The Effects And Mechanisms Of Baicalein On Mitochondrial Function Meliorating And Oxidative Stress Impairment Preventing In Human Vitiligo Melanocytes

Vitiligo is a common, depigmenting disorder of the skin with epidermal melanocytes loss.The prevalence of vitiligo is often referred to as 0.5-1% of the world’s population.Findings from one epidemiological study showed that almost 50% of people develop vitiligo before age 20 years. Vitiligolesions, without discomfort, but will seriously harm to the patient’s mental health, especially in adolescents and young adults, causing adverse effects such as low self-esteem, autism and etc.The clinical use of topical corticosteroids, immune modulators, and narrow bound-ultra violet B(NB-UVB) irradiation for the vitiligo treatment, has getten obvious therapeutic effect,which are based on the autoimmunity theory. However, these treatments have less effect on preventing the onset or spreading of the disease. In general opinions,autoimmunity, which only take place in post-onset phase,caused melanocyte damage. But the the exact mechanism of the activation of autoimmune response is unclear. Oxidative stress causing theory has been widely concerned in recent years, and has been supported by a series of experimental evidences. Some antioxidant treatment for vitiligo has achieved satisfactory outcomes in clinical study. Mitochondria are the major source of intracellular ROS, and they have a perfect antioxidant system under normal circumstances.Mitochondrial dysfunction causes intracellular ROS accumulation, which in turn damages mitochondrial. The cells in the lesion skin(keratinocytes) and perlesional skin(melanocytes and keratinocytes) have mitochondria ultrastructure damage in patients with vitiligo. these findings further support the oxidative stress theory.Baicalein is one of flavonoids extracted from the scutellaria baicalensis Georgi. It is found to have activities of antioxidation, anti-inflammatory,antivities, antibacterial. Our previous study confirmed that baicalein protects normal human melanocyte cell line(PIG1) against oxidative stress damage by inhibiting the p38 mapk pathway. We also found that baicalein can decrease the release of Cyt C, and inhibit mitochondrial apoptotic pathway to protect PIG1 cells, althrow the specific mechanism is still unclear. Dose BE protect cell under oxidative stress by improving mitochondrial dysfunction? And what’s the exact mechanism? Is there a similar effect on vitiligo melanocytes cell line(PIG3V)?NF-E2-related Factor2(Nrf2) is an important antioxidant transcription factor in cell, which protect melanocytes against oxidative stress by upregulation of HO-1. Is there any other mechanisms by which Nrf2 protects melanocytes? In order to solve these problems, we propose that baicalein improves mitochondrial dysfunction, promotes mitochondrial biogenesis through Nrf2-NRF1-TFAM pathway,as a resultenhances the antioxidant capacity of melanocytes under oxidative stress. In this study, we observed BE’s effects on the mitochondrial dysfunction and antioxidation ability of melanocytesin vitiligo melanocytes cell line(PIG3V). Furthermore, we explored the possible mechanism. This study will shed new light on the antioxidant mechanism of BE, and lay the foundations of BE’s clinical using for vitiligo treatment.Experimental design:1. BE’seffect on morphology, survival cell rate, mitochondrial ultrastructure, mitochondrial biosynthesis and functionin PIG3 V cells under oxidative stress;2. BE’seffect on Nrf2 pathway and mitochondrial synthesis related genein PIG3 V cells under oxidative stress;3. Effect of Nrf2 overexpression on mitochondrial function and biogenesisin PIG3 V cells.Methods:1. To observe the effect of BE pretreatment in an oxidative stress model of PIG3 V cells, we conducted an oxidative stress modle in PIG3 V. Study groups: control group: 254 medium; BE group: 40μM/L BEfor 1h, then switch to 254 medium; H2O2group: 0.75 m M/L H2O2; BE+H2O2group: 40 μM/L BE for 1h, then switch to 0.75 m M/L H2O2. In order to verify the anti-oxidative stress effect of BE in PIG3 V cells,we investigated the morphological changes of cell under the microscope, and detected the survival cell rate by CCK-8 assay. Electron microscope was used to observe the ultrastructural changes of mitochondria; Mitochondrial activity was detected by MTT assay. Intracellular ATP level was detected by ENLITEN ATP assay kit; mitochondrial membrane potential(MMP) and intracelluar ROS level was detected by flow cytometry; mitochondrial DNA(mt DNA) copy number was detected by real-time PCR(RT-PCR).2. Western blot was used to evaluate the level of Nrf2 and mitochondrial synthesis related proteins(NRF1, TFAM) in PIG3 V cells, which were treated by BE or H2O2;3. Then we used p EX-1-Nrf2 to upregulate Nrf2 expression in PIG3 V to establism a Nrf2 overexpression modle. Fluorescence and western blot wereused to confirm whether the modle was successful. In this modle,NRF1 and TFAM were estimated by RT-PCR and western blot. Mitochondrial DNA copy number was estamated by RT-PCR. Intracellular ATP level was detectes by ENLITEN®ATP detection kit. Furthermore, we detectedthe MMP.Result:1. Morphological observation and survival cell rate showed that BE protects PIG3 V cell under oxidative stress againstoxidative injury;2. BE pretects PIG3 V against the mitochondria ultrastructure damage by oxidative stress;3. BE ameliorates mitochondrial dysfunction in PIG3 V cell under oxidative stress by enhancingmitochondrail activity, improving MMP, promoting the ATP generation and decreasing intracellular ROS level.4. BE promotes the mitochondria biosynthesis in PIG3 V cell under oxidative stress;5. BE upregulates Nrf2, and increases NRF1, TFAM protein levels in PIG3 V cells under oxidative stress;6. Nrf2 ameliorates mitochondrial function in PIG3 V cell by improving MMP and promoting ATP generation.7. Nrf2 promotes the mitochondria biosynthesis in PIG3V: upregulating NRF1 and TFAM, and increasing mt DNA copy number.Conclusion:1. BEimproves the dysfunction of mitochondria, promotes mitochondrial biogenesis, and improves the anti-oxidative ability of PIG3 V cells under oxidative stress;2. BE upregulates NRF1,TFAM to improve mitochondrial dysfunctionand promote mitochondrial biogenesis through Nrf2 pathway in PIG3 V.