The Antioxidant Effects of L-carnitine on D-galactose Induced Aging Mice
LUAN Hai-yun, YANG Ming*, WANG Gui-hua, LI Na
Institute of Materia Medica, College of Pharmacy, Binzhou Medical University, Yantai 264003, China)
Abstract [Objective] To study the antioxidant effects of LC on D-galactose induced aging mice. [Method] 50 mice were randomly divided into blank control group, D-galactose model group, LC treatment groups at three levels of 0.25, 0.5 and 1.0 g/(kg·d), a total of 5 groups. Using the subacute D-galactose induced aging models, the mice were killed after 6 weeks of experiment and the activity of Superoxide dismutase (SOD), Glutathione peroxidase (GSH-PX) and Malondialdehyde (MDA) content in plasma, brain and liver tissue were determined. [Result] The activity of SOD and GSH-Px in plasma, brain and liver tissue were significantly increased after treated with LC, while the content of MDA were decreased with a dose-dependent manner. [Conclusion] LC had anti-aging effects in mice, and the mechanism may be correlated with the antioxidation.
Key words L-carnitine; Anti-aging; D-galactose
L-carnitine (LC) is a kind of amino acid extensively in the body and also a endogenic nutrinent, its main function is to participate in the energy metabolism in mammals and promote the β-oxidization of fatty acid[1-3], also with the function of antioxidant and cleaning free radical and so on[4-5]. In this research, the aging mice models induced by D-galactose were established and given different doses of LC. Then the activity of Superoxide dismutase (SOD), Glutathione peroxidase (GSH-PX) and Malondialdehyde (MDA) content in plasma, brain and liver tissue of mice were determined to further explore LC anti-aging mechanism.
1. Materials and Methods
1.1. Equipments LT-224S electronic balance (Beijing Sartorius Co.), GL-88 B-type Vortex mixer (Taicang Science Instrument Factory), DK-98-1 Electric heating constant temperature water bath pot (Tianjin Taisite Instrument Co., Ltd.), centrifuge 5804 (Eppendorf, Germany), Model 680 Enzyme mark instrument (BIO-RAD, America).
1.2. Reagents L-carnitine crude drug (Northeast General Pharmaceutical Factory); D-gal (Sigma); SOD kit, GSH-Px kit, MDA kit and Coomassie brilliant blue protein quantitative determination kit (Nanjing Jiancheng Bioengineering Institute).
1.3. Animals 50 Kunming mice of half gender, (18±4) g, clean grade, were provided by the Animal Center of Shandong Lukang pharmaceutical Co., Ltd. (number of animal license SCXK 2005-0017). They were fasting for 12 h before the test, but free to drink water.
1.4. Methods
1.4.1. Group and administration 50 Kunming mice were randomly and equally divided into 5 groups namely blank control group, D-gal model group, LC treatment groups (low-, middle-, and high-dose group). Apart from the control group, other groups of mice were subcutaneously injected with 200 mg/kg D-gal per day on the nape, 1 times a day, for 6 consecutive weeks, while the control group were injected with same volume of physiologic saline on the nape. From the 5th week on, the LC treatment groups continued to be injected with D-gal, meanwhile with 3 different doses of LC (0.25, 0.5, 1.0 g/kg), respectively, blank control group and model group were injected with same volume of saline.
1.4.2. Tissue processing 24 h after the last administration, blood was obtained from eyeballs, subsequently the mice were put to death by neck dislocation. The mice brain tissue and liver were rapidly took from the ice bag and washed by cold saline, after blotted with filter paper and precise weigh, 10% tissue homogenate were made to centrifuge at 4000 r/min and 4℃ for 10 min, the supernates were packed in 1.5 ml centrifuge tubes and cryopreserved in -20℃ refrigerator to be analyzed. All the indicators were determined strictly according to Kit instructions.
1.5. Statistical treatment The data were analyzed by SPSS 13.0 statistics software.
2. Results
2.1. Effects of LC on SOD, GSH-Px activity in plasma, brain and liver of aging mice SOD, GSH-Px activity in plasma, brain and liver of model group mice were significantly lower than that of the blank control group, the difference of group comparison was significant (P<0.01 or P<0.05). LC treatment groups [0.25, 0.5, 1.0 g/(kg·d)] could significantly increase the SOD, GSH-Px activity in plasma, brain and liver of aging mice, the difference compared with the model group was significant (P<0.05 or P<0.01), the result was shown in tables 1-3.
2.2. Effects of LC on MDA content in plasma, brain and liver of aging mice In LC treatment groups [0.25, 0.5, 1.0 g/(kg·d)] and blank control group, MDA content in plasma, brain and liver was significantly reduced with regard to that of the aging model group (P<0.05 or P<0.01), moreover, MDA content was more obviously reduced as the dosage of LC increased (P<0.05 or P<0.01), the results was shown in tables 1-3.
Table 1. Effects of LC on SOD, GSH-Px activity and MDA content in plasma of aging mice ( , n=10)
Groups
|
Dose (g/kg)
|
SOD (U/mg)
|
GSH-Px (U/mg)
|
MDA (nmol/mg)
|
Blank control group
|
|
80.07±10.11
|
2.61±0.88
|
3.65±0.72
|
Model group
|
|
45.73±5.22**
|
1.11±0.22*
|
6.31±0.81**
|
LC
|
0.25
|
59.92±4.44#
|
1.92±0.15##
|
4.87±1.25
|
LC
|
0.5
|
82.36±3.43##
|
2.15±0.21##
|
4.03±0.50#
|
LC
|
1.0
|
86.23±5.35##
|
2.45±0.26##
|
3.16±0.69##
|
Compared with blank control group: *P<0.05, ** P<0.01; Compared with model group: #P<0.05,## P<0.01. Similarly hereinafter.
Table 2. Effects of LC on SOD, GSH-Px activity and MDA content in brain of aging mice ( , n=10)
Groups
|
Dose (g/kg)
|
SOD (U/mg prot)
|
GSH-Px (U/mg prot)
|
MDA (nmol/mg prot)
|
Blank control group
|
|
107.77±6.22
|
2.73±0.58
|
4.19±0.77
|
Model group
|
|
61.20±6.57**
|
1.33±0.32*
|
8.91±0.64**
|
LC
|
0.25
|
87.89±7.79#
|
1.75±0.38
|
6.17±0.54##
|
LC
|
0.5
|
91.16±5.39##
|
1.87±0.38
|
4.86±0.82##
|
LC
|
1.0
|
92.65±6.29##
|
1.91±0.24#
|
4.05±0.73##
|
Table 3. Effects of LC on SOD, GSH-Px activity and MDA content in liver of aging mice ( , n=10)
Groups
|
Dose (g/kg)
|
SOD (U/mg prot)
|
GSH-Px (U/mg prot)
|
MDA (nmol/mg prot)
|
Blank control group
|
|
92.72±8.04
|
2.29±0.36
|
6.04±0.97
|
Model group
|
|
50.09±9.55**
|
1.25±0.26*
|
9.31±0.93*
|
LC
|
0.25
|
75.37±8.90#
|
1.58±0.23
|
6.56±0.52#
|
LC
|
0.5
|
80.42±8.51#
|
1.66±0.34
|
6.08±0.49##
|
LC
|
1.0
|
84.01±9.74#
|
1.71±0.15#
|
5.84±0.30##
|
3. Discussion
Through enzymatic system and non-enzymatic system, the organism produces free radical, too many of which can have peroxidation with lipoid substances in cell membranes, resulting in membrane damage and cell death; simultaneously, the decomposition products of lipid peroxide (MDA) can also combine with substances such as DNA, RNA, protein and phospholipid, thus damaging cell membranes and causing cell dysfunction, eventually making the organism appear old and feeble[6-7].
In order to defense injury and damage from metabolism free radical, body of anti-oxidation system is established and formed in tissues and cells, mainly including some antioxidases (such as SOD, GSH-Px), these enzyme often react together to clear internal lipid peroxide, and make the body avoid the injury of free radical, together maintaining the balance of generation and clearance of internal free radical[8-9]. However, MDA is important internal lipid peroxidation products, content of which can reflect the severity of body cell attacked by free radicals and indirectly reflect the degree of cell injury[10].
From the study on aging mice model, it can be observed that LC has a certain effect of anti-oxygen free radicals. The experiment results showed that anti-oxidation and lipid peroxidation abnormalities existed in D-galactose induced aging mice, in addition, LC could increase SOD, GSH-Px activity and reduce MDA content in plasma, brain and liver, presenting dose-dependent. It indicated that LC could reduce free radical damage effect through enhancing the ability of clearing oxygen free radicals and antioxidant function, thus to protect cells from oxidative damage. This research revealed from the free radical theory that LC could improve personal antioxidant capacity through the enhancement of endogenous antioxidant system in brain tissue, inhibit brain intracellular MDA production and reduce lipid peroxidation injury degree, thereby maintaining the stability of the intracellular environment, protecting the brain tissue and reducing free radical on brain tissue partial damage, finally to the effect of protecting the cells and delaying senility. However, it has yet to be further studied as for the question of how LC passes the blood-brain barrier, distributes in the brain and protects cranial neuron.
Acknowledgments
Yantai Science and Technology Development Plan (2011074).
About the author
LUAN Haiyun (1979-), female, P. R. China, Master, lecturer, working at research on anti-aging pharmacology. Tel: 15905355268. E-mail: bluehy2000@126.com.
*Corresponding author: YANG Ming (1976-), female, P. R. China, Doctor, Tel: 13396383355. E-mail: yt.yming@gmail.com.