SPINE Volume 40, Number 6, pp 363-368 ©2015, Wolters Kluwer Health, Inc. All rights reserved.
BASIC SCIENCE
Honokiol Downregulates Kruppel-Like Factor 4 Expression, Attenuates Inflammation, and Reduces Histopathology After Spinal Cord Injury in Rats Jia Liu, MD,*† Changmeng Zhang, MD,* Zhi Liu, MD,* Jianzheng Zhang, MD,* Zimin Xiang, MD,* and Tiansheng Sun, MD*
Study Design. Randomized experimental study. Objective. To investigate the neuroprotective effect of honokiol (HNK) on rats subjected to traumatic spinal cord injury (SCI) and the molecular mechanisms. Summary of Background Data. Inflammation contributes to the secondary injury to the spinal cord. Honokiol has been used as a neuroprotective agent because of its strong antioxidant and anti-inflammatory properties. Kruppel-like factor 4 (Klf4) is a newly identified critical target for the anti-inflammatory effect of HNK. Whether HNK can inhibit inflammatory response in rat model of SCI through mediating the expression of Klf4 has yet to be elucidated. Methods. Eighty-four adult female Sprague-Dawley rats were randomly divided into 4 groups as sham, SCI, SCI + Vehicle (0.1% propylene glycol in saline, intraperitoneally), and SCI + HNK (20 mg/kg, intraperitoneally) groups. The influences of HNK on the proinflammatory cytokines, microglial activation, neutrophil infiltration, histological changes, and improvement in motor function were assessed. Results. In the SCI group, proinflammatory cytokines, microglial activation, neutrophil infiltration, and Klf4 expression levels were increased compared with the sham group (P < 0.001). HNK intervention downregulated the expression of Klf4, reduced the production of proinflammatory cytokines, inhibited microglial activation, and neutrophil infiltration (P < 0.05). Furthermore, HNK also reduced histopathology and improved functional outcome after traumatic SCI. Conclusion. HNK reduces secondary tissue damage and improves locomotor function recovery after SCI through suppressing From the *Departmentof Institute of Orthopaedics, Chinese PLA (People's Liberation Army) Beijing Army General Hospital, Beijing, China; and †Department of Medical School of Chinese PLA, Beijing, China. Acknowledgment date: September 18, 2014. Acceptance date: December 3, 2014. The device(s)/drug(s) that is/are the subject of the manuscript is/are not intended for human use. National Natural Science Foundation of China (81150020) grants were received in support of this work. No relevant financial activities outside the submitted work. Address correspondence and reprint requests to Tiansheng Sun, MD, Institute of Orthopaedics, Chinese PLA Beijing Army General Hospital, Dongcheng District, Nanmencang No. 5, Beijing 100700, China; E-mail: [email protected] DOI: 10.1097/BRS.0000000000000758 Spine
inflammatory response, and can be used as a potential therapeutic agent for SCI. Key words: honokiol, Kruppel-like factor 4, spinal cord injury, rat, inflammatory response. Level of Evidence: NA Spine 2015;40:363–368
T
raumatic spinal cord injury (SCI) is one of the worldwide clinical problem, because of its complex pathophysiology.1 Primary trauma to spinal cord initiates a cascade of cellular and biochemical events, which lead to exacerbation of the injury and even influence the functional outcome of patients subjected to SCI.2,3 Inflammation is a key event in the secondary injury phase of SCI and marked by the excessive activation of inflammatory cells and production of proinflammatory mediators.4 Several laboratories have demonstrated that targeting the key regulator in inflammatory response, such as nuclear factor-κB, can improve functional recovery in rodent models of traumatic SCI.5,6 As a result, many anti-inflammatory agents have been explored for their potential neuroprotective effects on SCI.7 Honokiol (HNK), a small-molecule polyphenol extracted from the herb “Houpo,” exhibits anti-cancerous,8 antioxidant,9 and anti-inflammatory 10 properties. According to previous studies, the anti-inflammatory effects of honokiol act mainly via suppressing nuclear factor-κB activation and the subsequent cytokine production.11 Recently, an article reported that Kruppel-like factor 4 (Klf4), a zinc finger transcription factor that has been confirmed to play a critical role in several inflammatory cells mediated inflammation,12 was another key therapeutic target of HNK.13 So far, HNK has been explored as a neuroprotective agent in rodents model of several neurological diseases,14,15 but no study has been conducted to evaluate the possible therapeutic effects of this agent in SCI and explore the possible molecular mechanisms. In this study, we investigate whether HNK exerts a neuroprotective effect in rats subjected to SCI and the molecular mechanisms. Our results showed that HNK treatment inhibited the productions of proinflammatory factors, such as inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 www.spinejournal.com
363
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BASIC SCIENCE (COX-2), downregulated the expression of Klf4, attenuated secondary tissue destruction, and enhanced neurological recovery after SCI.
MATERIALS AND METHODS Animals and Groups All animal experiments followed the guidelines established by the Animal Ethics Committee of Beijing Military General Hospital. Eighty-four female Sprague-Dawley rats (220–250 g) were purchased from Beijing Haidian Thriving Experimental Animal Centre (Beijing, China) and housed in an environmentally controlled room with a 12:12-hour lightdark cycle. All the rats were randomly divided into 4 groups using a random number table. Sham group: rats received laminectomy only; SCI group: rats were subjected to SCI; SCI + Vehicle (Veh) group: rats were subjected to SCI with Veh treatment intraperitoneally (intraperitoneally, 0.1% propylene glycol in saline); SCI + HNK group: rats were subjected to SCI with intraperitoneal honokiol treatment (20 mg/kg; Sigma-Aldrich, St. Louis, MO) after SCI. The dose and timing of HNK administration were determined on the basis of findings of our preliminary experiments.
Surgical Procedure All rats were anesthetized via intraperitoneal injection of 10% chloral hydrate (3.0 mL/kg). Then, the injuries were performed as described in the previous text by Young.16 Briefly, a laminectomy was performed at the T9–T11 level to expose the cord underneath without disrupting the dura. The dorsal surface of the T10 level was subjected to a 25 g/cm impact. In the sham group, the vertebral plate at the same segment was removed, but the spinal cord was not injured. After operation, rats were injected with penicillin (intramuscularly, 400,000 unit/animal/d) to prevent infection and with buprenorphine (0.03 mg/kg) to relieve pain for 3 days. Manual bladder expression was performed twice a day until the rats were killed.
Behavioral Examination A hind limb functional test was performed in accordance with the Basso, Beattie, and Bresnahan criteria16 on days 1, 3, 7, 10, and 14 after SCI. Movement, step, and co-ordinated motor action of the lower limbs were rated on a 21-point Basso, Beattie, and Bresnahan scale. Two additional investigators, blinded to treatment assignment, assessed motor function in all the animals (n = 5 for each group).
Histological Analysis Rats were perfused via the left cardiac ventricle with physiological saline and subsequently with 4% paraformaldehyde in 0.1-M phosphate buffer (PB, pH = 7.4) at 24 hours in all groups (n = 4 for each group). Horizontal 10-μm sections of spinal cord tissues were cut with a cryostat and mounted onto polylysine-coated slides. All sections were kept at −80°C until used. 364
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Some sections were stained with hematoxylin and eosin and scored for edema, hemorrhage, and immune-cell infiltration. The histological score was performed as described in previous article.17 Extent of injury was rated on a 0- to 4-point scale: 0 for none or minor, 1 for limited, 2 for intermediate, 3 for prominent, and 4 for widespread.
Immunohistochemistry To examine the activation of microglia, some sections were incubated with rabbit anti-Iba1 antibody (1:200; Abcam, Cambridge, United Kingdom) overnight at 4 °C, rinsed with PBS, incubated with the appropriate secondary antibody (1:100) for 1 hour at 37 °C, rinsed with PBS again, and stained with diaminobenzidine (DAB; Sigma Chemical Company, St. Louis, MO) for visualization under an Olympus microscope (BX51; Olympus America Inc., Center Valley, PA).
Measurement of Myeloperoxidase Activity As myeloperoxidase (MPO) is a specific enzyme present in large quantities in granules of neutrophils, MPO activity is widely used to assess neutrophil infiltration and activation.18 In our study, the MPO activity was measured using a commercial kit (Jiancheng Co., Nanjing, China) according to instructions provided by the manufacturer, and was expressed as the number of units of MPO/mg of protein (n = 4 for each group).
Biochemical Analysis Spinal cord tissues at the lesion site (10 mm) were rapidly dissected at 6 hours after SCI, then homogenized in PBS and centrifuged at 4°C for 15 minutes at 1000 rpm. Tumor necrosis factor α, IL-1β, and IL-6 concentrations were measured in the collected supernatants using enzyme-linked immunosorbent assay kits (R&D Systems, Minneapolis, MN) (n = 4 for each group).
Extraction of the Protein and Western Blot Analysis Spinal cord tissues center the lesion site were dissected in all groups at 12 hours or 24 hours after SCI, and were homogenized in lysis buffer and centrifuged at 4°C for 15 minutes at 12000 rpm. Subsequently, the supernatant was collected and the protein levels were quantified using a bicinchoninic acid protein assay kit (Pierce, Thermo Scientific). Total protein (20 μg for each) was separated on 10% sodium dodecyl sulfate polyacrylamide gels and transferred onto nitrocellulose membranes. The membranes were first blocked with 5% skimmed milk and then incubated overnight at 4°C with either rabbit anti-Klf4 (1:500; Millipore, Bedford, MA), anti-iNOS (1:1,000; Millipore), anti-COX-2 (1:1000; Millipore), or anti-β-actin (1:1,000; Santa Cruz Biotechnology Inc., Santa Cruz, CA). After 5 washes in 1X PBST, membranes were incubated with the appropriate horseradish peroxidase-coupled secondary antibody (1:1000; Millipore) for 1 hour at 37°C, developed using an enhanced chemiluminescence kit (Thermo Scientific, Rockford, IL) for detection of proteins, and assessed for protein levels using imaging software (Quantity One; Bio-Rad Co. Ltd., Hercules, CA).
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Honokiol Alleviates Spinal Cord Injury • Liu et al
Statistical Analysis
All data are expressed as the mean ± standard error of the mean with 95% confidence interval. Comparisons between groups were analyzed using 1-way analysis of variance followed by the Dunnett post hoc test or Dunnett T3 post hoc test. All analysis was performed using SPSS software version 17.0 (SPSS Inc, Chicago, IL). Significance was accepted at P < 0.05.
RESULTS HNK Administration Promotes Functional Recovery After SCI To evaluate the effect of HNK on hind limb functional recovery after SCI, the Basso, Beattie, and Bresnahan score was measured on days 1, 3, 7, 10, and 14 after SCI (Figure 1). The data showed that both hind limbs of all rats subjected to SCI paralyzed immediately after operation and exhibited a partial functional recovery after weeks. When compared with the SCI group, rats in SCI + HNK group showed statistically significant functional recovery starting from the post-traumatic day 3 (P < 0.05).
HNK Administration Reduces the Severity of Histopathological Lesions To evaluate the protective effect of HNK on rats with SCI, histological changes in the spinal cord were assessed using light microscopy at 24 hours after intervention. Structures remained unchanged in the sham group, but were significantly changed in the SCI and SCI + Veh groups. The changes included edema, hemorrhage, neutrophil infiltration, and loosened tissue structure. The morphological changes in the SCI + HNK group were significantly less severe (Figure 2). The calculated histopathological scores are also shown in Figure 2.
HNK Administration Reduces Microglial Activation and Neutrophil Infiltration in the Spinal Cord We tested whether HNK is able to inhibit microglial activation after SCI. Activation of microglia was more marked in the SCI and SCI + Veh groups than that in the sham group at 24 hours after SCI. Activated microglia have a bushy appearance and a large number of processes. The number of
Figure 1. Effects of HNK on motor functional recovery after SCI. The recovery of hind limb function was accessed on days 1, 3, 7, 10, 14 after SCI by BBB score. The hind limb dysfunction was ameliorated with treatment of honokiol. Bars represent means ± SEM. †P < 0.001 vs. SCI group. SCI indicates spinal cord injury; HNK, honokiol; SEM, standard error of the mean; BBB, Basso, Beattie, and Bresnahan.
activated microglia in the SCI + HNK group was markedly reduced (Figure 3). We also investigated whether HNK modulates neutrophil infiltration via assessing the MPO activity in the spinal cord of rats. The MPO activity in the SCI and SCI + Veh groups (compared with the sham group) was significantly increased at 24 hours after injury. Moreover, this increase was significantly reduced by HNK (Figure 4).
HNK Administration Reduces Production the Production of Proinflammatory Enzymes and Cytokines To confirm whether HNK can reduce inflammatory response in injured spinal cord, we measured the levels of proinflammatory cytokines at 6 hours after injury, and important proinflammatory enzymes including iNOS and COX-2 at 24 hours after SCI. The results of the enzyme-linked immunosorbent assay analysis showed that SCI caused a significant increase in proinflammatory cytokines release in the SCI group. However, this increase was markedly downregulated upon
Figure 2. Effects of HNK on histological changes at 24 hours after SCI. A severe tissue structural damage was observed in the spinal cord of rats from the SCI group, and a significant protective effect of HNK was also observed in the SCI + HNK group (scale bar = 50 μm). Bars represent means ± SEM. *P < 0.001 vs. Sham group, †P < 0.001 vs. SCI group. SCI indicates spinal cord injury; HNK, honokiol; SEM, standard error of the mean. Spine
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Honokiol Alleviates Spinal Cord Injury • Liu et al
Figure 3. Effects of HNK on microglia activation at 24 hours after SCI. SCI induced the activation of microglia, which is characterized by a bushy appearance and increased number. In SCI + HNK group, the activation of microglia was inhibited obviously (scale bar = 50 μm). *P < 0.01 vs. Sham group, †P < 0.001 vs. SCI group. SCI indicates spinal cord injury; HNK, honokiol.
the intraperitoneal injection of honokiol in the SCI + HNK group (Figure 5). In addition, the results of the Western blot analysis showed that the SCI-induced iNOS and COX-2 protein expression was also decreased in the SCI + HNK group (Figure 6A).
HNK Administration Downregulates Klf4 Expression After SCI As Klf4 is a newly identified key factor involved in the process of inflammatory response and a new important target of the anti-inflammatory effect of HNK, we test the level of Klf4 using western blot analysis at 12 hours after SCI (Figure 6B). The results demonstrated that a significant increase of Klf4 level was induced by SCI at 12 hours after injury, and this increase can be inhibited by HNK administration in the SCI + HNK group (P < 0.05).
Figure 4. Effects of HNK on the MPO activity after SCI. Compared with rats in the sham group, the MPO activity in the SCI group was significantly increased. The increase was significantly reduced in the SCI + HNK group. Bars represent means ± SEM. *P < 0.01 vs. Sham group, †P < 0.001 vs. SCI group. SCI indicates spinal cord injury; HNK, honokiol; SEM, standard error of the mean; MPO, myeloperoxidase.
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DISCUSSION In the past few decades, Chinese herbal medicine has been widely used in the treatment of SCI in preclinical studies.17,19 HNK, a major active component of herb “Houpo,” has been shown to reduce cerebral infarction due to its various beneficial pharmacological properties.20 Moreover, previous pharmacokinetic and toxicological studies have revealed the desirable spectrum of bioavailability after intravenous administration in animal models and potential safety of it,21 thus making HNK a suitable agent for clinical trials. In this study, we demonstrated that treatment with HNK promoted functional recovery of the hind limbs, reduced tissue damage, microglial activation, and proinflammatory factors release. As one novel mechanism, HNK downregulated the expression of Klf4 in rats subjected to SCI. In the progress of SCI-induced inflammation, different types of cells were recruited to injury sites. First, microglia, the resident macrophage-like population in the CNS, are
Figure 5. Effects of HNK on proinflammatory cytokines after SCI. SCI induced the increase of TNF-α, IL-6, and IL-1β concentrations at 6 hours after SCI, but treatment with HNK decreased the level of above production. Bars represent means ± SEM. *P < 0.001 vs. Sham group, †P < 0.05 vs. SCI group. SCI indicates spinal cord injury; HNK, honokiol; SEM, standard error of the mean; IL, interleukin; TNF α, tumor necrosis factor α.
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Honokiol Alleviates Spinal Cord Injury • Liu et al
Figure 6. Effects of HNK on the iNOS, COX-2, and Klf4 expressions after SCI. The levels of iNOS and COX-2 were upregulated in the SCI group compared with the sham group. The increase of iNOS and COX-2 was attenuated with HNK treatment as compared with the SCI group (A). SCI induced the upregulation of Klf4 at 12 hours after SCI, but treatment with HNK decreased above increase (B). Bars represent mean ± SEM. *P < 0.001 vs. sham group; †P < 0.001 vs. SCI group. SCI indicates spinal cord injury; HNK, honokiol; SEM, standard error of the mean; iNOS, inducible nitric oxide synthase; COX, cyclooxygenase; Klf4, Kruppel-like factor 4.
quickly activated by release of intracellular factors from damaged cells and changes in the extracellular ion activity after SCI.22 Yang et al23 have demonstrated that resident microglia, rather than infiltrating macrophages, are the primary source of the proinflammatory cytokines including IL-1β, IL-6, and tumor necrosis factor α immediately after SCI. Some recent studies have shown that suppressing microglial activity may reduce inflammation and improve neuronal survival or plasticity.24 Our data showed that the number of Iba-1 positive cells was obviously increased at 24 hours after SCI, and HNK administration significantly reduced this number. Second, neutrophils are the first leucocytes to enter the injured spinal cord from the periphery and to be involved in secondary tissue damage.25 They may release reactive oxygen and nitrosyl radicals as well as cytokines, chemokines, and a variety of proteases, including metalloproteinases and neutrophil elastase, which are key determinants of early cell injury, axonal degeneration, and demyelination.26,27 In this study, we used MPO activity as a measure of neutrophil infiltration, the activity of which is an indicator of polymorphonuclear leucocyte accumulation. We found that the increased neutrophil infiltration in spinal cord tissues after SCI can be inhibited by treatment with HNK. Meanwhile, proinflammatory cytokines and enzymes the lesion site of spinal cord contribute to enhance vascular permeability,28,29 increase leukocyte infiltration, and even induce the apoptosis of neurons and oligodendrocytes directly.30,31 Consistent with previous studies,23 we detected increased protein levels of proinflammatory cytokines and enzymes after traumatic SCI. Furthermore, we found that HNK treatment significantly reduced the levels of tumor necrosis factor α, IL-1β, IL-6, iNOS, and COX-2 in the rat model of SCI. Spine
Importantly, Klf4 is one novel transcription factor participating inflammatory responses besides nuclear factor-κB, and has been newly reported as a important therapeutic target of honokiol.13 No article has reported its role in inflammatory conditions after SCI. Our study showed that SCI induced dynamic changes of Klf4 expression (data not shown) and HNK treatment resulted in significantly reduced Klf4 level. Further studies on signaling and regulation mechanisms of Klf4 expression by HNK after SCI are ongoing in our laboratory.
CONCLUSION Our study has shown that HNK significant inhibited the inflammatory responses and subsequently attenuated the tissue damage after SCI in rats, and this effect might be attributed to its powerful ability of downregulating the expression of Klf4.
➢ Key Points Inflammation contributes significantly to the secondary pathogenesis in traumatic injured spinal cord. Traumatic SCI results in an increase in the expression of Klf4 that is a newly identified key regulator in inflammatory pathways and important target for HNK. HNK treatment may suppress inflammatory response, reduce secondary tissue damage, and improve functional recovery of SCI rats partly by downregulating the expression of Klf4.
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BASIC SCIENCE References
1. van den Berg ME, Castellote JM, Mahillo-Fernandez I, et al. Incidence of spinal cord injury worldwide: a systematic review. Neuroepidemiology 2010;34:184–92. 2. Dumont RJ, Okonkwo DO, Verma S, et al. Acute spinal cord injury, part I: pathophysiologic mechanisms. Clin Neuropharmacol 2001;24:254–64. 3. Oyinbo CA. Secondary injury mechanisms in traumatic spinal cord injury: a nugget of this multiply cascade. Acta Neurobiol Exp (Wars) 2011;71:281–99. 4. Carlson SL, Parrish ME, Springer JE, et al. Acute inflammatory response in spinal cord following impact injury. Exp Neurol 1998;151:77–88. 5. Han X, Lu M, Wang S, et al. Targeting IKK/NF-kappaB pathway reduces infiltration of inflammatory cells and apoptosis after spinal cord injury in rats. Neurosci Lett 2012;511:28–32. 6. Yao AH, Jia LY, Zhang YK, et al. Early blockade of TLRs MyD88dependent pathway may reduce secondary spinal cord injury in the rats. Evid Based Complement Alternat Med 2012;2012:591298. 7. Cox A, Varma A, Banik N. Recent advances in the pharmacologic treatment of spinal cord injury [published online ahead of print May 16, 2014]. Metab Brain Dis. doi: 10.1007/s11011-014-9547-y. 8. Fried LE, Arbiser JL. Honokiol, a multifunctional antiangiogenic and antitumor agent. Antioxid Redox Signal 2009;11:1139–48. 9. Chiang J, Shen YC, Wang YH, et al. Honokiol protects rats against eccentric exercise-induced skeletal muscle damage by inhibiting NF-kappaB induced oxidative stress and inflammation. Eur J Pharmacol 2009;610:119–27. 10. Li CY, Chao LK, Wang SC, et al. Honokiol inhibits LPS-induced maturation and inflammatory response of human monocytederived dendritic cells. J Cell Physiol 2011;226:2338–49. 11. Chao LK, Liao PC, Ho CL, et al. Anti-inflammatory bioactivities of honokiol through inhibition of protein kinase C, mitogen-activated protein kinase, and the NF-kappaB pathway to reduce LPS-induced TNFalpha and NO expression. J Agric Food Chem 2010;58:3472–8. 12. Kaushik DK, Gupta M, Das S, et al. Kruppel-like factor 4, a novel transcription factor regulates microglial activation and subsequent neuroinflammation. J Neuroinflammation 2010;7:68. 13. Kaushik DK, Mukhopadhyay R, Kumawat KL, et al. Therapeutic targeting of Kruppel-like factor 4 abrogates microglial activation. J Neuroinflammation 2012;9:57. 14. Chen CM, Liu SH, Lin-Shiau SY. Honokiol, a neuroprotectant against mouse cerebral ischaemia, mediated by preserving Na+, K+-ATPase activity, and mitochondrial functions. Basic Clin Pharmacol Toxicol 2007;101:108–16. 15. Qiang LQ, Wang CP, Wang FM, et al. Combined administration of the mixture of honokiol and magnolol and ginger oil evokes antidepressant-like synergism in rats. Arch Pharm Res 2009;32:1281–92.
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16. Young W. Spinal cord contusion models. Prog Brain Res 2002;137: 231–55. 17. Yin X, Yin Y, Cao FL, et al. Tanshinone IIA attenuates the inflammatory response and apoptosis after traumatic injury of the spinal cord in adult rats. PloS One 2012;7:e38381. 18. Christie SD, Comeau B, Myers T, et al. Duration of lipid peroxidation after acute spinal cord injury in rats and the effect of methylprednisolone. Neurosurg Focus 2008;25:E5. 19. Hellal F, Hurtado A, Ruschel J, et al. Microtubule stabilization reduces scarring and causes axon regeneration after spinal cord injury. Science 2011;331:928–31. 20. Zhang P, Liu X, Zhu Y, et al. Honokiol inhibits the inflammatory reaction during cerebral ischemia reperfusion by suppressing NFkappaB activation and cytokine production of glial cells. Neurosci Lett 2013;534:123–7. 21. Wu X, Chen X, Hu Z. High-performance liquid chromatographic method for simultaneous determination of honokiol and magnolol in rat plasma. Talanta 2003;59:115–21. 22. David S, Kroner A. Repertoire of microglial and macrophage responses after spinal cord injury. Nat Rev Neurosci 2011;12: 388–99. 23. Yang L, Jones NR, Blumbergs PC, et al. Severity-dependent expression of pro-inflammatory cytokines in traumatic spinal cord injury in the rat. J Clin Neurosci 2005;12:276–84. 24. Wang X, Arcuino G, Takano T, et al. P2×7 receptor inhibition improves recovery after spinal cord injury. Nat Med 2004;10:821–7. 25. Chatzipanteli K, Yanagawa Y, Marcillo AE, et al. Posttraumatic hypothermia reduces polymorphonuclear leukocyte accumulation following spinal cord injury in rats. J Neurotrauma 2000;17: 321–32. 26. Dusart I, Schwab ME. Secondary cell death and the inflammatory reaction after dorsal hemisection of the rat spinal cord. Eur J Neurosci 1994;6:712–24. 27. Noble LJ, Donovan F, Igarashi T, et al. Matrix metalloproteinases limit functional recovery after spinal cord injury by modulation of early vascular events. J Neurosci 2002;22:7526–35. 28. Schnell L, Fearn S, Schwab ME, et al. Cytokine-induced acute inflammation in the brain and spinal cord. J Neuropathol Exp Neurol 1999;58:245–54. 29. Lopez-Vales R, Garcia-Alias G, Guzman-Lenis MS, et al. Effects of COX-2 and iNOS inhibitors alone or in combination with olfactory ensheathing cell grafts after spinal cord injury. Spine 2006;31:1100–6. 30. Beattie MS. Inflammation and apoptosis: linked therapeutic targets in spinal cord injury. Trends Mol Med 2004;10:580–3. 31. Wang XJ, Kong KM, Qi WL, et al. Interleukin-1 beta induction of neuron apoptosis depends on p38 mitogen-activated protein kinase activity after spinal cord injury. Acta Pharmacol Sin 2005;26:934–42.
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Honokiol Downregulates Kruppel-Like Factor 4 Expression, Attenuates Inflammation, and Reduces Histopathology After Spinal Cord Injury in Rats Jia Liu, MD,*† Changmeng Zhang, MD,* Zhi Liu, MD,* Jianzheng Zhang, MD,* Zimin Xiang, MD,* and Tiansheng Sun, MD*
Study Design. Randomized experimental study. Objective. To investigate the neuroprotective effect of honokiol (HNK) on rats subjected to traumatic spinal cord injury (SCI) and the molecular mechanisms. Summary of Background Data. Inflammation contributes to the secondary injury to the spinal cord. Honokiol has been used as a neuroprotective agent because of its strong antioxidant and anti-inflammatory properties. Kruppel-like factor 4 (Klf4) is a newly identified critical target for the anti-inflammatory effect of HNK. Whether HNK can inhibit inflammatory response in rat model of SCI through mediating the expression of Klf4 has yet to be elucidated. Methods. Eighty-four adult female Sprague-Dawley rats were randomly divided into 4 groups as sham, SCI, SCI + Vehicle (0.1% propylene glycol in saline, intraperitoneally), and SCI + HNK (20 mg/kg, intraperitoneally) groups. The influences of HNK on the proinflammatory cytokines, microglial activation, neutrophil infiltration, histological changes, and improvement in motor function were assessed. Results. In the SCI group, proinflammatory cytokines, microglial activation, neutrophil infiltration, and Klf4 expression levels were increased compared with the sham group (P < 0.001). HNK intervention downregulated the expression of Klf4, reduced the production of proinflammatory cytokines, inhibited microglial activation, and neutrophil infiltration (P < 0.05). Furthermore, HNK also reduced histopathology and improved functional outcome after traumatic SCI. Conclusion. HNK reduces secondary tissue damage and improves locomotor function recovery after SCI through suppressing From the *Departmentof Institute of Orthopaedics, Chinese PLA (People's Liberation Army) Beijing Army General Hospital, Beijing, China; and †Department of Medical School of Chinese PLA, Beijing, China. Acknowledgment date: September 18, 2014. Acceptance date: December 3, 2014. The device(s)/drug(s) that is/are the subject of the manuscript is/are not intended for human use. National Natural Science Foundation of China (81150020) grants were received in support of this work. No relevant financial activities outside the submitted work. Address correspondence and reprint requests to Tiansheng Sun, MD, Institute of Orthopaedics, Chinese PLA Beijing Army General Hospital, Dongcheng District, Nanmencang No. 5, Beijing 100700, China; E-mail: [email protected] DOI: 10.1097/BRS.0000000000000758 Spine
inflammatory response, and can be used as a potential therapeutic agent for SCI. Key words: honokiol, Kruppel-like factor 4, spinal cord injury, rat, inflammatory response. Level of Evidence: NA Spine 2015;40:363–368
T
raumatic spinal cord injury (SCI) is one of the worldwide clinical problem, because of its complex pathophysiology.1 Primary trauma to spinal cord initiates a cascade of cellular and biochemical events, which lead to exacerbation of the injury and even influence the functional outcome of patients subjected to SCI.2,3 Inflammation is a key event in the secondary injury phase of SCI and marked by the excessive activation of inflammatory cells and production of proinflammatory mediators.4 Several laboratories have demonstrated that targeting the key regulator in inflammatory response, such as nuclear factor-κB, can improve functional recovery in rodent models of traumatic SCI.5,6 As a result, many anti-inflammatory agents have been explored for their potential neuroprotective effects on SCI.7 Honokiol (HNK), a small-molecule polyphenol extracted from the herb “Houpo,” exhibits anti-cancerous,8 antioxidant,9 and anti-inflammatory 10 properties. According to previous studies, the anti-inflammatory effects of honokiol act mainly via suppressing nuclear factor-κB activation and the subsequent cytokine production.11 Recently, an article reported that Kruppel-like factor 4 (Klf4), a zinc finger transcription factor that has been confirmed to play a critical role in several inflammatory cells mediated inflammation,12 was another key therapeutic target of HNK.13 So far, HNK has been explored as a neuroprotective agent in rodents model of several neurological diseases,14,15 but no study has been conducted to evaluate the possible therapeutic effects of this agent in SCI and explore the possible molecular mechanisms. In this study, we investigate whether HNK exerts a neuroprotective effect in rats subjected to SCI and the molecular mechanisms. Our results showed that HNK treatment inhibited the productions of proinflammatory factors, such as inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 www.spinejournal.com
363
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BASIC SCIENCE (COX-2), downregulated the expression of Klf4, attenuated secondary tissue destruction, and enhanced neurological recovery after SCI.
MATERIALS AND METHODS Animals and Groups All animal experiments followed the guidelines established by the Animal Ethics Committee of Beijing Military General Hospital. Eighty-four female Sprague-Dawley rats (220–250 g) were purchased from Beijing Haidian Thriving Experimental Animal Centre (Beijing, China) and housed in an environmentally controlled room with a 12:12-hour lightdark cycle. All the rats were randomly divided into 4 groups using a random number table. Sham group: rats received laminectomy only; SCI group: rats were subjected to SCI; SCI + Vehicle (Veh) group: rats were subjected to SCI with Veh treatment intraperitoneally (intraperitoneally, 0.1% propylene glycol in saline); SCI + HNK group: rats were subjected to SCI with intraperitoneal honokiol treatment (20 mg/kg; Sigma-Aldrich, St. Louis, MO) after SCI. The dose and timing of HNK administration were determined on the basis of findings of our preliminary experiments.
Surgical Procedure All rats were anesthetized via intraperitoneal injection of 10% chloral hydrate (3.0 mL/kg). Then, the injuries were performed as described in the previous text by Young.16 Briefly, a laminectomy was performed at the T9–T11 level to expose the cord underneath without disrupting the dura. The dorsal surface of the T10 level was subjected to a 25 g/cm impact. In the sham group, the vertebral plate at the same segment was removed, but the spinal cord was not injured. After operation, rats were injected with penicillin (intramuscularly, 400,000 unit/animal/d) to prevent infection and with buprenorphine (0.03 mg/kg) to relieve pain for 3 days. Manual bladder expression was performed twice a day until the rats were killed.
Behavioral Examination A hind limb functional test was performed in accordance with the Basso, Beattie, and Bresnahan criteria16 on days 1, 3, 7, 10, and 14 after SCI. Movement, step, and co-ordinated motor action of the lower limbs were rated on a 21-point Basso, Beattie, and Bresnahan scale. Two additional investigators, blinded to treatment assignment, assessed motor function in all the animals (n = 5 for each group).
Histological Analysis Rats were perfused via the left cardiac ventricle with physiological saline and subsequently with 4% paraformaldehyde in 0.1-M phosphate buffer (PB, pH = 7.4) at 24 hours in all groups (n = 4 for each group). Horizontal 10-μm sections of spinal cord tissues were cut with a cryostat and mounted onto polylysine-coated slides. All sections were kept at −80°C until used. 364
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Some sections were stained with hematoxylin and eosin and scored for edema, hemorrhage, and immune-cell infiltration. The histological score was performed as described in previous article.17 Extent of injury was rated on a 0- to 4-point scale: 0 for none or minor, 1 for limited, 2 for intermediate, 3 for prominent, and 4 for widespread.
Immunohistochemistry To examine the activation of microglia, some sections were incubated with rabbit anti-Iba1 antibody (1:200; Abcam, Cambridge, United Kingdom) overnight at 4 °C, rinsed with PBS, incubated with the appropriate secondary antibody (1:100) for 1 hour at 37 °C, rinsed with PBS again, and stained with diaminobenzidine (DAB; Sigma Chemical Company, St. Louis, MO) for visualization under an Olympus microscope (BX51; Olympus America Inc., Center Valley, PA).
Measurement of Myeloperoxidase Activity As myeloperoxidase (MPO) is a specific enzyme present in large quantities in granules of neutrophils, MPO activity is widely used to assess neutrophil infiltration and activation.18 In our study, the MPO activity was measured using a commercial kit (Jiancheng Co., Nanjing, China) according to instructions provided by the manufacturer, and was expressed as the number of units of MPO/mg of protein (n = 4 for each group).
Biochemical Analysis Spinal cord tissues at the lesion site (10 mm) were rapidly dissected at 6 hours after SCI, then homogenized in PBS and centrifuged at 4°C for 15 minutes at 1000 rpm. Tumor necrosis factor α, IL-1β, and IL-6 concentrations were measured in the collected supernatants using enzyme-linked immunosorbent assay kits (R&D Systems, Minneapolis, MN) (n = 4 for each group).
Extraction of the Protein and Western Blot Analysis Spinal cord tissues center the lesion site were dissected in all groups at 12 hours or 24 hours after SCI, and were homogenized in lysis buffer and centrifuged at 4°C for 15 minutes at 12000 rpm. Subsequently, the supernatant was collected and the protein levels were quantified using a bicinchoninic acid protein assay kit (Pierce, Thermo Scientific). Total protein (20 μg for each) was separated on 10% sodium dodecyl sulfate polyacrylamide gels and transferred onto nitrocellulose membranes. The membranes were first blocked with 5% skimmed milk and then incubated overnight at 4°C with either rabbit anti-Klf4 (1:500; Millipore, Bedford, MA), anti-iNOS (1:1,000; Millipore), anti-COX-2 (1:1000; Millipore), or anti-β-actin (1:1,000; Santa Cruz Biotechnology Inc., Santa Cruz, CA). After 5 washes in 1X PBST, membranes were incubated with the appropriate horseradish peroxidase-coupled secondary antibody (1:1000; Millipore) for 1 hour at 37°C, developed using an enhanced chemiluminescence kit (Thermo Scientific, Rockford, IL) for detection of proteins, and assessed for protein levels using imaging software (Quantity One; Bio-Rad Co. Ltd., Hercules, CA).
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Statistical Analysis
All data are expressed as the mean ± standard error of the mean with 95% confidence interval. Comparisons between groups were analyzed using 1-way analysis of variance followed by the Dunnett post hoc test or Dunnett T3 post hoc test. All analysis was performed using SPSS software version 17.0 (SPSS Inc, Chicago, IL). Significance was accepted at P < 0.05.
RESULTS HNK Administration Promotes Functional Recovery After SCI To evaluate the effect of HNK on hind limb functional recovery after SCI, the Basso, Beattie, and Bresnahan score was measured on days 1, 3, 7, 10, and 14 after SCI (Figure 1). The data showed that both hind limbs of all rats subjected to SCI paralyzed immediately after operation and exhibited a partial functional recovery after weeks. When compared with the SCI group, rats in SCI + HNK group showed statistically significant functional recovery starting from the post-traumatic day 3 (P < 0.05).
HNK Administration Reduces the Severity of Histopathological Lesions To evaluate the protective effect of HNK on rats with SCI, histological changes in the spinal cord were assessed using light microscopy at 24 hours after intervention. Structures remained unchanged in the sham group, but were significantly changed in the SCI and SCI + Veh groups. The changes included edema, hemorrhage, neutrophil infiltration, and loosened tissue structure. The morphological changes in the SCI + HNK group were significantly less severe (Figure 2). The calculated histopathological scores are also shown in Figure 2.
HNK Administration Reduces Microglial Activation and Neutrophil Infiltration in the Spinal Cord We tested whether HNK is able to inhibit microglial activation after SCI. Activation of microglia was more marked in the SCI and SCI + Veh groups than that in the sham group at 24 hours after SCI. Activated microglia have a bushy appearance and a large number of processes. The number of
Figure 1. Effects of HNK on motor functional recovery after SCI. The recovery of hind limb function was accessed on days 1, 3, 7, 10, 14 after SCI by BBB score. The hind limb dysfunction was ameliorated with treatment of honokiol. Bars represent means ± SEM. †P < 0.001 vs. SCI group. SCI indicates spinal cord injury; HNK, honokiol; SEM, standard error of the mean; BBB, Basso, Beattie, and Bresnahan.
activated microglia in the SCI + HNK group was markedly reduced (Figure 3). We also investigated whether HNK modulates neutrophil infiltration via assessing the MPO activity in the spinal cord of rats. The MPO activity in the SCI and SCI + Veh groups (compared with the sham group) was significantly increased at 24 hours after injury. Moreover, this increase was significantly reduced by HNK (Figure 4).
HNK Administration Reduces Production the Production of Proinflammatory Enzymes and Cytokines To confirm whether HNK can reduce inflammatory response in injured spinal cord, we measured the levels of proinflammatory cytokines at 6 hours after injury, and important proinflammatory enzymes including iNOS and COX-2 at 24 hours after SCI. The results of the enzyme-linked immunosorbent assay analysis showed that SCI caused a significant increase in proinflammatory cytokines release in the SCI group. However, this increase was markedly downregulated upon
Figure 2. Effects of HNK on histological changes at 24 hours after SCI. A severe tissue structural damage was observed in the spinal cord of rats from the SCI group, and a significant protective effect of HNK was also observed in the SCI + HNK group (scale bar = 50 μm). Bars represent means ± SEM. *P < 0.001 vs. Sham group, †P < 0.001 vs. SCI group. SCI indicates spinal cord injury; HNK, honokiol; SEM, standard error of the mean. Spine
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Figure 3. Effects of HNK on microglia activation at 24 hours after SCI. SCI induced the activation of microglia, which is characterized by a bushy appearance and increased number. In SCI + HNK group, the activation of microglia was inhibited obviously (scale bar = 50 μm). *P < 0.01 vs. Sham group, †P < 0.001 vs. SCI group. SCI indicates spinal cord injury; HNK, honokiol.
the intraperitoneal injection of honokiol in the SCI + HNK group (Figure 5). In addition, the results of the Western blot analysis showed that the SCI-induced iNOS and COX-2 protein expression was also decreased in the SCI + HNK group (Figure 6A).
HNK Administration Downregulates Klf4 Expression After SCI As Klf4 is a newly identified key factor involved in the process of inflammatory response and a new important target of the anti-inflammatory effect of HNK, we test the level of Klf4 using western blot analysis at 12 hours after SCI (Figure 6B). The results demonstrated that a significant increase of Klf4 level was induced by SCI at 12 hours after injury, and this increase can be inhibited by HNK administration in the SCI + HNK group (P < 0.05).
Figure 4. Effects of HNK on the MPO activity after SCI. Compared with rats in the sham group, the MPO activity in the SCI group was significantly increased. The increase was significantly reduced in the SCI + HNK group. Bars represent means ± SEM. *P < 0.01 vs. Sham group, †P < 0.001 vs. SCI group. SCI indicates spinal cord injury; HNK, honokiol; SEM, standard error of the mean; MPO, myeloperoxidase.
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DISCUSSION In the past few decades, Chinese herbal medicine has been widely used in the treatment of SCI in preclinical studies.17,19 HNK, a major active component of herb “Houpo,” has been shown to reduce cerebral infarction due to its various beneficial pharmacological properties.20 Moreover, previous pharmacokinetic and toxicological studies have revealed the desirable spectrum of bioavailability after intravenous administration in animal models and potential safety of it,21 thus making HNK a suitable agent for clinical trials. In this study, we demonstrated that treatment with HNK promoted functional recovery of the hind limbs, reduced tissue damage, microglial activation, and proinflammatory factors release. As one novel mechanism, HNK downregulated the expression of Klf4 in rats subjected to SCI. In the progress of SCI-induced inflammation, different types of cells were recruited to injury sites. First, microglia, the resident macrophage-like population in the CNS, are
Figure 5. Effects of HNK on proinflammatory cytokines after SCI. SCI induced the increase of TNF-α, IL-6, and IL-1β concentrations at 6 hours after SCI, but treatment with HNK decreased the level of above production. Bars represent means ± SEM. *P < 0.001 vs. Sham group, †P < 0.05 vs. SCI group. SCI indicates spinal cord injury; HNK, honokiol; SEM, standard error of the mean; IL, interleukin; TNF α, tumor necrosis factor α.
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Figure 6. Effects of HNK on the iNOS, COX-2, and Klf4 expressions after SCI. The levels of iNOS and COX-2 were upregulated in the SCI group compared with the sham group. The increase of iNOS and COX-2 was attenuated with HNK treatment as compared with the SCI group (A). SCI induced the upregulation of Klf4 at 12 hours after SCI, but treatment with HNK decreased above increase (B). Bars represent mean ± SEM. *P < 0.001 vs. sham group; †P < 0.001 vs. SCI group. SCI indicates spinal cord injury; HNK, honokiol; SEM, standard error of the mean; iNOS, inducible nitric oxide synthase; COX, cyclooxygenase; Klf4, Kruppel-like factor 4.
quickly activated by release of intracellular factors from damaged cells and changes in the extracellular ion activity after SCI.22 Yang et al23 have demonstrated that resident microglia, rather than infiltrating macrophages, are the primary source of the proinflammatory cytokines including IL-1β, IL-6, and tumor necrosis factor α immediately after SCI. Some recent studies have shown that suppressing microglial activity may reduce inflammation and improve neuronal survival or plasticity.24 Our data showed that the number of Iba-1 positive cells was obviously increased at 24 hours after SCI, and HNK administration significantly reduced this number. Second, neutrophils are the first leucocytes to enter the injured spinal cord from the periphery and to be involved in secondary tissue damage.25 They may release reactive oxygen and nitrosyl radicals as well as cytokines, chemokines, and a variety of proteases, including metalloproteinases and neutrophil elastase, which are key determinants of early cell injury, axonal degeneration, and demyelination.26,27 In this study, we used MPO activity as a measure of neutrophil infiltration, the activity of which is an indicator of polymorphonuclear leucocyte accumulation. We found that the increased neutrophil infiltration in spinal cord tissues after SCI can be inhibited by treatment with HNK. Meanwhile, proinflammatory cytokines and enzymes the lesion site of spinal cord contribute to enhance vascular permeability,28,29 increase leukocyte infiltration, and even induce the apoptosis of neurons and oligodendrocytes directly.30,31 Consistent with previous studies,23 we detected increased protein levels of proinflammatory cytokines and enzymes after traumatic SCI. Furthermore, we found that HNK treatment significantly reduced the levels of tumor necrosis factor α, IL-1β, IL-6, iNOS, and COX-2 in the rat model of SCI. Spine
Importantly, Klf4 is one novel transcription factor participating inflammatory responses besides nuclear factor-κB, and has been newly reported as a important therapeutic target of honokiol.13 No article has reported its role in inflammatory conditions after SCI. Our study showed that SCI induced dynamic changes of Klf4 expression (data not shown) and HNK treatment resulted in significantly reduced Klf4 level. Further studies on signaling and regulation mechanisms of Klf4 expression by HNK after SCI are ongoing in our laboratory.
CONCLUSION Our study has shown that HNK significant inhibited the inflammatory responses and subsequently attenuated the tissue damage after SCI in rats, and this effect might be attributed to its powerful ability of downregulating the expression of Klf4.
➢ Key Points Inflammation contributes significantly to the secondary pathogenesis in traumatic injured spinal cord. Traumatic SCI results in an increase in the expression of Klf4 that is a newly identified key regulator in inflammatory pathways and important target for HNK. HNK treatment may suppress inflammatory response, reduce secondary tissue damage, and improve functional recovery of SCI rats partly by downregulating the expression of Klf4.
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