Role Of Necroptosis In UVB-induced Acute Skin Photodamage

Objective:To explore the role of necroptosis in UVB-induced acute skin photodamage.Methods:(1)HaCaT cells were irradiated with 120 m J/cm~2 doses of UVB to establish the photodamage model of HaCaT cells.After 24 hours of culture,the morphological changes of HaCaT cells were observed under a microscope,and the cell viability,necroptosis and ROS levels of HaCaT cells exposed to UVB radiation were examined by CCK-8 assay and Flow cytometry,respectively.The m RNA expression levels of inflammatory factors associated with acute photodamage were detected by q RT-PCR.The protein expression levels of RIPK3,p-RIPK3,MLKL and p-MLKL,were detected by Western Blot.(2)Silencing RIPK3 and MLKL gene respectively by the si RNA,and the expressions of RIPK3 and MLKL were detected by q RT-PCR to verify the transfection efficiency.UVB irradiation was performed on HaCaT cells after RIPK3 and MLKL knockdown.The effects of si-RIPK3/si-MLKL on cell viability,necroptosis and ROS levels of HaCaT cells exposed to UVB radiation were examined by CCK-8 assay and Flow cytometry,respectively.The m RNA expression levels of inflammatory factors associated with acute photodamage were detected by q RT-PCR.(3)C57BL/6 mice were used to establish an animal model of acute skin photodamage induced by UVB,and the expression levels of TNF-α,IL-1β,were detected by q RT-PCR,the expression levels of RIPK3 and MLKL were detected by immunohistochemistry.In the photodamage model,Necrosulfonamide(NSA),a cell necroptosis inhibitor,was evaluated its role in acute skin photodamage by intradermal injection of0.01mg/m L NSA into the back of mice.Results:(1)After UVB treatment of HaCaT cells,the shape of HaCaT cells became round,the adhesion ability decreased,the intercellular space increased,the number of floating cells increased significantly.CCK-8assay and Flow cytometry showed that UVB irradiation induced HaCaT cell activity rate decreased,necroptosis and ROS production increased,the m RNA expression of TNF-αand IL-1βrelated inflammatory factors in acute photodamage were increased,the protein expression of necroptosis effector molecules RIPK3,p-RIPK3,MLKL and p-MLKL were significantly up-regulated,and the proportion of P-RIPK3/RIPK3 and P-MLKL/MLKL increased.(2)Si RNA-RIPK3-03 and si RNA-MLKL-01 with the highest knockout efficiency were used to silence RIPK3 and MLKL respectively in subsequent experiments.CCK-8 assay and Flow cytometry showed that si RNA knockdown the expression of RIPK3/MLKL before UVB irradiated HaCaT cells could reduce the proportion of necroptosis and ROS of HaCaT cells,and down-regulate the expression of inflammatory factors.(3)Acute skin photodamage induced by UVB radiation on the back of mice was shown as the skin erythema,skin roughness and hypertrophy,accompanied by desquamation,and wrinkles.HE staining showed that the epidermal layer of mice irradiated by UVB thickened,with hyperkeratosis,exfoliation of epidermis,flattening of papilla layer,obvious thickening of dermis,thickening and disordered arrangement of collagen fibers in dermis.QRT-PCR showed that the expression levels of TNF-αand IL-1β,the inflammatory factors associated with acute photodamage,were increased.Immunohistochemistry showed that the expression levels of RIPK3 and MLKL,the key molecules of necroptosis,were up-regulated.The acute skin photodamage induced by UVB was alleviated by NSA pretreatment before UVB irradiation,and the difference was statistically significant.Conclusion:1.The activation of RIPK3-MLKL,a key molecule of necroptosis,participates in UVB-induced acute photodamage of HaCaT cells and mice skin.2.Inhibition of cell necroptosis,alleviates the UVB-induced acute photodamage of HaCaT cells and mice skin.