Effects And Mechanism Of β₂ Adrenergic Receptor Agonist On Psoriasis-Like Skin Disease

Objective:Psoriasis is a chronic,immune-mediated inflammatory disease that affects approximately 3%of the population worldwide.Treatments for psoriasis include topical therapies,phototherapy,and conventional and biological systemic therapies.However,many patients are still suffering from frequent relapse,adverse drug reactions,and huge costs of treatment.Keratinocytes play an essential role during the development of psoriasis.Cyclic adenosine monophosphate（c AMP）,an important second messenger molecule,can regulate the differentiation and proliferation of keratinocytes.Apremilast,a selective phosphodiesterase 4（PDE4）inhibitor has been approved for the treatment of psoriasis,and the effect is related to the enhancement of the c AMP pathway.Keratinocytes highly expressβ₂-adrenergic receptor（β₂-AR）,which can increase intracellular c AMP levels when activated.Therefore,in this study,we observed the long-actingβ2AR agonist salmeterol alone or combined with PDE4 inhibitor to regulate the proliferation and apoptosis of keratinocytes.We also investigate the effect on imiquimod-induced psoriasis-like skin lesions in mice by topical administration.Methods:1.Cell experiment:Human immortalized keratinocyte cell line（Ha Ca T）was cultured in DMEM complete medium containing 10%fetal bovine serum,and divided into（1）Vehicle（Veh）;（2）Salmeterol（Sal）high,medium,or low-dose groups（100n M,10n M,1n M）;（3）Roflumilast（Rof）high or low dose groups（100nM,10nM）;（4）High-dose salmeterol+high-dose roflumilast group（Sal+Rof）;（5）Forskolin（FSK）group.Ha Ca T cells were incubated with tumor necrosis factor-alpha（TNF-α,10ng/ml）to simulate a psoriasis-like cell model,and the cells were divided into（1）TNF-α;（2）TNF-αand high-dose Salmeterol group（TNF-α+Sal group）;（3）TNF-α+Sal+H89 group;（4）TNF-α+Sal+Rof+H89group;（5）TNF-α+FSK group.After the above groups were treated for 48 hours,CCK8,Edu incorporation test,and TUNEL were performed to detect the effects on cell viability,proliferation,and apoptosis.2.Animal experiment:C57/BL6J female mice（7-8 weeks old,16-20g）were randomly assigned into（1）Vehicle group（Veh）;（2）Model group（IMQ）;（3）Salmeterol group（IMQ+Sal）;（4）Roflumilast group（IMQ+Rof）;（5）Salmeterol combined with Roflumilast group（IMQ+Sal+Rof）.100μl of Saline containing 1%DMSO or above drugs were injected subcutaneously,and approximately 62.5 mg of petrolatum or 5%imiquimod cream was given 4 hours later for 5 days.The mice were sacrificed on the 6th day.The severity of skin lesions was evaluated with psoriasis area and severity index（PASI）.HE staining was performed and epidermal thickness was measured.Ki67 staining was used to detect cell proliferation,and TUNEL was used to detect apoptosis.The expression of proliferation and apoptosis-related proteins was measured by Western blot.Results:1.Cell experiment:Salmeterol alone,roflumilast alone,or the combination of the two drugs can inhibit viability and proliferation of Ha Ca T,and promote cell apoptosis under either serum culture conditions or the stimulation of TNF-α.However,the combination of the two drugs did not show a synergistic effect.PKA inhibitors can reverse the effects of Salmeterol alone and roflumilast alone on inhibiting cell proliferation and promoting cell apoptosis,but cannot reverse the inhibition of cell viability when the two drugs are used in combination.These data suggest that signal pathways other than c AMP/PKA are involved in this effect.2.Animal experiment:Compared with the model group,using salmeterol or roflumilast alone,or the combination of the two drugs can alleviate the severity of psoriasis,reduce the PASI score,and improve the degree of epidermal thickening in mice.Increased apoptosis and decreased proliferation in the epidermis were observed,but there was no synergistic effect in the combination of the two drugs.Western blot results showed that the expression of K17 was up-regulated and the ratio of Bax/Bcl2 was decreased in the model group,which was consistent with the increase in proliferation and the decrease in apoptosis in the skin lesions.Drug treatment can improve the expression of these markers,but no synergistic effect was observed in the combination of the two drugs.Our data suggest that both salmeterol alone and combination with roflumilast can inhibit excessive proliferation and promote apoptosis in the epidermis,and alleviate the severity of psoriasis.Besides,salmeterol alone or roflumilast alone only partially reverses the decreased phosphorylation of（c AMP-response element binding protein,CREB）caused by imiquimod,and the combination medication cannot further increase the phosphorylation of CREB,suggesting that the c AMP/PKA signaling pathway is only partially involved in this process.Conclusion:Using salmeterol or roflumilast alone,or the combination of the two drugs can alleviate the severity of psoriasis by inhibiting the proliferation and promoting the apoptosis in keratinocytes.The c AMP/PKA signaling pathway is partially involved in this process.However,no synergistic effect was observed in the combination medication.