Effect of Zhutan Tongluo Tang on fibrinolytic activity following intracerebral hemorrhage in rats＊★

Yongxi Jin, Xu Li, Gaowen Li, Lei Guo, Fang Li, Guoying Liu, Songfang Chen, Shijue Liu

1Department of Rehabilitation, Wenzhou Municipal Hospital of Traditional Chinese Medicine, Wenzhou 325005, Zhejiang Province, China

2Department of Physiology, Wenzhou Medical College, Wenzhou 325035, Zhejiang Province, China

3Department of Pharmacology, Ningbo TianYi Vocational and Technical College, Ningbo 315100, Zhejiang Province, China

4Department of Urology, Central Hospital of Xinyang, Xinyang 464000, Henan Province, China

5The Second Affiliated Hospital of Wenzhou Medical College, Wenzhou 325027, Zhejiang Province, China

lNTRODUCTlON

The fibrinolytic system has been shown to reduce hematomas[1-2]. It has been reported that there is a significant reduction of fibrinolytic system activities at the early stages of intracerebral hemorrhage (ICH)[3-6]. In addition to the initial hemorrhagic impact,secondary brain injury also contributes to poor clinical outcomes following ICH, and apoptosis contributes to the injury[7-8].

In our previous studies, Zhutan Tongluo Tang(ZTTLT) had protective effects on focal cerebral ischemia[9], and its effect was involved in fibrinolysis[10]. Moreover, ZTTLT has been proven to protect against ICH[11]. However, it is unknown whether this effect is involved in the function of the fibrinolytic system and its neuroprotective effect. The present study was designed to elucidate how ZTTLT ameliorates fibrinolytic activity in rats subjected to ICH, and to determine its neuroprotective effect.

RESULTS

Quantitative analysis of experimental animals

A total of 144 adult, male, Sprague-Dawley rats were randomly divided into three groups(n = 48): sham-surgery, model, and ZTTLT groups. Each group was assigned into four subgroups according to the intervention time-points (1, 2, 3, 7 days, n = 8). All the animals were included in the behavioral tests (forelimb placing test and corner turning test). Furthermore, six rats in each group were utilized for collection of plasma and brain tissues for assays to determine the concentrations of tissue plasminogen activator (t-PA), plasminogen activator inhibitor-1 (PAI-1), D-Dimer, catalase (CAT), and glutathione (GSH). The remaining six rats were perfused with formaldehyde to determine the expression of Bcl-2. All 144 rats were included in the final analysis. GSH,CAT, and Bcl-2 were only observed on days 3 and 7 according to a previously described method[12].

ZTTLT alleviated neural function and behavioral impairment in lCH rats

Forelimb placing test and corner turning test were performed to assess behavioral function. Rats in the model group exhibited a serious neural functional disorder on day 1 in both tests (Table 1). The percentage of forelimb placing at all time points significantly decreased, compared with the sham-surgery group (P ＜ 0.01). In the corner turning test, the percentage of animals turning right at all time points increased compared with the sham-surgery group (P ＜ 0.01).

Rats treated with ZTTLT had increased level of activity in the forelimb placing test and a decreased level of activity in the corner turning test, compared with the model group(P ＜ 0.01).

Table 1 Effects on behaviors after intracerebral hemorrhage (%)

ZTTLT activated the fibrinolytic system in rats subjected to lCH

Plasma t-PA, PAI-1, and D-Dimer were analyzed by enzyme linked immunosorbent assay (ELISA) to evaluate the activity of the fibrinolytic system. The plasma concentration of t-PA and D-Dimer in the model group rats gradually increased, while the PAI-2 concentration initially increased and then decreased. As shown in Table 2, the concentration of t-PA increased slowly in the model group from the 1stday, and there were significant differences compared with the sham-surgery group at days 3 and 7(P ＜ 0.01). The levels sharply increased when treated with ZTTLT, compared with the model group from the 2ndday to the 7thday (P ＜ 0.05 or P ＜ 0.01). In addition, the concentration of PAI-1 in the model group initially increased, and then a slowly decreased; reaching a minimum on day 7.

After drug therapy, the concentration of PAI-1 significantly decreased compared with the model group, and there were significant differences on days 3 and 7 (P ＜ 0.05 or P＜ 0.01; Table 2). The change in D-Dimer resembled the changes seen with t-PA (Table 2).

Antioxidant effect of ZTTLT

The whole brain GSH and CAT levels were analyzed to investigate the antioxidant action of ZTTLT on days 3 and 7. Results showed that both the level of GSH and the activity of CAT in model rats significantly decreased compared with the sham-surgery group (P ＜ 0.01).ZTTLT reversed these decreases on days 3 and 7(P ＜ 0.05, P ＜ 0.01; Table 3).

Table 2 Effect of ZTTLT on the concentration of peripheral plasma t-PA, PAI-1, and D-Dimer after intracerebral hemorrhage (ng/mL)

Table 3 Effect of ZTTLT on the levels of GSH (mg/kg) and CAT (U/g)

ZTTLT increased Bcl-2 expression in various regions of the rat hippocampus

The pyramidal layer and the dentate gyrus in the hippocampus of sham-surgery rats showed stronger Bcl-2 expression (Figure 1), while less expression was seen in the model rats, both in the CA1 and CA3 regions,and the dentate gyrus. This effect could be reversed(Table 4, Figure 1).

Figure 1 Effect of Zhutan Tongluo Tang on Bcl-2 expression in various regions of the hippocampus after intracerebral hemorrhage (immunohistochemistry). Red arrow indicates positive Bcl-2 expression. The sham-surgery group displays normally stained Bcl-2 expression (A: day 3,B: day 7), while the model group has a significant decrease in Bcl-2 on days 3 (C) and 7 (D) (brown-stained granules are decreased). Following treatment with Zhutan Tongluo Tang there is increased expression of Bcl-2 on days 3 (E) and 7(F). Scale bar: 100 μm.

ZTTLT significantly increased the expression of Bcl-2,both in the CA1 region and the dentate gyrus (P ＜ 0.01).

Table 4 Effect of ZTTLT on Bcl-2 expression in various regions of the hippocampus after intracerebral hemorrhage(%)

DlSCUSSlON

ZTTLT consists of several Chinese herbal components,which have been proven to show protection against ICH.For example, rheinic acid was shown to prevent apoptosis in the brain[13]. Rhubarb and turmeric extracts have been shown to be effective against ischemic injury by reducing the reactive oxygen species after ICH[14-16].

Gastrodin was reported to be therapeutic against cerebral edema[17]. ZTTLT has been shown to be effective against ICH by reversing excessive inflammation[11]. In this compound recipe, rhubarb may be used as the principal component to activate blood flow and resolve phlegm, while the other components can produce an additive effect.

Previous studies have shown that collagenase-induced ICH in rats is a highly reproducible model for the study of the pathophysiology of intracranial hematoma, and can be used to evaluate different therapeutic approaches to assess treatments for the alleviation of neurological deficits following ICH[18-19]. Here we replicated the same disabling behaviors described by Peeling[20]using the collagenase-induced ICH model. Following treatment with ZTTLT, the disabilities were significantly alleviated,which suggests that the rats subjected to ICH and ZTTLT had reduced hematoma-induced parenchymatous impairment after ICH.

In the present study, both plasma t-PA and D-Dimer levels in the model group were higher than that in the sham-surgery group. This was especially the case with t-PA, which suggests that there is an enhancement of the fibrinolytic system, but this enhancement is slow. Some researchers have suggested that this result is due to stress and an increase in synthesis of t-PA in the liver[21].

Results showed that the activity of the fibrinolytic system was augmented from the 2ndor 3rdday after ICH, and these changes were more pronounced after drug treatment. This suggests that ZTTLT can accelerate the activation of the fibrinolytic system to degrade the blood clot and absorb the hematoma.

Apoptosis can occur after ICH, and the peak time is typically from the 3rdto the 7thday[12]. Excessive oxidative stress initiates activation of apoptosis mediators, such as caspase-3[22]. The injury induced by oxygen radicals in vivo is responsible for the activation of apoptosis[23]. In the present study, lower expression of Bcl-2 was found in various subhippocampal regions in the model rats. The expression of Bcl-2 in CA1, CA3, and the dentate gyrus was significantly decreased; especially at day 3. ZTTLT significantly increased the expression of Bcl-2 in the CA1 region and the dentate gyrus on days 3 and 7. ZTTLT also increased the levels of both GSH and the activity of ACT in the brains on days 3 and 7. These findings suggest that ZTTLT has a neuroprotective effect through its antioxidant and apoptosis inhibitory properties, which may protect against secondary brain injury after ICH.

In conclusion, ZTTLT activated the fibrinolytic system to absorb the hematoma, and had a neuroprotective effect via antioxidant and apoptosis inhibitory actions. Currently,it is unclear if these findings can be extended to human subjects. Therefore, further studies are needed to determine the effects on humans following ICH.

MATERlALS AND METHODS

Design

A randomized, controlled, animal experiment.

Time and setting

The experiments were performed at the Institute of Experimental Neurobiology, Wenzhou Medical College,China from September 2009 to March 2010.

Materials

ZTTLT contains a series of Chinese herbs, such as Rhubarb (6 g), Turmeric (12 g), Arisaema Cum Bile (6 g),Concretio Silicea Bambusae (6 g), Acorus Tatarinowii (6 g),Lumbricus (15 g), Scorpio (15 g) and Rhizoma Gastrodiae (10 g). These raw materials were obtained from the Second Affiliated Hospital of Wenzhou Medical College, China, and stored in an environment of normal atmospheric pressure and decoction at 100°C for 30 minutes, and then the residues were discarded. Following filtration through an 8-layer gauze, the final decoction concentration was fixed at 1.25 g/mL. The decoction was stored at 4°C.

A total of 144 male Sprague Dawley rats (8-12 weeks old, 250-280 g) were bred, and 4-5 rats per cage were maintained at a 12 hour light/dark cycle at ambient temperature (22°C) and relative humidity (55 ± 5%).

Rats were obtained from the Experimental Animals Center of Wenzhou Medical College, China, with the license No. SYSXK (Zhejiang) 2005-006. All experiments were performed according to the National Institutes of Health Guide for the Care and Use of Laboratory Animals[24]and the Guidelines for the Care and Use of Animals in Neuroscience Research by the Society for Neuroscience, and approved by the Institutional Animal Care and Use Committee of Wenzhou Medical College,China.

Methods

Establishment of ICH models

As described previously, the ICH model was established by injecting collagenase (Sigma, St. Louis, MO, USA)into the hemi-caudate nucleus. Rats were intraperitoneally anesthetized with a dose of 10% chloral hydrate(0.1 mL/100 g). Then they were placed in a positioner(Stoelting Co., IL, USA), and a small visual field was cut over the head (supplementary Figure 1 online). The caudate nucleus was located (bregma caudal 0.5 mm,sagittal right 3 mm, depth 6 mm), and slowly injected with collagenase, 1.2 U in 5 minutes; retaining the needle for 5 minutes. Rats in the sham-surgery group were subjected to surgery as mentioned above, but received the same volume of saline injection, instead of collagenase[25]. Functional behaviors were used to assess whether the model was duplicated successfully[26].

Drug treatment

A total of 144 rats were randomly divided into three groups (n = 48): sham-surgery (injected with the same volume of plain water in the hemi-caudate nucleus),model (injected with collagenase in the hemi-caudate nucleus), and drug treated (in addition to the same operation with model rats; ZTTLT was administered).

Each group was assigned into four subgroups according to the time-points (1, 2, 3, and 7 days). Rats in the drug group were administered ZTTLT (2 g/mL), and plain water was given to the animals in the sham-surgery and model groups. Administration began 2 hours before tests or sacrifice after the model was replicated, and treatment lasted for 1, 2, 3 and 7 days, respectively.

Observation of behaviors

For analysis of corner turning, rats were put into a corner area with a slope of 30°, and frequencies were recorded respectively as rats turned left or right in the corner.

Experiments were duplicated 10-15 times, with an interval of 30 seconds. Only entire turning was recorded,and the percentage of times the animals turned to the right was calculated[27].

For forelimb placing, the rat body was held in position to allow the forelimb to be suspended, and moved up and down to relax the muscles. The rat’s whiskers were scraped with the corner of a desk. In this paradigm, the control rats would rapidly put their ipsilateral forelimb on the desk, while rats subjected to ICH would use the forelimb opposite of the hemorrhage site[28]. Each rat was tested 10 times and the percentage of successful trials was calculated.

Plasma t-PA, PAI-1 and D-Dimer concentrations detected by ELISA

The t-PA, PAI-1, and D-Dimer kits were purchased from R&D Systems (Minneapolis, USA). Detection of plasma t-PA concentration was as follows: heparinized blood from six rats in each group was collected and then centrifuged at 3 000 r/min for 10 minutes. The supernatant was collected, and then stored at -80°C.Rabbit anti-rat t-PA antibody (Boster, Wuhan, China) was added, 0.1 mL/well in the empty wells. A total of 1 000,500, 250, 125, 62.5, 31.3, and 15.6 ng/mL (sample dilution of an equal volume served as a zero standard)standard preparations (each 0.1mL) were added sequentially to a 96-well plate. Subsequently, 0.1 mL plasma sample was added. The samples were incubated at 37°C for 120 minutes. The liquid was removed from the plate prior to the addition of biotinylated goat anti-rabbit t-PA antibody (1: 40, 0.1 mL/well) and incubated for 60 minutes at 37°C. The wells were rinsed three times with 0.01 mol/L PBS. An avidin-biotin complex solution (0.1 mL/well) was added and incubated for 30 minutes at 37°C. The wells were then washed five times with 0.01 mol/L PBS for 2 minutes each time.

Subsequently, 3, 3’, 5, 5’-tetramethylbenzidine colored solution (0.1 mL/well) was added for 25 minutes at 37°C,in the dark. The reaction was terminated by the addition of 3, 3’, 5, 5’-tetramethylbenzidine stopping solution(0.1 mL/well). Absorbance values were measured at 492 nm with a plate reader (Bio-Rad, Hercules, CA,USA). The 3, 3’, 5, 5’-tetramethylbenzidine blank wells served as control. Absorbance values were calculated by subtracting the absorbance value of the zero standard from the absorbance values of all the standard curve preparations and samples. Taking concentration of t-PA as the X-axis and concentration of absorbance value as the Y-axis to draw the standard curve, the curve was used to calculate the concentration of t-PA.

All data were expressed as the common logarithm (lg).

Plasma PAI-1 and D-Dimer concentrations were determined similarly.

Bcl-2 expression in the hippocampus as detected by immunohistochemical staining

The detection of Bcl-2 immunoreactivity was performed on free-floating sections using a conventional avidin-biotin-immunoperoxidase technique modified from published protocols[29]. Briefly, sections were pretreated with 0.3% H2O2in methanol for 30 minutes to block endogenous peroxidase activity, followed by washes with PBS. Samples were incubated for 1 hour at room temperature with a blocking solution composed of 5%normal serum, 1% bovine serum albumin and 1% Triton X-100 in PBS. Sections were then incubated with rabbit anti-rat Bcl-2 antibody (Boster, Wuhan, China) at a dilution of 1: 50, in PBS containing 0.5% Triton X-100 and 1% normal serum, for 48 hours at 41°C, followed by 2 hours at room temperature. The primary antibodies were localized using goat anti-rabbit IgG (Boster, Wuhan,China), and the reaction products were developed using 0.025% 3, 3-diaminobenzidine as the chromogen, diluted in Tris-HCl-buffered saline (0.05 mol/L, pH 7.6), and 0.03% H2O2as the substrate. Immunohistochemical controls were performed as above, except for the omission of the primary antibodies. No positive immunostaining was found in the controls. Pictures were obtained by light microscope (Olympus, Tokyo, Japan).

Cell counting was performed by ImagePro Plus 5.0 software (MediaCybernetics, Silver Spring, MD, USA) to count the density of Bcl-2 staining.

Determination of the GSH level and CAT activity

The determinations of GSH and CAT were done according to instructions from assay kits (Jiancheng Bioengineering Institute, Nanjing, China).

Statistical analysis

Data were expressed as mean ± SD and analysis was performed by a SPSS 13.0 software (SPSS, Chicago, IL,USA). One-way analysis of variance, followed by Post-Hoc analysis, was used for significance with the Student-Newman-Keuls multiple comparison test. A P ＜0.05 was regarded as statistically significant.

Author contributions:Shijue Liu designed and executed this study, and also provided funds. Yongxi Jin, Lei Guo, and Gaowen Li performed the experiments and wrote the manuscript. Xu Li obtained and analyzed the data. Fang Li and Songfang Chen revised this manuscript. Guoying Liu participated in the authorization of this paper. All authors read and approved the manuscript.

Conflicts of interest:None declared.

Funding:This work is funded by the Wenzhou Science and Technology Bureau (No.Y20060700).

Ethical approval:The experiments were performed according to the National Institutes of Health Guide for the Care and Use of Laboratory Animals (publication no. 85–23, revised 1985)and the Guidelines for the Care and Use of Animals in Neuroscience Research by the Society for Neuroscience and approved by the Institutional Animal Care and Use Committee of Wenzhou Medical College in China.

Supplementary information:Supplementary data associated with this article can be found, in the online version, by visiting www.nrronline.org, and entering Vol. 6, No. 21, 2011 after selecting the “NRR Current Issue” button on the page.

[1]Qureshi AI, Mendelow AD, Hanley DF, et al. Intracerebral hemorrhage. Lancet. 2009;373(9675):1632-1644.

[2]Rohde V, Rohde I, Thiex R, et al. Fibrinolysis therapy achieved with tissue plasminogen activator and aspiration of the liquefied clot after experimental intracerebral hemorrhage: rapid reduction in hematoma volume but intensification of delayed edema formation. J Neurosurg. 2002;97(4):954-962.

[3]Thiex R, Mayfrank L, Rohde V, et al. The role of endogenous versus exogenous tPA on edema formation in murine ICH. Exp Neurol.2004;189(1):25-32.

[4]Caplan LR. Tissue plasminogen activator for acute ischemic stroke.N Engl J Med. 1999;340(23):1781-1787.

[5]Findlay JM, Jacka MJ. Cohorts study of intraventricular thrombolysis with recombinant tissue plasminogen activator for aneurismal intraventricular hemorrhage. Neurosurgery. 2004;55(3):532-537.

[6]Adams H, Adams R, Del Zoppo G, et al. Guidelines for the early management of patients with ischemic stroke: 2005 guidelines update-a scientific statement from the Stroke Council of the American Heart Association/American Stroke Association. Stroke.2005;36(4):916-923.

[7]Belayer L, Saul I, Curbelo K, et al. Experimental intracerebral hemorrhage in the mouse: histological, behavioral, and hemodynamic characterization of a double-injectiong model. Stroke.2003;34(9):2221-2227.

[8]Qureshi AI, Suri MF, Ostrow PT, et al. Apoptosis as a form of cell death in intracerebral hemorrhage. Neurosurgery. 2003,52(5):1041-1047.

[9]Zhou J, Liu SJ, Wang XT, et al. Protective effect of ZhuTan TongLuo Tang on Focal Cerebral Ischemia in Rats. Zhonghua Zhongyiyao Xuekan. 2009,9(27):1937-1939.

[10]Zhou J, Liu SJ, Zheng GQ, et al. The effect of ZhuTan TongLuo Tang on fibrinolysis system in focal cerebral ischemia in rats.Wenzhou Yixueyuan Xuebao. 2010;40(3):256-258.

[11]Guo L, Jing YX, Liu SJ. Effect of Zhutan Tongluo Tang on the expression of il-1β and il-6 in acute cerebral hemorrhage rat.Zhejiang Zhongyiyao Daxue Xuebao. 2010;1(34):32-34.

[12]Matsushita K, Meng W, Wang X, et al. Evidence for apoptosis after intracerebral hemorrhage in rat striatum. J Cereb Blood Flow Metab.2000;20(2):396-404.

[13]Chen L, Han Z, Hu WH, et al. Effect of rhubarb on neurological injury and apoptosis in ICH rats. Zhongguo Jiceng Yiyao.2010;17(1):26-27.

[14]Li J, Liu J, Wang D, et al. Effects of rhubarb aglycone combined with thrombolysis on brain microvascular basement membrance impairment in rats with thrombus-occluded cerebral ischemia.Zhongguo Zhongyao Zazhi. 2010;35(21):2908-2911.

[15]Gu J, Zhang X, Fei Z, et al. Rhubarb extracts in treating complication of severe cerebral injury. Chin Med J (Engl). 2000;113(6):529-531.

[16]Di JB, Gu ZL. Advances in studies on antioxidant and anti-inflammation of curcumin. Zhong Cao Yao. 2010;41(5):18-21.

[17]Zhang SH, Zhang ZD, Cai ZQ, et al. Observe the therapeutic effect of gastrodin on patients with acute cerebral hemorrhage. Zhongguo yiliao Qianyan. 2010;5(7):54-55.

[18]Xu D, Wen YJ, Zhang LX, et al. An animal model of collagenase-induced intracerebral hemorrhage in mice. Zhongguo Shiyan Dongwu Xuebao. 2006;14(1):36-39.

[19]Wasserman JK, Schlichter LC. Neuron death and inflammation in a rat model of intracerebral hemorrhage:effects of delayed minocycline treatment. Brain Res. 2007;1136(1):208-218.

[20]Lindgren A, Lindoff C, Norrving B, et al. Tissue plasminogen activatorand plasminogen activator inhibitor-1 in stroke patients.Stroke. 1996;27(6):1066-1071.

[21]de Baer JP, Abbink JJ, Brouwer MC, et al. PAI-1 synthesis in the human hepatoma cell line Hep G2 is increased by cytokines-evidence that the liver contributes to acute phase behaviour of PAI-1. Thromb Haemost. 1991;65(2):181-185.

[22]Namura S, Zhu J, Fink K, et al. Activation and cleavage of caspase-3 in apoptosis induced by experimental cerebral ischemia.J Neurosci. 1998;18(10):3659-3668.

[23]Wang J, Zhuang H, Dore S. Heme oxygenase 2 is neuroprotective against intracerebral hemorrhage. Neurobiol Dis. 2006;22(3):473-476.

[24]The Ministry of Science and Technology of the People’s Republic of China. Guidance Suggestions for the Care and Use of Laboratory Animals. 2006-09-30.

[25]Del Bigio MR, Yan HJ, Campbell TM, et al. Effects of fucoidan on collagenase-induced intracerebral hemorrhage in rats. Neurol Res.1999;21(4):415-419.

[26]Hua Y, Schallert T, Keep RF, et al. Behavioral tests after intracerebral hemorrhage in the rat. Stroke. 2002;33(10):2478-2484.

[27]Peeling J, Yan HJ, Corbett D, et al. Effect of FK-506 on inflammation and behavioral outcome following intracerebral hemorrhage in rat. Exp Neurol. 2001;167(2):341-347.

[28]Schallert T, Fleming SM, Leasure JL, et al. CNS plasticity and assessment of forelimb sensorimotor outcome in unilateral rat models of stroke,cortical ablation, parkinsonism and spinal cord injury. Neuropharmacology. 2000;39(5):777-787.

[29]Xu H, Qing H, Lu W, et al. Quetiapine attenuates the immobilization stressinduced decrease of brain-derived neurotrophic factor expression in rat hippocampus. Neurosci Lett. 2002;321(1-2):65-68.