Mechanism Study On The Restorative Effect Of Notoginsenoside R1 To RNA Methylation Mediated Skin Ultraviolet Damage

Objective: UVB can cause acute damage to the skin,local and systemic immune suppression,pigmentation,DNA damage,and even skin cancer.The implementation of targeted therapies for skin ultraviolet damage has been challenging.Notoginsenoside R1(NGR1)can be used in the UVB mediated skin damages,however,it is unknown for the rationale of pharmacodynamic performance and transdermal transport.The outcomes of this paper will provide general guidance on how to develop pharmacological activity of NGR1 after transdermal administration and promote their preparations and productions upgrades in the future.Methods: 1.Using animal models,investigation the therapeutic effects of NGR1 on acute UVB-induced skin damage.HE staining was used to investigate the protective effects of NGR1 on skin tissue.ELISA was used to examine the inhibition of inflammatory factors and promotion of the antioxidant capacity by NGR1.IHC was used to assess the pharmacological effects of NGR1 on cyclobutane pyrimidine dimers(CPD)in UVB-mediated nude mouse skin.2.We measured the expression profiles of m6 A “writers” methyltransferase like 3(METTL3),methyltransferase like 14(METTL14),and WT1 associated protein(WTAP)and “erasers” alpha-ketoglutarate dependent dioxygenase(FTO)and alk B homolog 5,demethylase(ALKBH5)in UVB and NGR1 treated mice skin.Investigating the changes in the BCRP transporter protein and m RNA levels in UVB-mediated skin damage in relation to NGR1.3.To investigate the transdermal transport mechanism of NGR1,the LC-MS/MS was established to determine the content of NGR1 in cells.Then,we conducted an in vitro experiment on the cellular accumulation of NGR1.We used KO143 to inhibit the transport ability of BCRP in Ha Ca T cells,using this KO143-Ha Ca T model,we investigated the interaction between NGR1 and the uptake transporter BCRP from an exogenous perspective.4.We established a UVB-induced Ha Ca T cell photodamage model to investigate whether NGR1 upregulates the expression of m6 A,WTAP,BCRP,and DDB2.Additionally,we examined NGR1 effect on the repair of CPD in UVB-mediated Ha Ca T cells damage.We designed specific si RNA targeting WTAP to investigate the effect of NGR1 on the expression of BCRP and DDB2,as well as its therapeutic efficacy in CPD repair,under WTAP inhibition.5.In vivo,the reparative effect of the m6 A RNA-modifying agonist FB23-2 in combination with NGR1 was investigated after UVB-induced damage.Results: 1.To elucidate the pharmacological effect of NGR1 in UVB-induced skin damage in nude mice,We measured the levels of inflammatory and antioxidant factors in nude mouse serum and found that NGR1 down-regulated the levels of pro-inflammatory factors TNF-α,IL-1β,and IL-6,while up-regulating the level of anti-inflammatory factor IL-10.NGR1 significantly reduced the level of MDA and increased the activities of SOD,T-AOC,and CAT.Histological analysis showed that UVB irradiation induced mice sunburn,NGR1 mice were resistant to UVB-induced sunburn.In parallel,we found that UVB irradiation increased the levels of CPD,NGR1 facilitates CPD repair.2.NGR1 exhibited no significant effect on the gene expression of UVB-mediated m6 A methyltransferases,demethylases,and BCRP transporter.NGR1 had no significant impact on the downregulation of METTL14 methyltransferase protein induced by UVB.However,it significantly upregulated the expression of WTAP and BCRP proteins.3.NGR1 accumulation in Ha Ca T cells was inhibited(1-2 times)by the BCRP-specific inhibitor KO143.The efflux rate of NGR1 in Ha Ca T cell monolayer cells was 1.50-1.82,significantly higher than the efflux rate on the membrane of KO143-Ha Ca T cell monolayer cells(0.92-1.10).The efflux ratio of NGR1 in normal Ha Ca T cells was consistently greater than 1.5 at different concentrations.However,after the addition of the inhibitor KO143,the net efflux rate decreased to approximately 1.0,indicating a significant reduction in efflux activity.4.NGR1 upregulated the expression of BCRP,WTAP,m6 A,and DDB2 in UVB-induced Ha Ca T cell photodamage.Specific si RNA targeting WTAP were designed,and quantitative analysis of m6A/A showed that si WTAP RNA effectively inhibited WTAP expression in UVB-induced Ha Ca T cells.Knocking down WTAP resulted in reduced expression levels of BCRP,DDB2,and m6 A in the cells.In the presence of WTAP inhibition,NGR1’s efficacy in repairing CPD was significantly suppressed,indicating that NGR1 promotes the expression of DDB2 in the NER pathway through WTAP-m6 A,thus facilitating the repair of UVB-induced DNA damage.5.Combining the agonist FB23-2 with NGR1 significantly promoted the expression of BCRP and DDB2,but had no significant effect on WTAP expression.Additionally,it was also confirmed that FB23-2 promoted the expression of DDB2 by enhancing m6 A expression.The combination of NGR1 and the agonist FB23-2 significantly promoted the repair of CPDs in mice skin.Conclusion: This study revealed the relationship between WTAP-m6 A and the expression of drug transporter protein BCRP and the transport function of NGR1.It also demonstrated that NGR1 can regulate the NER repair pathway through WTAP-m6 A,promoting the repair of CPD in skin DNA damage.Furthermore,the combination of NGR1 and an m6 A agonist FB23-2 significantly enhanced the repair of CPD,which is crucial for improving the in vivo therapeutic efficacy of NGR1 and enhancing its transdermal transport ability.This study provides scientific evidence for the therapeutic effect of NGR1 in repairing UVB-induced skin damage through transdermal administration and promotes the application of Panax notoginseng transdermal preparations.