Eotaxin在皮肤过敏性疾病中的表达调控及苦参方提取物与eotaxin siRNA的经皮治疗研究
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摘要
皮肤过敏性疾病是高发的变态反应型疾病,是当皮肤暴露于外界过敏原时,由全身免疫系统参与而症状产生于局部的病理过程。除了在普通人群中常见外,对于在边防、海岛等恶劣环境下进行工作与生活的部队官兵,特别如遇战事、灾害等严峻情况,影响尤为严重。因此,深入探讨皮肤变态反应性疾病的病理过程与机制,发现新的药物作用靶点和新的治疗药物,具有重要的科学价值与军事意义。
变态反应型疾病发病的确切机制目前仍不十分清楚,但炎症细胞的募集是最终导致过敏性炎症发生和发展原因。嗜酸粒细胞(eosinophils, Eos)是过敏性疾病中重要的效应细胞,其浸润于过敏部位,释放毒性蛋白,造成组织损伤,导致炎症发生。造成Eos局部浸润的最重要的趋化因子是嗜酸粒细胞趋化因子(eotaxin/CCL11),因此eotaxin的表达与调控是过敏性疾病的重要环节和药物治疗靶点。
Eotaxin参与了皮肤过敏性疾病(如特异性皮炎、接触性皮炎、药物过敏性皮肤疾病等)的进程已得到证实。而人体皮肤由表皮、真皮和皮下组织构成,eotaxin在过敏皮肤中的确切表达部位及表达的动态过程目前还没有证实。本研究中,我们以小鼠接触性皮炎(ACD)模型为基础,探讨了eotaxin在过敏环境下皮肤中的表达部位及动态特征。同时对皮肤过敏反应的免疫学特征进行了观察,我们对CD4+和CDS+T淋巴细胞的浸润的程度和时程进行了比较。此外,为了阐明eotaxin表达的调控机制,我们以构成表皮层的角质形成细胞(KC)株HaCaT细胞和构成真皮层的真皮成纤维细胞(FBs)为基础,使用TNF-a和IL-4刺激两种细胞,观察eotaxin分泌量的改变,并检测与两种因子相关的转录因子NF-κB和STAT6的激活情况,探讨eotaxin表达的转录机制。
皮肤过敏性疾病的治疗是本课题关注的重要内容。相对于化学药物治疗,传统药物更显示出开发成本低、疗效好、副作用少、重视免疫平衡等优点。苦参方为一治疗过敏性皮肤病的传统中医药方剂,其提取物的主要成份为苦参碱(Mat)、氧化苦参碱(Omt)、槐果碱(Sop)、氧化槐果碱(Osp)以及荆芥挥发油(SVO)。我们考察了苦参方提取物(KS)凝胶剂对小鼠接触性皮炎的治疗效果,同时检测其对eotaxin的抑制作用,并于细胞水平对其提取物抑制TNF-a和IL-4诱导的eotaxin表达的信号转导机制进行进一步阐述。
基因治疗是目前疾病治疗的一个研究热点,我们尝试性地对皮肤过敏性疾病的经皮基因治疗进行了探索。微乳是经皮给药的一个良好剂型,将经稳定性修饰的eotaxin特异性siRNA制成W／O型微乳剂型,经皮给药,观察其对小鼠接触性皮炎治疗效果及对eotaxin的在皮肤组织中表达的抑制作用。
第一部分：Eotaxin在皮肤过敏性疾病中的表达调控
(一) Eotaxin在小鼠接触性皮炎(ACD)中的表达规律研究
1.小鼠接触性皮炎模型的建立：以BALB/c小鼠为模型动物,用DNFB涂抹鼠耳,于第1、2天致敏和强化致敏,第5天激发,可造成鼠耳皮肤过敏反应；小鼠耳肿胀度呈现一动态过程,肿胀高峰出现在24h-48h,肿胀度可达0.26±0.04mm,炎症待续5天左右,皮肤组织病理检测结果、血液炎症细胞计数证实了这一结论。其中Eos计数检测中,激发前小鼠血Eos为0.19±0.042×107/L,激发后于1h-2h开始升高(1.94±0.12×107/L),至24h达到最高(6.63±1.12×107/L),72h开始减弱至5d恢复正常。此外,采用流式细胞术检测血中CD4+和CD8+T细胞数量比,免疫组织化学方法检测组织中CD4+T细胞和CD8+T细胞的量,发现在过敏进程中,血液和组织中的CD4+和CD8+T细胞数量都有不同幅度的增长。
2.采用原位杂交实验技术检测小鼠过敏皮肤中eotaxin mRNA表达情况,观察到其主要产生于真皮层,少量产生于表皮层,同时血管内皮和软骨组织也有表达,由此证明真皮成纤维细胞是eotaxin在过敏状态下的主要来源。
3.分别采用real-time PCR、免疫组织化学和western-blotting检测法检测eotaxin在组织中表达的动态规律,及用ELISA检测血清中eotaxin的浓度。发现激发后2h在组织中已经开始有eotaxin的表达,到6h-12h表达量到最大并持续到24h,之后有所下降,到72h,基本恢复到最初状态。
(二) Eotaxin在皮肤细胞中产生的调控机制研究
1.采用不同浓度的TNF-α(1 ng/mL、10 ng/mL、100 ng/mL)和IL-4(2ng/mL、10 ng/mL,50ng/mL)分别与HaCaT细胞株和真皮成纤维细胞(FBs)共同孵育12h,用real-time PCR和ELISA方法检测eotaxin mRNA和蛋白表达的情况,发现TNF-α和IL-4均能上调eotaxin的表达,并呈剂量依赖性。而且无论是基础表达还是在两种细胞因子刺激下,FBs分泌eotaxin的能力远远高于HaCaT细胞。当同时使用TNF-α100 ng/mL和IL-4 10 ng/mL时,发现TNF-α和IL-4对两种细胞中eotaxin mRNA和蛋白的上调均有协同作用。
2.分别或同时用100 ng／mL TNF-a和10 ng／mL IL-4与两种细胞共同孵育,用real-time PCR和流式细胞术观察eotaxin mRNA和蛋白在72h内动态表达的动力学过程,发现FBs比HaCaT细胞较早地表达eotaxin,也较快达到高峰,同时衰减也较快,而HaCaT细胞表达eotaxin则较为缓慢和平稳,可持续较长时间。
3.通过EMSA和western-blotting分别检测转录因子NF-κB核转位和STAT6磷酸化的情况。在两种皮肤细胞中,TNF-α和IL-4可以分别激活NF-κB和STAT6发生核转位,而NF-κB特异性拮抗剂PDTC和转录因子STAT6特异的siRNA可以抑制TNF-α和IL-4引起的eotaxin上调,从而证实两种细胞受TNF-α和IL-4刺激后,均可通过NF-κB和STAT6转录导致eotaxin的表达。
本部分研究证实了eotaxin在小鼠接触性皮炎模型皮肤组织中表达有明显上调,并确定了表达的具体部位及动态过程。同时在离体细胞的研究中也证实和在体情况相似的表达特征,真皮成纤维细胞表达eotaxin的能力要高于表皮角质形成细胞。并且TNF-α和IL-4可以通过NF-κB和STAT6调节eotaxin的表达。
第二部分：苦参方提取物小鼠接触性皮炎的治疗作用及对eotaxin抑制作用研究
(一)苦参方提取物(KS)治疗小鼠ACD的疗效及机制研究
1.苦参方治疗小鼠ACD疗效观察：采用检测小鼠耳厚度的方法观察药物治疗效果。致敏前耳厚度：0.37±0.03 mm；致敏24h：0.66±0.05 mm；1%苦参方凝胶干预组：0.47±0.04 mm；5%苦参方凝胶干预组：0.49±0.06 mm；西替利嗪干预组：0.50±0.04 mm；地塞米松干预组：0.41±0.04 mm。证实苦参方凝胶组可以显著改善小鼠耳肿胀度(P<0.01)。同时通过HE染色的病理切片观察,也充分证明了药物对炎症细胞浸润的改善作用。
2.采用流式细胞术和免疫组化分别对血液和组织中CD4+和CD8+的T淋巴细胞进行了检测,考察苦参方对机体免疫状态的调节作用。证明苦参方可以调节机体的免疫状态,可降低CD4+和CD8+T细胞的组织浸润和在血液中的募集。
3.小鼠全血Eos计数检测。苦参方凝胶组可以显著降低过敏导致的小鼠Eos升高(P<0.01),小鼠致敏前Eos计数：0.19±0.04×107/L；致敏24 h：6.63±1.12×107,/L；1%苦参方凝胶干预组：4.78±0.66×107/L；5%苦参方凝胶干预组：2.24±0.58×107/L；西替利嗪组：5.22±0.73×107/L；地塞米松组：0.96±0.22×107/L
4. Real-time PCR、western-blotting、免疫组化法检测eotaxin组织表达情况。结果表明苦参方凝胶剂可抑制eotaxin的表达,证明eotaxin可能为苦参方的作用靶点之一。
(二)KS抑制皮肤细胞中eotaxin表达机制研究
1.MTT法检测苦参提取物(KS)、荆芥挥发油(SVO)、Mat、Omt、Sop、Osp、盐酸西替利嗪(Cet)、地塞米松磷酸钠(Dex)对HaCaT细胞和FBs生存率的影响,证明KS和SVO≤500μg/mL, Mat、Omt、Sop、Osp≤1000μM、Cet≤200μM, Dex≤100μM时,对细胞生存率无显著影响(≥90%),亦无刺激分裂和增殖作用。
2.通过real-time PCR、ELISA检测KS、SVO、Mat、Omt、Sop、Osp对TNF-α和IL-4诱导HaCaT细胞和FBs产生的eotaxin的抑制作用,同时使用Cet、Dex进行干预,作为对照实验。发现KS、SVO、Mat、Omt、Sop、Osp对eotaxin均有不同程度的抑制作用,抑制程度的顺序为KS> Mat> Omt≈Sop> Osp≈SVO。
3.通过EMSA和western-blotting分别检测在两种皮肤细胞中,Mat、Omt、Sop、Osp、SVO对TNF-a和IL-4诱导NF-κB和STAT6活化的抑制作用,证实Mat、Omt、Sop、Osp均可对NF-κB和STAT6的活化发生不同程度的抑制作用,但SVO只能够部位抑制NF-κB的核转位,而对STAT6磷酸化没有明显影响。
本部分研究证实了苦参方提取物对小鼠接触性皮炎具有较好的治疗效果,对小鼠过敏皮肤中eotaxin的表达及Eos的募集有明显抑制作用,并可调节机体的免疫状态。并且证明苦参方提取物及其中主要有效成分可通过NF-κB和STAT6两条信号转导通路抑制TNF-a和IL-4引起的eotaxin上调。
第三部分：Eotaxin特异siRNA微乳治疗小鼠接触性皮炎初步研究
1. Eotaxin特异siRNA设计与筛选。设计三对小鼠eotaxin基因特异的siRNA,使用转染试剂将siRNA转染至小鼠成纤维细胞株NIH3T3,采用real-time PCR检测TNF-a诱导下eotaxin被抑制的情况,得出eotaxin siRNA UTR为敲除效果最好的siRNA.采用ELISA检测法证实siRNA抑制eotaxin分泌的效果。
2. Eotaxin siRNA微乳(ESM)的制备。eotaxin siRNA经稳定性修饰(甲氧修饰：糖的2'-OH变成2'-Ome)成stable eotaxin siRNA。处方：10% stable eotaxin siRNA(40.μg/mL或200μg／mL)水溶液,16% plurol oleique cc 497,16% labrasol,58% labrafac cc。考察了微乳的粒径、zeta电位、粘度等性状,并借助冷冻蚀刻电镜观察了微乳存在的状态、使用激光共聚焦显微镜证明了经荧光修饰的ESM (FAM-ESM)的包封效果。稳定性考察证实,微乳本身可以存放于4℃、-20℃三个月不会发生性状改变。且贮存于4℃, siRNA活性可以保持3个月,贮存于-20℃可以保持4个月。
3.ESM透皮动力学考察。利用小动物整体成像系统观察裸鼠FAM-ESM透过皮肤和皮下扩散的动态过程。ESM透皮吸收1h-2h可达平衡,并可在皮内维持5h左右。与水溶液皮下注射组相比,微乳组的siRNA在皮内可停留更长的时间。
4.ESM药效学观察。将ESM于小鼠ACD模型激发前24h涂于鼠耳,每日3次给药,可以明显改善过敏状态下的小鼠耳肿胀程度(正常小鼠耳厚度：0.37±0.03mm,过敏小鼠(激发后24h)：0.66±0.05 mm,低剂量治疗组：0.44±0.04 mm,P<0.01；高剂量治疗组：0.46±0.06 mm,P<0.01),并无明显不良反应发生。且其对血液和组织中CD4+和CD8+T细胞的影响较小。
5.ESM对eotaxin的抑制作用考察。ESM可以显著抑制过敏状态下eotaxin的上调,但对外周血Eos影响不大,且对炎症细胞的组织浸润影响较小。而提前48h给药比提前药24h给药,在炎症过程中对eotaxin有更好的敲除效果。
本部分研究制备了eotaxin特异siRNA微乳,证实了其透皮和治疗效果,ESM对小鼠接触性皮炎具有一定的治疗效果,可以显著抑制小鼠过敏皮肤中eotaxin的分泌。
通过本课题的研究,进一步证实了eotaxin在过敏性皮肤疾病病理过程中的重要意义,揭示了eotaxin在过敏性皮肤疾病中的表达规律及调控机制。证明了中药方剂苦参方治疗过敏性皮肤疾病的治疗效果和作用机制。初步探讨了以RNAi为基础的治疗皮肤疾病的新的基因治疗方式。
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 disease
A. Eotaxin expression in mouse contact dermatitis
1. 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 cells
1. 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 extract
A. Mechanism of Kushen Recipe extract in mouse ACD therapy
1. 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 cells
1. 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 ACD
1. 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.
变态反应型疾病发病的确切机制目前仍不十分清楚,但炎症细胞的募集是最终导致过敏性炎症发生和发展原因。嗜酸粒细胞(eosinophils, Eos)是过敏性疾病中重要的效应细胞,其浸润于过敏部位,释放毒性蛋白,造成组织损伤,导致炎症发生。造成Eos局部浸润的最重要的趋化因子是嗜酸粒细胞趋化因子(eotaxin/CCL11),因此eotaxin的表达与调控是过敏性疾病的重要环节和药物治疗靶点。
Eotaxin参与了皮肤过敏性疾病(如特异性皮炎、接触性皮炎、药物过敏性皮肤疾病等)的进程已得到证实。而人体皮肤由表皮、真皮和皮下组织构成,eotaxin在过敏皮肤中的确切表达部位及表达的动态过程目前还没有证实。本研究中,我们以小鼠接触性皮炎(ACD)模型为基础,探讨了eotaxin在过敏环境下皮肤中的表达部位及动态特征。同时对皮肤过敏反应的免疫学特征进行了观察,我们对CD4+和CDS+T淋巴细胞的浸润的程度和时程进行了比较。此外,为了阐明eotaxin表达的调控机制,我们以构成表皮层的角质形成细胞(KC)株HaCaT细胞和构成真皮层的真皮成纤维细胞(FBs)为基础,使用TNF-a和IL-4刺激两种细胞,观察eotaxin分泌量的改变,并检测与两种因子相关的转录因子NF-κB和STAT6的激活情况,探讨eotaxin表达的转录机制。
皮肤过敏性疾病的治疗是本课题关注的重要内容。相对于化学药物治疗,传统药物更显示出开发成本低、疗效好、副作用少、重视免疫平衡等优点。苦参方为一治疗过敏性皮肤病的传统中医药方剂,其提取物的主要成份为苦参碱(Mat)、氧化苦参碱(Omt)、槐果碱(Sop)、氧化槐果碱(Osp)以及荆芥挥发油(SVO)。我们考察了苦参方提取物(KS)凝胶剂对小鼠接触性皮炎的治疗效果,同时检测其对eotaxin的抑制作用,并于细胞水平对其提取物抑制TNF-a和IL-4诱导的eotaxin表达的信号转导机制进行进一步阐述。
基因治疗是目前疾病治疗的一个研究热点,我们尝试性地对皮肤过敏性疾病的经皮基因治疗进行了探索。微乳是经皮给药的一个良好剂型,将经稳定性修饰的eotaxin特异性siRNA制成W／O型微乳剂型,经皮给药,观察其对小鼠接触性皮炎治疗效果及对eotaxin的在皮肤组织中表达的抑制作用。
第一部分：Eotaxin在皮肤过敏性疾病中的表达调控
(一) Eotaxin在小鼠接触性皮炎(ACD)中的表达规律研究
1.小鼠接触性皮炎模型的建立：以BALB/c小鼠为模型动物,用DNFB涂抹鼠耳,于第1、2天致敏和强化致敏,第5天激发,可造成鼠耳皮肤过敏反应；小鼠耳肿胀度呈现一动态过程,肿胀高峰出现在24h-48h,肿胀度可达0.26±0.04mm,炎症待续5天左右,皮肤组织病理检测结果、血液炎症细胞计数证实了这一结论。其中Eos计数检测中,激发前小鼠血Eos为0.19±0.042×107/L,激发后于1h-2h开始升高(1.94±0.12×107/L),至24h达到最高(6.63±1.12×107/L),72h开始减弱至5d恢复正常。此外,采用流式细胞术检测血中CD4+和CD8+T细胞数量比,免疫组织化学方法检测组织中CD4+T细胞和CD8+T细胞的量,发现在过敏进程中,血液和组织中的CD4+和CD8+T细胞数量都有不同幅度的增长。
2.采用原位杂交实验技术检测小鼠过敏皮肤中eotaxin mRNA表达情况,观察到其主要产生于真皮层,少量产生于表皮层,同时血管内皮和软骨组织也有表达,由此证明真皮成纤维细胞是eotaxin在过敏状态下的主要来源。
3.分别采用real-time PCR、免疫组织化学和western-blotting检测法检测eotaxin在组织中表达的动态规律,及用ELISA检测血清中eotaxin的浓度。发现激发后2h在组织中已经开始有eotaxin的表达,到6h-12h表达量到最大并持续到24h,之后有所下降,到72h,基本恢复到最初状态。
(二) Eotaxin在皮肤细胞中产生的调控机制研究
1.采用不同浓度的TNF-α(1 ng/mL、10 ng/mL、100 ng/mL)和IL-4(2ng/mL、10 ng/mL,50ng/mL)分别与HaCaT细胞株和真皮成纤维细胞(FBs)共同孵育12h,用real-time PCR和ELISA方法检测eotaxin mRNA和蛋白表达的情况,发现TNF-α和IL-4均能上调eotaxin的表达,并呈剂量依赖性。而且无论是基础表达还是在两种细胞因子刺激下,FBs分泌eotaxin的能力远远高于HaCaT细胞。当同时使用TNF-α100 ng/mL和IL-4 10 ng/mL时,发现TNF-α和IL-4对两种细胞中eotaxin mRNA和蛋白的上调均有协同作用。
2.分别或同时用100 ng／mL TNF-a和10 ng／mL IL-4与两种细胞共同孵育,用real-time PCR和流式细胞术观察eotaxin mRNA和蛋白在72h内动态表达的动力学过程,发现FBs比HaCaT细胞较早地表达eotaxin,也较快达到高峰,同时衰减也较快,而HaCaT细胞表达eotaxin则较为缓慢和平稳,可持续较长时间。
3.通过EMSA和western-blotting分别检测转录因子NF-κB核转位和STAT6磷酸化的情况。在两种皮肤细胞中,TNF-α和IL-4可以分别激活NF-κB和STAT6发生核转位,而NF-κB特异性拮抗剂PDTC和转录因子STAT6特异的siRNA可以抑制TNF-α和IL-4引起的eotaxin上调,从而证实两种细胞受TNF-α和IL-4刺激后,均可通过NF-κB和STAT6转录导致eotaxin的表达。
本部分研究证实了eotaxin在小鼠接触性皮炎模型皮肤组织中表达有明显上调,并确定了表达的具体部位及动态过程。同时在离体细胞的研究中也证实和在体情况相似的表达特征,真皮成纤维细胞表达eotaxin的能力要高于表皮角质形成细胞。并且TNF-α和IL-4可以通过NF-κB和STAT6调节eotaxin的表达。
第二部分：苦参方提取物小鼠接触性皮炎的治疗作用及对eotaxin抑制作用研究
(一)苦参方提取物(KS)治疗小鼠ACD的疗效及机制研究
1.苦参方治疗小鼠ACD疗效观察：采用检测小鼠耳厚度的方法观察药物治疗效果。致敏前耳厚度：0.37±0.03 mm；致敏24h：0.66±0.05 mm；1%苦参方凝胶干预组：0.47±0.04 mm；5%苦参方凝胶干预组：0.49±0.06 mm；西替利嗪干预组：0.50±0.04 mm；地塞米松干预组：0.41±0.04 mm。证实苦参方凝胶组可以显著改善小鼠耳肿胀度(P<0.01)。同时通过HE染色的病理切片观察,也充分证明了药物对炎症细胞浸润的改善作用。
2.采用流式细胞术和免疫组化分别对血液和组织中CD4+和CD8+的T淋巴细胞进行了检测,考察苦参方对机体免疫状态的调节作用。证明苦参方可以调节机体的免疫状态,可降低CD4+和CD8+T细胞的组织浸润和在血液中的募集。
3.小鼠全血Eos计数检测。苦参方凝胶组可以显著降低过敏导致的小鼠Eos升高(P<0.01),小鼠致敏前Eos计数：0.19±0.04×107/L；致敏24 h：6.63±1.12×107,/L；1%苦参方凝胶干预组：4.78±0.66×107/L；5%苦参方凝胶干预组：2.24±0.58×107/L；西替利嗪组：5.22±0.73×107/L；地塞米松组：0.96±0.22×107/L
4. Real-time PCR、western-blotting、免疫组化法检测eotaxin组织表达情况。结果表明苦参方凝胶剂可抑制eotaxin的表达,证明eotaxin可能为苦参方的作用靶点之一。
(二)KS抑制皮肤细胞中eotaxin表达机制研究
1.MTT法检测苦参提取物(KS)、荆芥挥发油(SVO)、Mat、Omt、Sop、Osp、盐酸西替利嗪(Cet)、地塞米松磷酸钠(Dex)对HaCaT细胞和FBs生存率的影响,证明KS和SVO≤500μg/mL, Mat、Omt、Sop、Osp≤1000μM、Cet≤200μM, Dex≤100μM时,对细胞生存率无显著影响(≥90%),亦无刺激分裂和增殖作用。
2.通过real-time PCR、ELISA检测KS、SVO、Mat、Omt、Sop、Osp对TNF-α和IL-4诱导HaCaT细胞和FBs产生的eotaxin的抑制作用,同时使用Cet、Dex进行干预,作为对照实验。发现KS、SVO、Mat、Omt、Sop、Osp对eotaxin均有不同程度的抑制作用,抑制程度的顺序为KS> Mat> Omt≈Sop> Osp≈SVO。
3.通过EMSA和western-blotting分别检测在两种皮肤细胞中,Mat、Omt、Sop、Osp、SVO对TNF-a和IL-4诱导NF-κB和STAT6活化的抑制作用,证实Mat、Omt、Sop、Osp均可对NF-κB和STAT6的活化发生不同程度的抑制作用,但SVO只能够部位抑制NF-κB的核转位,而对STAT6磷酸化没有明显影响。
本部分研究证实了苦参方提取物对小鼠接触性皮炎具有较好的治疗效果,对小鼠过敏皮肤中eotaxin的表达及Eos的募集有明显抑制作用,并可调节机体的免疫状态。并且证明苦参方提取物及其中主要有效成分可通过NF-κB和STAT6两条信号转导通路抑制TNF-a和IL-4引起的eotaxin上调。
第三部分：Eotaxin特异siRNA微乳治疗小鼠接触性皮炎初步研究
1. Eotaxin特异siRNA设计与筛选。设计三对小鼠eotaxin基因特异的siRNA,使用转染试剂将siRNA转染至小鼠成纤维细胞株NIH3T3,采用real-time PCR检测TNF-a诱导下eotaxin被抑制的情况,得出eotaxin siRNA UTR为敲除效果最好的siRNA.采用ELISA检测法证实siRNA抑制eotaxin分泌的效果。
2. Eotaxin siRNA微乳(ESM)的制备。eotaxin siRNA经稳定性修饰(甲氧修饰：糖的2'-OH变成2'-Ome)成stable eotaxin siRNA。处方：10% stable eotaxin siRNA(40.μg/mL或200μg／mL)水溶液,16% plurol oleique cc 497,16% labrasol,58% labrafac cc。考察了微乳的粒径、zeta电位、粘度等性状,并借助冷冻蚀刻电镜观察了微乳存在的状态、使用激光共聚焦显微镜证明了经荧光修饰的ESM (FAM-ESM)的包封效果。稳定性考察证实,微乳本身可以存放于4℃、-20℃三个月不会发生性状改变。且贮存于4℃, siRNA活性可以保持3个月,贮存于-20℃可以保持4个月。
3.ESM透皮动力学考察。利用小动物整体成像系统观察裸鼠FAM-ESM透过皮肤和皮下扩散的动态过程。ESM透皮吸收1h-2h可达平衡,并可在皮内维持5h左右。与水溶液皮下注射组相比,微乳组的siRNA在皮内可停留更长的时间。
4.ESM药效学观察。将ESM于小鼠ACD模型激发前24h涂于鼠耳,每日3次给药,可以明显改善过敏状态下的小鼠耳肿胀程度(正常小鼠耳厚度：0.37±0.03mm,过敏小鼠(激发后24h)：0.66±0.05 mm,低剂量治疗组：0.44±0.04 mm,P<0.01；高剂量治疗组：0.46±0.06 mm,P<0.01),并无明显不良反应发生。且其对血液和组织中CD4+和CD8+T细胞的影响较小。
5.ESM对eotaxin的抑制作用考察。ESM可以显著抑制过敏状态下eotaxin的上调,但对外周血Eos影响不大,且对炎症细胞的组织浸润影响较小。而提前48h给药比提前药24h给药,在炎症过程中对eotaxin有更好的敲除效果。
本部分研究制备了eotaxin特异siRNA微乳,证实了其透皮和治疗效果,ESM对小鼠接触性皮炎具有一定的治疗效果,可以显著抑制小鼠过敏皮肤中eotaxin的分泌。
通过本课题的研究,进一步证实了eotaxin在过敏性皮肤疾病病理过程中的重要意义,揭示了eotaxin在过敏性皮肤疾病中的表达规律及调控机制。证明了中药方剂苦参方治疗过敏性皮肤疾病的治疗效果和作用机制。初步探讨了以RNAi为基础的治疗皮肤疾病的新的基因治疗方式。
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 disease
A. Eotaxin expression in mouse contact dermatitis
1. 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 cells
1. 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 extract
A. Mechanism of Kushen Recipe extract in mouse ACD therapy
1. 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 cells
1. 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 ACD
1. 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.
引文
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