Gastrodin alleviates microglia-mediated inflammatory responses in neonatal mice with hypoxic-ischemic brain damage by regulating CCR5/AKT signaling

doi:10.12122/j.issn.1673-4254.2024.10.02

Abstract

Abstract:

Objective To investigate the mechanism behind the protective effects of gastrodin against microglia-mediated inflammatory responses following hypoxic-ischemic brain damage (HIBD) in neonatal mice. Methods Thirty-six 10-day-old C57BL/6J mice were randomized into sham-operated group, HIBD (induced by ligation of the left common carotid artery followed by hypoxia for 40 min) group, and HIBD with gastrodin treatment groups (n=12). In gastrodin treatment group, 100 mg/kg gastrodin was injected intraperitoneally 1 h before and at 2 and 12 h after hypoxia. After the treatments, the expressions of CCR5, AKT, p-AKT, and TNF-α and the co-expression of IBA1 and CCR5 in the corpus callosum of the mice were detected with Western blotting and immunofluorescence double staining. In a BV2 microglial cell model of oxygen-glucose deprivation (OGD), the effects of pretreatment with gastrodin and Maraviroc (an CCR5 antagonist) on protein expressions of CCR5, AKT, p-AKT, TNF-α and IL-1β were evaluated using Western blotting and immunofluorescence double staining. Results The neonatal mice with HIBD showed significantly increased expressions of CCR5 and TNF-α with lowered p-AKT expression in the brain tissues, and GAS treatment obviously reversed these changes. HIBD also significantly increased the co-expression of IBA1 and CCR5 in the corpus callosum of the mice, which was obviously lowered by gastrodin treatment. In BV2 cells, OGD significantly increased the expressions of CCR5, TNF‑α, and IL-1β and decreased the expression of p-AKT, and these changes were inhibited by treatment with gastrodin, Maraviroc or their combination; the inhibitory effect of the combined treatment did not differ significantly from that of gastrodin or Maraviroc alone. Conclusion Gastrodin can produce neuroprotective effects in neonatal mice with HIBD by inhibiting inflammatory cytokine production and activate AKT phosphorylation via inhibiting CCR5.

Key words: gastrodin, hypoxic-ischemic brain damage, oxygen-glucose deprivation, microglia, CCR5/AKT

Jinsha SHI, Haonan ZHANG, Xinglin ZHANG, Haolong SHI, Hanjun ZUO, Tao GUO, Zhao WANG, Hang YU, Juanjuan LI. Gastrodin alleviates microglia-mediated inflammatory responses in neonatal mice with hypoxic-ischemic brain damage by regulating CCR5/AKT signaling[J]. Journal of Southern Medical University, 2024, 44(10): 1850-1857.

Figures/Tables 5

Fig.1 Western blotting for detecting the protein expressions of AKT, p-AKT, CCR5 and TNF-α in the corpus callosum of the mice at 1 day (A-D) and 3 days (E-H) after HIBD and gastrodin (GAS) treatment (Mean±SD, n=3). HIBD: Hypoxic-ischemic brain damage. *P<0.05, **P<0.01, ***P<0.001 vs Sham group; #P<0.05, ##P<0.01 vs HIBD group.

Fig.2 Co-expression of CCR5 and IBA1 proteins (white arrows) in the corpus callosum of the mice detected by immunofluorescence staining (Scale bar=50 μm). A: 1 day after HIBD and GAS treatment. B: 3 days after HIBD and GAS treatment.

Fig.3 Expression levels of CCR5, p-AKT, TNF-α and IL-1β proteins in BV2 microglia after oxygen-glucose deprivation (OGD) and GAS treatment (Mean±SD, n=3). *P<0.05, **P<0.01, ***P<0.001 vs Control group; #P<0.05, ##P<0.01 vs OGD group.

Fig.4 Expression levels of CCR5 (A)and p-AKT (B) proteins in BV2 microglia after OGD and GAS treatment detected by immunofluorescence staining (Scale bar=20 μm).

Fig.5 Expression levels of AKT, p-AKT, TNF-α and IL-1β proteins in BV2 microglia after OGD and Maraviroc treatment detected by Western blotting (Mean±SD, n=3). M: Maraviroc. #P<0.05 vs OGD.

References 32

1	Kamino D, Almazrooei A, Pang EW, et al. Abnormalities in evoked potentials associated with abnormal glycemia and brain injury in neonatal hypoxic-ischemic encephalopathy[J]. Clin Neurophysiol,2021, 132(1): 307-13.
2	汤易, 刘影. 评估新生儿缺血缺氧性脑损伤MRI的临床应用与研究进展[J]. 磁共振成像, 2013, 4(3): 228-31. DOI: 10.3969/j.issn.1674-8034.2013.03.014
3	Sankaran R, Radhakrishnan K, Sundaram K R. Hyperbaric oxygen therapy in patients with hypoxic ischemic encephalopathy[J]. Neurol India, 2019, 67(3): 728-31.
4	Parakalan R, Jiang B, Nimmi B, et al. Transcriptome analysis of amoeboid and ramified microglia isolated from the corpus callosum of rat brain[J]. BMC Neurosci, 2012, 13: 64.
5	Pont-Lezica L, Beumer W, Colasse S, et al. Microglia shape corpus callosum axon tract fasciculation: functional impact of prenatal inflammation[J]. Eur J Neurosci, 2014, 39(10): 1551-7.
6	Yao YY, Ling EA, Lu D. Microglia mediated neuroinflammation- signaling regulation and therapeutic considerations with special reference to some natural compounds[J]. Histol Histopathol, 2020,35(11): 1229-50.
7	Festa BP, Siddiqi FH, Jimenez-Sanchez M, et al. Microglial-to-neuronal CCR5 signaling regulates autophagy in neurodegeneration[J]. Neuron, 2023, 111(13): 2021-37.
8	Cowell RM, Xu H, Parent JM, et al. Microglial expression of chemokine receptor CCR5 during rat forebrain development and after perinatal hypoxia-ischemia[J]. J Neuroimmunol, 2006, 173(1-2): 155-65.
9	Bokhari SM, Yao H, Bethel-Brown C, et al. Morphine enhances Tat-induced activation in murine microglia[J]. J Neurovirol, 2009, 15(3): 219-28.
10	Lin CS, Hsieh PS, Hwang LL, et al. The CCL5/CCR5 axis promotes vascular smooth muscle cell proliferation and atherogenic phenotype switching[J]. Cell Physiol Biochem, 2018, 47(2): 707-20.
11	Li X, Zhang J, Zhu X, et al. Progesterone reduces inflammation and apoptosis in neonatal rats with hypoxic ischemic brain damage through the PI3K/Akt pathway[J]. Int J Clin Exp Med,2015,8(5):8197-8203.
12	Wei ZZ, Zhang JY, Taylor TM, et al. Neuroprotective and regenerative roles of intranasal Wnt-3a administration after focal ischemic stroke in mice[J]. J Cereb Blood Flow Metab, 2018, 38(3):404-21.
13	Wang Z, Qiu Z, Gao C, et al. 2,5-hexanedione downregulates nerve growth factor and induces neuron apoptosis in the spinal cord of rats via inhibition of the PI3K/Akt signaling pathway[J]. PLoS One,2017, 12(6): e0179388.
14	Zhang BG, Hu L, Zang MD, et al. Helicobacter pylori CagA induces tumor suppressor gene hypermethylation by upregulating DNMT1 via AKT-NFkappaB pathway in gastric cancer development[J]. Oncotarget, 2016, 7(9): 9788-800.
15	王妍, 阮毓卿, 崔璨, 等. 交泰丸通过激活PI3K/AKT信号通路改善阿尔茨海默病模型小鼠大脑的葡萄糖代谢[J]. 南方医科大学学报, 2024, 44(5): 894-903. DOI: 10.12122/j.issn.1673-4254.2024.05.11
16	艾艳萍. 天麻素对癫痫合并偏头痛病人血清炎症因子、脑源性神经营养因子及氧化应激水平的影响[J]. 中西医结合心脑血管病杂志,2018, 16(11): 1592-5. DOI: 10.12102/j.issn.1672-1349.2018.11.038
17	Guan J, Wang L, Jin J, et al. Simultaneous determination of calycosin-7-O-beta-D-glucoside, cinnamic acid, paeoniflorin and albiflorin in rat plasma by UHPLC-MS/MS and its application to a pharmacokinetic study of Huangqi Guizhi Wuwu Decoction[J]. J Pharm Biomed Anal, 2019, 170: 1-7.
18	Qin B, Luo N, Li Y, et al. Protective effect of gastrodin on peripheral neuropathy induced by anti-tumor treatment with vincristine in rat models[J]. Drug Chem Toxicol, 2021, 44(1): 84-91.
19	Li S, Yang Q, Zhou Z, et al. Gastrodin protects retinal ganglion cells from ischemic injury by activating phosphatidylinositol 3-kinase/protein kinase B/nuclear factor erythroid 2-related factor 2 (PI3K/AKT/Nrf2) signaling pathway[J]. Bioengineered,2022,13(5):12625-36.
20	Levene ML, Kornberg J, Williams TH. The incidence and severity of post-asphyxial encephalopathy in full-term infants[J]. Early Hum Dev, 1985, 11(1): 21-6.
21	Finer NN, Robertson CM, Richards RT, et al. Hypoxic-ischemic encephalopathy in term neonates: perinatal factors and outcome[J]. J Pediatr, 1981, 98(1): 112-7.
22	Yu L, Huang L, Zhao Y, et al. Atorvastatin promotes pro/anti-inflammatory phenotypic transformation of microglia via Wnt/β‑catenin pathway in hypoxic-ischemic neonatal rats[J]. Mol Neurobiol, 2024, 61(6): 3559-77.
23	Renz P, Schoeberlein A, Haesler V, et al. A novel murine multi-Hit model of perinatal acute diffuse white matter injury recapitulates major features of human disease[J]. Biomedicines,2022,10(11):2810.
24	Yan J, Xu W, Lenahan C, et al. CCR5 activation promotes NLRP1-dependent neuronal pyroptosis via CCR5/PKA/CREB pathway after intracerebral hemorrhage[J]. Stroke, 2021, 52(12): 4021-32.
25	Martin-Blondel G, Brassat D, Bauer J, et al. CCR5 blockade for neuroinflammatory diseases-beyond control of HIV[J]. Nat Rev Neurol, 2016, 12(2): 95-105.
26	Wu Y, Che J, Dong J, et al. CCR5 antagonist maraviroc alleviates doxorubicin-induced neuroinflammation and neurobehavioral deficiency by regulating NF-κB/NLRP3 signaling in a breast cancer mouse model[J]. Neuropharmacology, 2024, 254: 109981.
27	Laudati E, Curro D, Navarra P, et al. Blockade of CCR5 receptor prevents M2 microglia phenotype in a microglia-glioma paradigm[J]. Neurochem Int, 2017, 108: 100-8.
28	Xiong Z, Yu S, Xie Z, et al. Cancer-associated fibroblasts promote enzalutamide resistance and PD-L1 expression in prostate cancer through CCL5-CCR5 paracrine axis[J]. iScience,2024,27(5):109674.
29	校欢, 马晓娇, 承欧梅, 等. NO-cGMP-PKG信号通路在天麻素促进脑缺血后海马神经发生中的作用[J]. 中国中药杂志, 2019, 44(24):5451-6.
30	吴芳, 左涵珺, 任雪琪, 等. 天麻素对氧糖剥夺诱导的小胶质细胞Notch信号通路的影响[J]. 神经解剖学杂志, 2022, 38(1): 38-44.
31	Guo J, Zhang XL, Bao Z R, et al. Gastrodin regulates the Notch signaling pathway and Sirt3 in activated microglia in cerebral hypoxic-Ischemia neonatal rats and in activated BV-2 microglia[J]. Neuromolecular Med, 2021, 23(3): 348-62.
32	Chen YX, Yang H, Wang DS, et al. Gastrodin relieves cognitive impairment by regulating autophagy via PI3K/AKT signaling pathway in vascular dementia[J]. Biochem Biophys Res Commun,2023, 671: 246-54.

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[10]	. Macrophage migration inhibitory factor meditates MPP+/MPTP-induced NLRP3 inflammasome activation in microglia cells [J]. Journal of Southern Medical University, 2021, 41(7): 972-979.
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[14]	. [J]. Journal of Southern Medical University, 2013, 33(04): 463-.
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