| ObjectiveHypoxia is a common physiological and pathological factor of many diseases. Long-term and chronic hypoxia causes injury to body in various aspects, which attracts more and more attention in research and clinic. Several research have shown the organism could behave as adipose metabolic disorder in long-term and chronic hypoxia state. Its mechanism is related to the key enzyme metabolizing the blood fat in liver, which is affected by hypoxia. The adipose metabolic disorder is the primary and independent risk factor of artherosclerosis and coronary heart disease. Obesity, adipose metabolic disorder and cardiovascular diseases are closely related to each other. Currently, there are few research about the effect of hypoxia on lipometabolism. And those researches mainly focus on cell injury and related lipometabolism, but lack of the observation on whole level. Especially for the chronic and continuous hypoxia, few related articles was found during literature retrieval. Our experiment is aimed to mimic the hypoxia state in vivo, exploring the effect of hypoxia on weight, blood glucose and blood fat on the whole level of rats. We also obeseve the mRNA change of hypoxia and liponetabolism related gene in liver, adipose tissue and skeleton muscle on the tissue level, so as to find the different expression level of target gene in different organs. Therefore we could know the relationship between hypoxia and adipose metabolism. Then we will explore the recovery and its mechanism of the pathological change after reoxygenation, and find the hypoxia-sensitive target organs and index, so as to provide new insight and experimental reference to the early diagnose and treatment of the disease.Methods56three-week male wistar rats were randomly divided into three categories or seven groups (8rats for each group). We treated these rats as following methods: normoxia-3group (euthanized after given normal oxygen for3weeks), hypoxia-3group (euthanized after given hypoxia for3weeks), normoxia-6group (euthanized after given normoxia for6weeks), hypoxia-6group (euthanized after given hypoxia for6weeks), reoxygenation-3group (given hypoxia for3weeks following normoxia for3weeks, then euthanized), normoxia-12group (euthanized after given normoxia for12weeks), reoxygenation-12group (given hypoxia for6weeks following normoxia for6weeks, then euthanized)After hypoxia treatment, we measured the weight, blood glucose, blood fat, serum high molecular weight adiponectin and interleukin-6(IL-6) of all rats. Using real-time PCR for liver, subcutaneous fat, brown fat, visceral fat and skeletal muscle tissue of all rats, we detected the mRNA change of hypoxia and adipose metabolic related gene6weeks after the experiment started.Results1. After3weeks, the erythrocyte counting (7.18±0.41vs.6.52±0.29×1012/L, P<0.01) and the hemoglobin in hypoxia-3group (187.50±13.43vs.141.63±6.12g/L, P<0.01) were significantly higher compared to those in nomoxia-3group, which is1.10and1.30times more than those in nomoxia-3group, respectively. The data obtained after6weeks showed similar results, the erythrocyte counting and hemoglobin were1.13and1.42times more than those in nomoxia-6group, respectively (P<0.05, P<0.01). The erythrocyte counting (6.62±0.5×1012/L) and hemoglobin (144.57±13.00g/L) significantly decreased in reoxygenation-6group to the level, which showed no significant difference compared to those in normoxia-6group, indicating that these index completely returned back to the nomoxia level. Similar results were found in reoxygenation-12group that the two index showed no significant difference compared to those in nomoxia-12group (P=0.19, P=0.28). The results above proved that the hypoxia rat-model were developed successfully.2. The whole levela) After3weeks, the rat weight in hypoxia-3group decreased by about20%compared to that in normoxia-3group (170.59±7.30vs.213.96±13.42g, P<0.01). After6weeks, the weight in hypoxia-6group decreased by about40%compared to that in normoxia-6group (P<0.01). The weight in reoxygenation-6group (278.00±10.46g) significantly increased compared to that in hypoxia-6group (P<0.01), but did not return to that in normoxia group (P<0.01). After12weeks, the weight in reoxygenation-12group were still13%lowered than that in the normoxia-12group (P<0.01). b) After6weeks, the fasting blood glucose level in hypoxia-6group significantly decreased compared to that in normoxia-6group (3.52±0.59vs.8.80±1.64mmol/L, P<0.01). The index significantly increased in reoxygenation-6group compared to that in hypoxia-6group (P<0.05) and showed no significant difference compared to that in normoxia-6group, indicating that it returned completely to the normoxia level. However, after12weeks, the index in reoxygenation-12group were still lowered than that in normoxia-12group (8.68±1.38vs.11.03±1.50mmol/L, P<0.01).c) The total blood cholesterol level (CHO) in hypoxia group significantly decreased compared to that in normoxia group (3weeks:1.66±0.14vs.2.09±0.18mmol/L;6weeks:1.16±0.10vs.1.65±0.24mmol/L, P<0.01). The index in reoxygenation-6group significantly increased (1.63±0.05vs.1.16±0.10mmol/L, P<0.01) to the level which showed no significant difference compared to that in normoxia-6group, indicating that it returned completely to the normoxia level. Similar to the effect of hypoxia on the CHO, the high-density lipoprotein (HDL-C) showed the same tendency. The HDL-C level in hypoxia group significantly decreased compared to that in normoxia group (P<0.01) and returned to normoxia level after reoxygenation. In contrast, the low-density lipoprotein (LDL-C) level and free fatty acid (FFA) level significantly increased compared to those in normoxia-6group (P<0.01) and returned completely to normoxia level in reoxygenetion-6group. However, the blood triglyceride (TG) level only decreased significantly in hypoxia-6group (P<0.01), but did not restore after reoxygenation.d) Using ELISA to measure the serum high molecular weight adiponectin and interlukin-6, we found that the serum high molecular weight adiponectin level in reoxygenation-12group significantly increased compared to that in normoxia-12group (17.08±2.35vs14.47±1.73μg/mL, P<0.05). No significant difference were observed between other groups. The interlukin-6level did not show significant difference between any groups either.3. The tissue levela) The hypoxia related gene:the relative expression level of HIF-la for subcutaneous fat in hypoxia-6group was significantly up regulated compared to that in normoxia-6group (276.87±2.77vs100±4.03, P<0.05). The index for brown fat in reoxygenation-6group significantly increased compared to that in hypoxia-6group and showed no significant difference compared to normoxia level. The relative expression level of HO-1significantly increased for the liver (236.54±2.53vs100±1.41, P<0.05) and the skeletal muscle (131.59±1.19vs100±1.23, P<0.01) in hypoxia-6group. After3-week reoxygenation, the index significantly reduced to the level which showed no significant difference to that in the normoxia-6group, indicating it completely restored to the normoxia level.b) Cytokine:the relative expression level of liponectin for skeletal muscle in hypoxia-6group significantly increased compared to that in normoxia-6group (131.59±1.19vs.100±1.23, P<0.01). After3-week reoxygenation, the index significantly reduced to the level which showed no significant difference to that in the normoxia-6group, indicating it completely restored to the normoxia level. The relative expression level of IL-6for visceral fat in hypoxia-6group reduced compared to that in normoxia-6group (51.91±2.31vs.100±2.02, P<0.05).c) Signal factors:the expression of AMPK (259.88±3.03vs.100±2.95, P<0.05) and NF-κB (184.41±2.17vs.100±1.56, P<0.05) for subcutaneous fat was up regulated in hypoxia, and was not significantly down regulated after reoxygenation. While the index for brown fat was down regulated (48.93±1.71vs.100±1.55, P<0.01) in hypoxia, and restored to normoxia level after reoxygenation (P<0.01).Conclusion1. By activating NF-kB, hypoxia induces the expression of inflammatory factors TNF-α and IL-6, causes the release of these two factors, which is probably the critical bridge connected NF-kB to the adipose metabolic disorder.2. By establishing reoxygenation groups, we confirm the pathologic injury caused by hypoxia could be recover in various degree after reoxygenation. Most index could restore to nomoxia level after3-week reoxygenation.3. Different tissues have different sensitive levels to the hypoxia stimulation. HIF-1α〠AMPK and NF-κB for subcutaneous fat and HO-1for liver and skeletal muscle are probably the sensitive targets of hypoxia stimulation. |
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