Skip to main content
SpringerLink
Log in

JLX001 Modulated the Inflammatory Reaction and Oxidative Stress in pMCAO Rats via Inhibiting the TLR2/4-NF-κB Signaling Pathway

  • Original Paper
  • Published:
Neurochemical Research Aims and scope Submit manuscript

Abstract

Inflammatory reactions and oxidative stress play critical roles in cerebral ischemic injuries. Microglia are activated after ischemic injury. Activated microglia produce neurotoxic proinflammatory factors and reactive oxygen species (ROS), which have been demonstrated closely related TLR2/4-NF-κB signal pathways. This study was to evaluate the effect of JLX001 against ischemic injury and investigate the mechanisms. The permanent middle cerebral artery occlusion (pMCAO) model was employed in rats. The neurobehavioral score, brain infarction rate, brain water content, pathological changes, immunohistochemical staining, biochemical index (T-AOC, SOD, and MDA), proinflammatory factors (IL-1β, TNF-α, and NO), expression of TLR2/4 and nuclear translocation of NF-κB p65 were determined. To explore probable underlying mechanism of the neuroprotective effect of JLX001, BV-2 cells were exposed to in oxygen–glucose deprivation (OGD) for 4 h to mimic ischemic injury in vitro. The result showed that JLX001 significantly decreased neurological deficit score, infarct size, and brain edema, attenuated pathological changes, inhibited the activation of microglia, improved the process of oxidative stress, reduced the release of proinflammatory cytokines and downregulated TLR2/4-NF-κB signal pathway. Moreover, OGD reduced BV2 cell viability, induced oxidative damage, increased the release of proinflammatory factors and activated TLR2/4-NF-κB signal pathway, which was significantly reversed by the intervention of JLX001. This study demonstrates that JLX001 is effective in protecting the brain from ischemic injury, which may be mediated by regulating oxidative stress, inflammation and inhibiting TLR2/4-NFκB signal pathway.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7
Fig. 8
Fig. 9
Fig. 10
Fig. 11

Similar content being viewed by others

Abbreviations

CVB-D:

Cyclovirobuxine D

JLX001:

Cyclovirobuxine hydrochloride D

pMCAO:

Permanent middle cerebral artery occlusion

OGD:

Oxygen–glucose deprivation

TLRs:

Toll-like receptors

MyD88:

Myeloid differentiation primary response 88

NF-κB:

Nuclear factor-κB

IL-1β:

Interleukin-1β

TNF-α:

Tumor necrosis Factor-α

NO:

Nitric oxide

ROS:

Reactive oxygen species

T-AOC:

Total antioxidant capacity

NADPH:

Nicotinamide adenine dinucleotide phosphate

GBDI:

Ginkgo biloba diterpene lactone meglumine injection

ICA:

Internal carotid artery

ECA:

External carotid artery

References

  1. Grossini E, Battaglia A, Brunelleschi S, Mary DA, Molinari C, Viano I, Vacca G (1999) Coronary effects of cyclovirobuxine D in anesthetized pigs and in isolated porcine coronary arteries. Life Sci 65(5):PL59–65

    Article  CAS  PubMed  Google Scholar 

  2. Shan P, Mao RB, Xu JM, Li JX (1984) The beneficial effects of cyclovirobuxine D (CVBD) in coronary heart disease. A double blind analysis of 110 cases. J Tradit Chin Med 4(1):15-19

    Google Scholar 

  3. Yan M (1990) Congestive heart failure treated by a combination of cyclovirobuxine D with digoxin. Zhong Xi Yi Jie He Za Zhi 10(2):88–89

    CAS  PubMed  Google Scholar 

  4. Guo Q, Guo J, Yang R, Peng H, Zhao J, Li L, Peng S (2015) Cyclovirobuxine D attenuates doxorubicin-induced cardiomyopathy by suppression of oxidative damage and mitochondrial biogenesis impairment. Oxid Med Cell Longev. https://doi.org/10.1155/2015/151972

  5. Yu B, Fang TH, Lu GH, Xu HQ, Lu JF (2011) Beneficial effect of Cyclovirobuxine D on heart failure rats following myocardial infarction. Fitoterapia 82(6):868–877

    Article  CAS  PubMed  Google Scholar 

  6. Yan YY, Ao LY, Zhou L, Li CY, Fang WR, Shen WY, Liang BW, Zhu X, Li YM (2018) Therapeutic effects of JLX001 on cerebral ischemia through inhibiting platelet activation and thrombus formation in rats. Biomed Pharmacother 106:805–812

    Article  CAS  PubMed  Google Scholar 

  7. del Zoppo GJ (2010) Acute anti-inflammatory approaches to ischemic stroke. Ann N Y Acad Sci 1207:143–148

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Tang SC, Arumugam TV, Xu X, Cheng A, Mughal MR, Jo DG, Lathia JD, Siler DA, Chigurupati S, Ouyang X, Magnus T, Camandola S, Mattson MP (2007) Pivotal role for neuronal Toll-like receptors in ischemic brain injury and functional deficits. Proc Natl Acad Sci U S A 104(34):13798

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Taylor R, Sansing L (2013) Microglial responses after ischemic stroke and intracerebral hemorrhage. Clin Dev Immunol 2013:746068

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Kawagoe T, Sato S, Matsushita K, Kato H, Matsui K, Kumagai Y, Saitoh T, Kawai T, Takeuchi O, Akira S (2008) Sequential control of Toll-like receptor-dependent responses by IRAK1 and IRAK2. Nat Immunol 9(6):684–691

    Article  CAS  PubMed  Google Scholar 

  11. Huang J, Upadhyay UM, Tamargo RJ (2006) Inflammation in stroke and focal cerebral ischemia. Surg Neurol 66(3):232–245

    Article  PubMed  Google Scholar 

  12. Yenari MA, Kauppinen TM, Swanson RA (2010) Microglial activation in stroke: therapeutic targets. Neurotherapeutics 7(4):378–391

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Zhu XC, Jiang T, Zhang QQ, Cao L, Tan MS, Wang HF, Ding ZZ, Tan L, Yu JT (2015) Chronic metformin preconditioning provides neuroprotection via suppression of NF-kappaB-mediated inflammatory pathway in rats with permanent cerebral ischemia. Mol Neurobiol 52(1):375–385

    Article  CAS  PubMed  Google Scholar 

  14. Wu LR, Liu L, Xiong XY, Zhang Q, Wang FX, Gong CX, Zhong Q, Yang YR, Meng ZY, Yang QW (2017) Vinpocetine alleviate cerebral ischemia/reperfusion injury by down-regulating TLR4/MyD88/NF-kappaB signaling. Oncotarget 8(46):80315–80324

    PubMed  PubMed Central  Google Scholar 

  15. Wang SL, Duan L, Xia B, Liu Z, Wang Y, Wang GM (2017) Dexmedetomidine preconditioning plays a neuroprotective role and suppresses TLR4/NF-kappaB pathways model of cerebral ischemia reperfusion. Biomed Pharmacother 93:1337–1342

    Article  CAS  PubMed  Google Scholar 

  16. Kleinschnitz C, Grund H, Wingler K et al (2010) Post-stroke inhibition of induced NADPH oxidase type 4 prevents oxidative stress and neurodegeneration. PLoS Biol 8(9):e100479

    Article  CAS  Google Scholar 

  17. Shang YZ, Miao H, Cheng JJ, Qi JM (2006) Effects of amelioration of total flavonoids from stems and leaves of Scutellaria baicalensis Georgi on cognitive deficits, neuronal damage and free radicals disorder induced by cerebral ischemia in rats. Biol Pharm Bull 29(4):805–810

    Article  CAS  PubMed  Google Scholar 

  18. Wicha P, Tocharus J, Janyou A, Jittiwat J, Changtam C, Suksamrarn A, Tocharus C (2017) Hexahydrocurcumin protects against cerebral ischemia/reperfusion injury, attenuates inflammation, and improves antioxidant defenses in a rat stroke model. PLoS ONE 12(12):e0189211

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Tao X, Sun X, Xu L, Yin L, Han X, Qi Y, Xu Y, Zhao Y, Wang C, Peng J (2016) Total flavonoids from Rosa laevigata Michx Fruit ameliorates hepatic ischemia/reperfusion injury through inhibition of oxidative stress and inflammation in rats. Nutrients 8(7):148

    Article  CAS  Google Scholar 

  20. Khandelwal P, Yavagal DR, Sacco RL (2016) Acute ischemic stroke intervention. J Am Coll Cardiol 67(22):2631–2644

    Article  PubMed  Google Scholar 

  21. Eady TN, Khoutorova L, Anzola DV, Hong SH, Obenaus A, Mohd-Yusof A, Bazan NG, Belayev L (2013) Acute treatment with docosahexaenoic acid complexed to albumin reduces injury after a permanent focal cerebral ischemia in rats. PLoS ONE 8(10):e77237

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Luo YP, Zhang H, Hu HF, Cao ZY, Zhang XZ, Cao L, Wang ZZ, Xiao W (2017) Protective effects of Ginkgo Terpene Lactones Meglumine Injection on focal cerebral ischemia in rats. Zhongguo Zhong Yao Za Zhi 42(24):4733–4737

    PubMed  Google Scholar 

  23. Ford G, Xu Z, Gates A, Jiang J, Ford BD (2006) Expression Analysis Systematic Explorer (EASE) analysis reveals differential gene expression in permanent and transient focal stroke rat models. Brain Res 1071(1):226–236

    Article  CAS  PubMed  Google Scholar 

  24. Xu Z, Croslan DR, Harris AE, Ford GD, Ford BD (2006) Extended therapeutic window and functional recovery after intraarterial administration of neuregulin-1 after focal ischemic stroke. J Cereb Blood Flow Metab 26(4):527–535

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  26. Joo SP, Xie W, Xiong X, Xu B, Zhao H (2013) Ischemic postconditioning protects against focal cerebral ischemia by inhibiting brain inflammation while attenuating peripheral lymphopenia in mice. Neuroscience 243:149–157

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Li D, Wang C, Yao Y, Chen L, Liu G, Zhang R, Liu Q, Shi FD, Hao J (2016) mTORC1 pathway disruption ameliorates brain inflammation following stroke via a shift in microglia phenotypefrom M1 type to M2 type. FASEB J 30(10):3388–3399

    Article  CAS  PubMed  Google Scholar 

  28. Arauchi R, Hashioka S, Tsuchie K et al (2018) Gunn rats with glial activation in the hippocampus show prolonged immobility time in the forced swimming test and tail suspension test. Brain Behav 8(8):e01028

    Article  PubMed  PubMed Central  Google Scholar 

  29. Chen H, Yoshioka H, Kim GS, Jung JE, Okami N, Sakata H, Maier CM, Narasimhan P, Goeders CE, Chan PH (2011) Oxidative stress in ischemic brain damage: mechanisms of cell death and potential molecular targets for neuroprotection. Antioxid Redox Signal 14(8):1505–1517

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Xu YN, Wang Y, Bo S, Jiang Y, Liu XD, Shen XC (2014) Ameliorated effects of cyclovirobuxine D on oxidative stress and energy metabolism in experimental cardiac injured rats induced by sympathetic overactivity in vivo. Zhong Yao Cai 37(7):1213–1217

    CAS  PubMed  Google Scholar 

  31. Tu XK, Yang WZ, Chen JP, Chen Y, Ouyang LQ, Xu YC, Shi SS (2014) Curcumin inhibits TLR2/4-NF-kappaB signaling pathway and attenuates brain damage in permanent focal cerebral ischemia in rats. Inflammation 37(5):1544–1551

    Article  CAS  PubMed  Google Scholar 

  32. Wang X, An F, Wang S, An Z, Wang S (2017) Orientin attenuates cerebral ischemia/reperfusion injury in rat model through the AQP-4 and TLR4/NF-kappaB/TNF alpha signaling pathway. J Stroke Cerebrovasc Dis 26(10):2199–2214

    Article  PubMed  Google Scholar 

  33. Hwang JH, Kumar VR, Kang SY, Jung HW, Park YK (2018) Effects of Flower Buds Extract of Tussilago farfara on Focal Cerebral Ischemia in Rats and Inflammatory Response in BV2 Microglia. Chin J Integr Med 24(11):842–852

    Article  CAS  Google Scholar 

  34. Guo D, Li JR, Wang Y, Lei LS, Yu CL, Chen NN (2014) Cyclovirobuxinum D suppresses lipopolysaccharide-induced inflammatory responses in murine macrophages in vitro by blocking JAK-STAT signaling pathway. Acta Pharmacol Sin 35(6):770–778

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Block ML, Zecca L, Hong J-S (2007) Microglia-mediated neurotoxicity: uncovering the molecular mechanisms. Nat Rev Neurosci 8(1):57–69

    Article  CAS  PubMed  Google Scholar 

  36. Kreutzberg GW (1996) Microglia: a sensor for pathological events in the CNS. Trends Neurosci 19(8):312–318

    Article  CAS  PubMed  Google Scholar 

  37. Ridder D, Schwaninger M (2009) NF-κB signaling in cerebral ischemia. Neuroscience 158(3):995–1006

    Article  CAS  PubMed  Google Scholar 

  38. Zeng WX, Han YL, Zhu GF, Huang LQ, Deng YY, Wang QS, Jiang WQ, Wen MY, Han QP, Xie D, Zeng HK (2017) Hypertonic saline attenuates expression of Notch signaling and proinflammatory mediators in activated microglia in experimentally induced cerebral ischemia and hypoxic BV-2 microglia. BMC Neurosci 18(1):32

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Chen J, Wang Z, Zheng Z, Chen Y, Khor S, Shi K, He Z, Wang Q, Zhao Y, Zhang H, Li X, Li J, Yin J, Wang X, Xiao J (2017) Neuron and microglia/macrophage-derived FGF10 activateneuronal FGFR2/PI3K/Akt signaling and inhibit microglia/macrophages TLR4/NF-kappaB-dependent neuroinflammation to improve functional recovery after spinal cord injury. Cell Death Dis 8(10):e3090

    Article  PubMed  PubMed Central  Google Scholar 

  40. Wang PF, Xiong XY, Chen J, Wang YC, Duan W, Yang QW (2015) Function and mechanism of toll-like receptors in cerebral ischemic tolerance: from preconditioning to treatment. J Neuroinflammation 12:80

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Koizumi S, Hirayama Y, Morizawa YM (2018) New roles of reactive astrocytes in the brain; an organizer of cerebral ischemia. Neurochem Int 119:107–114

    Article  CAS  PubMed  Google Scholar 

  42. Kanazawa M, Ninomiya I, Hatakeyama M, Takahashi T, Shimohata T (2017) Microglia and monocytes/macrophages polarization reveal novel therapeutic mechanism against stroke. Int J Mol Sci 18(10):2135

    Article  CAS  PubMed Central  Google Scholar 

Download references

Acknowledgements

This work was supported by National Major Scientific and Technological Special Project for “Significant New Drugs Development” during the Thirteenth Five-year Plan Period (Nos. 2018ZX09301043-00 and 2016ZX09101031, respectively) and the “Double First-Class” Construction Technology Innovation Team Project of China Pharmaceutical University (No. CPU2018GY23).

Author information

Author notes

  1. Yanying Qiu and Qiyang Yin have contributed equally to this work.

Authors and Affiliations

  1. State Key Laboratory of Natural Medicines, China Pharmaceutical University, No. 24, Tongjia Alley, Gulou District, Nanjing, 210009, Jiangsu, China

    Yanying Qiu, Qiyang Yin, Yuxiang Fei, Weirong Fang & Yunman Li

  2. School of Pharmacy, China Pharmaceutical University, No. 24, Tongjia Alley, Gulou District, Nanjing, 210009, Jiangsu, China

    Yize Li & Hongfei Huang

  3. School of Science, China Pharmaceutical University, No. 24, Tongjia Alley, Gulou District, Nanjing, 210009, Jiangsu, China

    Weiyang Shen

  4. Jiangsu Jinglixin Pharmaceutical Technology Company Limited, No. 18, Zhilan road, Jiangning University Town, Jiangning District, Nanjing, 211100, Jiangsu, China

    Bingwen Liang & Xiong Zhu

  5. Medicine & Chemical Institute, China Pharmaceutical University, No. 24, Tongjia Alley, Gulou District, Nanjing, 210009, Jiangsu, China

    Xiong Zhu

Authors
  1. Yanying Qiu
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Qiyang Yin
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Yuxiang Fei
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Yize Li
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Hongfei Huang
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Weirong Fang
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Weiyang Shen
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Bingwen Liang
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Xiong Zhu
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Yunman Li
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding authors

Correspondence to Xiong Zhu or Yunman Li.

Ethics declarations

Conflict of interest

The authors declare that they have no conflict of interest

Research Involving Animals

SPF male SD rats were purchased from Qinglongshan Animal Farm (Nanjing, China, license number: SYXK (Su) 2017-0001). Rats were housed in coops on a 12 h light–12 h dark schedule at the controlled temperature of 23 ± 2.5 °C with free access to a normal diet and tap water. All animals were cared for and treated according to institutional guidelines of China Pharmaceutical University, which conforms to the European Community guidelines (EEC Directive of 1986: 86/609/EEC). Efforts were made to minimize the number of rats and their suffering.

Additional information

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Qiu, Y., Yin, Q., Fei, Y. et al. JLX001 Modulated the Inflammatory Reaction and Oxidative Stress in pMCAO Rats via Inhibiting the TLR2/4-NF-κB Signaling Pathway. Neurochem Res 44, 1924–1938 (2019). https://doi.org/10.1007/s11064-019-02826-0

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s11064-019-02826-0

Keywords

Search

Navigation