https://www.frontiersin.org/articles/10.3389/fneur.2018.00253/full?
Yanzhe Wang
Lei Li,
Zhiyi He- Department of Neurology, The First Affiliated Hospital of China Medical University, Shenyang, China
Toll-like receptors (TLRs) play key roles in cerebral ischemia and reperfusion injury by inducing the production of inflammatory mediators, such as interleukins (ILs) and tumor necrosis factor-alpha (TNF-α). According to recent studies, ursolic acid (UA) regulates TLR signaling and exhibits notable anti-inflammatory properties. In the present study, we explored the mechanism by which UA regulates inflammation in the rat middle cerebral artery occlusion and reperfusion (MCAO/R) model. The MCAO/R model was induced in male Sprague–Dawley rats (MCAO for 2 h, followed by reperfusion for 48 h). UA was administered intragastrically at 0.5, 24, and 47 h after reperfusion. The direct high mobility group box 1 (HMGB1) inhibitor glycyrrhizin (GL) was injected intravenously after 0.5 h of ischemia as a positive control. The degree of brain damage was estimated using the neurological deficit score, infarct volume, histopathological changes, and neuronal apoptosis. We assessed IL-1β, TNF-α, and IL-6 levels to evaluate post-ischemic inflammation. HMGB1 and TLR4 expression and phosphorylation of nuclear factor kappa-light-chain-enhancer of activated B cell (NFκB) were also examined to explore the underlying mechanism. UA (10 and 20 mg/kg) treatment significantly decreased the neurological deficit scores, infarct volume, apoptotic cells, and IL-1β, TNF-α, and IL-6 concentrations. The infarct area ratio was reduced by (33.07 ± 1.74), (27.05 ± 1.13), (27.49 ± 1.87), and (39.74 ± 2.14)% in the 10 and 20 mg/kg UA, GL, and control groups, respectively. Furthermore, UA (10 and 20 mg/kg) treatment significantly decreased HMGB1 release and the TLR4 level and inactivated NFκB signaling. Thus, the effects of intragastric administration of 20 mg/kg of UA and 10 mg/kg of GL were similar. We provide novel evidence that UA reduces inflammatory cytokine production to protect the brain from cerebral ischemia and reperfusion injury possibly through the HMGB1/TLR4/NFκB signaling pathway.
Introduction and Background
Ischemic stroke, which occurs as a result of the sudden occlusion of a blood vessel by a thrombus or embolism, is a common cause of death and disability worldwide (1). Currently, thrombolysis therapy within the therapeutic window and mechanical thrombectomy in stroke patients are widely accepted for the treatment of sudden cerebral ischemia (2, 3). However, an inflammatory response has been shown to occur after thrombolysis, exacerbating the reperfusion injury (4–6). Therefore, studies aiming to identify an effective adjunct to treatments for cerebral ischemia and reperfusion injury deserve more attention.
Toll-like receptor 4 (TLR4) plays a key role in cerebral ischemia and reperfusion injury by inducing the production of inflammatory mediators, such as interleukins (ILs) and tumor necrosis factor-alpha (TNF-α) (7, 8). TLR4 were initially identified as receptors for endogenous ligands known as damage-associated molecular patterns (DAMPs), particularly high mobility group box 1 (HMGB1), during brain injury. HMGB1 is a ubiquitous DNA-binding nuclear protein that is either passively released from necrotic cells or actively secreted in response to inflammatory signals (9, 10). In addition, overactive microglia and reactive astrocytes in the ischemic region can aggravate ischemic damage after activation of the TLR4 signaling pathways (11). Therefore, strategies that modulate post-ischemic TLR4 signaling in the brain may suppress inflammation induced by cerebral ischemia and provide new therapies for stroke.
Ursolic acid (UA: 3b-hydroxy-urs-12-ene-28-oic acid), a natural pentacyclic triterpenoid, has been reported to exhibit biological activities in the brain, including anti-oxidative, anti-tumor, anti-rheumatic, anti-viral, and anti-inflammatory effects (12). Furthermore, UA also inhibited microglial and astrocyte activation and decreased the levels of TNF-α, IL-1β, and IL-6 in lipopolysaccharide-induced brain inflammation in mice with cognitive deficits (13). However, researchers have not determined whether UA protects against ischemia and reperfusion injury by antagonizing the HMGB1/TLR4 signaling pathway. In this study, we used glycyrrhizin (GL) as a positive control drug. GL is a direct HMGB1 inhibitor and the effective dose for treating cerebral ischemia and reperfusion injury has been established (14).
In the present study, we used the rat middle cerebral artery occlusion and reperfusion (MCAO/R) model with UA and GL to examine the mechanism by which UA regulates the inflammation response induced by ischemia and reperfusion. We investigated whether UA reduced inflammatory cytokine production to protect the brain from cerebral ischemia and reperfusion injury possibly though the HMGB1/TLR4/NFκB signaling pathway.
Materials and Methods
Animal Preparation and Drug Administration
All experimental protocols involving animals were performed according to the guidelines of the National Institutes of Health Guide for the Care and Use of Laboratory Animals (Publication No. 85-23, revised 1985), the UK Animals Scientific Procedures Act 1986 or the European Communities Council Directive of 24 November 1986 (86/609/EEC) and the “Guiding Principles in the Use of Animals in Toxicology,” adopted by the Society of Toxicology in 1989. All procedures used in animal experiments were approved by the Institutional Animal Care and Use Committee of China Medical University. Ninety male Sprague–Dawley rats were purchased from the Liaoning Changsheng Biotechnology Company (Benxi, China). These rats were housed under a 12-h light/12-h day cycle with free access to food and water ad libitum. Rats weighing between 250 and 280 g were randomly divided into five groups. (1) In the sham group (n = 18), rats underwent the same surgical procedures as rats in the MCAO/R group without filament insertion and received the vehicle. (2) In the control group (n = 18), rats underwent the MCAO/R surgical procedures and received vehicles both intragastrically (i.g.) and intravenously (i.v.) when the other treatment groups were administered UA or GL. (3) In the low-dose UA (L-UA) group (n = 18), 10 mg/kg UA (purity ≥ 95.0%, Sigma-Aldrich, St. Louis, MO, USA) in distilled water containing 0.5% Tween-80 (ddH2O/0.5% Tween-80) was administered by oral gavage at 0.5, 24, and 47 h after reperfusion, according to previous studies clarifying the oral absorption rate and drug action time (15–17). (4) The high-dose UA (H-UA) group (n = 18) was administered 20 mg/kg UA. (5) The GL group rats (n = 18) were i.v. administered 10 mg/kg of GL (purity ≥ 95.0%, Sigma-Aldrich, St. Louis, MO, USA) in a volume of 0.5 ml of distilled water containing 0.5% Tween-80 (ddH2O/0.5% Tween-80) via the tail vein 0.5 h after ischemia and before reperfusion as a positive control (18–21).
According to previous studies clarifying the oral absorption rate and drug action time, UA-administered mice had a lethal dose 50 of 60 mg/kg and a rat-to-mouse dosing ratio of 6.3/9.1. The final dose of UA was 5, 10, and 20 mg/kg. Since UA is insoluble in water, 0.1% Tween-80 is used as a solubilizer and 0.1% Tween-80 is used to dilute UA to 1 mg/ml, and the pH is adjusted to 7.4 to avoid the acid and alkali caused by the drug stimulate.
Forty-eight hours after reperfusion, 18 rats in each group were randomly divided into three groups by a researcher who was unaware of the neurological deficits in these rats. Six rats were decapitated to obtain fresh brain tissue samples for biochemical analyses. The ischemic cortex, which was defined as the penumbra, was collected for ELISA and western blotting analyses based on methods modified from Jiang et al. (22). The brains of six rats were stained to determine the infarct volume; six rats were perfused with fixative for histological preparation and analysis of the brains. The brain samples from each animal were sectioned into three slices beginning 3 mm from the anterior tip of the frontal lobe in the coronal plane. The slices were 3-, 4-, and 3-mm thick from front to back, respectively. The middle slices were embedded in paraffin and sliced into 5-μm thick sections for Nissl staining, immunohistochemical staining, immunofluorescence staining, and double-labeling using terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) and neuronal nuclei (NeuN). To ensure that the positive cells were counted at the same coronal level, we collected ten 5-μm thick coronal sections of the dorsal hippocampus (−3.3 to −4.5 mm from the bregma). The number of positive cells in each section was averaged from three non-overlapping fields at the same site of the middle cerebral artery blood supply in the ischemic (right) cortex within the penumbral area based on methods modified from previous studies (23, 24).
The success rate of model preparation in this experiment was 83.3%. No neurological impairment was observed in 3 of them, 3 with score of 4, 2 died in surgery, 14 with subarachnoid hemorrhage. Subarachnoid hemorrhage accounted for 77.8% of excluded factors, as the main excluded factor.
Experimental Transient Middle Cerebral Artery Occlusion Model
Surgical procedures for MCAO/R were performed in rats using the intracranial suture method, as previously described (25). Briefly, a 5-cm nylon monofilament (diameter, 0.26 mm) with a rounded tip coated with silicon (Guangzhou Jialing Biotechnology Company) was inserted into the right internal carotid artery to block the origin of the MCA (approximately 18 ± 2 mm) and maintained for 120 min. Rats in the sham group underwent the same surgical procedures without the insertion of a filament. The rectal temperature was maintained above 36.5°C during and after the surgery with a heating pad. Cerebral blood flow (CBF) was monitored throughout the entire operation. The success of the MCAO/R model was defined as a decrease in CBF by at least 80% during MCA occlusion and a return to 80% CBF after reperfusion.
Analysis of Neurological Deficits
A five-point scale of neurologic deficit scores was used to evaluate neurological behavior. The neurological deficits were scored 48 h after reperfusion by other investigators who were blinded to the experimental groups (n = 18 in each group). The scoring criteria for neurological deficits have been described previously by Longa et al. (25) and Bederson et al. (26, 27).
Infarct Volume Measurements
Infarct volume was assessed 48 h after reperfusion (n = 6 per group) with 2,3,5-triphenyltetrazolium chloride (TTC, Sigma), as previously described in detail (28, 29). The stained slices were photographed and quantified using ImagePro Plus 6.0. Lesion volumes were corrected using the following formula to compensate for the effect of post-ischemic edema on the volume of the injury (26, 30):
Nissl Staining
Sections were deparaffinized and then incubated with a 1% cresyl violet (Sigma) solution for Nissl staining. Images were captured using a light microscope (at 400× magnification). In the Nissl-stained sections, only intact neurons were counted.
Double-Labeling Using TUNEL and NeuN
A TUNEL assay was performed according to the manufacturer’s instructions (Roche Molecular Biochemicals, Inc., Mannheim, Germany). Sections were incubated with rabbit anti-NeuN antibody (Cell Signaling Technology, Danvers, MA, USA) in PBS/0.2% TX-100 and then incubated with the TUNEL reaction mixture to verify the neuronal identity of the TUNEL-positive cells. Finally, 4′,6-diamidino-2-phenylindole (DAPI) was added. The total number of TUNEL-positive neurons was counted by an investigator who was blinded to the study protocol.
Immunohistochemical Staining of HMGB1 and TLR4
Immunohistochemical staining of HMGB1 and TLR4 was performed using paraffin-embedded brain samples from each animal (n = 6 per group), which were sectioned and deparaffinized. The sections were incubated with an anti-HMGB1 monoclonal antibody (diluted 1:400, Cell Signaling Technology, Danvers, MA, USA) and an anti-TLR4 monoclonal antibody (diluted 1:100, Abcam PLC, Cambridge, UK). Binding was detected using the streptavidin-peroxidase kit (Maixin, Fuzhou, China). The positive cells were identified, counted, and analyzed in the sections with the ImageJ software.
Immunofluorescence Staining of Iba-1 and GFAP
Immunofluorescence staining of the microglial marker Iba-1 and the astrocytic marker GFAP were performed using paraffin-embedded brain samples of rats (n = 6 per group) that had been sectioned and deparaffinized. Sections were incubated with primary antibodies (goat anti-Iba-1, 1:100, Abcam, Cambridge, UK, or rabbit anti-GFAP, 1:200, Abcam, Cambridge, UK) and then with secondary antibodies labeled with fluorescent dyes (rabbit anti-goat, 1:200, Santa Cruz Biotechnology, CA, USA, or mouse anti-rabbit, 1:200, Santa Cruz Biotechnology, CA, USA). Photomicrographs were quantified performed by converting the images to gray scale, inverting their color, and quantifying the staining intensity in each field with ImageJ software.
Measurement of the IL-1β, TNF-α, IL-6, and Plasma HMGB1 Levels by ELISA
The IL-1β, IL-6, and TNF-α levels in the ischemic cortex and the HMGB1 levels in the plasma samples were determined using ELISA kits (USCN Life Science Inc., Wuhan, China) according to the manufacturer’s instructions.
Isolation of Protein and Western Blot Analysis for HMGB1, TLR4, IκB, Phospho-IκB, NFκB p65, and Phospho-NFκB p65
Cytosolic and nuclear proteins from the ischemic cortex were prepared with the Nuclear/Cytosol Fractionation Kit (BioVision, Mountain View, CA, USA) for western blotting analysis. As previously described in detail, the protein samples were separated using 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and then transferred to a polyvinylidene fluoride membrane (Millipore Corporation, Billerica, MA, USA). The membrane was incubated with the following antibodies: anti-HMGB1 antibody (diluted 1:1,000, Cell Signaling Technology), anti-TLR4 antibody (diluted 1:200, Abcam), anti-NFκB p65 antibody (diluted 1:500; Abcam), anti-IκB antibody (diluted 1:500, Abcam), anti-phospho-IκB antibody (diluted 1:500; Abcam), and phospho-NFκB p65 antibody (diluted 1:500; Abcam). To confirm equal loading, we used an anti-GAPDH antibody (1:500 dilution, Santa Cruz Biotechnology) and an anti-lamin A antibody (diluted 1:1,000, Abcam). The density of each band was quantified using ImageJ.
Statistical Analysis
All data are expressed as mean ± SD and analyzed with one-way analysis of variance using SPSS20.0. P < 0.05 was defined as statistically significant. The neurological deficit scores among the different groups were compared using the Kruskal–Wallis test. When the Kruskal–Wallis test showed a significant difference, the Dunn’s multiple comparisons test was applied. Given the simple size of six animals per group, actual power was performed with the G*Power 3.1.9.2 software at 5% significance level. We got a power greater than 0.9.
Results
Effect of UA on Neurological Deficits in Rats With MCAO/R
After 48 h of reperfusion, neurological deficit scores were significantly increased in the control group (Figure 1B). The UA-treated group (10 and 20 mg/kg) and the GL-treated group displayed significant improvements in their general condition and in neurological deficits compared with the control group (Figure 1B). Moreover, rats treated with 20 mg/kg UA displayed lower median neurological deficit scores than rats treated with GL.
No comments:
Post a Comment