No.45 September 2005 |
|||
print view |
Introduction
Exercise induced fatigue is a common phenomenon in the process of endurance training. It is an important limitation of performance improving and physiological well being. In order to delay the occurrence of exercise induced fatigue and accelerate its elimination, we adopted acupoint iontophoresis as a possible method to achieve this. Acupoint iontophoresis is an organic combination of Traditional Chinese Medicine and modern physical method that is utilised on acupoints. According to previous research, pure electric stimulation on acupoints in endurance training can increase the movable platform fatigue time and exhausting time of rat [1-3]. In order to observe the effect of acupoint iontophoresis on an endurance training rat, we designed this experiment. Since previous research shows that PKC (Protein Kinase C, PKC) has the function of cardiac protection [4,5], we take myocardial PKC expression as an evaluation window, separately from protein and mRNA level, so as to find out if there is any possible molecular mechanism to explain the rats’ longer exhausting time during the experiment.
Materials and methods Materials and methods
Materials
Immunohistochemistry Kit (Boster Co., China), TRIZOL Reagent (Gibco), one step RNA PCR Kit (Takara). The primer of PKC and β-actin were synthesis by Bocai Biotechnology Corporation.
Animals
36 SD rats were used, aged 2 months old, weight 200~220g all provided by the experiment animal centre of Shanghai University of TCM. In terms of experimental purpose, all rats were divided into 5 groups at random: 1) contrast group (n=4); 2) endurance training group (ET, n=8), rats in this group receive exercise training on animal platform everyday and 6 days a week, the load is increasing during the whole 8 weeks; 3) endurance training and dosing group (ED, n=8), rats in this group take Chinese herbs everyday based on training; 4) endurance training and acupoint iontophoresis group (EAI, n=8), rats in this group; 5)endurance training, dosing and acupoint iontophoresis group (EDAI, n=8), rats in this group were treated having both Chinese herbs and acupoint iontophoresis everyday, 6 days a week.
Training method
Each group (except the contrast group) received exercise training on animal platform everyday and 6 days a week, the load was increased during the whole 8 weeks. Before the test, the experimental animals received one-week adaptive training. The load increased from the second week as follow: 20m/minx20min, 25m/minx20min, 25m/minx25min, 25m/minx30min, 30m/minx25min, 30m/minx30min, and 35m/minx30min.
Harvesting specimen
At the end of the experiment, all rats were sacrificed by intravenous injection of a lethal dose of barbiturate. The left ventricles were extracted and fixed in 10% neutral buffered formaldehyde (pH 7.2) for 48 hours. After being dehydrated, the specimens were embedded in paraffin. Sections of up to 5μm were made and specimens were stained with an immunohistochemistry Kit.
Immunohistochemistry
5μm sections were deparaffinized in xylene and then passed through decreasing gradients of ethanol. Endogenous peroxidase activity was blocked with 1% hydrogen peroxide. The sections were then immersed in 20% normal horse serum to block nonspecific reactions and were incubated in moisture chamber at 4 degrees Celsius overnight with Mouse monoclonal antibody against PKC, an intrinsic protein expressed yellow identically in myocardial cell. 0.01M phosphate-buffered saline solution was applied as a control.
RT-PCR
Immediately extracted 100mg heart point organization when the rats were sacrificed, and preserved in liquid nitrogen. The total of myocardial RNA were extracted by TRIZOL, and take 2μg RNA to carry on RT-PCR according to operation manual. After amplification, the products of RT-PCR did electrophoresis on gel, then analysed the result using KS400 image analysis system.
Statistics
All data were analysed with ANOVA. Significance was set at p< 0.05. Analysis was performed using the statistical software package SAS 6.12 for Windows.
Results
PKC can be seen on the cell membrane and in the cell nucleus after endurance training while it mainly exists in plasma among contrast group. Both PKC protein and mRNA expression are higher in ET group, ED group or EAI group than in contrast group (P<0.05), especially in EDAI group (P<0.01). The results summarized in Tables 1 and 2 and could been seen in Figures 1 and 2.
Table 1. Effects of acupoint iontophoresis on endurance training
rat myocardial PKC protein expression
The index of immunohistochemistry = expression strength × expression area/260000; *compared with control group: P<0.05; # compared with * group: P<0.05; 1 compared with *# group: P<0.05. ![]() Figure 1. Result of immunohistochemistry of rat myocardial PKC protein
A. Control group: PKC mainly exit in cell plasma, expression strength weak and expression area small.
B. ET group: PKC could been seen both in cell membrane and in the cell nucleus, expression strength were stronger than Control group, and the expression area was also enlarged.
C. ED group: PKC’ expression strength and area were much higher than ET group.
D. EAI group: PKC’ expression strength and area weren’t changed than ED group.
E. EDAI group: PKC could been seen in cell membrane and nucleus clearly, expressed extensively in sight.
Table 2. Effects of acupoint iontophoresis on endurance training
rat myocardial PKC mRNA expression
The index of immunohistochemistry = expression strength × expression area/260000;
*compared with control group: P<0.05; # compared with * group: P<0.05; 1 compared with *# group: P<0.05. ![]() Figure 2. Result of RT-PCR of rat myocardial PKC mRNA T
Discussion
Exercise can be taken as a kind of preconditioning that cause relative ischemia and hypoxia in myocardium. Repetitions enable athletes to adapt to the load and therefore engage in longer and more vigorous exercise. Such kind of adaptation can partly be explained by some endocrine change, such as incretion of atrial natriuretic factor, catecholamine, endothelins, angiotensin, etc. it can also cause some initial response gene (such as c-fos, c-myc, Egr-1, myb, ebrA, CuZn-SOD, et al.) and the subsequent response gene (such as MHC, MLC-2, Actin, et al.) to express [6-9]. Meanwhile, tumor correlation gene, cytoskeleton protein gene, G protein signal transduction gene and CD98 gene is also present in a different expression [10]. All of these molecular changes could result in myocardium hypertrophy and function reinforcement. On the other hand, if the heart load is beyond its bearing capability, it would result in ischemia and hypoxia locally and produce free radicals, heat shock protein and cytokine, which could damage myocardium structure and function. Therefore, we combined the Chinese herb, acupoint and modern physical methods organically, expecting it could increase some protective gene, protein or endogenous protective medium to express.
PKC is a calcium and phospholipid dependence phosphorylase, constituted by types of isoenzyme of serine/threonine protein kinase family and distributing widely in organic tissue. It can transmit signals from the nucleus to the out-cell, therefore its activation plays an important role in across membrane signal delivering processes [11]. It can protect cells and adjust its growth, contraction, excretion, conduction, permeability, extracellular matrix and gene expression via catalysising many protein phosphoricacidulated.
In this study, it could be observed that PKC transferred after the endurance training when having Chinese herbs and acupoint iontophoresis. Whereas in the control group it existed mainly in the cell plasma. This illustrates that endurance training with Chinese herbs or acupoint iontophoresis can activate PKC and accelerate its protein expression, although the expression level is different. The results also showed that having Chinese herbs or acupoint iontophoresis had a stronger effect than endurance training and that there is a synergism between these two methods. Moreover, we found that PKC mRNA expressed in low amounts in normal myocardium and expressed significantly higher amounts after endurance training and with Chinese herbs or acupoint iontophoresis than in the control group. Especially after combining the two methods (Chinese herbs and acupoint iontophoresis (P<0.05)). This explains why activated PKC might be an important pathway for acupoint iontophoresis to protect heart and prevent cardiogenic fatigue.
Till now, researchers have detected nPKC-d, nPKC-e, nPKC-a, nPKC-? et al isoforms existing in myocardial cell by utilising the immunoblot technique. However, there are still many areas which need further investigation. The exact mechanism of PKC cardioprotection may contribute to a more appropriate application of external facilities.
Conclusion
Endurance training can activate PKC to transfer, increase its protein and mRNA expression, but the effects are not as strong as when having Chinese herbs and acupoint iontophoresis; the effect seems stronger than one method used on its own.
Since the heart function is the main restricting factor in an endurance event, activating PKC may be one of the main mechanisms that acupoint iontophoresis protects the myocardium and prevents it from exercise induced fatigue.
References
[1] Yi Y, Zhanghua L, Jinsen H, et al. Effects of Acupoint Transcutaneus Electrical Nerve Stimulation on Endurance Training in rats. Chin J Rehabilitation Theory and Practice. 2004,10(7):408-410
[2] Lei L, Xiaojin X. The effect of electric acupuncture on free radical metabolism and function of rat’s kidney mitochondria in exhausting swimming. Chin Acupuncture and Moxibustion. 2001,21(6): 366-368
[3] Xunjie G, The improvement effect of electric acupuncture on mouse performance. Clinical J of Acupuncture and Moxibustion. 1998,14(12): 9-10
[4] Ding HL, Zhu HF, Dong JW, et al. Intermittent hypoxia protects the rat heart against ischemia/reperfusion injury by activating protein kinase C. Life Sci. 2004 Oct 8;75(21):2587-603.
[5] Sarre A, Lange N, Kucera P, et al. MitoKATP channel activation in the post-anoxic developing heart protects E-C coupling via NO, ROS and PKC dependent pathways. Am J Physiol Heart Circ Physiol. 2004 Nov 18 doi:10.1152
[6] Baldwin KM, Haddad F. Effects of different activity and inactivity paradigms on myosin heavy chain gene expression in striated muscle. J Appl Physiol, 2001,90(1): 345-357
[7] Yun C, Zhongying Z. The expression of cardiac MLC-2 in endurance trained rats. Chin J Sports Medicine, 2000,19(2):132-134,144
[8] Min Z, Junzong P, Quan Z. The effect of exercise preconditioning on the Cu-Zn gene expression in ratheart. Chin J Sports Medicine, 2001, 20(3): 239-240,309
[9] Jin H,Yang R,Li W,et al. Effects of exercise training on cardiac function,gene expression,and apoptosis in rats. Am J Physiol Heart Circ Physiol,2000,279(6):H2994-3002
[10] Zhenjun T, Zhiqi Z, Liang T, et al. Study on differentially expressed gene of exercise-induced cardiac hypertrophy mice using cDNA microarray. Chin J Sports Medicine, 2002, 21(2): 122-126
[11] Wang Yanlin, Wang Chenyao, Zeng Rui, et al. Effect of angelica on protein kinase C during myocardial ischemia reperfusion injury in rat. Chin J Anesthesiol, 2000, 20(8): 490-491
[12] Peter J. Parker, Judith Murray-Rust.Cell PKC at a glance. J Cell Sci. 2004 Jan 15;117(Pt 2):131-2.
Nishizuka Y. Protein kinase C and lipid signaling for sustained cellular responses. FASEB, 1995;9(7):484
[13] Liu WS, Heckman CA.The sevenfold way of PKC regulation. Cell Signal.1998;10(8):529
[14] Neckar J, Markova I, Novak F, et al. Increased expression and altered subcellular distribution of PKC isoform {delta} in chronically hypoxic rat myocardium: involvement in cardioprotection. Am J Physiol Heart Circ Physiol. 2004 Dec 2; [Epub ahead of print]
[15] Kim MJ, Moon CH, Kim MY, et al. Role of PKC-delta during Hypoxia in Heart-Derived H9c2 Cells. Jpn J Physiol. 2004 Aug;54(4):405-14.
[16] Cleveland JC, Meldium DR, Rowland RT, et al. The obligate role of protein kinase C in mediating clinically accessible cardiac preconditioning. Surgery, 1996,120(2): 345-353
[17] Light PE, Sabir AA, Alien BG, et al. Protein kinase C-induced changes in the stoichiometry of ATP binding activate cardiac ATP-sensitive K+ channels. A possible mechanistic link to ischemic preconditioning. Circ Res, 1996,79(3): 399-406
[18] Wang Y, Hirar K, Ashraf M, et al. Activation of mitochondrial ATP sensitive K channel for cardiac protection against injury is dependant on protein kinase C activity. Circ Res, 1999,85(8): 731-741
Yi Yang
Wuhan Institute of Physical Education Wuhan, China 430079 Tel: +86-27-87190188 Fax:+86-27-87190851 E-mail: yangyi999999@yahoo.com ![]() http://www.icsspe.org/portal/index.php?w=1&z=5 |