Wang CB et al / Acta Pharmacol Sin 2002 Sep; 23 (9): 813-818

Protective effect of polypeptide from Chlamys farreri on hairless mice damaged by ultraviolet A¹

WANG Chun-Bo², YAO Ru-Yong³, LIU Zhan-Tao, ZHONG Wei-Zhen, LIU Xiao-Ping, WANG Yue-Jun⁴

Medical College Qingdao University, Qingdao 266021;
³Medical College Hospital of Qingdao University, Qingdao 266003;
⁴Yellow Sea Fishery Research Institute, Qingdao 266071, China

¹ Supported by the National Natural Science Foundation of China, ¡í 39970638.

² Correspondence to Prof WANG Chun-Bo. Phn 86-532-383-8480, ext 3756.

Received 2001-12-29 Accepted 2002-05-29

KEY WORDS Chlamys farreri; ultraviolet rays; antioxidants; hairless mice; bcl-2 genes; nitric-oxide synthase; immunohistochemistry; topical administration

ABSTRACT

AIM: To study the protective effect of the polypeptide isolated from Chlamys farreri (PCF) on hairless mice skin damaged by ultraviolet A. METHODS: Enzymes and malondialdehyde (MDA) were determined by biochemical methods; the expressions of Bcl-2 protein and NOS protein were examined by immunohistochemical technique. The ultra-structure of the skin was observed through electronic microscope. RESULTS: PCF could enhance the activities of glutathione peroxidase (GSH-px), superoxide dismutase (SOD), and total anti-oxidative capacity (T-AOC). Also PCF could reduce the amount of MDA, increase the expression of Bcl-2 protein, and inhibit the expression of NOS protein. The ultra-structure of epidermis and fibroblasts remained normal in 20 % PCF groups; there were vacuoles in smooth endoplasm reticulum in epidermis of mice and the number of rough endoplasm reticulum in fibroblasts was decreased in model group. CONCLUTION: PCF had the protective effects on hairless mice skin damaged by ultraviolet A via its anti-oxidative mechanisms.

INTRODUCTION

Many reports have shown the oxidative damages of ultraviolet on skin as well as on cells^[1]. Ultraviolet A (UVA) can cause more damages on the skin for its great penetrating capability^[2]. UVA can penetrate the epidermis and the dermis and is the most intense light in ultraviolet.

Natural antioxidants can inhibit the oxidative injuries caused by UVA. They mainly come from plants or herbs and have been used in medicine and health care^[3]. The reports mostly concerned about the oxidative damage models as nude mice or naked mice (immunodeficiency animal) and some kinds of cells. For instance, they are fibroblasts and horny cells, etc. We seldom see the reports on polypeptides as antioxidants, especially those from marine products and the reports on hairless mice (animal with normal immunity) as oxidative model^[4,15]. In this study, we established the continuous oxidative damage model of hairless mice and then probed the protective effect of the polypeptides isolated from Chlamys farreri, on the mice skin.

MATERIALS AND METHODS

Drugs and reagents Polypeptides isolated from Chlamys farreri (PCF, M_r 800-1000), was purified and analyzed by HPLC, stored at 4 ¡æ, and isolated using biological engineering technique. Hairless mice: Kunming species, male, 18 g±2 g, bought from Beijing Medical University. Malondialdehyde (MDA) test kits and enzyme test kits including glutathione peroxidase (GSH-px), superoxide dismutase (SOD), and total anti-oxidative capacity (T-AOC): bought from Nanjing Jiancheng Bioengineering Institute, lot number: 20001102. Bcl-2 and NOS protein immunohistochemistry test kits: bought from Beijing Zhongshan Corporation, lot number: 20000102. Transmission electronic microscope: JEM-1200ES. Image analysis system: VIDAS-21, made in Japan. Radiometer and ultraviolet A light source: bought from Beijing Normal University and tissue homogenizer: Unicorn Medical Instrument Factory.

Establishment of oxidative model for hairless mice skin damaged by UVA Hairless mice (n=40) were randomly divided into five groups (8 per group). They were control group, model group, 5 % PCF group, 20 % PCF group, and 10 % vitamin C group. After routine breeding for about one week, saline, PCF, and vitamin C were smeared on the backs of the mice (1 mL per mouse a day) respectively^[6]. One hour later, the mice were exposed to UVA (radiant intensity: 151.68 J·cm^-2·d^-1) except those of control group. Thirty days later (total radiant intensity: 4556.4 J·cm^-2), the mice were put to death and the skin radiated by UVA was used to make the homogenate^[7].

Observation of skin ultra-structure We used 5 % glutaral dehyde to fix the skin for 24 h and then dehydrated it with ethanol grade-by-grade, saturated the skin in embedding medium: epoxy resin-618. Then we cut it into very thin slices and stained them. Finally we observed the ultrastructure of the skin through transmission electronic microscope^[8].

Immunohistochemistry detection of Bcl-2 protein and NOS protein First, we routinely prepared the skin tissue slices. Then the expression of Bcl-2 protein (the first antibody is rabbit-anti-mouse Bcl-2 IgG) and NOS protein (the first antibody is rabbit-anti-mouse NOS IgG) were examined by immunochemical technique following the kits directions and the results were processed by image processing system^[9].

Effects of PCF on the antioxidative index of hairless mice skin damaged by UVA First, we prepared the homogenate of the skin by using cold PBS (pH 7.4, in ice bath) through tissue homogenizer. Second, we centrifuged the skin homogenate and get the supernatants for biochemical tests. After determining the amount of total protein in the supernatants, we detected the enzymes that include GSH-px, SOD as well as T-AOC and MDA by biochemistry method using the supernatants following the kits directions^[10].

RESULTS

Bcl-2 immunohistochemical staining of epidermis in 20 % PCF group was deep compared with model group. NOS immunohistochemical staining of epidermis in 20 % PCF group was light compared with model group. Compared with model group, the expression of Bcl-2 protein in PCF groups rose obviously, while that of NOS in PCF groups decreased (P<0.05, Tab 1, Fig 1). PCF enhanced the enzyme activities and decreased the content of MDA in the skin of hairless mice
(Tab 2).

Tab 1. Effects of PCF on the expressions of Bcl-2 protein and NOS protein in hairless mice skin damaged by UVA. n=8 mice. Mean±SD. ^cP<0.01 vs model group.

Group	Bcl-2	NOS
Control	0.20¡À0.03	0.25¡À0.03
Model	0.14¡À0.06	0.34¡À0.07
5 % PCF	0.26¡À0.05c	0.23¡À0.03c
20 % PCF	0.31¡À0.08c	0.22¡À0.03c
10 % Vit C	0.22¡À0.03c	0.24¡À0.04c

Fig 1. Immunohistochemical staining in epidermis of hairless mice. A) Bcl-2 in model group. B) Bcl-2 in 20 % PCF group. C) NOS in model group. D) NOS in 20 % PCF group. ×400.

Tab 2. Effects of PCF on the enzyme activities and MDA amount of skin homogenate of hairless mice damaged by UVA. n=8 mice. Mean±SD. ^bP<0.05, ^cP<0.01 vs model group.

Group	GSH-px/U	SOD/	T-AOC/	MDA/
		mU¡ÁL-1	U¡Ág-1	mmol¡ÁL-1
Control	25.88¡À0.10	5.97¡À0.22	291¡À30	5496¡À1022
Model	26.09¡À0.11	5.1¡À0.3	194¡À40	13592¡À866
5 % PCF	26.46¡À0.13b	6.1¡À0.6c	323¡À42c	8941¡À530c
20 % PCF	26.61¡À0.12b	6.4¡À0.5c	382¡À63c	7826¡À596c
10 % Vit C	26.36¡À0.09c	6.19¡À0.28c	314¡À22c	10746¡À539c

After epidermis cells were exposed to UVA, we could see the damage with vacuoles in cytoplasm obviously. The ridges of mitochondria in epidermis cells in model group were broken and rough endoplasm reticulum expanded into vacuoles. The interspace between cells in epidermis of model group was broadened and there were some vacuoles in cytoplasm. The amounts of rough endoplasm reticulum, Golgi complex, and mitochondria of fibroblasts decreased dramatically and some nuclei became pycnosis (Fig 2A, B, C), which showed that the activity of the cell was weakened.

Fig 2. Ultrastructure in epidermis and dermis of hairless mice. A) Ridges of mitochondria and rough endoplasm in epidermis cells in model group. ×30 000. B) The interspace between cells and some vacuoles in cytoplasm in epidermis of model group. ×80 000. C) The big fibroblast of dermis of model group. ×40 000. D) Normal structure of epidermis in 20 % PCF group. ×40 000. E) Normal structure of fibroblasts in dermis of 20 % PCF group. ×40 000. F) Structure of epidermis in 5 % PCF group. ×30 000. G) Structure of fibroblasts in dermis of 5 % PCF group. ×30 000.

The constructions of epidermis and fibroblasts in dermis of 20 % PCF group were normal. The mitochondria and rough endoplasm reticula were distinct. The rough reticula were plentiful. The tonofilaments and intercellular bridges could be clearly observed in epidermis (Fig 2D, E).

The structure of epidermis and fibroblasts in dermis in 5 % PCF group were normal. The endoplasm tonofilaments were easily seen in cells but the mitochondria were obscure. The rough endoplasm reticula were plentiful in fibroblasts of dermis (Fig 2F, G).

DISCUSSION

From all the results, we could see that in model group UVA induced the deformation of the epidermis and dermis, inhibited the expression of Bcl-2 protein, and enhanced that of NOS. At the same time, UVA decreased the activities of antioxidative enzymes in the skin and increased the lipid peroxide. All this had made it clear that we had established the continuous UVA damage model on mice successfully.

Electron microscope plays an important role in modern medical science. It is the most reliable method in observing the shape of cells. Under the electron microscope, we may study the character of the cell and see the small changes of the cell ultrastructure^[11]. Our results showed that UVA could damage the epidermis as well as dermis of the mice. PCF protected the mice from oxidative damage caused by UVA thus remained the ultrastructure of the cells in skin normal.

Fibroblasts in dermis can produce collagen fibers, elastic fibers, argyrophilic fiber, and dermis matrix, which have the function of maintaining the elasticity and the strength of the skin. When the fibroblasts in dermis are in dysfunction, the effect of skin will be in disturbance^[12]. PCF could protect the fibroblasts in dermis thus maintained the skin function normal.

In 1988, Veux reported the function of Bcl-2 protein firstly. Bcl-2 is a kind of inhibitor of the cell apoptosis. It can prevent the apoptosis induced by free radicals and lipid peroxidation^[13]. Bcl-2 has the antioxidative characteristics in cells through participating the reduction action and inhibiting the formation of active oxygen^[14]. PCF could up-regulate the expression of Bcl-2 protein thus inhibit the lipid peroxidation induced by UVA.

Recently, many reports showed that NO· could induce the apoptosis of cells^[15]. NO· combined with free radicals can perform nitroso which may causes the damage of DNA in cells. NO· itself also can injure the cells^[16]. The expression of NOS protein indicates the amounts of NO· produced in cells. Our results showed that PCF inhibited the expression of NOS therefore prevented the damage caused by NO.

Several antioxidative enzymes including GSH-px, SOD, and CAT etc scavenge free radicals produced by ultraviolet^[17]. The activities of the antioxidative enzymes reflect the potential anti-oxidative capability of the cell. UVA causes the cell of the skin to produce lipid peroxide^[18]. The amounts of MDA reflect the level of lipid peroxidation in vivo^[19]. Our results showed that PCF enhanced the activities of GSH-px and SOD and claimed that PCF was a potential antioxidant against ultraviolet damage. PCF can stimulate enzymes to remove free radicals therefore protect cells from the injury of radicals. Also PCF can enhance the activity of T-AOC that reflects the anti-oxidative capability of the skin. At the same time, PCF decrease the amounts of MDA which also means PCF reduces the lipid peroxidation of the cell and keeps the cells in skin out of the damage of the radicals.

In summary, on the background of successfully establishment of the continuous UVA damage model (irradiation intensity: 4556.4 J·cm^-2) of hairless mice, the results of this study indicated that PCF had the protective effects on hairless mice damaged by ultraviolet A. At the concentration from 5 % to 20 %, PCF obviously increased the activities of GSH-Px, T-AOC, and SOD, inhibited the lipid peroxidation, up-regulated the expression of Bcl-2 protein, and down-regulated that of NOS. Thus we observed the normal ultrastructures of epidermis and fibroblasts of mice in PCF group. PCF could protect not only the epidermis but also the dermis from the injury caused by UVA.

REFERENCES

• 1 Black HS. Potential involvement of free radical reaction in ultraviolet light mediated cutaneous damage. Photochem Photobiol 1987; 46: 213-21.
• 2 Kligman LH, Akin FJ, Kligman AM. The contribution of UVA and UVB to connective tissue damage in hairless mice. J Invest Dermatol 1985; 84: 272-84.
• 3 Wei H, Zhang X, Zhao JF, Wang ZY, Bickers D, Lebwohl M. Scavenging of hydrogen peroxide and inhibition of ultraviolet light-induced oxidative DNA damage by aqueous extracts from green and black teas. Free Radic Biol Med 1999; 26: 1427-35.
• 4 Skoog ML, Ollinger K, Skogh M. Microfluorometry using fluorescein diacetate reflects the integrity of the plasma membrane in UVA-irradiated cultured skin fibroblasts. Photodermatol Photoimmunol Photomed 1997; 13: 37-42.
• 5 Noonan FP, Webber LJ, De Fabo EC, Hoffman HA, Bendich A, Mathews-Roth M. Dietary beta-carotene and ultraviolet induced immunosuppression. Clin Exp Immunol 1996; 103: 54-60.
• 6 Burke KE, Clive J, Combs GF Jr, Commisso J, Keen CL, Nakamura RM. Effects of topical and oral vitamin E on pigmentation and skin cancer induced by ultraviolet irradiation in Skh: 2 hairless mice. Nutr Cancer 2000; 38: 87-97.
• 7 Xu SY, Bian RL, Chen X. Experiment methodology of pharmacology. Beijing: People's Medical Publishing House; 1994. p 135-221.
• 8 Liu SC, Zhai S, Lawler J, Palek J. Hemin-medical dissociation of erythrocyte membrane skeletal proteins. J Biochem 1985; 260: 122-34.
• 9 Hong JT, Kim EJ, Ahn KS, Jung KM, Yun YP, Park YK, et al. Inhibitory effect of glycolic acid on ultraviolet-induced skin tumorigenesis in SKH-1 hairless mice and its mechanism of action. Mol Carcinog 2001; 31: 152-60.
• 10 Goth L. A simple method for determination of serum catalase activity and revision of reference range. Clin Chem Acta 1991; 196: 143-52.
• 11 Bromage TG. The scanning electron microscope in craniofacial remodeling research: application of the topographic principle. Prog Clin Biol Res 1982; 101: 143-53.
• 12 Fryer MJ. Evidence for the photoprotective effects of vitamin E and vitamin C. Photochem Photobiol 1993; 58: 304-12.
• 13 Hockenbery D, Nuriez G, Milliman C. Bcl-2 is an inner mitochondrial membrane protein that blocks programmed cell death. Nature 1990; 348: 334-36.
• 14 Veux G. Location of the Bcl-2 protein within haemopoietic neoplasms. Blood 1988; 81: 3091-96.
• 15 Palluy O, Rigaud M. Nitric oxide induces cultured cortical neuron apoptosis. Neurosci Lett 1996; 208: 1-4.
• 16 Albina JE, Cui S, Mateo RB, Reichner JS. Nitric oxide-mediated apoptosis in murine peritoneal macrophages. J Immunol 1993; 150: 5080-5.
• 17 Lebel CP, Bondy SC. Oxidative damage and cerebral aging. Prog Neurobiol 1992; 38: 601-9.
• 18 Kalka K, Mukhtar H, Turowski-Wanke A, Merk H. Biomelanin antioxidants in cosmetics: assessment based on inhibition of lipid peroxidation. Skin Pharmacol Appl Skin Physiol 2000; 13: 143-9.
• 19 Zhao BL, Qu BJ, Zhang CA, Zhang JZ, Xin WJ. Study on the interaction between photoradiation-hematoporphyrin derivative and liposome with spin label and ESR spectrum. Acta Pharm Sin 1985; 20: 89-93.