# Purpurogallin, a Natural Phenol, Attenuates High-Mobility Group Box 1 in Subarachnoid Hemorrhage Induced Vasospasm in a Rat Model

Authors: Chih-Zen Chang; Chih-Lung Lin; Shu-Chuan Wu; Aij-Lie Kwan
Journal: International Journal of Vascular Medicine
 (2014)

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## Abstract

High-mobility group box 1 (HMGB1) was shown to be an important extracellular mediator involved in vascular inflammation of animals following subarachnoid hemorrhage (SAH). This study is of interest to examine the efficacy of purpurogallin, a natural phenol, on the alternation of cytokines and HMGB1 in a SAH model. A rodent double hemorrhage SAH model was employed. Basilar arteries (BAs) were harvested to examine HMGB1 mRNA and protein expression (Western blot). CSF samples were to examine IL-1β, IL-6, IL-8, and TNF-α (rt-PCR). Deformed endothelial wall, tortuous elastic lamina, and necrotic smooth muscle were observed in the vessels of SAH groups but were absent in the purpurogallin group. IL-1β, IL-6, and TNF-α in the SAH only and SAH plus vehicle groups were significantly elevated (P<0.01). Purpurgallin dose-dependently reduced HMGB1 protein expression. Likewise, high dose purpurogallin reduced TNF-α and HMGB1 mRNA levels. In conclusion, purpurogallin exerts its neuroinflammation effect through the dual effect of inhibiting IL-6 and TNF-α mRNA expression and reducing HMGB1 protein and mRNA expression. This study supports purpurogallin could attenuate both proinflammatory cytokines and late-onset inflammasome in SAH induced vasospasm.


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## Body

## 1. Introduction

After decades of study, subarachnoid hemorrhage (SAH) induced cerebral vasospasm persists to be a major cause of morbidity and mortality in patients who suffered from aneurism rupture [1–7]. Because of the lack of effective therapies to change this condition, the precise mechanisms of this disease content, in a multifaceted way, require further investigation. An ongrowing body of researches summarize two main hypotheses focusing on the role of endothelins/nitric oxide [8] and another role on SAH induced oxidative stress and inflammation in the development and maintenance of delayed vasoconstriction [1, 9–14]. As we know, once SAH appeared, the presence of oxy-hemolysate will elicit a cascade of cellular and molecular events in the subarachnoid space and these culminate in a vigorous inflammatory response [14–16]. The putative importance of inflammatory activity has not been fully emphasized, even though its role has been recognized throughout the pathogenesis of cerebral vasospasm. By now, various inflammasomes, including adhesion molecules, cytokines, leukocytes, immunoglobulins, and complements, were observed in the pathogenesis of SAH induced apoptosis and cerebral vasospasm [1, 4, 13, 17–19].The levels of proinflammatory cytokines and adhesion molecules have been found increased in cerebrospinal fluid (CSF) after SAH in our previous study [20]. However, the benefits of inflammation development after SAH remain unclear. High-mobility group box 1 (HMGB1) is a ubiquitously expressed nuclear protein released by activated monocytes, macrophages, circulating neutrophils, and platelets [21]. HMGB1 mediates vascular monocyte chemotaxis, neuron dendrite outgrowth, and the proinflammatory reaction of endothelial cells [22–25]. The kinetics of HMGB1 release is late-onset to tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β). Lu et al. [26] stated that HMGB1, through binding glycation end products (RAGE) and toll-like receptor (TLR)-2 and TLR-4 receptors, is able to activate nuclear factor-κB (NF-κB) and extracellular regulated kinase (ERK) 1 and ERK 2 [21, 27, 28]. As a late mediator, HMGB1 induces the expression of vascular cell-adhesion molecule (VCAM), intercellular adhesion molecule (ICAM), and E-selectin and leads to upregulating the recruitment of leukocytes [29, 30]. It may be reasonable to postulate that HMGB1 is involved in cerebral inflammation and plays a putative role in SAH induced vasoconstriction.Purpurogallin, a naturally occurring phenol, extracted from the plants ofQuercus spp. [31–34] has been reported as being able to protect both ventricular myocytes and aortic endothelial cells of rats against proxy radical damage through inhibiting glutathione S-transferase, xanthine oxidase, and catechol methyltransferase [31, 35, 36]. In Puurunen et al.’s study, purpurogallin, as an antioxidant, could prevent high cholesterol diet-induced atherosclerosis in rabbits [37]. In addition, this compound also exerts an apoptotic effect on the culture of murine fibrosarcoma L-929 and human U-87 MG glioblastoma cells [31]. Recent studies indicate that single administration of purpurogallin exhibited antilipopolysaccharide (LPS) mediated inflammation in human umbilical vein endothelial cell culture (HUVECs) study [38].Given the importance of purpurogallin on atherosclerotic lesion formation and its various effects of proinflammatory cytokine stimulation on endothelial dysfunction, the rat SAH model was used to test the hypothesis that purpurogallin attenuates SAH related vasospasm and HMGB1 associated inflammation.


## 2. Methods

### 2.1. Materials

Purpurogallin has been characterized as a naturally occurring potent antioxidant and was bought from Sigma Laboratory, Taipei, Taiwan. Monoclonal anti-rat IL1-β, IL-6, IL-8, and TNF-α antibodies were obtained from Abcam (Cambridge, MA 02139, USA), BD Transduction Lab (BD Biosciences, San Jose, CA 95060, USA), Upstate Biotech (Lake placid, NY 12946, USA), and Santa Cruz Biotech (Santa Cruz Biotechnology, Inc. Santa Cruz, CA 95060, USA). Rabbit anti-rat HMBG1 antibody was purchased from Biochiefdom international Co., Ltd, Taipei 11659, Taiwan, distributing Abchem Biochemicals, Cambridge, MA 02139, USA. CNM protein extraction kits were from Biochain (Hayward, CA 94545, USA). Purpurogallin was prepared by Ms. Wu SC (Kaohsiung Medical University Hospital, Kaohsiung 807, Taiwan). Dimethyl sulfoxide (DMSO) was used as a vehicle.


### 2.2. Induction of Experimental SAH

Fifty-four male Sprague-Dawley rats (250–350 g; bought from BioLasco Taiwan Co., Ltd., authorized by Charles River Lab) were enclosed in this study. All the protocols were approved and supervised by the University of Kaohsiung Medicine Animal Research Committee and were compliant with the Declaration of Helsinki (1964). The rats received anesthesia by an intraperitoneal injection of a mixture of 0.9 mg/100 gm xylazine and 5.5 mg/100 gm KetaVed. An amount of 0.3 mL fresh arterial blood was withdrawn and injected into the cisterna magna via a stereotactic apparatus (Stoelting, Wood Dale, IL 60191, US). Animals were placed in ventral recumbent position for 20 minutes to allow clot formation. After monitoring for respiratory distress and giving mechanical ventilation if needed, the animals were returned to the vivarium with a 12 hr light-dark cycle and an access to food and water ad libitum.


### 2.3. General Design of Experiments and Treatment Groups

The animals were randomly divided into the following groups (Number = 9 rats):(1) sham operated (no SAH); (2) SAH only; (3) SAH plus vehicle; (4) SAH plus purpurogallin (100 ug/kg/day); (5) SAH plus treatment with purpurogallin (200 ug/kg/day); and (6) treatment with 400 ug/kg purpurogallin in SAH rats. According to our pilot study, the dosage was adjusted to be devoid of hepatic-renal toxicity. The administration was initialized at 1 hr after induction of SAH by using an osmotic mini-pump (Alzet Corp., Palo Alto, CA 94301, USA). After the animals received reanesthesia 48 hr after first SAH, 2 mL fresh blood was injected through a 30-gauge needle into the cranial-cervical junction to maintain the tendency of vasoconstriction. The animals were sacrificed by perfusion-fixation 72 hr after second SAH. Cortical tissue samples were obtained by inserting a 24-gauge needle 5 mm into the skull bone (N=5) through a burr hole craniectomy (2 mm apart from the bregma).


### 2.4. Perfusion-Fixation

By 72 hr after second SAH, the animals were reanesthetized by administration of pancuronium bromide (0.3 mg/kg). Perfusion-fixation was performed by opening the chest, then catheterizing with a NO16 needle into the left ventricle as well as the clamped descending aorta, and incising the right atrium. One hundred miniliters of 80 mm Hg of 0.01 M phosphate buffer (pH 7.4) was dripped, followed by 120 mL of 2% paraformaldehyde in the PBS solution at 36°C. The brain was obtained and immersed in a fixative at 4°C overnight. Visual inspection made sure that blood clots formed over the basilar artery (BA) in all SAH animals.


### 2.5. Hemodynamic Measurements

Heart rate, blood pressure, and rectal temperature were monitored before and after the administration of purpurogallin as well as at interval of 12 hr after the induction of SAH by a tail-cuff method (SC1000 Single Channel System, Hatteras Instruments, NC 27518, USA) and rectal thermometer (BIO-BRET-2-ISO, FL 33780, USA). The femoral artery was catheterized to obtain blood samples to determine arterial blood gas, Na+, K+, glutamate oxaloacetate transaminase (GOT), glutamate pyruvate transaminase (GPT), blood urea nitrogen (BUN), and creatinine levels.


### 2.6. Basilar Artery Morphometric Studies

Five BA cross sections of each animal (one of the middle third segment) were analyzed by two investigators blinded to the experiment set. The cross-sectional area of BA was automatically evaluated via a computer-assisted morphometer (Image-1/Metamorph Imaging System, Universal Imaging Corp. Sunnyvale, CA 94089, USA). The average of five cross sections of a given animal was collected for this animal. Group data are expressed as the means ± standard deviation.


### 2.7. Neurological Assessment

A modified limb-placing test (MLPT) [39] was performed before and at a 48 hr interval after the animals were subjected to SAH. The behavioral assessment was composed of forelimbs and hindlimbs ambulation and placing/stepping reflex examinations, which was performed before and after animals were subjected to SAH. A motor deficit index (MDI) was calculated for each rat at an interval of 24 hr. The final index was the sum of the scores (walking with lower extremities and placing/stepping reflex). Animals with MDI score more than three were considered as paraplegia, whereas MDI score less than three was esteemed as neurological deficit.


### 2.8. Quantification of mRNA Expression of IL-1β, IL-6, IL-8, and TNF-α

To examine the CSF cytokines mRNA, the ABI PRISM 7900 System was applied (Applied Biosystems, Foster City, CA 94404, USA). As stated by the manufacturer’s instructions, the mRNAs for IL-1β, IL-6, IL-8, and TNF-α expression were established, while 18S was designated as a standard control owing to its stable expression under the stimuli of SAH. Each sample was launched into a TaqMan Human Cytokine Card that enclosed probes and primers for specific targets. This procedure was performed via an affixed filling reservoir and a vacuum loading process via the ABI PRISM Card Filling Station.Target mRNAs were standardized according to the reference gene (18S), and final data were expressed as a relative fold from the baseline. Comparative mRNA expression was set by the Livak and SchmittgenΔCT method [40]. The results were analyzed if a 5-fold increase in the mRNA levels compared with the baseline to allow for data consistency.


### 2.9. HMGB1 Protein Analyzed by Western Blotting

The cortical homogenates (20μg) were stirred with LDS sample buffer (contains 40% glycerol, 4% lithium dodecyl sulfate (LDS), 0.8 M triethanolamine-Cl pH 7.6, 4% Ficoll-400, 0.025% phenol red, 0.025% coomassie G250, and 2 mM EDTA disodium, NuPAGE LDS Sample Buffer (4x) NP0007; Invitrogen, Carlsbad, CA 92008, USA). Samples were loaded for 8% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then separated after being centrifuged at 12,000 rpm for 10 min twice. The specimen was mounted onto a polyvinylidene difluoride membrane and incubated in blocking buffer (5% nonfat dry milk in tris-buffered saline with 0.2% tween 20) at room temperature. Rabbit anti-rat HMGB1 monoclonal antibody (1 : 50,000; Biochiefdom International Co., Ltd, Wenshen District, Taipei 11659, Taiwan, distributing Abcam Biochemicals, Cambridge, MA 02139, USA) was used, while β-actin (monoclonal anti-β-actin, dilution 1 : 40,000; Sigma-Aldrich, Taipei 116, Taiwan) was used as a loading control. A secondary antibody conjugated with horseradish peroxidase (HRP) in TBS-t at room temperature for 1 hr. The immunoblots are developed via a GS-700 digital scan and Molecular Analyst (a GS-700 digital densitometer, GMI, Ramsey, MN 55303, USA). Relative optical densities are obtained by comparison between the measured values and the mean values from the vehicle plus SAH groups.


### 2.10. Detection of HMGB1 mRNA by rt-PCR

The levels of activated HMGB1 mRNA in the cortical homogenates were determined by TriPure RT-PCR Reagent (Roche Diagnostics Corp., IN 46256, USA). According to the manufacturer's instructions, the PCR primer sequences were designed according to the HMGB1 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH as a standard control) gene sequences in GenBank. HMGB1: 285 bp; upstream: 5′-AGTTCAAGGACCCCAATG-3′; downstream: 5′-TGCTCTTCTCAGCCTTGACCA-3′. GAPDH: 347 bp; upstream: 5′-GGAGCCAAAAGGGTCATC-3′; downstream: 5′-CCAGTGAGTTTCCCGTTC-3′. Incubated with Avian Myeloblastosis Virus Reverse Transcriptase (AMV RT) (Promega, WI 53718, USA), the HMGB1 and GAPDH cDNA were amplified. The amplified cDNA fragments, when swirled with 1μL ethidium bromide, were detected via agarose gel electrophoresis [35]. A comet assay method was used to examine the intensity of the gene bands. GAPDH mRNA was multiplexed as a housekeeping gene owing to its stability. The tissue of five animals was used for rt-PCR and an average of three measurements for each animal was performed to obtain a mean value.


### 2.11. Statistical Analysis

Data are expressed as the means ± standard deviation. For comparison among groups, all statistical analyses were performed using SPSS 19.0 software (SPSS Inc., Chicago, IL 60614, USA). Difference was considered significant at a probability value less than 0.01.


## 2.1. Materials

Purpurogallin has been characterized as a naturally occurring potent antioxidant and was bought from Sigma Laboratory, Taipei, Taiwan. Monoclonal anti-rat IL1-β, IL-6, IL-8, and TNF-α antibodies were obtained from Abcam (Cambridge, MA 02139, USA), BD Transduction Lab (BD Biosciences, San Jose, CA 95060, USA), Upstate Biotech (Lake placid, NY 12946, USA), and Santa Cruz Biotech (Santa Cruz Biotechnology, Inc. Santa Cruz, CA 95060, USA). Rabbit anti-rat HMBG1 antibody was purchased from Biochiefdom international Co., Ltd, Taipei 11659, Taiwan, distributing Abchem Biochemicals, Cambridge, MA 02139, USA. CNM protein extraction kits were from Biochain (Hayward, CA 94545, USA). Purpurogallin was prepared by Ms. Wu SC (Kaohsiung Medical University Hospital, Kaohsiung 807, Taiwan). Dimethyl sulfoxide (DMSO) was used as a vehicle.


## 2.2. Induction of Experimental SAH

Fifty-four male Sprague-Dawley rats (250–350 g; bought from BioLasco Taiwan Co., Ltd., authorized by Charles River Lab) were enclosed in this study. All the protocols were approved and supervised by the University of Kaohsiung Medicine Animal Research Committee and were compliant with the Declaration of Helsinki (1964). The rats received anesthesia by an intraperitoneal injection of a mixture of 0.9 mg/100 gm xylazine and 5.5 mg/100 gm KetaVed. An amount of 0.3 mL fresh arterial blood was withdrawn and injected into the cisterna magna via a stereotactic apparatus (Stoelting, Wood Dale, IL 60191, US). Animals were placed in ventral recumbent position for 20 minutes to allow clot formation. After monitoring for respiratory distress and giving mechanical ventilation if needed, the animals were returned to the vivarium with a 12 hr light-dark cycle and an access to food and water ad libitum.


## 2.3. General Design of Experiments and Treatment Groups

The animals were randomly divided into the following groups (Number = 9 rats):(1) sham operated (no SAH); (2) SAH only; (3) SAH plus vehicle; (4) SAH plus purpurogallin (100 ug/kg/day); (5) SAH plus treatment with purpurogallin (200 ug/kg/day); and (6) treatment with 400 ug/kg purpurogallin in SAH rats. According to our pilot study, the dosage was adjusted to be devoid of hepatic-renal toxicity. The administration was initialized at 1 hr after induction of SAH by using an osmotic mini-pump (Alzet Corp., Palo Alto, CA 94301, USA). After the animals received reanesthesia 48 hr after first SAH, 2 mL fresh blood was injected through a 30-gauge needle into the cranial-cervical junction to maintain the tendency of vasoconstriction. The animals were sacrificed by perfusion-fixation 72 hr after second SAH. Cortical tissue samples were obtained by inserting a 24-gauge needle 5 mm into the skull bone (N=5) through a burr hole craniectomy (2 mm apart from the bregma).


## 2.4. Perfusion-Fixation

By 72 hr after second SAH, the animals were reanesthetized by administration of pancuronium bromide (0.3 mg/kg). Perfusion-fixation was performed by opening the chest, then catheterizing with a NO16 needle into the left ventricle as well as the clamped descending aorta, and incising the right atrium. One hundred miniliters of 80 mm Hg of 0.01 M phosphate buffer (pH 7.4) was dripped, followed by 120 mL of 2% paraformaldehyde in the PBS solution at 36°C. The brain was obtained and immersed in a fixative at 4°C overnight. Visual inspection made sure that blood clots formed over the basilar artery (BA) in all SAH animals.


## 2.5. Hemodynamic Measurements

Heart rate, blood pressure, and rectal temperature were monitored before and after the administration of purpurogallin as well as at interval of 12 hr after the induction of SAH by a tail-cuff method (SC1000 Single Channel System, Hatteras Instruments, NC 27518, USA) and rectal thermometer (BIO-BRET-2-ISO, FL 33780, USA). The femoral artery was catheterized to obtain blood samples to determine arterial blood gas, Na+, K+, glutamate oxaloacetate transaminase (GOT), glutamate pyruvate transaminase (GPT), blood urea nitrogen (BUN), and creatinine levels.


## 2.6. Basilar Artery Morphometric Studies

Five BA cross sections of each animal (one of the middle third segment) were analyzed by two investigators blinded to the experiment set. The cross-sectional area of BA was automatically evaluated via a computer-assisted morphometer (Image-1/Metamorph Imaging System, Universal Imaging Corp. Sunnyvale, CA 94089, USA). The average of five cross sections of a given animal was collected for this animal. Group data are expressed as the means ± standard deviation.


## 2.7. Neurological Assessment

A modified limb-placing test (MLPT) [39] was performed before and at a 48 hr interval after the animals were subjected to SAH. The behavioral assessment was composed of forelimbs and hindlimbs ambulation and placing/stepping reflex examinations, which was performed before and after animals were subjected to SAH. A motor deficit index (MDI) was calculated for each rat at an interval of 24 hr. The final index was the sum of the scores (walking with lower extremities and placing/stepping reflex). Animals with MDI score more than three were considered as paraplegia, whereas MDI score less than three was esteemed as neurological deficit.


## 2.8. Quantification of mRNA Expression of IL-1β, IL-6, IL-8, and TNF-α

To examine the CSF cytokines mRNA, the ABI PRISM 7900 System was applied (Applied Biosystems, Foster City, CA 94404, USA). As stated by the manufacturer’s instructions, the mRNAs for IL-1β, IL-6, IL-8, and TNF-α expression were established, while 18S was designated as a standard control owing to its stable expression under the stimuli of SAH. Each sample was launched into a TaqMan Human Cytokine Card that enclosed probes and primers for specific targets. This procedure was performed via an affixed filling reservoir and a vacuum loading process via the ABI PRISM Card Filling Station.Target mRNAs were standardized according to the reference gene (18S), and final data were expressed as a relative fold from the baseline. Comparative mRNA expression was set by the Livak and SchmittgenΔCT method [40]. The results were analyzed if a 5-fold increase in the mRNA levels compared with the baseline to allow for data consistency.


## 2.9. HMGB1 Protein Analyzed by Western Blotting

The cortical homogenates (20μg) were stirred with LDS sample buffer (contains 40% glycerol, 4% lithium dodecyl sulfate (LDS), 0.8 M triethanolamine-Cl pH 7.6, 4% Ficoll-400, 0.025% phenol red, 0.025% coomassie G250, and 2 mM EDTA disodium, NuPAGE LDS Sample Buffer (4x) NP0007; Invitrogen, Carlsbad, CA 92008, USA). Samples were loaded for 8% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then separated after being centrifuged at 12,000 rpm for 10 min twice. The specimen was mounted onto a polyvinylidene difluoride membrane and incubated in blocking buffer (5% nonfat dry milk in tris-buffered saline with 0.2% tween 20) at room temperature. Rabbit anti-rat HMGB1 monoclonal antibody (1 : 50,000; Biochiefdom International Co., Ltd, Wenshen District, Taipei 11659, Taiwan, distributing Abcam Biochemicals, Cambridge, MA 02139, USA) was used, while β-actin (monoclonal anti-β-actin, dilution 1 : 40,000; Sigma-Aldrich, Taipei 116, Taiwan) was used as a loading control. A secondary antibody conjugated with horseradish peroxidase (HRP) in TBS-t at room temperature for 1 hr. The immunoblots are developed via a GS-700 digital scan and Molecular Analyst (a GS-700 digital densitometer, GMI, Ramsey, MN 55303, USA). Relative optical densities are obtained by comparison between the measured values and the mean values from the vehicle plus SAH groups.


## 2.10. Detection of HMGB1 mRNA by rt-PCR

The levels of activated HMGB1 mRNA in the cortical homogenates were determined by TriPure RT-PCR Reagent (Roche Diagnostics Corp., IN 46256, USA). According to the manufacturer's instructions, the PCR primer sequences were designed according to the HMGB1 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH as a standard control) gene sequences in GenBank. HMGB1: 285 bp; upstream: 5′-AGTTCAAGGACCCCAATG-3′; downstream: 5′-TGCTCTTCTCAGCCTTGACCA-3′. GAPDH: 347 bp; upstream: 5′-GGAGCCAAAAGGGTCATC-3′; downstream: 5′-CCAGTGAGTTTCCCGTTC-3′. Incubated with Avian Myeloblastosis Virus Reverse Transcriptase (AMV RT) (Promega, WI 53718, USA), the HMGB1 and GAPDH cDNA were amplified. The amplified cDNA fragments, when swirled with 1μL ethidium bromide, were detected via agarose gel electrophoresis [35]. A comet assay method was used to examine the intensity of the gene bands. GAPDH mRNA was multiplexed as a housekeeping gene owing to its stability. The tissue of five animals was used for rt-PCR and an average of three measurements for each animal was performed to obtain a mean value.


## 2.11. Statistical Analysis

Data are expressed as the means ± standard deviation. For comparison among groups, all statistical analyses were performed using SPSS 19.0 software (SPSS Inc., Chicago, IL 60614, USA). Difference was considered significant at a probability value less than 0.01.


## 3. Results

### 3.1. General Observation

In the whole study, no significant differences were observed in the recorded physiological parameters, including GOT, GPT, BUN, creatinine, pH, blood pressure, and arterial blood gas analysis among the experimental groups. This proved that purpurogallin administration in the selected dosage was devoid of hepatic and renal toxicity.


### 3.2. Tissue Morphometry

The internal elastic lamina (IEL) in the BAs of SAH and SAH + vehicle groups showed substantial corrugation when compared with that obtained from the controls (Figure1, upper panel). IEL disruption was less prominent in the purpurogallin treatment groups. The cross-sectional areas of BAs in the SAH and SAH + vehicle groups were significantly reduced when compared with the control group (0.36±0.18 and 0.41±0.24). Purpurogallin dose-dependently attenuated SAH induced vasoconstriction (Figure 1, lower panel, P<0.01).Figure 1
Comparison of lumen cross-sectional capacities of the basilar artery (bottom panel). Top panel represents micrographs of the BAs obtained from the healthy controls (A), the SAH only rats (B), the vehicle-treated SAH rats (C), SAH rats received 100 ug/kg/day purpurogallin treatment (D), SAH rats received 200 ug/kg/day (E), and 400 ug/kg/day purpurogallin treatment (F). Standard bar = 200μm. P*<0.01: compared with the SAH and sham operated group. P#>0.05: comparison between the SAH group and the controls. P##,###>0.05, P*<0.01: purpurogallin + SAH groups compared with the SAH group. Data are means ± SEM (n=9/group).


### 3.3. Neurological Deficit

MLPT score was obtained to examine the motor-sensory incorporation of the forelimb and hindlimb and placing/stepping reflex as a reflective response to tactile and proprioceptive stimuli. The sum from these two tests is considered as motor deficit index (MDI). The mean MDI in the SAH and SAH + vehicle groups was2.78±0.30 and 2.86±0.32, compared with the healthy control. Treatment with purpurogallin (at 400 ug/kg) significantly improved the MDI in the SAH groups (Table 1). Likewise, MDI ≥ 3 was substantially decreased in the purpurogallin treatment groups when compared with the SAH animals.Table 1
Modified limb-placing test (MLPT).
Group treatment
Ambulation
Placing/stepping reflex
MDI
Normal
0
0
0
SAH
1.28 ± 0.17
1.50 ± 0.13
2.78 ± 0.30
SAH + vehicle
1.28 ± 0.11
1.58 ± 0.21
2.86 ± 0.32
SAH + purpurogallin
100 ug/kg/day
0.91 ± 0. 60
1.54  ± 0.25
2.45  ± 0.85
200 ug/kg/day
0. 82  ± 0.25
0.83 ± 0.43*
1.65 ± 0.68
400 ug/kg/day
0.56 ± 0.22*
0.65 ± 0.20*
1.21 ± 0.42*
Results are expressed as the means ± SEM,n = 9; *P<0.05 versus SAH condition by Mann-Whitney test.


### 3.4. mRNA Expression of IL-1β, IL-6, IL-8, and TNF-α

Subsequent to the induction of SAH, the CSF concentrations of IL-1β, IL-6, IL-8, and TNF-α were found to increase 1000- and 3000-fold at 24 hr and 72 hr, when compared with the sham operated group. Administration of purpurogallin reduced cytokine levels by 12%, 34%, 52%, and 11% for IL-1β, IL-6, IL-8, and TNF-αrelative to SAH groups at 48 hr after first SAH (Figure 2, left column). Levels of IL-1βand TNF-α were reduced in the purpurogallin treatment groups in a dose-dependent mechanism. By the way, treatment with purpurogallin failed to reduce IL-8 level to statistical difference from the rats subject to SAH (Figure 2, P>0.05).Figure 2
Bar graph depicting purpurogallin on the time-course change of proinflammatory cytokines after the induction of SAH. Data are depicted for IL-1β, IL-6, IL-8, and TNF-α at 48 hr after first SAH and 72 hr after second SAH. Data in the figure are presented as means ± SD (n=9). P*,**<0.01, and P#,##,###>0.05 when compared with the SAH group.


### 3.5. HMGB1 Protein Expression

HMGB1 was demonstrated to play a critical role in the onset of delayed and systemic inflammation. The expression of HMGB1 protein was not significantly difference among the experimental groups at 48 hr after the induction of SAH (Figure3, left column, P>0.05). In this study, purpurogallin (at medium and high doses) reduced the expression of HMGB1 protein at 72 hr after second SAH, when compared with the SAH group (Figure 3, right column, P<0.01).Figure 3
High-mobility group protein B1 (HMGB1) expression on 24 hr after first SAH and 72 hr after second SAH, respectively (Western blot). (A) SAH; (B) SAH + vehicle; (C) 100 ug/kg/day purpurogallin + SAH; (D) 200 ug/kg/day purpurogallin + SAH; and (E) 400 ug/kg/day purpurogallin in SAH rats. Purpurogallin tended to decrease HMGB1 expression in 48 hr after the induction of SAH. By the end of second SAH, purpurogallin dose-dependently attenuated HMGB1 protein expression.P*,**<0.01, experimental animals compared with the SAH group. P#,##,###>0.05, compared with the animals subject to SAH.


### 3.6. The Activation of HMGB1 mRNA

To observe the neuronal inflammation following SAH, HMGB1 mRNA was examined. The activated HMGB1 mRNA was observed to be increased in the SAH groups when compared with the sham operated groups (P<0.01). Purpurogallin tends to decrease HMGB1 mRNA in the 48 hr SAH rats (Figure 4, left column, P>0.05). By 72 hr after second SAH, purpurogallin dose-dependently reduced HMGB1 mRNA when compared with the SAH groups (Figure 4, right column, P<0.01).Figure 4
HMRB1 mRNA expression on 48 hr after first SAH and 72 hr after second SAH, respectively (rt-PCR). All groups are identical to those shown in the legends of Figure3. P#,##>0.05; P*,**<0.01: represents purpurogallin treatment (48 hr after first SAH); purpurogallin dose-dependently (72 hr after second SAH) attenuated HMGB1 mRNA transcription, when compared with the SAH groups.


## 3.1. General Observation

In the whole study, no significant differences were observed in the recorded physiological parameters, including GOT, GPT, BUN, creatinine, pH, blood pressure, and arterial blood gas analysis among the experimental groups. This proved that purpurogallin administration in the selected dosage was devoid of hepatic and renal toxicity.


## 3.2. Tissue Morphometry

The internal elastic lamina (IEL) in the BAs of SAH and SAH + vehicle groups showed substantial corrugation when compared with that obtained from the controls (Figure1, upper panel). IEL disruption was less prominent in the purpurogallin treatment groups. The cross-sectional areas of BAs in the SAH and SAH + vehicle groups were significantly reduced when compared with the control group (0.36±0.18 and 0.41±0.24). Purpurogallin dose-dependently attenuated SAH induced vasoconstriction (Figure 1, lower panel, P<0.01).Figure 1
Comparison of lumen cross-sectional capacities of the basilar artery (bottom panel). Top panel represents micrographs of the BAs obtained from the healthy controls (A), the SAH only rats (B), the vehicle-treated SAH rats (C), SAH rats received 100 ug/kg/day purpurogallin treatment (D), SAH rats received 200 ug/kg/day (E), and 400 ug/kg/day purpurogallin treatment (F). Standard bar = 200μm. P*<0.01: compared with the SAH and sham operated group. P#>0.05: comparison between the SAH group and the controls. P##,###>0.05, P*<0.01: purpurogallin + SAH groups compared with the SAH group. Data are means ± SEM (n=9/group).


## 3.3. Neurological Deficit

MLPT score was obtained to examine the motor-sensory incorporation of the forelimb and hindlimb and placing/stepping reflex as a reflective response to tactile and proprioceptive stimuli. The sum from these two tests is considered as motor deficit index (MDI). The mean MDI in the SAH and SAH + vehicle groups was2.78±0.30 and 2.86±0.32, compared with the healthy control. Treatment with purpurogallin (at 400 ug/kg) significantly improved the MDI in the SAH groups (Table 1). Likewise, MDI ≥ 3 was substantially decreased in the purpurogallin treatment groups when compared with the SAH animals.Table 1
Modified limb-placing test (MLPT).
Group treatment
Ambulation
Placing/stepping reflex
MDI
Normal
0
0
0
SAH
1.28 ± 0.17
1.50 ± 0.13
2.78 ± 0.30
SAH + vehicle
1.28 ± 0.11
1.58 ± 0.21
2.86 ± 0.32
SAH + purpurogallin
100 ug/kg/day
0.91 ± 0. 60
1.54  ± 0.25
2.45  ± 0.85
200 ug/kg/day
0. 82  ± 0.25
0.83 ± 0.43*
1.65 ± 0.68
400 ug/kg/day
0.56 ± 0.22*
0.65 ± 0.20*
1.21 ± 0.42*
Results are expressed as the means ± SEM,n = 9; *P<0.05 versus SAH condition by Mann-Whitney test.


## 3.4. mRNA Expression of IL-1β, IL-6, IL-8, and TNF-α

Subsequent to the induction of SAH, the CSF concentrations of IL-1β, IL-6, IL-8, and TNF-α were found to increase 1000- and 3000-fold at 24 hr and 72 hr, when compared with the sham operated group. Administration of purpurogallin reduced cytokine levels by 12%, 34%, 52%, and 11% for IL-1β, IL-6, IL-8, and TNF-αrelative to SAH groups at 48 hr after first SAH (Figure 2, left column). Levels of IL-1βand TNF-α were reduced in the purpurogallin treatment groups in a dose-dependent mechanism. By the way, treatment with purpurogallin failed to reduce IL-8 level to statistical difference from the rats subject to SAH (Figure 2, P>0.05).Figure 2
Bar graph depicting purpurogallin on the time-course change of proinflammatory cytokines after the induction of SAH. Data are depicted for IL-1β, IL-6, IL-8, and TNF-α at 48 hr after first SAH and 72 hr after second SAH. Data in the figure are presented as means ± SD (n=9). P*,**<0.01, and P#,##,###>0.05 when compared with the SAH group.


## 3.5. HMGB1 Protein Expression

HMGB1 was demonstrated to play a critical role in the onset of delayed and systemic inflammation. The expression of HMGB1 protein was not significantly difference among the experimental groups at 48 hr after the induction of SAH (Figure3, left column, P>0.05). In this study, purpurogallin (at medium and high doses) reduced the expression of HMGB1 protein at 72 hr after second SAH, when compared with the SAH group (Figure 3, right column, P<0.01).Figure 3
High-mobility group protein B1 (HMGB1) expression on 24 hr after first SAH and 72 hr after second SAH, respectively (Western blot). (A) SAH; (B) SAH + vehicle; (C) 100 ug/kg/day purpurogallin + SAH; (D) 200 ug/kg/day purpurogallin + SAH; and (E) 400 ug/kg/day purpurogallin in SAH rats. Purpurogallin tended to decrease HMGB1 expression in 48 hr after the induction of SAH. By the end of second SAH, purpurogallin dose-dependently attenuated HMGB1 protein expression.P*,**<0.01, experimental animals compared with the SAH group. P#,##,###>0.05, compared with the animals subject to SAH.


## 3.6. The Activation of HMGB1 mRNA

To observe the neuronal inflammation following SAH, HMGB1 mRNA was examined. The activated HMGB1 mRNA was observed to be increased in the SAH groups when compared with the sham operated groups (P<0.01). Purpurogallin tends to decrease HMGB1 mRNA in the 48 hr SAH rats (Figure 4, left column, P>0.05). By 72 hr after second SAH, purpurogallin dose-dependently reduced HMGB1 mRNA when compared with the SAH groups (Figure 4, right column, P<0.01).Figure 4
HMRB1 mRNA expression on 48 hr after first SAH and 72 hr after second SAH, respectively (rt-PCR). All groups are identical to those shown in the legends of Figure3. P#,##>0.05; P*,**<0.01: represents purpurogallin treatment (48 hr after first SAH); purpurogallin dose-dependently (72 hr after second SAH) attenuated HMGB1 mRNA transcription, when compared with the SAH groups.


## 4. Discussion

In this study, purpurogallin, a polyphenolic compound, has been shown to attenuate SAH induced IL-1β and TNF-α expressions at the initial stage of SAH. However, how to maintain the intensity of neuroinflammation in delayed vasoconstriction remained unknown. HMGB1, a late-produced immunity mediator, showed a clue to investigate the mechanism of SAH induced delayed cerebral ischemia. In Kim et al.’s study, purpurogallin is demonstrated to modulate the vascular permeability and expression of adhesion molecules involving monocyte adhesion and migration through endothelial cells [33]. This finding corresponds to our previous observation of brain blood barrier disruption after the induction SAH, which was observed in the Evan blue test in the SAH rats. Besides, owing to the antioxidant activity, purpurogallin was able to retard the development of hypercholesterolemic atherosclerosis without lowering blood cholesterol level [38]. In the study of TNF-α induced human umbilical vein endothelial cells (HUVECs), purpurogallin is demonstrated to inhibit both the activities and production of thrombin and FXa. Hemostatic events are triggered in response to the vascular wall damage by exposure to blood existing in the endothelial extracellular matrix [38], which led to upregulate a series of coagulation cascades, including activated platelets, conversion of fibrinogen to fibrin, and amplified coagulation [41]. Our result supports the fact that purpurogallin, in a selected dose, is devoid of hepatic and renal adverse effect. This compound was proved to be able to reduce proinflammatory cytokines in the early stage of SAH induced vascular deformity.In the pilot study, elevation in the mRNA levels for IL-1β, IL-6, IL-8, TNF-α, and adhesion molecules have been observed imposed in the pathogenesis of aneurysmal SAH induced vasospasm in rats [8]. However, the relationship among the development of inflammatory response, early brain injury, and delayed cerebral ischemia in the brain after SAH needs to be clarified. Bowman et al. [9] demonstrated that a polyclonal antibody targeted against IL-6 was able to alleviate SAH induced vasoconstriction in a femoral artery study. The cumulative findings indicate that the upregulation of proinflammatory cytokines is antecedent to radiographic vasospasm (peaks at 4th to 14th day after aneurismal SAH in Human) [3], and attenuation of cytokines tends to minimize vascular constriction and reduced brain ischemia in animals. In this study, IL-1β and TNF-α (at 48 hr after 1st SAH) levels were suppressed by the administration of purpurogallin, while IL-6 level was reduced at high dose purpurogallin treatment group at 72 hr after second SAH.High-mobility group box 1 (HMGB1), a trigger of inflammation, attracting inflammatory cells of tissue repair, recruiting stem cells, and promoting their proliferation, is released by activated monocytes, macrophages, neutrophils, platelets, and microglia [21, 24, 26, 42]. HMGB1 mediates the immediate and delayed inflammatory responses of vascular endothelial cells [24]. HMGB1 served as a late mediator and participated in the pathogenesis of chronic and prefunded inflammation after the early immunity response has resolved [25]. It further bonds to transmembrane receptors, like receptor for advanced glycation end products (RAGE), toll-like receptor (TLR)-2, TLR-4, activated nuclear factor-κB (NF-κB), and extracellular regulated kinase (ERK) 1 and ERK 2 [21]. Otherwise, HMGB1 also induced the expression of vascular cell-adhesion molecule (VCAM), intercellular adhesion molecule (ICAM), and E-selectin from the activated platelets and microglia to upregulate the recruitment of inflammation. In Xiong et al.’s study [43], inhibiting HMGB1 expression is able to reduce focal cerebral ischemia insult. Via inhibiting the HMGB1/RAGE axis, angiotensin receptor blockers (ARBs), telmisartan, irbesartan, and candesartan, were able to prevent and treat atherosclerosis associated stroke. In this study, purpurogallin (at medium and high doses) is able to restrict HMGB1 mRNA transcription and protein expression in SAH induced delayed neuroinflammation by 72 hr after the induction of second SAH.In summary, the results of this study show that administration of purpurogallin, at a therapeutic dosage, is safe and efficacious in the prevention of experimental vasoconstriction and meritorious of further investigation. Decreased levels of IL-1β, TNF-α (at 48 hr), and HMGB 1 protein (at 72 hr after second SAH) as well as IL-6 (at 24 apart from first SAH and 72 hr after second SAH) may contribute to the antidelayed vasospasm effect of this compound. Besides, purpurogallin, by reducing HMGB1 mRNA activation, exerts a dual effect on microglia related T-cell transmigration and IL-6 related delayed inflammatory cascade.


## 5. Conclusions

The outcome of SAH patients revealed to be devastating and stood still after decades of research. The acuminated results arouse interest to consider the pathogenesis of SAH induced acute and delayed neuroinflammation and its effect dictates on the patient’s outcome. The breakout of T-cell immigration accompanying SAH may be a critical and complicated pathway underlying the development and maintenance of delayed neurologic deficits. This study shows that administration of purpurogallin may diminish SAH induced early proinflammation and subsequent HMGB1 stimulation in a rodent model of SAH. This study suggests that purpurogallin, a naturally occurring polyphenol, could prove to be clinically useful in preventing and treating SAH-induced vasospasm. This study is limited owing to its lack of in vitro mechanistic data to correspond to those found in this study. Further SAH induced vascular remodeling should be considered as a variable factor.



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*Source: 254270-2014-11-17.xml*