LI Yan(李巖),DONG Ji-rui(董繼銳),MA Xiao-ke(馬曉科),FENG Hui-hong(馮會(huì)紅),CHEN Xiao(陳曉),ZHAO Jian-pu(趙劍璞),GUO Nan(郭楠),LIU Pin-duo(劉品多)

(1.School of Life Science, Beijing Institute of Technology, Beijing 100081, China;2.Cancer Center, The First Hospital of Jinlin University, Changchun 130000, China)

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Effect of juglone on immunity response and oxidative stress in mice

LI Yan(李巖)1,DONG Ji-rui(董繼銳)1,MA Xiao-ke(馬曉科)1,FENG Hui-hong(馮會(huì)紅)1,CHEN Xiao(陳曉)2,,ZHAO Jian-pu(趙劍璞)1,GUO Nan(郭楠)1,LIU Pin-duo(劉品多)1

(1.School of Life Science, Beijing Institute of Technology, Beijing 100081, China;2.Cancer Center, The First Hospital of Jinlin University, Changchun 130000, China)

To research juglone’s immunology regulation, several perspectives including immunology regulation, lymphocyte proliferation, and cytokines were presented.The index of thymus and spleen, total supperoxide dismutase activity (T-SOD), serum malondialdehyde (MDA),content of nitrogen oxide (NO), the anti-superoxide anion ability, the suppressing hydroxy radical ability, contents of reduced glutathione (GSH) in thymus, total antioxidationability (T-AOC) and the level of mouse lysozyme (LZM) in plasma were measured. LPS-induced B thymocytes proliferation was measured by MTT assay.In the low-immunity model group, MDA and NO levels were decreased (p<0.001). Juglone improved lysozyme (LZM), GSH contents in thymus, T-AOC, T-SOD activity, the anti-superoxide anion ability (anti- O2-) (p<0.001).In the stimulation immunity model group, MDA and NO levels (p<0.05), anti- O2- and T-SOD activity (p<0.05) were up-regulated, whereas LZM, T-AOC contents were down-regulated.juglone has a significant effect, which promotes cell regeneration and function recovery, also may alleviate oxidative damage; juglone possesses a dual regulating effect on humoral immunity in mice.

juglone; immunity; oxidative stress; mice

Juglone, as one of the typical naphthoquinones, has many uses including anticancer, analgesia, and anti-inflammatory[1-3].Researches have shown that juglone has such a variety of bio-activities which may be because of its outstanding immunology regulation[4-5].This paper studies the effects of juglone on mice with different immune status and the antioxidant capacity to explore whether there are correlations between the immune-modulatory effects of juglone and the body’s free radical reactions.To this purpose we adopted dexamethasone sodium phosphate injection to establish low immunity model and built a mice immune stimulation model induced by sheep red blood cells. By intraperitoneal injection of cyclophosphamide before the stimulation of the antigen, the antigen-antibody reaction can be significantly enhanced. It may be due to the suppression and elimination of the precursors of Ts cells and also to reducing the negative regulation of Ts cells in the immune response[6].

1 Materials and methods

1.1 Laboratory animals animal science

The study complied with the “Guide for the Care and use of Laboratory Animals” published by the US National Institutes of Health (NIH publication No. 85-23, revised in 1985).

And all animals were approved by the Institutional Animal Experiments Committee. Adult KM mice (20±2 g body weight), all male, SPF level, were purchased from Department of Laboratory Animal Science of Peking University Health Science center (Beijing, China). Animals were maintained under 12 h light/dark cycles at a temperature of approximately 24±1 ℃ with food and water ad libitum.

1.2 Materials

Juglone (purity 95%) was extracted from Juglans mandshurica Maxim (collected by Professor Yan Li at Mountain Shi, 857, Mudanjiang agricultural Authority, Heilongjiang Province, China) in the Pharmacology Lab. Coomassie brilliant blue protein assay kit, LZM detection kit, malondialdehyde assay kit (thiobarbituric acid, TBA method), T-AOC kit, reduced GSH detection kit, NO test kit, T-SOD kit and anti-superoxide anion radicals test kit were purchased from Nanjing Jiancheng Bioengineering Institute. Mice phospholipase A2 (PLA2) ELISA kit, mice cyclooxygenase-2 (COX-2) ELISA kit, mice arachidonic acid lipid oxygenation enzyme ELISA kit were purchased from R&D Systems China Co.,Ltd.

1.3 Establishment of animal models of Low-immunity and immune enhancement

Forty-eight Kunming (KM) mice were divided into 6 groups (n=8 for each group), all male. They were intraperitoneal administrated with different solution respectively for 10 consecutive days (Tab.1).

[a]:Low-immunity model is developed through peritoneal injecting dexamethasone sodium phosphate under the dose 0.2 mL/10 g for seven days.[b]:Cyclophosphamide(Cy)-induced stimulation immunity model is developed through peritoneal injecting 2% (v/v, prepared with normal saline) hematocrit 0.2 mL SRBC to immunization and peritoneal injecting 250 mg/kg cyclophosphamide three days before it.

1.4 Detection of immune organ index

All mice were weighed on Day 11. After taking blood by the removal of the eyeball, serum was obtained. All mice were put to death by cervical vertebra dislocation, and then there thymus and spleen were taken. The ratio of thymus weight (g) and spleen weight (g) to the mice weight (g) were used as the thymus index and spleen index, which is calculated as follows:

Thymus (spleen) index = thymus weight (g) / body weight (g)

1.5 Peripheral blood detection

0.2 mL blood per mice was obtained by the orbital blooding and then they were collected in a centrifuge tubewith EDTAK2 as anticoagulant, mixed and inspected immediately.

LPS-induced B cell proliferationwas detected by 3-[4, 5-dimehyl-2-thiazolyl]-2, 5-diphenyl-2H-tetrazolium bromide (MTT) assay at 490 nm wavelength.

The detection and methods of antioxidant capacity index was the same as in the detection of mice immune organ index.

Concentrations of thymus and spleen protein were detected by Coomassie Brilliant Blue; Serumof LZM, MDA, T-AOC, GSH in thymus, Spleen NO levels, T-SOD and capacity of Spleen superoxide anion radical (anti-O2-) were also measured by using their own assay kits respectively, according to the introduction.

1.6 Determination of inflammatory factors

The serum was used for testing. Blood sample processing methods: 1 mL blood was got by the removal of the eyeball. Then the blood was stilled for 1 h and centrifuged at 3 000 r/min for 20 min to get serum.

Mice PLA2 content, COX-2 and the content of arachidonic acid lipoxygenase (ALOX-5) in mice were measured by using their own assay kit according tothe introduction.

2 Results and discussion

Juglone, also called 5-hydroxy-1, 4-naphthalenedione (IUPAC) or 5-hydroxynaphthoquinone, is an organic compound. Juglone exerts its effect by inhibiting certain enzymes needed for metabolic function. Dama’s research shows that juglone has anthelmintic activity on mature and immature Hymenolepis nana in mice[7]. It is also showed antimicrobial activity of napthoquinonic compounds[8]and effect of naturally occurring napthoquinones on root- knot nematode Meloidogyne spp[9].

However,we have done lots of work on the effect of juglone on immunity response and oxidative stress in mice. As in Tab.2 and Tab.3, compared with the normal control group, the spleen index and thymus index of the model control group are significantly decreased(P<0.01). And in the serum of control group, T-AOC and LZM are decreased significantly(P<0.05) while the concentration of MDA is significantly increased(P<0.01). The concentration of NO in the spleen is significantly increases(P<0.01), T-SOD is slightly decreased. Anti-O2- is significantly decreased (P<0.01). Also, the concentration of GSH in thymus is significantly decreased (P<0.01). All the data shown above proves that dexamethasone sodium phosphate is useful in developing the low-immunity rat model.

Tab.2 Effect of juglone on immune organ

Compared with normal control group, *p<0.05; compared with Low-immunity control group,#p<0.05,##p<0.001, compared with stimulation control group,△p<0.05,△△p<0.001.

Tab.3 Effect of juglone on different indexes of laboratory mice

Compared with normal control group, *p<0.05, **p<0.001; compared with Low-immunity control group,#p<0.05,##p<0.001, compared with stimulation control group,△p<0.05,△△p<0.001.

Compared with the two control groups, both of the juglone groups’ spleen index and thymus index are improved; and the concentration of MDA in juglone groups are siginficantly deceased(P<0.01) compared with the corresponding control group, the concentration of T-AOC and LZM in the serum are significantly increased(P<0.01); in addition, the juglone groups’ spleens have a low significant concentration in NO(P<0.01) and a relatively high one in T-SOD and anti-O2-(P<0.01); furthermore, the concentration of GSH in the thymus has an significantly increase(P<0.05). All these phenomena show that juglone could improve the whole immunity through improving the concentration of LZM and the ability of oxidation resistance under either the normal condition or the low-immunity condition.

The results show that juglone increases the spleen index, the thymus index, raises the content of GSH in thymus, improves T-AOC and T-SOD activity, improves the anti-superoxide anion ability, and reduces the level of LPO and the content of NO.

Peritoneal injecting dexamethasone sodium phosphate can damage the immune function in mice. Thymus index and spleen index in mice are significantly decreased and low-immunity model is successfully developed. Through peritoneal injecting SRBC to immunization and cyclophosphamide three days before, thymus index and spleen index of mice are enhanced. And the immune function is significantly improved.

Juglone has a significant effect to promote cell regeneration and function recovery and alleviate oxidative damage. Juglone possesses a dual regulating effect on humoral immunity in mice.

Juglone prominently antagonizes immune suppression induced by dexamethasone. It improves and enhances immunologic function through improving the structure of immuneorgan, as well as regulating levels of free radical and the transduction of the reduction-oxidation signal.

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(Edited by Wang Yuxia)

10.15918/j.jbit1004-0579.201524.0120

R 961.1 Document code: A Article ID: 1004- 0579(2015)01- 0139- 04

Received 2013- 09- 10

E-mail: leeyan@bit.edu.cn