Effects and mechanism of Lactococcus lactis thermo-sensitive hydrogel on the wound healing of full-thickness skin defects in diabetic mice
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摘要:
目的 探讨乳酸乳球菌温敏水凝胶对糖尿病小鼠全层皮肤缺损创面愈合的影响及其机制。 方法 (1)按照菌与培养基体积比为1∶100用M17GS液体培养基培养约5×108集落形成单位/mL乳酸乳球菌(浓度下同)，分别于培养0(即刻)、2、4、6、8、10、12 h用酶标仪观测其生长情况。另取该菌菌落同前处理并于相同时间点收集培养基分离细菌培养上清液，用台式pH计测定pH值，并用L-乳酸检测分析试剂盒测定培养0(即刻)、2、4、8、12 h的L-乳酸浓度(样本数为3)。(2)将泊洛沙姆温敏聚合物与M17GS液体培养基按照质量与体积比为0.2 g∶1 mL充分混匀，制备单纯温敏水凝胶。按照菌与水凝胶体积比1∶100在单纯温敏水凝胶中加入乳酸乳球菌，充分混匀后制备乳酸乳球菌温敏水凝胶，分别在4、37 ℃孵育以及成胶后再4 ℃孵育观察其形态；通过流变仪测定乳酸乳球菌温敏水凝胶在10～40 ℃的储能模量与损耗模量，同时观察成胶温度；将乳酸乳球菌温敏水凝胶冷冻干燥后，通过扫描电子显微镜观察其表面及其中乳酸乳球菌的形态结构。(3)将小鼠巨噬细胞系Raw264.7细胞用终质量浓度分别为100、10 ng/mL内毒素/脂多糖与γ干扰素培养24 h刺激M1型极化，将细胞分为空白对照组(不做其他处理)、乳酸乳球菌温敏水凝胶组和乳酸组。乳酸乳球菌温敏水凝胶组细胞加入1 mL乳酸乳球菌温敏水凝胶，乳酸组细胞加入终物质的量浓度为30 mmol/L乳酸，37 ℃培养24 h后，通过实时荧光定量反转录PCR(RT-PCR)法检测M2型巨噬细胞标志物精氨酸酶1、CD206的mRNA表达量(样本数为3)，采用免疫荧光法检测精氨酸酶1和CD206的蛋白定位与表达。(4)取15只8～10周龄雌性BALB/c小鼠，通过链脲佐菌素联合高糖高脂饲料的方法诱导为糖尿病小鼠模型后，在每只小鼠背部制作直径6 mm全层皮肤缺损创面，采用随机数字表法将小鼠分为空白对照组(不做其他处理)、单纯温敏水凝胶组和乳酸乳球菌温敏水凝胶组，每组5只。水凝胶处理2组小鼠伤后即刻分别滴加200 μL相应水凝胶至创面，每天更换水凝胶。水凝胶处理2组小鼠处理0(即刻)、3、6、9、12 d后，观察创面愈合情况，测量创面面积；处理12 d后，取创面组织，行苏木精-伊红染色观察肉芽组织厚度，采用免疫荧光法观测CD206、M1型巨噬细胞标志物诱导型一氧化氮合酶(iNOS)阳性细胞。空白对照组小鼠于前述相同时间点进行相应观测。(5)取9只8～10周龄雌性BALB/c小鼠同实验(4)方法诱导糖尿病小鼠模型后，采用随机数字表法分为正常皮肤组(不做其他处理)、单纯创面组、乳酸乳球菌温敏水凝胶组，每组3只。单纯创面组与乳酸乳球菌温敏水凝胶组小鼠按照实验(4)方法制成全层皮肤缺损创面，前一组小鼠伤后不做其他处理，后一组小鼠伤后即刻滴加200 μL乳酸乳球菌温敏水凝胶至创面。水凝胶处理组小鼠处理1 d后，取创面组织，通过实时荧光定量RT-PCR法检测白细胞介素1β(IL-1β)、肿瘤坏死因子α(TNF-α)和核因子κB mRNA表达量；眼球取血后，通过全自动血细胞分析仪检测外周血白细胞计数、淋巴细胞计数和单核细胞计数，通过L-乳酸检测分析试剂盒测定血清L-乳酸浓度。于前述相同时间点，取正常皮肤组小鼠相应部位正常皮肤组织、单纯创面组小鼠创面组织及2组小鼠血液进行相应检测。对数据行单因素方差分析、重复测量方差分析、Tukey和Dunnett检验。 结果 (1)乳酸乳球菌于培养约6 h生长达到平台期。乳酸乳球菌培养上清液中，pH值逐渐降低，至培养8 h下降至最低值4.9左右；L-乳酸浓度逐渐升高，至培养8 h达到最高值约70 mmol/L。(2)乳酸乳球菌温敏水凝胶在4 ℃为液体溶胶态，在37 ℃为固体凝胶态，成胶后于4 ℃孵育后再次变为液体溶胶态；成胶温度约为25 ℃，成胶后储能模量约为3 000 Pa、损耗模量约为1 000 Pa。扫描电子显微镜下可见，乳酸乳球菌温敏水凝胶为疏松三维多孔结构，乳酸乳球菌为椭球形并被包裹在水凝胶内部。(3)培养24 h，与空白对照组比较，乳酸乳球菌温敏水凝胶组、乳酸组巨噬细胞中精氨酸酶1表达量显著升高(
q ＝11.620、15.250，
P <0.01)、CD206 mRNA表达量显著升高(
q ＝16.770、19.030，
P <0.01)，定位于细胞膜的CD206蛋白和定位于细胞质的精氨酸酶1蛋白表达明显升高；乳酸组巨噬细胞中精氨酸酶1、CD206 mRNA表达量与乳酸乳球菌温敏水凝胶组相近(
q ＝3.629、2.259，
P >0.05)。(4)处理3～12 d后，与空白对照组和单纯温敏水凝胶组比较，乳酸乳球菌温敏水凝胶组小鼠创面愈合速度更快，创面面积明显缩小，创缘炎症减轻。乳酸乳球菌温敏水凝胶组小鼠处理3、6、9、12 d后创面面积[(25.8±5.9)、(21.2±4.6)、(16.0±2.4)、(8.4±2.4)mm2]较空白对照组[(31.8±5.3)、(28.0±3.4)、(22.6±3.7)、(17.0±1.0)mm2]显著缩小(
q ＝3.506、3.973、3.856、5.025，
P <0.05或
P <0.01)，处理3、6 d后创面面积较单纯温敏水凝胶组显著缩小(
q ＝3.739、3.739，
P <0.05)。处理12 d后，与空白对照组和单纯温敏水凝胶组比较，乳酸乳球菌温敏水凝胶组小鼠创面肉芽组织更厚，创面组织中iNOS阳性细胞明显减少且CD206阳性细胞明显增多。(5)处理1 d后，单纯创面组小鼠创面组织中IL-1β、TNF-α和核因子κB mRNA表达量显著高于正常皮肤组小鼠正常皮肤组织(
q ＝9.253、4.819、6.020，
P <0.01)，与乳酸乳球菌温敏水凝胶组相近(
q ＝2.850、2.735、2.556，
P >0.05)；单纯创面组小鼠外周血白细胞计数、淋巴细胞计数与单核细胞计数显著高于正常皮肤组(
q ＝3.523、5.373、5.279，
P <0.05或
P <0.01)，与乳酸乳球菌温敏水凝胶组相近(
q ＝0.621、1.240、1.293，
P >0.05)；3组小鼠血清L-乳酸浓度均保持在正常范围内且组间总体比较差异无统计学意义(
F ＝4.095，
P >0.05)。 结论 乳酸乳球菌温敏水凝胶在糖尿病全层皮肤缺损小鼠创面局部使用安全，能够通过原位生产投递乳酸，促进巨噬细胞从M1型向M2型极化，重塑创面愈合微环境，促进创面的高效愈合。
Abstract:Objective To explore the effects and mechanism of
Lactococcus lactis (
L .
lactis ) thermo-sensitive hydrogel on the wound healing of full-thickness skin defects in diabetic mice. Methods (1) According to the volume ratio of bacteria to medium of 1∶100, about 5×108 colony forming units/mL (the same concentration below)
L .
lactis was cultured in M17GS liquid medium. The growth conditions were observed at 0 (immediately), 2, 4, 6, 8, 10, and 12 h of culture with a microplate reader. In addition, another colony of the bacteria was taken and cultured under the same condition mentioned above. The culture medium was collected at the same time points as mentioned above, and the supernatant of bacterial culture was isolated. With the supernatant, the pH value was measured with a desktop pH meter, and the concentration of L-lactic acid at 0 (immediately), 2, 4, 8, and 12 h of culture was determined by the L-lactic acid detection and analysis kit (
n =3). (2) To prepare a simple thermo-sensitive hydrogel, the poloxamer thermo-sensitive polymer and M17GS liquid medium were mixed thoroughly according to the mass-volume ratio of 0.2 g∶1 mL.
L .
lactis was added to the simple thermo-sensitive hydrogel according to the volume ratio of bacteria to hydrogel of 1∶100, and the
L .
lactis thermo-sensitive hydrogel was prepared after thorough mixing. Afterwards, the morphology of
L .
lactis thermo-sensitive hydrogel was observed after 4 ℃, 37 ℃ incubation and again at 4 ℃ incubation after gelation. The storage modulus and loss modulus of the
L .
lactis thermo-sensitive hydrogel at 10-40 ℃ were measured by rheometer, and the gel forming temperature was observed. After freeze-drying the
L .
lactis thermo-sensitive hydrogel, the surface and the morphological structure of
L .
lactis in the hydrogel were observed by scanning electron microscope. (3) Mouse macrophages Raw264.7 cells were M1-type polarization stimulated by culturing with lipopolysaccharide and interferon γ in the final mass concentration of 100 and 10 ng/mL respectively for 24 h. The cells were divided into blank control group (without other treatment),
L .
lactis thermo-sensitive hydrogel group, and lactic acid group.
L .
lactis thermo-sensitive hydrogel in the volume of 1 mL was added to the cells of
L .
lactis thermo-sensitive hydrogel group, while lactic acid with the final molarity of 30 mmol/L was added to the cells in lactic acid group. After being cultured at 37 ℃ for 24 h, mRNA expressions of the markers arginase 1 and CD206 of M2-type macrophages were detected by real-time fluorescence quantitative reverse transcription polymerase chain reaction (RT-PCR) (
n =3), and the immunofluorescence method was used to detect the protein localization and expression of arginase 1 and CD206. (4) Fifteen female BALB/c mice aged 8-10 weeks were induced into diabetic mouse models by the method of streptozotocin combined with high-sugar and high-fat diet, and a full-thickness wound with the diameter of 6 mm was made on the back of each mouse. The mice were divided into blank control group (without other treatment), thermo-sensitive hydrogel alone group, and
L .
lactis thermo-sensitive hydrogel group according to the random number table, with 5 mice in each group. The mice in the hydrogel treatment two groups were dripped with 200 μL corresponding hydrogel to the wound surface immediately after injury, and the hydrogel was replaced every day. After treatment for 0 (immediately), 3, 6, 9, and 12 days in the hydrogel treatment two groups, wound healing was observed, and wound area was measured. After 12 days of treatment, the wound tissue was taken to observe the thickness of granulation tissue by hematoxylin-eosin staining and CD206 and the marker of M1-type macrophages of inducible nitric oxide synthase (iNOS) positive cells by immunofluorescence method. The mice in blank control group were observed at the same time points as mentioned above. (5) Nine female BALB/c mice aged 8-10 weeks were induced into diabetic mouse models by the same method of experiment (4). Then, they were divided into normal skin group (without other treatment), wound alone group, and
L .
lactis thermo-sensitive hydrogel group according to the random number table, with 3 mice in each group. Mice in wound alone group and
L .
lactis thermo-sensitive hydrogel group were prepared with full-thickness skin defect wounds according to the method of experiment (4). Mice in the former group was left untreated after injury, and in the latter group, 200 μL
L .
lactis thermo-sensitive hydrogel was dripped onto the wound surface immediately after injury. After treatment for 1 day in hydrogel treatment group, the wound tissue of mice was taken, and the mRNA expressions of interleukin 1β (IL-1β), tumor necrosis factor α (TNF-α), and nuclear factor κB were detected by real-time fluorescence quantitative RT-PCR; after the eyeball blood was collected, the leukocyte count, lymphocyte count, and monocyte count in peripheral blood were measured by an automatic blood cell analyzer, and the serum L-lactic acid concentration was measured by the L-lactic acid detection and analysis kit. At the same time point mentioned above, normal skin tissue was taken from the corresponding parts of mice in normal skin group, wound tissue was taken from mice in wound alone group, and blood was taken from mice of the two groups for corresponding detection. Data were statistically analyzed with one-way analysis of variance, analysis of variance for repeated measurement, Tukey, and Dunnett test. Results (1) The growth of
L .
lactis reached the plateau in about 6 h of culture. In the culture supernatant of
L .
lactis, the pH value gradually decreased, reaching the nadir about 4.9 after 8 h of culture, and the L-lactic acid concentration gradually increased, which peaked about 70 mmol/L after 8 h of culture. (2) The
L .
lactis thermo-sensitive hydrogel was a liquid at 4 ℃, and a solid gel at 37 ℃. After gelation, it became a liquid again after incubating at 4 ℃. The gel forming temperature was about 25 ℃. The storage modulus was about 3 000 Pa, and the loss modulus was about 1 000 Pa after gelation. Under the scanning electron microscope, the
L .
lactis thermo-sensitive hydrogel showed a loose three-dimensional porous structure, and the
L .
lactis had an ellipsoidal shape being wrapped inside the hydrogel. (3) After 24 h of culture, compared with those in blank control group, the expression of arginase 1 increased significantly (
q =11.620, 15.250,
P <0.01), the expression of CD206 mRNA increased significantly (
q =16.770, 19.030,
P <0.01), and the expression of CD206 protein located in the cell membrane and arginase 1 protein located in the cytoplasm increased significantly in the macrophages of
L .
lactis thermo-sensitive hydrogel group and lactic acid group. The expressions of arginase 1 and CD206 mRNA in the macrophages between lactic acid group and
L .
lactis thermo-sensitive hydrogel group were similar (
q =3.629, 2.259,
P >0.05). (4) After 3-12 days of treatment, compared with those in blank control group and thermo-sensitive hydrogel alone group, the wound of mice in
L .
lactis thermo-sensitive hydrogel group healed faster, the wound area was significantly reduced, and the inflammation of the wound edge tissue was reduced. After treatment of 3, 6, 9, 12 days, the wound areas of mice in
L .
lactis thermo-sensitive hydrogel group were (25.8±5.9), (21.2±4.6), (16.0±2.4), (8.4±2.4) mm2 respectively, which were significantly smaller than (31.8±5.3), (28.0±3.4), (22.6±3.7), (17.0±1.0) mm2 in blank control group (
q =3.506, 3.973, 3.856, 5.025,
P <0.05 or
P <0.01). After treatment of 3 and 6 days, the wound areas of mice in
L .
lactis thermo-sensitive hydrogel group were significantly smaller than those in thermo-sensitive hydrogel alone group (
q =3.739, 3.739,
P <0.05). After 12 days of treatment, compared with those in blank control group and thermo-sensitive hydrogel alone group, the wound granulation tissue of mice in
L .
lactis thermo-sensitive hydrogel group was thicker, with significantly reduced iNOS positive cells and increased CD206 positive cells in wound tissue. (5) After 1 day of treatment, the mRNA expressions of IL-1β, TNF-α, and nuclear factor κB in the wound tissue of mice in wound alone group were significantly higher than those of normal skin tissue of mice in normal skin group (
q =9.253, 4.819, 6.020,
P <0.01) but similar to those in
L .
lactis thermo-sensitive hydrogel group (
q =2.850, 2.735, 2.556,
P >0.05). The peripheral blood leukocyte count, lymphocyte count, and monocyte count of mice in wound alone group were significantly higher than those in normal skin group (
q =3.523, 5.373, 5.279,
P <0.05 or
P <0.01) but similar to those in
L .
lactis thermo-sensitive hydrogel group (
q =0.621, 1.240, 1.293,
P >0.05). The serum L-lactic acid concentration of mice in the three groups remained within the normal range and the overall comparison among them was not statistically significant (
F =4.095,
P >0.05). Conclusions The
L .
lactis thermo-sensitive hydrogel was safe to use locally on the wounds of diabetic mice with full-thickness skin defects. It can produce and deliver lactic acid in situ, promote the polarization of macrophages from M1 to M2, reshape the wound healing microenvironment, and promote efficient wound healing.
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Key words:
- Diabetes mellitus /
- Wound healing /
- Hydrogel /
- Macrophages /
- Lactococcus lactis
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