The Regulation Of Hypothalamus-pituitary-adrenal Axis And Growth Hormone Axis In Experimental Intermittent Hypoxia Rats And Obstructive Sleep Apnea Syndrome Patients

ObjectiveBy researching hypothalamus - puitary - adrenal axis , growth hormone axis and orexin system variation of rats in intermittent hypoxia and OSAS patients, To explore whether intermittent hypoxia , the important pathophysiological change of obstructve sleep apnea syndrome , can lead to the hypothalamus - puitary - adrenal axis , growth hormone axis and orexin system variation or not ; In OSAS patients; to explore the relationship of hypothalamus - puitary - adrenal axis , growth hormone axis and sleep structure; to evaluate the effect of intermittent hypoxia in OSAS patients.Methods1 materials63 male mature Wistar rats , 300 - 350 g, gas exchange control sysytem, mixed gas; Trizol solution; ultraviolet light instrument; PCR cycler; MMLV RT - PCR kit; Fluorchem V2. 0 Stand Alonemicrosoftware; CRH, GHRH ELISA kit; ACTH, growth hormone radioimmunology kit; polysomnography; OSAS patients 33 cases including 28 males and 5 females,average age :male:49.6 ±9.9 years old, female: 47. 6 ±8.7 years old , BMI ( body mass index, BMI) : male: 27.9 ± 1.5.female:26. 9 ±2. 7 kg/m2. pure obesity without chronic respiratoryand circulation disease 1 lcases including 9 males and 2 females,age;male:46. 2 ± 10. 3years old,female;45. 5 ± 13.6 years old,BMI:male;27.2 ± 1.4,female; 25. 3 ±9. 9kg/m . There is no difference in age and BMI,there is no significant statistic difference in BMI and age among the same sex.2 Experiment procedure(1) Rats were randomly divided into three groups, 9 sub - groups, each group of rats were placed within a glass computer - controlled environmental chamber, in which the moment - to - moment desired oxygen concentration of the chamber was programmed and adjusted automatically. Group A; the IH profile consisted of alternating room air and 10% oxygen every 90 seconds at least 8 hours during the light phase. The input valve were controlled by a gas control system. According to the days after experiment , animals were divided to 1 day group ( A1) ,3 days group (A2) ,7 days group ( A3) and 4 weeks group (A4) ; Group B(CH) ; matched control animals were exposed to 10% oxygen continuously at the same time ,and divided to B1 ,B2,B3,B4 group respectively. Group C; animals were exposed to circulating normoxic gas (21% oxygen) in one of the chambers . Oxygen concentration were measured by an oxygen analyzer. Animals were kept on a 12; 12 - hours cycle . Food and water were available ad libitum.(2) Every OSAS patients are no chronic respiratory and circulation disease, no endocrinal and metabolic disease, no trauma and illness recently, routine diet and normal emotion before test. The monitor procedure is at least 7 hours, before and after sleep, vein blood 5ml were collected,and plasma were separated.3 Measurement methods.(1) Animals were killed after anaesthesia. The brains were immediately removed ,and the hypothalami were dissected. The samples were put into 1ml Trizol solution and stored in an ultra -low -temperature freezer.(2) Total RNA was extracted with CHC13, isopropanol, ethanol, DEPC and RNase - free water to determined reaction system, primers were added. RT -PCR reaction system was constructed according to the reference of MMLV RT -PCR kit. The reactions for amplified were conducted, Gels were run at a 2% agarose gel, 10 V for 2 hours and photographed under ultraviolet light using acamera. The images were captured and stored in a computer for densitometric a-nalysis using Fluorchem V2. 0 Stand Alone software.(3) CRH, GHRH of rats plasma were measured by ELISA and ACTH, GH , cortisol by RIA(4) OSAS patients'CRH, GHRH were measurement by ELISA and ACTH, GH and cortisol by radioimmunology. Calculate the difference and average between before and after sleep, calculate the ratio of CRH and GHRH. Statistics All data are expressed as means ± SE and were subjected to one - way ANOVA , paired one samples t - test.RESULTSIThe variation of CRH,GHRH mRNA in rats hypothalamus after hypoxia: compare to controls,CRH mRNA of hypothalamus increased and GHRH mRNA decreased after 1 day, 3 day,and 7days;After4 weeks,CRH increased and GHRH decreased in intermittent hypoxia, but in continuous hypoxia that is similar to normal.2 ACTH, cortisol and growth hormone of plasma results: ACTH and cortisol increased, GH no difference after 4 weeks intermittent hypoxia, but which in continuous hypoxia is similar to normal.3 The prepo - orexin mRNA levels in rats hypothamus which exposed to intermittent hypoxia were decreased significantly and OXR increased than those exposed to continuous hypoxia , as well as normal control after 4 weeks, but there were no difference between continuous hypoxia and normal control. After 1 day,3days, and 1 week, prepo -orexin mRNA levels decreased respectively and OXR increased than normal control, but there were no difference between IH and CH.4 The results of OSAS patients measurement: compare to pure obesity, OSAS patients'AHI, average awake duration, the maximal apnea duration, awake index increased significantly, average blood oxygen and the minimal blood oxygen ,3+4 stage sleep ratio decreased, but snore index no difference. Hormone level :compare to control, OSAS patients' CRH,cortisol increased;GHRH de-creased,and growth hormone no difference; the variation of CRH,GHRH before and after sleep is increased; and the ratio of the variation of them also. The multiple factor analysis of OSAS indicates: there is positive relation inACRH/ AGHRH and average awake duration, r =0. 882, negative relation to average blood oxygen r = -0. 696, negative relation to minimal blood oxygen; average blood oxygen is negative to average awake duration,r = -0.729, GHRH is positive relation to 3 + 4 stage sleep, r = 0. 852. The linear analysis ofACRH/ AGHRH ( Y) and average awake duration (X!) and blood oxygen (X2) ; regression equation are; Y = -109.821 +27.314X,; Y =4860.062 -51.375X2.Conclusion1 Intermittent hypoxia is the beginning factor of OSAS, and an important pathophysiological change, can lead to special neuroendocrinal variation which is different to that of other hypoxia mode.2 Hypoxia as a stress acted at bodies, CRH has beginning effect, and changed in mRNA level, HP A axis was activated, meanwhile GHRH was inhibited.3 Acute intermittent and continuous hypoxia can lead to similar HPA axis and GH axis level change, chronic continuous hypoxia have no significant effect on HPA and GH axis .chronic intermittent can lead HPA axis to be on a high level, GH axis was inhibited , feedback turbulence. Maybe it is the cause of abnormal hormone in OSAS patients which is discriminated to other respiratory disease with hypoxia .4 chronic intermittent hypoxia can lead to prepo - orexin reduced and it's receptor increased , while acute continuous hypoxia can change them , but chronic CH lead to no significant change in prepo - orexin. It can be the reason of OSAS patients'sleepiness and obesity.5 The cortisol and CRH level of OSAS patients increase,but ACTH normal, and affect on sleep structure, lead to awake increased during night and deep sleep decreased. GHRH decrease, the ratio of CRH and GHRH increase, and positive related to awake during sleep.