Effects Of Heat Stress On Calcium Signal Transduction Of Lymphocytes From Broilers And The Regulation By Chromium Picolinate

In vivo and in vitro experiments were conducted to investigate the effects of heat stress on calcium signal transduction of lymphocytes from broilers chickens. We explored the mechanism of heat stress modulate immune response at molecular level, in order to provide a basis for research into the nutritional strategies of reducing the negative effects of heat stress on broilers. This thesis was devoted to makes the research from three respects:1. Effect of heat stress on Ca2+ concentration of lymphocyte and immune response from broiler chickens(1) We investigated the effect of acute heat stress on Ca2+ concentration and cellular immunity in splenic lymphocytes from broiler chickens. Eighty 1-d-old Arbor Acres male broiler chickens were randomly allotted to 2 treatments, each treatment represented by 4 replicates of 10 birds each. At 49 d, the chickens of 2 treatments were housed in two controlled environment chambers, which temperature were kept 22℃and 35℃for 3 h, respectively. The result shows that acute heat stress caused a significant increase the [Ca2+]i and the secretion of interleukin-2 (IL-2) in lymphocytes,enhanced the ConA-stimulated lymphocyte proliferation significantly, increased the ratio of CD4+ to CD8+ in lymphocytes, and promoted the transition of activated T cells from the G1 to the S phase . These results suggest that acute heat stress could enhance immune function.(2) The experiment was conducted to investigate the effect of chronic heat stress on Ca2+ concentration and cellular immunity in splenic lymphocytes from broiler chickens. One hundred and fifty 1-d-old Arbor Acres male broiler chickens were randomly allotted to 3 treatments, each treatment represented by 5 replicates of 10 birds each. At 35 d, the normal thermal groups (NT) and paired-feeding group (PF) were housed in controlled environment chambers at 22℃.The high-temperature group (HT) was housed in a controlled environment chamber with daily cyclic high temperature (27℃—35℃—27℃), the experiment last two weeks. The result shows that chronic heat stress increased the [Ca2+]i of lymphocyte. After 7-d heat exposure,the secretion of IL-2 by splenic lymphocytes was increased significantly, and ConA-stimulated lymphocyte proliferation was suppressed significantly. The 14-d heat stress decrease the S phase proportion, and increase the G1 phase proportion of lymphocyte. In addition, the indicators mentioned above were not associated with feed intake of heat stress broiler chickens.2.Effect of high temperature on calcium signal transduction of lymphocyte from broiler chickens(1) We investigated effect of high temperature (44℃) and calcium channel blockers on the [Ca2+]i of lymphocyte . The rusult shows that expousure to high temperature induced an increase in the [Ca2+]i of lymphocyte. And the increase in [Ca2+]i was dependent mainly on the intracellular Ca2+ stores; Incubation at 40℃for 1.5 h, following heating for 15min at 44℃, resulted in partial recovery of the lymphocytes [Ca2+]i, but it did not return to the basal level; The increase in [Ca2+]i was induced by high temperature could be inhibited by Inositol 1,4,5-trisphosphate receptor (IP3R) blocker 2-APB, but not be influenced by calcium release activated channel blocker SKF-96365 and ryanodine receptor antagonists ruthenium red. The rusults suggest that the heat-induced increase in [Ca2+]i was dependent mainly on the IP3R-mediated calcium release channel on the endoplasmic reticulum(2) It was investigated that the effect of the Ca2+ ionophore and the membrane-permeant Ca2+ chelator on the IL-2 mRNA expression and IL-2 protein secretion. The rusult shows that when the Ca2+ ionophore A23187 increased the lymphocytes [Ca2+]i, the IL-2mRNA expression and IL-2 protein secretion of lymphocyte were increased; when the membrane-permeant Ca2+ chelator BAPTA-AM decreased the lymphocytes [Ca2+]i, the IL-2mRNA expression and IL-2 protein secretion of lymphocyte were decreased. The results suggest that the lymphocytes [Ca2+]i directly related with IL-2 mRNA expression and IL-2 protein secretion.(3) We investigated effect of the receptor blockers under high temperature on [Ca2+]i and the IL-2 mRNA expression in lymphocyte. The result shows that high temperature induce an increase in [Ca2+]i and the IL-2 mRNA expression of lymphocytes, and the increasement could be inhibited by the phosphatidylinositol-specific phospholipase C inhibitor U73122, IP3 receptor blocker 2-APB and CaM-dependent protein kinase (CaM-PK) antagonist W7. It suggests that the IP3/Ca2+ signal transduction plays an important role in the process of Ca2+ to IL-2 mRNA expression.3. Effect of Chromium Picolinate on Ca2+ concentration of lymphocyte and immune response from heat stress broiler chickens(1) In order to investigate the effect of chromium picolinate (CrPic) on Ca2+ concentration of lymphocyte and immune response from heat stress broiler chickens, three hundred 1-d-old Arbor Acres male broiler chickens were randomly allotted to 5 treatments (Ⅰ-Ⅴ), each treatment represented by 5 replicates of 12 birds each. At 15 d,the broilers in treatmentⅠ-Ⅳwere pre-fed 0, 400,800,1200μg /kg Cr of diet for 2 weeks, then they were housed in 3 controlled environment chambers with daily high cyclic temperature (27℃—35℃—27℃) for 14 d, the broilers in treatmentⅤwere kept in 22℃as the control group. The result shows that supplemental chromium (800-1200μg/kg Cr) decreased serum insulin concentration, supressed the increasement of the lymphocytes [Ca2+]i and IL-2 concentration, increased the lymphocyte proliferation. The results suggets that the CrPic could alleviate the negative effect on the cellular immune response of heat stress broiler chickens.(2) In vitro experiments were conducted to investigate the effects insulin, CrPic and insulin+CrPic on calcium homostasis of lymphocyte.The result shows that insulin+CrPic treatment caused more significant increase in [Ca2+]i recovery rate than CrPic or insulin treatment. It suggest that the CrPic could modulate the lymphocytes [Ca2+]i in potentiating insulin action.