MEBT/MEBO對(duì)小鼠燒傷創(chuàng)面組織中K19表達(dá)的影響及作用機(jī)制

李 雄 彭 浩 賀全勇

角蛋白(keratin)主要用于維持細(xì)胞結(jié)構(gòu)的完整性和穩(wěn)定性，其中角蛋白19(keratin 19，K19)陽(yáng)性表達(dá)的表皮干細(xì)胞可增殖、分化為皮膚各層組織細(xì)胞予以修復(fù)創(chuàng)面。部分研究顯示，在燒傷創(chuàng)瘍?cè)偕t(yī)療技術(shù)(moist exposed burn therapy/moist exposed burn ointment，MEBT/MEBO)治療深度燒傷創(chuàng)面的過程中，K19陽(yáng)性表達(dá)的干細(xì)胞可發(fā)生從無(wú)到有并不斷增加的變化[1-2]，但具體機(jī)制尚不明確。為此，筆者于本研究中觀察了MEBT/MEBO對(duì)小鼠燒傷創(chuàng)面組織中K19、自噬相關(guān)蛋白LC3及Beclin-1表達(dá)的影響，并分析了可能的作用機(jī)制，以期為MEBT/MEBO的臨床應(yīng)用提供理論依據(jù)。

1 材料與方法

1.1 實(shí)驗(yàn)動(dòng)物

隨機(jī)選取15只體重為19～21 g的SPF級(jí)健康雄性昆明小鼠作為受試對(duì)象。所有小鼠均由湖北省實(shí)驗(yàn)動(dòng)物研究中心提供，飼養(yǎng)環(huán)境清潔，室溫(25.0±3.0)℃，自由進(jìn)食進(jìn)水。本研究經(jīng)中南大學(xué)湘雅三醫(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 主要試劑與藥物

免疫組織化學(xué)SP試劑盒及DAB顯色試劑盒，LC3(bs-8878R)、K19(bs-2190R)及GAPDH(bs-2188R)抗體：北京博奧森生物技術(shù)有限公司生產(chǎn)；Beclin-1(sc-48341)抗體：Santa Cruz Biotechnology公司生產(chǎn)；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。

1.3 模型建立與分組

隨機(jī)選取10只小鼠，采用4%水合氯醛(400 mg/kg，即0.2 mL/20 g)腹腔注射麻醉及背部脫毛備皮后，將自制的帶有2 cm×2 cm孔洞的木板置于備皮處，并經(jīng)木板孔洞將備皮區(qū)置于(92.0±1.0)℃的熱水中浸泡20 s，建立面積約20% TBSA的燙傷創(chuàng)面(經(jīng)燙傷組織病理切片證實(shí)為深Ⅱ度燙傷)；燙傷后立即予以腹腔注射生理鹽水抗休克(20 mL/kg，即0.4 mL/20 g)。小鼠蘇醒后能夠正常進(jìn)食進(jìn)水即表明建模成功。建模成功后，按照隨機(jī)數(shù)表法隨機(jī)分為模型組與治療組，每組5只；其余5只未建模的小鼠背部脫毛后作為對(duì)照組。

1.4 局部處理

對(duì)照組：正常進(jìn)食進(jìn)水，備皮處不做任何處理。

治療組：正常進(jìn)食進(jìn)水，創(chuàng)面均勻涂抹濕潤(rùn)燒傷膏，每2 h換藥1次，連續(xù)治療1周。治療過程中，保證創(chuàng)面始終處于濕潤(rùn)狀態(tài)；每次換藥前拭凈創(chuàng)面分泌物及殘余藥膏。

模型組：正常進(jìn)食進(jìn)水，創(chuàng)面采用生理鹽水沖洗，每2 h沖洗1次，連續(xù)沖洗1周。

1.5 樣本采集

定時(shí)觀察并拍照記錄治療組與模型組小鼠的創(chuàng)面變化。治療1周后，取對(duì)照組小鼠的皮膚組織及治療組與模型組小鼠的創(chuàng)面組織均分為3份，1份用于免疫組織化學(xué)染色檢測(cè)，2份置于-80 ℃冰箱內(nèi)保存?zhèn)溆谩?/p>

1.6 免疫組織化學(xué)染色法檢測(cè)Beclin-1、LC3及K19的表達(dá)水平

待所有標(biāo)本采集完畢，取新鮮組織進(jìn)行石蠟包埋、切片、脫蠟及3% H2O2處理10 min、PBS沖洗5 min后行微波修復(fù)；修復(fù)后，滴加正常山羊血清并于37 ℃環(huán)境下封閉10 min，然后分別相應(yīng)加入Beclin-1抗體、LC3抗體及K19抗體(1∶200)4 ℃孵育過夜；孵育過夜后，PBS沖洗3次(每次5 min)，加入二抗孵育10 min；二抗孵育后，PBS沖洗3次(每次5 min)，加入HRP標(biāo)記復(fù)合物(三抗)孵育10 min；三抗孵育后，PBS沖洗3次(每次5 min)，然后依次進(jìn)行DAB顯色，蘇木素復(fù)染，酒精脫水，二甲苯透明，中性樹膠封片；每張切片于顯微鏡下隨機(jī)選取5個(gè)高倍視野檢測(cè)LC3、K19及Beclin-1的平均光密度，并取其均值進(jìn)行統(tǒng)計(jì)學(xué)分析。

1.7 Western blot法檢測(cè)Beclin-1、LC3及K19的表達(dá)水平

待所有標(biāo)本采集完畢，取1份備用組織進(jìn)行RIPA裂解、勻漿機(jī)研磨，提取總蛋白，并經(jīng)SDS聚丙烯酰胺凝膠電泳分離及轉(zhuǎn)膜后，TBST洗膜3次；洗膜后，5%脫脂奶粉封閉，4 ℃孵育相應(yīng)目的抗體(LC3、K19、BECN-1，1∶1000或GADPH 1∶2000)過夜；孵育過夜后，TBST洗膜3次，室溫孵育HRP-抗兔二抗(1∶5000)；孵育二抗后，TBST洗膜3次，ECL顯影。

1.8 Q-PCR技術(shù)檢測(cè)K19 mRNA的表達(dá)水平

待所有標(biāo)本采集完畢，取1份備用組織按照試劑盒說明書中的Trizol法提取總RNA，并檢測(cè)質(zhì)量及濃度(A260 ∶A280在1.8～2.1之間視為RNA質(zhì)量合格)后，置于-80 ℃冰箱內(nèi)保存?zhèn)溆谩?/p>

按照逆轉(zhuǎn)錄試劑盒說明書以總RNA為模板逆轉(zhuǎn)錄合成cDNA后進(jìn)行RT-q PCR檢測(cè)，并采用熒光定量統(tǒng)計(jì)軟件進(jìn)行分析，結(jié)果以待測(cè)基因與內(nèi)參基因GAPDH的表達(dá)水平比值表示。其中K19引物長(zhǎng)度為138 bp，序列為上游-CAGATAAGCAAGACCGAAG，下游-CAGCTGGACTCCATAACG；β-actin引物序列為上游-GAGGGAAATCGTGCGTGAC，下游-CTGGAAGGTGGACAGTGAG。

1.9 統(tǒng)計(jì)學(xué)處理

2 結(jié)果

2.1 大體觀創(chuàng)面愈合情況

治療組：治療第1～7天，創(chuàng)面清潔，并始終保持濕潤(rùn)狀態(tài)，且隨著治療時(shí)間的延長(zhǎng)逐漸呈現(xiàn)上皮化狀態(tài)；模型組：治療第1～7天，創(chuàng)面逐漸加深，并出現(xiàn)感染征象(圖1)。

2.Results

2.1.Overview on wound healing condition

Treatment group: during the treatment duration of day 1-7, the wounds were clean and always moist, presenting epithelialization gradually with time. Model group: during the treatment duration of day 1-7, the wounds gradually deepened in depth and showed signs of infection(Fig.1).

2.2 免疫組織化學(xué)染色法檢測(cè)結(jié)果

免疫組織化學(xué)染色法檢測(cè)Beclin-1、LC3及K19的結(jié)果顯示，3組小鼠皮膚或創(chuàng)面組織細(xì)胞內(nèi)均可見棕黃色顆粒分布，且以治療組小鼠創(chuàng)面組織細(xì)胞內(nèi)分布最多(圖2)；Beclin-1、LC3及K19平均光密度值對(duì)比，治療組>模型組>對(duì)照組，P<0.01，差異具有統(tǒng)計(jì)學(xué)意義；3組小鼠皮膚或創(chuàng)面組織中Beclin-1、LC3及K19平均光密度值兩兩對(duì)比，P均<0.01，差異具有統(tǒng)計(jì)學(xué)意義(圖3，表1)。

2.3 Western blot法檢測(cè)結(jié)果

Western blot法檢測(cè)Beclin-1、LC3及K19的結(jié)果顯示，3組小鼠皮膚或創(chuàng)面組織中Beclin-1、LC3及K19表達(dá)水平對(duì)比，治療組>模型組>對(duì)照組，P<0.01，差異具有統(tǒng)計(jì)學(xué)意義；除治療組與模型組小鼠創(chuàng)面組織中LC3表達(dá)水平對(duì)比，P>0.05外，其余各組小鼠皮膚或創(chuàng)面組織中Beclin-1、LC3及K19表達(dá)水平兩兩對(duì)比，P均<0.05，差異具有統(tǒng)計(jì)學(xué)意義(圖4，表2)。

圖2 3組小鼠皮膚或創(chuàng)面組織中Beclin-1、LC3及K19免疫組織化學(xué)染色結(jié)果典型圖；圖3 免疫組織化學(xué)染色法檢測(cè)Beclin-1、LC3及K19平均光密度直方圖(a表示與對(duì)照組對(duì)比P<0.01，b表示與模型組對(duì)比P<0.01)

Fig.2 The typical pictures of the immunohistochemical staining results of Beclin-1, LC3 and K19 in the three groups； Fig.3 The histogram of mean optical density of Beclin-1, LC3 and K19 detected by the immunohistochemical staining (a represents comparison with the control group,P<0.01; b represents comparison with the model group,P<0.01)

表1 3組小鼠皮膚或創(chuàng)面組織中Beclin-1、LC3及K19平均光密度值對(duì)比Table 1 Comparison of mean optical density of Beclin-1, LC3 and K19 in skin or wound tissues of the three

注：3組小鼠皮膚或創(chuàng)面組織中Beclin-1、LC3及K19平均光密度值對(duì)比，P均<0.01，差異具有統(tǒng)計(jì)學(xué)意義。3組小鼠皮膚或創(chuàng)面組織中Beclin-1、LC3及K19平均光密度值兩兩對(duì)比，其中與對(duì)照組對(duì)比，aP<0.01，差異具有統(tǒng)計(jì)學(xué)意義；與模型組對(duì)比，bP<0.01，差異具有統(tǒng)計(jì)學(xué)意義

Note: Statistically significant differences were observed when the mean optical densities of Beclin-1, LC3 and K19 in skin or wound tissues of the three groups were compared, allP<0.01. The mean optical densities of Beclin-1, LC3 and K19 in skin or wound tissues of the three groups were compared in pairs, in which the comparisons with the control group and with the model group all showed statistically significant differences (respectivelyaP< 0.01 andbP<0.01)

2.4 Q-PCR技術(shù)檢測(cè)結(jié)果

Q-PCR技術(shù)檢測(cè)K19 mRNA的結(jié)果顯示，3組小鼠皮膚或創(chuàng)面組織中K19 mRNA表達(dá)水平對(duì)比，治療組>模型組>對(duì)照組，P<0.01，差異具有統(tǒng)計(jì)學(xué)意義；3組小鼠皮膚或創(chuàng)面組織中K19 mRNA表達(dá)水平兩兩對(duì)比，P均<0.01，差異具有統(tǒng)計(jì)學(xué)意義(圖5，表3)。

圖4 Western blot法檢測(cè)Beclin-1、LC3及K19表達(dá)水平直方圖(a表示與對(duì)照組對(duì)比P<0.05，b表示與模型組對(duì)比P<0.05)

Fig.4 The histogram of the expression levels of Beclin-1, LC3 and K19 according to the Western blot analysis (a represents comparison with the control group,P<0.05; b represents comparison with the model group,P<0.05)

表2 3組小鼠皮膚或創(chuàng)面組織中Beclin-1、LC3及K19表達(dá)水平對(duì)比Table 2 Comparison of the expression levels of Beclin-1, LC3 and K19 in skin or wound tissues of the three

注：3組小鼠皮膚或創(chuàng)面組織中Beclin-1、LC3及K19表達(dá)水平對(duì)比，P均<0.01，差異具有統(tǒng)計(jì)學(xué)意義。3組小鼠皮膚或創(chuàng)面組織中Beclin-1、LC3及K19表達(dá)水平兩兩對(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: Statistically significant differences were observed when the expression levels of Beclin-1, LC3 and K19 in skin or wound tissues were compared among the three groups, allP<0.01. The expression levels of Beclin-1, LC3 and K19 in skin or wound tissues were compared in pairs among the three groups, in which statistically significant differences were observed in comparisons respectively with the control group (aP<0.05) and with the model group (bP<0.05)

3 討論

研究表明，角蛋白是表皮細(xì)胞的主要結(jié)構(gòu)蛋白，可在細(xì)胞內(nèi)形成廣泛的網(wǎng)狀結(jié)構(gòu)，對(duì)表皮具有重要的保護(hù)作用，其中K19是表皮干細(xì)胞的特異性標(biāo)志物，其單克隆抗體被廣泛應(yīng)用于表皮干細(xì)胞的檢測(cè)[3-4]。此外，K19還可通過促進(jìn)細(xì)胞外基質(zhì)的降解或細(xì)胞移動(dòng)提高細(xì)胞的轉(zhuǎn)移能力，進(jìn)而促進(jìn)創(chuàng)面的再生修復(fù)[5-8]。有研究證實(shí)，MEBT/MEBO治療深度燒傷創(chuàng)面的過程中，K19陽(yáng)性表達(dá)干細(xì)胞可出現(xiàn)從無(wú)到有、從少到多，再?gòu)亩嗟缴?、從少到無(wú)的變化規(guī)律，但具體機(jī)制尚不明確。

圖5 Q-PCR技術(shù)檢測(cè)K19 mRNA表達(dá)水平直方圖(a表示與對(duì)照組對(duì)比P<0.01，b表示與模型組對(duì)比P<0.01)

Fig.5 The histogram of K19 mRNA expression levels tested by the Q-PCR technique (a represents comparison with the control group,P<0.01; b represents comparison with the model group,P<0.01)

表3 3組小鼠皮膚或創(chuàng)面組織中K19 mRNA表達(dá)水平對(duì)比Table 3 Comparison of K19 mRNA expression level in skin or wound tissues among the three

注：3組小鼠皮膚或創(chuàng)面組織中K19 mRNA表達(dá)水平對(duì)比，P<0.01，差異具有統(tǒng)計(jì)學(xué)意義。3組小鼠皮膚或創(chuàng)面組織中K19 mRNA表達(dá)水平兩兩對(duì)比，其中與對(duì)照組對(duì)比，aP<0.01，差異具有統(tǒng)計(jì)學(xué)意義；與模型組對(duì)比，bP<0.01，差異具有統(tǒng)計(jì)學(xué)意義

Note: Statistically significant difference was observed when the K19 mRNA expression levels in skin or wound tissues were compared among the three groups,P<0.01. The expression levels of K19 mRNA in skin or wound tissues were compared in pairs among the three groups, in which statistically significant differences were observed in comparisons respectively with the control group(aP<0.01) and with the model group(bP<0.01)

筆者在臨床研究中發(fā)現(xiàn)，燒傷創(chuàng)面應(yīng)用MEBT/MEBO治療后，創(chuàng)面壞死組織可無(wú)損傷地液化排除，作用溫和無(wú)刺激，并可減輕局部炎癥反應(yīng)，同時(shí)對(duì)殘存細(xì)胞具有保護(hù)作用，這與作為程序性細(xì)胞死亡機(jī)制及細(xì)胞自我防御機(jī)制的自噬介導(dǎo)的細(xì)胞代謝的動(dòng)態(tài)平衡及自我保護(hù)[9-13]具有異曲同工之處，而已有研究證實(shí)，MEBT/MEBO治療的創(chuàng)面修復(fù)主要依賴于K19陽(yáng)性表達(dá)的表皮干細(xì)胞的分化、增殖、遷移及融合，且K19還可抑制創(chuàng)傷造成的炎癥反應(yīng)[14]。因此，在本研究中，筆者對(duì)比觀察了MEBT/MEBO對(duì)小鼠燒傷創(chuàng)面組織中自噬相關(guān)蛋白Beclin-1、LC3及K19表達(dá)的影響。結(jié)果顯示，治療1周后，3組小鼠皮膚或創(chuàng)面組織中Beclin-1、LC3、K19及K19 mRNA表達(dá)水平對(duì)比，治療組>模型組>對(duì)照組，P均<0.01，差異具有統(tǒng)計(jì)學(xué)意義；除Western blot法檢測(cè)結(jié)果中顯示治療組與模型組小鼠創(chuàng)面組織中LC3表達(dá)水平對(duì)比，P>0.05外，其余各檢測(cè)結(jié)果中均顯示小鼠皮膚或創(chuàng)面組織中Beclin-1、LC3、K19及K19 mRNA表達(dá)水平兩兩對(duì)比，P<0.01，差異具有統(tǒng)計(jì)學(xué)意義。即MEBT/MEBO可明顯提高小鼠燒傷創(chuàng)面組織中自噬相關(guān)蛋白Beclin-1、LC3及K19的表達(dá)水平，促進(jìn)創(chuàng)面的愈合。由此推斷，MEBT/MEBO治療的創(chuàng)面組織中K19的表達(dá)水平明顯升高可能與自噬相關(guān)[15-19]。

綜上所述，MEBT/MEBO促進(jìn)燒傷創(chuàng)面的愈合與提高創(chuàng)面組織中K19的表達(dá)水平有關(guān)，且誘導(dǎo)自噬相關(guān)蛋白的高表達(dá)可能是其作用機(jī)制之一。因本研究設(shè)計(jì)限制，其對(duì)自噬相關(guān)通路的調(diào)控機(jī)制仍需進(jìn)一步深入研究探討。

(Received on June 10, 2018)

Keratin is mainly for maintaining the integrity and stability of cell structure, while epidermal stem cells containing the positively expressed keratin 19 (K19) can proliferate and differentiate into skin tissues of all layers, and further to repair skin wounds. Some studies have showed that during the treatment course of deep burn wounds with moist exposed burn therapy/moist exposed burn ointment (MEBT/MEBO), the number of stem cells with positively expressed K19 presented a change from nothing to something and continued to increase[1-2], for which, however, the exact mechanism remains unclear. To this end, this study observed the effects of MEBT/MEBO on the expression of K19, and autophagy-related proteins LC3 and Beclin-1 in mouse burn wounds, and also analyzed the possible action mechanism, with a view to providing theoretical basis for the clinical application of MEBT/MEBO.

1.Materialsandmethods

1.1.Experimental animals

Fifteen SPF healthy male Kunming mice, weighing 19-21 g, were randomly selected as subjects. All subjects, provided by Hubei Experimental Animal Research Center, are kept in a clean environment and at room temperature (25.0±3.0)℃, with free access to food and water. This study was approved by the Animal Ethics Committee of The Third Xiangya Hospital of Central South University and was in line with the ethical requirements of animal experiments.

1.2.Main reagents and drugs

Immunohistochemical SP kit and DAB Substrate kit; antibodies of LC3 (bs-8878R), K19 (bs-2190R) and GAPDH (bs-2188R) are manufactured by Beijing Bioss Bio-technology Co., Ltd.; Beclin-1 (sc-48341) antibody by Santa Cruz Biotechnology; Moist Exposed Burn Ointment (MEBO) by Shantou MEBO Pharmaceutical Co., Ltd. (SFDA Approval No.: Z20000004).

1.3.Modeling and Grouping

Anesthetize 10 mice selected randomly from the 15 subjects by intraperitoneal injection of 4% Chloral Hydrate (400 mg/kg, i.e., 0.2 mL/20 g), remove their back hair to prepare skin, put the self-made plank with a 2 cm×2 cm hole onto the prepared skin, and then soak the prepared skin through the plank hole into hot water (92.0±1.0) ℃ for 20 s to establish a scald wound of about 20% TBSA (identified as deep second degree scalds according to scalding tissue pathological sections). Immediately after the scalding injury, inject normal saline intraperitoneally to prevent subsequent shock (20 mL/kg, i.e., 0.4 mL/20 g). The models were established successfully if the mice can have water and food normally when they wake up. After successful modeling, the 10 mice were assigned into a model group and a treatment group according to the random number table, 5 in each group, while the left 5 mice only with back hair removal were included into the control group.

1.4.Local management

Control group: eat and drink normally, no intervention to the prepared skin.

Treatment group: eat and drink normally, apply a layer of MEBO onto the wound surface, change dressing every two hours continuously for one week. During the treatment course, keep the wound surface moist all the time, and remove the secretion and residual drug on the wound surface before each dressing change.

Model group: eat and drink normally, rinse the wound surface with normal saline every two hours continuously for one week.

1.5.Sample collection

Regularly observe and record the wound changes in the treatment group and the model group. One week after treatment, collect skin tissue sample of each mouse in the control group, wound tissue sample of each mouse in the treatment group and the model group. Each tissue sample is divided evenly to three samples, one is used for the immunohistochemical staining and the other two are stored in -80℃ refrigerator for later use.

1.6.Test the expression levels of Beclin-1, LC3 and K19 by the immunohistochemical staining

After sample collection, take the fresh tissue sections to undergo paraffin embedding, slicing and de-waxing and 3% H2O2treatment for 10 min and PBS rinsing for 5 min, followed by microwave repair. After repairing, normal goat serum was added into the sections, and keep them blocked for 10 min at 37℃, and then add Beclin-1 antibody, LC3 antibody and K19 antibody (1∶200) in turn, incubate at 4℃ for overnight followed by PBS wash three times (5 min each), and then add secondary antibody to incubate for 10 min. After second incubation, rinse the sections with PBS for 3 times (5 min each), add HRP labeled complex (third antibody) to incubate for 10 min followed by PBS wash for 3 times (5 min each). Proceed sequentially with DAB stain, Hematoxylin counterstain, ethanol dehydration, transparency with xylene and mounting with neutral gum. Randomly select 5 high power fields in each section under microscope to detect the mean optical density of LC3, K19 and Beclin-1, and calculate the mean value for statistical analysis.

1.7.Test the expression levels of Beclin-1, LC3 and K19 by the Western blot method

After sample collection, take out one prepared tissue sample to undergo RIPA lysis and Homogeniser grinding for the extraction of total proteins. The total proteins were separated by using SDS-polyacrylamide gel electrophoresis and transferred onto a membrane, followed by TBST wash three times. After washing, use 5% nonfat dry milk to block, incubate the targeted antibodies (LC3, K19, BECN-1, 1∶1000 or GADPH, 1∶2000) at 4 ℃ for overnight. And then, do TBST wash three times and incubate HRP-anti-rabbit secondary antibody (1∶5000) at room temperature, followed by TBST wash again for three times and ECL development.

1.8.Test the expression level of K19 mRNA with the Q-PCR technique

After sample collection, take out the other prepared tissue sample to extract the total RNA with the Trizol method as specified in the kit manual and store the extracted total RNA in a refrigerator of -80℃ for later use after its quality and density have been detected qualified (A260∶A280 ranging between 1.8-2.1 is regarded as right RNA quality).

According to the kit manual, generate cDNA by reverse transcription with the total RNA as template to proceed with the RT-q PCR test. Quantitative fluorescence statistical software was used for analysis, of which the results were expressed as the ratio of measured gene expression level to the expression level of the reference gene GAPDH. The length of K 19 primer was 138bp, with upstream sequence as -CAGATAAGCAAGACCGAAG and the downstream as -CAGCTGGACTCCATAACG, while the upstream sequence of β-actin primer was -GAGGGAAATCGTGCGTGAC and the downstream sequence was -CTGGAAGGTGGACAGTGAG.

1.9.Statistical analysis

2.2.Results of the immunohistochemical staining

The immunohistochemical staining of Beclin-1, LC3 and K19 showed that there were brown granules scattering in the skin or wound tissue cells of the three groups, with the most in the treatment group (Fig.2). The comparison of mean optical density of Beclin-1, LC3 and K19 among the three groups showed statistically significant difference (treatment group>model group>control group,P<0.01). The pairwise comparison of mean optical density of Beclin-1, LC3 and K19 among the three groups all showed statistically significant differences,P<0.01(Fig.3, Table 1).

2.3.Results of the Western blot analysis

According to the Western blot analysis, the expression levels of Beclin-1, LC3 and K19 in skin or wound tissues of the three groups showed a pattern of the treatment group being higher than the model group, with the latter being higher than the control group, presenting a statistically significant difference,P<0.01. The comparison of LC3 expression levels between the treatment group and the model group showed no statistically significant difference,P>0.05. Except that, all the other pairwise comparisons of the expression levels of Beclin-1, LC3 and K19 among the three groups showed statistically significant differences, allP<0.05 (Fig.4, Table 2).

2.4.Results of the Q-PCR technique

According to the detection results of the Q-PCR technique, the expression level of K19 mRNA in skin or wound tissues in the three groups showed a pattern of the treatment group being higher than the model group, with the latter being higher than the control group, presenting a statistically significant difference,P<0.01. The pairwise comparisons of K19 mRNA expression levels among the three groups all showed statistically significant differences, allP<0.01 (Fig.5, Table 3).

3.Discussion

Studies have indicated that keratin, as the main structural protein of epidermal cells, can form a wide mesh structure in cells, playing an important role of protecting epidermis. Keratin 19 (K19) is a specific marker of epidermal stem cells, and its monoclonal antibody has been widely applied in the detection of epidermal stem cells[3-4]. Moreover, K19 can improve cell migration by accelerating the degradation of extracellular matrices, or cell movement, and further promote the regenerative repair of wounds[5-8]. Some studies have confirmed that in the treatment of deep burns with MEBT/MEBO, the stem cells with the positively expressed K19 presented a change from nothing to something, from less to more, and then gradually decreasing to nothing, for which the exact mechanism is still not clear.

In clinical studies, the authors found that after the application of MEBT/MEBO on burn wounds, the necrotic tissues on wound surface can liquefy and be removed without damage to residual tissues, being very mild and non-irritating, and also the local inflammatory responses can be relieved and residual cells can be protected well, which realized the same satisfactory results as the dynamic balance and self-protection of autophagy-mediated cell metabolism as a mechanism of procedural cell death and cellular self-defense, though in different approaches[9-13]. And also, studies have confirmed that in wound treatment with MEBT/MEBO, the wound repair mainly depends on the differentiation, proliferation, migration and fusion of K19-positively expressed epidermal stem cells and, moreover, K19 can inhibit the inflammatory responses caused by traumas[14]. Thus, in this study, the effects of MEBT/MEBO on the expression of autophagy-related proteins such as Beclin-1, LC3 and K19 were investigated in mouse burn wounds. The results showed that after one week of treatment, the expression levels of Beclin-1, LC3 and K19 in mouse skin or wound tissues presented a statistically significant difference among the three groups, with the treatment group being higher than the model group and the latter being higher than the control group,P<0.01. No statistically significant difference was observed when the LC3 expression levels were compared between the treatment group and the model group according to the Western blot method(P>0.05), while based on all the other detection results the pairwise comparisons of the expression levels of Beclin-1, LC3, K19 and K19 mRNA among the three groups showed statistically significant differences, allP<0.01. This means, MEBT/MEBO can markedly improve the expression levels of autophagy-related proteins including Beclin-1, LC3 and K19 in mouse burn wounds, and promote wound healing. It can be concluded that the significantly elevated expression level of K19 in wound tissues treated with MEBT/MEBO may be associated with autophagy[15-19].

To sum up, MEBT/MEBO in promoting the healing of burn wounds is related to improving the expression level of K19 in wound tissues, of which inducing the high expression of autophagy-related proteins may be one mechanism. In this study, due to design limitations, the regulatory mechanism of MEBT/MEBO in autophagy-related signaling pathways remains to be further explored in the future.