The Role And Mechanism Of Costimulatory Molecule B7-H3 In UVB-induced Photodamage Of Human Skin Keratinocytes

Objective:To explore the role and mechanism of costimulatory molecule B7-H3 in the acute injury of keratinocytes induced by UVB.Methods:1.Collect clinical human skin tissue samples and divide them into normal group,sunlight exposure group,natural aging group and photoaging group according to age and exposure location,and use immunohistochemistry to detect the expression of B7-H3.2.Cultivate the immortalized human epidermal keratinocyte line HaCaT cells,and use different doses(0,30,60,90,150,300 mJ/cm2)of UVB to induce acute light damage in HaCaT cells,and use Western Blot to detect the expression of B7-H3.3.3.HaCaT cells were transfected with lentivirus to silence B7-H3 expression,and flow cytometry was used to detect B7-H3 expression to verify the silencing effect.Divide HaCaT cells into shNon control group and shB7-H3 group to receive the above-mentioned different doses of UVB irradiation and detect the following indicators:(1)Detect cell cycle changes by flow cytometry;(2)Detect the changes of intracellular reactive oxygen species(ROS)by flow cytometry and fluorescent confocal microscopy;(3)Detect the changes of B7-H3,p53,p21 mRNA expression levels by real-time PCR(real-time PCR);(4)Detect changes in the expression levels of B7-H3,P21,and P53 proteins by Western Blot;(5)Detect changes in collagen damage-related factors including matrix metalloproteinase-1,9,10 and TGF-β1 mRNA expression levels by real-time PCR..Results:1.B7-H3 is highly expressed in epidermal tissues irradiated by sunlight.Compared with normal non-exposed areas,B7-H3 expression levels in exposed skin tissues and photoaging skin tissues are significantly higher,and the difference is statistically significant(P<0.05).2.Used different doses of UVB irradiation to establish a HaCaT cell acute light damage model.24 h after irradiation,it can be observed that cell proliferation activity decreases,cell density decreases,morphological changes,cytoplasmic retraction and other damaging manifestations.In HaCaT cells with acute light damage,when the UVB dose is lower than 150mJ/cm2,the expression level of B7-H3 increases in a dose-dependent manner.When the UVB dose exceeds 150mJ/cm2,B7-H3 no longer increases.3.The results of flow cytometry showed that the expression level of B7-H3 in HaCaT cells after lentivirus transfection was significantly lower than that of the no-load control group,and it could be cultured stably.4.UVB radiation causes G1 phase blockage in HaCaT cells,up-regulation of ROS levels,and inhibition of antioxidant enzyme SOD and GSH-Px activities;silencing the expression of B7-H3 can significantly alleviate the above changes.5.Low-dose(<150mJ/cm2)UVB radiation up-regulates the expression of B7-H3 and simultaneously induces the activation of P53 and P21.After silencing the expression of B7-H3,the expression of P53 and P21 is significantly inhibited;high-dose(≤150 mJ)/cm2)UVB radiation down-regulates B7-H3,P53 and P21.Silencing B7-H3 has no obvious effect on P53 and P21.6.30,60,90,150,300mJ/cm2 UVB radiation can significantly rincrease the level of MMP-1,9,10,and decrease the level of TGF-β;while silencing B7-H3 can inhibit the expression of MMPs and significantly increase the expression level of TGF-β..Conclusion:B7-H3 is related to the photodamage process of skin tissue;UVB radiation can up-regulate the expression of B7-H3 in epidermal cells,which in turn produces cell cycle arrest,oxidative damage,and collagen damage;silencing the expression of B7-H3 can reduce the photodamage of epidermal cells and promote it The mechanism of repair may be related to the regulation of P53/P21,the promotion of antioxidant enzyme and transforming growth factor activity,and the reduction of collagenase activity.