不同溫度MEBO 對大鼠超脈沖CO2 點陣激光術(shù)后皮膚屏障功能的影響

隨著醫(yī)療水平的不斷提高， 超脈沖CO2點陣激光聯(lián)合濕潤燒傷膏 （moist exposed burn ointment， MEBO） 在臨床中的應(yīng)用也越發(fā)廣泛， 而臨床研究發(fā)現(xiàn)， 不同溫度的MEB0 對超脈沖CO2點陣激光術(shù)后創(chuàng)面的修復(fù)效果也存在差異［1］， 但具體作用機(jī)制尚不完全明確。 為進(jìn)一步探討其作用機(jī)制， 本研究采用不同溫度的MEBO 治療大鼠超脈沖CO2點陣激光術(shù)后創(chuàng)面，并通過測定經(jīng)皮水分丟失（transepidermal water loss， TEWL） 值的變化情況探討不同溫度MEBO對大鼠皮膚屏障功能的影響， 現(xiàn)報告如下。

1 實驗材料

1.1 實驗動物及分組

健康雄性Wistar 大鼠30 只（南方醫(yī)科大學(xué)動物中心提供）， 體質(zhì)量200 ～250 g， 按照隨機(jī)數(shù)表法將其隨機(jī)分為空白對照組、 正常對照組、 冷敷組、 MEBO 低溫組、 MEBO 常溫組，每組6 只， 均于21 ～25 ℃室溫、 自然光照下適應(yīng)性喂養(yǎng)1 周。

1.2 主要儀器與藥物

超脈沖CO2點陣激光機(jī)： 成都國雄光電技術(shù)有限公司生產(chǎn)； 皮膚屏障測定儀 （GPSkin Barrier）： 韓國G-Power 公司生產(chǎn)； MEBO： 汕頭市美寶制藥有限公司生產(chǎn)。

2 方法

2.1 模型建立

所有大鼠稱重后均給予3% 戊巴比妥鈉（30 mg／kg） 腹腔注射麻醉。 麻醉成功后， 空白對照組大鼠僅做背部備皮處理， 并檢測TEWL 值； 正常對照組、 冷敷組、 MEBO 低溫組及MEBO 常溫組大鼠于背部備皮處理及檢測TEWL 值后固定于實驗臺上， 對備皮部位行超脈沖CO2點陣激光燒灼處理 （能量設(shè)置為20 mJ／s， 光斑 設(shè) 置 為10.0 mm × 10.0 mm），每只大鼠每側(cè)背部燒灼2 處創(chuàng)面， 即每只大鼠4 處創(chuàng)面、 每組大鼠24 處創(chuàng)面。 激光術(shù)后， 正常對照組大鼠創(chuàng)面不做任何處理； 冷敷組大鼠創(chuàng)面予以4 ℃水袋外敷30 min， 每天1 次；MEBO 低溫組大鼠創(chuàng)面外涂4 ℃MEBO， 每天換藥1 次； MEBO 常溫組大鼠創(chuàng)面外涂28 ℃MEBO， 每天換藥1 次。

2.2 TEWL 檢測

采用皮膚屏障測定儀測定各組大鼠激光術(shù)前及激光術(shù)后即刻、 12 h、 24 h 與7 d 的TEWL值。 每次檢測前使用生理鹽水清洗創(chuàng)面， 自然晾干后進(jìn)行檢測。

2.3 統(tǒng)計學(xué)處理

采用SPSS 25.0 統(tǒng)計軟件對所得數(shù)據(jù)進(jìn)行統(tǒng)計學(xué)分析， 計量資料以均數(shù)±標(biāo)準(zhǔn)差（±s）表示， 采用方差分析及LSD 法進(jìn)行對比分析，均以P ＜0.05 為差異具有統(tǒng)計學(xué)意義。

3 結(jié)果

激光術(shù)前， 各組大鼠皮膚TEWL 值對比，P 均＞0.05， 差異無統(tǒng)計學(xué)意義， 具有可比性。激光術(shù)后， 除空白對照組大鼠皮膚TEWL 值無明顯變化外， 其余各組大鼠創(chuàng)面TEWL 值均呈先升高后降低的趨勢， P 均＜0.05， 差異具有統(tǒng)計學(xué)意義； 至激光術(shù)后7 d， 正常對照組與MEBO 常溫組大鼠創(chuàng)面TEWL 值均恢復(fù)至與術(shù)前相當(dāng)?shù)乃剑≒ 均＞0.05）， 冷敷組與MEBO低溫組大鼠創(chuàng)面TEWL 值仍高于術(shù)前（P 均＜0.05）。 激光術(shù)后即刻， 正常對照組、 冷敷組、MEBO 低溫組、 MEBO 常溫組大鼠創(chuàng)面TEWL值均明顯升高， 與空白對照組相比， P 均＜0.05， 差異具有統(tǒng)計學(xué)意義； 激光術(shù)后12 h、24 h， 正常對照組、 冷敷組、 MEBO 低溫組、MEBO 常溫組大鼠創(chuàng)面TEWL 值仍明顯高于空白對照組， 且冷敷組與MEBO 低溫組大鼠創(chuàng)面TEWL 值明顯高于正常對照組， P 均＜0.05，差異具有統(tǒng)計學(xué)意義， 而MEBO 常溫組大鼠創(chuàng)面TEWL 值與正常對照組相當(dāng)（P ＞0.05）； 激光術(shù)后7 d， 除MEBO 常溫組大鼠創(chuàng)面TEWL值恢復(fù)至空白對照組水平（P ＞0.05）， 且明顯低于正常對照組、 冷敷組與MEBO 低溫組（P均＜0.05） 外， 其余各組大鼠創(chuàng)面TEWL 值仍明顯高于空白對照組， P 均＜0.05， 差異具有統(tǒng)計學(xué)意義（表1）。 可見常溫MEBO 有利于創(chuàng)面皮膚屏障功能的恢復(fù)。

表1 各組大鼠皮膚或創(chuàng)面TEWL 值對比（g·h -1·m -2， ±s）Table 1 Comparison of skin or wound TEWL values in each group （g·h -1·m -2， ±s）

表1 各組大鼠皮膚或創(chuàng)面TEWL 值對比（g·h -1·m -2， ±s）Table 1 Comparison of skin or wound TEWL values in each group （g·h -1·m -2， ±s）

注： 與空白對照組比較， aP ＜0.05， 差異具有統(tǒng)計學(xué)意義； 與正常對照組比較， bP ＜0.05， 差異具有統(tǒng)計學(xué)意義； 與冷敷組比較，cP ＜0.05， 差異具有統(tǒng)計學(xué)意義； 與MEBO 低溫組比較， dP ＜0.05， 差異具有統(tǒng)計學(xué)意義。 與同組激光術(shù)前比較， eP ＜0.05， 差異具有統(tǒng)計學(xué)意義； 與同組激光術(shù)后即刻比較， fP ＜0.05， 差異具有統(tǒng)計學(xué)意義； 與同組激光術(shù)后12 h 比較， gP ＜0.05， 差異具有統(tǒng)計學(xué)意義； 與同組激光術(shù)后24 h 比較， hP ＜0.05， 差異具有統(tǒng)計學(xué)意義Note： The comparisons with the blank control group showed statistically significant differences （aP ＜0.05）； the comparisons with the normal control group showed statistically significant differences （bP ＜0.05）； the comparisons with the cold compress group showed statistically significant differences（cP ＜0.05）；the comparisons with MEBO low temperature group showed statistically significant differences（dP ＜0.05）；the comparisons with the TEWL values before the laser operation within each group showed statistically significant differences（eP ＜0.05）；the comparisons with the TEWL values immediately after the laser operation within each group showed statistically significant differences （fP ＜0.05）； the comparisons with the TEWL values at 12 h after the laser operation within each group showed statistically significant differences（gP ＜0.05）；the comparisons with the TEWL values at 24 h after the laser operation within each group showed statistically significant differences （hP ＜0.05）

組別Group n激光術(shù)前Before the laser operation激光術(shù)后即刻Immediately after the laser operation激光術(shù)后12 h 12 h after the laser operation激光術(shù)后24 h 24 h after the laser operation激光術(shù)后7 d Day 7 after the laser operation F 值F value P 值P value空白對照組Blank control group 24 2.79 ±2.30 2.83 ±2.30 2.67 ±2.10 2.46 ±1.89 2.88 ±2.40 0.139 0.968正常對照組Normal control group 24 2.45 ±2.57 27.05 ±5.83ae 18.58 ±8.31aef 14.58 ±7.56aef 6.61 ±1.95afgh 66.740 0.000冷敷組Cold compress group 24 2.00 ±1.25 25.29 ±6.62ae 26.54 ±5.42abe 22.88 ±7.79abe 8.31 ±2.12aefgh 106.100 0.000 MEBO 低溫組MEBO low temperature group 24 1.68 ±0.95 28.14 ±9.29ae 26.04 ±6.36abe 22.08 ±10.00abef 7.75 ±2.44aefgh 70.860 0.000 MEBO 常溫組MEBO room temperature group 24 1.57 ±1.03 25.11 ±7.08ae 21.96 ±6.79ae 17.92 ±6.95aef 4.31 ±1.66bcdfgh 91.080 0.000 F 值F value 1.233 61.590 60.490 30.400 28.140 - -P 值P value 0.301 0.000 0.000 0.000 0.000 - -

4 討論

超脈沖CO2點陣激光的波長為10.6 μm，能夠被組織中的水分吸收而產(chǎn)生大量熱量， 并通過局灶性光熱作用啟動皮膚組織的再生修復(fù)機(jī)制， 促進(jìn)皮膚各層組織的再生修復(fù)， 改善皮膚老化等狀況［2-3］， 被廣泛應(yīng)用于痤瘡、 瘢痕、光老化等皮膚疾病的治療。 然而， 光熱作用也可使角蛋白及酶蛋白等變性， 破壞角質(zhì)層的正常結(jié)構(gòu)， 影響酶促反應(yīng)， 導(dǎo)致保濕因子及脂質(zhì)生成代謝障礙， 進(jìn)而破壞皮膚的“磚墻結(jié)構(gòu)”，降低皮膚對外界刺激的抵御能力。 近年來， 部分研究學(xué)者鑒于MEBO 在多種類型創(chuàng)面的修復(fù)中均取得了顯著的臨床療效， 故將其應(yīng)用于超脈沖CO2點陣激光術(shù)后創(chuàng)面的治療， 但臨床研究發(fā)現(xiàn)， 不同溫度的MEBO 對超脈沖CO2點陣激光術(shù)后創(chuàng)面的修復(fù)效果存在差異， 遂筆者于本研究中對比分析了不同溫度MEBO 對大鼠超脈沖CO2點陣激光術(shù)后皮膚屏障功能的影響。

TEWL 值是評價皮膚屏障功能的重要指標(biāo)［4］， 其可準(zhǔn)確、 穩(wěn)定地反映皮膚屏障功能的恢復(fù)過程［5］。 本研究發(fā)現(xiàn)， 激光術(shù)后即刻， 正常對照組、 冷敷組、 MEBO 低溫組、 MEBO 常溫組大鼠創(chuàng)面TEWL 值均明顯升高， 與空白對照組相比， P 均＜0.05， 差異具有統(tǒng)計學(xué)意義；激光術(shù)后7 d， MEBO 常溫組大鼠創(chuàng)面TEWL 值基本恢復(fù)至與空白對照組相當(dāng)?shù)乃?（P ＞0.05）， 且明顯低于正常對照組、 冷敷組與MEBO低溫組（P 均＜0.05）。 可見， 激光術(shù)后創(chuàng)面的皮膚屏障被破壞， 皮膚角質(zhì)層水分丟失增多， 而常溫MEBO 有利于創(chuàng)面皮膚屏障功能的恢復(fù)。 與吳樹毅等的不同溫度MEBO 對超脈沖CO2點陣激光術(shù)后創(chuàng)面修復(fù)影響的研究結(jié)果一致［1］。 MEBO 內(nèi)含有的β-谷甾醇、 黃芩甙、小檗堿等成分具有抗炎、 改善局部血液循環(huán)、增強(qiáng)免疫力等作用， 可促進(jìn)創(chuàng)面的再生修復(fù)［6］。 作為一種低熔點軟膏制劑， MEBO 可在皮膚的溫化作用下液化排除創(chuàng)面壞死組織， 發(fā)揮自動循環(huán)引流作用［7-8］， 而溫度過低將延長藥膏液化時間， 影響藥效的發(fā)揮［9-10］。 本研究還發(fā)現(xiàn)， 激光術(shù)后7 d， 冷敷組與MEBO 低溫組大鼠創(chuàng)面TEWL 值仍明顯高于術(shù)前， 而正常對照組與MEBO 常溫組大鼠創(chuàng)面TEWL 值均恢復(fù)至與術(shù)前相當(dāng)?shù)乃剑?提示低溫對創(chuàng)面愈合有不利影響， 原因可能在于低溫可引起皮膚水合作用降低， 創(chuàng)面毛細(xì)血管收縮， 散熱減少，進(jìn)而抑制促炎因子的釋放， 并減緩血液循環(huán)，影響創(chuàng)面修復(fù)［1］。

綜上所述， 超脈沖CO2點陣激光術(shù)可損害皮膚屏障功能， 常溫（28 ℃） MEBO 可加快創(chuàng)面愈合， 促進(jìn)皮膚屏障功能快速恢復(fù)， 而低溫（4 ℃） MEBO對創(chuàng)面愈合無明顯促進(jìn)作用。

With the continuous development of medicine， the combined application of ultra pulsed CO2fractional laser and moist exposed burn ointment （MEBO） is becoming more and more extensive in clinical practice. Clinical studies have found that MEB0 at different temperatures have different effects on the wound repair following the ultra pulsed CO2fractional laser operation［1］， but its specific mechanism of action remains unclear. To further explore the mechanism of action，this study used MEBO at different temperatures to treat wounds caused by the ultra pulsed CO2laser operation in rats，and also investigated the effect of MEBO at different temperatures on the skin barrier function of rats by measuring changes of transepidermal water loss（TEWL） values.

1.Experimental material

1.1.Experimental animals and grouping

Thirty healthy male Wistar rats （provided by Animal Center of Southern Medical University）， each weighing 200 - 250 g， were divided， according to the random number table， into blank control group， normal control group， cold compress group， MEBO low temperature group and MEBO room temperature group， 6 rats in each group. All the rats were fed at room temperature of 21 -25 ℃ for 1 week under natural light.

1.2.Main equipment and medicines

Ultra pulsed CO2fractional laser machine （produced by Chengdu Guoxiong Optoelectronic Technology Co.， Ltd. ）， GPSkin Barrier（produced by G-Power， Korea）， MEBO （produced by Shantou MEBO Pharmaceutical Co.， Ltd.）.

2.Methods

2.1.Modelling

After weighing， intraperitoneal injection of 3% pentobarbital sodium （30 mg／kg） was given to all the rats. For the rats in the blank control group， back skin was prepared and skin TEWL value was measured； for rats in the normal control group， cold compress group， MEBO low temperature group and MEBO room temperature group， after the back skin preparation and TEWL value measurement， they were fixed on test beds to be cauterized with ultra pulsed CO2fractional laser on the prepared skin （energy： 20 mJ／s， light spot：10 mm × 10 mm）. Two wounds were made on each of the two back sides of a rat， that is， there were 4 wounds in each rat and 24 wounds in each group. After the laser operation， the wounds in the normal control group were not given any treatment； the wounds in the cold compress group were managed with water bags of 4 ℃for 30 min every day； the wounds in MEBO low temperature group were smeared with MEBO （4 ℃） and the dressing was changed once a day； and the wounds in MEBO room temperature group were smeared with MEBO （28 ℃） and the dressing change was also once a day.

2.2.TEWL value measurement

The TEWL values in each group were measured with GPSkin Barrier respectively at the time points of before the laser operation，immediately after the laser operation，12 h，24 h and day 7 after the laser operation. The wounds should be washed with normal saline and dried naturally before each testing.

2.3.Statistical analysis

The SPSS 25.0 software was adopted to analyze the study data，in which analysis of variance and LSD test were used to manage the measurement data expressed with mean value ± standard deviation（±s）. P ＜0.05 was considered as statistically significant difference.

3.Results

Before the laser operation， the skin or wound TEWL values in each group were compared and the results showed no statistically significant difference （all P ＞0.05）， and thus， they are comparable.After the laser operation，no big change of TEWL values was observed in the blank control group， but the TEWL values in all the other four groups all presented a tendency of increasing first and then decreasing， the differences were all statistically significant （all P ＜0.05）.Immediately after the laser operation， the TEWL values in the normal control group， cold compress group， MEBO low temperature group

and MEBO room temperature group all increased significantly and the differences as compared with that in the blank control group were statistically significant （all P ＜0.05）. At 12 h and 24 h after the laser operation， the TEWL values in the normal control group， cold compress group， MEBO low temperature group and MEBO room temperature group were still significantly higher than that in the blank control group， and the TEWL values in the cold compress group and MEBO low temperature group were markedly higher than that in the normal control group， and the differences were all statistically significant（P ＜0.05）； On day 7 after the laser operation， the TEWL values in the MEBO room temperature group returned to the level in the blank control group （P ＞0.05） and were significantly lower than that in the normal control group， cold compress group and MEBO low temperature group （P ＜0.05）. In contrast， the TEWL values in the normal control group， cold compress group and MEBO low temperature group were all significantly higher than that in the blank control group，presenting statistically significant differences （all P ＜0.05） （Table 1）. It can be concluded that MEBO at room temperature can facilitate the recovery of skin barrier function of wounds.

4.Discussion

The ultra pulsed CO2fractional laser has a wavelength of 10.6 μm， and such a wavelength can be absorbed by water in tissues to generate a large amount of heat. The fractional heat and light produced by laser can activate the regenerative restoration mechanism of skin tissues to promote the regenerative repair of all layers of tissues and alleviate the symptoms of skin aging［2-3］， and thus the laser is widely applied in the treatment of such skin diseases as acne， scars and photoaging. However， the photothermy of laser can also denature keratin and zymoproteins， destroy the normal structure of the stratum corneum and affect enzymatic reaction， resulting in metabolism disturbance for the production of moisturizing factors and lipids， and further decreasing the defence ability of skin to external stimulus. In recent years， having witnessed the significant clinical efficacy of MEBO in repairing various types of wounds， some researchers applied it to the treatment of wounds caused by the ultra pulsed CO2fractional laser operation. Clinical studies have found that MEBO at different temperatures have different effects on the repair of such wounds. In this study，the authors compared the effects of MEBO at different temperatures on rats’ skin barrier function after they underwent the ultra pulsed CO2fractional laser operation.

TEWL value is an important indicator of evaluating skin barrier function［4］. It can accurately reflect the recovery of skin barrier function in a continuous manner［5］. This study found that， immediately after the laser operation， the TEWL values in the normal control group， cold compress group， MEBO low temperature group and MEBO room temperature group all increased sharply and the differences were statistically significant as compared to that in the blank control group （all P ＜0.05）. On day 7 after the laser operation， the TEWL values in MEBO room temperature group basically returned to the similar level as in the blank control group （P ＞0.05）， and were significantly lower than that in the normal control group， cold compress group and MEBO low temperature group （all P ＜0.05）.Therefore， it can be seen that after the laser operation， the barrier function of skin following the laser operation is destroyed，and the water loss from the stratum corneum is increased. Fortunately， MEBO at room temperature can facilitate the recovery of the barrier function.This conclusion is consistent with the results of Wu Shuyi’s study“Effect of MEBO on Repair of Postoperative Wound of Ultra Pulsed CO2Fractional Laser operation at Different Temperatures”［1］. The ingredients contained in MEBO such as β-sitosterol， baicalin and berberine， etc. have the effects of fighting inflammation， improving the local blood circulation and enhancing the immunity function， thus facilitating the regenerative restoration of wounds［6］. MEBO， as a type of low-melting ointment， can liquefy the skin necrotic tissues under the skin warming，and help achieve automatic drainage of necrotic tissues［7-8］. However， if the temperature is too low， the liquefaction time of MEBO ointment will be delayed， impairing its therapeuticeffect［9-10］. The study also founds that， on day 7 after the laser operation， the TEWL values in the cold compress group and MEBO low temperature group were still significantly higher than that before the operation， while the TEWL values in the normal control group and MEBO room temperature group all returned to their corresponding preoperative levels， which indicates that low temperature has an adverse effect on wound healing. The reason might be that low temperature reduces the skin hydration， leading to the contraction of capillaries in wounds and the decrease of heat dissipation， thereby inhibiting the release of pro-inflammatory factors and slowing down blood circulation， negatively affecting wound repair［1］.

In summary， MEBO （28 ℃） can accelerate wound healing to recover skin barrier function after the function is impaired as a result of the ultra pulsed CO2fractional laser operation. However， MEBO at low temperature （4 ℃） isn’t helpful for the healing of such wounds.