| Preliminary studies confirmed that different wavelengths of monochromatic light can affect the proliferation and differentiation of skeletal muscle satellite cells during chick embryo incubation. Insulin-like growth factor (IGF-1) is an important factor in regulating of muscle growth and development, however the effects of light color on chick embryo liver IGF1-secretion and its mechanism are still unclear. In this study, the chick embryos were incubated under LED light shines stimulation with red light (660nm), green light (560nm), blue light (480nm), white light (400-760nm) and the dark condition (control group). We explore the effects of different monochromatic lights on IGF-1secretion and development of chick embryo liver, determine the role of melatonin and its receptors mediated antioxidant function, and related intracellular signaling pathways. The main findings are as follows:I:Monochromatic light affects IGF-1secretion of chick embryo liverFrom E15to E21, hepatic IGF-1secretion gradually increased with age of embryo (62.96%-114.83%, p<0.001). At E15, there was no significant difference; at E18, the IGF-1in GL was higher than that in other group by6.26%～14.11%, and compared with RL significant difference was found (p=0.036); at E21, IGF-1in GL was higher than that of other group by9.54%～28.43%(p=0.000-0.044), while RL was lower than Dark group and BL by12.01%～14.71%(p=0.000-0.020). Similarly, the expression of IGF-1mRNA in liver tissue gradually increased from E18to E21, but GL gained the largest increase, up to82.56%(p<0.001).Ⅱ:Monochromatic light affects the development of chick embryo liverFrom E15to E18, liver index increased by8.57%(Dark group),3.81%(RL),9.86%(GL),6.54%(BL) and6.13%(WL), respectively; from E18to E21, only in GL and BL, there was an increase (4.27%-0.44%), while decreased in RL (3.21%). At E15there was no significant difference; at E18, GL was the highest and RL was the lowest, GL was higher than RL by7.34%(p=0.007); at E21, GL was higher than other group by6.55%～15.64%(p=0.000-0.037). PCNA reflects liver cell proliferative activity. At E18and E21, the ratio of PCNA positive cells in GL was the highest and that in RL was the lowest, GL was higher than other group by4.10%～4.48%(Dark group),5.17%～10.58%(RL),1.75%～3.27%(BL) and3.57%～5.88%(WL).m:Monochromatic light affects melatonin secretion and mealtonin receptoer expression in chick embryo liverFrom E15to E21, the plasma melatonin gradually increased by83.45%～121.26%(p<0.001). At E15there was no significant difference; at E18, GL was higher than RL, BL and WL by5.57%～12.93%with difference (p=0.000-0.048); at E21, GL was higher than other group by6.71%～22.99%(p=0.000-0.039), RL and WL were lower than Dark group and BL signifcantly (p=0.000-0.024). Melatonin in plasma and liver IGF-1levels at E18and E21gained a significant correlation (r=0.895-0.931). The melatonin receptor protein level in chick embryo liver was detected by Western Blot method. At E21, Mellc was significantly higher than Mel la and Mellb. For Mella, GL was higher than the other group by32.00%～85.92%(p=0.000-0.019); RL was lower than Dark group and BL by26.80%v29.00%(p=0.028-0.040). For Mellb, GL wase the highest and only with RL there was a significant difference (37.50%, p=0.014). For Mellc, GL was higher than other groups by12.70%～111.94%(p=0.000-0.117). In addition, RL and WL were lower than Dark group and BL significantly (p=0.000-0.007). The trend of melatonin receptor mRNA was similar with protein results. Immunohistochemistry showed, Mel la was expressed in the hepatic artery endothelial cells, and Mellc was highly expressed in cell cytoplasm and Mellb significant positive signal was not detected.Ⅳ:Monochromatic light affects the antioxidant function in chick embryo liverFrom E15to E21, the antioxidant capacity of chick embryo liver gradually increased and lipid peroxidation product malondialdehyde (MDA) gradually decreased. At E15, there was no significantly difference (p>0.05). At E18green light can enhance the antioxidant capacity of chick embryo liver and at E21this effect was more significant (p<0.05). RL and WL significantly inhibited liver antioxidant capacity (p<0.05) at E21. Between GSH-Px, T-SOD, T-AOC, MDA and IGF-1level in liver there was a significant correlation (r=0.893-0.947).V:The receptor pathway of melatonin-mediated IGF-1secretion and antioxidant function in hepatocytes of chick embryoIn vitro,250pg/mL exogenous melatonin can significantly enhance IGF-1secretion of liver cells (22.57%, p=0.003), improve the levels of GSH-Px, T-SOD and T-AOC (17.82%～26.40%, p=0.009-0.022), while decreased the content of MDA by20.25%(p=0.017). However, this effects were inhibited by0.1μM Mellc specific receptor blocker Prazosin (p=0.009-0.031), not Mella/Mellb non-specific receptor blocker (Luzindole) and Mellb specific receptor blocker (4P-PDOT)(p>0.05).VI:The intracellular signaling pathways in monochromatic light-affected IGF-1secretion in chick embryo liverIn vivo, At E21, GL significantly inhibited the phosphorylation levels of JAK2/STAT3signals and the generation of reactive oxygen species in chick embryo liver (p<0.05); RL and WL activated JAK2/STAT3signals with much more ROS product (p<0.05). Meanwhile, GL decreased Caspase-3protein expression by22.00%～37.60%than other light group (P=0.021-0.045), increased Bcl-2expression in chicken embryo liver (p=0.000-0.022). JAK2/STAT3, Caspase-3and Bcl-2levels and IGF-1content in the liver showed a significant correlation (r=0.861-0.967). In vitro,250pg/mL exogenous melatonin can significantly inhibit the phosphorylation of JAK2/STAT3(26.00%～31.00%, p<0.001) and Caspase-3expression, increase Bcl-2level in liver cells; however this effects were blocked by0.1μM Prazosin (p<0.05). In H2O2-induced hepatic cells oxidative stress model, the pathway of melatonin was similar with above.Conclusion:During the incubation of chick embryo irradiated by monochromatic light, green light stimulation can significantly promote the development of the chick embryo liver and IGF1-secretion. The mechanism is that monochromatic green light can enhance melatonin secretion of chick embryo and melatonin receptor expression in liver. Melatonin inhibited the phosphorylation of JAK2/STAT3signal through Mellc receptor, and then enhanced antioxidant enzyme activities (GSH-Px, T-SOD) and improved antioxidant capacity, improved the proliferative activity and Bcl-2expression in liver cell, finally, affectd the developmentprocess of liver cll and IGF-1secretion. |
