The Proliferation And Apoptosis Effect Of Erianin On HaCaT Cells And The Study Of Dendritic Mesoporous Silica Nanosphere On Skin Delivery

Psoriasis is a recurrent skin disease described as keratinocyte hyperproliferation and aberrant differentiation.Erianin,a bibenzyl compound extracted from Dendrobium chrysotoxum,has displayed antitumor and anti-angiogenesis effects.However,the effects of erianin on a human keratinocyte cell line(HaCaT)are not fully understood.Meanwhile,more and more nano-drug carriers are applied to the topical administration of skin,which greatly increases the therapeutic effect of drugs on skin diseases and reduces unnecessary toxicity caused by drugs entering the system circulation.Recently,mesoporous silica nanoparticles have drawn the attention as carrier system for the topical delivery of active chemical compounds because of their unique properties such as extremely high surface area,ordered porosity,and tunable pore volume.In the present study,we explored the effect of erianin on proliferation and apoptosis in HaCaT cells.To improve its poor water-solubility,increase antiproliferation activity,and enhance the skin delivery,erianin loaded dendritic mesoporous silica nanospheres(E/DMSNs)were employed.In this paper,the effect of erianin on HaCaT cell viability was evaluated by MTT assay,and the effect of erianin on HaCaT cells apoptosis was analyzed by Annexin V-FITC / PI staining.The levels of intracellular reactive oxygen species ROS in HaCaT cells were detected with DCFH-DA fluorescence.The expression levels of apoptosis-related proteins cleaved PARP and cleaved caspase-3,and JNK/c-Jun and AKT/m TOR signaling pathways were measured using western blot.We synthesized two DMSNs with similar diameters but different pore diameters of central radioactive hollow channels by one-step biphasic method,and characterized their physical and chemical properties,including average particle size,zeta potential,high-resolution transmission electron microscope,nitrogen adsorption and desorption curve.We used the rotary evaporation method to load erianin into DMSNs and characterize it by FTIR and XRD.TGA and UPLC were used to measure the content of erianin.The dialysis bag was used to determine the in vitro release behavior,and vertical Franz cells was used to determine the permeation performance of E/DMSNs loaded carbomer gel through pig skin.We synthesized FITC labeled DMSNs,used confocal microscopy and flow cytometry to analyze its cell uptake behavior,and explored its possible cell uptake pathways.MTT method,Annexin V-FITC/PI double staining flow cytometry analysis method,JC-1 staining method and fluo-4 AM staining method were used to detect the effects on the survival rate,apoptosis rate,mitochondrial membrane potential and intracellular calcium ion concentration of HaCaT cells,compare the E/DMSNs and pure erianin.The western blot was used to detect signaling pathways(Bcl-2,Bax,cytochrome c,cleaved caspase-3,and cleaved PARP)with the endoplasmic reticulum pressure signaling pathways(PERK,ATF6,IRE1?,and CHOP).Our results indicated that treatment with erianin inhibited proliferation and induced apoptosis of HaCaT cells.In addition,erianin-induced apoptosis was accompanied by elevated ROS.The ROS scavenger NAC attenuated this elevation.Moreover,treatment with erianin induced activation of the JNK/c-Jun signaling pathway and suppressed the AKT/m TOR signaling pathway,while pretreatment with NAC also reversed these effects.DMSNs with pore size of 3.5 nm and 4.6 nm were fabricated and E/DMSNs showed pore-size-dependent,significantly stronger anti-proliferative and pro-apoptotic effect than free erianin on HaCaT cells,resulting from higher cellular uptake efficiency.In addition,compared to free erianin,treatment with E/DMSNs was more effective in reducing mitochondrial membrane potential and increasing cytoplasmic calcium levels,which were accompanied by regulation of mitochondria and ERS pathway.Porcine skin was utilized in the ex vivo accumulation and permeation studies,and the results indicated higher drug retention and less drug penetration in the skin when administered as the E/DMSNs-loaded hydrogel compared to the erianin-loaded hydrogel.