Protective effect of 4-methyl-2-{[4-(toluene-4-sulfonyl)-thiomorpholine-3-carbonyl]-amino}-pentanoic acid isopropyl ester against cerebral ischemia

Tinghui Zhng †,Yihn Wng †,Xin Li ,Jinwei Di ,Qunkun Zhung ,Xirui Song ,Junhi Xio,Lili Wng,Yuyng Zhng＊

a School of Life Science and Biopharmaceutics,Shenyang Pharmaceutical University,Shenyang 110016,China;

b Laboratory of Computer-Aided Drug Design &Discovery;State Key Laboratory of Toxicology and Medical Countermeasures,Institute of Pharmacology and Toxicology,Academy of Military Medial Sciences,Beijing 100850,China;

c Laboratory of the drug screening,Institute of Pharmacology and Toxicology,Academy of Military Medial Sciences,Beijing 100850,China

AbstractTo investigate the effects of 4-methyl-2-{[4-(toluene-4-sulfonyl)-thiomorpholine-3-carbonyl]-amino}-pentanoic acid isopropyl ester (HD5-6) against cerebral ischemia in rodents,the models with global and focal ischemia were induced by bilateral common carotid artery occlusion plus hypotension (BCAOH) and permanent cerebral artery occlusion (p-MCAO) in mice (n=10 -12 per group in BCAOH;n=8 per group in p-MCAO) and rats (n=10 -11 in each group).HD5-6 prolonged lifetime and improved neurological function.Neurological deficits score in HD5-6 (30 mg/kg) decreased significantly.Malonaldehyde (MDA) in HD5-6-treated mice with ischemia considerably dropped.The infarction volume of the HD5-6-treated rats with MCAO-induced ischemia decreased significantly in the high dose group (P ＜ 0.05,i.g.and P ＜ 0.01,i.v.).Immunohistochemistry showed that Brain derived neurotrophic factor (BDNF) in the ipsilateral hemisphere increased and Vascular endothelial growth factor (VEGF) decreased with HD5-6 treatment.HD5-6 has protective effects against experimental cerebral ischemia in rodents and the action mechanism may involve anti-oxidation and neurogenesis.

Keywords:HD5-6;global ischemia;focal ischemia;MDA;BDNF;VEGF

1 Introduction

4-methyl-2-{[4-(toluene-4-sulfonyl)-thiomorpholine-3-carbonyl]-amino}-pentanoic acid isopropyl ester (HD5-6) Cerebral ischemia is a common disease,which leads to more than 9% mortality and is one of the major causes of permanent disability worldwide.However,there are few therapeutic options for patients with cerebral ischemia,and new insights on treatment are urgently needed.Extensive studies in recent years have shown that neuroprotection is effective for the sequence of events in ischemic cascade,so it is considered as the principal therapeutic strategy for ischemic stroke [1,2].

Although there are various mechanisms of neuroprotection,neurogenesis is the primary neuroprotection strategy.BDNF is one of many neurotrophic factors that promote neurogenesis.It plays a key role in regulating the viability of neurons and the proliferation and differentiation of nerve cells in the brain.Under some pathologic conditions such as focal cerebral ischemia,it can prevent the death of nerve cells and reduce the volume of brain injury [3,4].The increase of endogenous BDNF also affects the activation of molecular and cell repair mechanisms [5].Moreover,exogenous BDNF supplement or other methods to increase BDNF are beneficial to the recovery of brain injury [6].VEGF also plays an important role in neuroprotection,which is associated with angiogenesis,neurogenesis,axonal plasticity,neuronal survival and vascular permeability [7].Some studies have indicated that VEGF is an interesting therapeutic agent,which can prevent neuronal cell death after cerebral ischemia [8].However,whether the expression of VEGF is closely related to blood brain barrier (BBB) leakage in the acute ischemic brain remains controversial [9].Inhibition of VEGF in the early phase of cerebral ischemia has been proved to effectively prevent the injury after cerebral ischemia [8,10].

FK506 binding protein (FKBP) is the main immunophilin of FKBP family,which mainly binds FK506 and has peptidylprolyl cis/trans isomerase activity [11].Obviously,the ligand for FKBPs,also known as neuroimmunophilin ligand,is neuritotrophic,neuroprotective or neuroregenerative [12].FK506,a potent immunosuppressant,showed neuroprotective effect in experimental models of cerebral ischemia and also inhibited microglial activation and peripheral immune response [13].In addition,the specific FKBP38 inhibitor has potent neuroprotective and neurotrophic properties against cerebral ischemia [14].However,their development is limited by the powerful immunosuppressant activity.Therefore,novel non-immunosuppressive FKBP ligands obviously are in urgent need of development.

HD5-6 is a potent,nonimmunosuppressive neurotrophic agent

in vitro

[15].The present study determined the protective effect of HD5-6 in the experimental cerebral ischemia models and investigated whether the resulting neuroprotection involved the anti-oxidation and/or the regulation of neurotrophic factors.

2 Materials and methods

2.1 Chemicals and drugs

HD5-6 (ee＞99%) was provided by the Laboratory of Computer-Aided Drug Design &Discovery,Academy of Military Medical Sciences(Beijing,China).Trichloroacetic acid and chloral hydrate were purchased from Sinopharm Chemical Reagent Co.,Ltd.(Shenyang,China).Silicone 732 is the product of Dow Corning (USA).2-thiobarbituric acid (TBA) was obtained from SIGMA.2,3,5

-

Triphenyl tetrazolium chloride(TTC) was obtained from Tianjin Bodi Chemical Technology Co.,Ltd.VEGF antibody was purchased from Wuhan Boster Biotechnology Co.,Ltd.Rabbit Streptavidin-Peroxidase (SP) and DAB Kit were bought from Beijing Zhongshan Gold Bridge Biotechnology Co.,Ltd.

2.2 Animals

The subjects are male Kunming adult mice and male Sprague-Dawley rats which were obtained from Animal Center of Shenyang Pharmaceutical University.The weight of Kunming mouse or the rat ranges from 27 to 33 g and 220 to 300 g,respectively.They were group-housed under standard environmental conditions (22±1 ℃,humidity 60±5%,12 h light:12 h dark cycle) with free access to standard commercial diet and water

ad libitum

,and were allowed at least one week of acclimatization before application.This animal experiment was conducted in accordance with the animal experiment rules and the Guidelines for Care and Use of Laboratory Animals of Shenyang Pharmaceutical University.The protocol followed the rules of the local Animal Ethics Committee.

2.3 Acute global ischemia in mice

2.3.1 Induction of incomplete global cerebral ischemia in mice and pharmacological intervention

The mice were randomly divided into groups of sham

(

n

=10),ischemia

(

n

=12),and ischemia plus HD5-6 treated

(

n

=12).Before ischemia was induced,the mice were intragastrically and daily administered(7.5,15 and 30 mg/kg body weight) with compound solution (suspended in 0.7% CMC-Na) for 3 d.The surgery of bilateral common carotid artery occlusion plus hypotension (BCAOH) was performed according to reference [16] 1 h after administration on the last day.After the mean arterial blood pressure was reduced to 30% of total blood volume by eye venous exsanguinations [17],the mice were placed on an isothermic customized operating table with their necks extended.Their skin was cut with a midline vertical incision and the underlying submandibular gland was bluntly dissected in the midline to produce left and right lobes.The omohyoid muscle was separated and the right sternocleidomastoid muscle was dissected to expose the common carotid artery (CCA),from which the vagus nerve was separated.The CCA was then ligated at both ends with two sutures.The same treatment was performed on the second CCA.When the last CCA was ligated,the timer was started to record the time of bilateral common carotid artery occlusion.Thereafter,each CCA was cut at the midpoint between the two sutures to ensure complete ligation [18].Their skin was closed with clinical sutures and the animals were allowed to recover in pre-warmed cages.Sham control was treated with the same volume of vehicle and underwent similar surgical procedures without BCAOH.The survival time of each mouse that died of cerebral ischemia within 6 h was recorded.The animal protocol was approved by Shenyang Pharmaceutical University Animal Care and Use Committee.The experiments were in accordance with the Guidelines for Care and Use of Laboratory Animals.

2.3.2 Neurological evaluation of the global ischemia

After global cerebral ischemia surgery,neurological deficits were tested in the mice according to the following scoring table (Table 1),which was established on the basis of both the previous laboratory work and available literature [16].An investigator blinded to the experimental groups conducted the behavioral assessment.Measurement at T6 h after surgery showed that deficits in mice were different,mainly including flexion of forelimbs,head moving more than 10 degrees from the vertical axis,and disabled limbs in placing test.

Table 1 Modified neurological severity score in the behavioral test of mice with BCAOH-induced ischemia

(to be continued)

Continued table 1

2.3.3 Determination of lipid peroxidation in mouse brain

MDA was used as the indicator of lipid peroxidation in this study.After the 6 h ischemia,brain tissues were removed and the level of MDA was determined by general spectrophotometry.Brains were homogenized in normal standard saline.Then,1.2 milliliters of homogenate was incubated at 37 ℃ in HH-S11.2 type electric water bath pot(Jiangsu Dongtai Electrical Appliance Factory,China) and stirred slightly for 1 h.After that,0.6 mL of 20% trichloroacetic acid was mixed with them in a TDL80-2B centrifuge (Shanghai Anting Scientific Instrument Factory,China).The supernatant was collected and mixed with 0.6 mL of 0.67%2-thiobarbituric acid,and boiled at 100 ℃ for 10 min.MDA was colorimetrically determined at 532 nm with a spectrophotometer (Shanghai Analytical Instrument Factory,China).Level of MDA in the tested samples was calculated against the standard and expressed as nmol/g wet tissue.

2.4 Induction of focal ischemia with MCAO

The surgery was performed according to the method of intraluminal vascular occlusion described in reference [19] with slight modification.The animals were intraperitoneally (i.p.) anesthetized with chloral hydrate (400 mg/kg in mice and 400 mg/kg in rats).During the operation,the body temperature was kept at 37 ± 0.5 ℃ by means of a heating pad.The right common carotid artery(CCA) was exposed through the midline incision of the neck.The muscles around the neck were further separated to expose external carotid artery(ECA) and internal carotid artery (ICA).A filament(Kumyang company,Shenyang),one end of which was wound with Silicone 732 in advance,was introduced into ECA through a small nick and pushed gently through ICA to block the origin of middle cerebral artery (MCA).Once the thread was in place,the timer was started to record the time of occlusion.Sham-operated control group was subjected to the same surgical procedure and the thread was inserted without occluding MCA.

2.4.1 Drug treatment and experimental protocol in mice

The mice were randomly divided into groups of sham,MCAO ischemia,and ischemia plus HD5-6 (1.4,9.8 and 19.6 mg/kg,i.g.) (

n

=8 in each group).Except for the sham group,the other groups underwent the MCAO surgery as described above.Mice in sham control group and MCAO ischemia group were intragastrically given the same volume of vehicle (0.5% CMC-Na),while those in other groups were treated with HD5-6 solution (suspended in 0.5% CMC-Na).After MCAO operation,the mice were treated with drugs T2.5 h and T7.5 h (Fig.1).

2.4.2 Drug treatment and experimental protocol in rats

The rats were randomly divided into groups of sham

(

n

=10),MCAO ischemia

(

n

=10),and ischemia plus HD5-6 (1,7 and 14 mg/kg,i.g.or i.v.) (i.g.:

n

=10 in 1 and 7 mg/kg HD5-6 group;

n

=11 in 14 mg/kg HD5-6 group;i.v.:

n

=11 in 1 and 7 mg/kg HD5-6 group;

n

=10 in 14 mg/kg HD5-6 group).All groups except the group treated with sham underwent MCAO as described above.Rats in sham control group and MCAO ischemia group were intragastrically or intravenously given the same volume of vehicle (0.5% CMC-Na or 5%DMSO),while those in other groups were treated with compound solution (suspended in 0.5% CMCNa for intragastric routes and in 5% DMSO saline solution for intravenous route).After MCAO,the rats were treated with drugs at T2.5 h and T7.5 h(Fig.1).

Fig.1 Experiment protocol.Animals are subjected to MCAO according to the method of intraluminal vascular occlusion described in literature.After MCAO,the ischemic animals were administrated with tested drugs at T2.5 h and T7.5 h.Neurological deficits are determined at T24 h

2.4.3 Neurological evaluation of the focal ischemia

Neurological deficits were measured among all animals according to a five-point scale [19] at T24 h after MCAO surgery (Table 2).An investigator who is not privy to the experimental groups assessed behaviors of the animals.If the mouse scored 1-3,it would be used in the subsequent experiment.Dead mice and mice with the score of 0 or 4 were excluded from statistical analysis.

Table 2 Neurological severity score in the behavioral test of animals with MCAO-induced ischemia

2.5 Measurement of infarct volume

Rats were killed at T24 h after MCAO and their brains were taken out to check macroscopically whether the suture was in the appropriate place.Brains with the suture in the right place were used to detect infarct volume.Each brain was sectioned into five coronal slices with a rat brain matrix (Beijing Pharmaceutical Research Service Center,Beijing)and stained with 2% TTC in dark at 37 ℃ for 10 min [20].The slices were scanned and the infarct areas were analyzed using IPP software (Image-Pro Plus 6.0,Media Cybernetics Co.).The percentage of infarct volume was calculated according to the following equation:infarct volume%=(contralateral volume -ipsilateral non-ischemic volume)/contralateral volume × 100%.

2.6 Immunohistochemical staining of BDNF and VEGF

After neurological evaluation,the mice were sacrificed for immunohistochemistry.They were intracardially perfused with PBS and 4%paraformaldehyde before their brains were removed from the skull.The excised brains were fixed in 4%paraformaldehyde overnight,and then immersed in 20% and 30% sucrose solution at 4 ℃ to be dehydrated.The snapped frozen brain tissues were cryo-sectioned to a 15

μ

m thickness.The brain slices were balanced at room temperature for 30 min and washed with 0.01 M PBS for 15 min.Then they were repaired with 0.01 M citric acid buffer for 20 min.Afterward,they were put into 0.3% HO-methanol solution for 20 min and washed with 0.01 M PBS for 15 min.To identify the expression level of BDNF or VEGF,the tissues were probed with anti-BDNF antibody or anti-VEGF antibody (Wuhan Boster Biotechnology Co.,Ltd).After incubation with the specific antibody,DAB chromogenic agent was added to the incubation solution containing the brain slices.The slices were then washed with distilled water and dehydrated with ethanol and dimethylbenzene.Finally,they were sealed with neutral gum.Brownyellow granules in cytoplasm or nuclei were recognized as immunohistochemical positive staining of BDNF or VEGF.Slices were evaluated by detecting the optical density (OD).Five randomly selected areas on each slice were observed under an optical microscope.All measurements were made using the image analyzer Image-Pro Plus 6.0.Positive staining was expressed as density per unit area.

2.7 Statistical analysis

Data were expressed as means ± SD.Differences among groups were tested by oneway ANOVA.LSD was used to test homogeneity of variance and Dunnett’s T3 was used to test heteroscedastic variance,after estimating whether the variance of data was homogeneous with the homogeneity test of variance.All statistical calculation and analysis were completed with SPSS 19.0.Statistical significance was defined as

P

＜ 0.05.

3 Results

3.1 Suppression of HD5-6 on global ischemia induced by BCAOH in mice

The survival time of each mouse and neurological score was recorded.As shown in Fig.2-A,BCAOH (vehicle) could shorten the survival time.There was no significant statistical difference in survival time between the vehicle group and the HD5-6 treated groups.Survival time varied greatly among different groups of mice after BCAOH.The standard deviations of survival time in each group were large.Neurological scores showed that mice with ischemia had neurological and functional deficits due to ischemic insult (Fig.2-B,

P

＜ 0.01).Mice treated with HD5-6 (30 mg/kg) showed signs of improvement in the behavioral test (Fig.2-B,*

P

＜ 0.05).After 6-h ischemia,brains were dissected in an ice bath to determine the levels of MDA.As shown in Fig.2-C,compared with the group of control,BCAOH significantly increased the level of MDA(

P

＜ 0.05).Compound treatment dose-dependently inhibited the ischemia-induced increase of lipid peroxidation (compared to the ischemia group,*

P

＜ 0.05 and **

P

＜ 0.01,Fig.2-C).

Fig.2 Effects of HD5-6 on survival time,neurological deficits and the level of MDA in the brains of the mice subjected to incomplete global ischemia induced by BCAOH.(A) Survival time;(B) Neurological deficit scores;(C)MDA content in brain tissues.Data were expressed as mean ± SD (n =10 in control group; n =12 in vehicle group and the HD5-6 treated group).Values were significant between the control group and the ischemia (#P ＜ 0.05 and##P ＜ 0.01) and between the ischemia+HD5-6 and the ischemia (*P ＜ 0.05 and **P ＜ 0.01).Differences among the groups were analyzed by one-way ANOVA,followed by LSD or Dunnett’s T3 with SPSS 19.0

3.2 Suppression of HD5-6 on focal ischemia induced by MCAO in mice

3.2.1 HD5-6 reduces neurological deficit

At T24 h after ischemia induced by MCAO,the neurological function score of mice treated with 19.6 mg/kg of HD5-6 was significantly decreased(*

P

＜ 0.05,Fig.3),compared with that of the model group (vehicle) mice.

Fig.3 Effect of HD5-6 (1.4,9.8 and 19.6 mg/kg,i.g.) on neurological deficit scores of mice at T24 h after MCAO.*P ＜ 0.05 represents significant difference between the drug treatment group and the vehicle group.Differences among the groups were analyzed by one-way ANOVA,followed by LSD or Dunnett’s T3 with SPSS 19.0.The short bar represents mean ± SD.n =8 in each group

3.2.2 HD5-6 increases the expression of BDNF in brain areas injured

As shown in Fig.4,the results of immumohistochemical staining indicated that HD5-6 (1.4,9.8 and 19.6 mg/kg) dose-dependently increased the level of BDNF expression in striatum,cortex and hippocampus of ipsilateral hemisphere at T24 h after ischemia stroke,while moderate to high doses (9.8 and 19.6 mg/kg) of the drug significantly increased the levels of the factor in the nucleus (Fig.4-B).

Fig.4 Immunohistochemical analysis of BDNF expression in hippocampus,cortex and striatum of ipsilateral hemisphere at T24 h after ischemia. (A) Immunohistochemistry of BDNF in hippocampus,cortex and striatum.Images represent eight mice in each group and the magnification is 200 ×;(B) The histogram below represents the quantification of the BDNF expression.Slices were evaluated by detecting the optical density.Five randomly selected areas on each slice were observed under an optical microscope.All measurements were made using the image analyzer Image-Pro Plus 6.0.The positive staining was expressed as density per unit area.*P ＜ 0.05,** P ＜0.01,*** P ＜ 0.001 compared with the vehicle group

(to be continued)

Continued fig.4

3.2.3 HD5-6 decreases the expression of VEGF in brain areas injured

As shown in Fig.5,the results of immumohistochemical staining indicated that the treatment with HD5-6 (1.4,9.8 and 19.6 mg/kg) lowered the levels of VEGF expression in cortex and striatum of ipsilateral hemisphere at T24 h after ischemia stroke,while high dose (19.6 mg/kg) of the drug significantly decreased the level of the factor in hippocampus nucleus.

Fig.5 Immunohistochemical analysis of VEGF expression in hippocampus,cortex and striatum of ipsilateral hemisphere at T24 h after ischemia. (A) Immunohistochemistry of VEGF in hippocampus,cortex and striatum.Images represent eight mice per group and magnification is 200 ×;(B) The histogram below represents the quantification of VEGF expression.Slices were evaluated by detecting the optical density.Five randomly selected areas on each slice were observed under an optical microscope.All measurements were made using the image analyzer Image-Pro Plus 6.0.The positive staining was expressed as density per unit area.** P ＜ 0.01 compared to the vehicle group

(to be continued)

Continued fig.5

3.3 HD5-6 decreases infarct volume of rats with focal ischemia

High dose of HD5-6 (14 mg/kg),given through intragastric (i.g.) route,significantly reduced the infarction volume of rats at T24 h after MCAO(Fig.6),while moderate to high doses (7 and 14 mg/kg),given through intravenous (i.v.) route,were significant (Fig.7).

Fig.6 HD5-6 (1,7 and 14 mg/kg),given through intragastric (i.g.) route,dose-dependently reduced the infarction volume of rats at T24 h after MCAO.(A) Representative cerebral infarct areas (in white) were determined by TTC staining;(B) Histogram represents quantification of the cerebral infarct volume (the percentage of infarct volume).The infarct areas (white part) were analyzed using IPP software and the percentage of infarct volume was calculated.Data were expressed as mean ± SD.n =10 in control group,in vehicle group and in 1 and 7 mg/kg HD5-6 group;n=11 in 14 mg/kg HD5-6 group.* P ＜ 0.05 represents significant difference between drug treatment and the vehicle.Differences among the groups were analyzed by one-way ANOVA,followed by LSD or Dunnett’s T3 with SPSS 19.0

Fig.7 HD5-6 (1,7 and 14 mg/kg),given through intravenous (i.v.) route,dose-dependently reduced the infarction volume of rats at T24 h after MCAO.(A) Representative cerebral infarct areas (in white) were determined by TTC staining;(B) Histogram represents the quantification of cerebral infarct volume (the percentage of infarct volume).Infarct areas (white part) were analyzed using IPP software and the percentage of infarct volume was calculated.Data were expressed as mean ± SD.n=10 in control group,in vehicle group and in 14 mg/kg HD5-6 group,respectively;n =11 in 1 and 7 mg/kg HD5-6 group.* P ＜ 0.05 and ** P ＜ 0.01 represent significant difference between drug treatment and the vehicle.Differences among the groups were analyzed by one-way ANOVA,followed by LSD or Dunnett’s T3 with SPSS 19.0

4 Discussion

In the present study,the protective effect of HD5-6 in two animal ischemia models,MCAO and BCAOH,was investigated.We have used the models to mimic and analyze the damage caused by ischemic stroke [16,18,21].In the global ischemia model induced by BCAOH in mice,HD5-6 improved neurological function and significantly reduced the level of MDA.MDA has been interpreted as an index to evaluate lipid peroxidation,and determination of MDA in tissue or serum is critical to assess the anti-ischemic effect of the tested drugs because the level of MDA in tissue or serum can indirectly reflect the information of oxidant,anti-oxidant and the degree of tissue damage due to peroxidation [22-24].Previous studies have shown that FK506 can inhibit the accumulation of lipid peroxidation products and oxidative DNA damage,indicating that the neuroprotection of FK506 from ischemia-reperfusion injury is achieved through antioxidative pathways [25].Results in the present study showed that HD5-6 dose-dependently restrained the level of MDA,suggesting that the effect of the drug against global cerebral ischemia might involve antioxidative mechanism.

In the MCAO models of mice,it was found that the neurological function of ischemic mice was effectively improved after HD5-6 treatment.Gabryel,B.and Bernacki,J.demonstrated that FK506 and cyclosporine A effectively upregulated BDNF in ischemic astrocytes

in vitro

experiments and showed significant protective effect on rats with transient forebrain ischemia [26].Results of the present study showed that the expression of BDNF in hippocampus,cortex and striatum of ipsilateral hemisphere was upregulated in the mice with MCAO-induced ischemia after HD5-6 treatment.Obviously,BDNF is necessary for neuronal proliferation,survival,and differentiation.Some studies have found that BDNF is involved in neuronal plasticity and synaptic restructuring associated with ischemia.However,endogenous BDNF seems insufficient to overcome barriers and facilitate functional recovery from brain injury [27].Reaching the threshold BDNF level is essential to maintain neuronal plasticity and promote post-stroke recovery [28].Others tried to improve recuperation from brain injury by exogenous BDNF therapy or other methods to increase BDNF [5,29].Results in the present study showed that the protective effect of HD5-6 against cerebral ischemia might be related to the exogenous increase of BDNF.

Besides,it was also found that the expression of VEGF in cortex and striatum of ipsilateral hemisphere of the ischemic mice decreased in a dose-dependent manner after HD5-6 treatment.Traditionally,VEGF is another factor considered to play a critical role in angiogenesis and has direct effect on migration,neuritic outgrowth,axon guidance and survival of neuronal cells [30].However,VEGF is not only a secreted mitogen associated with angiogenesis but also a potent vascular permeability factor [9].Zhang et al.believed that early inhibition of VEGF might have significant potential against cerebral ischemia [10].In the present study,HD5-6 treatment at 2.5 h and 7.5 h after MCAO was found to inhibit the expression of VEGF,indicating that the inhibition of VEGF expression might be involved in the neuroprotection of HD5-6 against cerebral ischemic injury.

FK506 inhibits the function of the immunophilins and plays neuroprotective and neuroregenerative roles in a variety of experimental paradigms including experimental models of stroke,Parkinson’s disease,neuropathies,and other neurologic disorders [31].However,if it is used as a neuroprotective agent,the immunosuppressive effects cause lots of trouble.HD5-6,a derived compound of the novel ligand of FKBP (FK506-binding protein),has few immunosuppressive effects.It is one of the compounds with similar mother structure to FK506 and is the FKBP12-binding domain analogue of FK506.The purpose of designing this series of compounds was to discard the immunosuppressant adverse effects of FKBP when used as neuroprotective agents.Their non-immunosuppressions have been assured in the previous study [15].The present study proved that HD5-6 protected ischemic animals from brain damage,which might be due to the basic pharmacological properties of neuroprotection,which are also mentioned in other studies [32,33].Besides,the mechanisms of the actions are largely related to promoting angiogenesis and/or neural regeneration,involving the increase of some factors such as BDNF and VEGF,and inhibiting oxidative stress as well [34,35].

In this study,rats with MCAO-induced ischemia were given HD5-6 through intragastric and intravenous routes.Results showed that,despite similar therapeutic results,HD5-6 given through intravenous routes was more effective than that through oral route in reducing the infarction volume.

In conclusion,it is confirmed that HD5-6 has protective effects against global and focal cerebral ischemia in rodents.The compound can effectively improve the neurological deficit,lengthen the lifetime,reduce the infarction volume,decrease the level of MDA,increase the BDNF expression and inhibit early VEGF expression in the ischemia animals.All these results suggest that HD5-6 has the potential to treat cerebral ischemia.The underlying action mechanism may involve anti-oxidation and neurogenesis,which needs to be studied further.

Acknowledgments

This work is supported by a grant from the national science and technology major project,a candidate drug for neurodegenerative diseases targeting FKBPs,(2009ZX09103-024).