不同温度MEBO 对大鼠超脉冲CO2 点阵激光术后皮肤屏障功能的影响

随着医疗水平的不断提高， 超脉冲CO2点阵激光联合湿润烧伤膏 （moist exposed burn ointment， MEBO） 在临床中的应用也越发广泛， 而临床研究发现， 不同温度的MEB0 对超脉冲CO2点阵激光术后创面的修复效果也存在差异［1］， 但具体作用机制尚不完全明确。 为进一步探讨其作用机制， 本研究采用不同温度的MEBO 治疗大鼠超脉冲CO2点阵激光术后创面，并通过测定经皮水分丢失（transepidermal water loss， TEWL） 值的变化情况探讨不同温度MEBO对大鼠皮肤屏障功能的影响， 现报告如下。

1 实验材料

1.1 实验动物及分组

健康雄性Wistar 大鼠30 只（南方医科大学动物中心提供）， 体质量200 ～250 g， 按照随机数表法将其随机分为空白对照组、 正常对照组、 冷敷组、 MEBO 低温组、 MEBO 常温组，每组6 只， 均于21 ～25 ℃室温、 自然光照下适应性喂养1 周。

1.2 主要仪器与药物

超脉冲CO2点阵激光机： 成都国雄光电技术有限公司生产； 皮肤屏障测定仪 （GPSkin Barrier）： 韩国G-Power 公司生产； MEBO： 汕头市美宝制药有限公司生产。

2 方法

2.1 模型建立

所有大鼠称重后均给予3% 戊巴比妥钠（30 mg／kg） 腹腔注射麻醉。 麻醉成功后， 空白对照组大鼠仅做背部备皮处理， 并检测TEWL 值； 正常对照组、 冷敷组、 MEBO 低温组及MEBO 常温组大鼠于背部备皮处理及检测TEWL 值后固定于实验台上， 对备皮部位行超脉冲CO2点阵激光烧灼处理 （能量设置为20 mJ／s， 光斑 设 置 为10.0 mm × 10.0 mm），每只大鼠每侧背部烧灼2 处创面， 即每只大鼠4 处创面、 每组大鼠24 处创面。 激光术后， 正常对照组大鼠创面不做任何处理； 冷敷组大鼠创面予以4 ℃水袋外敷30 min， 每天1 次；MEBO 低温组大鼠创面外涂4 ℃MEBO， 每天换药1 次； MEBO 常温组大鼠创面外涂28 ℃MEBO， 每天换药1 次。

2.2 TEWL 检测

采用皮肤屏障测定仪测定各组大鼠激光术前及激光术后即刻、 12 h、 24 h 与7 d 的TEWL值。 每次检测前使用生理盐水清洗创面， 自然晾干后进行检测。

2.3 统计学处理

采用SPSS 25.0 统计软件对所得数据进行统计学分析， 计量资料以均数±标准差（±s）表示， 采用方差分析及LSD 法进行对比分析，均以P ＜0.05 为差异具有统计学意义。

3 结果

激光术前， 各组大鼠皮肤TEWL 值对比，P 均＞0.05， 差异无统计学意义， 具有可比性。激光术后， 除空白对照组大鼠皮肤TEWL 值无明显变化外， 其余各组大鼠创面TEWL 值均呈先升高后降低的趋势， P 均＜0.05， 差异具有统计学意义； 至激光术后7 d， 正常对照组与MEBO 常温组大鼠创面TEWL 值均恢复至与术前相当的水平（P 均＞0.05）， 冷敷组与MEBO低温组大鼠创面TEWL 值仍高于术前（P 均＜0.05）。 激光术后即刻， 正常对照组、 冷敷组、MEBO 低温组、 MEBO 常温组大鼠创面TEWL值均明显升高， 与空白对照组相比， P 均＜0.05， 差异具有统计学意义； 激光术后12 h、24 h， 正常对照组、 冷敷组、 MEBO 低温组、MEBO 常温组大鼠创面TEWL 值仍明显高于空白对照组， 且冷敷组与MEBO 低温组大鼠创面TEWL 值明显高于正常对照组， P 均＜0.05，差异具有统计学意义， 而MEBO 常温组大鼠创面TEWL 值与正常对照组相当（P ＞0.05）； 激光术后7 d， 除MEBO 常温组大鼠创面TEWL值恢复至空白对照组水平（P ＞0.05）， 且明显低于正常对照组、 冷敷组与MEBO 低温组（P均＜0.05） 外， 其余各组大鼠创面TEWL 值仍明显高于空白对照组， P 均＜0.05， 差异具有统计学意义（表1）。 可见常温MEBO 有利于创面皮肤屏障功能的恢复。

表1 各组大鼠皮肤或创面TEWL 值对比（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 各组大鼠皮肤或创面TEWL 值对比（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： 与空白对照组比较， aP ＜0.05， 差异具有统计学意义； 与正常对照组比较， bP ＜0.05， 差异具有统计学意义； 与冷敷组比较，cP ＜0.05， 差异具有统计学意义； 与MEBO 低温组比较， dP ＜0.05， 差异具有统计学意义。 与同组激光术前比较， eP ＜0.05， 差异具有统计学意义； 与同组激光术后即刻比较， fP ＜0.05， 差异具有统计学意义； 与同组激光术后12 h 比较， gP ＜0.05， 差异具有统计学意义； 与同组激光术后24 h 比较， hP ＜0.05， 差异具有统计学意义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）

组别Group n激光术前Before the laser operation激光术后即刻Immediately after the laser operation激光术后12 h 12 h after the laser operation激光术后24 h 24 h after the laser operation激光术后7 d Day 7 after the laser operation F 值F value P 值P value空白对照组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正常对照组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冷敷组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 低温组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 常温组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 讨论

超脉冲CO2点阵激光的波长为10.6 μm，能够被组织中的水分吸收而产生大量热量， 并通过局灶性光热作用启动皮肤组织的再生修复机制， 促进皮肤各层组织的再生修复， 改善皮肤老化等状况［2-3］， 被广泛应用于痤疮、 瘢痕、光老化等皮肤疾病的治疗。 然而， 光热作用也可使角蛋白及酶蛋白等变性， 破坏角质层的正常结构， 影响酶促反应， 导致保湿因子及脂质生成代谢障碍， 进而破坏皮肤的“砖墙结构”，降低皮肤对外界刺激的抵御能力。 近年来， 部分研究学者鉴于MEBO 在多种类型创面的修复中均取得了显著的临床疗效， 故将其应用于超脉冲CO2点阵激光术后创面的治疗， 但临床研究发现， 不同温度的MEBO 对超脉冲CO2点阵激光术后创面的修复效果存在差异， 遂笔者于本研究中对比分析了不同温度MEBO 对大鼠超脉冲CO2点阵激光术后皮肤屏障功能的影响。

TEWL 值是评价皮肤屏障功能的重要指标［4］， 其可准确、 稳定地反映皮肤屏障功能的恢复过程［5］。 本研究发现， 激光术后即刻， 正常对照组、 冷敷组、 MEBO 低温组、 MEBO 常温组大鼠创面TEWL 值均明显升高， 与空白对照组相比， P 均＜0.05， 差异具有统计学意义；激光术后7 d， MEBO 常温组大鼠创面TEWL 值基本恢复至与空白对照组相当的水平 （P ＞0.05）， 且明显低于正常对照组、 冷敷组与MEBO低温组（P 均＜0.05）。 可见， 激光术后创面的皮肤屏障被破坏， 皮肤角质层水分丢失增多， 而常温MEBO 有利于创面皮肤屏障功能的恢复。 与吴树毅等的不同温度MEBO 对超脉冲CO2点阵激光术后创面修复影响的研究结果一致［1］。 MEBO 内含有的β-谷甾醇、 黄芩甙、小檗碱等成分具有抗炎、 改善局部血液循环、增强免疫力等作用， 可促进创面的再生修复［6］。 作为一种低熔点软膏制剂， MEBO 可在皮肤的温化作用下液化排除创面坏死组织， 发挥自动循环引流作用［7-8］， 而温度过低将延长药膏液化时间， 影响药效的发挥［9-10］。 本研究还发现， 激光术后7 d， 冷敷组与MEBO 低温组大鼠创面TEWL 值仍明显高于术前， 而正常对照组与MEBO 常温组大鼠创面TEWL 值均恢复至与术前相当的水平， 提示低温对创面愈合有不利影响， 原因可能在于低温可引起皮肤水合作用降低， 创面毛细血管收缩， 散热减少，进而抑制促炎因子的释放， 并减缓血液循环，影响创面修复［1］。

综上所述， 超脉冲CO2点阵激光术可损害皮肤屏障功能， 常温（28 ℃） MEBO 可加快创面愈合， 促进皮肤屏障功能快速恢复， 而低温（4 ℃） MEBO对创面愈合无明显促进作用。

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.