Calcitonin gene-related peptide cooperates with substance P to inhibit melanogenesis and induces apoptosis of B16F10 cells.

Skin is the largest organ in human body and works as biologically active barrier to provide critical preservation of body homeostasis. The skin is highly innervated by a plenitude of nerve fiber subpopulations, each carrying one or more neuronal mediators. Melanocyte itself also intimately contact with nerve fibers to form 'synaptic-like structure' and its functions may be directly regulated by the mediators contained in terminals of intra-epidermal nerve fibers. Clinical and biochemical studies have suggested that calcitonin gene-related peptide (CGRP) is involved in vitiligo skin. The present study was designed to investigate the effect of CGRP on epidermal melanocytes. After treatment with CGRP ranging from 0 to 500 ng/mL for 48 h, tyrosinase activity and melanogenesis were with little changes compared to treatment with medium only in B16F10 cells. Treatment with 500 ng/mL of CGRP cooperates with substance P (SP) (0.1-10 nM) to decrease tyrosinase activity and decrease melanin biosynthesis in B16F10 cells in a concentration-dependent manner. Furthermore, CGRP (8-37) antagonizes the synergistic effect of CGRP. The effect of CGRP on the cell apoptosis was examined. Treatments with 0-500 ng/mL of CGRP for 24 h, the expression levels of cleaved caspase-3, total caspase-3, cleaved caspase-9 and total caspase-9 were increased in a concentration-dependent manner. And 500 ng/mL of CGRP induced B16F10 cell apoptosis showed by TUNEL assay. In addition, Bax expression was up-regulated and Bcl-2 down-regulated in response to CGRP treatment. Hence, the Bax/Bcl-2 ratio was significantly increased. These in vitro observations indicate the pro-apoptotic impact of CGRP on B16F10 cell.

Interleukin-18 directly protects cortical neurons by activating PI3K/AKT/NF-κB/CREB pathways.

Interleukin-18 (IL-18), a member of the IL-1 family of cytokines, was initially identified as an interferon (IFN)-γ-inducing factor. IL-18 is expressed in both immune and non-immune cells and participates in the adjustment of multitude cellular functions. Nonetheless, the effects of IL-18 on cortical neurons have not been explored. The present study was conducted to investigate the influence of IL-18 on rat primary cortical neurons and elucidate the underlying mechanisms. We proved that rrIL-18 increased the brain-derived neurotrophic factor (BDNF) expression in a time-dependent manner. Treatment with rrIL-18 (50 ng/ml) deactivated phosphatase and tensin homolog deleted on chromosome 10 (PTEN) by facilitating its phosphorylation, enhanced the expression of Phosphoinositide 3-OH kinase (PI3K) and p-Akt, standing for the activation of the PI3K/Akt pathway. As its pivotal downstream pathways, nuclear factor-kappa B (NF-κB), cAMP-responsive element binding protein (CREB)/Bcl-2 and glycogen synthase kinase-3ß (GSK-3ß) were examined in further steps. Our data revealed that rrIL-18 stimulated NF-κB activation, improved p-CREB and anti-apoptotic Bcl-2 expression levels. But rrIL-18 had little or no effect on GSK-3ß pathway. Besides, rrIL-18 increased levels of BDNF and Bcl-2/Bax ratio and decreased cleaved caspase-3 expression to protect cortical neurons from damage induced by oxygen-glucose deprivation (OGD). These results in vitro showed the protection of IL-18 on cortical neurons. And this direct neuroprotective effect of IL-18 is crippled by PI3K inhibitor wortmannin.

Tanshinone IIA reduces apoptosis induced by hydrogen peroxide in the human endothelium-derived EA.hy926 cells.

ETHNOPHARMACOLOGICAL RELEVANCE: Salvia Miltiorrhiza Bunge (also known as herb Danshen in Chinese) is a widely used Chinese herbal medicine. Tanshinone IIA (TSN IIA) is considered to be the most important bioactive ingredient in Danshen and exhibits an anti-atherosclerotic activity. AIM OF STUDY: To evaluate the protective effect of TSN IIA on the human endothelial EA.hy926 cells injured by hydrogen peroxide in vitro and its possible mechanism. MATERIALS AND METHODS: The EA.hy926 cells were incubated for 24h with different concentrations of TSN IIA (5, 10 and 20 µg/µL ) or DMEM. Subsequently, cells were treated with 300 µmol/L H(2)O(2) for another 4h. Then, the percentage of cell viability was evaluated by 3-(4, 5-di-methylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) assay. The apoptosis of EA.hy926 cells was detected by flow cytometry with AnnexinV-FITC/PI double staining and laser scanning spectral confocal technique. The generation of intracellular reactive oxygen species (ROS) generation was analyzed by flow cytometry. The mRNA expressions of caspase-3, Bcl-2 and Bax were tested by real time-reverse transcription polymerase chain reaction (real time RT-PCR). The protein expression of Bcl-2 and Bax was determined by Western blotting. MDA levels, NO production, LDH leakage, and SOD as well as caspase-3 activities were also measured using standard methods. RESULTS: Loss of cell viability and excessive cell apoptosis were observed in EA.hy926 cells after 4h of challenge with H(2)O(2) (300 µmol/L). However, cell apoptosis was attenuated in different concentrations of TSN IIA (5, 10 and 20 µg/µL) pretreated cells. Furthermore, TSN IIA markedly inhibited the elevation of ROS evoked by H(2)O(2). Real time RT-PCR and Western blotting analysis showed that TSN IIA significantly decreased the expressions of pro-apoptotic proteins (Bax and caspase-3) while significantly increased the expression of anti-apoptotic protein Bcl-2, and resulted in obvious reduction of Bax/Bcl-2 ratio in EA.hy926 cells induced by H(2)O(2). CONCLUSION: These observations provide preliminary evidence that TSN IIA protects EA.hy926 cells against H(2)O(2) damage, which is mainly associated with the ROS generation, followed by the imbalance of the Bax/Bcl-2 ratio, and caspase-3 activation leading to apoptosis.

[Effect of tanshinone II A on TLR4 and TNF-α of endothelial cells induced by LPS].

AIM: To observe the influence of tanshinone II A on the expression of toll like receptor 4(TLR4) and tumor necrosis factor-α(TNF-α) in cultured human umbilical veins endothelial cells EA.hy926 induced by LPS, and to investigate the molecular mechanism of tanshinone II A against atherosclerosis(AS). METHODS: The EA.hy926 cells were cultured in vitro. The mRNA and protein expression of TLR4 were tested by RT-PCR and immunocytochemistry technique respectively. The mRNA and protein expression of TNF-α were detected by RT-PCR and ELISA technique respectively. RESULTS: The expression of TLR4 and TNF-α were both increased compared to normal group after stimulation of LPS(P<0.05). The expression of TLR4 and TNF-α in tanshinone II A group were obviously inhibited compared to stimulation group(P<0.05). CONCLUSION: The molecular mechanism of tanshinone II A against AS is probably to inhibit the expression of TLR4 and TNF-α.