BVD protects against UVB-induced HaCaT keratinocytes photodamage through reactivating Nrf2 antioxidative stress signaling CURRENT STATUS: POSTED

Background : To investigate the protective role and mechanism of exogenous biliverdin (BVD) on ultraviolet B (UVB) irradiated human keratinocytes (HaCaT cells). Methods : Cultured HaCaT cells were divided into control group, UVB group, and BVD + UVB group. Morphological changes in the HaCaT cells were observed, the cell viability of each group was detected, the mean fluorescence intensity (reactive oxygen species, ROS) of the cells was detected, the superoxide dismutase (SOD) and malondialdehyde (MDA) levels of the cells were detected, and the protein expression levels of nuclear factor erythroid 2-related factor 2 (Nrf2), matrix metalloproteinase-1 (MMP-1), and matrix metalloproteinase-3 (MMP-3) were determined. Moreover, Nrf2 gene transfection was performed in HaCaT cells. Results : UVB irradiation induced apoptosis of HaCaT cells, decreased Nrf2 protein expression and increased MMP-1 and MMP-3 protein expression, which increased ROS and MDA levels and decreased SOD levels. Nrf2 gene enhancement increased UVB-irradiated HaCaT cell viability. Conclusion : Exogenous BVD plays a protective role in UVB-induced HaCaT cell damage, and this effect may be related to the Nrf2 antioxidant signaling pathway. in the UVB and the statistically significant < 0.05). These results illustrate that gene enhancement increased the viability in UVB-irradiated HaCaT cells.

are mainly concentrated in fields related to the protection of the nervous system, the cardiovascular system, and organ transplantation [17,18]. However, there are no other researchers who have reported on the action of BVD in skin photoaging and cell photodamage. It has been reported in studies that BVR in skin fibroblasts decreases with age [19]; therefore, it may play a protective role in skin photoaging through the related metabolites BVD/BIL [20,21]. In a previous animal experiment, we confirmed that exogenous BVD can delay the UV-induced chronic skin photoaging in nude mice [22]. In the current study, we studied the antioxidative and antiaging role of BVD in UVB-induced HaCaT cell damage, and explored the mechanism of BVD antagonization of skin photodamage by observing the effect of BVD on Nrf2. In addition, we observed the severity of cellular damage from UV rays through activating or silencing the Nrf2 gene to further prove that Nrf2 may be related to the antioxidative stress pathway.
fluorescence intensity of the cells in the UVB group clearly increased, indicating that more ROS was generated in the cells after UVB irradiation, which caused certain damage to the cells. The ROS level (MEAN) of the cells incubated with BVD prior to UVB irradiation was clearly lower than that of the UVB group, indicating a dose-dependent correlation with BVD concentration. The results in Fig. 3g show that, compared with the control group, the SOD activity in the cells in the UVB group clearly decreased and the SOD activity in the cells incubated with added BVD before UVB irradiation was clearly increased, indicating a dose-dependent correlation with BVD concentration; the difference for each group was statistically significant (p < 0.05). The result in Fig. 3h shows that, compared with the control group, the MDA content in the cells in the UVB group increased, with a statistically significant difference (p < 0.05), and that the MDA content of HaCaT cells in each group irradiated by UVB after BVD pretreatment was reduced. However, only the difference between the 10 µmol/L BVD + UVB group and the UVB group was statistically significant (p < 0.05).

Effect of BVD on MMP-1 and MMP-3 protein expression in UVB-irradiated HaCaT cells
The western blot results showed that, compared with the control group, MMP-1 (Fig. 4a)  increased. The CCK-8 results showed that the cell viability of experimental groups was 100%, 21.06%, 71.25%, and 43.04% (Fig. 6b), respectively. Compared with the control group (no transfection, no UVB irradiation), cell activity in the UVB group was clearly reduced; the viability of cells in the shRNA + UVB group was clearly increased compared with that of cells in the UVB group, and the difference was statistically significant (p < 0.05). These results illustrate that Nrf2 gene enhancement increased the viability in UVB-irradiated HaCaT cells.

Discussion
Oxidative stress plays a key role in the pathogenesis of photoaging,and some studies have confirmed that antioxidants are very effective to prevent skin photoaging in recent years [23,24]. In this experient, the defense mechanism of BVD on the photodamage caused by UVB irradiation was explored in HaCaT cell. Both BVD and the metabolite BIL have antioxidation effects [25]. Under these in vitro conditions, BVD scavenges peroxyl radicals more effectively than unconjugatedor conjugated BIL: each molecule of BIL and BVD scavenges 1.9 and 4.7 molecules of peroxyl radicals, respectively.
In addition BVD/BIL have been reported to scavenge a number of other ROS, suggesting that BIL and BVD can scavenge both 1e-and 2e-oxidants, similar toascorbate [26]. By comparison, many other antioxidants are effective against only one class of oxidants. For example, atocopherol reacts rapidly with 1e-oxidants, but is a poor scavenger of 2e-oxidants [27]. In this study, we found that BVD had a protective effect against UVB-induced photodamage. Compared with cells in the control group, after 30 mJ/cm 2 UVB irradiation and a 24-hour culture, HaCaT cells became smaller and rounded, and the number of cells was clearly reduced. Moreover, as the BVD concentration increased, the cells pretreated with BVD had less cell damage, showing a dose correlation, which fully proved that BVD is able to increase the viability of UVB-irradiated cells.
Overexposure to UVB irradiation results in the overproduction of free radicals, playing a potentially important role in damaged skin, such as photo-damage, photo-aging and skin cancer. Free radicals can diminish the protection of antioxidant enzymatic activities, resulting in increased oxidative stress.
During oxidative stress, ROS and MDA are continuously being produced, and SOD is being decreased [28][29][30]. In this study, it was shown that the ROS in cells clearly increased after UV irradiation but that ROS decreased significantly in cells incubated for one hour with BVD before UV irradiation, indicating a dose-dependent correlation with BVD concentration. In this study, it was also shown that intracellular SOD activity clearly reduced after UV irradiation; after adding BVD at a certain concentration, SOD activity increased. In addition, this study showed that UVB irradiation increased the MDA levels in HaCaT cells and that the MDA levels in the UVB group pretreated with BVD were clearly reduced. The above results show that the ROS and MDA levels in cells in the BVD pretreatment groups were clearly reduced and the SOD activity was significantly increased, thereby proving that BVD plays an antioxidative role in UVB-induced photodamaged cells.
MMPs are secreted by keratinocytes and dermal fibroblasts after UV irradiation. wherein the abnormal expression of MMPs plays a major role in the pathophysiological mechanism of skin photoaging [31,32]. Long-term exposure to UV can reconstruct the extracellular matrix (ECM), degrade collagen, damage connective tissue, and generate a series of ROS, leading to premature photoaging of the skin [33]. The changes start from the activation of MMPs, ultimately leading to the generation of skin wrinkles [34]. Therefore, inhibiting MMPs generation has become an important strategy in the prevention of photodamage, and reducing the natural materials produced by MMPs can similarly prevent photodamage [35,36]. In this study, it was found that after keratinocytes were UV irradiated, MMP-1 and MMP-3 protein expression increased, whereas MMP-1 and MMP-3 protein expression in cells in the UVB group pretreated with BVD was clearly inhibited, showing a dose correlation, which further confirms that BVD has a protective effect against UVB-induced cell photodamage.
Nrf2 participates in the regulation of the cellular antioxidant response through the Nrf2/ARE pathway [37]. When the external redox environment changes or is induced by the signal transduction system, this pathway can be activated to switch on downstream target genes that play roles in antioxidation, antiinflammation, antiapoptosis, maintaining the stability of the internal environment, and so on. The Nrf2/ARE pathway plays a role in numerous diseases associated with the oxidative stress response [38][39][40]. In this study, we found that after enhancing the Nrf2 gene in keratinocytes is via lentivirus transfection, Nrf2 protein expression was significantly increased, and Nrf2 protein expression and viability of cells in the shRNA + UVB group were clearly higher than those of the cells in the UVB group, which further illustrates that the application of Nrf2 activators can increase the level of intracellular Nrf2 protein expression and the viability of UVB-irradiated cells. In a study by Soeur J et al., after UV irradiation was performed on Nrf2 gene knockout mice and antioxidative gene expression was reduced, the oxidative stress damage was clearly enhanced, thereby proving that the Nrf2-ARE pathway is important for regulating the intracellular redox status [13], which is consistent with our study. In this study, after the keratinocytes were UVB irradiated, Nrf2 protein expression was reduced, and cell viability was clearly reduced; however, Nrf2 protein expression was clearly increased and cell viability was clearly increased after UVB-irradiated cells were pretreated with BVD. This illustrates that BVD can activate the Nrf2 protein to antagonize the cell photodamage caused by UVB irradiation and that its mechanism may be related to the Nrf2 antioxidative stress pathway.

Conclusion
In summary, UVB irradiation can lead to a clear decrease in the viability of HaCaT cells, but the viability of cells in the UVB group pretreated with BVD was significantly increased, suggesting that BVD can antagonize the cellular photodamage caused by UVB irradiation. The Nrf2 gene has a protective effect against photodamage. As an activator of Nrf2, BVD has a protective effect in UVBirradiated HaCaT cells, and its mechanism is related to the Nrf2 antioxidative stress pathway. and 5% CO2 in a humid environment.

Cell Grouping And Prossing
HaCaT cells were divided into five groups: control group: no BVD added, no UVB irradiation; UVB group: 30 mJ/cm 2 UVB irradiation, no BVD added; and three BVD groups − 100 nmol/L, 1 µmol/L, or 10 µmol/L BVD + UVB groups: corresponding concentration of BVD was added one hour before 30 mJ/cm 2 UVB irradiation.
HaCaT cells were divided into four groups: control group: no transfection, no UVB irradiation; UVB group: 30 mJ/cm 2 UVB irradiation, no transfection; shRNA group:lentivirus transfection and gene enhancement, no UVB irradiation; and shRNA + UVB group: lentivirus transfection and gene enhancement before 30 mJ/cm 2 UVB irradiation.

UVB Irradiation
A bank of two UVB lamps (Nanjing huaqiang, China) was used as the UVB source. These lamps emit UV light at wavelengths ranging from 290 to 320 nm (mainly UVB), with the peak emission at 313 nm.
The intensity of irradiation by the lamp (3.0 mW/m 2 ) was measured using a Waldmann UV meter (Waldmann, Medizintechnik, Germany). The cells to be irradiated by UVB were prepared. When their confluence reached more than 80%, the culture solution was aspirated, PBS was used to wash the cells once, and a small amount of PBS was added to cover the bottom surface of the plate; UVB irradiation (total irradiation dose of 30 mJ/cm 2 ) was applied with the culture plate 15 cm from the light source. After irradiation, the PBS was discarded, and fresh DMEM was added, and the cells were cultured for 24 hours, after which the cells were collected for analysis.

Virus Transfection
The infection method and infection parameters were confirmed by an interference pretest.   Cell viability of each group Compared with the control group, cell activity in the UVB group was clearly reduced p<0.05 ; cell activity in the BVD+UVB groups was clearly increased, vs the UVB group p<0.05 ; cell activity in three BVD+UVB groups was statistically significant (p<0.05).