Molecular Mechanism Of MiR-98-5p-mediated Isovitexin Intervention Of HaCaT Cells On Apoptosis And Inflammation

Ultraviolet radiation,smoke and other factors can cause oxidative damage and aging of skin cells,and even lead to the loss of skin corneum,namely keratinocyte apoptosis.The main site of skin apoptosis is the stratum corneum,and inflammatory factors such as tumor necrosis factor（TNF-α）play an important role in this process.Screening out natural active components that inhibit inflammation and apoptosis of keratinocytes is expected to prevent skin damage and delay skin aging.Isovitexin is a flavonoid contained in many food-borne plants（such as mung beans and Ziziphus jujuba seeds）,which has antioxidant,anti-inflammatory and anti-apoptosis effects,but its mechanism of protecting skin keratinocytes is still unclear.Therefore,in this paper,a model of human keratinocytes（HaCaT）damaged by H₂O₂ was constructed,and the inhibitory effect of isovitexin on skin inflammation and apoptosis was systematically analyzed,and the potential protective mechanism of isovitexin was clarified from the perspective of microRNA.The main results are as follows:（1）Isovitexin protects HaCaT keratinocytes from oxidative damage.MTT results showed that the survival rate of HaCaT cells decreased with the increase of H₂O₂ concentration.When the concentration was close to 900μM,the IC₅₀ value of cell survival rate was reached.When the concentration of isovitexin was less than 40μM,there was no significant difference compared with CK group,which proved that isovitexin below 40μM was not toxic to HaCaT cells.Therefore,human keratinocytes damaged by H₂O₂ were treated with different concentrations（5,10,20μM）.The results showed that in the range of 5-20μM,the higher the concentration of isovitexin,the higher the cell survival rate.20μM isovitexin can significantly reduce ROS level and MDA content,and increase the activities of antioxidant enzymes SOD and GSH-Px.Isovitexin can also significantly reduce the apoptosis induced by H₂O₂.（2）Isovitexin alleviates H₂O₂-induced apoptosis through Bcl-xL/Bcl-2/Caspase-3signaling pathway.In order to reveal the anti-apoptosis mechanism of isovitexin,the m RNA and protein expression mechanism of Bcl-xL/Bcl-2/Caspase-3 signaling pathway were detected by fluorescence quantitative PCR and Western Blot.The results showed that H₂O₂ activated the apoptosis signal pathway,and different concentrations of isovitexin（5,10,20μm）could decrease the expression of p53 gene and protein,and then increase the expression of Bcl-2 and Bcl-xL,thus inhibiting the apoptosis-promoting effects of Bax,Caspase-9 and Caspase-3.（3）MiR-98-5p mediates the protective effect of isovitexin on HaCaT cells.MiRNA was screened by using ENCORI bioinformatics software and PCR technology.HaCaT human keratinocytes were pretreated with miR-98-5p mimics and inhibitors for 24 h,respectively,to determine the targeting relationship between miR-98-5p and Bcl-xL and Bcl-2.The results showed that at the molecular level,miR-98-5p mimics decreased the expression of Bcl-2 and Bcl-xL,while miR-98-5p inhibitor increased the expression of Bcl-2 and Bcl-xL.In order to deeply analyze the internal mechanism of mimir-98-5p mediating the protective effect of isovitexin,mimics of mimir-98-5p was pre-transfected.The results showed that mimir-98-5p inhibited the promoting effect of isovitexin on Bcl-2 and Bcl-xL and the inhibiting effect on Caspase-9 and Caspase-3.In addition,miR-98-5p mimics reversed the changes of cell survival rate,ROS level and apoptosis rate induced by isovitexin.（4）Isovitexin alleviates H₂O₂-induced inflammatory reaction through MAPKs/NF-κB signaling pathway.The expression of inflammatory cytokines was tested by PCR and ELISA.The results showed that H₂O₂ treatment significantly increased the gene expression level and protein release of inflammatory cytokines IL-6,IL-1βand TNF-α,and isovitexin significantly recovered their expression levels in a dose-dependent manner.Therefore,isovitexin may play a protective role through anti-inflammatory pathway.The Western Blot experiment showed that H₂O₂ treatment increased the protein expression of MAPK p38,JNK and NF-κB phosphorylation,while isovitexin treatment decreased the protein expression of MAPK p38,JNK and NF-κB phosphorylation.The results showed that isovitexin played a protective role by inhibiting the activation of MAPKs/NF-κB signaling pathway.