Effect of monochromatic lights combinations on morphological change of bursa
To investigate whether monochromatic lights combinations promoted bursa development, we examined the morphological changes in the bursa. The organ index is shown in Fig. 1a. At P42, the organ index of G→B was the highest and was significantly larger by 27.61–95.45% (P = 0.000–0.038) than that of WW, RR, GG, BB, and R→B, but no significant difference was found between G→B and B→G (P = 0.075). The H&E staining results showed that the bursa of the G→B had the largest follicle area and the highest lymphocyte density in the medulla compared to that of WW, RR, GG, BB, and R→B, respectively (Fig. 1b-j). However, there was no significant difference between G→B and B→G (P = 0.090–0.747). These results indicated that a green and blue monochromatic light combination could better promote the bursa of Fabricius development than WW, RR, GG, BB, or R→B.
Effect of monochromatic lights combinations on PCNA, cyclin D1 expression, and B-lymphocyte proliferation in the bursa
Next, we assessed whether a combination of monochromatic light could affect bursa B-lymphocyte proliferation in vivo and in vitro. In vivo, B-lymphocyte proliferation was detected by PCNA immunohistochemistry. The PCNA-positive cells were scattered in the bursa medulla and cortex. As shown in the Fig. 1k-r, the IOD of PCNA-positive cells was higher in G→B than in WW, RR, GG, BB, and R→B (31.72–170.44%, P = 0.000–0.025). Similarly, the proliferation of bursa lymphocytes in response to LPS treatment was highest in the G→B group at P42 (Fig. 2a). There was no significant difference between the G→B and B→G (P = 0.353), but G→B was higher than that of B→G by 1.29%. Additionally, we tested the protein level of cyclin D1, which plays an important role in the G1 phase progression of the cell cycle in proliferating cells. As shown in Fig. 2b, cyclin D1 expression was higher in G→B than in WW, RR, GG, BB, and R→B (38.78–61.31%, P = 0.009–0.028). However, there was no significant differences between G→B and B→G (P = 0.225). These results showed that a green and blue monochromatic light combination could enhance B-lymphocyte proliferation in the chick bursa.
Effect of monochromatic lights combinations on plasma melatonin level
The plasma melatonin levels of chickens after exposure to various monochromatic lights are shown in Fig. 2c. By 42 d, the chickens that were exposed to G→B showed significantly higher levels of circulatory melatonin compared to chickens reared under WW, RR, GG, and R→B (P = 0.005–0.045). There was no significant difference between the G→B and B→G (P = 0.502), but G→B was higher than that of B→G by 8.89%. Additionally, there was a strong correlation between the melatonin concentration in the plasma and the stimulation index of B lymphocytes (r = 0.9674, P = 0.000). These results suggested that melatonin plays an important role in the monochromatic lights combinations-induced B lymphocyte proliferation in response to LPS.
Effect of exogenous melatonin on B-lymphocyte proliferation in vitro
To further verify whether exogenous melatonin promoted the proliferation of B lymphocytes in vitro, we used an MTT assay to determine the effect of 0 pg/mL to 2 000 pg/mL of melatonin on B-lymphocyte proliferation after 44 h of culture. As shown in Fig. 2d, when the concentration of exogenous melatonin was ≤ 250 pg/mL, there was a strong correlation to strengthen the statement of melatonin enhanced lymphocyte proliferation in a dose-dependent manner (r = 0.9994, P = 0.022). According to the results of the MTT assay, we chose 250 pg/mL of melatonin for the follow-up experiments. These results indicated that melatonin has the proliferation promotion effect on B lymphocyte.
Effects monochromatic lights combinations on melatonin receptor expression in the bursa
The classic melatonin signaling was induced by binding to its membrane receptor (Mel1a, Mel1b and Mel1c), then activated melatonin membrane receptors initiated downstream intracellular signaling pathways. To investigate the effects of a combination of monochromatic light on the melatonin membrane receptors, we measured the expression levels of Mel1a, Mel1b, and Mel1c mRNA and protein. As shown in Fig. 3a-c, G→B significantly upregulated Mel1a, Mel1b, and Mel1c expression, while RR suppressed the Mel1a, Mel1b, and Mel1c expression at the mRNA levels. However, there were no differences found among G→B and B→G (P = 0.089–0.315). Similar results were seen in western blot analysis (Fig. 3d-f), although no differences were found among G→B, and B→G (P = 0.182–0.473). Additionally, a strong correlation between B-lymphocyte proliferation and the protein expression of Mel1a (r = 0.9111, P = 0.0043), Mel1b (r = 0.9307, P = 0.0023), and Mel1c (r = 0.8949, P = 0.0065) was noted. These findings implied that Mel1a, Mel1b, and Mel1c may be involved in a combination of monochromatic light-induced-B lymphocyte proliferation and bursal development, but they may play different roles in this process.
Effects of melatonin receptors on the combinations of monochromatic lights-induced bursa B-lymphocyte proliferation
To determine the involvement of melatonin receptors on B-lymphocyte proliferation, primary cultures were pretreated with luzindole (a nonselective antagonist of both Mel1a and Mel1b), 4-P-PDOT (a selective antagonist of Mel1b), and prazosin (a selective antagonist of Mel1c). As shown in Fig. 5a and 5c, we found that pretreatment of B lymphocytes with luzindole or prazosin in response to LPS + melatonin significantly decreased the B lymphocyte stimulating index and cyclin D1 protein expression by 13.01–15.93% (P = 0.000–0.001) and 22.93–31.29% (P = 0.000–0.007) compared with the LPS + melatonin co-treated group, respectively. However, 4P-PDOT (0.1 µM), which was co-incubated with LPS and melatonin, showed no statistical significance when compared with the LPS + melatonin co-treated group (P = 0.120). Thus, melatonin-induced B-lymphocyte proliferation was mediated by Mel1a and Mel1c, but not Mel1b.
Effects of a combination of monochromatic light-induced p-AKT, p-PKC, p-ERK, p-GSK-3β, and β-catenin protein expression in the bursa
To determine the molecular mechanisms underlying the effects of a combination of monochromatic light-induced B lymphocyte proliferation, we examined the expression of PI3K/AKT signaling and PKC/ERK signaling related proteins in chick bursa under WW, RR, GG, BB, G→B, B→G, and R→B. Western blot analysis revealed that the p-AKT, p-PKC, p-ERK, p-GSK-3β, and β-catenin protein were significantly upregulated in G→B (Fig. 4a-e), but no significant difference was found between G→B and B→G (P = 0.054–0.772). These results indicated that AKT, GSK-3β, PKC, and ERK cascade may participate in a combination of monochromatic light-induced Mel1a-and Mel1c-mediated B-lymphocyte proliferation.
Gi coupled to Mel1a is involved in melatonin-mediated a combination of monochromatic light-induced B-lymphocyte proliferation
Melatonin receptors are typically coupled to Gi, Gq or Gs proteins. To identify which specific G proteins coupled to Mel1a or Mel1c for the relay of melatonin to downstream activation in B lymphocytes, we isolated B lymphocytes from G→B chickens bursa and treated them with melatonin, LPS, and melatonin receptor antagonists either alone or together. Then, cAMP production was determined by ELISA. As shown in Fig. 5b, the melatonin + LPS co-treated group showed significantly decreased cAMP levels (36.10%) compared to that of the control group (P = 0.000), and this response was prevented by luzindole and not affected by 4P-PDOT or prazosin. Taken together, these data indicated that Gi might couple to Mel1a and be involved in B-lymphocyte proliferation in G→B chickens. Notably, pretreatment of B cells with Mel1c antagonists (prazosin) before melatonin addition did not affect melatonin-induced inhibition of cAMP levels, indicating that Mel1c might not depend on Gi or Gs to activate B-lymphocyte proliferation.
Mel1a-activated PI3K/AKT through Gi involved in melatonin-mediated a combination of monochromatic light-induced B-lymphocyte proliferation
To further confirm that the PI3K/AKT pathway was involved in melatonin-induced B lymphocyte proliferation, we treated cells with LY294002, a PI3K inhibitor, and HY102, an AKT inhibitor. We found that the treatment of isolated B lymphocytes with LPS + melatonin for 30 min markedly elevated the ratios of p-AKT/total-AKT. However, LY294002 and HY102 significantly abrogated the melatonin induced-upregulation of the p-AKT/total-AKT ratio (Fig. 6a) and inhibited B-lymphocyte proliferation (Fig. 6f). Additionally, the observed melatonin-induced upregulation in the ratio of p-AKT/total-AKT was abrogated by luzindole and not affected by 4P-PDOT or prazosin (Fig. 5d). Taken together, these results indicated that melatonin activated PI3K/AKT through Mel1a in LPS-stimulated B-lymphocyte proliferation.
Mel1c-activated PKC/ERK in melatonin-mediated a combination of monochromatic light-induced B-lymphocyte proliferation
Regulation of ERK activity by melatonin has been reported to play an important role in the proliferation, migration, and differentiation of a variety of cell types. Phospholipase C (PLCβ)/protein kinase C (PKC), which is a typical upstream transduction factor of ERK, can stimulate the ERK module by direct phosphorylation or by indirect C-Raf stimulation. We blocked the activity of PKC and ERK using the specific inhibitors Go9863 and PD98059 to determine whether the Mel1c-mediated B-lymphocyte proliferation is PKC/ERK-dependent or independent. The MTT and western blots assays showed that Go9863 or PD98059 significantly inhibited B-lymphocyte proliferation and decreased the ratio of p-ERK/total-ERK compared with the LPS + melatonin co-treated group (Fig. 6b, 6f). Additionally, melatonin-induced upregulation of p-PKC and p-ERK1/2 protein expression was abrogated by the Mel1c antagonist and not affected by 4P-PDOT or luzindole (Fig. 5e–5f). These data implied that Mel1c-activated PKC/ERK in melatonin-mediated a combination of monochromatic light-induced B-lymphocyte proliferation.
Mel1a/Gi/PI3K/AKT and Mel1c/PKC/ERK signaling pathways are involved in melatonin-mediated a combination of monochromatic light-induced B-lymphocyte proliferation
To better understand the different intracellular signal dependency of B lymphocytes by Mel1a and Mel1c, we determined the downstream pathways involved in PI3K/AKT and PKC/ERK. It was previously reported that GSK-3β/β-catenin pathways are important candidates as downstream mediators of AKT protein. As expected, the results showed that LPS + melatonin co-treatment had markedly elevated ratios of p-GSK-3β /total- GSK-3β ratio and the protein level of β-catenin by 69.33–69.62% compared with the control cells (P = 0.000) (Fig. 6c-6d). Additionally, the phosphorylation of GSK-3β and β-catenin evoked by treatment with melatonin was markedly inhibited by treatment with luzindole, LY294002, or HY102 (Fig. 5g-5h, 6c-6d). Consistent with this, the promoting effect of melatonin on B-lymphocyte proliferation and cyclin D1 protein expression was significantly strengthened by the GSK inhibitor TWS119 (Fig. 6e-6f). Unexpectedly, prazosin, Go9863, and PD98059 all significantly inhibited the p-GSK-3β/total- GSK-3β ratio, β-catenin, and cyclin D1 protein expression compared with the LPS + melatonin co-treatment (Fig. 5g-5h, 6c-6e). These results provide strong evidence that Mel1a mediates the-PI3K/AKT signal pathway and the Mel1c-mediated-PKC/ERK signaling pathway has a cooperative action in promoting melatonin-induced B-lymphocyte proliferation.