濕潤(rùn)燒傷膏對(duì)尋常型銀屑病小鼠IL23／Th17軸及其相關(guān)因子表達(dá)水平的影響

任曉燕 王李雯 張麗麗 孫麗萍

作者單位：712000 陜西 咸陽(yáng)，陜西中醫(yī)藥大學(xué)附屬醫(yī)院皮膚科

尋常型銀屑病是一種常見(jiàn)的慢性、復(fù)發(fā)性、炎癥性皮膚病［1］。研究發(fā)現(xiàn)，白細(xì)胞介素-23（IL-23） ／輔助性T細(xì)胞17（Th17） 軸失調(diào)是銀屑病的主要發(fā)病機(jī)制之一［2］， IL23／Th17軸相關(guān)因子參與炎癥反應(yīng)與銀屑病的復(fù)發(fā)及持續(xù)密切相關(guān)［3］。臨床上針對(duì)銀屑病IL-23／Th17通路的靶向藥物已被證實(shí)有效［4］，但卻存在癥狀反復(fù)、藥物不良反應(yīng)較多等弊端［5］。近年來(lái)，部分研究學(xué)者將濕潤(rùn)燒傷膏應(yīng)用于銀屑病的治療，取得了一定的臨床療效，且未見(jiàn)明顯不良反應(yīng)［6］，但具體作用機(jī)制尚不明確。本研究通過(guò)觀察濕潤(rùn)燒傷膏對(duì)尋常型銀屑病小鼠皮損組織中白細(xì)胞介素-17（IL-17）、白細(xì)胞介素-23（IL-23）、 腫瘤壞死因子-α （TNF-α） 及干擾素-γ（IFN-γ）水平的影響，探討了其對(duì)尋常型銀屑病的作用機(jī)制，以期為濕潤(rùn)燒傷膏的臨床應(yīng)用提供理論依據(jù)。

Psoriasis vulgaris is a common chronic，recurrent and inflammatory skin disease［1］.Studies have shown that the imbalance of interleukin-23 （IL-23） ／helper T cell 17 （Th17） axis is one of the main pathogenesis of psoriasis［2］.The involvement of IL23 ／Th17 axisrelated factors in the inflammatory reactions is closely related to the recurrence and persistence of psoriasis［3］.Targeted drugs for IL-23 ／Th17 pathway in psoriasis have been proven effective in clinical practice［4］， but such drugs still have some drawbacks， because they may lead to the recurrence of the disease and some adverse drug reactions［5］.In recent years， some research scholars have adopted MEBO to treat psoriasis.Certain clinical effect has been obtained and no obvious adverse reactions have been observed［6］.However， the specific mechanism of action of MEBO remains unclear.In this study，the effect of MEBO on the expression levels of interleukin-17（IL-17）， interleukin-23 （IL-23）， tumor necrosis factor-α （TNF-α）and interferon-γ （IFN-γ） in skin tissues on the backs of mice with psoriasis vulgaris was observed and the mechanism of its action on psoriasis vulgaris was explored with a view to provide experimental evidence for its clinical application.

1 材料與方法

1.1 實(shí)驗(yàn)動(dòng)物

SPF級(jí)健康雌性近交系 BALB／c小鼠40只，6～7周齡，體重 （18±2）g，成都達(dá)碩實(shí)驗(yàn)動(dòng)物有限公司提供，動(dòng)物合格證號(hào)SCXK（川）2015-030，飼養(yǎng)于SPF級(jí)動(dòng)物室，自由進(jìn)食水。本研究經(jīng)陜西中醫(yī)藥大學(xué)附屬醫(yī)院動(dòng)物倫理委員會(huì)批準(zhǔn)，符合動(dòng)物實(shí)驗(yàn)的倫理學(xué)要求。

1.2 主要藥品及試劑

濕潤(rùn)燒傷膏：汕頭市美寶制藥有限公司生產(chǎn)，國(guó)藥準(zhǔn)字Z20000004；卡泊三醇軟膏：香港澳美制藥有限公司生產(chǎn)，批準(zhǔn)文號(hào)HC20150020；5%咪喹莫特乳膏：四川明欣藥業(yè)有限責(zé)任公司生產(chǎn)，國(guó)藥準(zhǔn)字H20030128；兔多抗 IL-17 （ab79056）、 兔多抗 IL-23（ab45420）、 兔多抗 TNF-α （ab9739）、 兔多抗IFN-γ（ab9657）：英國(guó)Abcam公司生產(chǎn)；小鼠單抗β-actin：武漢博士德生物工程有限公司生產(chǎn)，貨號(hào)BM0627。

1.3 模型建立

用硫化鈉脫去小鼠背部面積約3 cm×2 cm的毛發(fā)，裸露背部皮膚并涂抹5%咪喹莫特乳膏，每次62.5 mg，每天1次，連續(xù)涂抹7 d［7］。隨機(jī)選取5只小鼠在無(wú)菌操作下取其背部皮損組織，10%福爾馬林溶液固定，制備石蠟切片，并于HE染色后，光鏡下觀察皮膚角化程度，顆粒層、棘層、表皮突、真皮乳頭層厚度及排列情況，淋巴細(xì)胞、中性粒細(xì)胞分布情況等病理變化。

模型評(píng)價(jià)：小鼠背部可見(jiàn)暗紅色或紅褐色斑塊，表面覆有較厚銀白色鱗屑，有不同程度浸潤(rùn)增厚，符合尋常型銀屑病的皮損表現(xiàn) （圖1）；病理切片顯示皮膚角化不全，顆粒層變薄，棘層明顯增厚，表皮突延長(zhǎng)，嗜中性粒細(xì)胞浸潤(rùn) （圖2），類(lèi)似人尋常型銀屑病的病理組織學(xué)表現(xiàn)。

1.4 分組與給藥

模型建立成功后，采用隨機(jī)數(shù)表法將35只小鼠隨機(jī)分為對(duì)照組、模型組、MEBO低劑量組、MEBO中劑量組及MEBO高劑量組，每組7只。

對(duì)照組：小鼠背部裸露皮膚均勻涂抹卡泊三醇乳膏 （1 mg／cm2）；模型組：小鼠背部裸露皮膚均勻涂抹適量麻油；MEBO低劑量組：小鼠背部裸露皮膚均勻涂抹濕潤(rùn)燒傷膏與麻油混合物 （混合比例為1∶3，厚約1 mm）；MEBO中劑量組：小鼠背部裸露皮膚均勻涂抹濕潤(rùn)燒傷膏與麻油混合物 （混合比例為1∶1，厚約1 mm）；MEBO高劑量組：小鼠背部裸露皮膚均勻涂抹濕潤(rùn)燒傷膏 （厚約1 mm）。5組小鼠均每天換藥2次，連續(xù)用藥28 d。

1.Materials and methods

1.1.Experiment animals

The experiment animals were forty 6-to 7-week-old specificpathogen free （SPF） healthy inbred BALB／c female mice， each weighing（18±2） g，provided by Chengdu Dossy Experimental Animals Co.， LTD.Animal certificate No.： SCXK （Sichuan） 2015-030.They were kept in SPF animal room with sufficient food and water.This study has been approved by The Animal Ethics Committee of Affiliated Hospital of Shaanxi University of Chinese Medicine and complies with the ethical requirements of animal experiments.

1.2.Main drugs and reagents

MEBO：produced by Shantou MEBO Pharmaceutical co.，LTD，SFDA approval number： Z20000004； Capotriol cream： produced by BrightFuture PharmaceuticalLaboratoriesLtd.， approvalNo.HC20150020；5%imiquimod cream： produced by MED.SHINE，China， SFDA approval No.： H20030128； Rabbit polyclonal antibody IL-17 （ab79056），rabbit polyclonal antibody IL-23（ab45420），rabbit polyclonal antibody TNF-α （ab9739） and rabbit polyclonal antibody IFN-γ （ab9657）： produced by British Abcam company； mouse monoclonal antibody： produced by Boster Biological Technology Co.，LTD in Wuhan，article No.BM0627.

1.3.Model establishment

3 cm×2 cm area of hairs on the backs of the mice was removed with sodium sulfide to expose their back skin，on which 5% imiquimod cream was applied， 62.5 mg each time， once a day for 7 days［7］.Five mice were randomly selected.Tissues of skin lesions on their backs were taken under aseptic operation and then fixed with 10%formalin solution to prepare paraffin sections.The degree of skin keratinization， the thickness and order of the granular layer， spinous layer， trochanterellus and dermal papilla layer， and the distribution of lymphocytes and neutrophils were observed under the light microscope after HE staining.

Model evaluation：dark red or reddish-brown plaques could be seen on the backs of the mice，the surface of their back skin was covered with thick silvery white scales，varying degrees of infiltration and thickening could be observed，and all of these symptoms were consistent with skin lesions in psoriasis vulgaris（Fig.1）； pathological sections showed that the skin keratinization was incomplete，the granular layer got thinned， the spinous layer became significantly thickener，the trochanterellus prolonged and the neutrophil infiltration was found（Fig.2）， which were similar to the pathohistological manifestations of human psoriasis vulgaris.

1.4.Grouping and drug administration

After the models were established， 35 mice were divided， according to random number table， into a control group， a model group，a MEBO low-dose group， a MEBO medium-dose group， and a MEBO high-dose group.

The control group： calpatriol cream （1 mg／cm2） was evenly applied on the exposed back skin of the mice； the model group： appropriate volume of sesame oil was evenly applied on the exposed back skin of the mice； the MEBO low-dose group： the mixture of MEBO and sesame oil（mixing ratio＝1∶3， thickness＝1 mm） was evenly applied on the exposed back skin of the mice； the MEBO medium-dose group： the mixture of MEBO and sesame oil（mixing ratio＝1∶1， thickness＝1 mm） was evenly applied on the exposed back skin of the mice；the MEBO high-dose group：MEBO was evenly applied on the exposed back skin of the mice（thickness＝1 mm）.Dressing change was performed twice a day for 28 days in the five groups of mice.

圖1 典型尋常型銀屑病小鼠模型皮損表現(xiàn)；圖2 典型尋常型銀屑病小鼠模型皮損組織病理表現(xiàn)（HE， ×100）Fig.1 Skin lesions of typical mouse model of psoriasis vulgaris； Fig.2 Pathological manifestations of skin lesions of typical mouse model of psoriasis vulgaris（HE， ×100）

1.5 標(biāo)本采集與檢測(cè)

1.5.1 皮損嚴(yán)重程度評(píng)分 分別于治療第7、14天，參照銀屑病皮損面積與嚴(yán)重程度指數(shù) （psoriasis area and severity index， PASI）評(píng)分標(biāo)準(zhǔn)［8］評(píng)估皮損嚴(yán)重程度，PASI評(píng)分標(biāo)準(zhǔn)包含紅斑、鱗屑及浸潤(rùn)增厚程度3項(xiàng)，每項(xiàng)0～4分，3項(xiàng)之和為皮損嚴(yán)重程度總評(píng)分，分值越高表示皮損越嚴(yán)重。

1.5.2 Western blotting法檢測(cè)IL-17、IL-23、TNF-α及IFN-γ蛋白表達(dá)水平 分別于治療第14、28天，每組分別選取4只、3只小鼠以脫頸法處死后，在無(wú)菌操作下取其背部皮損組織置于-80℃冰箱中保存?zhèn)溆?。待所有?biāo)本收集完畢，取出冷凍組織置于冰上稱(chēng)重后，置于研缽內(nèi)研磨至粉末狀；將研磨后的組織粉末置于EP管中行RIPA裂解，提取總蛋白，并采用紫外分光光度計(jì)測(cè)定總蛋白濃度，繪制蛋白濃度標(biāo)準(zhǔn)曲線(xiàn)。取蛋白上樣經(jīng)SDS-聚丙烯酰氨凝膠 （SDS-PAGE）電泳分離及轉(zhuǎn)模后，TBST洗膜3次；洗膜后，5%脫脂奶粉封閉2 h，4℃孵育相應(yīng)目的抗體（IL-23、 IL-17、 TNF-α 混合比例均為1∶1000；IFN-γ 混合比例為1∶2000； β-actin混合比例為1∶200）過(guò)夜；孵育過(guò)夜后，TBST洗膜3次，室溫孵育 HRP-羊抗鼠二抗 （混合比例為1∶5000）2 h；二抗孵育后，TBST洗膜3次，ECL顯影，并用BandScan 5.0軟件分析目的條帶灰度值。每組小鼠標(biāo)本重復(fù)檢測(cè)3次，取均值進(jìn)行對(duì)比分析。

1.5.Specimen collection and detection

1.5.1.Skin lesion severity score On day 7 and day 14 of treatment，skin lesion severity was assessed based on the psoriasis area and severity index （PASI） scoring system［8］.Under the PASI score criteria， erythema， scales， and infiltration and thickening are scored between 0 to 4 points.The sum of the 3 items is total score of skin lesion severity.Higher score means more severe lesion.

1.5.2.Detection of the protein expression levels of IL-17， IL-23， TNF-α and IFN-γ with Western blotting On day 14 and day 28 of treatment， 4 and 3 mice were selected from each group and killed by cervical dislocation.After that，the skin tissues on their backs were taken under aseptic operation and stored in a refrigerator at the temperature of-80℃.After all the specimens were collected，the frozen tissues were taken out and put on ice to be weighted and then grounded into powder in a mortar.The grounded tissue powder was placed in the EP tube to be cracked with RIPA lysis buffer for total protein extraction.Ultraviolet spectrophotometer was used to measure the concentration of total protein，based on which the standard curve for protein concentration was drawn.The protein was loaded and then separated by using SDS-PAGE gel electrophoresis and then transferred to membrane，which was washed 3 times with TBST； after washing， it was sealed for 2 hours with 5%skim milk powder，and the corresponding target antibody was incubated overnight at the temperature of 4 ℃ （the mixing ratio of IL-23，IL-17， and TNF-α was 1∶1000； the mixing ratio of IFN-γ was 1∶2000； the mixing ratio of β-actin was 1∶200） ； after overnight incubation， the membrane was washed with TBST for 3 times，and HRP-goat anti-mouse secondary antibody（mixing ratio＝1∶5000） was incubated at room temperature for 2 hours； after secondary antibody incubation， the membrane was washed with TBST for 3 times， ECL was used for its development， and Band-Scan 5.0 software was adopted to measure the gray value of target band.The specimens of each group of mice were detected three times and the mean value of the test was taken for comparative analysis.

1.6 統(tǒng)計(jì)學(xué)處理

采用SPSS 19.0統(tǒng)計(jì)軟件對(duì)所得數(shù)據(jù)進(jìn)行統(tǒng)計(jì)學(xué)分析，計(jì)量資料符合正態(tài)分布用均數(shù)±標(biāo)準(zhǔn)差 （）表示，多樣本間比較采用單因素方差分析；均以P＜0.05為差異具有統(tǒng)計(jì)學(xué)意義。

2 結(jié)果

2.1 5組小鼠背部皮損PASI評(píng)分對(duì)比

治療第7天，與模型組相比，對(duì)照組、MEBO中劑量組及MEBO高劑量組小鼠背部皮損紅斑顏色較淺，浸潤(rùn)增厚不明顯，鱗屑較少，PASI評(píng)分均較低 （P均＜0.05），且對(duì)照組、MEBO中劑量組及MEBO高劑量組組間兩兩對(duì)比，PASI評(píng)分無(wú)明顯差異 （P均＞0.05）。治療第14天，5組小鼠背部皮損癥狀均明顯改善，斑塊顏色呈淡紅色，無(wú)明顯浸潤(rùn)增厚，可見(jiàn)極少量鱗屑，PASI評(píng)分無(wú)明顯差異 （P＞0.05），詳見(jiàn)表1。

2.2 5組小鼠背部皮損組織中IL-17、IL-23、

TNF-α及IFN-γ蛋白表達(dá)水平對(duì)比

治療第14天，小鼠背部皮損組織中IL-17、 IL-23、 TNF-α及 IFN-γ蛋白表達(dá)水平對(duì)比，對(duì)照組顯著低于其他各組 （P均 ＜0.05），且MEBO高劑量組顯著低于模型組（P均＜0.05），詳見(jiàn)表2、圖3。治療第28天，小鼠背部皮損組織中 IL-17、IL-23、TNF-α及IFN-γ蛋白表達(dá)水平對(duì)比，對(duì)照組顯著低于模型組、MEBO低劑量組及MEBO中劑量組 （P均 ＜0.05），與 MEBO高劑量組無(wú)明顯差異 （P均＞0.05）；MEBO高劑量組和MEBO中劑量組顯著低于模型組和MEBO低劑量組 （P均＜0.05），且MEBO高劑量組與MEBO中劑量組無(wú)明顯差異 （P均＞0.05）， 詳見(jiàn)表3、 圖3。

1.6.Statistical processing

SPSS 19.0 statistical software was used to analyze the obtained data.The measurement data conforming to the normal distribution were expressed as mean±standard deviation（）， and multi-sample comparison was performed using one-way ANOVA；P＜0.05 was considered as statistically significant.

2.Results

2.1.Comparison of PASI scores of skin lesions on the backs of the five groups of mice

On day 7 of treatment， the back skin lesions in the control group，the MEBO medium-dose group and the MEBO high-dose group was lighter in color， had less obvious infiltration and thickening and fewer scales，and lower PASI score（all P＜0.05） compared with that in the model group， and pairwise comparison among the control group， the MEBO medium-dose group and the MEBO high-dose group showed no significant difference in PASI scores（all P ＞0.05）.On day 14 of treatment， the skin lesions on the back of the five groups of mice improved significantly：the plaques were reddish in color， no obvious infiltration and thickening，only a very small amount of scales were observed，and no significant difference was observed in PASI score（P＞0.05），as shown in table 1.

2.2.Comparison of the protein expression levels of IL-17， IL-23， TNF-α and IFN-γ in the skin lesions on the backs of the five groups of mice

On day 14 of treatment， the protein expression levels of IL-17，IL-23， TNF-α and IFN-γ in the skin tissues on the backs of the five groups of mice were compared：their levels in the control group was significantly lower than other groups（all P＜0.05） and their levels in the MEBO group were significantly lower than the model group（all P＜0.05）.See Table 2 and Fig.3 for details.On day 28 of treatment， the protein expression levels of IL-17， IL-23， TNF-α and IFN-γ in the skin tissues on the backs of the five groups of mice were compared：their levels in the control group was significantly lower than the model group，the MEBO low-dose group and the MEBO medium-dose group（all P＜0.05）and no significant difference was observed between the control group and MEBO high-dose group （all P ＞0.05）；their levels in the MEBO high-dose group and MEBO medium-dose group were significantly lower than the model group and MEBO low-dose group （all P ＜0.05），but there was no significant difference between MEBO high-dose group and MEBO medium-dose group （all P ＞0.05）.See Table 2 and Fig.3 for details.

表1 5組小鼠背部皮損PASI評(píng)分對(duì)比 （）Table 1 Comparison of PASI scores of skin lesions on the backs of the five groups of mice（）

表1 5組小鼠背部皮損PASI評(píng)分對(duì)比 （）Table 1 Comparison of PASI scores of skin lesions on the backs of the five groups of mice（）

注：5組小鼠背部皮損PASI評(píng)分組間兩兩對(duì)比，其中與對(duì)照組對(duì)比，aP＜0.05，差異具有統(tǒng)計(jì)學(xué)意義；與模型組對(duì)比，bP＜0.05，差異具有統(tǒng)計(jì)學(xué)意義；與MEBO低劑量組對(duì)比，cP＜0.05，差異具有統(tǒng)計(jì)學(xué)意義Note：Pairwise comparison was made among the five groups in the PASI scores.Among them，comparison with that of the control group（aP＜0.05）showed statistically significant difference；comparison with that of the model group（bP ＜0.05） showed statistically significant difference；comparison with that of the MEBO low-dose group（cP＜0.05） showed statistically significant difference

組別Group鼠數(shù) （只）Number of mice （n）第7天 （分）Day 7 （point）第14天 （分）Day 14 （point）對(duì)照組Control group 7 5.29±0.75 1.71±0.49模型組Model group 7 7.00±0.81a 2.00±0.82 MEBO低劑量組MEBO low-dose group 7 6.86±0.69a 1.86±0.69 MEBO中劑量組MEBO medium-dose group 7 5.86±0.69bc 1.71±0.76 MEBO高劑量組MEBO high-dose group 7 5.71±0.95bc 1.57±0.54 F值F value 6.346 0.415 P值P value 0.001 0.797

表2 治療第14天5組小鼠背部皮損組織中IL-17、IL-23、TNF-α及IFN-γ蛋白表達(dá)水平對(duì)比 （）Table 2 Comparison of the protein expression levels of IL-17， IL-23， TNF-α and IFN-γ in the skin lesions on the backs of the five groups of mice on day 14 of treatment（）

表2 治療第14天5組小鼠背部皮損組織中IL-17、IL-23、TNF-α及IFN-γ蛋白表達(dá)水平對(duì)比 （）Table 2 Comparison of the protein expression levels of IL-17， IL-23， TNF-α and IFN-γ in the skin lesions on the backs of the five groups of mice on day 14 of treatment（）

注：治療第14天，5組小鼠背部皮損組織中IL-17、IL-23、TNF-α及IFN-γ蛋白表達(dá)水平組間兩兩對(duì)比，其中與對(duì)照組對(duì)比，aP＜0.05，差異具有統(tǒng)計(jì)學(xué)意義；與模型組對(duì)比，bP＜0.05，差異具有統(tǒng)計(jì)學(xué)意義Note： Pairwise comparison in the expression levels of IL-17， IL-23， TNF-α and IFN-γ in the skin tissues on the backs of the five groups of mice was made，comparison with that of the control group（aP＜0.05） showed statistically significant difference，comparison with that of the model group（bP＜0.05）showed statistically significant difference

組別Group鼠數(shù) （只）Number of mice （n） IL-17 IL-23 TNF-α IFN-γ對(duì)照組Control group 4 0.104±0.012 0.102±0.060 0.275±0.068 0.139±0.058模型組Model group 4 0.344±0.043a 0.272±0.037a 0.598±0.044a 0.484±0.062a MEBO低劑量組MEBO low-dose group 4 0.307±0.053a 0.258±0.027a 0.586±0.083a 0.451±0.125a MEBO中劑量組MEBO medium-dose group 4 0.294±0.037a 0.232±0.023a 0.539±0.081a 0.425±0.091a MEBO高劑量組MEBO high-dose group 4 0.248±0.036ab 0.205±0.023ab 0.507±0.105ab 0.408±0.077ab F值F value 17.565 10.302 8.158 7.761 P值P value 0.000 0.001 0.003 0.004

3 討論

尋常型銀屑病臨床以紅斑、鱗屑為主要表現(xiàn)，是由多種免疫細(xì)胞和細(xì)胞因子介導(dǎo)的自身免疫性疾病［9］，且與皮膚屏障功能不全密切相關(guān)［10］。相關(guān)研究證實(shí)， IL-23／Th17軸失調(diào)是銀屑病的主要發(fā)病機(jī)制之一［11］，IL-23作為重要的致炎因子，可通過(guò)促進(jìn)Th17細(xì)胞的分化及活化而促使IL-17、IL-22、IL-21等細(xì)胞因子的大量釋放，進(jìn)而導(dǎo)致角質(zhì)形成細(xì)胞過(guò)度增生［12］，并向真皮層生長(zhǎng)，逐漸衍變?yōu)槠つw炎性損害，形成銀屑?。?3］；IL-17可通過(guò)促進(jìn)炎癥反應(yīng)的發(fā)生，刺激角質(zhì)形成細(xì)胞分泌IL-6、TNF-α及IFN-γ［14］而加快中性粒細(xì)胞的增殖、成熟和趨化，進(jìn)而導(dǎo)致表皮細(xì)胞過(guò)度增殖。濕潤(rùn)燒傷膏作為一種油膏制劑，因其內(nèi)含有的有效成分可抑制TNF-α與IL-6等細(xì)胞因子的產(chǎn)生與釋放，減輕局部與全身炎癥反應(yīng)［15-17］而被應(yīng)用于尋常型銀屑病的治療，療效較為滿(mǎn)意，但具體作用機(jī)制并未被證實(shí)。

3.Discussion

Psoriasis vulgaris is an autoimmune disease mediated by a variety of immune cells and cytokines［9］.Its main clinical manifestations are erythema and scales and its occurrence is closely related to skin barrier dysfunction［10］.Studies have confirmed that the imbalance of IL-23 ／Th17 axis is one of the main pathogenesis of psoriasis［11］.As an important inflammatory factor，IL-23 can promote the differentiation and activation of Th17 cells，leading to the release of large amount of cell factors like IL-17， IL-22 and IL-21.As a result， keratinocytes proliferate excessively［12］and start growing towards dermis， causing inflammatory skin lesions and finally psoriasis［13］； IL-17 can accelerate the proliferation，maturation and chemotaxis of neutrophils by promoting inflammatory reactions and stimulating keratinocytes to secrete IL-6，TNF-α and IFN-γ［14］， resulting in excessive proliferation of epidermal cells.MEBO is a kind of ointment and has been applied in the treatment of psoriasis vulgoriasis thanks to its active ingredients，which can inhibit the production and release of cytokines such as TNF-α and IL-6 and reduce local and systemic inflammatory reactions［15-17］.Despite of its satisfactory effect，its specific mechanism of action has not been proven.

表3 治療第28天5組小鼠背部皮損組織中IL-17、IL-23、TNF-α及IFN-γ蛋白表達(dá)水平對(duì)比 （）Table 3 Comparison of the protein expression levels of IL-17， IL-23， TNF-α and IFN-γ in the skin lesions on the backs of the five groups of mice on day 28 of treatment（）

表3 治療第28天5組小鼠背部皮損組織中IL-17、IL-23、TNF-α及IFN-γ蛋白表達(dá)水平對(duì)比 （）Table 3 Comparison of the protein expression levels of IL-17， IL-23， TNF-α and IFN-γ in the skin lesions on the backs of the five groups of mice on day 28 of treatment（）

注：治療第28天，5組小鼠背部皮損組織中IL-17、IL-23、TNF-α及IFN-γ蛋白表達(dá)水平組間兩兩對(duì)比，其中與對(duì)照組對(duì)比，aP＜0.05，差異具有統(tǒng)計(jì)學(xué)意義；與模型組對(duì)比，bP＜0.05，差異具有統(tǒng)計(jì)學(xué)意義；與MEBO低劑量組對(duì)比，cP＜0.05，差異具有統(tǒng)計(jì)學(xué)意義Note： The protein expression levels of IL-17， IL-23， TNF-α and IFN-γ in the skin lesions on the backs of the five groups of mice were compared between each two groups.Among them，comparison with that of the control group（aP＜0.05） showed statistically significant difference；comparison with that of the model group（bP＜0.05）showed statistically significant difference；compared with the MEBO low-dose group（cP＜0.05）showed statistically significant difference

組別Group鼠數(shù) （只）Number of mice （n） IL-17 IL-23 TNF-α IFN-γ對(duì)照組Control group 3 0.133±0.038 0.104±0.031 0.164±0.069 0.189±0.086模型組Model group 3 0.369±0.429a 0.356±0.043a 0.497±0.026a 0.521±0.077a MEBO低劑量組MEBO low-dose group 3 0.322±0.029a 0.276±0.031a 0.418±0.066a 0.432±0.062a MEBO中劑量組MEBO medium-dose group 3 0.245±0.004abc 0.198±0.018abc 0.321±0.025abc 0.380±0.045abc MEBO高劑量組MEBO high-dose group 3 0.187±0.026bc 0.144±0.012bc 0.251±0.021bc 0.302±0.028bc F值F value 28.428 36.995 24.137 12.106 P值P value 0.000 0.000 0.000 0.001

圖3 5組小鼠背部皮損組織中IL-17、IL-23、TNF-α及IFN-γ蛋白表達(dá)條帶圖Fig.3 Histogram of protein expression levels of IL-17， IL-23， TNF-α and IFN-γ in the skin lesions on the backs of the five groups of mice

本研究通過(guò)觀察濕潤(rùn)燒傷膏對(duì)尋常型銀屑病小鼠背部皮損組織中 IL-17、IL-23、TNF-α及IFN-γ水平的影響探討其對(duì)尋常型銀屑病的作用機(jī)制。結(jié)果顯示，治療第7天，對(duì)照組、MEBO中劑量組及MEBO高劑量組小鼠背部皮損PASI評(píng)分均顯著低于模型組，且對(duì)照組、MEBO中劑量組及MEBO高劑量組組間兩兩對(duì)比，PASI評(píng)分均無(wú)明顯差異?？梢?jiàn)，濕潤(rùn)燒傷膏能夠有效改善尋常型銀屑病小鼠紅斑、鱗屑、浸潤(rùn)增厚等皮損癥狀，且能夠達(dá)到臨床已證實(shí)有效的卡泊三醇軟膏同等的療效。Western blotting檢測(cè)結(jié)果顯示，治療第14天，小鼠背部皮損組織中IL-17、IL-23、TNF-α及 IFN-γ蛋白表達(dá)水平對(duì)比，對(duì)照組顯著低于其他各組，且MEBO高劑量組顯著低于模型組；治療第28天，小鼠背部皮損組織中IL-17、IL-23、TNF-α及IFN-γ蛋白表達(dá)水平對(duì)比，對(duì)照組顯著低于模型組、MEBO低劑量組及MEBO中劑量組，與MEBO高劑量組無(wú)明顯差異，MEBO高劑量組和MEBO中劑量組顯著低于模型組和MEBO低劑量組，且MEBO高劑量組與MEBO中劑量組無(wú)明顯差異?？梢?jiàn)，濕潤(rùn)燒傷膏可有效降低IL-23／Th17軸相關(guān)因子的表達(dá)水平，抑制炎癥反應(yīng)及角質(zhì)形成細(xì)胞過(guò)度增生，且隨著治療時(shí)間的延長(zhǎng)，其對(duì)炎癥反應(yīng)的抑制作用加強(qiáng)，并達(dá)到卡泊三醇軟膏同等的作用水平；另外，本結(jié)果可見(jiàn)濕潤(rùn)燒傷膏的作用效果具有劑量及時(shí)間依賴(lài)性，即在一定條件下，藥物濃度越高、治療時(shí)間越長(zhǎng)，其治療作用越強(qiáng)。

綜上所述，濕潤(rùn)燒傷膏治療銀屑病的療效與卡泊三醇軟膏相當(dāng)，且調(diào)節(jié)IL-23／Th17軸及其相關(guān)因子的表達(dá)水平，抑制炎癥反應(yīng)可能是其作用機(jī)制。

In this study， the effect of MEBO on the levels of IL-17， IL-23，TNF-α and IFN-γ in the skin tissues on the backs of mice was investigated so as to explore its mechanism of action on psoriasis vulgoriasis.The results showed that， on day 7 of treatment， the PASI scores of the skin lesions on the backs of mice in the control group，the MEBO mediumdose group，and the MEBO high-dose group were significantly lower than the model group.Pairwise comparison in the PASI scores among the control group，the MEBO medium-dose group and the MEBO high-dose group was made and no significant difference was observed.It can be concluded that MEBO can alleviate the skin lesions of psoriasis vulgaris on mice such as erythema， scales， and infiltration and thickening and is clinically as effective as calcipotriol cream.The result of western blotting test showed that on day 14 of treatment，the protein expression levels of IL-17， IL-23， TNF-α and IFN-γ in the skin lesions on the backs of the mice were compared：their levels in the control group was significantly lower than other groups and their levels in the MEBO high-dose group was significantly lower than the model group； on day 28 of treatment， the protein expression levels of IL-17， IL-23， TNF-α and IFN-γ in the skin tissues on the backs of the mice were compared：their levels in the control group was significantly lower than the model group，the MEBO lowdose group and the MEBO medium-dose group，no significant difference was observed between the control group and the MEBO high-dose group；their levels in the MEBO high-dose group and the MEBO medium-dose group were significantly lower than the model group and the MEBO lowdose group and no significant difference was observed between the MEBO high-dose group and the MEBO medium-dose group.It can be concluded that MEBO can reduce the expression levels of IL-23／Th17 axis-related factors，inhibit inflammatory response and excessive proliferation of keratinocytes.As treatment time increases，inhibitory effect of MEBO on inflammatory reactions can be strengthened to the same level as calcipotriol cream；in addition，the results showed that the effect of MEBO is closely related to its dosage amount and action time， that is， under certain conditions，the higher the drug concentration and the longer the treatment time are the stronger its therapeutic effect will be.

In summary，the efficacy of MEBO in treating psoriasis is equivalent to calcipotriol cream，and its mechanism of action may lie in its ability to regulate the expression levels of the IL-23／Th17 axis and its related factors and inhibit inflammation.

（收稿日期：2019-12-09）