To Optimize the Therapeutic Dose and Time Window of Picroside II in Cerebral Ischemic Injury in Rats by Orthogonal Test

doi:10.4236/nm.2013.43027

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Neuroscience & Medicine, 2013, 4, 166-171

http://dx.doi.org/10.4236/nm.2013.43027 Published Online September 2013 (http://www.scirp.org/journal/nm)

To Optimize the Therapeutic Dose and Time Window of

Picroside II in Cerebral Ischemic Injury in Rats by

Orthogonal Test

Hui Huang1, Li Sun1*, Ling Wang1, Lei Fang1, Li Zhao2, Yan Li2

1Department of Neurology, The Second Affiliated Hospital, Qingdao University Medical College, Qingdao, China; 2Institute of Inte-

grative Medicine, The Second Affiliated Hospital, Qingdao University Medical College, Qingdao, China.

Email: *[email protected]

Received July 4th, 2013; revised August 5th, 2013; accepted August 22nd, 2013

which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

ABSTRACT

The paper aims to optimize the therapeutic dose and time window of picroside II by orthogonal test in cerebral ischemic

injury in rats. The forebrain ischemia models were established by bilateral common carotid artery occlusion (BCCAO)

methods. The successful models were randomly divided into sixteen groups according to orthogonal experimental de-

sign and treated by injecting picroside II intraperitonenally at different ischemic time with different dose. The concen-

trations of neuron-specific enolase (NSE), neuroglial marker protein S100B and myelin basic protein (MBP) in serum

were determined by enzyme linked immunosorbent assay to evaluate the therapeutic effect of picroside II in cerebral

ischemic injury. The results indicated that best therapeutic time window and dose of picroside II in cerebral ischemic

injury were ischemia 1.5 h with 20 mg/kg body weight according to the concentrations of NSE, S100B and MBP in se-

rum. It is concluded that according to the principle of lowest therapeutic dose with longest time window, the optimized

therapeutic dose and time window are injecting picroside II intraperitonenally with 20 mg/kg body weight at ischemia

1.5 h in cerebral ischemic injury in rats.

Keywords: Picroside II; Cerebral Ischemia; Therapeutic Dose; Time Window; Rats

1. Introduction

S100B, a neuroglial marker protein, participates in cell

multiplication, cytoskeleton regulation and other bio-

logical activities. When there are plentiful S100B be-

tween cells, it may promote the expression of inflamma-

tory reaction factors, and induce neuron apoptosis [1].

Clinical study [2] has found that S100B, a neuroglial

marker protein, elevates significantly in the serum of

patients with ischemic stroke, and S100B concentration

is closely related to ischemic stroke type, severity, infarc-

tion volume and mortality [3]. Neuron-specific enolase

(NSE), which exists specifically in the neurons and neu-

roendocrine cells [4], involves in the formation of mem-

brane structures and the repair of brain cells [5]. The

level of NSE in CSF reaches its peak on the third day after

cerebral ischemia in rats, and is positively correlated with

the final infarction volume [6]. The up-regulation of NSE

level in serum lags behind that in brain tissue for 2 hours

in permanent cerebral ischemia model [7,8]. Myelin ba-

sic protein (MBP) is an important myelin structural pro-

tein. It is beneficial to stabilize the structure and function

of the central nerve system (CNS) [9], and its level

change can reflect the severity of the injury in CNS and

myelin damage [10]. The mRNA expression of MBP is a

small amount in normal adult rat brain, and the expres-

sion decreases after cerebral ischemic injury, however, as

ischemic time goes on, the expression of MBP mRNA

increases slowly [11]. Our previous studies have shown

that in rats with cerebral ischemia for 1.5 h, intraperito-

neal injection of picroside II (20 mg/kg) can suppress the

expression of inflammatory cytokines and neuronal apop-

tosis [12-15]. This study attempts to detect the changes

of NSE, S100B and MBP levels in serum to further

explore the optimal therapeutic dose and the time win-

dow of picroside II for treatment of cerebral ischemic

injury.

*Corresponding author.

To Optimize the Therapeutic Dose and Time Window of Picroside

II in Cerebral Ischemic Injury in Rats by Orthogonal Test

167

2. Material and Methods

2.1. Animal Model Establishment

Thirty healthy, male, specific-pathogen free, Wistar rats,

weighting 230 - 250 g, were provided by the Laboratory

Animal Center of Qingdao Drug Inspection Institute

(certificate No. SCXK (Lu) 20100010). Five rats were

selected for the sham control group randomly, and the

remaining 25 were used to establish the model of cere-

bral ischemia. The rats were deprived of food for 12

hours before surgery, and anesthetized by intraperitoneal

injection of 10% chloral hydrate (0.3 ml/kg), and the

bilateral carotid arteries were isolated and occluded to

establish forebrain ischemia model [16]. Body tempera-

ture was monitored using a rectal probe, and was main-

tained at 36˚C - 37˚C using a homeothermic blanket con-

trol unit during surgery. 4 rats were excluded for uncon-

sciousness in two hours after the surgery or death, and

the remaining 21 successful models were included in the

study. The sham control group also had the operation, but

no occlusion of the bilateral carotid arteries were per-

formed.

2.2. Group Design

The 21 successful animal models were randomly divided

into 5 for model group and 16 for treatment group. Ani-

mal models of the treatment group were grouped by four

levels and two factors [L16(45)] orthogonal experimental

design. Therapeutic time window was factor A, and four

levels were set including cerebral ischemia for 1.0 h, 1.5

h, 2.0 h, 2.5 h. Therapeutic dose was factor B, and 5, 10,

20, 40 mg/kg body weight were set as four levels.

2.3. Interventions

Picroside (CAS ⅡNo.: 39012-20-9, purity> 98%) was

provided by the Tianjin Kuiqing pharmaceutical com-

pany, and was diluted to 1% solution with saline solution.

According to [L16(45)] orthogonal experimental design,

appropriate dose of picroside was injected intraⅡperi-

toneally on corresponding ischemic time. Equal amount

of saline were injected at cerebral ischemia 2 h for sham

control group and model group (Table 1).

2.4. ELISA

The rats were anesthetized by intraperitoneal injection of

10% chloral hydrate (0.3 ml/kg) 24 hours after drug ad-

ministration. 4 ml blood was taken through the heart and

centrifuged at 4000 r/min for 10 minutes, and the serum

were separated. And then the levels of NSE (E02N0025),

S100B (E02S0042), MBP (E02M0034) were measured

in serum by the enzyme-linked immunosorbent assay kit

(Blue Gene biotech company). The ELISA plate coated

Table 1. [L16(45)] orthogonal experimental design.

Therapeutic

dose

Ischemia

1.0 h (A1)

Ischemia

1.5 h (A2)

Ischemia

2.0 h (A3)

Ischemia

2.5 h (A4)

5 mg/kg (B1)1.0 × 5 1.5 × 5 2.0 × 5 2.5 × 5

10 mg/kg (B2)1.0 × 10 1.5 × 10 2.0 × 10 2.5 × 10

20 mg/kg (B3)1.0 × 20 1.5 × 20 2.0 × 20 2.5 × 20

40 mg/kg (B4)1.0 × 40 1.5 × 40 2.0 × 40 2.5 × 40

with anti NSE, S100 and MBP specific antibody was

used, 100 μl standard solution was added in the blank

micropores according to the order of the samples, 100 μl

samples were added in the blank micropores, and 100 μl

distilled water was added in the blank control, and 50 μl

enzyme marker solution was added in each hole (the

blank control holes were not included); the enzyme label

plate was sealed with sealing compound, and incubated

for 1 hour at 37˚C. The microtiter plate was washed five

times using distilled or de-ionized water, and dried thor-

oughly with absorbent paper; 50 μl substrate A & B was

added to each well (the blank control holes were not in-

cluded) and incubated for 10 minutes in darkness at 20˚C

- 25˚C, followed by addition of 50 μl stop solution to

each well; The OD value was calculated by an enzyme

monitor (Bio-Rad 680, USA) at 450 nm for each set of

reference standards and samples; the concentration can

be found in the standard curve according to the OD value

of the samples, expressed in ng/ml.

2.5. Statistical Analysis

SPSS 17.0 statistical software was applied to process

statistical analysis, to analyze the contribution of differ-

ent levels of ischemia (drug administration) time and

dose to the test indicators, as well as the interaction of

ischemia time and dose to the test indicators, and comes

to the best dose and the combination of the time window

according to the results.

3. Results

3.1. Test Results

The model animal group had significantly elevated serum

NSE (6.773 ± 0.812), S100B (0.762 ± 0.110) and MBP

(0.675 ± 0.083) levels compared with the sham control

group, in which the serum NSE, S100B and MBP levels

were 2.368 ± 0.532, 0.234 ± 0.051 and 0.227 ± 0.042 (t =

2.79 - 5.97，P < 0.05). And the model animal group af-

ter treatment had more decreased serum NSE (5.613 ±

1.362), S100B

（0.634 ± 0.153）

and MBP (0.305 ± 0.099)

levels than before (t = 2.33 - 3.91, P < 0.05).

Table 2 is an orthogonal experiment of [L16(45)].

To Optimize the Therapeutic Dose and Time Window of Picroside

II in Cerebral Ischemic Injury in Rats by Orthogonal Test

168

Table 2. [L16(45)] orthogonal design and test results.

Rank No. Serum Serum Serum

Test

No. A B C D E NSE S100 MBP

1 1 1 1 1 1 4.784 0.631 0.565

2 1 2 2 2 2 4.976 0.651 0.655

3 1 3 3 3 3 4.494 0.640 0.517

4 1 4 4 4 4 5.074 0.710 0.573

5 2 1 2 3 4 5.268 0.696 0.598

6 2 2 1 4 3 4.122 0.537 0.387

7 2 3 4 1 2 4.076 0.415 0.341

8 2 4 3 2 1 5.385 0.461 0.404

9 3 1 3 4 2 7.086 0.753 0.756

10 3 2 4 3 1 6.396 0.409 0.623

11 3 3 1 2 4 3.290 0.375 0.526

12 3 4 2 1 3 7.478 0.664 0.603

13 4 1 4 2 3 7.319 0.780 0.759

14 4 2 3 1 4 7.523 0.783 0.722

15 4 3 2 4 1 5.433 0.740 0.726

16 4 4 1 3 2 7.098 0.891 0.823

Ⅰ 19.328 24.457 19.294 23.861 21.998 89.802 10.136 9.578

Ⅱ 18.851 23.017 23.155 20.970 23.236

Ⅲ 24.250 17.293 24.488 23.256 23.413

Ⅳ 27.373 25.035 22.865 21.715 21.155

SS 12.544 9.407 3.696 1.343 0.857

Test No. are rat models (No. 1 - 16) and Rank No. are

impact factor’s levels. Data listed in the table of serum

NSE, S100 and MBP are the experimental results of cor-

responding rat models. Data listed in line I, II, III, IV and

SS are the ANOVA result of serum NSE, the ANOVA

results of Serum S100 and MBP are omitted.

3.2. NSE Analysis

Significant difference was showed in the content of NSE

in serum (Table 3) on different levels of administration

time (factor A) and dosage (factor B) (P < 0.05), but the

interaction of administration time and dosage (factor C)

showed no significant difference (P > 0.05). With the

method of least significant difference (LSD) in each two

groups, only in the levels of administration time of 1.0 h

(A1) and 1.5 h (A2), 2.0 h (A3) and 2.5 h (A4), the con-

tent of NSE were not different significantly (P > 0.05),

and other levels of administration time all showed sig-

Table 3. Variance analysis of the content of NSE in serum.

source of variation SS df MS F P

ischemia time 12.5443 4.181 11.410.01

drug dose 9.4073 3.136 8.55 0.01

time × dose 3.6963 1.232 3.36 0.10

deviation 2.1996 0.367

nificant difference (P < 0.05); no significance was found

in administration dose of 5 mg/kg (B1) and 10 mg/kg

(B2), 10 mg/kg (B2) and 40 mg/kg (B4), 5 mg/kg (B1)

and 40 mg/kg (B4) (P > 0.05), and other levels of ad-

ministration dose all showed significant difference (P <

0.05). From the point of view of the maximum therapeu-

tic time window and minimum administration dose,

A2B3 is the optimal combination, in other words, the

best treatment time window and dose were ischemia for

To Optimize the Therapeutic Dose and Time Window of Picroside

II in Cerebral Ischemic Injury in Rats by Orthogonal Test

169

1.5 h, 20 mg/kg, respectively.

3.3. S100B Analysis

Significant difference was showed in the content of

S100B (Table 4) on different levels of factor A (P =

0.05), but the factor B, as well as time-dose interaction

(C), had not taken effect on serum S100B levels after

ischemia significantly (P > 0.05). With the method of

least significant difference (LSD) in each two groups,

only in the levels of administration time of 1.5 h (A2)

and 2.5 h (A4), 2.0 h (A3) and 2.5 h (A4), the content of

NSE were different significant (P < 0.05) , and none of

the other levels of administration time showed any dif-

ference (P > 0.05); significance (P < 0.05) was found in

administration dose of 5 mg/kg (B1) and 20 mg/kg (B3),

and other levels of administration dose showed no dif-

ference (P > 0.05). From the point of view of the maxi-

mum therapeutic time window and minimum administra-

tion dose, A2B3 is the optimal combination, namely, the

best treatment time window and dose were ischemia for

1.5 h, 20 mg/kg, respectively.

3.4. BMP Analysis

Significant difference was showed in the content of BMP

(Table 5) on different levels of factor A (P < 0.05), but

no significance was found between BMP levels after

ischemia, and there were no significant difference in

time-dose interaction (C) (P > 0.05). With the method of

least significant difference (LSD) in each two groups,

only in the levels of administration time of 1.0 h (A1)

and 2.0 h (A3), the content of NSE were not different

significantly (p > 0.05), and other levels of administra-

tion time all showed significant difference (p < 0.05);

Table 4. Variance analysis of the content of S100B in serum.

source of variation SS df MS F P

ischemia time 0.184 3 0.061 5.87 0.03

drug dose 0.075 3 0.025 2.38 0.17

time × dose 0.029 3 0.010 0.93 0.48

deviation 0.063 6 0.010

Table 5. Variance analysis of the content of BMP in serum.

source of

variation SS df MS F P

ischemia time 0.216 3 0.072 15.080.01

drug dose 0.040 3 0.013 2.81 0.13

time × dose 0.013 3 0.004 0.93 0.48

deviation 0.029 6 0.005

significance was found in administration dose of 5 mg/kg

(B1) and 20 mg/kg (B3) (p < 0.05), and other levels of

administration dose showed no difference (p > 0.05).

From the point of view of the maximum therapeutic time

window and minimum administration dose, A2B3 is the

optimal combination, that is, the best treatment time

window and dose were ischemia for 1.5 h, 20 mg/kg,

respectively.

4. Discussion

S100B mainly exists in the astrocytes and Schwann cells

of nervous system. Tracing S100B may advance the ex-

tension of neuron axons and enhance the survival rate of

neurons, while a great deal of it may generate toxic and

side-effect [1]. A large number of activated S100B pro-

tein which is produced by glial cells is released into the

extracellular after brain tissue injury [17], and then leaks

into CSF and blood through damaged blood brain barrier,

and S100B level in serum is positively correlated with

the severity of the injury of cerebral ischemia [18].

Brouns and his group have also found that the concentra-

tion of S100B, which is in the cerebral spinal fluid (CSF)

of the patients with acute ischemic stroke, is highly cor-

related to the severity of the injury and the prognosis of

cerebral ischemia [19]. Under normal circumstances,

NSE content is not high in blood, however, along with

the necrosis of neurons and the disintegration of nerve

myelin, NSE is released into CSF from the cells after

cerebral ischemia injury, and then leaks into blood

through the blood brain barrier. The more severe the

brain tissue is injured, the more nerve cells are damaged,

and the more NSE is released into the blood, therefore,

the detection of changes of NSE levels in CSF or in se-

rum becomes diagnosis markers of neuronal damage [20].

Normal MBP content in CSF is less than 6.95 mg/L,

however, when the ischemic cerebral injury happens,

cerebral ischemia and hypoxia may be due to oligoden-

drocyte death and demyelination. At the same time, MBP

will flow into the CSF and further into blood with the

damage of the blood brain barrier, which is caused by

brain injuries. Otherwise, the increase of MBP synthesis

may be stimulated by stress conditions themselves, in-

cluding ischemia, hypoxia and others [21]. So to a certain

degree, the detection of the serum MBP can show

whether there are brain injuries, and MBP becomes a

specific protein marker of demyelination [22]. Our study

confirmed that the content of MBP in serum of the model

group was significantly higher than that in the sham con-

trol group, and reduced in different degrees after treat-

ment. Our results showed that the contents of NSE,

S100B and MBP in serum of the model group were sig-

nificantly higher compared with that of the sham control

group, and decreased significantly after treatment, sug-

To Optimize the Therapeutic Dose and Time Window of Picroside

II in Cerebral Ischemic Injury in Rats by Orthogonal Test

170

gesting a protective role of picroside II on both ische-

mic brain tissue and blood brain barrier.

This experiment was a [L16(45)] orthogonal experi-

ment design, in which four time points of 1 h, 1.5 h, 2 h

and 2.5 h after brain ischemia were set and four levels of

picroside II dose were given (5 mg/kg, 10 mg/kg, 20 mg/kg

and 40 mg/kg). The expressions of NSE, S100B and

MBP in serum were measured, and the optimal therapeu-

tic dosage and time window of picroside II were analysed.

These results showed that there was a significant differ-

ence for the therapeutic effects of picroside II in admini-

stration time and the dose. From the principle of the low-

est therapeutic dose with the longest time window, A2B3

was the optimal combination and the best treatment time

window and dose were ischemia for 1.5 h and intraperi-

toneal injection of 20 mg/kg. For the mechanism of cere-

bral ischemic injury was very complicated, only four

indexes, as mentioned here, were observed in this study,

which must bring some variances. Therefore, the exact

mechanism and optimal therapeutic time window and

dosage of picroside II should be evaluated combined

with other detection indexes.

5. Acknowledgements

This study was supported by grant-in-aids for The Na-

tional Natural Science Foundation of China (No. 8104

1092, 81274116).

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