A valuable model of UVB-induced oxidative damage in HaCat cells
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Presented by: Xin Yang
At the moment of "effective skincare", the biological effect of UV has been paid more and more attention. Numerous studies have shown that UV is the main environmental factor leading to skin photoaging. Excessive UV exposure can cause a variety of skin damage, such as sunburn, skin darkening, skin photosensitivity, etc. Many cosmetic companies are working on skin care products that can effectively resist UV light damage. In order to establish a biological evaluation system for the cell-level evaluation method of cosmetic raw materials stably responding to photoaging, we have developed a UV simulator for cells that contain both UVB and UVA light. This simulator simulates UVA and UVB ultraviolet radiation in sunlight and designed for in vitro evaluation of chemical and cosmetic phototoxicity safety and anti-ROS effect. We measured the oxidative damage marker ROS level, the release level of cellular inflammatory factors, and cell apoptosis, and cell cycle changes in human keratinocyte HaCat after UVB irradiation, and comprehensively evaluated the physiological changes of HaCat cells after UVB irradiation. A model of UV-induced oxidative stress in skin-related cells was successfully established, and the antioxidant capacity of vitamin E was verified by this oxidative stress model.
Keywords: UVB; ROS; inflammatory factors; apoptosis; cell cycle
Research purposes: A UV-induced oxidative stress model of skin-related cells was established to provide an efficacy evaluation method for the development of cosmetic raw materials that can effectively resist UV damage and photoaging.
Research methods: 1. The UVA/UVB simulator uses LED lamp beads, which have the advantages of good wavelength controllability. By setting a number of sensors to collect the radiation intensity, the device can provide real-time feedback and accurately calibrate the irradiation power of the LED lamp beads to realize the uniformity and controllability of the illumination in the irradiated areas. 2. Keratinocytes HaCat were cultured in DMEM medium containing 10% fetal bovine serum. The cells were quantitatively seeded in 6-well plates and cultured in a 37°C, 5% CO2 incubator. Human keratinocytes HaCat were irradiated with a certain dose of UVB after 24 hours. 3. The apoptosis degree, cell cycle, and reactive oxygen species level of HaCat cells after UVB irradiation were detected by flow cytometry. 4. The effects of UVB irradiation on the production of inflammatory factors TNF-α, IL-6, and IL-8 in HaCat cells were determined by qPCR. 5. The ability of vitamin E to scavenge ROS in UV-induced HaCat cells was determined by flow cytometry.
Research results: 1.UV simulator has a compact structure with three independent irradiation areas, each of which can accommodate a standard culture plate. The irradiation intensity and energy of UVA and UVB of three independent irradiation areas can be precisely adjusted and controlled. For the traceability of test data, the device is equipped with an experimental process recording module, to record the operating parameters after each startup for subsequent experimental analysis. 2. Compared with the control group, the HaCat cell cycle did not change in the 900mJ/cm2 UVB group, but its ROS production increased significantly (P=0.0143), and its apoptotic rate increased by 5%, so a ROS-positive model was successfully established. 3. Compared with the control group, TNF-α, IL-6, and IL-8 inflammatory factors in HaCat cells in the 900mJ/cm2 UVB group increased, indicating that 900mJ/cm2 UVB can stimulate HaCat cells to secrete inflammatory factors TNF-α, IL-6, and IL-8. 4. The constructed ROS-positive model was used to detect the antioxidant capacity of vitamin E, and the results showed that 100ppm of the vitamin had significant ROS scavenging ability(P=0.0103).
Conclusion: We developed a UV simulator for cells and conducted an experimental study of UVB-induced oxidative stress in HaCat cells. The UVB-induced HaCat cell ROS-damage model was successfully constructed, providing a powerful evaluation method for the development of cosmetic raw materials that effectively resist UV damage and photoaging.
Keywords: UVB; ROS; inflammatory factors; apoptosis; cell cycle
Research purposes: A UV-induced oxidative stress model of skin-related cells was established to provide an efficacy evaluation method for the development of cosmetic raw materials that can effectively resist UV damage and photoaging.
Research methods: 1. The UVA/UVB simulator uses LED lamp beads, which have the advantages of good wavelength controllability. By setting a number of sensors to collect the radiation intensity, the device can provide real-time feedback and accurately calibrate the irradiation power of the LED lamp beads to realize the uniformity and controllability of the illumination in the irradiated areas. 2. Keratinocytes HaCat were cultured in DMEM medium containing 10% fetal bovine serum. The cells were quantitatively seeded in 6-well plates and cultured in a 37°C, 5% CO2 incubator. Human keratinocytes HaCat were irradiated with a certain dose of UVB after 24 hours. 3. The apoptosis degree, cell cycle, and reactive oxygen species level of HaCat cells after UVB irradiation were detected by flow cytometry. 4. The effects of UVB irradiation on the production of inflammatory factors TNF-α, IL-6, and IL-8 in HaCat cells were determined by qPCR. 5. The ability of vitamin E to scavenge ROS in UV-induced HaCat cells was determined by flow cytometry.
Research results: 1.UV simulator has a compact structure with three independent irradiation areas, each of which can accommodate a standard culture plate. The irradiation intensity and energy of UVA and UVB of three independent irradiation areas can be precisely adjusted and controlled. For the traceability of test data, the device is equipped with an experimental process recording module, to record the operating parameters after each startup for subsequent experimental analysis. 2. Compared with the control group, the HaCat cell cycle did not change in the 900mJ/cm2 UVB group, but its ROS production increased significantly (P=0.0143), and its apoptotic rate increased by 5%, so a ROS-positive model was successfully established. 3. Compared with the control group, TNF-α, IL-6, and IL-8 inflammatory factors in HaCat cells in the 900mJ/cm2 UVB group increased, indicating that 900mJ/cm2 UVB can stimulate HaCat cells to secrete inflammatory factors TNF-α, IL-6, and IL-8. 4. The constructed ROS-positive model was used to detect the antioxidant capacity of vitamin E, and the results showed that 100ppm of the vitamin had significant ROS scavenging ability(P=0.0103).
Conclusion: We developed a UV simulator for cells and conducted an experimental study of UVB-induced oxidative stress in HaCat cells. The UVB-induced HaCat cell ROS-damage model was successfully constructed, providing a powerful evaluation method for the development of cosmetic raw materials that effectively resist UV damage and photoaging.