| A large number of researches showed that chronic intermittent hypobaric hypoxia(CIHH) has numerous benefits on the body, such as tolerance enhancement of organism to ischemia and anoxia, protection on heart, nerves and liver against ischemia/reperfusion or hypoxia/ reoxygenation injury, anti-hypertention effect. Meanwhile, CIHH has been widely used in sports training to enhance the tolerance of organism or tissues to anoxia. The limited literatures reported that CIHH could influence immune function of the body. Recently, our study showed that CIHH could antagonize the immunological dysfunction caused by acute hypoxia. Also, there are reports that CIHH had a positive treatment effect on some immunological diseases, such as chronic obstructive bronchitis and bronchial asthma. Therefore, it is reasonable to speculate that CIHH can prevent or treatment for some immunological diseases, such as rheumatoid arthritis.Rheumatoid arthritis (RA), a chronic systemic autoimmune disease, is characterized as progressive, symmetrical, multi-synovium arthritis and extra-articular lesions, simultaneously accompanied with cardiovascular, nervous and metabolic diseases. The study on the mechanism and treatment of RA has both theoretical significance and practice value. The Collagen-induced arthritis (CIA) rat model, a success experimental animal model for arthritis, is an ideal animal model of autoimmune arthritis because the pathological changes of blood and articular tissue in the model are similar to those in human RA. The CIA rat model is widely used in human RA research. The mechanism of RA, however, is not fully understood yet and there is no specific and effective therapy for RA treatment. The aim of present study was to investigate the effect of CIHH on CIA and to explore the underlying immunological mechanism, using flow cytometry, immunohistochemistry, ELISA and molecular biology methods in cell, tissue and whole body levels.This study consists of four parts: (1) To observe the protective effect of CIHH against CIA in rat and explore the mechanism of CIHH anti-inflammatory effect using ELISA, HE staining and immunohisto- chemical staining methods. (2) To investigate the effect of CIHH on T lymphocyte subsets in CIA rats and the immunological mechanism using flow cytometry and immunohistochemistry methods. (3) To investigate the effect of CIHH on apoptotic rate and the mechanism of apoptosis in CIA rat using Terminal dUTP nick end labeling (TUNEL), flow cytometry and immunohistochemistry methods. (4) To explore effect of CIHH on protein expression of nuclear factor-kappa B (NF-κB) and hypoxia- inducible factor 1 (HIF-1α) in synovial tissue and the signal transduction using Western blotsmethod.â… The protective effect of chronic intermittent hypobaric hypoxia against collagen-induced arthritis in ratObjective: To investigate effect of CIHH on CIA in rat and explore the mechanism of anti-inflammatory effect using ELISA, HE staining and immunohistochemical staining.Methods: Fifty male adult Sprague-Dawley rats were randomly divided into 5 groups: CIHH pre-treatment group(Pre-T), pre-control group(Pre-C), CIHH post-treatment group(Post-T), post-control group(Post-C)and blank control group(Con). The rats in Pre-T and Post-T groups were exposed to 28 days hypobaric hypoxia (simulated 3000m altitude, 5 h per day, PO2=108.8mmHg,O2:14%) in a hypobaric chamber before and after CIA, respectively. The rats in Pre-C and Post-C groups were experienced CIA only. The rats in Con group were not given any treatment. The thickness of two-hind paw in rat was measured and the degree of arthritis was evaluated by arthritis index (AI). The morphological change of synovial tissue of ankle joint in rat was observed by HE staining. TNF-αand IL-6 expression in synovial tissue of joint were examined by immunohistochemistry SP method. And ELISA method was used to measure TNF-αand IL-6 level in serum.Results: 1 Incidence rate of CIA in Pre-T rats was decreased significantly compared with Pre-C rats(P<0.05),but there was no significant change of incidence rate of CIA between Post-T and Post-C rats.2 The paw thickness and AI value in Pre-T and Post-T rats were lower than those in Pre-C and Post-C rats(P < 0.05).3 In Pre-C and Post-C rats, there were hyperplasia of synovial cell, pannus formation, infiltration with inflammatory cell, and destroyed cartilage and bone in ankle joint. On the contrary, pathological changes of ankle joint were alleviated significantly in Pre-T and Post-T rats.4 TNF-αand IL-6 in synovial tissue of joint and serum in Pre-CIHH and Post-CIHH rats were decreased significantly compared with Pre-Con and Post-Con rats,(P < 0.05).Conclusion: CIHH treatment decreases the prevalence of CIA and alleviate the symptoms of CIA, and reduces the pathological damage of joint tissue significantly,which suggests that the protective effect of CIHH against CIA was related to the inhibition on production of inflammatory cell factors TNF-аand IL-6.â…¡The cytoimmunity mechanism of T lymphocyte in protective effect of CIHH against CIAObjective: To observe effects of CIHH on T lymphocyte subsets in CIA rats and explore the cytoimmunity mechanism using flow cytometry and immunohistochemistry.Methods: Fifty male adult Sprague-Dawley rats were randomly divided into 5 groups: CIHH pre-treatment group(Pre-T), pre-control group(Pre-C), CIHH post-treatment group(Post-T), post-control group(Post-C)and blank control group(Con). The rats in Pre-T and Post-T groups were exposed to 28 days hypobaric hypoxia (simulated 3000m altitude, 5 h per day, PO2=108.8mmHg,O2:14%) in a hypobaric chamber before and after CIA, respectively. The rats in Pre-C and Post-C groups were experienced CIA only. The rats in Con group were not given any treatment. Flow cytometry techenique was used to measure CD4 and CD8 T lymphacytes in blood; ELISA method was used to measure IFN-γ, IL-4 and IL-17 level in serum; IFN-γ, IL-4 and IL-17 expression in synovial tissue of joint were detected by immunohistochemistry SP method.Results:1 Compared with Pre-C and Post-C rats, CD4 T lymphocyte in peripheral blood in Pre-T and Post-T rats was decreased significantly(P < 0.05), but CD8 T lymphocyte was increased, and ratio of CD4/CD8 was reduced(P < 0.05). There were no obvious changes of T lymphocyte in Pre-T and Post-T rats compared with Con rats.2 Compared with Pre-C rats, IFN-γ, IL-4, ratio of IFN-γ/IL-4 and IL-17 in serum of Pre-T rats were decreased significantly(P < 0.05);. Compared with Post-C rats, IFN-γ, ratio of IFN-γ/IL-4 and IL-17 in serum of Post-T rats were decreased, but IL-4 was increased significantly(P < 0.05).3 Compared with Pre-C rats, IFN-γ, IL-4 and IL-17 in synovial tissue of joint in Pre-T rats were decreased significantly(P < 0.05);. Compared with Post-C rats, IFN-γand IL-17 in synovial tissue of joint in Post-T rats were decreased, but IL-4 was increased significantly(P < 0.05). Conclusion: CIHH has an effect on stabilizing proliferation of CD4+T cell, keeping the homeostasis of CD4/CD8 and Th1/Th2 of T lymphocyte subset, and reducing the genesis of inflammatory cytokines of IL-17. The cytoimmunity regulation may be one of machanisms of CIHH against CIA.â…¢Apoptotic mechanism of protective effect of CIHH against CIAObjective: To investigate the effect of apoptotic rate in synovial cells and spleen lymphocytes of CIHH on CIA in rat and explore the apoptotic mechanism.Methods: Fifty male adult Sprague-Dawley rats were randomly divided into 5 groups: CIHH pre-treatment group(Pre-T), pre-control group(Pre-C), CIHH post-treatment group(Post-T), post-control group(Post-C)and blank control group(Con). The rats in Pre-T and Post-T groups were exposed to 28 days hypobaric hypoxia (simulated 3000m altitude, 5 h per day, PO2=108.8mmHg,O2:14%) in a hypobaric chamber before and after CIA, respectively. The rats in Pre-C and Post-C groups were experienced CIA only. The rats in Con group were not given any treatment. The apoptotic rate in synovial tissue of knee joint was measured by Terminal dUTP nick end labeling (TUNEL); and the apoptotic rate of CD3+ T lymphocyte in spleen was measured by flow cytometry techenique; the protein expression of Bcl-2 and Bax was measured using immunohistochemistry SP method.Results: 1 Compared with Pre-C and Post-C rats, apoptotic rates of synovial cell and T lymphocyte in Pre-T and Post-T rats were increased (P < 0.05).2 Compared with Pre-C and Post-C rats, decreased Bcl-2 expression and increased Bax expression of joint synovial cells and spleen lymphocytes in Pre-T and Post-T rats (P < 0.05), respectively.Conclusion: CIHH possesses a protective effect against collagen- induced arthritis in rat by enhancing apoptosis of synovial cells and T lymphocytes, which is related to the inhibition of Bcl-2 protein expression and the promotion of Bax protein expression.â…£Signal transduction of protective effect of CIHH against CIAObjective: To explore effect of CIHH on protein expression of nuclear factor-kappa B (NF-κB) and hypoxia-inducible factor 1 (HIF-1α) in synovial tissue and the signal transduction using Western blots technique.Methods: Fifty male adult Sprague-Dawley rats were randomly divided into 5 groups: CIHH pre-treatment group(Pre-T), pre-control group(Pre-C), CIHH post-treatment group(Post-T), post-control group(Post-C)and blank control group(Con). The rats in Pre-T and Post-T groups were exposed to 28 days hypobaric hypoxia (simulated 3000m altitude, 5 h per day, PO2=108.8mmHg,O2:14%) in a hypobaric chamber before and after CIA, respectively. The rats in Pre-C and Post-C groups were experienced CIA only. The rats in Con group were not given any treatment. The protein expression of VEGF and TGF-βin synovial tissue was measured using immunohistochemistry SP method; The synovial tissues were homogenated and the protein of HIF-1αand NF-κB was measured using Western blots method.Results: 1 Compared with Pre-C and Post-C rats, the protein expression of VEGF and TGF-βin Pre-T and Post-T rats was decreased (P < 0.05), respectively.2 Compared with Pre-C and Post-C rats, the protein expression of HIF-1αand NF-κB in Pre-T and Post-T rats was decreased (P < 0.05), respectively. The protein expression of NF-κB in Pre-T rats was increased compared withControl rats (P < 0.05). The protein expression of HIF-1αand NF-κB in Post-T rats was increased compared with Control rats (P < 0.05). Conclusion: CIHH possesses a protective effect against collagen- induced arthritis in rat by inhibiting the protein expression of HIF-1αand NF-κB and in turn inhibiting the protein expression of VEGF and TGF-β.
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