Yu X, Dai X, et al. Scalp acupuncture attenuates brain damage after intracerebral hemorrhage through enhanced Mitophagy and reduced apoptosis in rats. Front Aging Neurosci. 2021;13:718631.

[17] Rosenberg GA, Mun-Bryce S, Wesley M, Kornfeld M. Collagenase-induced intracerebral hemorrhage in rats. Stroke. 1990;21(5):801-807.

[18] Belayev L, Alonso OF, Busto R, et al. Middle cerebral artery occlusion in the rat by intraluminal suture. Neurological and pathological evaluation of an improved model. Stroke. 1996;27(9):1616-1623.

[19] De Ryck M, Van Reempts J, Borgers M, et al. Photochemical stroke model: flunarizine prevents sensorimotor deficits after neocortical infarcts in rats. Stroke. 1989;20(10):1383-1390.

[20] Bederson JB, Pitts LH, Tsuji M, Nishimura MC, Davis RL, Bartkowski H. Rat middle cerebral artery occlusion: evaluation of the model and development of a neurologic examination. Stroke. 1986;17(3):472-476.

[21] Zhao XY, Zhang XL. Establishment of composite experimental animal model of brain edema after cerebral hemorrhagic. Ann Palliat Med. 2021;10(1):362-371.

[22] Kalisvaart ACJ, Abrahart AH, Coney AT, Gu S, Colbourne F. Intracranial pressure dysfunction following severe intracerebral hemorrhage in middle-aged rats. Transl Stroke Res. 2022;14:970-986.

[23] Riento K, Ridley AJ. Rocks: multifunctional kinases in cell behaviour. Nat Rev Mol Cell Biol. 2003;4(6):446-456.

[24] Amano M, Nakayama M, Kaibuchi K. Rho-kinase/ROCK: a key regulator of the cytoskeleton and cell polarity. Cytoskeleton. 2010;67:545-554.

[25] Shi J, Wei L. Rho kinase in the regulation of cell death and survival. Arch Immunol Ther Exp (Warsz). 2007;55:61-75.

[26] Chen J, Sun L, Ding GB, et al. Oxygen-glucose deprivation/reoxygenation induces human brain microvascular endothelial cell hyperpermeability via VE-cadherin internalization: roles of RhoA/ROCK2. J Mol Neurosci. 2019;69(1):49-59.

[27] Feng S, Zou L, Wang H, He R, Liu K, Zhu H. RhoA/ROCK-2 pathway inhibition and tight junction protein upregulation by catalpol suppresses lipopolysaccaride-induced disruption of blood-brain barrier permeability. Molecules. 2018;23(9):2371.

[28] Fu Z, Chen Y, Qin F, et al. Increased activity of rho kinase contributes to hemoglobin-induced early disruption of the blood-brain barrier in vivo after the occurrence of intracerebral hemorrhage. Int J Clin Exp Pathol. 2014;7(11):7844-7853.

[29] Yu Y, Qin J, Liu M, et al. Role of rho kinase in lysophosphatidic acid-induced altering of blood-brain barrier permeability. Int J Mol Med. 2014;33(3):661-669.

[30] Wu Y, Yue Z, Wang Q, et al. BK_Ca compensates impaired coronary vasoreactivity through RhoA/ROCK pathway in hind-limb unweighted rats. FASEB J. 2019;33(12):13358-13366.

[31] Shimizu T, Pastuhov SI, Hanafusa H, Matsumoto K, Hisamoto N. The C. Elegans BRCA2-ALP/enigma complex regulates axon regeneration via a rho GTPase-ROCK-MLC phosphorylation pathway. Cell Rep. 2018;24(7):1880-1889.

[32] Orgaz JL, Crosas-Molist E, Sadok A, et al. Myosin II reactivation and cytoskeletal remodeling as a Hallmark and a vulnerability in melanoma therapy resistance. Cancer Cell. 2020;37(1):85-103.e9.

[33] Russ PK, Kupperman AI, Presley SH, Haselton FR, Chang MS. Inhibition of RhoA signaling with increased Bves in trabecular meshwork cells. Invest Ophthalmol Vis Sci. 2010;51(1):223-230.

[34] Tang ST, Su H, Zhang Q, et al. Melatonin attenuates aortic endothelial permeability and arteriosclerosis in Streptozotocin-induced diabetic rats: possible role of MLCK-and MLCP-dependent MLC phosphorylation. J Cardiovasc Pharmacol Ther. 2016;21(1):82-92.

[35] Xue Y, He JT, Zhang KK, Chen LJ, Wang Q, Xie XL. Methamphetamine reduces expressions of tight junction proteins, rearranges F-Acti. cytoskeleton and increases the blood brain barrier permeability via the RhoA/ROCK-dependent pathway. Biochem Biophys Res Commun. 2019;509(2):395-401.

[36] Yang C, Hawkins KE, Doré S, Candelario-Jalil E. Neuroinflammatory mechanisms of blood-brain barrier damage in ischemic stroke. Am J Physiol Cell Physiol. 2019;316(2):C135-C153.

[37] Langen UH, Ayloo S, Gu C. Development and cell biology of the blood-brain barrier. Annu Rev Cell Dev Biol. 2019;35:591-613.

[38] Huang Q, Zhong W, Hu Z, Tang X. A review of the role of cav-1 in neuropathology and neural recovery after ischemic stroke. J Neuroinflammation. 2018;15(1):348.

[39] Minshall RD, Sessa WC, Stan RV, Anderson RGW, Malik AB. Caveolin regulation of endothelial function. Am J Physiol Lung Cell Mol Physiol. 2003;285(6):L1179-L1183.

[40] Pradhan BS, Prószyński TJ. A role for Caveolin-3 in the pathogenesis of muscular dystrophies. Int J Mol Sci. 2020;21(22):8736.

[41] Grande-García A, Echarri A, de Rooij J, et al. Caveolin-1 regulates cell polarization and directional migration through Src kinase and rho GTPases. J Cell Biol. 2007;177(4):683-694.

[42] Cao D, Li B, Cao C, et al. Caveolin-1 aggravates neurological deficits by activating neuroinflammation following experimental intracerebral hemorrhage in rats. Exp Neurol. 2023;368:114508.

[43] Pan CS, Yan L, Lin SQ, et al. QiShenYiQi pills attenuates ischemia/reperfusion-induced cardiac microvascular Hyperpermeability implicating Src/Caveolin-1 and RhoA/ROCK/MLC signaling. Front Physiol. 2021;12:753761.

[44] Sweeney MD, Zhao Z, Montagne A, Nelson AR, Zlokovic BV. Blood-brain barrier: from physiology to disease and Back. Physiol Rev. 2019;99(1):21-78.

[45] De Ieso ML, Kuhn M, Bernatchez P, et al. A role of Caveolae in trabecular meshwork mechanosensing and contractile tone. Front Cell Dev Biol. 2022;10:855097.

[46] Abdullahi W, Tripathi D, Ronaldson PT. Blood-brain barrier dysfunction in ischemic stroke: targeting tight junctions and transporters for vascular protection. Am J Physiol Cell Physiol. 2018;315(3):C343-C356.

[47] Liu WY, Wang ZB, Zhang LC, Wei X, Li L. Tight junction in blood-brain barrier: an overview of structure, regulation, and regulator substances. CNS Neurosci Ther. 2012;18(8):609-615.

[48] Li Y, Zhang J. Animal models of stroke. Animal Model Exp Med. 2021;4(3):204-219.

[49] Arvanitis CD, Ferraro GB, Jain RK. The blood-brain barrier and blood-tumour barrier in brain tumours and metastases. Nat Rev Cancer. 2020;20(1):26-41.

[50] Anthony DP, Hegde M, Shetty SS, Rafic T, Mutalik S, Rao BSS. Targeting receptor-ligand chemistry for drug delivery across blood-brain barrier in brain diseases. Life Sci. 2021;274:119326.

[51] Ye F, Garton HJL, Hua Y, Keep RF, Xi G. The role of thrombin in brain injury after hemorrhagic and ischemic stroke. Transl Stroke Res. 2021;12(3):496-511.

[52] Zhou Z, Guo F, Dou Y, Tang J, Huan J. Guanine nucleotide exchange factor-H1 signaling is involved in lipopolysaccharide-induced endothelial barrier dysfunction. Surgery. 2013;154(3):621-631.

[53] Lee HW, Xu Y, Zhu X, et al. Endothelium-derived lactate is required for pericyte function and blood-brain barrier maintenance. EMBO J. 2022;41(9):e109890.

[54] Liu Z, Khalil RA. Evolving mechanisms of vascular smooth muscle contraction highlight key targets in vascular disease. Biochem Pharmacol. 2018;153:91-122.

[55] Yu Y, Wu Y, Wei J, et al. NMDA mediates disruption of blood-brain barrier permeability via rho/ROCK signaling pathway. Neurochem Int. 2022;154:105278.

[56] Lagendijk AK, Hogan BM. VE-cadherin in vascular development: a coordinator of cell signaling and tissue morphogenesis. Curr Top Dev Biol. 2015;112:325-352.

[57] Holmes T, Brown AW, Suggitt M, et al. The influence of hypoxia and energy depletion on the response of endothelial cells to the vascular disrupting agent combretastatin A-4-phosphate. Sci Rep. 2020;10(1):9926.

[58] Mehta D, Malik AB. Signaling mechanisms regulating endothelial permeability. Physiol Rev. 2006;86(1):279-367.

RIGHTS & PERMISSIONS

2024 The Authors. Animal Models and Experimental Medicine published by John Wiley & Sons Australia, Ltd on behalf of The Chinese Association for Laboratory Animal Sciences.