湿润烧伤膏外敷联合红外线照射对寻常型天疱疮患者血清细胞因子表达水平的影响

戚世玲 方铭恒 谢振谋 底大可 薛汝增 何仁亮

寻常型天疱疮是临床最常见、最严重的天疱疮类型,其典型表现为正常皮肤或黏膜上出现松弛性水疱,且尼氏征阳性,水疱易破溃糜烂引发感染、低蛋白血症、恶病质而危及患者生命[1],目前临床尚无明显有效的治疗方法,致使创面愈合困难,严重影响了患者的生活质量。为探寻一种较为理想的治疗该疾病的方法,笔者对22例寻常型天疱疮患者采用了湿润烧伤膏 （moist exposed burn ointment,MEBO）外敷联合红外线照射治疗,观察了其临床疗效及患者血清细胞因子表达水平的变化情况,并与依沙吖啶溶液外敷联合红外线照射治疗者进行了对比,以期为寻常型天疱疮的治疗提供理论依据,现报告如下。

1 临床资料

1.1 一般资料

选取2013年1月至2018年6月广州医科大学附属第五医院及南方医科大学皮肤病医院收治的44例寻常型天疱疮患者作为研究对象,并按照随机数表法将其随机分为观察组 （22例患者,261处创面）与对照组 （22例患者,253处创面）,其中观察组男性12例、女性10例,年龄 （56.7±12.5）岁,病程 （10.5±4.8） 个月,皮损面积 （37.5±16.4）%TBSA,糖皮质激素初始剂量 （61.4±14.3）mg,皮损程度为轻症 （皮损面积≤10%TBSA）者2例、中症 （皮损面积＞10%TBSA且≤50%TBSA）者11例、重症 （皮损面积＞50%TBSA[2]）者9例,创面细菌感染者17例、真菌感染者5例；对照组男性9例、女性13例,年龄 （59.3±13.9）岁,病程 （11.4±5.6）个月,皮损面积 （40.2±18.1）%TBSA,糖皮质激素初始剂量 （63.5±16.8）mg,皮损程度为轻症者4例、中症者8例、重症者10例,创面细菌感染者19例、真菌感染者3例。所有患者均伴有不同程度的口腔糜烂,水疱处尼氏征均呈阳性。两组患者性别、年龄、病程等一般资料对比,P均＞0.05,差异无统计学意义 （表1）,具有可比性。本研究经广州医科大学附属第五医院及南方医科大学皮肤病医院伦理委员会批准,且所有患者均签署了知情同意书。

1.2 纳入与排除标准

纳入标准：（1）符合寻常型天疱疮的诊断标准者；（2）对本研究知情,并自愿签署知情同意书者。

排除标准：（1）合并有糖尿病、急性心力衰竭、败血症、严重低蛋白血症及贫血等疾病者；（2）合并有恶性肿瘤等疾病者；（3）合并有严重肝、肾、肺等脏器功能不全者；（4）依从性较差,无法按要求完成全程治疗者。

表1 两组患者一般资料对比Table 1 Comparison of general data between the two groups

2 方法

2.1 治疗方法

观察组：在给予患者抗感染、营养支持等全身综合治疗的同时,局部创面于生理盐水冲洗后用红外线照射20 min；照射后,创面均匀涂抹MEBO（厚约1 mm）,并依次覆盖MEBO药纱及2层无菌纱布包扎、固定。根据创面渗出情况,每4～8 h用红外线照射并换药1次。

对照组：在给予患者抗感染、营养支持等全身综合治疗的同时,局部创面于高锰酸钾溶液 （浓度约为1∶8000）冲洗后用0.1%依沙吖啶溶液湿敷20 min,每天2次；湿敷后,予以红外线持续照射,全天照射10 h以上。

2.2 标本采集与检测

分别于治疗前及治疗第14天取患者早8点空腹静脉血10 mL,静置2 h后3000 r/min离心2 min,取上层血清置于-70℃冰箱内备检。

标本采集完成后,按照说明书采用ELISA双抗体夹心法检测血清 IL-6、IL-10、IL-17、IL-23、IFN-γ及TNF-α的表达水平。

2.3 观察指标及判定标准

对比观察两组患者治疗第14天的临床疗效和血清 IL-6、 IL-10、 IL-17、 IL-23、 IFN-γ 及TNF-α的表达水平及住院时间。

疗效判定标准：痊愈,水疱全部消退,创面完全愈合；显效,创面愈合率≥60%；好转,创面愈合率≥30%且＜60%；无效,创面愈合率＜30%或扩大；有效率= （痊愈例数+显效例数+好转例数） /总例数×100%[1]。创面愈合率=愈合创面面积/创面总面积×100%。

2.4 统计学处理

采用SPSS 16.0统计软件对所得数据进行统计学分析,其中计数资料以频数或百分比表示,采用卡方检验或秩和检验；计量资料以均数±标准差 （x±s） 表示,采用t检验或t'检验；均以P＜0.05为差异具有统计学意义。

3 结果

3.1 临床疗效及住院时间对比

治疗第14天,观察组患者中创面痊愈40处、显效84处、好转88处、无效49处,有效率为81.23%；对照组患者中创面痊愈23处、显效63处、好转59处、无效108处,有效率为57.31%,两组对比,P＜0.05,差异具有统计学意义。观察组患者住院时间为 （21.9±7.8）d,对照组患者住院时间为 （32.8±11.5）d,两组对比,P＜0.05,差异具有统计学意义 （表2）。可见,MEBO外敷联合红外线照射治疗者的临床疗效明显优于依沙吖啶溶液外敷联合红外线照射治疗者,且住院时间明显短于依沙吖啶溶液外敷联合红外线照射治疗者。

3.2 血清细胞因子表达水平对比

治疗前,两组患者血清 IL-6、IL-10、IL-17、 IL-23、 IFN-γ及 TNF-α 表达水平对比,P均＞0.05,差异无统计学意义,具有可比性；治疗第14天,两组患者血清IL-10及TNF-α表达水平对比,P均＞0.05,差异无统计学意义,血清 IL-6、IL-17、IL-23及 IFN-γ表达水平对比,P均＜0.05,差异具有统计学意义 （表3）。可见,与依沙吖啶溶液外敷联合红外线照射相比,MEBO外敷联合红外线照射可更有效抑制血清细胞因子 IL-6、IL-17及 IL-23的分泌,促进IFN-γ的分泌。

表2 两组患者临床疗效及住院时间对比Table 2 Comparison of clinical efficacy between the two groups

表3 两组患者血清细胞因子表达水平对比 （ng/L，x ±s）Table 3 Comparison of the expression levels of serum cytokines between the two groups（ng/L， x±s）

3.3 不良反应发生情况对比

治疗过程中,观察组患者中有3例患者局部皮损创面出现轻微刺痛、瘙痒,持续时间均在5 min内,未予特殊处理,自行缓解；对照组患者均无明显不良反应发生。两组患者不良反应发生情况对比采用四格表资料的卡方检验,χ2=3.220,P=0.073,P＞0.05,差异无统计学意义。

4 讨论

寻常型天疱疮是一种严重的自身免疫性疾病,目前临床多采用激素、免疫抑制剂、生物制剂、创面敷料等予以治疗,但临床疗效不佳,尤其是传统的干性疗法易使创面脱水、结痂,不利于上皮细胞的爬行,创面愈合速度缓慢,严重影响了患者的生活质量[3-4]。本研究中,对照组患者采用依沙吖啶溶液外敷联合红外线照射治疗,临床疗效明显不及采用MEBO外敷联合红外线照射治疗的观察组,创面愈合时间明显长于观察组。

天疱疮创面持续暴露于红外线中,虽有利于创面的消炎、镇痛,改善局部血液循环,提高组织细胞的营养代谢,但创面迅速干燥结痂后,可造成创面疼痛,且无法隔绝细菌入侵,易形成痂下脓肿[1]。而MEBO外敷联合红外线照射治疗,红外线照射20 min后,血液循环明显改善,此时应用MEBO,可促进MEBO向组织深层渗入,进而发挥更大的药效。MEBO内含有的黄芩甙、小檗碱等成分可破坏细菌生长环境,抑制其活性及毒性,有效防治创面感染[5-6]；其有效成分可激活创面组织内的潜能再生细胞,并将其转化为干细胞,再在原位增殖、分化为创面各层组织细胞,原位再生修复创面[7-8]；可提高创面组织中 VEGF、bFGF、EGF基因及其蛋白的表达水平,促进成纤维细胞及血管内皮细胞的增殖分化,从而加快肉芽组织生长,再生修复创面；其为创面营造的生理性湿润环境可预防组织细胞脱水,加速血管生成,增强生长因子与其靶细胞的相互作用,促进创面愈合[9-10]。

天疱疮是一种自身抗体介导的免疫性疾病,而T细胞分泌的细胞因子可通过介导T细胞亚群间的相互作用影响机体免疫系统的功能。如IL-6含量上升时可诱导更多的初始T细胞向Th17分化,进而促进IL-17等细胞因子的分泌,进一步加重组织炎性损害[9,11]；IL-10可抑制Thl细胞的增殖,进而减少IL-2、TNF-α等细胞因子的生成。本研究结果显示,MEBO外敷联合红外线照射治疗组患者在治疗第14天时IL-6、IL-17、IL-23及 TNF-α的表达水平明显降低,IL-10及IFN-γ的表达水平明显升高,且与依沙吖啶溶液外敷联合红外线照射相比,MEBO外敷联合红外线照射可更有效地抑制细胞因子IL-6、IL-17及IL-23的分泌,促进IFN-γ的分泌,两组对比,P＜0.05,差异具有统计学意义。可见,MEBO外敷联合红外线照射可有效缩短天疱疮创面的愈合时间,促进天疱疮的消退可能与其能够调节IL-6、IL-17、IL-23及IFN-γ等细胞因子的分泌有关,有待进一步深入研究探讨。

综上所述,MEBO外敷联合红外线照射可促进天疱疮创面的愈合,疗效显著,可能与其能够调节天疱疮患者血清IL-6、IL-17、IL-23及IFN-γ等细胞因子的表达水平有关,值得临床进一步推广应用与验证。

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.