ФЛОУЦИТОМЕТРИЧЕСКОЕ ИССЛЕДОВАНИЕ КОЛИЧЕСТВА Т-ЛИМФОЦИТОВ И НАТУРАЛЬНЫХ КИЛЛЕРОВ В ПЕРИФЕРИЧЕСКОЙ КРОВИ УЧИТЕЛЕЙ «ИСКУССТВА ЖИЗНИ», ЗДОРОВЫХ ЛЮДЕЙ И ОНКОЛОГИЧЕСКИХ БОЛЬНЫХ

Всеиндийский институт медицинских наук, Нью-Дели

Как известно, Т-лимфоциты (клетки-хелперы и клетки-супрессоры) и натуральные киллеры являются важным клеточным компонентом иммунной системы. Клетки-хелперы опосредованно вызывают иммунный ответ, обеспечивая выработку антител В-лимфоцитами, а также, активизируясь, продуцируют растворимые факторы, которые усиливают иммунную реакцию носителя. С другой стороны клетки-супрессоры/цитотоксические клетки способны уничтожать опухолевые и инфицированные клетки. Натуральные киллеры – это клетки-наблюдатели иммунной системы, которые могут очень эффективно напрямую уничтожать опухолевые и инфицированные клетки без предварительной сенсибилизации. Учитывая их значение в формировании иммунной реакции, было проведено настоящее исследование по подсчету этих клеток в периферической крови учителей «Искусства Жизни» (ИЖ), здоровых людей контрольной группы и онкологических больных методом флоуцитометрии с целью обнаружения различий показаний в этих группах.

Исследование показало, что общее количество Т-лимфоцитов и клеток-хелперов было значительно выше у учителей ИЖ и испытуемых контрольной группы по сравнению с онкологическими больными. Однако значительной разницы в количестве этих клеток у учителей ИЖ и здоровых людей не наблюдалось. Существенная разница была обнаружена в количестве натуральных киллеров, число которых было намного выше (p< 0,01) у учителей ИЖ по сравнению со здоровыми людьми и онкологическими больными. Больших различий в популяции натуральных киллеров у здоровых людей и онкологических больных не отмечено. Поскольку другие факторы не отличаются у здоровых испытуемых и учителей ИЖ, более высокий уровень натуральных киллеров у учителей ИЖ можно объяснить практиками ИЖ (Сударшан Крийа).

Objective: Oxidative stress or free radicals may contribute to the pathophysiology of atherosclerosis and other chronic diseases associated with aging. Because psychosocial stress has been shown to increase oxidative stress, we conducted an exploratory study to investigate the effect of Sudarshan Kriya (SK) program, on Superoxide dismutase (SOD), Catalase, Glutathione and blood lactate levels in long term practitioners of Sudarshan Kriya.

Method: Ten normally healthy subjects aged 21-27 years were recruited from Police Training College, New Delhi, India. They had been doing SK for 5 months. Their blood samples prior, during and after SK were analyzed for Superoxide dismutase (SOD), Catalase, Glutathione and lactate levels. Differences at 0 min, 45 min and 65 min in these parameters were analysed by t-test and correlations between different variables was done using Pearson's correlation coefficient.

Results: Significantly lower levels of blood lactate were found at 45 min and 65 min of SK and the antioxidants Glutathione and SOD increased at 65 min of SK. The Catalase activity was found to be increased at 45 min of SK in practitioners.

Conclusions: The findings of this exploratory study suggest that lower levels of blood lactate and a better antioxidant status in practitioners are associated with SK technique. However, this study needs to be done on a larger sample size to confirm this effect of SK.

The human body is constantly under the attack of reactive Oxygen species (ROS) either generated during the normal metabolic process in the cells or by external agents such as radiation, ultraviolet light, cigarette smoke, environmental pollutants like asbestos, pesticides, etc (1,2). ROS/ Free radicals can damage cellular functions and cause peroxidation of lipids in the cellular membrane (3,4,5).

The hypotheses that free radical mediated oxidation (i.e. oxidative stress) may contribute to the pathophysiology of atherosclerosis, coronary heart disease (CHD), other chronic diseases (e.g. cancer and rheumatoid arthritis) (6,7,8) and the aging process have gained increased acceptance (9). There is increasing body of evidence that chronic psychosocial stress may increase oxidative stress. Thus, psychosocial stress may contribute to the etiology of CHD, other chronic diseases and aging through free radical mechanisms (10).

Sudarshan Kriya (SK) is a breathing technique introduced by Sri Sri Ravi Shankarji and involves breathing in three different rhythms. It is preceded by Ujjayi Pranayam (long and deep breaths with constriction at the base of throat) and Bhastrika (fast and forceful breaths through nose alongwith arm movements) (Fig 1).

There are reports to suggest that stress reduction with the Transcendental Meditation (TM) program reduces psychosocial stress (11). However, to date, the effects of stress reduction with such stress management techniques on oxidative stress has been poorly investigated. Therefore, we conducted an initial exploratory study to evaluate antioxidant levels and blood lactate in practitioners of Sudarshan Kriya.

The 10 subjects were normal, healthy males. Since blood lactate and antioxidant enzymes may vary depending upon age, sex, diet, physical exercise and emotional stress, it was decided to have a homogenous group. The group consisted of ten males in the age range of 21-27 years from Police Training College (PTC), Delhi, India having same diet, physical exercise and living conditions. The experimental group had practiced Sudarshan Kriya for a period of five months before they were included in the study.

Blood was collected in EDTA containing tubes from the practitioners for estimating the antioxidant enzymes. The plasma was used for catalase estimation whereas the RBCs were used for SOD and glutathione estimation. 0.5 ml of plain blood was collected for lactate estimation and transferred to a tube containing lml of 5% perchloric acid. The supernatant was used for lactate estimation.

SOD estimation: SOD was estimated using a modified method of Marklund and Marklund (12). This method utilizes the inhibition of autoxidation of pyrogallol by SOD enzyme. 2.8 ml of Tris buffer containing 50 uM Tris & lmM EDTA was added to 100 ul of the sample to which 100 ul of pyrogallol was then added. For the control sample 2.9 ml of Tris buffer was added to lOOul of pyrogallol. The O.D. was then taken at 420 nm every 30 seconds for 5 minutes. The concentration of pyrogallol was adjusted to get the difference in the absorbance (0.020-0.030 nm) at the interval of one minute. The SOD activity was calculated as follows:

Glutathione estimation: Glutathione was estimated using the method of Tietze (13). 100 ul of 25% Trichloroacetic acid (TCA) was added to 0.4 ml of plasma. The solution was then centrifuged at 5000 rpm at 4°C for 15 min. 100 ul of protein free solution was then taken from the supernatant to which 2 ml of 0.6 mM Dithionitrobenzidine (DTNB) solution in 0.2M phosphate buffer, pH 8.0 was added. The volume was made to 3 ml with 0.2M phosphate buffer. Blank containing lOOul of 5% TCA and glutathione standard in the range of nanograms were run simultaneously. The Optical Density (O.D.) was taken at 412 nm. The amount of glutathione was then calculated from the standard curve, and the results expressed as nmoles of glutathione per mg protein. Protein estimation was done using the Bradford's method (14).

Assay of Catalase activity: Plasma Catalase was estimated using the method of Brannan et al (15). Briefly, 50 ul of Imidazole buffer (17.14 mM Imidazole plus 1% Triton x 100, 0.7% BSA) containing 12nM H202was added to 10 ul of plasma and the volume was made to 100 ul with distilled water. Sample containing lOul of lOmM sodium azide and blank containing plasma with only imidazole buffer but no H202 was also taken. The reaction mixtures were incubated at 25°C for 25 min. The reaction was stopped by adding 750 ul of stop buffer containing 0.1M potassium phosphate buffer, pH 7.4, peroxidase (800U/100ml), 6nM 0-dianisidine dimethoxybenzidine and 100% ethanol. The catalase activity was calculated as follows:

Lactate estimation: Lactate was estimated using the method of Pesce et al (16). 1 ml of perchloric acid was added to 0.5 ml of blood immediately after collection. It was then centrifuged at 4000 rpm for 15 min. 200 ul of supernatant was added to 2 ml glycine buffer containing 20 ul Lactate dehydrogenase (LDH). Then 200 ul of Nicotinamide Adenine Dinucleotide (NAD) was be added to the solution at 4°C. The reaction mixture was then incubated for lh at 25°C. The O.D. was taken at 340 nm against reagent blank containing only perchloric acid.

Statistical analysis - Mean difference between the various groups was assessed by unpaired and paired 't' test. Correlation between different parameters was calculated using Pearson's correlation coefficient.

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