| Vitiligo is an acquired, progressive depigmenting disorder characterized by the loss of functional melanocytes from the epidermis. Apoptosis of melanocytes in vitiligo is supported by histologic findings. Although mechanism for loss of melanocytes is still not well understood, accumulating evidence suggests that the occurrence of oxidative stress plays a key role in development of vitiligo. Elevated level of dopamine (DA), an initiator of oxidative stress, reportedly is found in patients with vitiligo and induces oxidative stress and apoptosis in melanocyte in vitro. DA-treated melanocytes have been used as a model to search for antioxidants for treating vitiligo. Most traditional chinese medicines (TCM) derive from plant, which ingredients reveal antioxidants with anti-apoptosis property. Thus, seeking antioxidants from extracts of TCM for treating vitiligo suggests a new orientation in research of vitiligo.Objective:1.To establish the model of dopamine-induced oxidative stress-triggered apoptosis in melanocyte in vitro.2.Using this model, to detect the effects of 6 ingredients of TCMs, reportedly with anti-oxidative and anti-apoptotic properties, against oxidative stress-induced apoptosis in melanocytes.3.To investigative the effects of apigenin, the ingredient among the 6 ones which revealed a protective effect for melanocytes, on DA-induced oxidative stress and related signaling pathways including JNK(c-Jun N-terminal Kinase), p38 and PI3K (phosphatidylinositol 3-kinase) /Akt (v-akt murine thymoma viral oncogene homolog) in melanocytes, to detect its underlying mechanism.Methods: 1. Human melanocytes were isolated from foreskin of healthy children by primary culture. S-100 protein was dectected by immunocytochemical method to identify the melanocytes. MTT assays were used to measure the effects of dopamine at various concentrations and various time points on melanocytes viability. To identify the apoptosis of melanocytes induced by DA, the proportion of apoptotic cells was dectected by Annexin-V/PI staining flow cytometry and levels of cleaved caspase 3 and poly ADP-ribose polymerase (PARP) were measured by Western Blotting. DCFH-DA(2’,7’-dichloro?uorescin diacetate)method was used to measure the effects of DA on ROS (reactive oxygen species) generation in melanocytes. 2. MTT assays were used to detect the protective effects of 6 ingredients of TCMs including apigenin against DA-induced decrease of melanocyte viability. The effects of these ingredients on the proportion of apoptotic cells were dectected by Annexin-V/PI staining flow cytometry. 3. The effects of apigenin on DA-induced activation of caspase 3 and PARP were dectected by Western Blotting. And DCFH-DA method was used to measure the effects of apigenin on DA-induced ROS (reactive oxygen species) generation. 4. DA-triggered activation of JNK, p38 and Akt in melanocytes was investigated by Western Blotting. Annexin-V/PI staining flow cytometry analyse was used to detect the effects of pretreatment with p38 inhibitor SB203580, the JNK inhibitor SP600125 or the Akt inhibitor LY294002 on DA-induced apoptosis in melanocytes. And the effects of apigenin on DA-triggered activation of JNK, p38 and Akt were measured by Western Blotting.Results:1. The establishment of the model of dopamine-induced oxidative stress-triggered apoptosis in melanocyte in vitro.The purified melanocytes isolated by primary culture showed distinguished features of melanocytes and were identified as human melanocytes with high purity by detection of S-100 protein by immunocytochemical method. DA could inhibit melanocytes viability in a dose- and time-dependent manner. After exposure to 500μM DA for 12h, the melanocyte viability was decreased by approximately 50 %. And DA treatment for 12h could dose-dependently increase the proportion of apoptotic cells in Annexin-V/PI staining flow cytometry. The results of Western Blot analyse showed that treatment with DA for 6 or 12h significantly activated caspase 3 and PARP. And the levels of cleaved caspase 3 and PARP in 12h group were significantly higher than that in 6h group. After treatment of DA, the ROS generation in melanocytes was significantly increased. These results suggest that treatment with 500μM DA for 12h significantly triggered apoptosis in melanocytes.2. The screening of TCM-dereived antioxidants for protection of melanocytes against apoptosis According to the previous reports, 6 TCM-dereived anioxidants with anti-apoptosis property were selected, including apigenin, paeoniflorin, puerarin, acteoside, ginsenoside Rb1 and curcumin. Results showed that after pretreatment of various concentrations of the 6 antioxidants, only apigenin protected the melanocytes against DA-induced decrease of cell viability and inhibited the apoptosis induced by DA. Other antioxidants under concentrations used in present study showed no significant effects on cell viability and apoptosis.3. The effects of apigenin on DA-induced oxidative stress and apoptosis in melanocytesApigenin at 0.3~10μM protected the melanocytes against DA-induced desrease of cell viability and inhibited the apoptosis induced by DA in a dose-dependent manner. Pretreatment with apigenin inhibited the activation of PARP and caspase 3 as well as ROS generation induced by DA.4. The effects of apigenin on oxidative stress-related apoptotic signaling in melanocytesDA at 500μM increased the levels of phosphorylated-p38, JNK and Akt in melanocytes in a time-dependent manner, while inhibitors of p38, JNK and Akt significantly decreased DA-induced melanocyte apoptosis. This confirmed that DA-induced apoptosis in melanocytes depends on the activation of p38, JNK and Akt. Pretreatment with apigenin significantly inhibited DA-triggered activation of p38, JNK and Akt, suggesting the involvement of p38, JNK and Akt in the protective effects of apigenin against DA-induced cytotoxicity.Conclusion:1. Treatment with 500μM of DA significantly induced oxidative stress and apoptosis in melanocytes.2. Apigenin could inhibit DA-induced ROS generation and apoptosis in melanocytes, while paeoniflorin, puerarin, acteoside, ginsenoside Rb1 and curcumin at the concentrations used in present study showed no significant protective effects against DA-treated cell viability and apoptosis.3. DA-induced apoptosis in melanocytes depends on the activation of p38, JNK and Akt. The p38, JNK and Akt signalings were involved in the protective effects of apigenin against DA-induced apoptosis in melanocytes. |
