1003-1057(2012)2-132-04 synthesis of an active peptide from carapax trionycis and its inhibitory.pdf

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Journal of Chinese Pharmaceutical Sciences http://www.jcps.ac.cn 132

Synthesis of an active peptide from Carapax Trionycis and its inhibitoryeffect on the proliferation of hepatic stellate cells

Chun-Ling Hu, Yin-Ping Tang, Yan-Wen Liu*

School of Pharmacy, Hubei University of Traditional Chinese Medicine, Wuhan 430065, China

Abstract: This study was aimed to synthesize an active peptide from Carapax Trionycis and to investigate its effect on the proliferation

of hepatic stellate cells (HSCs). An active peptide from Carapax Trionycis, which was shown to have significant anti-hepatic

fibrosis activity, was synthesized by solid phase method and characterized by MALDI-TOF MS analysis. The HSCs in log

growth phase was treated with the synthetic peptide at different concentrations. Viability and apoptosis of hepatic stellate cells

were determined by MTS assay and Annexin V-FITC/PI staining, respectively. The active peptide showed strong inhibition of

proli feration and inducti on of apoptosis of HSC-T6 in a concentration-dependent manner. The results suggest that the active

peptide from Carapax Trionycis could be synthesized efficiently and has significant inhibitory effect on the proliferation of HSC-T6. Keywords: Hepatic stellate cells; Synthetic peptide; Carapax Trionycis; MTS; Apoptosis

CLC number: R916 Document code: A Article ID: 1003–1057(2012)2–132–04

Received date: 2011-09-08.*Corresponding author. Tel.: 86-027-88920834;

E-mail: [email protected]

doi:10.5246/jcps.2012.02.016

1. Introduction

Liver fibrosis is a wound-healing response to

various chronic liver injuries, including alcoholism,

persistent viral and helminthic infections, and

hereditary metal overload[1,2]. Activation of hepatic

stellate cells plays a crucial role in the development

of liver fibrosis[3–5]. During the activation process,

hepatic stellate cells (HSCs) undergo phenotype

transformation from vitamin-A-storing quiescent

cells to myofibroblast-like activated cells. Activated

HSCs are proliferative and fibrogenic, with accumu-

lation of extracellular matrix (ECM). Thus, ways to

eliminate HSCs such as inhibition of proliferation

and induction of apoptosis have become importantstrategies for the treatment of liver fibrosis [6].

Carapax Trionycis, a traditional Chinese medicine,

is originated from the shell of Trionyx sinensis

Wiegmann. It has such activities as replenishing “yin”,

restraining “yang”, softening and resolving hard

masses, reducing fever and removing steam. Clinical

experience indicated that traditional Chinese medicine

containing Carapax Trionycis showed curative effect

when used for the treatment of liver fibrosis [7].

Previous studies have demonstrated that extracts of

Carapax Trionycis were able to protect liver against

fibrosis in CCl4 animal models[8]. Moreover active

peptides were first isolated from Carapax Trionycis

and shown to have significant anti-hepatic fibrosis

activities by our group[9,10]. However, the natural

source of Carapax Trionycis is very limited and

extractive cost is very high, which restrict the use

of these active peptides.

In this study, we selected one of the active

peptides from Carapax Trionycis whose sequence is

HGRFG (molecular weight: 572.3) and synthesized

it by solid-phase method. We determined whether the

synthetic peptide affects the proliferation and apoptosis

of HSCs by MTS assay and Annexin V-FITC/PI

staining. Our results may provide experimental

support in the search of new anti-hepatic fibrosis

agents.

2. Materials and methods

2.1. Materials

The HSC-T6 cell line was kindly provided by

Prof. Hang-Pin Yao (Zhejiang University, Hangzhou).

The fetal calf serum (FCS) was purchased from Sanli

Biological Co., China. Trypsin and High-DMEM were



133 C. L. Hu et al. / Journal of Chinese Pharmaceutical Sciences 21 (2012) 132–135

from Gibcol Co., USA. MTS (3-(4,5-dimethylthiazol-

2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-

2 H -tetrazolium) was from Sigma Chemical Co., USA.

Annexin V-FITC/PI assay kit was supplied by Rose

Co., USA. Fmoc-AA-OH, Wang resin and TBTU were

obtained from Gil Biochemical Shanghai limited Co.,

China. MALDI-TOF MS was supplied by Shimadzu

Co., Japan. Flow cytometry was supplied by

Beckman-counter Inc., USA.

2.2. Solid-phase synthesis of an active peptide

from Carapax Trionycis

The introduction of solid phase peptide synthesis(SPPS) in 1963 by Bruce Merrifield opened the door

for researchers to prepare structurally diverse peptides.

In this study, the SPPS was carried out on Wang resin

as the carrier and N -Fmoc protected α-amino acid

as the starting materials. After condensed with the

mix reagents of TBTU/NMM and deprotected with

20% piperidine, the crude synthetic peptide products

were cleaved from the Wang resin by the cleavage

reagents TFA/TIS/H2O. We finally obtained the

active peptide of the sequence HGRFG.

2.3. Cell culture and MTS assay

HSC-T6 cells were maintained in Dulbecco’s

modified Eagle’s medium with 10% FCS and incu-

bated at 37 ºC under 5% CO2 humidified atmosphere.

HSCs were digested with 0.25% trypsin and adjusted

to 6×104 cells/mL when the HSCs were in exponential

growth phase. The cells were planted into 96-well

plate at 0.2 mL/well and 5-wells/group and treated

separately with serum containing the synthetic pepti de. After 2 h, the cells were added 800 pg/mL

TGF-β1 in each well.

The control group was cultured only in serum

medium. The stimulant group was cultured in TGF-β1

medium. The experimental group was treated with

the synthetic peptide at different concentrations (0.01,

0.05, 0.1 and 0.5 mg/mL) in TGF-β1 medium. After

cells were incubated for 72 h, the culture medium

was removed, and 20 μL MTS/PMS was added in

each well for 1 h. OD values at 490 nm weremeasured. The rate of inhibition (IR) was calculated

as follows IR (%) = [(control value – blank) – (test

value – blank)]/(control value – blank) × 100.

2.4. Annexin V-FITC/PI analysis

HSC-T6 cells were cultured in 25 cm2 flasks under

humidified 5% CO2 atmosphere at 37 ºC to 80%confluence and then treated with the synthetic peptide

before analysis. The control group was cultured only

in serum medium. The experimental group was treated

with the synthetic peptide at different concentrations

(0.5, 1.0, 2.0 and 4.0 mg/mL) in serum medium.

After 24 h, the culture medium was removed

and the cells were washed in phosphate-buffered

saline (PBS) and resuspended. Subsequently, 100 μL

(2×106 cells/mL) of the supernatant was reacted with

5 μL (1 μg/mL) FITC-conjugated annexin V and

10 μL (2 μg/mL) PI. The cells were incubated at room

temperature for 15 min in the dark and analysed

by flow cytometry. Only fluorescein-positive cells

without PI staining were regarded as apoptotic cells.

2.5. Statistical analysis

Each experiment was repeated a minimum of three

times. Results were expressed as mean±SD. Statistical

analysis was performed by one-way ANOVA test.

The P values lower than 0.05 were considered statisti-

cally significant.

3. Results

3.1. Analysis of the synthetic peptide

The active peptide from Carapax Trionycis was

synthesized by classical solid phase method. After

purification by RP-HPLC, the purity of the synthetic

peptide was over 98%. A typical chromatogram is

shown in Figure 1.

Figure 1. An HPLC chromatogram of crude peptide HGRFG.

1 3

. 9 2 6

1 4

. 6 1 6

1 7

. 9 3 4

2 0

. 3 2 2

2 1

. 6 5 8

mAU

120

100

80

60

40

20

0

–20

–40

0 5 10 15 20 25 30

t (min)



C. L. Hu et al. / Journal of Chinese Pharmaceutical Sciences 21 (2012) 132–135 134

The molecular weight of the synthetic peptide

was determined by MALDI-TOF MS (Fig. 2). The

[M+H]+ peak was at m/z 573.04, indicating the syn-

thetic peptide has the same molecular weight as the

active peptide HGRFG (molecular weight: 572.3).

3.2. Inhibitory effect of the synthetic peptide on

the proliferation of HSC-T6 cells

Results from MTS assay demonstrated that the

synthetic peptide at 0.01–0.5 mg/mL significantly

inhibited HSC-T6 proliferation in a concentration-

dependent manner ( P <0.05, Table 1).

3.3. Induction of apoptosis in HSC-T6 cells

Flow cytometry is a widely used and important

method for research and clinical applications. The

apoptosis of HSCs was determined using a flow-

cytometric annexin V-FITC/PI assay. The annexin

V-FITC/PI assay showed that 24 h after the synthetic

peptide was administer ed, the apoptosis rate of

cultured HSC-T6 cells increased significantly in a

concentration-dependent manner. The results of the

annexin V-FITC/PI assays for the different groups

are shown in Figure 3A–E, and the changes in the

apoptosis rate are shown in Figure 3F.

GroupSynthetic peptide (mg/mL)

Control TGF-β1 stimulant0.01 0.05 0.1 0.5

OD490 value 2.2533±0.0513 2.0733±0.0961 1.8467±0.0971* 1.2400±0.0634* 2.3400±0.0655 2.8067±0.0847

Inhibitory rate (%) 3.85 12.11 20.94 47.01 –20.09

Table 1. Inhibition of the proliferation of HSC-T6 cells by the synthetic peptide (mean±SD)

* P <0.05 compared with the control group (n = 4).

Figure 2. Mass spectrum of the synthetic peptide HGRFG.

Intens.[a.u.]

8000

6000

4000

2000

0

200 400 600 800 1000 1200 1400 m/ z

352.814 529.043

630.064

729.213

573.040

Figure 3. Detection of the synthetic peptide induced apoptosis in HSC-T6 by flow cytometry. Panels A–E represent the control and synthetic

peptide tr eatments a t 0.5, 1.0, 2.0 and 4.0 mg/mL, res pectively. F, bar graph showing t he i ncreased HSC-T6 cell apoptosis rate (%) 24 h after the

synthetic peptide treatment. ** P <0.05 compared with the control group (n = 4).

(C)(B)(A)

F L 2 - H

1 0

0

1 0 1

1 0 2

1 0 3

1 0 4

100 101 102 103 104

FL1-H

F L 2 - H

1 0

0

1 0 1

1 0 2

1 0 3

1 0 4

100 101 102 103 104

FL1-H

F L 2 - H

1 0

0

1 0 1

1 0 2

1 0 3

1 0 4

100 101 102 103 104

FL1-H

Control 0.5 1 2 4Synthetic peptide of Carapax Trionycis (mg/mL)

A p o p t o s i s r a t e ( % )

90

80

70

60

50

40

30

20

10

0

**

**

**(F)(E)(D)

F L 2 - H

1 0 0

1 0 1

1 0 2

1 0 3

1 0 4

100 101 102 103 104

FL1-H

F L 2 - H

1 0 0

1 0 1

1 0 2

1 0 3

1 0 4

100 101 102 103 104

FL1-H



C. L. Hu et al. / Journal of Chinese Pharmaceutical Sciences 21 (2012) 132–135 135

4. Discussion

In recent years, the use of peptides as therapeutic

agents has gained momentum. Chemical synthesis

of active anti-hepatic fibrosis peptides from Carapax

Trionycis, which were considered as lead compounds

in drug development, is an important way in the

search of anti-hepatic fibrosis agents. In this study

we synthesized an active peptide from Carapax

Trionycis by Fmoc solid phase method. The results

showed that the synthetic peptide could be obtained

by SPPS and was identical to the natural peptide.

Hepatic stellate cells play a central role in fibro-genesis and fibrolysis[11,12]. Activation and proliferation

of HSCs is the key to fibrogenesis while apoptosis

of HSC is associated with resolution of fibrosis[6,13].

Therefore, inhibition of HSCs activation and prolifera-

tion and induction of apoptosis of activated HSCs have

been proposed as potential anti-fibrotic strategies[11,14].

In this study we found that the synthetic peptide at

0.01–0.5 mg/mL inhibited HSC-T6 proliferation in

a concentration-dependent manner by MTS assay;

Annexin V-FITC/PI assay showed the early apoptosis

rate after treatment with 1.0, 2.0 and 4.0 mg/mL

synthetic peptide were significantly higher than

control ( P <0.05).

In summary, our study showed that an active

peptide from Carapax Trionycis could be synthesized

efficiently by solid phase method and the synthetic

peptide has significant proliferation inhibition activity

against HSCs.

Acknowledgments

We thank Dr. Yin-Ping Tang for her expert help in

preparing the manuscript and Professor Yan-Wen Liufor his excellent technical assistance.

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[4] Friedman, S.L. Physiol. Rev. 2008, 88, 125–172.

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鳖甲活性多肽的合成及对肝星状细胞增殖的抑制作用

胡春玲, 唐尹萍, 刘焱文 *

湖北中医药大学药学院, 湖北武汉 430065

要: 探讨鳖甲活性多肽的固相合成及对肝星状细胞 (HSC-T6) 增殖的抑制作用。根据鳖甲活性多肽的序列结构 ,

采用固相方法对多肽进行全合成, 并用MALDI-TOF MS质谱对合成多肽进行分析鉴定; 肝星状细胞 (HSC-T6) 培养至

对数生长期后, 加入不同浓度合成多肽作用肝星状细胞株HSC-T6, 采用MTS法分析HSC-T6生长的抑制作用; 使用Annexin

V-FITC/PI法检测HSC凋亡率。结果显示通过固相合成可以得到与原序列结构一致的鳖甲合成多肽 ; 鳖甲合成多肽浓度

依赖性地抑制肝星状细胞增殖, 并能显著提高肝星状细胞早期凋亡率。因此, 可以成功地合成鳖甲活性多肽, 且对肝星状

细胞增殖有明显的抑制作用。

键词: 肝星状细胞; 合成多肽; 鳖甲; MTS法; 凋亡

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