Regulation On Eotaxin Expression In Cutaneous Allergy Reaction And Related Transdermal Delivery Therapy By Kushen Recipe Extract And Eotaxin SiRNA

Allergic skin diseases occur when the body's immune system over-reacts to some environmental substances known as allergens, which could produce additive effects as itchy skin lesions. As a prevalent health problem, allergic skin diseases also happen to soldiers and become threaten for them in the severe environment, especially when warfare or disaster happens. Therefore, to investigate the immunologic and pathologic mechanisms of allergic skin diseases might have implications for potential targets of future therapeutic interventions, which take on significant meaning for both research and military.Although the pathogenesis of allergic diseases is not fully understood at present, the inflammatory cells infiltrate in the allergy sites is the reason that eventually lead to the occurrence and development of allergic inflammation. Eosinophils are important mediators of allergic responses and are associated with disease severity. Following activation by an immune stimulus, eosinophils in blood migrate to inflammatory sites in tissues in response to chemokines and degranulate to release an array of cytotoxic granule cationic proteins that are capable of inducing tissue damage and dysfunction. However, eotaxin is the most important chemokine for eosinophils recruitment, and its expression and regulation are meaningful for allergic diseases. Therefore, eotaxin could be a new drug therapy target.It is reported that eotaxin expressed in the allergic skin diseases, such as atopic dermatitis, contact dermatitis and drug allergic skin diseases. Human skin are composed of epidermis, dermis and subcutaneous tissue, however, the exact site where eotaxin express and how this progress performed in the allergic skin diseases have not been proved. The present study was to determine the location where eotaxin expressing in the allergic skin and its kinetics features by mouse contact dermatitis model, and demonstrate the immunological characteristics in the skin allergic responses including comparing the migration degree and kinetics between CD4+ and CD8+ T lymphocytes. We used interleukin (IL)-4 or tumor necrosis factor-alpha (TNF-α) to stimulate human keratinocytes (HaCaT) and dermal fibroblasts (FBs) to detect eotaxin production changes, and investigate eotaxin expression mechanism by detecting transcription factor nuclear factor-κappaB (NF-κB) as well as transducer and activator of transcription6 (STAT6) which are respectively TNF-αand IL-4 related transcription factor.Sophorae Flavescentis Radix decoction was used for the treatment of pruritus and eczema since ancient China, and its major components from extraction are matrine (Mat), oxymatrine (Omt), sophocarpine (Sop), oxysophocarpine (Osp) and schizonepeta tenuifolia volatile oils (SVO). We detected the therapeutic efficacy of mouse contact dermatitis by Kushen recipe (KS) micro-emulsion and investigated the signal transduction mechanism of KS inhibition for TNF-αand IL-4 induced eotaxin expression.We also adopted RNA interference (RNAi) which already revolutionized experimental biology by specifically knocking down molecular targets. Since micro-emulsion is ideal for percutaneous drug delivery, we made stability modified eotaxin specific siRNA into W/O micro-emulsion formulation, and evaluated the therapy efficacy on mouse contact dermatitis model.Part I:Regulation on eotaxin expression in allergic skin diseaseA. Eotaxin expression in mouse contact dermatitis1. Mouse contact dermatitis model establishment. BALB/c mice were adopted in the contact hypersensitivity mouse model. On day 1 and 2 mice were sensitized by applying 1-fluoro-2,4-dinitrobenzene (DNFB) to each side of ears. At day 5 mice were challenged by applying DNFB again, leading to allergic responses on the mice ears. After the treatment, the inflammation lasted for about 5 days, ear swelling assessed by measuring ear thickness, displayed a maximal value of 0.26±0.04 mm, between 24h to 48h, that result is coincident with the tissue pathological testing and inflammatory cell count results. Eosinophils were counted in the whole mice blood. It was found that after challenge 2 hours, eosinophils increased from 0.19±0.042×107/L to 1.94±0.12×107/L, the peak (6.63×1.12×107/L) appeared at 24 h, after that at 72 h it started to reduce and return to the initial at the 5 day. Furthermore we detected the ratio of CD4+to CD8+T lymphocytes from blood and tissue by flow cytometry and immunohistochemistry respectively. It was found that during the hypersensitivity CD4+and CD8+T lymphocytes in blood or tissue have similar increase tendency.2. Eotaxin mRNA expression detection in the mice hypersensitive skin by situ hybridization. It was found that majority located at dermis and little amount at epidermis, blood vessel endothelium and cartilaginous tissue. Therefore, when cutaneous allery occurs, dermal fibroblasts are the main source of eotaxin.3. Eotaxin expression investigation by real-time PCR, immunohistochemistry, western blot and ELISA. The dynamic expression from mRNA to protein showed that eotaxin secretion started from the first 2 hours, and reached the peak between 6 to 12h and last until 24h, then reduced to initial between 48 to 72h.B. Regulation on eotaxin expression in skin cells1. HaCaT cells and dermal fibroblasts (FBs) were stimulated with different concentrations of TNF-α(1 ng/mL,10 ng/mL,100 ng/mL) and IL-4 (2 ng/mL,10 ng/mL,50 ng/mL) for 12 h. Real-time PCR and ELISA results demonstrated that both TNF-αand IL-4 could increase eotaxin expression in dose-dependence manner. FBs produced more eotaxin before and after stimulation. And It showed synergy effect treated with TNF-α(100 ng/mL) and IL-4 (10 ng/mL) both in HaCaT cells and fibroblasts.2. Dynamic progress of eotaxin expression during 72 hours after treated with 100 ng/mL TNF-αand 10 ng/mL IL-4 detected by real-time PCR and flow cytometry. The results showed that fibroblasts synthesis eotaxin and reached the peak prior to HaCaT cells, while HaCaT lasted longer.3. To determine nuclear translocation of NF-κB and STAT6 phosphorylation, EMSA and western blot were used respectively. It was found that TNF-αcould induce NF-κB to conduct nuclear translocation, while IL-4 lead STAT6. Meanwhile, PDTC, a specific antagonist for NF-κB, could inhibit the TNF-α-induced eotaxin upregulation, as well as STAT6 siRNA for IL-4. Therefore, it was demonstrated that after application of TNF-αand IL-4, more eotaxin expressed due to the activation of NF-κB and STAT6 respectively.In this part of study, it was proved that eotaxin expression was up-regulated in the mice contact dermatitis, and determined the exact expression site and its dynamic progress, which was in agreement with the cellular experiments results. Fibroblasts produced more eotaxin than HaCaT cells. TNF-αand IL-4 regulated eotaxin through NF-κB and STAT6 respectively.Part II:Therapeutic effect and inhibition effect on eotaxin secretion in mouse ACD treated with Kushen Recipe extractA. Mechanism of Kushen Recipe extract in mouse ACD therapy1. Therapeutic efficacy was assessed by measuring ear thickness 24 hours after sensitization. The results showed that KS could lighten mice ear swelling (P<0.01), ear thickness before sensitization was 0.37±0.03 mm while 24 hours after sensitization was 0.66±0.05 mm. Ear thickness of 1% KS-treated group was 0.47±0.04 mm,5%KS-treated group was 0.49±0.06 mm, Cet-treated group was 0.50±0.04 mm, Dex-treated group was 0.41±0.04 mm. The inflammatory cells infiltration assessed by HE stain confirmed these results.2. CD4+ and CD8+ T lymphocytes in tissue were measured by immunohistochemistry and in blood by flow cytometry to evaluate KS effect on immunity regulation. The results showed that KS could regulate CD4+ and CD8+ T lymphocytes ratio to normal condition when allergy take place.3. Eosinophils count in mice whole blood. Before treatment the eosinophils amount was 0.19±0.04x107/L,24 hours after sensitization it was 6.63±1.12x107/L, 1%KS-treated group was 4.78±0.66×107/L,5%KS-treated group was 2.24±0.58×107/L, Cet-treated group was 5.22±0.73×107/L, and Dex-treated group was 0.96±0.22×107/L. Therefore, KS could moderate allergy-induced eosinophils recruitment in mice (P<0.01)4. Real-time PCR, western-blotting and immunohistochemistry results indicated that eotaxin expression in tissue as well as in blood serum by ELISA was suppressed by KS, therefore, eotaxin is one of KS functional targets.B. Mechanism of eotaxin expression inhibited by Kushen Recipe extract in skin cells1. KS, SVO, Mat, Omt, Sop, Osp, Cet, Dex effect for HaCaT and fibroblasts viability was investigated by MTT. The results indicated that the toxicity threshold of KS and SVO was 500μg/mL, Mat, Omt, Sop and Osp was 1000μM, Cet was 200μM and Dex was 100μM.2. Evaluation of the inhibition effect for TNF-α/IL-4-induced eotaxin expression in HaCaT and fibroblasts of KS, SVO, Mat, Omt, Sop and Osp by real-time PCR and ELISA. Cet and Dex were applied as positive control. It was found that inhibition effect of all the treated group ordered descendingly was KS>Mat>Omt≈Sop>Osp≈SVO.3. EMSA and western-blotting were used to determine the inhibition of TNF-α/ IL-4-induced NF-κB and STAT6 nuclear translocation by Mat, Omt, Sop, Osp and SVO respectively. It was demonstrated that all had effect on NF-κB and STAT6 except SVO which had no effect on STAT6.The present study found that Kushen Recipe extract had therapeutic effect on mice contact dermatitis, and confirmed its inhibition of eotaxin expression and eosinophils recruitment in hypersensitive skin, also its regulation of immune response. It was demonstrated that Kushen Recipe extract as well as its major effective ingredient could suppress TNF-α/IL-4-induced eotaxin expression by NF-κB and STAT6 signal transduction pathways.PartⅢ:Therapeutic action of eotaxin siRNA micro-emulsion on mouse ACD1. Eotaxin siRNA design and optimizing. We designed three pairs of mice eotaxin siRNA. They were delivered into mice fibroblasts cell line-NIH3T3 by transfection reagent. Then we used real-time PCR to detect eotaxin mRNA expression with or without TNF-αinduction, and ELISA to confirm the knockdown effect. It was indicated that UTR had the best knockdown efficiency.2. Eotaxin siRNA micro-emulsion (ESM) preparation. Methoxy-modification, substitute 2'-OH ribosyl for 2'-O-methyl ribosyl, was applied to stabilize eotaxin siRNA with equal interference efficiency and safeness. ESM contained 10% stable eotaxin siRNA dissolved in water,18% plurol oleique cc 497,18% labrasol and 54% labrafac cc. Properties of ESM including grain diameter, zata potential and viscosity were evaluated. By freeze-etching electron microscopy, the status of micro-emulsion was confirmed, also encapsulation effects of fluorescent modified ESM (FAM-ESM) were demonstrated by laser scanning confocal microscope. The stability assessment indicated that micro-emulsion could conserve for 3 months at 4℃or-20℃. For siRNA, it could preserve for 3 months at 4℃, or 5 months at-20℃.3. ESM delivery kinetics measurement. Dynamic FAM-ESM transdermal and diffusion progress in nude mice were recorded by small animal imaging system. It was found that it took 1-2 hour for ESM transdermal delivery and reaching the equilibrium concentration, and ESM could maintain in skin for 5 h. Compared to hypodermic injection, siRNA in mirco-emulsion could conserve longer in skin.4. ESM therapeutic action. After applied ESM on mice ears 24 hours before challenge,3 times a day, ears thickness were measured. The results showed that the ear thickness of blank-control group was 0.44±0.04 mm, non-treated group was 0.66±0.05 mm, ESM-treated group was 0.46±0.06 mm, P<0.01. Therefore, ESM could ease mice ear swelling in hypersensitivity without adverse effect. But it rarely influenced peripheral blood eosinophils, inflammatory cells infiltration and CD4+/CD8+T lympocytes ratio in blood and tissue.5. Inhibition effect of ESM on eotaxin. ESM could suppress the eotaxin upregulation in allergy. What's more, ESM was more effective in inhibiting eotaxin expression when administrated at 48 h before challenge than 24 h.This partial study demonstrated the therapeutic effect of ESM on mice contact dermatitis, also its suppression on eotaxin expression.This study revealed eotaxin expression and regulation mechanism in the allergic skin diseases, confirmed the therapeutic effect and mechanism of Kushen Recipe for cutaneous hypersensitivity. We demonstrated the potential of siRNA encapsulated into micro-emulsion in the treatment of allergic skin diseases by transdermal delivery.