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. 2015 Mar 29;35(7):921–930. doi: 10.1007/s10571-015-0187-5

Protective Effect of Pyrroloquinoline Quinone (PQQ) in Rat Model of Intracerebral Hemorrhage

Hongjian Lu 1,2,#, Jiabing Shen 3,4,#, Xinjian Song 2, Jianbin Ge 2, Rixin Cai 4, Aihua Dai 3,4, Zhongli Jiang 1,✉
PMCID: PMC11486243  PMID: 25820784

Abstract

Pyrroloquinoline quinone (PQQ) has invoked considerable interest because of its presence in foods, antioxidant properties, cofactor of dehydrogenase, and amine oxidase. Protective roles of PQQ in central nervous system diseases, such as experimental stroke and spinal cord injury models have been emerged. However, it is unclear whether intracerebral hemorrhage (ICH), as an acute devastating disease, can also benefit from PQQ in experimental conditions. Herein, we examined the possible effect of PQQ on neuronal functions following ICH in the adult rats. The results showed that rats pretreated with PQQ at 10 mg/kg effectively improved the locomotor functions, alleviated the hematoma volumes, and reduced the expansion of brain edema after ICH. Also, pretreated rats with PQQ obviously reduced the production of reactive oxygen species after ICH, probably due to its antioxidant properties. Further, we found that, Bcl-2/Bax, the important indicator of oxidative stress insult in mitochondria after ICH, exhibited increasing ratio in PQQ-pretreated groups. Moreover, activated caspase-3, the apoptotic executor, showed coincident alleviation in PQQ groups after ICH. Collectively, we speculated that PQQ might be an effective and potential neuroprotectant in clinical therapy for ICH.

Keywords: PQQ, ICH, ROS, Neuroprotectant, Rat

Introduction

Intracerebral hemorrhage (ICH) is associated with a high mortality rate and severe morbidity (Yang et al. 2014). Rupture of blood vessels within the brain parenchyma leads to primary and secondary injuries (Kuramatsu et al. 2013). During the processes of ICH, cytotoxicity of blood, inflammation, oxidative stress and so on all contribute to irreversible injuries, and eventually cause the disability or even the death (Ke et al. 2013; Sukumari-Ramesh et al. 2012). Evidence during the past decades has demonstrated that the fatality rate of ICH at 1 month is approximately 40 %, which likely results from a lack of effective therapeutic schedule after ICH (Anderson et al. 2013; Barratt et al. 2014). Therefore, effective therapeutic methods will be urgent for patients who suffer from ICH in social activities.

Pyrroloquinoline quinone (PQQ) is a redox cycling planar orthoquinone, which has been proven to exist in various fruits, vegetables, milk, and even tissues of mammalian animal (Kasahara and Kato 2003). PQQ has been drawing attention from both the nutritional and the pharmacological view point (Stites et al. 2000). PQQ can antagonize the oxidative stress-induced cell damage, including reoxygenation injury of heart, hyperoxia-caused cognitive deficit, and ethanol-induced liver damage (Ohwada et al. 2008; Singh et al. 2014; Tao et al. 2007). In recent years, emerging evidences indicate its protective role in CNS diseases, such as inhibiting 6-hydroxydopamine-induced neurotoxicity in rat model of Parkinson (Hara et al. 2007), attenuating the gene expression of inducible nitric oxide synthase (iNOS) in the injured spinal cord (Hirakawa et al. 2009), protecting rat brain from reversible middle cerebral artery occlusion (Zhang et al. 2006), and promoting the behavioral recovery after traumatic brain injury that reported in our laboratory previously (Zhang et al. 2012). PQQ can also modulate NMDA receptor by directly oxidizing its redox modulatory site and inhibit glutamate-induced ROS production in cultured cortical neurons (Scanlon et al. 1997). However, little is known about the effect of PQQ following ICH. Due to its inherent properties mentioned above, we speculate PQQ may also play a positive role on neuronal activities after ICH.

Therefore, we evaluated the role of PQQ in rat ICH models. We discovered PQQ could efficiently improve the locomotor function after ICH. It reduced the expansion of hematoma and alleviated brain edema when rats were subjected to ICH. As an antioxidant, PQQ inhibited the production of ROS in PQQ-treated groups when compared to vehicle-treated group. Further, PQQ increased the ratio of Bcl-2/Bax, and inhibited the activity of caspase-3. All of the above demonstrate its neuroprotective effect in the processes of ICH.

Experimental Procedures

Animal Model

All animal care and surgical procedures were carried out based on guide for the care and use of laboratory animals promulgated by the National Research Council in 1996, and supported by the Chinese National Committee to use of experimental animals for medical purposes, Jiangsu Branch. Male Sprague–Dawley rats with an average body weight of 250 g (ranging from 220 to 275 g) were used. All animals were maintained in a temperature controlled room (22 ± 1 °C) on a 12 h light–dark cycle and the food and water were available ad libitum. The number of animals studied was the minimum to obtain significant results, and all efforts were made to minimize their discomfort caused by the experimental procedures.

Pyrroloquinoline quinone (PQQ, Sigma, St. Louis, MO) was dissolved in sterile phosphate-buffered saline (PBS), and administered intraperitoneally into the animals (5.0, 10.0 mg/kg) every day. Two weeks later, rats were subjected to ICH. The dosage of PQQ used in the experimental procedures was referred to the previous study (Zhang et al. 2006). The administration was terminated until the day rats sacrificed. The vehicle-treated animals received PBS without PQQ.

For ICH model, the rats were anesthetized intraperitoneally with 10 % chloral hydrate and then positioned in a stereotaxic frame. Autologous whole blood (50 μL) collected from caudal vein was quickly introduced into right basal ganglia (coordinates: 0.2 mm anterior, 5.5 mm ventral, and 3.5 mm lateral to the bregma). The sham group only had a needle insertion, and other procedures were same as ICH-operated group. Throughout the experiment, body and brain temperature were maintained until the animal was completely recovered from anesthesia and returned to its cage. All animals showed a left side weakness (circling and/or falling to right) when they recovered from anesthesia, verifying ICH-induced neurological deficits. The animals were sacrificed at indicative time points (1, 2, 3, 5, and 7 days) for necessary experiments. The study utilized a total of 150 rats that were randomly assigned to either ICH operation with PQQ pretreatment (n = 60)/without PQQ pretreatment (n = 60) or sham operation (n = 30).

Neurobehavioral Deficit Scoring

Neurobehavioral deficit scoring was referred to the 18-point scale described by Garcia et al. (1995). Briefly, neurological status was scored in each rat daily for 7 days. They were examined in the late afternoon to avoid any effect of circadian rhythm. The individual evaluating neurobehavioral deficits were blinded as to whether vehicle or PQQ was treated. The neurobehavioral scale consisted of the following six tests:

1. Spontaneous activity (0–3 points: 0 = none; 1 = barely moves; 2 = moves but does not approach at least three sides of cage; and 3 = moves and approaches at least three sides of cage).

2. Symmetry in the movement of four limbs (0–3 points: 0 = no movement in left side; 1 = slight movement in left side; 2 = moves slowly in left side; and 3 = move symmetrically in both sides).

3. Symmetry of forepaw outstretching while held by tail (0–3 points: 0 = no outstretching in left side; 1 = slight movement to outreach in left side; 2 = moves and outreaches less than right; and 3 = symmetrically outreach).

4. Climbing (1–3 points: 1 = fail to climb; 2 = left side is weak; and 3 = normal climbing).

5. Body proprioception (1–3 points: 1 = no response on left side; 2 = weak response on left side; and 3 = symmetrical response).

6. Response to vibrissae touch (1–3 points: 1 = no response on left side; 2 = weak response on left side; and 3 = symmetrical response).

Sections and Hemorrhagic Injury Analysis

Rats after ICH were anesthetized and perfused with 500 ml of 0.9 % saline, followed by 4 % paraformaldehyde. After perfusion, the brains were removed and post-fixed in the same fixative for 24 h and transferred to 20 % sucrose for 2–3 days, followed by immersion in 30 % sucrose for another 2–3 days. The tissues were then cut at 2 mm with a cryostat and the sections were stored at −20 °C until use.

Hematoxylin/eosin (H&E) staining was performed to visualize the hematoma. Briefly, sections were washed twice in PBS and kept in hematoxylin for 2 min at room temperature (RT). They were washed twice in tap water, 70 % ethanol with 1 % hydrochloride acid for 3 s, followed by three washes in tap water. After placed in eosin for 1 min, they were washed in PBS, dehydrated in gradient ethanol and mounted. Lesion volumes were calculated from summed and measured areas [determined by computer-assisted image analysis program (OPTIMAS 5.1, Optimas Inc)] of hemorrhagic tissues in mm2 multiplied by 2-mm slice thickness. The individual measuring the hematoma size was also blinded as to whether vehicle or PQQ was administered.

Evaluation of Brain Water Content

Brain water content (brain edema) was measured via the wet/dry weight method as previously described (Tang et al. 2004). Briefly, rats under deep anesthesia were decapitated at indicative time points (1, 2, and 3 days) after ICH, and brain specimens were quickly removed. These brain specimens were divided into the ipsi-and contralateral hemisphere. The cerebellum was additionally collected as an internal control. All tissue samples were weighed using an analytical microbalance (APX-60, Denver Instrument, Bohemia, NY) in order to obtain the wet weight. The samples were then dried at 100 °C for 24 h before determining the dry weight. Brain water content (%) was calculated as (wet weight−dry weight)/wet weight × 100 %.

Western Blot Analysis

Rats were sacrificed at different time points by injecting overdose of chloral hydrate. Tissues surrounding the hematoma (2 mm) from the ICH or the corresponding areas from the sham group were dissected and flash-frozen at −80 °C. For brain tissue proteins, the samples were weighted and cut into pieces, then homogenized in modified RIPA lysis buffer (150 mM NaCl, 50 mM Tris, pH 7.5, 1 % Nonidet P-40, 1 % sodium dodecyl sulphate (SDS), 1 % sodium deoxycholate, 5 mM EDTA, phosphatases inhibitor cocktails, and protease inhibitor cocktail tablet). The supernatant was collected by centrifuging tissue homogenate at 12,000 rpm for 15 min in a microcentrifuge at 4 °C. After the determination of concentration with the Bradford assay (Bio-Rad), the samples were subjected to SDS–polyacrylamide gel electrophoresis and transferred to a polyvinylidene difluoride filter membrane by a transfer apparatus at 300 mA for 1.5 h. The membranes were blocked with 5 % nonfat milk and incubated with anti-Bcl-2 (1:500, Santa Cruz), anti-Bax (1:1000, Santa Cruz), and anti-β-actin (1:1000, Santa Cruz) overnight. After washing with TBST, the blots were incubated with secondary antibodies for 1.5 h at RT and detected using either an enhanced chemiluminescence kit (Pierce) or Odyssey infrared imaging system (LI-COR Bioscience). All results shown are representative of at least three independent experiments.

Measurement of Caspase-3 Activity

The caspase-3 activity was measured by caspase-3/CPP32 colorimetric assay kit according to the manufacturer’s specifications (Promega, Madison, WI, USA). The brain extracts were homogenized in modified RIPA lysis buffer and quantified by BCA analysis. The 50 μL of supernatant was mixed with 50 μl of 2× loading buffer (containing 10 mM dithiothreitol) and 200 μM DEVD-pNA substrate. The reaction was maintained in a 37 °C water bath for 2 h, and the absorbance was measured at 405 nm with an ELx-800 microplate reader. Data were expressed as the relative activity over control.

Measurement of ROS Production

Brain samples after ICH were collected at the indicated times. Total ROS in brains was measured with the Oxiselect in vitro ROS/RNS assay kit (Cell Bio-labs, San Diego, CA), following the manufacturer’s instructions. Fluorescence of dichlorofluorescein was measured using a microplate reader and normalized to sham animals.

Statistical Analysis

All experiments were repeated at least three times and the results were found to be reproducible. Student’s t test was used to calculate the significance of differences between the groups. The level of statistical significance was set at p < 0.05.

Results

Effect of PQQ on Rat’s Locomotor Function Following ICH

Recovery of the locomotor function of the left limbs after the injury was evaluated by using neurobehavioral deficit scoring according to previous studies (Garcia et al. 1995; Zhang et al. 2006). Rats suffered from ICH resulted in serious motor deficit of the left limbs compared to normal or sham-operated groups. Compared with the sham-operated group, the ICH group exhibited significantly functional deficits in the first 3 days (*p < 0.05). By 5 days and thereafter, neurological test scores of the rats in the ICH group went back to baseline (Fig. 1a). Recovery from the motor deficit was significantly improved by the intraperitoneal administration of PQQ (10 mg/kg) compared with that of the vehicle-treated group at 1, 2, and 3 days after ICH (Fig. 1b). However, PQQ at 5 mg/kg exhibited no significant improvement of functional scores (Fig. 1c). Moreover, PQQ treatment at the same time or after ICH showed no locomotor functional improvement, even PQQ concentration was increased to 20 mg/kg (data not shown).

Fig. 1.

Fig. 1

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Effect of treatment with PQQ on neurobehavioral scores. a The ICH group exhibited distinctly functional deficits compared with the sham-operated or normal group over the first 3 days (*p < 0.05, significantly different from the sham-operated or normal group), but had no significant difference at baseline for 5 days later. b Treatment with 10 mg/kg PQQ resulted in improved neurobehavioral scores at 1, 2, and 3 days after ICH (*p < 0.05). c No significant effect on neurobehavioral scores of ICH when treated with 5 mg/kg PQQ. Values are mean ± SEM; n = 6

Pretreatment of PQQ Reduced the Hematoma Volumes Following ICH

Volumes of hematoma after ICH were visualized by H&E staining. In vehicle-treated rats, the hematoma volumes were 27.2 ± 3.21 mm3 at 1 day after ICH, expanding largely in next two days (34.8 ± 3.95 mm3 at day 2 and 45.2 ± 3.23 mm3 at day 3) (Fig. 2a, b, c). Three days later, it became absorbed gradually (Fig. 2a, d, e). In PQQ pretreated groups, the hematoma size of ICH was totally alleviative than that of vehicle-treated group at the first 3 days of ICH. It was 16.2 ± 3.67 mm3 at 1 day after ICH, and proceeded slightly for following 2 days (22.3 ± 3.18 mm3 at day 2, and 27.5 ± 2.78 mm3 at day 3), while no significant changes happened at 5 days or later (Fig. 2a–e). Figure 2c represents the changes of hematoma volumes treated by vehicle or PQQ (*p < 0.05). These data suggest that PQQ pretreatment may affect the progression of the hematoma.

Fig. 2.

Fig. 2

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Hematomas of right cerebral hemisphere at various time points after ICH were visualized by H&E staining. a a 1 day after ICH of vehicle group, b 2 days after ICH of vehicle group, c 3 days after ICH of vehicle group, d 5 days after ICH of vehicle group, e 7 days after ICH of vehicle. b a–e: rats from PQQ-treated group were visualized by H&E staining at corresponding time point of vehicle group. c Size of hematoma from vehicle or PQQ-treated group was measured at each time point of ICH as described in material and method. Values are mean ± SEM; n = 6. *p < 0.05; compared with vehicle group at corresponding time point. Dashed lines encircle the hematoma

Pretreatment of PQQ Alleviated Brain Edema Induced by Hematoma Following ICH

Injections of autologous whole blood obviously evoked the formation of brain edema (brain water content) after ICH. Brain water content in ipsilateral cerebral hemispheres of vehicle group was 81.3 ± 0.8 % at 1 day after ICH, and enlarged progressively in next two days (83.1 ± 0.9 % at day 2 and 84.6 ± 0.8 % at day 3 after ICH) (Fig. 3a–c, vehicle groups). Compared with vehicle groups, PQQ at 10 mg/kg pretreatment of ipsilateral hemispheres significantly reduced the expansion of brain edema after ICH. It was 80 ± 0.8 % at day 1, 81.5 ± 0.7 % at day 2, and 82.4 ± 0.9 % at day 3 after ICH (Fig. 3a–c, PQQ groups). However, contralateral hemispheres in either vehicle or PQQ-treated groups showed no obvious changes of brain water content after ICH (Fig. 3a–c). And neither contralateral nor ipsilateral hemispheres in sham-operated groups exhibited the elevation of brain water content after experimental injuries (Fig. 3a–c). The data suggest that PQQ pretreatment may alleviate the elevation of brain edema after ICH.

Fig. 3.

Fig. 3

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Brain edema was evaluated via the wet/dry weight method. The brain specimens were divided into the ipsi- and contralateral hemisphere. Brain edema at 1 a, 2 b, and 3 days c after ICH was measured, respectively. The cerebellum was collected as an internal control. *p < 0.05 versus vehicle-treated group of corresponding time point

PQQ Inhibited the Production of ROS After ICH

To further explore the role of PQQ in the process of ICH, ROS production was measured. As an antioxidant, PQQ was reported to effectively reduce the production of ROS in previous studies (Pandya et al. 2013; Singh et al. 2014). ROS assay showed that ROS production in vehicle groups significantly increased in the prior 3 days, then progressively decreased in following two days after ICH. However, when giving pretreatment with PQQ at 10 mg/kg, it significantly alleviated the production of ROS after ICH (Fig. 4a). Thus, we demonstrated that PQQ exerts its protective role through inhibiting the production of ROS in the process of ICH.

Fig. 4.

Fig. 4

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Quantification of ROS changes in the perihematomal region in sham, vehicle, and PQQ pretreated groups at indicative time points. a ROS production was progressively increased in the first 3 days after ICH, and then returned to baseline gradually. PQQ at 10 mg/kg pretreated significantly alleviated ROS production induced by ICH. b western blot was applied to detect the expression of Bcl-2 and Bax in vehicle or PQQ-treated groups at different time points after ICH. c The change of Bcl-2/Bax ratio was shown in a histogram. β-actin was used for equal protein loading. *p < 0.05 versus vehicle-treated group of corresponding time point

PQQ Affected the Expression of Bcl-2 and Bax in Rat ICH Models

As known, neuronal apoptosis is regarded as one of the most crucial events after ICH (Wu et al. 2008). Bcl-2 family members include both anti-apoptotic (e.g., Bcl-2 and Bcl-xl) and pro-apoptotic (e.g., Bax, Bad, Bak, and Bid) proteins. The ratio between the two subsets determines the susceptibility of cells to a death signal (Zhang et al. 2011). Additionally, changes in ratio of Bcl-2/Bax could also reflect the oxidative stress insult in mitochondrial after ICH. Western blot analysis showed that rats from vehicle groups exhibited down-regulation of Bcl-2 and up-regulation of Bax, thus decreasing the Bcl-2/Bax ratio (Fig. 4b). However, pretreated with PQQ at 10 mg/kg, decreased ratio of Bcl-2/Bax was significantly reversed (Fig. 4b, c). The ratio of Bcl-2/Bax that represented the results of Western blot is shown in Fig. 4c. The effect of PQQ on the Bcl-2/Bax ratio might probably constitute an important element responsible for antioxidant insult of PQQ.

PQQ Inhibited the Activation of Caspase-3

To further explore the anti-apoptotic effect of PQQ in rat ICH model, we detected the activity of caspase-3, the executor of cells apoptosis. Brain extracts from vehicle rats and PQQ-treated rats were performed to detect caspase-3 activity according to the manufacturer’s specification. Caspase-3 activity in vehicle-treated groups progressively increased, peaked at day 2, and gradually returned to baseline. However, pretreatment with PQQ (10 mg/kg) significantly slowed down the caspase-3 activity compared to the vehicle groups at corresponding time points (Fig. 5). Collectively, we inferred that PQQ exerts the protective role in the process of ICH probably through reducing ROS production and inhibiting neuronal apoptosis.

Fig. 5.

Fig. 5

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Effect of PQQ on the activation of caspase-3 after rats subjected to ICH treatment. Brain extract from vehicle-treated and PQQ-treated rats was measured with a microplate reader. Caspase-3 activity was increased after ICH, and peaked at day 2 in vehicle-treated group. 10 mg/kg PQQ treatment significantly reduced the increasing trend in prior 3 days of ICH. Values are mean ± SEM; n = 6. *p < 0.05, compared with vehicle-treated rats at corresponding time point

Discussion

In the present study, we attempted to prove that PQQ has the protective effect in neuronal injury following ICH. From neurobehavioral deficit scoring, PQQ was proved to be effective on functional recovery of rat left limbs that induced by ICH. PQQ could alleviate hematoma volume and reduce expansion of brain edema after ICH, which always reflected the extent of neuronal injury in clinical ICH patients (Krafft et al. 2014). As an antioxidant, PQQ effectively reduced the production of ROS in PQQ-treated groups when compared with vehicle-treated groups at corresponding time point of the prior 3 days after ICH. To further examine oxidative stress insult that due to the production of ROS, apoptosis-relate proteins (such as Bcl-2 and Bax et al.) were detected by western blot. It showed that the levels of Bcl-2 increased, while Bax down-regulated in PQQ-treated groups when compared with the vehicle groups. The ratio of Bcl-2/Bax was then calculated, and it significantly reversed in PQQ pretreatment. Caspase-3 activity, which indicates early apoptosis, also appeared with reduced expression in PQQ-treated rats. Thus, we conclude that PQQ pretreatment has a protective role in experimental ICH models.

ICH is a dynamic process, generally consisted of initial hemorrhage, hematoma expansion, and formation of perihematomal brain edema (Xi et al. 2002). Hematoma within brain parenchyma triggers a series of events leading to secondary injury and severe neurological deficits (Zhao et al. 2007). The mechanism of this secondary injury is complex, but it is caused primarily by the cytotoxic effect of extravasated blood and by cytotoxic substances released by activated neuroglia and hematogenous cells that invade the brain (Zhao et al. 2009). Therapies preventing expansion could thus provide a key opportunity to decrease final ICH volume. In our experiments, PQQ could effective promote the recovery of locomotor function, and reduce the expansion of hematoma after ICH. Subsequently, there are reports indicating that formation of brain edema after ICH is highly associated with its poor outcome, and it has been regarded as the main predictor of neuronal injuries (Krafft et al. 2014). As anticipated, the level of brain edema was significantly alleviated after PQQ pretreated in rat ICH models.

However, the mechanism of PQQ functions in ICH is still not illustrated. Several properties of PQQ might be involved in the protection of neurological diseases. The first and most important is that PQQ functions as an antioxidant, which has been demonstrated to protect mitochondrial functions from oxidative damage (Kumar and Kar 2014; Zhang et al. 2009). Pretreatment of PQQ prevents acute EtOH-induced oxidative damage by reducing lipid peroxidation in blood and liver and increasing hepatic-reduced glutathione in rats (Singh et al. 2014). Nunome et al. (2008) indicated that PQQ prevented oxidative stress-induced neuronal death probably through changing oxidative status of DJ-1, a causative gene product for a familial form of Parkinson’s disease (Nunome et al. 2008). And in our experiments, we uncovered that pretreatment of PQQ effectively reduced the production of ROS after ICH. Second, PQQ may potentially oxidize the NMDA receptor redox site and thereby decreases its activity (Aizenman et al. 1994; Sanchez et al. 2000; Zhou et al. 2014). It is known that pathological activation of NMDA receptors has been implicated in various CNS disorders, such as ischemia, ICH, Alzheimer’s disease, and so on. In these diseases, it functions probably through exacerbating several calcium-dependent pathways, also called excitotoxicity, that cause oxidative stress and apoptosis (Liu et al. 2010; Mokrushin and Pavlinova 2013; Sharp et al. 2008). Third, PQQ has been reported to increase production of nerve growth factor (NGF) in some cell lines (Nakano et al. 2013; Yamaguchi et al. 1993). NGF is required for peripheral sympathetic and sensory neuron function, and aids in protecting the magnocellular cholinergic neurons in the basal forebrain nuclei (Yamaguchi et al. 1996).

It is well known that oxidative stress-induced DNA damage could trigger cell apoptosis by inducing mitochondrial permeability transition pore (MPTP). MPTP is considered the “point of no return” for apoptotic cell death, which is able to switch cells to apoptotic death via oxidative stress-responsive signaling cascades (Thomas et al. 2011). In addition, MPTP also can be directly induced by decreased Bcl-2/Bax ratio, leading to the release of cytochrome c along with other proapoptotic proteins (Park et al. 2013). In consequence, the balance between Bcl-2 and Bax determines the fate of survival or death of cells in response to oxidative stress insults (Zha and Reed 1997). In this study, we found that PQQ pretreatment increased the ratio of Bcl-2/Bax in ICH-injured neuronal functional damage, suggesting that PQQ rescued brain neurons from cell apoptosis possibly through regulation of apoptosis-related proteins.

Critically, it is important to point out that compounds of PQQ that engage in redox cycling can also be effective free radical initiators. Although toxicity studies are limited, nephrotoxicity and oxidative damage have been reported when 35 mmol or more per kg body weight was administered daily by injections (i.p) to rats over a 4–5 days period (Singh et al. 2014; Watanabe et al. 1989). PQQ has also been demonstrated to initiate DNA damage in vitro (Hiraku and Kawanishi 1996). However, relatively high concentrations of PQQ are required for DNA damage relative to the concentrations of PQQ needed to cause cell proliferation (Naito et al. 1993). Certainly, it is also dependent on the species of animals or experimental environment that used in their process of studies.

On the whole, all the data of present research certificated a protective role of PQQ in the process of ICH. Thus, PQQ, which acts as an essential nutrient, antioxidant, and redox modulator in a variety of systems, produces an effective neuroprotection and represents a new class of naturally occurring agents with potential use in the therapy of adult ICH. Nevertheless, further studies remain to be done to seek the underlying cellular and molecular mechanisms for ICH.

Acknowledgments

We thank Dr. Aiguo Shen of Nantong University for his advice and great help on this paper. This work was supported by the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD).

Conflict of interest

The authors declare no conflict of interest.

Footnotes

Hongjian Lu and Jiabing Shen contributed equally to this work.

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