| Artificial controlled-illumination provides an important means of increasing efficient for modern poultry raising, and directly affects production performance and health of poultry. Our previous studies have demonstrated that green light enhances the humoral and cellular immunity in chickens and that is positive relationship with melatonin secretion. However, its mechanism is still unclear. In this study, post-hatching day (P) 0 Arbor Acre male broilers were randomly exposed to white light (WL,400-700 nm), red light (RL,660 nm), green light (GL,560 nm) or blue light (BL,480 nm) for 42 days, in this period the development of their spleens and the intracellular signal pathways of splenocyte proliferation were investigated. These results were as follows.1. The dynamic changes of development, plasma melatonin level and melatonin receptor expression in the spleen of broiler.The spleen growth of broiler was faster than weight gain before P7, and this change was opposite after P7. Periarterial lymphatic sheath (PALS) had appeared in the spleen of broilerwhen chick hatched out, however, splenic nodule didn’t appear until P14. In the following days both of them rapidly developed with an increase in age.The immunohistochemical detection of melatonin receptors (Mella, Mellb and Mellc) showed that Mella positive cells were predominantly located in the capillaries and red pulp of broiler spleen, Mellb positive cells were mainly distributed in splenic nodule and PALS, and Mellc positive cells were widespread distributed in splenic nodule, capillaries, PALS and red pulp. The mRNA and protein expressions of three melatonin receptor subtypes in the broiler spleen were increased by an age-dependent manner. Furthermore, plasma melatonin concentration also elevated with age, and positively correlated with the protein levels of Mella (r=0.938, P=0.005), Mellb (r=0.912, P=0.011), and Mellc (r=0.906, P=0.012) in the broiler spleen.2. Mekwonin mediated the effect of monochromatic light on the spleen development of broiler.At P14, spleen weight, spleen index, area of PALS, splenic nodule diameter, the IOD of PCNA positive cells and T-lymphocyte proliferation in the GL control group was higher than that in the WL control group (3.47%-30%, P=0.007-0.989), the RL control group (6.66%-39.29%, P=0.001-0.059) or the BL control group (2.71%-22.96%, P=0.000-0.292); Meanwhile, compared to other three roups, GL group markedly had a increase the melatonin concentration (10.93%-19.15%, P=0.004-0.029) in the plasma of broilers. However, when chickens were performed pinealectomy, the increased plasma melatonin levels were down by 16.07%-24.11%(P=0.002-0.011) in all monochromatic light treated groups, and accompanied with the decline of these spleen development index by different degrees (5.84%-30.86%, P=0.000-0.666). By in vitro and in vivo experiments we suggested that green light promoted the development of broiler spleen by increasing plasma melatonin concentration.3. Melatonin receptors mediated the promoting-effect of monochromatic green light on T-lymphocyte proliferation.In the broiler spleen, Mella mRNA expression was lower than that of Mellb or Mellc in all light control treatments group at P14. Both Mella and Mellc mRNA expressions of broiler spleen in the GL control group were higher than that in the WL control group (17.86%-40.43%, P=0.004-0.022) or in the RL control group (22.54%-33.85%, P=0.001-0.007). Likewise, the mRNA expression of Mellb in the GL control group was significantly elevated by 16.05%-30.56%(P=0.001-0.021) compared with WL, RL, and BL control groups. However, compared with the corresponding light treatment of the control and sham operation groups, the mRNA expression of Mella, Mellb and Mellc were downregulated between 42.42%-107.41%(P<0.05) after pinealectomy.The changes in protein expression of three melatonin receptor subtypes were similar to the changes in mRNA expression for each light treatment group. The protein level of Mella in the GL group was 16.67%-34.62%(P=0.000-0.044) higher than other light treatments. Moreover, Mellb and Mellc protein level in the GL control group were higher than the WL control group (19.44%-27.78%, P=0.007-0.009) and the RL control group (30.3%-39.39%, P=0.000-0.001). Pinealectomy decreased the protein level of three melatonin receptor subtypes by 37.50%-94.44%(P< 0.05) in all light treatments compared with the corresponding control light. These results suggested that green light markedly increased melatonin receptors expression.In vitro experiments showed that melatonin significantly increased T-lymphocyte proliferation by 9.7%(P=0.002). While using Mellb selective antagonist (4P-PDOT) or Mellc selective antagonist (prazosin) together with melatonin inhibited the promoting effect of melatonin on T-lymphocyte proliferation by 7.60%-11.19%(P=0.000); Furthermore, pretreatment with Mel 1 a/Mel lb non-selective antagonist (luzindole) did not affect T-lymphocyte proliferative response to melatonin treatment (P>0.005). These data indicated that Mellb and Mellc may involve the melatonin-induced T-lymphocyte proliferation.4. The intercellular signal pathway of melatonin receptors mediated the promoting-effect of monochromatic green light on T-lymphocyte proliferation.In vivo experiments showed ERK1/2 activity in the broiler spleen of GL group was 47.87% (P=0.048) and 50.17%(P=0.030) higher than of RL and BL group, respectively. However, performing pinealectomy remarkably inhibited the ERK1/2 activity by 40.15%(P=0.012) in the GL group. In vitro experiments showed T-lymphocyte proliferation which in broliers at P14 induced by melatonin treatment was accompanied with a parallel increase in ERK1/2 activity. However, pretreatment with a MEK-1 inhibitor (PD98059) decreased of T-lymphocyte proliferation and ERK1/2 activity by 11.96% (P=0.020) and 38.67%(P=0.000) in response to melatonin treatment. The stimulatory effect of melatonin on ERK1/2 activity was blocked by 4P-PDOT (38.67%, P=0.000) and prazosin (40%, P=0.000), but unaffected by luzindole. These results demonstrated that melatonin mediates green-light-induced T-lymphocyte proliferation via the Mellb and Mellc receptors followed by ERK1/2 activation.Besides, either an adenylyl cyclase activator forskolin, a PKA inhibitor H89, a PLC inhibitor U73122 or a PKC inhibitor Go6083, inhibited T-lymphocyte proliferation by 17.04%-28.15% (P=0.000-0.001) and ERK1/2 activity by 50.55%-70.32%(P=0.000) in response to melatonin, suggesting that the upregulation of T-lymphocyte proliferation and ERK1/2 activity by melatonin binding to Mellb and Mellc receptors were mediated through dual stimulation of the AC/PKA and PLC/PKC signal pathways.5. The non-receptor pathway of melatonin mediated the effect of monochromatic light on the development of broilers spleens.The activity of GSH-Px and SOD and T-AOC apacity in the broiler spleen of GL control group at P14 was higher than WL control group (13.35%-22.71%, P=0.000-0.029), RL control group (28.92%-35.63%, P=0.000-0.008) and BL control group (4.25%-16.48%, P=0.001-0.996). Furthermore, the content of MDA in GL control group was 25.28%-45.98%(P=0.000) lower than that in the other light treatment goups. However, pinealectomy caused the decrease of the activity of GSH-Px and SOD and T-AOC capacity (21%-70.01%, P=0.000-0.035), and the increase of MDA content (20.47%-59.78%, P=0.000-0.002) compared to the control group of the corresponding light treatment. These results demonstrated that green light increased the activity of SOD and GSH-Px in order to strengthen the antioxidant (T-AOC) capacity in the broiler spleen.Conclusion:Green light promoted the spleen development in young broilers exposed to the illumination of 15 lux. The research on its mechanism showed that green light enhanced the plasma melatonin level and the mRNA and protein expressions of three melatonin receptor subtypes (Mella, Mellb and Mellc) in young broiler spleen. Melatonin regulated ERK1/2 activity via the Mellb and Mellc receptors by triggering crosstalk between the cAMP/PKA and PLC/PKC signal pathways or improved the antioxidant capacity to enhanced spleen development and lymphocyte proliferation. |
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