The Study Of Hypoxia-Induced Immune Injury And Its Intervention Measure

More people now enter into high altitude with the development of western area and the rise of high altitude travel. Subjects who rapidly ascend to altitudes above 3000 m may develop acute mountain sickness(AMS), which symptoms are headache, nausea, lassitude, dizziness insomnia et al, the malignant form will be high altitude cerebral edema (HACE) and high altitude pulmonary edema (HAPE) .Hypoxia is principal feature of high altitude, which can cause serial changes in body and is also the pathological basis of development of many diseases, such as shock, cancer, and immunology diseases. Although several efforts have been devoted to study the effects of hypobaric hypoxia on respiratory, cardiovascular, nervous, and locomotor system, less emphasized is the injury of the immune system to adverse environmental condition. Epidemiologic data indicated that local resident had higher risk in lung infection, soldiers of high altitude were more susceptible to pneumonia than low altitude, the incidence and the death rate of pneumonia in local infant was higher. High altitude cold was difficult to heal and was easy to evolve to HAPE, which could be severe enough to lead to death. These observations suggest that the host defense mechanisms might be impaired in hypobaric hypoxia which makes persons more susceptible to infectious disease and facilitates the occurrence and the development of high altitude illness. This will seriously influence the health of people who immigrate to high altitude and their work efficiency, and it is an urgent problem. Thus the pathophysiology of hypoxia-induced immune injury and its intervention measure became the focus of attention.The ideal method of hypobaric hypoxia was used. The animals were exposed to a simulated altitude of 8000 m in a decompression for different time. The change of nonspecific cellular immunity, specific cellular immunity, humoral immunity and immune organ index were observed. The aim was intended to look for the indexes of marked change in order to research the characteristic of hypoxia-induced immune injury, and the possible mechanism of hypoxia-induced immune injury was analyzed from lymphocyte activation, lymphocyte apoptosis and whether lymphocytes redistribute to the important organ. New Compound Codonopsis Pilosula Tablet (NCCPT) and Tai Kang Capsule (TKC) were used to study the intervention effect on hypoxia-induced immune injury.I. Effect of hypoxia on immune system functionThe mice were exposed to a simulated altitude of 8000 m in a decompression chamber for varying periods-8h daily for 1 day, 6 days and 10 days, the effect of exposure to hypoxia on phagocytic activity of macrophage, proliferation of T-lymphocytes, delayed hypersensitivity, proliferation of B-lymphocytes and index of immune organ were investigated.Results showed that after exposed to 8000 m for 8 hours, calibration clearance index (carbon particle clearance test) was significantly increased compared with control group(P<0.05). After exposed to 8000 m for 6 days and 10 days, ear swelling (delayed type hypersensitivity) of hypoxic group was significantly decreased (P<0.01). Proliferate activity of spleen T-lymphocytes in hypoxic group was significant decreased compared with control group (P<0.05). Spleen was significantly enlarged, index of spleen was significantly increased (P<0.05), thymus was significant atrophy, index of thymus was significantly decreased compared with control group (P<0.05). Proliferation of B-lymphocytes was not significant change during the period of hypoxia. It is concluded that specific cellular immunity and immune organ (spleen and thymus) were significant injury in the early period of hypoxia.II. Study of the characteristic of hypoxia-induced immune injury and its mechanismsThe animals were exposed to a simulated altitude of 8000 m for varying periods-8h daily for 1 day, 3 days and 6 days, characteristic of hypoxia-induced immune injury was researched from lymphocyte subsets (CD3, CD4, CD8, CD19) of peripheral blood, spleen and thymus and the possible mechanism was analyzed from three aspects. 1. Effect of hypoxia on T lymphocyte subsets in peripheral blood, spleen and thymusExposure to hypoxia at 8000 m simulated altitude for 8 hours resulted in marked decrease in CD3+ lymphocyte percentage(P<0.01), marked increase in CD19+ lymphocyte percentage(P<0.01) of peripheral blood and marked decrease in CD4+CD8+ thymocytes (P<0.01), marked increase in CD4+CD8-, CD4-CD8+ thymocytes (P<0.01). After 3 days of hypoxia, aforesaid changes were further enlarged and the mice also had a much lower percentage of CD4+ T-cell (P<0.05) and the ratio of CD4+/CD8+ decreased significantly. After 6 days of hypoxia, CD3+,CD4+,CD8+ lymphocyte percentage of spleen were significantly decreased (P<0.01) compared with the other groups and there was no significant change in the percentage of CD8+ lymphocyte in peripheral blood and CD19+ lymphocyte in spleen during the whole hypoxia period. After exposed to hypoxia at 8000 m for 6 days, cellular immunity disorder mainly with the significant decrease of helper T lymphocyte was observed, at the same time a low function of generating mature lymphocyte in thymus (central immune organ) was found.2. Effect of hypoxia on activation of lymphocyte in peripheral bloodRats were exposed to hypoxia at 8000 m for 8 hours, and CD3+, CD8+, CD8+CD28- were significantly decreased (P<0.01). After 3 days of hypoxia, besides aforesaid change, CD4+, CD4+CD28+ also prominently decreased (P<0.01) and CD4+CD28- prominently increased (P<0.01). After 6 and 10 days of hypoxia, CD8+CD28+ significantly increased (P<0.01) while CD4+CD28-, CD4+CD28- nearly recovered as normal. The results showed that after exposed to hypoxia at 8000 m for 6 days, T lymphocyte activation of CD8+ and CD4+ was prominently decreased, while activation of CD8+ was significantly increased with the prolong of exposed time.3. Effect of hypoxia on apoptosis of lymphocyte in peripheral blood, spleen and thymusAccording to the change of cell membrane of early apoptotic cells, apoptosis were measured by Annexin-V/PI double labeling. Results showed that after simulated altitude of 8000m for 3 days, mice had a pronounced increase in rates of late apoptosis or necrosis of spleen lymphocyte and thymocyte (P<0.05). After 6 days of hypoxia, late apoptosis or necrosis lymphocytes of spleen and thymus were further increased (P<0.01), viable cell rates of spleen lymphocyte and thymocytes were markedly decreased (P<0.01), early apoptosis rates of spleen lymphocyte were markedly decreased (P<0.01) . It was inferred that after 6 days of hypoxia, lymphocytes possessing immunocompetence in thymus and spleen were significantly decreased, apoptosis and necrosis rates of lymphocyte was significantly increased.Lymphocytes were labeled by death factor (Fas) and results showed that after exposed to a simulated altitude of 8000 m for 8 hours, lymphocytes of CD3+CD95+ in peripheral blood and spleen were pronouncedly decreased (P<0.01). After 6 days of hypoxia, aforesaid change was further decreased (P<0.05). It was elucidated that after 6 days of hypoxia, apoptosis rate of lymphocyte in peripheral blood and spleen was pronouncedly decreased. After 6 days of hypoxia, lymphocytes of CD19+CD95 + in peripheral blood and spleen were pronouncedly increased (P<0.05). It indicated that after 6 days of hypoxia, apoptosis rate of lymphocyte in peripheral blood and spleen was pronouncedly increased.4. Effect of hypoxia on morphology of the important organAfter exposed to hypoxia at simulated altitude of 8000m for 6 days, the congestion of lobe of lung showed dark red under visual inspection. After 8 hours of hypoxia, a small quantity of red cell in alveolar space was observed under optical microscope. After 3 days of hypoxia, the infiltration of mononuclear cells besides red cells was found. After 6 days of hypoxia, a little pink diffusate in alveolar space and the hyperemia in kidney were observed.III. Study of intervention measure of hypoxia-induced immune injuryHypoxia-induced immune injury model was established on a simulated altitude of 8000 m for 6 days based on previous study, and the effectivity of pretreated NCCPT and TKC in modulating hypoxia-induced immune injury was discovered.1. Effect of NCCPT and TKC on anti-hypoxiaSurvival time in mice of all of TKC groups was significantly increased compared with control group (P<0.05), and there was dose-effect relationship. It demonstrated that TKC could improve hypoxia tolerance. 50mg/kg NCCPT could significantly increase the survival time in mice (P<0.05), and also there was dose-effect relationship. Medium dose of NCCPT combining with low dose of TKC had synergism, the survival time in mice was significantly increased (P<0.01) compared with single administration group. It showed that hypoxia tolerance was not reduced but improved when TKC combining with NCCPT.2. Immunomodulatory effect of TKC and NCCPT on hypoxia-induced immune injuryAfter exposed to hypoxia at simulated altitude of 8000m for 6 days, CD3+, CD4+ lymphocyte percentage of peripheral blood and CD4+ ,CD8+ lymphocyte percentage of spleen in mice were significantly decreased (P<0.01) , which showed the disorder of cellular immune function in mice. NCCPT, TKC and NCCPT+TKC could significantly increase the number of peripheral blood CD3+,CD4+ and spleen CD4+, but had no significant influence on the number of spleen CD8+. TKC, and TKC+NCCPT could significantly decrease the number of CD4+CD8+( P<0.01), increase CD4+CD8(-P<0.01), and had no significant influence on CD4-CD8+ in thymus. However, NCCPT didn't influence the component of thymocytes. Results indicated that both NCCPT and TKC could significantly increase lymphocytes of peripheral blood and spleen in hypoxia-induced immune injury mice, but the mechanism seemed to be difference.Therefore, we came to the conclusions:1. After exposed to hypoxia at simulated altitude of 8000m for 6 days, the characteristic of immune injury was the low specific cellular immunity accompanied with splenomegaly and atrophy in thymus. CD3+ T-Lymphocytes of peripheral blood were gradually decreased ,which was mainly reduction of helper T-cell (CD4+) accompanied with decreased of CD4+/CD8+ value in mice, the decrease of both CD4+and CD8+ cells in rats. CD3+ T-lymphocytes of spleen were significantly decreased induced by the decrease of CD4+ and CD8+, and CD4+/CD8+ value was not significant change. In the central immune organ, immature double positive cells in thymus were compensatory decreased in order to increase the mature single positive cells. However with prolonged exposure time, a low function of generating mature lymphocyte in thymus was formed.2. After exposed to hypoxia at simulated altitude of 8000m for 6 days, decrease of lymphocyte of periphery may be related with increase of apoptosis and necrosis of lymphocyte , increase of distribution of lymphocyte to lung in early period of exposure.3. NCCPT, TKC and NCCPT+TKC can significantly increase T-Lymphocytes cells of peripheral blood and spleen in hypoxia-induced immune injury mice, it shows that they have hopeful prospect in intervention study of hypoxia-induced immune injury.