(+)-Borneol is neuroprotective against permanent cerebral ischemia in rats by suppressing production of proinflammatory cytokines

Abstract

Stroke is one of the leading causes of disability and death globally. It occurs when a major artery is occluded in the brain and leads to death of cells within the injured tissue. (+)-Borneol, a simple bicyclic monoterpene extracted from traditional Chinese medicine, is widely used in various types of diseases. However, no study has proved the effects of (+)-borneol on functional recovery from permanent ischemic stroke and the mechanism is still unknown. Here, we report that in the rat model of permanent cerebral ischemia, we found that (+)-borneol (1.0 mg/kg) significantly ameliorated infarct size and neurological scoresvia reducing the expression of inducible nitric oxide synthase (iNOS) and tumor necrosis factor-alpha (TNF-α) in a dose dependent manner. Notably, (+)-borneol showed long-term effects on the improvement of sensorimotor functions in the photothrombotic model of stroke, which decreased the number of foot faults in the grid-walking task and forelimb asymmetry scores in the cylinder task, at least in part through reducing loss of dendritic spines in the length, brunch number and density. These findings suggest that (+)-borneol could serve as a therapeutic target for ischemic stroke.

Keywords

(+)-borneol / neuroprotective effects / permanent cerebral ischemia / anti-inflammation / functional recovery / dendritic spines

1 Introduction

Non-alcoholic steatohepatitis or NASH has become a significant health threat in a growing number of nations accompanying the global pandemic of obesity and type 2 diabetes^[ 1^]. Liver fibrosis is one of the many complications associated with NASH^[ 2^]. In response to nutrition surplus, several different types of cells including hepatic stellate cells and portal fibroblast cells trans-differentiate into pro-fibrogenic myofibroblasts and accelerate the production and deposition of extracellular matrix (ECM) proteins^[ 3- 4^]. Although considered a host defense mechanism, excessive fibrogenesis can disrupt normal liver structure and interfere with liver functionalities precipitating the development of end-stage liver diseases such as cirrhosis and hepatocellular carcinoma^[ 5^]. The mechanism accounting for liver fibrosis during NASH pathogenesis is not fully appreciated.

Inside the ECM-producing cells, fibrogenesis is dictated by a network of growth factors, cytokines, and transcription factors^[ 6^]. Transforming growth factor (TGF-β) is by far the most extensively studied pro-fibrogenic factor in the liver, signaling primarily through the SMAD family of transcription factors^[ 7^]. Upon binding to its receptor, TGF-β triggers the phosphorylation and nuclear translocation of SMAD3. SMAD3 in turn binds to the promoter regions of pro-fibrogenic genes (e.g., type I collagen) and activates transcription. The ability of SMAD3 to promote pro-fibrogenic transcription is in part impacted by its post-translational modification status. In addition to phosphorylation, SMAD3 can also be acetylated in its lysine residues; acetylation of SMAD3 by the histone acetyltransferase p300/CBP enhances its activity^[ 8^]. In contrast, deacetylation of SMAD3 by the lysine deacetylase SIRT1 dampens its activity^[ 9^].

Previously we have shown that protein inhibitor of activated STAT 4 (PIAS4) downregulates SIRT1 expression at the transcriptional level in response to hypoxia in cancer cells^[ 10- 11^]. Therefore, we hypothesized that PIAS4 could potentially contribute to liver fibrosis by modulating SIRT1-dependent SMAD3 (de)acetylation. Our data as summarized in this report support this hypothesis and indicate that targeting PIAS4 may provide novel therapeutic solutions against NASH-induced liver fibrosis.

2 Materials and methods

2.1 Animals

All animal protocols were approved by the NJMU Intramural Ethics Committee on Animal Studies. To induce steatohepatitis, 8 week-old male C57/BL6 mice were fed a high fat high carbohydrate (HFHC) diet (D12492, Research Diets) for 16 consecutive weeks^[ 12^]. To knock down PIAS4 , the mice were injected via the tail vein with purified lentiviral particles (1X10⁹ MOI) that carry short hairpin RNA (shRNA) targeting PIAS4 (5'-GTGCTGTACGGGAAGTACTT-3') or scrambled shRNA (SCR) every 10 days for the duration of the experiments.

2.2 Protein extraction and Western blotting assay

Tissue lysates were obtained as previously described^[ 13^]. Western blot analyses were performed with anti-SIRT1 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-type III collagen (Santa Cruz Biotechnology), anti-PIAS4 (Sigma), anti-β-actin (Sigma), anti-acetyl lysine (Cell Signaling Tech), anti-type I collagen (Rockland), anti-α-SMA (Abcam), and anti-SMAD3 (Abcam) antibodies.

2.3 Chromatin immunoprecipitation (ChIP)

ChIP assays were performed essentially as described before^[ 14^] with anti-SMAD3 antibody (Abcam). Precipitated genomic DNA was amplified by real-time PCR with primers as previously described^[ 3, 11, 15^].

2.4 Histology

Histological analyses were performed essentially as described before^[ 11, 13^]. Briefly, paraffin sections were stained with picrosirius red (Sigma) or Masson's trichrome (Sigma) according to standard procedures. Pictures were taken using an Olympus IX-70 microscope.

2.5 Statistical analysis

Data are presented as mean±SD. For experiments concerning multiple groups, one-way ANOVA with post-hoc Scheffe analyses were performed to evaluate the differences. The differences between two (control and experimental) groups were determined by two-sided, unpaired Student's t-test. P values smaller than 0.05 are considered significant. For the in vivo experiments, specific P values are spelled out.

3 Results

3.1 PIAS4 knockdown alleviates liver fibrosis in mice

We first examined the effect of PIAS4 on liver fibrosis in vivo. To induce liver fibrosis, C57/BL6 mice were fed with a HFHC diet for 16 weeks^[ 12^]. Picrosirius red (Fig. 1A) and Masson's trichrome (Fig. 1B) staining revealed extensive interstitial fibrosis in the livers of mice fed on the HFHC diet compared to the mice on a control (chow) diet. PIAS4 knockdown was achieved via lentivirus-mediated delivery of shRNA injected through the tail vein. Western blotting analysis showed that compared to mice injected with control shRNA (SCR), PIAS4-specific shRNA (shPias4) significantly downregulated PIAS4 levels in the liver (Fig. 2B). Histological measurements showed that PIAS4 silencing largely abrogated HFHC diet induced liver fibrosis (Fig. 1A and Fig. 1B).

Fig.1 PIAS4 knockdown alleviates liver fibrosis in mice. C57/BL6 mice were fed on a high fat high carbohydrate (HFHC)-diet or a chow diet for 16 weeks. Lentivirus carrying either PIAS4 targeting shRNA or a control shRNA was injected weekly via the tail vein. Picrosirius red (A) and Masson's trichrome (B) stainings were performed as described in Methods. Quantification was carried out using Image Pro. N = 5 mice for each group. Data are presented as mean±S.D. Scale bar, 50 mm.

Full size|PPT slide

3.2 PIAS4 depletion downregulates expression of pro-fibrogenic genes

Next, we examined the effects of PIAS4 depletion on the expression of pro-fibrogenic genes in the liver. Quantitative PCR analyses showed that the HFHC diet stimulated the synthesis of a panel of pro-fibrogenic genes, including type I collagen (col1a1 and co1a2), type III collagen (col3a1), alpha smooth muscle actin (acta2), tissue inhibitors of matrix metalloproteinase (timp1 and timp3), lysyl oxidase (lox), and integrin subunit alpha 1 (itga1). PIAS4 depletion, to varying extents, downregulated all the pro-fibrogenic genes examined here (Fig. 2A). Western blotting experiments confirmed the observation that PIAS4 depletion systemically downregulated the induction of pro-fibrogenic gene expression by the HFHC diet (Fig. 2B). Taken together, we were able to conclude that PIAS4 might be essential for liver fibrosis in an HFHC diet-induced model of NASH.

Fig.2 PIAS4 depletion downregulates expression of pro-fibrogenic genes. C57/BL6 mice were fed on an HFHC-diet or a chow diet for 16 weeks. Lentivirus carrying either PIAS4 targeting shRNA or a control shRNA was injected weekly via the tail vein. Expression levels of pro-fibrogenic genes were examined by qPCR (A) and Western blotting assays (B). N = 5 mice for each group. Data are presented as mean±S.D. *P<0.05.

Full size|PPT slide

3.3 PIAS4 modulates SMAD3 activity by influencing SIRT1-dependent deacetylation

SMAD3 is one of the most potent pro-fibrogenic transcription factors. We hypothesized that PIAS4 could modulate SMAD3 activity in the liver. ChIP assay showed that binding of SMAD3 to its target genes, including col1a1, col1a2, and acta2, was significantly upregulated in the livers of mice fed on the HFHC diet (Fig. 3A). On the contrary, PIAS4 silencing markedly dampened the occupancies of SMAD3 on its target promoters.

Fig.3 PIAS4 modulates SMAD3 activity by influencing SIRT1-dependent deacetylation. C57/BL6 mice were fed on an HFHC-diet or a chow diet for 16 weeks. Lentivirus carrying either PIAS4 targeting shRNA or a control shRNA was injected weekly via the tail vein. (A) ChIP assay was performed using liver homogenates with anti-SMAD3 antibody. Precipitated DNA was amplified using primers surrounding the indicated gene promoters. (B) Immunoprecipitation was performed with anti-SMAD3 using liver homogenates. Western blotting was performed with anti-SMAD3 or anti-acetyl lysine. n = 3 mice for each group. Data are presented as mean±S.D. *P<0.05.

Full size|PPT slide

It has been documented that SIRT1 suppresses SMAD3 activity by promoting its deacetylation^[ 9^]. As shown in Fig. 2B, HFHC diet feeding caused a decrease in SIRT1 expression, consistent with an increase in SMAD3 activity; PIAS4 knockdown, however, was able to normalize SIRT1 expression, again in keeping with suppressed SMAD3 activity. We then examined the acetylation status of SMAD3 in various settings. Immunoprecipitation combined with Western blotting showed that HFHC diet feeding resulted in a significant upregulation of SMAD3 acetylation, consistent with increased SMAD3 binding activity and decreased SIRT1 expression, in the liver (Fig. 3B). PIAS4 depletion, however, blocked the induction of SMAD3 acetylation, which was in agreement with restored SIRT1 expression and reduced SMAD3 binding on target promoters. Collectively, these data suggest that PIAS4 might contribute to liver fibrosis possibly by modulating SIRT1-dependent deacetylation of SMAD3.

4 Discussion

NASH serves as an intermediate disease state bridging the reversible and manageable steatosis and the irreversible and refractory cirrhosis^[ 1^]. Liver fibrosis is a hallmark event in the pathogenesis of NASH, the ineffective intervention of which may precipitate the development end-stage liver diseases and significantly dim the chance of patient survival. We report here that PIAS4 may play a critical role in liver fibrosis by modulating SMAD3 activity, likely through SIRT1-dependent deacetylation. Several previous investigations have implicated PIAS1 as a modulator of the TGF-β pathway although the conclusions seem to be contradictory. For instance, the Kurabayashi group has reported that PIAS1 is essential for TGF-β induced α-SMA trans-activation in smooth muscle cells by SUMOylating KLF4^[ 16^]. In contrast, Netherton and Bonni demonstrated that PIAS1 represses TGF-β initiated mesenchymal cell differentiation by SUMOylating SnoN^[ 17^]. Our data suggest that PIAS4 modulates TGF-β signaling in an indirect manner, namely, through SIRT1-dependent deacetylation of SMAD3. However, the possibility that PIAS4 could directly interact with and SUMOylate SMAD3 cannot be excluded especially in light of the finding that SMAD4, the common SMAD protein required for TGF-β signaling, have been found to be a direct substrate for SUMOylation^[ 18^]. In addition, it remains unclear whether other members of the PIAS family could play a non-redundant role in regulating liver fibrosis. Starkel et al. have shown that PIAS3 expression was progressively increased during the development of liver fibrosis/cirrhosis in patients infected with hepatitis C virus (HCV), suggesting that PIAS3 may also play a precipitating role in this process^[ 19^]. When further confirmed, our data add support to the argument of using a pan-PIAS inhibitor to stall or reverse fibrogenesis in the liver.

We show here that PIAS4 promotes liver fibrosis likely through SIRT1-dependent SMAD3 deacetylation. In fact, several alternative scenarios exist to interpret the data. First, PIAS4 has been known to modulate cellular response to hypoxia^[ 10- 11, 20^], which by itself is a risk factor for NASH and a promoter of liver fibrosis^[ 21^]. Second, liver fibrosis in the context of NASH often occurs as a result of excessive hepatic inflammation. PIAS4 can directly SUMOylate and thus activate NF-kB, the master regulator of cellular inflammation^[ 22^]. Therefore, our observation that PIAS4 knockdown attenuated liver fibrosis could be secondary to reduced hepatic inflammation as a result of NF-kB deactivation. Finally, we used a lentivirus delivery system that did not differentiate the liver from other organs or cells in the circulation. It is possible that PIAS4 might influence liver fibrosis by regulating circulating myeloid cells (e.g., macrophages), which are considered a driving force of liver fibrosis^[ 23^]. These remaining issues will have to be sorted out by future investigations.

In summary, we provide evidence that PIAS4 knockdown in a mouse model of NASH effectively attenuated liver fibrosis. Therefore, PIAS4 could become an attractive target for the development of novel therapeutic strategies to prevent excessive liver fibrogenesis.

References

[1] Blanco M, Castillo  J. Stroke in 2012: Major advances in the treatment of stroke[J]. Nat Rev Neurol, 2013, 9(2): 68–70

[2] Dirnagl U, Iadecola  C, Moskowitz MA . Pathobiology of ischaemic stroke: an integrated view[J]. Trends Neurosci, 1999, 22(9): 391–397

[3] Yan T, Chopp  M, Chen J . Experimental animal models and inflammatory cellular changes in cerebral ischemic and hemorrhagic stroke[J]. Neurosci Bull, 2015, 31(6): 717–734

[4] Liu R, Zhang  L, Lan X , Protection by borneol on cortical neurons against oxygen-glucose deprivation/reperfusion: involvement of anti-oxidation and anti-inflammation through nuclear transcription factor kappaB signaling pathway[J]. Neuroscience, 2011, 176: 408–419

[5] Ehrnhöfer-Ressler MM ,  Fricke K ,  Pignitter M , Identification of 1,8-cineole, borneol, camphor, and thujone as anti-inflammatory compounds in a Salvia officinalis L. infusion using human gingival fibroblasts[J]. J Agric Food Chem, 2013, 61(14): 3451–3459

[6] Tambe R, Jain  P, Patil S , Antiepileptogenic effects of borneol in pentylenetetrazole-induced kindling in mice[J]. Naunyn Schmiedebergs Arch Pharmacol, 2016, 389(5): 467–475

[7] Chen Z, Gong  X, Lu Y , Enhancing effect of borneol and muscone on geniposide transport across the human nasal epithelial cell monolayer[J]. PLoS One, 2014a, 9(7): e101414

[8] Chen J, Li  L, Su J , Synergistic apoptosis-inducing effects on A375 human melanoma cells of natural borneol and curcumin[J]. PLoS One, 2014b, 9(6): e101277

[9] Jiang J, Shen  YY, Li J , (+)-Borneol alleviates mechanical hyperalgesia in models of chronic inflammatory and neuropathic pain in mice[J]. Eur J Pharmacol, 2015, 757: 53–58

[10] Longa EZ, Weinstein  PR, Carlson S , Reversible middle cerebral artery occlusion without craniectomy in rats[J]. Stroke, 1989, 20(1): 84–91

[11] Clarkson AN, Huang  BS, Macisaac SE , Reducing excessive GABA-mediated tonic inhibition promotes functional recovery after stroke[J]. Nature, 2010, 468(7321): 305–309

[12] Chen J, Li  Y, Wang L , Therapeutic benefit of intravenous administration of bone marrow stromal cells after cerebral ischemia in rats[J]. Stroke, 2001, 32(4): 1005–1011

[13] Zhou L, Li  F, Xu HB , Treatment of cerebral ischemia by disrupting ischemia-induced interaction of nNOS with PSD-95[J]. Nat Med, 2010, 16(12): 1439–1443

[14] Blasi E, Barluzzi  R, Bocchini V , Immortalization of murine microglial cells by a v-raf/v-myc carrying retrovirus[J]. J Neuroimmunol, 1990, 27(2-3): 229–237

[15] Sims NR, Muyderman  H. Mitochondria, oxidative metabolism and cell death in stroke[J]. Biochim Biophys Acta, 2010, 1802(1): 80–91

[16] Xu P, Li  Y, Du SY , Comparative pharmacokinetics of borneol in cerebral ischemia-reperfusion and sham-operated rats[J]. J Zhejiang Univ Sci B, 2014, 15(1): 84–91

[17] Macrae IM. Preclinical stroke research--advantages and disadvantages of the most common rodent models of focal ischaemia[J]. Br J Pharmacol, 2011, 164(4): 1062–1078

[18] Yu CL, Zhou  H, Chai AP , Whole-scale neurobehavioral assessments of photothrombotic ischemia in freely moving mice[J]. J Neurosci Methods, 2015, 239: 100–107

[19] Hering H, Sheng  M. Dendritic spines: structure, dynamics and regulation[J]. Nat Rev Neurosci, 2001, 2(12): 880–888

[20] Kasai H, Matsuzaki  M, Noguchi J , Structure-stability-function relationships of dendritic spines[J]. Trends Neurosci, 2003, 26(7): 360–368

[21] Walberer M, Rueger  MA, Simard ML , Dynamics of neuroinflammation in the macrosphere model of arterio-arterial embolic focal ischemia: an approximation to human stroke patterns[J]. Exp Transl Stroke Med, 2010, 2(1): 22

[22] Amantea D, Micieli  G, Tassorelli C , Rational modulation of the innate immune system for neuroprotection in ischemic stroke[J]. Front Neurosci, 2015, 9: 147

[23] Kastrup A, Engelhorn  T, Beaulieu C , Dynamics of cerebral injury, perfusion, and blood-brain barrier changes after temporary and permanent middle cerebral artery occlusion in the rat[J]. J Neurol Sci, 1999, 166(2): 91–99

[24] Molina CA, Alvarez-Sabín  J. Recanalization and reperfusion therapies for acute ischemic stroke[J]. Cerebrovasc Dis, 2009, 27(Suppl 1): 162–167

[25] Bourne JN, Harris  KM. Balancing structure and function at hippocampal dendritic spines[J]. Annu Rev Neurosci, 2008, 31: 47–67

[26] Maiti P, Manna  J, Ilavazhagan G , Molecular regulation of dendritic spine dynamics and their potential impact on synaptic plasticity and neurological diseases[J]. Neurosci Biobehav Rev, 2015, 59: 208–237

[27] Fiala JC, Spacek  J, Harris KM . Dendritic spine pathology: cause or consequence of neurological disorders[J]? Brain Res Brain Res Rev, 2002, 39(1): 29–54

[28] Ahmed ME, Tucker  D, Dong Y , Methylene Blue promotes cortical neurogenesis and ameliorates behavioral deficit after photothrombotic stroke in rats[J]. Neuroscience, 2016, 336: 39–48

[29] Chimura T, Launey  T, Yoshida N . Calpain-mediated degradation of drebrin by excitotoxicity in vitro and in vivo[J]. PLoS One, 2015, 10(4): e0125119

[30] Sala C, Segal  M. Dendritic spines: the locus of structural and functional plasticity[J]. Physiol Rev, 2014, 94(1): 141–188

[31] Brown CE, Wong  C, Murphy TH . Rapid morphologic plasticity of peri-infarct dendritic spines after focal ischemic stroke[J]. Stroke, 2008, 39(4): 1286–1291

[32] Walberer M, Rueger  MA, Simard ML , Dynamics of neuroinflammation in the macrosphere model of arterio-arterial embolic focal ischemia: an approximation to human stroke patterns[J]. Exp Transl Stroke Med, 2010, 2(1): 22

[33] Zhang R, Zhao  M, Ji HJ , Study on the dynamic changes in synaptic vesicle-associated protein and axonal transport protein combined with LPS neuroinflammation model[J]. ISRN Neurol, 2013, 2013: 496079

[34] Santa-Cecília FV ,  Socias B ,  Ouidja MO , Doxycycline suppresses microglial activation by inhibiting the p38 MAPK and NF-kB signaling pathways[J]. Neurotox Res, 2016, 29(4): 447–459

[35] Chen XH, Lin  ZZ, Liu AM , The orally combined neuroprotective effects of sodium ferulate and borneol against transient global ischaemia in C57 BL/6J mice[J]. J Pharm Pharmacol, 2010, 62(7): 915–923

[36] Verheul HB, de Leeuw  FE, Scholten G , GABAA receptor function in the early period after transient forebrain ischaemia in the rat[J]. Eur J Neurosci, 1993, 5(7): 955–960

[37] Granger RE, Campbell  EL, Johnston GA . (+)- And (-)-borneol: efficacious positive modulators of GABA action at human recombinant α1β2g2L GABA(A) receptors[J]. Biochem Pharmacol, 2005, 69(7): 1101–1111

Acknowledgments

This work was supported by grants from National Natural Science Foundation of China (91232304, 31530091, 81571188 and 81222016), the Natural Science Foundation of Jiangsu Province (BK2011029) and Distinguished Young Scientists Fund (BK20130040), and the Collaborative Innovation Center for Cardiovascular Disease Translational Medicine.

RIGHTS & PERMISSIONS

2017 2017 by the Journal of Biomedical Research. All rights reserved