| Circadian rhythms can be found at levels of physiological function, biochemical metabolism and behavior change in human, animals and other living organism. Structural basis of generating circadian rhythm consists in the circadian biological clock. Most important genes used to regulate and to output the circadian signal are named clock genes. Various changes in the endogenous circadian clock activities can be reset by a series of external environmental factors (zeitgeber, e.g., illumination, temperature, etc. ) in order to reach a accordance each other between body`s function and environmental change.The molecular regulatory mechanisms of clock oscillation are involved in clock signal input, several important clock genes, clock-associated genes, clock-controlled genes and their correspondent protein products, via the interconnection of intracellular transcription– translation - posttranslation event, to organize a fundamental molecular framework named autoregulatory feedback loop of the clock oscillator for accurate clock signal output. Of core clock genes, the Clock and Bmal1 whose products shape a heterodimer serving as a crucial positive transcription regulator in the loop, have been evidenced the 24-hour rhythmic expressions not only in the central clock tissues such as the suprachiasmatic nucleus (SCN) and the pineal gland (PG), but also in various peripheral clock tissues including blood cells. CLOCK:BMAL1, binding to the E-box element in the promoter region of the clock genes such as Period (Per), Cryptochromes (Cry) or Timeless (Tim), activate the above-mentioned genes transcription and translation. Therefore, clock genes, the Clock and Bmal1 have been regarded to play a crucial role in the molecular oscillatory mechanisms of biological clock.Nowadays, circadian expression patterns of the nuclear clock genes, Clock and Bmal1 in different central and peripheral tissues in mammals have been reported. Characteristics of the circadian rhythmic transcription and photoresponses of two genes in human peripheral blood lymphocytes under specific geographicical environment in Antarctica, however, is still open so far, which is an aim of present study.ObjectiveThis study was conducted to investigate the circadian rhythmic transcription of core clock genes, Clock and Bmal1 in the chinese scientific expeditioners` peripheral blood lymphocytes before and after Antarctica, and to better understand the molecular regulatory mechanisms of the human peripheral immune clock action under the specific geographicical environment.Methods8 healthy male volunteers, age from 24～30 years (mean age 25 years), came from chinese scientific expeditioners for Antarctica., they previously lived under the circadian model condition (natural light regime, 16 h-light : 8 h-dark cycle, LD) for one week before Antarctica: room temperature 25±1 oC, time out of bed 7:00, bedtime (no photoperiod) 23:00～7:00, breakfast time 7:30～8:00, lunch time 11:30～12:00 and supper time 5:30～6:00. Intensity of illumination was less than 0.1 Lux during sleeping time and subjects drank water freely, and had no smoking and alcohol drinking, no time zone-spaning traveling within 6 months and no pharmacotherapy but the same food intake and daily activities. Then every 2 subjects selected at random were sampled for each peripheral blood 6 ml every 4 hours in a circadian day and blood sampling was finished at 4 discontinuous random day and night. Lymphocytes were separated from blood and the total RNA was extracted from each sample. The quantitative real time reverse transcription polymerase chain reaction (RT-PCR) was used to determine the temporal changes in mRNA levels of Clock gene and Bmal1 gene during different zeitgeber time (ZT, n=6 for 24 hours, each time point was n=8). Gel electrophoresis and melting curve analysis were used for data double check. The data circadian parameters were obtained and analyzed by both the cosine function, Clock Lab software and the amplitude F test to reveal the genes` circadian rhythm in the LD (16:08) condition.After the same 8 healthy male volunteers experienced the specific geographicical environment changes in Antarctica such as illumination difference during polar day and polar night, season and climate discrepancy, temperature and atmospheric pressure variance, etc., for one year or so, the same manipulation of circadian model living, blood collection, sampling time, experiment testing methods and statistical treatment was correspondently carried out.Results1. Circadian expression of core clock genes, Clock and Bmal1, in peripheral blood lymphocytes of the chinese scientific expeditioners before Antarctica1.1 Under the LD (16:08) light regime, both the Clock and Bmal1 mRNA transcription in the chinese scientific expeditioners` peripheral blood lymphocytes displayed a robust circadian oscillation (amplitude F test, P<0.05).1.2 For the Clock genes, circadian parameters peak phase was -335.85±13.80, amplitude was 3.46±1.27, mesor was -11.40±1.59, peak time was ZT22, trough time was ZT10, mRNA level at peak was -7.95±2.55 and mRNA level at trough was -14.85±1.32.1.3 For the Bmal1 genes, circadian parameters peak phase was -307.12±108.17, amplitude was 2.77±1.11, mesor was-5.50±1.32, peak time was ZT20, trough time was ZT8, mRNA level at peak was -2.74±0.71 and mRNA level at trough was -8.25±2.33.1.4 Comparison of the the Clock and Bmal1 circadian rhythmic transcription showed that circadian expression levels of the Clock were less than those of the Bmal1 at all 6 time points in a circadian day under the LD (16:08) light condition (P<0.05). Circadian parameters mesor, mRNA level at peak and mRNA level at trough of the Clock expression was also respectively lower than that of the Bmal1 (P<0.05) but differences of two genes` peak phase and amplitude were not obtained (P>0.05). The Clock expression peak time and trough time were delayed 2 hours or so compared with the Bmal1.2. Circadian expression of core clock genes, Clock and Bmal1, in peripheral blood lymphocytes of the chinese scientific expeditioners after Antarctica2.1 The mRNA transcription of 2 expeditioners` Clock and 3 expeditioners` Bmal1 in the peripheral blood lymphocytes showed a significant circadian oscillation (amplitude F test, P<0.05) among the same 8 chinese scientific expeditioners.2.2 For 2 expeditioners` Clock genes, circadian parameters peak phase was -42.28±5.27, amplitude was 0.79±0.29, mesor was -9.02±0.24, peak time was ZT3, trough time was ZT15, mRNA level at peak was -8.23±0.53 and mRNA level at trough was-9.81±0.05.2.3 For 3 expeditioners` Bmal1 genes, circadian parameters peak phase was -184.58±29.58, amplitude was 1.04±0.65, mesor was-8.50±0.45, peak time was ZT12, trough time was ZT24, mRNA level at peak was -7.46±0.41 and mRNA level at trough was -9.54±1.04.2.4 Comparison of the the Clock and Bmal1 circadian rhythmic transcription displayed that circadian expression levels and circadian parameter values of 2 expeditioners` Clock were almost similar to those of 3 expeditioners` Bmal1 at 6 time points (except ZT14) in a circadian day under the LD (16:08) light condition (P>0.05). 2.5 The above-mentioned two genes expressions within 24 hours in other chinese scientific expeditioners` peripheral blood lymphocytes did not show the significant circadian oscillation (amplitude F test, P>0.05), which expression levels within 24 hours of the Clock, except ZT22, were less than those of the Bmal1 at other time points (P<0.05).3. Comparison of circadian transcription of core clock genes, Clock and Bmal1, in peripheral blood lymphocytes of the chinese scientific expeditioners before and after Antarctica3.1 Comparison of circadian transcription of core clock gene, Clock in peripheral blood lymphocytes of the chinese scientific expeditioners before and after Antarctica displayed that the Clock circadian rhythmic expression levels after Antarctica, compared with those before Antarctica, except ZT18 and ZT22, were increased at other time points (P<0.05). The Clock circadian parameters after Antarctica, amplitude was decreased but mesor and mRNA level at trough were increased (P<0.05) and peak time and trough time were delayed 5 hours or so.3.2 Comparison of circadian transcription of core clock gene, Bmal1 in peripheral blood lymphocytes of the chinese scientific expeditioners before and after Antarctica showed that the Bmal1 circadian rhythmic expression levels after Antarctica, compared with those before Antarctica, except ZT10 and ZT14, were decreased at other time points (P<0.05). The Bmal1 circadian parameters after Antarctica, amplitude, mesor and mRNA level at peak were all decreased (P<0.05) and peak time and trough time were delayed 16 hours or so.Conclusion1. Under the LD (16:08) light condition, core clock genes, both the Clock and Bmal1 mRNA transcription in the chinese scientific expeditioners` peripheral blood lymphocytes before Antarctica has a significantly circadian oscillatory feature with the peak at ZT22, trough during ZT 10 for the Clock and with the peak during ZT20, trough at ZT8 for the Bmal1. The circadian expression levels, mesor, mRNA level at peak and mRNA level at trough of the Clock are correspondently lower than those of the Bmal1 in a circadian day, and the Clock expressive peak time and trough time are delayed 2 hours or so compared with the Bmal1.2. The above-mentioned two genes expressions within 24 hours in majority of the same 8 chinese scientific expeditioners` peripheral blood lymphocytes after Antarctica are not possessed of the significant circadian oscillatory characteristics any longer, which expression levels within 24 hours of the Clock, except ZT22, are less than those of the Bmal1 at other time points.3. The mRNA transcriptions of 2 expeditioners` Clock and 3 expeditioners` Bmal1 in the peripheral blood lymphocytes continuously sustain the significant circadian oscillation among the same 8 chinese scientific expeditioners after Antarctica with the peak at ZT3, trough during ZT15 for the Clock and with the peak during ZT12, trough at ZT24 for the Bmal1. The changes in circadian expression levels and circadian parameters of 2 expeditioners` Clock at 6 time points (except ZT14) in a circadian day are not different from those of 3 expeditioners` Bmal1.4. The expeditioners` Clock circadian rhythmic expression levels after Antarctica, compared with those before Antarctica, except ZT18 and ZT22, are increased at other time points. The Clock circadian parameters after Antarctica, amplitude is decreased but mesor and mRNA level at trough are increased, and the peak time and trough time are delayed 5 hours or so.5. The expeditioners` Bmal1 circadian rhythmic expression levels after Antarctica, compared with those before Antarctica, except ZT10 and ZT14, are decreased at other time points. The Bmal1 circadian parameters after Antarctica, amplitude, mesor and mRNA level at peak are all decreased, and the peak time and trough time are delayed 16 hours or so. |
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