戚世玲 方銘恒 謝振謀 底大可 薛汝增 何仁亮

尋常型天皰瘡是臨床最常見、最嚴(yán)重的天皰瘡類型,其典型表現(xiàn)為正常皮膚或黏膜上出現(xiàn)松弛性水皰,且尼氏征陽性,水皰易破潰糜爛引發(fā)感染、低蛋白血癥、惡病質(zhì)而危及患者生命[1],目前臨床尚無明顯有效的治療方法,致使創(chuàng)面愈合困難,嚴(yán)重影響了患者的生活質(zhì)量。為探尋一種較為理想的治療該疾病的方法,筆者對22例尋常型天皰瘡患者采用了濕潤燒傷膏 （moist exposed burn ointment,MEBO）外敷聯(lián)合紅外線照射治療,觀察了其臨床療效及患者血清細(xì)胞因子表達(dá)水平的變化情況,并與依沙吖啶溶液外敷聯(lián)合紅外線照射治療者進(jìn)行了對比,以期為尋常型天皰瘡的治療提供理論依據(jù),現(xiàn)報告如下。

1 臨床資料

1.1 一般資料

選取2013年1月至2018年6月廣州醫(yī)科大學(xué)附屬第五醫(yī)院及南方醫(yī)科大學(xué)皮膚病醫(yī)院收治的44例尋常型天皰瘡患者作為研究對象,并按照隨機數(shù)表法將其隨機分為觀察組 （22例患者,261處創(chuàng)面）與對照組 （22例患者,253處創(chuàng)面）,其中觀察組男性12例、女性10例,年齡 （56.7±12.5）歲,病程 （10.5±4.8） 個月,皮損面積 （37.5±16.4）%TBSA,糖皮質(zhì)激素初始劑量 （61.4±14.3）mg,皮損程度為輕癥 （皮損面積≤10%TBSA）者2例、中癥 （皮損面積＞10%TBSA且≤50%TBSA）者11例、重癥 （皮損面積＞50%TBSA[2]）者9例,創(chuàng)面細(xì)菌感染者17例、真菌感染者5例；對照組男性9例、女性13例,年齡 （59.3±13.9）歲,病程 （11.4±5.6）個月,皮損面積 （40.2±18.1）%TBSA,糖皮質(zhì)激素初始劑量 （63.5±16.8）mg,皮損程度為輕癥者4例、中癥者8例、重癥者10例,創(chuàng)面細(xì)菌感染者19例、真菌感染者3例。所有患者均伴有不同程度的口腔糜爛,水皰處尼氏征均呈陽性。兩組患者性別、年齡、病程等一般資料對比,P均＞0.05,差異無統(tǒng)計學(xué)意義 （表1）,具有可比性。本研究經(jīng)廣州醫(yī)科大學(xué)附屬第五醫(yī)院及南方醫(yī)科大學(xué)皮膚病醫(yī)院倫理委員會批準(zhǔn),且所有患者均簽署了知情同意書。

1.2 納入與排除標(biāo)準(zhǔn)

納入標(biāo)準(zhǔn)：（1）符合尋常型天皰瘡的診斷標(biāo)準(zhǔn)者；（2）對本研究知情,并自愿簽署知情同意書者。

排除標(biāo)準(zhǔn)：（1）合并有糖尿病、急性心力衰竭、敗血癥、嚴(yán)重低蛋白血癥及貧血等疾病者；（2）合并有惡性腫瘤等疾病者；（3）合并有嚴(yán)重肝、腎、肺等臟器功能不全者；（4）依從性較差,無法按要求完成全程治療者。

表1 兩組患者一般資料對比Table 1 Comparison of general data between the two groups

2 方法

2.1 治療方法

觀察組：在給予患者抗感染、營養(yǎng)支持等全身綜合治療的同時,局部創(chuàng)面于生理鹽水沖洗后用紅外線照射20 min；照射后,創(chuàng)面均勻涂抹MEBO（厚約1 mm）,并依次覆蓋MEBO藥紗及2層無菌紗布包扎、固定。根據(jù)創(chuàng)面滲出情況,每4～8 h用紅外線照射并換藥1次。

對照組：在給予患者抗感染、營養(yǎng)支持等全身綜合治療的同時,局部創(chuàng)面于高錳酸鉀溶液 （濃度約為1∶8000）沖洗后用0.1%依沙吖啶溶液濕敷20 min,每天2次；濕敷后,予以紅外線持續(xù)照射,全天照射10 h以上。

2.2 標(biāo)本采集與檢測

分別于治療前及治療第14天取患者早8點空腹靜脈血10 mL,靜置2 h后3000 r/min離心2 min,取上層血清置于-70℃冰箱內(nèi)備檢。

標(biāo)本采集完成后,按照說明書采用ELISA雙抗體夾心法檢測血清 IL-6、IL-10、IL-17、IL-23、IFN-γ及TNF-α的表達(dá)水平。

2.3 觀察指標(biāo)及判定標(biāo)準(zhǔn)

對比觀察兩組患者治療第14天的臨床療效和血清 IL-6、 IL-10、 IL-17、 IL-23、 IFN-γ 及TNF-α的表達(dá)水平及住院時間。

療效判定標(biāo)準(zhǔn)：痊愈,水皰全部消退,創(chuàng)面完全愈合；顯效,創(chuàng)面愈合率≥60%；好轉(zhuǎn),創(chuàng)面愈合率≥30%且＜60%；無效,創(chuàng)面愈合率＜30%或擴大；有效率= （痊愈例數(shù)+顯效例數(shù)+好轉(zhuǎn)例數(shù)） /總例數(shù)×100%[1]。創(chuàng)面愈合率=愈合創(chuàng)面面積/創(chuàng)面總面積×100%。

2.4 統(tǒng)計學(xué)處理

采用SPSS 16.0統(tǒng)計軟件對所得數(shù)據(jù)進(jìn)行統(tǒng)計學(xué)分析,其中計數(shù)資料以頻數(shù)或百分比表示,采用卡方檢驗或秩和檢驗；計量資料以均數(shù)±標(biāo)準(zhǔn)差 （x±s） 表示,采用t檢驗或t'檢驗；均以P＜0.05為差異具有統(tǒng)計學(xué)意義。

3 結(jié)果

3.1 臨床療效及住院時間對比

治療第14天,觀察組患者中創(chuàng)面痊愈40處、顯效84處、好轉(zhuǎn)88處、無效49處,有效率為81.23%；對照組患者中創(chuàng)面痊愈23處、顯效63處、好轉(zhuǎn)59處、無效108處,有效率為57.31%,兩組對比,P＜0.05,差異具有統(tǒng)計學(xué)意義。觀察組患者住院時間為 （21.9±7.8）d,對照組患者住院時間為 （32.8±11.5）d,兩組對比,P＜0.05,差異具有統(tǒng)計學(xué)意義 （表2）?？梢?MEBO外敷聯(lián)合紅外線照射治療者的臨床療效明顯優(yōu)于依沙吖啶溶液外敷聯(lián)合紅外線照射治療者,且住院時間明顯短于依沙吖啶溶液外敷聯(lián)合紅外線照射治療者。

3.2 血清細(xì)胞因子表達(dá)水平對比

治療前,兩組患者血清 IL-6、IL-10、IL-17、 IL-23、 IFN-γ及 TNF-α 表達(dá)水平對比,P均＞0.05,差異無統(tǒng)計學(xué)意義,具有可比性；治療第14天,兩組患者血清IL-10及TNF-α表達(dá)水平對比,P均＞0.05,差異無統(tǒng)計學(xué)意義,血清 IL-6、IL-17、IL-23及 IFN-γ表達(dá)水平對比,P均＜0.05,差異具有統(tǒng)計學(xué)意義 （表3）。可見,與依沙吖啶溶液外敷聯(lián)合紅外線照射相比,MEBO外敷聯(lián)合紅外線照射可更有效抑制血清細(xì)胞因子 IL-6、IL-17及 IL-23的分泌,促進(jìn)IFN-γ的分泌。

表2 兩組患者臨床療效及住院時間對比Table 2 Comparison of clinical efficacy between the two groups

表3 兩組患者血清細(xì)胞因子表達(dá)水平對比 （ng/L，x ±s）Table 3 Comparison of the expression levels of serum cytokines between the two groups（ng/L， x±s）

3.3 不良反應(yīng)發(fā)生情況對比

治療過程中,觀察組患者中有3例患者局部皮損創(chuàng)面出現(xiàn)輕微刺痛、瘙癢,持續(xù)時間均在5 min內(nèi),未予特殊處理,自行緩解；對照組患者均無明顯不良反應(yīng)發(fā)生。兩組患者不良反應(yīng)發(fā)生情況對比采用四格表資料的卡方檢驗,χ2=3.220,P=0.073,P＞0.05,差異無統(tǒng)計學(xué)意義。

4 討論

尋常型天皰瘡是一種嚴(yán)重的自身免疫性疾病,目前臨床多采用激素、免疫抑制劑、生物制劑、創(chuàng)面敷料等予以治療,但臨床療效不佳,尤其是傳統(tǒng)的干性療法易使創(chuàng)面脫水、結(jié)痂,不利于上皮細(xì)胞的爬行,創(chuàng)面愈合速度緩慢,嚴(yán)重影響了患者的生活質(zhì)量[3-4]。本研究中,對照組患者采用依沙吖啶溶液外敷聯(lián)合紅外線照射治療,臨床療效明顯不及采用MEBO外敷聯(lián)合紅外線照射治療的觀察組,創(chuàng)面愈合時間明顯長于觀察組。

天皰瘡創(chuàng)面持續(xù)暴露于紅外線中,雖有利于創(chuàng)面的消炎、鎮(zhèn)痛,改善局部血液循環(huán),提高組織細(xì)胞的營養(yǎng)代謝,但創(chuàng)面迅速干燥結(jié)痂后,可造成創(chuàng)面疼痛,且無法隔絕細(xì)菌入侵,易形成痂下膿腫[1]。而MEBO外敷聯(lián)合紅外線照射治療,紅外線照射20 min后,血液循環(huán)明顯改善,此時應(yīng)用MEBO,可促進(jìn)MEBO向組織深層滲入,進(jìn)而發(fā)揮更大的藥效。MEBO內(nèi)含有的黃芩甙、小檗堿等成分可破壞細(xì)菌生長環(huán)境,抑制其活性及毒性,有效防治創(chuàng)面感染[5-6]；其有效成分可激活創(chuàng)面組織內(nèi)的潛能再生細(xì)胞,并將其轉(zhuǎn)化為干細(xì)胞,再在原位增殖、分化為創(chuàng)面各層組織細(xì)胞,原位再生修復(fù)創(chuàng)面[7-8]；可提高創(chuàng)面組織中 VEGF、bFGF、EGF基因及其蛋白的表達(dá)水平,促進(jìn)成纖維細(xì)胞及血管內(nèi)皮細(xì)胞的增殖分化,從而加快肉芽組織生長,再生修復(fù)創(chuàng)面；其為創(chuàng)面營造的生理性濕潤環(huán)境可預(yù)防組織細(xì)胞脫水,加速血管生成,增強生長因子與其靶細(xì)胞的相互作用,促進(jìn)創(chuàng)面愈合[9-10]。

天皰瘡是一種自身抗體介導(dǎo)的免疫性疾病,而T細(xì)胞分泌的細(xì)胞因子可通過介導(dǎo)T細(xì)胞亞群間的相互作用影響機體免疫系統(tǒng)的功能。如IL-6含量上升時可誘導(dǎo)更多的初始T細(xì)胞向Th17分化,進(jìn)而促進(jìn)IL-17等細(xì)胞因子的分泌,進(jìn)一步加重組織炎性損害[9,11]；IL-10可抑制Thl細(xì)胞的增殖,進(jìn)而減少IL-2、TNF-α等細(xì)胞因子的生成。本研究結(jié)果顯示,MEBO外敷聯(lián)合紅外線照射治療組患者在治療第14天時IL-6、IL-17、IL-23及 TNF-α的表達(dá)水平明顯降低,IL-10及IFN-γ的表達(dá)水平明顯升高,且與依沙吖啶溶液外敷聯(lián)合紅外線照射相比,MEBO外敷聯(lián)合紅外線照射可更有效地抑制細(xì)胞因子IL-6、IL-17及IL-23的分泌,促進(jìn)IFN-γ的分泌,兩組對比,P＜0.05,差異具有統(tǒng)計學(xué)意義?？梢?MEBO外敷聯(lián)合紅外線照射可有效縮短天皰瘡創(chuàng)面的愈合時間,促進(jìn)天皰瘡的消退可能與其能夠調(diào)節(jié)IL-6、IL-17、IL-23及IFN-γ等細(xì)胞因子的分泌有關(guān),有待進(jìn)一步深入研究探討。

綜上所述,MEBO外敷聯(lián)合紅外線照射可促進(jìn)天皰瘡創(chuàng)面的愈合,療效顯著,可能與其能夠調(diào)節(jié)天皰瘡患者血清IL-6、IL-17、IL-23及IFN-γ等細(xì)胞因子的表達(dá)水平有關(guān),值得臨床進(jìn)一步推廣應(yīng)用與驗證。

Pemphigus vulgaris is the commonest and most serious type of pemphigus in the clinical practice.It is characterized by slack blisters on normal skin or mucous membranes with positive Nikolsky's sign.The blisters are susceptible to ulcerate and rot,which may cause infection, hypoproteinemia and cachexia, endangering patients'lives[1].At present,there is no obviously effective treatment modality for such disease,leaving the ulcerated wounds hard to heal and thus greatly affecting the quality of life of patients.In order to find a better way to treat this disease,the authors treated 22 cases of pemphigus vulgaris with the external application of MEBO combined with infrared irradiation to observe the influence on the expression level of serum cytokines and the clinical efficacy,and also,made a comparison with the treatment modality of the external application of ethacridine lactate solution combined with infrared irradiation,with the aim of providing theoretical evidence for the treatment of pemphigus vulgaris.The details are reported：

1.Clinical data

1.1.General data

44 patients with pemphigus vulgaris,admitted into The Fifth Affiliated Hospital of Guangzhou Medical University and Dermatology Hospital of Southern Medical University from January 2013 to June 2018,were divided,according to the random number table,into an observation group （22 patients,261 wounds） and a control group（22 patients,253 wounds）.In the observation group,12 males and 10 females,aged（56.7±12.5） years old,duration of disease-（10.5±4.8） months,skin damage area-（37.5±16.4）%TBSA,initial dose of glucocorticoids-（61.4±14.3） mg,2 cases of mildskin damage（damage area≤10%TBSA）,11 cases of moderate skin damage（skin damage area ＞10%TBSA and≤50%TBSA）,9 cases of severe skin damage（skin damage area ＞50%TBSA[2]）,17 cases with wound bacterial infection,5 cases with wound fungal infection；in the control group,9 males and 13 females,aged （59.3±13.9）years old,duration of disease-（11.4±5.6） months,skin damage area-（40.2 ±18.1）%TBSA,initial dose of glucocorticoids（63.5±16.8） mg,4 cases of mild skin damage,8 cases of moderate skin damage,10 cases of severe skin damage,19 cases with wound bacterial infection,3 cases with fungal infection.All patients in the two groups had oral erosion of different degrees with positive Nikolsky's sign in blisters.The general data such as gender,age,duration of disease and etc.were respectively compared between the two groups and the results showed no statistically significant difference（P ＞0.05） （Table 1）,presenting comparability between the two groups.The study was approved by the Ethics Committee of The Fifth Affiliated Hospital of Guangzhou Medical University and Dermatology Hospital of Southern Medical University,and all patients signed the informed consent form.

1.2.Inclusion and exclusion criteria

Inclusion criteria： （1） those who met the diagnostic criteria for pemphigus vulgaris； （2） those who are well informed of this study and voluntarily sign the informed consent form.

Exclusion criteria： （1） patients with pemphigus vulgaris but complicated with diabetes,acute heart failure,sepsis,severe hypoproteinemia and anemia； （2） patients with pemphigus vulgaris but complicated with malignant tumors； （3） patients with pemphigus vulgaris but complicated with severe dysfunction in liver,kidney,lung and etc.； （4） patients with poor compliance or those who are unable to complete the full course of treatment as required.

2.Methods

2.1.Treatment methods

Observation group：in addition to systemic comprehensive treatments such as anti-infection,nutritional support,infrared ray was adopted to irradiate the local wounds for 20 min after the wounds were rinsed with normal saline.After the irradiation,the wound surface was evenly smeared a layer of MEBO （about 1 mm thick） and then covered with MEBO-impregnated gauze,followed by wrapping-up and fixing the wounds with 2 layers of sterile gauze.Based on the wound exudation,infrared irradiation and dressing change were performed every 4-8 h.

Control group：in addition to systemic comprehensive treatments such as anti-infection,nutritional support,hydropathic compress with 0.1%ethacridine lactate solution was performed twice a day,20 min each time,after the wound was rinsed with potassium permanganate solution （concentration： 1∶8000）.After the hydropathic compress,infrared irradiation was performed for over 10 hours per day.

2.2.Sample collection and detection

Before the treatment and on day 14 of treatment,10 mL of fasting venous blood was taken at 8 o'clock in the morning.After 2 hours of standing time,the blood was centrifuged at the speed of 3000 r/min for 2 min.The upper serum was put in a refrigerator at-70℃for detection.

After the specimens were collected,the expression levels of serum IL-6,IL-10,IL-17,IL-23,IFN-γ and TNF-α were measured with ELISA double antibody sandwich method according to the instruction.

2.3.Observational indexes and assessment criteria of clinical efficacy

The clinical efficacy and the expression levels of serum IL-6,IL-10,IL-17,IL-23,IFN-γ and TNF-α on day 14 of treatment,and hospitalization time were observed and compared between the two groups.

Assessment criteria of clinical efficacy： cured： disappearance of all blisters and complete wound healing； markedly effective： wound healing rate≥60%；improved：wound healing rate≥30%and ＜60%；ineffective：wound healing rate ＜30%or wound area became larger.Total effective rate= （number of cured cases+number of markedly effective cases+number of improved cases）/total number of cases×100%.Wound healing rate=healed wound area/total wound area×100%.

2.4.Statistical analysis

The SPSS 16.0 software was adopted to analyze the obtained data,in which the chi-square test or the rank sum test was applied to manage the count data expressed with frequency or percentage while thettest ort'test was used to manage the measurement data expressed with（x±s）.P＜0.05 was considered as statistically significant difference.

3.Results

3.1.Comparison of clinical efficacy and hospitalization time

On day 14 of treatment,the total effective rate in the observationgroup was 81.23%,with 40 cases cured,84 cases showed markedly effective,88 cases improved and 49 cases ineffective,while the total effective rate in the control group was 57.31%,with 23 cases cured,63 cases markedly effective,59 cases improved and 108 cases ineffective,between which the comparison showed statistically significant difference（P ＜0.05）.The hospitalization time in the observation group was（21.9±7.8） d and was（32.8±11.5） d in the control group,and the comparison of the hospitalization time between the two groups showed statistically significant difference（P ＜0.05） （Table 2）.It can be concluded that the clinical efficacy of the external application of MEBO combined with infrared irradiation is much better than that of ethacridine lactate solution combined with infrared irradiation,and also the hospitalization time in the former treatment modality is obviously shorter than that in the latter one.

3.2.Comparison of expression levels of serum cytokines

Before the treatment,the expression levels of serum IL-6,IL-10,IL-17,IL-23,IFN-γ and TNF-α were respectively compared between the two groups,and the results all showed no statistically significant difference（P ＞0.05）,and thus the two groups were comparable.On day 14 of treatment,the expression levels of serum IL-10 and TNF-α were respectively compared between the two groups,and the results showed no statistically significant difference（P ＞0.05）,but the comparison of expression levels of serum IL-6,IL-17,IL-23 and IFN-γ between the two groups showed statistically significant differences（all P ＜0.05） （Table 3）.It can be concluded that,compared with the external application of ethacridine lactate solution combined with infrared irradiation,the external application of MEBO combined with infrared irradiation can better inhibit the secretion of serum cytokines-IL-6,IL-17 and IL-23 and promote the secretion of IFN-γ.

3.3.Comparison of adverse reactions

During the treatment course,3 patients in the observation group suffered slight sting and itching in partial damaged skin areas,and the symptoms lasted for less than 5 minutes and disappeared naturally without any treatment.None of the patients in the control group had any obvious adverse reactions.The Chi-square test of fourfold table was used for the comparison of adverse reactions between the two groups and the results showed no statistically significant difference（χ2=3.220,P=0.073,P ＞0.05）.

4.Discussion

Pemphigus vulgaris,as a kind of serious autoimmune diseases,is currently often treated with hormones,immunosuppressors,biological agents,wound dressings and etc.in clinical practice,but the clinical efficacy is poor.Traditional dry therapy,in particular,probably can dehydrate the wound and lead to incrustation,which is not conducive to the growth of epithelial cells.As a result,the wound will heal very slow,seriously affecting the quality of life of patients[3-4].In this study,patients in the observation group,treated with the external application of MEBO and infrared irradiation,got better therapeutic effect than that of the control group treated with the external application of ethacridine lactate solution combined with infrared irradiation.

Exposing pemphigus wounds to infrared ray continuously can realize such effects as anti-inflammation,easing pain,improving the local blood circulation and enhancing the nutritional metabolism of tissues and cells.However,it can also make the wound dry quickly and lead to wound incrustation,causing wound pain.Moreover,this method cannot isolate the wound from bacterial invasion,probably leading to subeschar abscess[1].In contrast,when the treatment modality of external application of MEBO combined with infrared irradiation was applied in pemphigus wounds,the blood circulation will obviously improve after 20 minutes of infrared irradiation,which will facilitate the penetration of MEBO into deep tissues after the following application of MEBO onto the wounds,thereby increasing the therapeutic effect of MEBO.The ingredients contained in MEBO such as baicalin and berberine can damage the growth environment of bacteria,inhibit their activity and toxicity,and effectively prevent wound infection[5-6].And the other active ingredients contained in MEBO can activate the potential regenerative cells（PRCs） in the wound tissues and convert them into stem cells which can proliferate and differentiate in situ into different types of cells in the wound tissues to restore the wound in situ[7-8].Besides,MEBO can improve the expression levels of VEGF,bFGF,EGF genes and the corresponding proteins in wound tissues,promote the proliferation and differentiation of fibroblasts and vascular endothelial cells,hence accelerating the growth of granulation tissues,and promoting tissue regeneration and wound repair.The physiologically moist environment created by MEBO for wounds can help prevent tissues and cells from dehydration,accelerate angiogenesis,and enhance the interaction between growth factors and their target cells,and thereby promoting wound healing[9-10].

Pemphigus is an autoantibody-mediated immune disease,and cytokines secreted by T cells can affect the normal function of the body's immune system by mediating interactions between T cell subsets.For example,when IL-6 level increases,more initial T cells can be induced to differentiate into Th17,promoting the secretion cytokines including IL-17,and further aggravating tissue inflammatory injury[9,11]；IL-10 can inhibit the proliferation of Th1 cell,further reducing the production of cytokines such as IL-2 and TNF-α.The results of this study showed that the expression levels of IL-6,IL-17,IL-23 and TNF-α of patients,when treated with the external application of MEBO combined with infrared irradiation,significantly decreased on day 14 of treatment,whereas the expression levels of IL-10 and IFN-γ increased substantially.Compared with the external application of ethacridine lactate solution combined with infrared irradiation,the external application of MEBO combined with infrared irradiationcan better inhibit the secretion of cytokines IL-6,IL-17 and IL-23 and promote the secretion of IFN-γ.The comparison between the two groups showed statistically significant difference（P ＜0.05）.It can be concluded that the external application of MEBO combined with infrared irradiation can effectively shorten the healing time of pemphigus wounds,and accelerate the fading away of pemphigus,which may be related to MEBO's effect in regulating the secretion of cytokines such as serum IL-6,IL-17,IL-23 and IFN-γ,deserving to be further studied in the future.

To sum up,the external application of MEBO combined with infrared irradiation can promote the healing of pemphigus wounds,which may be related to MEBO's effect in regulating the expression levels of serum cytokines such as IL-6,IL-17,IL-23,IFN-γ and etc.in patients with pemphigus,and such a treatment modality should be further promoted and validated in clinical practice.