Research Article
Preliminary Exploration of the Neuroprotective Effect of a Novel Z-Ligustilide Analogue on Intracerebral Hemorrhage in Mice
Li Han, Dong-Ling Liu, Chu Chen* and Jun-Rong Du
Corresponding Author: Chu Chen, Sichuan Academy of Chinese Medicine Sciences, No. 51, Section 4, South Renmin Road, Chengdu, 610041, China
Received: November 05, 2018; Accepted: January 16, 2019;
Citation: Han L, Liu DL, Chen C & Du JR. (2019) Preliminary Exploration of the Neuroprotective Effect of a Novel Z-Ligustilide Analogue on Intracerebral Hemorrhage in Mice. J Damage Assoc Mol Patterns, 1(1): 14-30.
Copyrights: ©2019 Han L, Liu DL, Chen C & Du JR. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
 

Background: Inflammatory mechanism is thought to play a critical role in hemorrhagic brain injury. Our previous study showed that Z-ligustilide (LIG) had potent anti-inflammatory effects and effectively alleviated brain injury following intracerebral hemorrhage (ICH). However, LIG is hard to be made into medication because of poor stability. Therefore, we synthesized a series of new phthalide compounds, which have the mother nuclear structure of phthalide as that of LIG but have higher stability than LIG. The present study made a tentative research on the neuroprotective potentials of a novel Z-Ligustilide analogue (named CD01) on experimental ICH in mice from the perspectives of inflammatory pathways.

Methods: ICH was induced in adult male CD-1 mice by injection of autologous blood into the right striatum. CD01, LIG and vehicle, respectively, were intraperitoneally administrated after ICH induction. Neurological dysfunction, brain water content, injury volume, the number of surviving/dying neurons, inflammatory cell profile and inflammatory gene expression were assessed at 3 days post-ICH.

Results: Neurological dysfunction, brain water content, neuronal damage, microglia and astrocytes activation as well as peripheral immune cells infiltration were all evidently ameliorated by CD01 relative to vehicle-treated animals. Besides, the expression of TLR4, p-NF-κB p65, TNF-α and IL-6, was obviously reduced by CD01 treatment. So was Prx1 expression and release. However, there was no significant difference in hemorrhagic injury volume between CD01-treated and vehicle-treated mice. All these results indicate that CD01 has the similar effects against the inflammatory brain injury following ICH to LIG.

Conclusion: CD01 provides the potent neuroprotective effect against hemorrhagic stroke as LIG. The mechanism may be involved in the inhibition of Prx1/TLR4/NF-κB signaling and the subsequent immune and neuroinflammation injury.

 

Keywords: Z-Ligustilide analogue, Intracerebral hemorrhage, Neuroinflammation, TLR4/NF-κB pathway, Peroxiredoxin 1 (Prx1)

INTRODUCTION

Stroke is the second most common cause of death worldwide following heart diseases and a leading cause of disability [1]. Intracerebral hemorrhage (ICH) is one of the most devastating subtypes of stroke. It accounts for merely 15% to 20% of all strokes cases, but has much higher disability and mortality rate than ischemic stroke [2]. Although there has been considerable progress in our understanding of ICH pathogenesis, presently available treatments for ICH are mainly limited to supportive medical treatments and surgeries. Effective therapy options still lack for secondary brain injury after ICH.

Large amounts of studies recently demonstrate that secondary brain injury after ICH is the leading cause of neurological deterioration in patients with ICH, including brain edema, neuroinflammation and cellular apoptosis [2-9], while inflammatory responses are highly thought to play a vital role in perpetuating brain injury induced by ICH [5,10,11]. Studies revealed that inflammatory brain injury after ICH was closely associated with the activation of glial cells, infiltration of peripheral inflammatory cells and the subsequent upregulation of proinflammatory cytokines, which all worked together and led to brain edema, blood-brain barrier (BBB) breakdown, substantial nerve cell death and neurological dysfunction [2,3,12,13]. Furthermore, several lines of evidence suggest that immunomodulation plays a pivotal role in neuroinflammation after ICH, which could be a consequence of activation of Toll-like receptors (TLRs), a family of highly conserved innate immunity receptors[3-5,13]. Among of TLRs, TLR4 is recognized as the key factor of the upstream in the immunological and inflammatory cascade in the brain. Increasing evidence indicates that cerebral hemorrhage stress forcefully induces TLR4/NF-κB signaling activation and the resulting inflammatory responses surrounding the hematoma by upregulating proinflammatory cytokines such us TNF-α, IL-6 and IL-1β [2,11,14-19]. Instead, obstructing TLR4/NF-κB pathway effectively alleviated neuroinflammatory injury in the perihematoma region following ICH [11,18,19]. Additionally, current research suggests that TLR4 can initiate a cascade of immune or inflammatory responses by identifying certain endogenous molecules from aseptic damaged tissues or cells such as ischemic/hemorrhagic brain that are described as damage-associated molecular patterns (DAMPs) [20]. Peroxiredoxin 1 (Prx1) is lately identified one of DAMPs involved in hemorrhagic stroke [21]. Study showed that cerebral hemorrhagic stress evoked Prx1 expression and extracellular release from dead or dying brain cells, while activation of extracellular Prx1-mediated TLR4/NF-κB pathway, in turn, contributed to inflammatory brain injury after ICH.

Z-ligustilide (3-butylidene-4, 5-dihydrophthalide, LIG) is the biologically active ingredient of traditional Chinese medicines Radix Angelicae sinensis and Ligusticum chuanxiong [22]. Large amounts of research have confirmed that LIG has potent anti-inflammatory [23-26] and neuroprotective effects towards multiple cerebral ischemic insults [22,27-31]. One research conducted by Chen et al. [32] suggested that LIG significantly improved brain damage following subarachnoid hemorrhage in rats and effectively reduced the mortality of SAH rats. Additionally, our research lately showed that LIG effectively alleviated inflammatory brain injury following autologous blood-induced ICH in mice via inhibiting Prxs/TLR4/NF-κB pathway activation and the resulting immunoreaction and neuroinflammation [33]. However, with a α, β-unsaturated lactone in its structure, LIG is extremely unstable for heat, light, oxidation and etc., and thus inclined to degrade in natural conditions [34,35], which makes LIG hard to be made into medication and limits its application greatly. Therefore, we synthesized a series of new phthalide compounds, CD01 and CD04, by transforming the chemical structure of LIG, which hold the mother nuclear structure of phthalide as that of LIG but have higher stability against heat, light and oxidation than LIG owing to the substitution of stable structure of benzene ring for α, β-unsaturated lactone. One recent study conducted in our research group found that CD01, not CD04, provided a significant neuroprotective effect on the global cerebral ischemia reperfusion (GCIR) in mice through inhibiting activation of astrocytes and microglia, release of inflammatory mediators such as TNF-α and IL-6 and the consequent inflammatory brain damage after GCIR (Supplementary Figures 1-4) [36]. View of this, we investigated the neuroprotective potentials and possible mechanisms of CD01 on experimental ICH in mice from the perspectives of Prx1/TLR4/NF-κB signaling pathway so as to look for a more effective candidate drug for ICH treatment.

MATERIALS AND METHODS

Reagents

Cresyl violet was obtained from Chroma-Gesellschaft Schmid GmbH & Co. (Köngen, Germany). Fluoro-Jade B was obtained from Histo-Chem, Inc. (Arkansas, USA). The primary antibodies used in this study, including allograft inflammatory factor-1 (AIF-1), glial fibrillary acidic protein (GFAP), CD3, myeloperoxidase (MPO), TLR4, the p65 subunit of NF-κB (NF-κB p65) and Prx1 are summarized in Table 1. 2-(4-Amidinophenyl)-6-indolecarbamidine dihydrochloride (DAPI) was purchased from Boster (Wuhan, China). The DeadEend Fluorimetric TUNEL System was purchased from Promega (Madison, USA). Enzyme-linked immunosorbent assay (ELISA) Kits for mouse TNF-α and IL-6 were obtained from Dakewe Biotech (Shenzhen, China). Bone wax and Triton X-100 were from Johnson & Johnson (New Jersey, USA) and Amresco (Colorado, USA), respectively. All other reagents were obtained from local commercial sources. 

CD01 was synthesized by Chu Chen in Sichuan Academy of Chinese Medicine Sciences, which is colorless crystalline 3-alkyl-5, 6-dimethoxy phthalide used in its racemic form. The synthetic processes of CD01 were as followed: 6.76 g of 5, 6-dimethoxyphthalide, 116 mg of benzoyl peroxide and 7.0 g of N-bromosuccinimide were dissolved in 88 ml of CCl4 and refluxed for 4 h. The reaction mixture was then kept at 4°C for 12 h. After filtered, the concentrated filtrate was dissolved in 30 ml of distilled water and refluxed again for 1.5 h. After kept at 4°C for 12 h, the reaction mixture was filtered, and the filter cake was washed with ice water and dried in vacuum for 4 h at 50°C to yield 4.38 g of CD01. The purity of CD01 was more than 98%, determined by HPLC analysis. LIG was prepared as described previously [37], which is faint yellow oil liquid 3-butylidene-4, 5-dihydro-1(3H)-isobenzofuranone. In this present study, LIG was freshly formulated in 3% Tween80 before administration as described before [33]. CD01 solution was prepared in 3% Tween80 before administration same as LIG at the day of operation and was placed at 4°C for reserve. The chemical structures of CD01 and LIG are as followed (Figure 1).

Animals

Specific-pathogen-free male CD-1 mice weighing 28 to 35 g (8-10 weeks old, n=85) were used in this study, which were obtained from Dossy of Laboratory Animal Co. Ltd., Chengdu, China. These mice were kept at constant ambient temperature and humidity under a 12 h/12 h light/dark cycle and given food and water ad libitum. All animal procedures were carried out in accordance with Animal Welfare Legislation in China and approved by the Committee on the Care and Use of Laboratory Animals of Sichuan University, and all experiments in this study were carried out according to the ethical guidelines of the Sichuan University Animal Research Committee.

ICH model

The autologous blood ICH model was established as described previously [38,39]. Concisely, mice were anesthetized with 4% chloral hydrate (400 mg/kg, intraperitoneally) and fixed on a mouse stereotaxic frame (SM-15, Narishige, Japan). A 0.5 mm burr hole in the skull was drilled 2.0 mm right lateral to the midline and 0.2 mm anterior to the bregma. Two or three large drops of arterial blood were collected from the central tail artery. 25 μL of autologous blood was subsequently withdrawn rapidly into a 30 gauge microsyringe and was then advanced stereotactically through the burr hole into the right striatum (coordinates: 0.2 mm anterior, 2.0 mm lateral to the bregma, 3.5 mm below the skull surface). Using a microinfusion pump (TJ-1A, LongerPump, China), 5 μL of autologous blood were first delivered at a rate of 2 μL/min. After a 5 min period, the remaining 20 μL autologous blood was delivered at the same rate of 2 μL/min. After left in place for 10 min, the microsyringe was slowly removed, the burr hole was sealed using bone wax and the skin incision was closed using 5-0 suture. The sham-operated animals merely received needle insertion. Rectal temperature was kept at 37 ± 0.5°C with a feedback-controlled animal body temperature maintaining instrument throughout the procedure. After procedure, mice recovered beside oil heater until being fully awake and then transferred in a temperature-controlled environment at a constant room temperature of 25°C, with free access to food and water. These mice that died for anesthesia were excluded.

Experimental protocol

Mice were randomly allocated to the following groups: 1) sham-operated group (n=21); 2) ICH+vehicle group (n=22); 3) ICH+LIG group (n=21); 4) ICH+CD01 group (n=21). Except for the sham-operated group, all other mice were subjected to ICH through autologous blood infusion as above described. 30 min after ICH induction, mice in the groups of ICH+LIG and ICH+CD01 were administrated intraperitoneally with an equimolar dose of LIG (10 mg/kg) and CD01 (14 mg/kg), respectively. Mice in the sham-operated and ICH+vehicle groups were treated with a volume-matched vehicle (sterilized normal saline containing 3% Tween 80) in the same manner. All treatments were given again at 24 h and 48 h after ICH induction for the next 2 days. These mice that died either on the operating table or within the first 3 h after operation were not assigned to any group. The protocol was according to a previous report [32]. The treatment doses and days of LIG and CD01 were determinate according to our previous report [33]. The detailed protocol for this present study is summarized in Figure 2.

Neurological deficit scoring

Neurological deficits in each mouse were estimated with 5 tests at day 1, 2 and 3 after autologous blood infusion. The tests included the neurological deficit scoring (NDS) system; the modified Bederson’s scoring system, the forelimb placing test, the beam-walking ability test and the coat hanger test. All behavioral tests were performed by two trained experimenters who were blinded to mice grouping.

In the NDS system, mice were scored according to a 28-point scoring scale including 7 items [40]: body symmetry, gait, climbing, circling behavior, front limb symmetry, compulsory circling and whisker response. Each item was graded from 0 to 4 and the maximum neurological deficit score was 28.

In the Bederson’s scoring system, mice were scored based on a 6-point scale which is modified by a previous report [41]: 0, no significant deficits; 1, failure to extend left forepaw fully without forelimb flexion; 2, failure to extend left forepaw fully with forelimb flexion; 3, decreased resistance to lateral push without circling; 4, circling to the right after slight stimulation of the tail of mice; 5, persistent spontaneous circling to the right; 6, failing to the left and inability to walk spontaneously.

The forelimb placing tests were carried out based on previous reports [42-44]. Briefly, mice were held by their torsos, allowing the forelimbs to hang free. Each independent testing of each forelimb for mice was induced by brushing the ipsilateral vibrissae on the corner edge of a countertop. Each mouse was measured 10 times for each forelimb. The percentage of trials in which the mouse placed the appropriate forelimb on the edge of the countertop in response to the ipsilateral vibrissae stimulation was computed. Mice were moved gently up and down before each test in order to facilitate muscle relaxation. Trials during which struggling, extreme muscle tension or placing of any of the limbs onto the experimenter’s hand occurred were not counted.

The beam-walking ability test for mice was performed on a beam (120 cm in length and 0.6 cm wide) that connected two platforms and was suspended approximately 60 cm above the surface. Mice were placed on either end of the beam. For an innate tendency to avoid falling, Mice walked along the beam to the opposite end. The number of foot faults, defined as the number of times that the forepaws and/or hind paws slipped from the horizontal surface of the beam over a predetermined number of steps (50 steps in this present study), was counted. Mice were subsequently scored on a 7-point scale as described previously [41,44,45]: 7, to traverse the beam with no more than two foot slips; 6, to traverse the beam and use the affected limbs to aid more than 50 percent of 50 steps along the beam; 5, to use the affected limbs in less than half of 50 steps along the beam; 4, to traverse the beam and at least once placed the affected hind paw on the horizontal surface of the beam; 3, to traverse the beam while dragging the affected hind limb; 2, unable to traverse the beam, but able to place the affected hind limb on the horizontal surface of the beam and maintain balance; 1, unable to traverse the beam and place the affected hind limb on the horizontal surface. The score of each trial was recorded, and the average value of the three trials scores was calculated for the final score of each mouse. Three days before surgery, each mouse was trained daily to traverse the beam until the animal could walk the length of the beam with no more than two foot slips. If unable to meet the requirements, mice were excluded from this study.

The evaluation of muscle strength and coordination was conducted through a fixed coat hanger. Mice were placed to hang at the center of the coat hanger’s horizontal bar (diameter 3 mm, length 35 cm, about 40 cm above the surface) with two forepaws. The body position of each mouse was observed for 30 s and scored as followed [46]: 0, falls off within 10 s; 1, hangs onto the bar by two forepaws; 2, attempts to climb onto the bar; 3, hangs onto the bar by two forepaws plus one or both hind paws; 4, hangs by all four paws plus the tail wrapped around the bar; 5, active escapes to the end of the bar. The average value of two attempts was recorded as the final score of each mouse.

Brain water content measurement

Measurement of Brain edema severity for each mouse was performed as described previously [42,47]. Concisely, 3 days after ICH induction, eight mice from each group after the neurobehavioral tests were overdosed with chloral hydrate and subsequently transcardially perfused with cold phosphate-buffered saline (PBS, pH 7.4) to measure brain water content. The brains were immediately harvested en bloc including the cerebellum. After olfactory bulb removal, five 2 mm coronal slices were obtained beginning 2 mm from the frontal pole. Each brain slice was divided into 2 hemispheres along the midline and each hemisphere was followed to carefully dissect into the cortex and the striatum. The cerebellum was detached to serve as a control. Thus, a total of 5 samples from each brain were obtained: the ipsilateral cortex, the contralateral cortex, the ipsilateral striatum, the contralateral striatum and the cerebellum. The five samples were weighed on an electronic analytical balance (AE260, Mettler, USA), respectively, to obtain the wet weight, and then placed onto pre-weighed silver paper and dried at 62°C for 72 h to obtain the dry weight. Brain water content (%) was computed as follows:

[(Wet weight - Dry weight)/Wet weight] × 100%

Assessment of histopathology

Three days after ICH induction, seven to eight mice from each group after the neurobehavioral tests were deeply anesthetized with chloral hydrate and subsequently perfused through the heart with cold PBS, followed by cold 4% paraformaldehyde. The brains were removed and placed in the same fixative for 24-48 h. The brain tissue at the level of bregma+2.0 mm ~ -2.0 mm was obtained, dehydrated and then embedded in paraffin. 5 μm consecutive sections were prepared on the coronal plane from the rostral to the caudal portion of the damaged brain areas, and then used for Nissl staining, Fluoro-Jade B (FJB) staining and Immunohistochemistry. All these results of histopathology were estimated blindly by an experienced investigator to minimize observation bias.

Nissl staining: Nissl staining in this study was used to evaluate the number of surviving neurons in the perihematoma area, the hemorrhagic injury volume as well as the hemispheric enlargement. The 5 μm coronal sections from the rostral to the caudal portion of the injured brain areas, which were spaced 200 μm apart, were stained with cresyl violet according to a previous report [48]. Normal neurons have relatively big cell body, rich in cytoplasm, with one or two big round nuclei. In comparison, injured cells show shrunken cell body, condensed nuclei, dark cytoplasm and many empty vesicles. Neuronal density was evaluated on the basis of the established method [33,48]. Sections were digitized and analyzed using ImagePro plus 6.0 software (Media Cybernetics, Bethesda, MD, USA). Striatal surviving neurons were counted at 400x magnification in four areas (squares) around the hematoma per hemisphere of the section with maximal lesion diameter as shown in Figure 3. The relative loss of neurons was computed by subtracting the average neuronal counts of the ipsilateral hemisphere from the contralateral side. The percent of the relative loss of neurons of the ipsilateral hemisphere was computed as follows:

[(The average neuronal counts of the ipsilateral - The average neuronal counts of the contralateral) / The average neuronal counts of the contralateral] × 100%

The hemorrhagic injury volume and the degree of hemispheric enlargement were quantified with the use of all the coronal sections stained with cresyl violet as described previously [33]. The whole section was digitized with the use of SMZ1000 stereomicroscope (Nikon, Japan). Using ImagePro Plus 6.0 software, the damaged area of each section stained by cresyl violet was traced and tabulated, so was the area of the right and left hemispheres of each section. The hemorrhagic injury area was computed by quantifying the Nissl staining-negative area in each section, and the hemorrhagic injury volume was calculated by summation of the Nissl staining-negative areas multiplied by the interslice distance (200 μm). The percentage of the ipsilateral hemispheric enlargement was computed as the following formula:

[(Ipsilateral hemisphere volume - Contralateral hemisphere volume) / Contralateral hemisphere volume] ×100%

FJB staining: FJB is a polyanionic fluorescein derivative that specifically binds to the degenerating neurons. Paraffin sections of the brain per mouse in the range of 200 μm surrounding the point of the needle were stained using FJB in order to quantify the number of dying neurons around the hematoma. Staining was conducted following the manufacturers’ instructions as described before [33]. Concisely, sections were initially incubated in a solution of 1% NaOH in 80% ethanol for 5 min and subsequently hydrated in 70% ethanol and distilled water for 2 min, respectively. They were then incubated in a solution of 0.06% potassium permanganate at room temperature for 10 min, rinsed in distilled water for 2 min, incubated in a 0.0004% FJB working solution for 20 min. Sections were observed and photographed under a fluorescence microscope (Ni-U, Nikon, Japan). Striatal FJB-positive cells were counted in four areas (squares) adjacent to the hematoma as shown in Figure 3 at a magnification of x400 using ImagePro plus 6.0 software. The mean value of FJB-positive cells in the four fields was computed as the final number of dying neurons in the perihematoma area per mouse.

Immunohistochemistry: Paraffin sections of the brain per mouse in the range of 200 μm surrounding the point of the needle were used for immunohistochemical staining as previously described [22,33]. Concisely, the brain sections (5 μm) were rinsed with 3% H2O2 for 20 min in order to block endogenous peroxide activity and incubated with 5% bovine serum albumin (BSA) for 1 h so as to block nonspecific binding. The sections were then incubated with primary antibodies, including AIF-1, GFAP, CD3, MPO, p-NF-κB p65 and TLR4, respectively, overnight at 4°C. Primary antibodies were subsequently recognized by a biotinylated secondary antibody (Boster) at 37°C for 1 h and detected by the streptavidin-biotin complex (Boster) at 37°C for 1 h. Immunoreactions were visualized using 3, 3’-diaminobenzidine tetrahydrochloride (DAB). Except p-NF-κB p65, the nuclei were all counterstained with haematoxylin. Negative control sections were stained only with secondary antibody to control for possible nonspecific staining. For the semi-quantitative analysis of immunohistochemical results, four microscopic fields in the perihematoma area in the section as shown in Figure 3 per mouse brain were acquired under a 40x objective. The immunoreactivity of the target proteins was quantified on the basis of the integrated optical density (IOD) of immunostaining-positive cells per field using ImagePro plus 6.0 software.

To determine the expression and the extracellular release of Prx1 around the hematoma, the brain sections surrounding the needle point were firstly stained with the DeadEnd fluorimetric apoptosis detection system (Promega, Madison, WI, USA) for TUNEL staining in accordance with the manufacturer's instructions, followed by Prx1 immunostaining and DAPI staining. Concisely, after TUNEL staining, the sections were treated with 0.3% Triton at room temperature for 30 min, blocked with 5% BSA at 37°C and incubated overnight at 4°C with Prx1 antibody (1:50). The sections were then incubated with TRITC-conjugated fluorescent secondary antibody (Boster) for 1 h at 37°C. The nuclear staining was finally conducted with DAPI solution (Boster). Prx1 localization was viewed with the use of a fluorescence microscope (Nikon, NI-U, Japan). Photomicrographs were digitized with a 40x objective and processed using the ImageJ software (National Institutes of Health, Bethesda, MD, USA) as described in our previous research [21,22].

Enzyme-linked immunosorbent assay

Three days following ICH induction, six mice per group were overdosed with chloral hydrate and perfused through the heart with cold PBS. Brains were rapidly removed and stored in -80°C. Perihematoma tissues collected from each mouse were homogenized and centrifuged, respectively, and the supernatant was subsequently collected for assay. The concentrations of proinflammatory cytokines including TNF-α and IL-6 were determinate with the use of ELISA reagent kits (Dakewe Biotech). All samples and standards were detected in determinated duplicate in accordance with the manufacturer’s instructions. Tissue chemokine concentrations were expressed as picograms of antigen per milligram of protein.

STATISTICAL ANALYSIS

All data are presented as mean ± SEM. Statistical analyses were conducted with SPSS 17.0 soft-ware. Data were firstly analyzed to ensure normal distribution through the Kolomogorov-Simirnov test. Statistical differences between the two groups were analyzed with the use of the t-test. Multiple comparisons were statistically analyzed with one-way analysis of variance followed by Student-Newman-Keuls test. Statistical significance was set at P<0.05.

RESULTS

Effects of CD01 on brain damage after ICH

Effects of CD01 on ICH-induced neurological deficits: To assess whether CD01 influences recovery from neurological deficits after ICH, multiple behavioral tests were carried out in this present study.

As a result, the NDS’s and Bederson’s scores of mice in the ICH+vehicle group were significantly increased on day 1 to 3 after ICH compared to that in the sham-operated group (all p<0.001) and the highest NDS’s and Bederson’s scores were observed on day 1 after ICH induction. However, intraperitoneal administration of CD01 (14 mg/kg) obviously decreased the NDS’s and Bederson’s scores on day 1 to 3 after ICH induction (all p<0.05). Although LIG (10 mg/kg) merely showed significant effect on the NDS’s and Bederson’s scores of mice on day 3 after ICH (p<0.001, p<0.05) compared to that in the ICH+vehicle group, the NDS’s and Bederson’s scores of mice in the ICH+LIG group 3 days after ICH induction had no significant difference compared with that in the ICH+CD01 group as shown in Figures 4A and 4B. Besides, the results in Figures 4C and 4D displayed that the vehicle-treated ICH mice had marked neurofunctional defects in the forelimb placing test as well as in the beam-walking ability test on day 1 to 3 after ICH induction compared with the sham-operated mice (p<0.001). Treatment of CD01 (14 mg/kg) notably improved the defects on day 1 to 3 after ICH in the two tests. In the forelimb placing test, mice performed better on day 1 after CD01 (14 mg/kg) administration (p<0.05) compared with LIG-treated mice, however, the CD01-treated mice did not show better performance on day 3 after ICH induction than LIG-treated mice. Similarly, there was no statistical difference in the beam-walking ability of mice between the ICH+CD01 group and the ICH+LIG group on day 3 after ICH induction as shown in Figure 4D. Finally, in the coat hanger test, the vehicle-treated ICH mice showed weak muscle strength and poor coordination as displayed by the score of the body position on day 1 to 3 after ICH (all p<0.001) as shown in Figure 4E. Treatment of CD01 (14 mg/kg) obviously improved the score of body position on day 1 to 3 after ICH induction. However, the score of body position demonstrated little differences between the CD01-treated and LIG-treated groups on day 1 to 3 after ICH. 

All the findings of behavioral tests indicate that CD01 can significantly ameliorate the post-ICH neuropathological defects, and there is little difference in the improvement of neurological function after ICH between mice by CD01 and LIG treatment.

Effects of CD01 on ICH-induced brain edema and hemorrhagic injury volume: Brain edema after ICH forms in consequence of BBB breakdown and perihematoma inflammation, which is regarded as a vital index for evaluating ICH severity as behavioral tests do. Here, we checked the brain water content and the degree of hemispheric enlargement 3 days after ICH in mice in order to assess the effect of CD01 against ICH-induced brain edema. These results suggested that intra-striatal injection of autologous whole blood (25 μL) evoked the remarkable rise of brain water content in the ipsilateral cortex and the ipsilateral striatum (83.86 ± 0.82 versus 81.75 ± 0.94, p<0.01, cortex and 83.09 ± 0.95 versus 80.34 ± 1.44, p<0.01, striatum; Figure 5A) and significant enlargement of the ipsilateral hemisphere (11.98% ± 1.64% versus 2.40% ± 0.84%, P<0.001; Figures 5B and 5C) 3 days after surgery compared to the sham-operated group. Administration of CD01 (14 mg/kg) markedly lowered the brain water content in the ipsilateral cortex and striatum and the degree of ipsilateral hemispheric enlargement (all p<0.01) compared to the ICH+vehicle group, so was LIG (10 mg/kg) treatment. However, there were no statistical difference in the influences on brain water content and hemispheric enlargement of mice after ICH between CD01 and LIG. These results reveal that CD01 can effectively alleviate brain edema formation after ICH same as LIG.

Additionally, the results as shown in Figures 5B and 5D displayed that the hemorrhagic injury volume 3 days after ICH induction was remarkably enhanced in the ICH+vehicle group compared to the sham-operated group (p<0.001). However, there was no statistical difference in the average hematoma volume between CD01-treated and vehicle-treated mice (P>0.05), which was similar to the effect of LIG on post-ICH hemorrhagic injury volume.

Effects of CD01 on striatal neurons after ICH: As illustrated in Figures 6A and 6C, a remarkable rise in neuronal density loss was observed in the caudate putamen 3 days after ICH in the ICH+vehicle group, compared with the sham-operated group (p<0.001). The rise in striatal neuronal density loss was effectively decreased by CD01 (14 mg/kg) and LIG (10 mg/kg) (both p<0.01). Besides, we also examined a marked increase in the number of FJB-positive neurons surrounding the hematoma in the ICH+vehicle group compared to the sham-operated group (note: FJB-positive neurons show the number of degenerating neurons, which were rarely viewed in the sham-operated mice apart from a handful of FJB-positive neurons occasionally observed along the needle track), which was significantly reduced by treatment of CD01 and LIG (p<0.01) as shown in Figures 6B and 6D. Clearly, the results of FJB staining were in consistent with that of Nissl staining. However, there was no significant difference in the influence on striatal neurons after ICH between CD01-treated and LIG-treated mice. Altogether, these findings indicate that CD01 can effectively improve neuronal damage after ICH same as LIG. 

Effects of CD01 on ICH-induced neuroinflammation

Numerous studies reveal that both activated glia and infiltrating leukocytes in the brain play a vital role in the development of neuroinflammation and neuronal injury after ICH. Here, we examined the neuroinflammatory alterations in the cerebra of mice 3 days after ICH induction. Immunostaining for GFAP and AIF-1 was used to assess the pathological changes of astrocytes and microglia, respectively, which are thought as parenchymal glia implicated in the neuroinflammatory processes after ICH. As displayed in Figures 7A-7C, there were merely a few GFAP and AIF-1-immunoreactive cells as well as very weak immunoreactivity of GFAP and AIF-1 adjacent to the hematoma in the striatum in the sham-operated group. The vehicle-treated mice had strikingly increased the number of GFAP and AIF-1-positive cells and the immunoreactivity of GFAP and AIF-1 around the hematoma, accompanied by the activated morphology of both glia (all p<0.001), which were effectively lowered by CD01 (14 mg/kg) and LIG (10 mg/kg) treatment compared to the vehicle-treated group (all p<0.01). Moreover, we also observed a marked rise in peripherally invading MPO-positive neutrophils and CD3-positive T-lymphocytes surrounding the hematoma in the ipsilateral striatum in the ICH+vehicle group compared to the sham-operated group (p<0.001), which was also significantly reduced by CD01 (14 mg/kg) and LIG (10 mg/kg) treatment (all p<0.05) as shown in Figures 7A, 7D and 7E. Furthermore, as previous studies [11,16], the results from ELISA suggested that the levels of proinflammatory mediators TNF-α and IL-6 were remarkably raised surrounding the hematoma in the ICH+vehicle group compared with the sham-operated group (p<0.01, p<0.001) (Figure 8). In agreement with the immunohistochemistry results, CD01 (14 mg/kg) and LIG (10 mg/kg) treatment remarkably decreased the levels of TNF-α and IL-6 in the cerebra of mice subjected to ICH (all p<0.05). However, CD01 showed no statistical difference in the expression of GFAP, AIF-1, MPO, CD3, TNF-α and IL-6 around the hematoma compared with LIG. Collectively, these present findings reveal that CD01 can effectively alleviate post-ICH glia activation, peripheral leukocyte infiltration and therefore restrain the expression and release of proinflammatory mediators, preventing the cerebra from being damaged in consequence of ICH, which is similar to LIG.

Effects of CD01 on Prx1/TLR4/NF-κB signaling in the ICH-induced brain damage

In this study, we examined the levels of TLR4/NF-κB in the perihematoma tissue with immunohistochemical staining in order to further investigate the mechanism that underlies the neuroprotective effect of CD01 against cerebral damage after ICH. As illustrated in Figure 9, cerebral hemorrhagic stress obviously evoked TLR4 expression increase and the subsequent activation of the signaling effector NF-κB, reflected by enhanced expression and the nuclear translocation surrounding the hematoma in the vehicle-treated ICH mice compared to the sham-operated mice (both p<0.001). Administration of CD01 (14 mg/kg) and LIG (10 mg/kg) remarkably suppressed the expression of TLR4 and the nuclear translocation of NF-κB in the perihematoma area, which indicates that the neuroprotective effect of CD01 is closely correlated with the inhibition of TLR4/NF-κB pathway around the hematoma after ICH. Similarly, CD01 and LIG had little difference in the expression of TLR4 and the nuclear translocation of NF-κB around the hematoma.

It is well documented that Prx1 expression is enhanced around the hemorrhagic region of rats suffered from autologous blood-induced ICH [49]. Our recent studies again substantiated this conclusion in autologous blood or collagenase-induced ICH in mice [21,33]. Here, we detected the effects of CD01 on the expression level as well as the extracellular release of Prx1 adjacent to the hematoma after ICH by immunofluorescent staining. As shown in Figure 10, Prx1 immunoreactivity was obviously increased in the perihematoma region of mice 3 days after ICH induced by infusion of autologous blood, whereas Prx1 was rarely detected in the striatum of the sham-operated mice. Moreover, we observed that CD01 at a dose of 14 mg/kg significantly lowered the immunoreactivity of Prx1 in the perihematoma region. To further check whether enhanced Prx1 in the hemorrhagic cerebral tissues acts as DAMPs outside cells in autologous blood-evoked ICH mice, the cellular localization of Prx1 3 days after ICH was determined with Prx1-TUNEL-DAPI triple-immunofluorescent staining. Unpredictably, we observed that strong Prx1 expression around the hematoma of ICH mice mostly appeared in the location of nucleus and nearly totally overlapped with TUNEL-positive cells (Figure 10), which indicates that merely a few of Prx1 are released extracellularly 3 days after autologous blood-induced ICH. Furthermore, in this current study, we examined that administration of CD01 reduced the number of apoptotic cells in the perihematoma region, accompanied with decrease of Prx1 expression. Additionally, we also observed that all these findings about the influence of CD01 on the expression and extracellular release of Prx1 after ICH was similar to LIG as reported in our recent research.

Altogether, these present findings indicate that CD01 can significantly inhibit the activation of Prx1/TLR4/NF-κB pathway evoked by ICH induced by autologous blood infusion as LIG did.

DISCUSSION

Extensive experimental and clinical experiments determine that neuroinflammation is an essential event for secondary injury of cerebral ischemia. However, emerging evidence indicated that extravasated whole blood into the brain parenchyma could induce a greater degree of cell death and inflammatory injury compared to ischemic lesions of similar size [50]. Besides, substantial researches revealed that brain inflammatory responses occurred after ICH worsened ICH-evoked cerebral damage through inducing blood-brain barrier disruption, cerebral edema and massive nerve cell death [3,5,12,13,51], which are thus considered the key orchestrator and one of the main mechanisms of secondary cerebral damage after ICH [2]. Consequently, therapeutic agents that target neuroinflammatory cascades are thought as the promising treatment options for ICH.

LIG is a phthalide derivative and the main effective constituent isolated from the essential oil extract of Radix Angelicae sinensis and Ligusticum chuanxiong, which are traditional Chinese medicine clinically administered to treat cardio-cerebrovascular diseases for thousands of years. It reported that LIG provided potent neuroprotective effects on multiple cerebral ischemic insults. Moreover, the neuroprotective effects of LIG against brain damage after brain ischemia were confirmed tightly linked to its powerful inhibition of inflammatory responses induced by brain ischemic stress. Recent research indicated that administration of LIG effectively alleviated brain damage following ICH via inhibiting Prx1/TLR4/NF-κB signaling and the resulting immune inflammatory responses [33]. However, LIG is difficult to be made into medication because of poor stability. CD01 has the mother nuclear structure of phthalide as that of LIG but have higher stability against heat, light and oxidation than LIG. It was documented that many of naturally occurring phthalides apart from LIG displayed the anti-inflammatory effects [52-54]. As a phthalide derivative as well as LIG analogue, it was observed that CD01 effectively improved inflammatory brain damage after GCIR. However, to this day, it remains unclear whether CD01 could exhibit the neuroprotective effect towards ICH as LIG did.

To date a great deal of research has confirmed that neuroinflammation is also an essential event for secondary injury of cerebral hemorrhage. Studies showed that following ICH, production of inflammatory mediators from extravasated blood elements or lysates in the brain parenchyma activated resident glia (i.e., astrocytes and microglia) and infiltrating immune cells (i.e., leucocytes) [2] and subsequently initiated immune-inflammatory responses and cerebral damage via evoking diverse proinflammatory cytokines release from activated glia as well as infiltrating leukocytes in cerebra [5,16,51,55-60]. Several lines of evidence indicated that resident glia cells (i.e., astrocytes and microglia), infiltrating leukocytes (such as neutrophils and T-lymphocytes) and proinflammatory cytokines (e.g. TNF-α and IL-6) contributed to brain inflammation and secondary brain damage after ICH, which were all found excess aggregation and activation in the perihematoa tissues following ICH. In this study, we found that administration of CD01 remarkably ameliorated neurological outcomes 3 days after ICH induction in mice. To further confirm the neuroprotective property of CD01, we checked the influences of the LIG analogue towards brain histopathological alterations 3 days after ICH. In accordance with previous studies [11,33,61-65], ICH brought about increases in brain water content, injury volume, neuronal damage and positive immunoreactivity of different biomarkers of glial cells and leukocytes (i.e., GFAP for astrocytes, AIF-1 for microglia, CD3 for T lymphocytes and MPO for neutrophils) adjacent to the hematoma of mice 3 days after ICH. While CD01 treatment significantly improved the overall post-ICH neuropathological changes except for the hematoma volume. It indicates that CD01 is incapable to effectively improve the hematoma resolution, which is in accord with our findings about LIG. Similarly, in agreement with previous studies [11,33], we further observed that ICH led to rise in the protein levels of neurotoxic cytokines TNF-α and IL-6 surrounding the hematoma of mice 3 days after ICH, which were markedly reduced by administration of CD01. However, the differences in the effects on neurological dysfunction, cerebral histopathological alterations and expression of TNF-α and IL-6 3 days after ICH were not observed between CD01 and LIG in our present study. Altogether, these present findings reveal that CD01 have significant neuroprotective effect towards ICH insults as LIG via suppressing activation of resident glial cells, invasion and infiltration of peripheral leukocytes into the cerebra and the resulting production of TNF-α and IL-6.

Emerging evidence reveals that both innate and adaptive immunity, which were found to play deleterious roles in the pathogenesis of neuroinflammation and brain damage evoked by hemorrhagic stroke as well as ischemic stroke, are highly regulated by DAMP/TLRs signaling pathway [21,22]. TLRs, a family of evolutionarily highly conserved innate immunity receptors, were confirmed to play a vital role in innate immunity and inflammatory responses [66,67]. Of TLRs, TLR4, as the first TLR identified in mammals, are generally recognized as the core factor of the upstream in the immunological and inflammatory cascade and therefore widely investigated in cerebral hemorrhagic stroke as well as in cerebral ischemic stroke. It has been suggested that TLR4 is constitutively expressed on the membrane of various cerebral cells and peripheral inflammatory cells [68,69]. And, large amounts of studies showed that TLR4 activation contributed to the detrimental inflammatory responses in cerebra and evoked poor functional outcome following ICH [14,16,70,71]. In addition, it reported that upregulation of TLR signaling stimulated by ICH brought about the activation of the transcription factors NF-κB and ERK1/2, and the subsequent excessive expression of the proinflammatory cytokines TNF-α, IL-6 and IL-1β from the resident/infiltrating immune cells, ultimately inducing significant inflammatory brain lesions following ICH [11,16]. Conversely, inhibition of the TLR4/NF-κB signaling effectively weakened the inflammatory responses in the preihematoma tissue and the ensuing acute brain injury evoked by ICH [11,16]. In this present study, we detected that ICH elicited activation of TLR4/NF-κB signaling, reflected by increases in TLR4 expression and the nuclear translocation of NF-κB in perihematomal brain tissue. Whereas, systemic administration of CD01 significantly restrained TLR4/NF-κB signaling activation surrounding the hematoma, revealed by downregulation of TLR4 expression and the nuclear translocation of NF-κB. It indicates that the improvement of CD01 on the neuroinflammatory responses after ICH is closely connection with inhibition of TLR4/NF-κB signaling.

As well known, inhibition of the initial upstream event that triggers the ensuing inflammatory responses can effectively relieve the resulting inflammatory damage. DAMPs are called damage-associated molecular patterns, which were thought some endogenous molecules released from aseptic injured tissues or cells including the ischemic or hemorrhagic brain. Recent researches have shown that DAMPs can be specifically combined by TLRs, and in turn trigger a cascade of innate immune and inflammatory responses through the activation of TLR signaling effectors, NF-κB, ERK1/2, STAT3 and etc. Up to now, different DAMPs from dead or dying cells in the cerebra or blood elements have been gradually identified [2,21,22,72]. Peroxiredoxins (Prxs), including six subtypes (Prx1-6), are newly discovered DAMPs involved in ischemic/hemorrhagic stroke[21,22,72], which were originally described as anti-oxidative enzymes responsible for elimination of hydrogen peroxides together with catalase and glutathione peroxidase in mammalian cells, thus regulating oxidative stress-related apoptosis [73]. Previous study showed that intracellular Prxs prevented ischemic brain injury by inactivating hydrogen peroxide [21]. However, in vivo studies lately suggested that once released from dead or dying neural cells due to cerebral ischemia or hemorrhage, Prxs lose their antioxidant capacity and in turn function as DAMPs of TLRs to evoke immune and inflammatory responses through activating TLR signaling effectors, and ultimately bring about neuroinflammation and brain damage after ischemic/hemorrhagic stroke [21,22,72]. Among Prxs, Prx1 was found to stimulate secretion of proinflammatory cytokines by binding TLR4 [74] and was thought a major hemorrhagic stress-inducible subtype of Prxs in ICH rats [49]. In our previous study, we observed that collagenase-induced ICH in mice stimulated Prx1 expression and extracellular release from dead or dying cells in the cerebra, accompanied by TLR4/NF-κB pathway activation [21], which indicates that Prx1 is probably to play a deleterious role in inflammatory brain damage after ICH. However, in our present research, we found that although autologous blood-induced ICH in mice also induced increased Prx1 expression around the hematoma, positive immunoreactivity of Prx1 was mainly detected in the nucleus and merely a few of Prx1 were found in the extracellular compartment of dead or dying cells in the brain, which might be the reason that autologous blood-induced ICH brings about a lighter brain lesion compared with collagenase-induced ICH [75]. Besides, enhanced apoptotic cells were checked in the perihematoma region, which is in accordance with previous studies [32,33]. However, consistent with LIG treatment, CD01 treatment also decreased Prx1 expression 3 days after autologous blood-induced ICH, whether in the nucleus or extracellularly, as well as the number of apoptotic cells surrounding the hematoma, indicating the neuroprotective effect of CD01 towards ICH might be closely related to significant inhibition of apoptosis and production or release of Prx1 from injured brain cells. Nevertheless, enhanced Prx1 expression mostly appears in the nucleus in the cerebra of mice subjected to autologous blood-induced ICH, yet TLR4 is a membrane protein, further studies are thus needed to elucidate how CD01 suppresses the production or release of Prx1 and whether Prx1 in cerebrospinal fluid after ICH induced by injection of autologous blood is apparently increased, how CD01 blocks the interaction between TLR4 and Prx1, or how Prx1 worsens brain damage after ICH induction by autologous blood.

In summary, our findings revealed that CD01 conferred a protective effect towards intracerebral hemorrhage whereby they restrained Prx1/TLR4/NF-κB signaling and the ensuing immune inflammatory damage after ICH and benefited the experimental hemorrhagic outcome. It indicates that CD01 might be a better candidate drug for ICH treatment than LIG in view of its higher stability against heat, light, oxidation and etc. Nevertheless, our present study merely carried out the methods of ELISA and immunostaining to check the levels of multiple inflammatory markers, which is not optimal for this study. Hence, in our future study, additional molecular techniques (such as western blot or RTqPCR) for inflammatory markers would be performed so as to better reveal our conclusions.

Additionally, since inflammatory responses are the common pathogenesis of multiple diseases and the anti-inflammatory property of CD01 may not be specifically for ICH, CD01 might be also a promising compound for treatment of other pathological conditions.

CONFLICT OF INTEREST

The authors have no conflict of interest.

ACKNOWLEDGEMENT

This study was funded by Science Foundation for Excellent Youth Scholars in Sichuan Province (No. 2017JQ0014). 

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