Influences and mechanism of berberine on wound healing of full-thickness skin defects in diabetic mice
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摘要:
目的 探讨小檗碱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 采用实验研究方法。配制含小檗碱终质量浓度分别为0（不含小檗碱）、1.25、2.50、5.00、10.00、20.00、40.00、80.00、160.00 μg/mL的小鼠真皮成纤维细胞（MDF）正常糖完全培养基（以下简称常规培养基）。取原代MDF，分别用常规培养基和MDF高糖（30 mmol/L葡萄糖）完全培养基（以下简称高糖培养基）培养，收集第3~6代细胞进行以下实验。取采用常规培养基培养的细胞，饥饿处理12 h后更换为含不同浓度小檗碱的常规培养基培养48 h，采用细胞计数试剂盒8（CCK-8）筛选小檗碱最适作用浓度（样本数为6），用于后续细胞实验。取2种培养基培养的细胞，将采用常规培养基培养的细胞纳入正常对照组，将采用高糖培养基培养的细胞按照随机数字表法（分组方法下同）分为单纯高糖组和高糖+小檗碱组。分别用相应的培养基培养48 h后，采用CCK-8检测细胞活力，采用划痕试验和Transwell实验检测细胞24 h迁移能力，采用实时荧光定量反转录PCR法和蛋白质印迹法分别检测细胞中血小板源性生长因子（PDGF）、血管内皮生长因子（VEGF）、转化生长因子β 1（TGF-β 1）、基质金属蛋白酶9（MMP-9）和胱天蛋白酶3（caspase-3）的mRNA和蛋白表达水平，前述实验的样本数分别为6、3、9、3、6。取15只8周龄雄性BALB/c小鼠，构建糖尿病小鼠模型后在其背部制作全层皮肤缺损创面，分为单纯糖尿病组、糖尿病+低浓度（25 μg/mL）小檗碱组和糖尿病+高浓度（75 μg/mL）小檗碱组，分别作相应处理，每组5只鼠。伤后0（即刻）、3、7、14、21 d，采用ImageJ软件测量创面面积。伤后21 d，采用苏木精-伊红染色和Masson染色分别检测创面组织病理学变化及胶原生成情况，采用免疫组织化学法和实时荧光定量反转录PCR法分别检测创面组织中MMP-9、PDGF、TGF-β 1、VEGF、CD31和caspase-3的蛋白和mRNA表达水平。动物实验的样本数均为5。对数据行单因素方差分析、独立样本 t检验、Tukey检验、析因设计方差分析。 结果 培养48 h，筛选出小檗碱质量浓度为20.00 μg/mL时细胞的活力最大。培养48 h后，与正常对照组相比，单纯高糖组、高糖+小檗碱组细胞活力均明显减弱（ P<0.05）；与单纯高糖组相比，高糖+小檗碱组细胞活力明显增强（ P<0.05）。培养48 h后，划痕试验结果显示，与正常对照组相比，单纯高糖组、高糖+小檗碱组细胞24 h迁移率均明显下降（ P<0.05）；与单纯高糖组相比，高糖+小檗碱组细胞24 h迁移率明显升高（ P<0.05）。培养48 h后，Transwell实验结果显示，与正常对照组细胞24 h迁移数（141±7）个相比，单纯高糖组、高糖+小檗碱组细胞24 h迁移数的（28±3）、（86±6）个均明显减少（ t值分别为117.28、31.80， P<0.05）；与单纯高糖组相比，高糖+小檗碱组细胞24 h迁移数明显增多（ P<0.05）。培养48 h后，与正常对照组相比，单纯高糖组、高糖+小檗碱组细胞中PDGF、VEGF、TGF-β 1、MMP-9 mRNA水平均明显降低（ P<0.05），caspase-3 mRNA水平均明显升高（ P<0.05）；与单纯高糖组相比，高糖+小檗碱组细胞中PDGF、VEGF、TGF-β 1、MMP-9 mRNA水平均明显升高（ P<0.05），caspase-3 mRNA表达水平明显降低（ P<0.05）。培养48 h后，与正常对照组相比，单纯高糖组、高糖+小檗碱组细胞中PDGF、VEGF、TGF-β 1、MMP-9蛋白表达水平均明显降低（ P<0.05），caspase-3蛋白表达水平均明显升高（ P<0.05）；与单纯高糖组相比，高糖+小檗碱组细胞中PDGF、VEGF、TGF-β 1、MMP-9蛋白表达水平均明显升高（ P<0.05），caspase-3蛋白表达水平明显降低（ P<0.05）。与单纯糖尿病组相比，糖尿病+低浓度小檗碱组小鼠伤后14、21 d及糖尿病+高浓度小檗碱组小鼠伤后3、7、14、21 d创面面积均明显减小（ P<0.05）；与糖尿病+低浓度小檗碱组相比，糖尿病+高浓度小檗碱组小鼠伤后3、7、14、21 d创面面积均明显减小（ P<0.05）。伤后21 d，单纯糖尿病组小鼠创面组织尚未形成上皮，真皮层有大量炎症细胞和肉芽组织；糖尿病+低浓度小檗碱组小鼠创面组织大部分完成上皮化，真皮层有大量炎症细胞；糖尿病+高浓度小檗碱组小鼠创面大部分完成上皮化，真皮层有少量毛囊和炎症细胞。伤后21 d，与单纯糖尿病组相比，糖尿病+低浓度小檗碱组、糖尿病+高浓度小檗碱组小鼠创面胶原面积均明显增加（ P<0.05）；与糖尿病+低浓度小檗碱组相比，糖尿病+高浓度小檗碱组小鼠创面胶原面积明显增加（ P<0.05）。伤后21 d，与单纯糖尿病组相比，糖尿病+低浓度小檗碱组、糖尿病+高浓度小檗碱组小鼠创面组织中MMP-9、PDGF、TGF-β 1、VEGF和CD31蛋白表达水平均明显升高（ P<0.05），caspase-3蛋白表达水平均明显降低（ P<0.05）；与糖尿病+低浓度小檗碱组相比，糖尿病+高浓度小檗碱组小鼠创面组织中MMP-9、PDGF、TGF-β 1、VEGF和CD31蛋白表达水平均明显升高（ P<0.05），caspase-3蛋白表达水平明显降低（ P<0.05）。伤后21 d，与单纯糖尿病组相比，糖尿病+低浓度小檗碱组、糖尿病+高浓度小檗碱组小鼠创面组织中MMP-9、PDGF、TGF-β 1、VEGF和CD31 mRNA水平均明显升高（ P<0.05），caspase-3 mRNA水平均明显降低（ P<0.05）；与糖尿病+低浓度小檗碱组相比，糖尿病+高浓度小檗碱组小鼠创面组织中MMP-9、PDGF、TGF-β 1、VEGF和CD31 mRNA水平均明显升高（ P<0.05），caspase-3 mRNA水平明显降低（ P<0.05）。 结论 小檗碱可以通过上调MMP-9、PDGF、TGF-β 1、VEGF等因子的表达，下调促凋亡因子caspase-3的表达，从而促进MDF的增殖和迁移以及糖尿病小鼠全层皮肤缺损创面的愈合。 Abstract:Objective To investigate the influences and mechanism of berberine on wound healing of full-thickness skin defects in diabetic mice. Methods The experimental research method was adopted. Mouse dermal fibroblasts (MDF) conventional-glucose complete medium (hereinafter referred to as conventional medium) were prepared with final mass concentrations of berberine of 0 (no berberine), 1.25, 2.50, 5.00, 10.00, 20.00, 40.00, 80.00, and 160.00 μg/mL, respectively. Primary MDF were cultured using conventional medium and MDF high-glucose (30 mmol/L glucose) complete medium (hereinafter referred to as high-glucose medium), and the 3 rd to 6 th passage cells were collected for the following experiments. Cells cultured in conventional medium were taken and subjected to starvation treatment for 12 hours, and then cultured in conventional media containing different concentrations of berberine for 48 hours to screen out the optimal working concentration of berberine using the cell counting kit 8 (CCK-8), the sample number was 6, and the selected optimal berberine concentration was used for subsequent cell culture experiments. Cells cultured in 2 media were taken, of which the cells cultured in conventional medium were included to the normal control group; cells cultured in high-glucose medium were divided into high-glucose alone group and high-glucose+berberine group according to the random number table (the same grouping method below). After 48 h of cultivation, cell viability was detected by CCK-8, cell migration capacity was evaluated by scratch test and Transwell assays, and mRNA and protein expression levels of platelet-derived growth factor (PDGF), vascular endothelial growth factor (VEGF), transforming growth factor-β 1 (TGF-β 1), matrix metalloproteinase 9 (MMP-9), and cysteine aspartic acid specific protease (caspase-3) in the cells were detected by real-time fluorescence quantitative reverse transcription polymerase chain reaction and Western blotting, respectively, and the sample numbers of the aforementioned experiments were 6, 3, 9, 3, and 6, respectively. Fifteen 8-week-old male BALB/c mice were used to establish diabetic mouse model, then full-thickness skin defect wounds on their backs were made and divided into diabetes alone group, diabetes+low-concentration berberine group (25 μg/mL), and diabetes+high-concentration berberine group (75 μg/mL) for corresponding treatments, with 5 mice in each group. The wound areas were measured using ImageJ software on post injury day (PID) 0 (immediately), 3, 7, 14, and 21. On PID 21, histological changes and collagen formation in the wound tissue were detected by hematoxylin-eosin and Masson staining, respectively, protein expression and mRNA levels of MMP-9, PDGF, TGF-β 1, VEGF, CD31, and caspase-3 in the wound tissue were detected by immunohistochemistry and real-time fluorescence quantitative reverse transcription polymerase chain reaction, respectively, the sample number of animal experiments was all 5. Data were statistically analyzed with one-way analysis of variance, independent sample t-test, Tukey's test, and factorial design analysis of variance. Results After 48 hours of cultivation, the cell viability was the highest when the mass concentration of berberine was 20.00 μg/mL. After 48 h of cultivation, compared with that in normal control group, cell viability in both high-glucose alone group and high-glucose+berberine group reduced significantly ( P<0.05); compared with that in high-glucose alone group, the cell viability in high-glucose+berberine group was significantly enhanced ( P<0.05). After 48 h of cultivation, scratch test results showed that, the cell migration rates in 24 h in both high-glucose alone group and high-glucose+berberine group were significantly decreased than that in normal control group ( P<0.05); compared with that in high-glucose alone group, the cell migration rate in 24 h in high-glucose+berberine group was significantly enhanced ( P<0.05). After 48 h of cultivation, the results of Transwell experiments showed that, compared with (141±7) of cells migrating in 24 h in normal control group, the number of cells migrating in 24 h in high-glucose alone group and high-glucose+berberine group were 28±3 and 86±6, respectively, which were significantly decreased ( P<0.05); compared with that in high-glucose alone group, the number of cells migration in 24 h in high-glucose+berberine group was significantly increased ( P<0.05). After 48 h of cultivation, compared with those in normal control group, the mRNA levels of PDGF, VEGF, TGF-β 1, and MMP-9 of cells in high-glucose alone group and high-glucose+berberine group were significantly decreased ( P<0.05), the mRNA levels of caspase-3 were significantly increased ( P<0.05); compared with those in high-glucose alone group, the mRNA levels of PDGF, VEGF, TGF-β 1, and MMP-9 of cells in high-glucose+berberine group were significantly increased ( P<0.05), the mRNA expression level of caspase-3 was significantly decreased ( P<0.05). After 48 h of cultivation, compared with those in normal control group, the protein expression levels of PDGF, VEGF, TGF-β 1, and MMP-9 of cells in high-glucose group and high-glucose+berberine group were significantly decreased ( P<0.05), the protein expression levels of caspase-3 were significantly increased ( P<0.05); compared with those in high-glucose alone group, the protein expression levels of PDGF, VEGF, TGF-β 1, and MMP-9 of cells in high-glucose+berberine group were significantly increased ( P<0.05), and the protein expression level of caspase-3 was significantly decreased ( P<0.05). Compared with those in diabetes alone group, the wound areas of mice in diabetes+low-concentration berberine group on PID 14 and 21 and in diabetes+high-concentration berberine group on PID 3, 7, 14, and 21 were significantly decreased ( P<0.05); compared with that in diabetes+low-concentration berberine group, the wound area in diabetes+high-concentration berberine group was significantly decreased on PID 3, 7, 14, and 21 ( P<0.05). On PID 21, the wound of mice in diabetes alone group was not epithelialized with a large number of inflammatory cells and granulation tissue in the dermis; most of the wound tissue of mice in diabetes+low-concentration berberine group was already epithelialized, although there was a large number of inflammatory cells in the dermis; and most of the wound tissue of mice in diabetes+high-concentration berberine group had completed epithelialization with a small number of hair follicles and inflammatory cells in the dermis. On PID 21, compared with that in diabetes alone group, the collagen area of wound of mice in diabetes+low-concentration berberine group and diabetes+high-concentration berberine group was significantly increased ( P<0.05); compared with that in diabetes+low-concentration berberine group, the collagen area of wound of mice in diabetes+high-concentration berberine group was significantly increased ( P<0.05). On PID 21, compared with those in diabetes alone group, the protein expression levels of MMP-9, PDGF, TGF-β 1, VEGF, and CD31 in wound tissue of mice in diabetes+low-concentration berberine group and diabetes+high-concentration berberine group were significantly increased ( P<0.05), the protein expression levels of caspase-3 were significantly decreased ( P<0.05); compared with those in diabetes+low-concentration berberine group, the protein expression levels of MMP-9, PDGF, TGF-β 1, VEGF, and CD31 in wound tissue of mice in diabetes+high-concentration berberine group were significantly increased ( P<0.05), the protein expression level of caspase-3 was significantly decreased ( P<0.05). On PID 21, compared with those in diabetes alone group, the mRNA levels of MMP-9, PDGF, TGF-β 1, VEGF, and CD31 in wound tissue of mice in diabetes+low-concentration berberine group and diabetes+high-concentration berberine group were significantly increased ( P<0.05), the mRNA levels of caspase-3 were significantly decreased ( P<0.05); compared with those in diabetes+low-concentration berberine group, the mRNA levels of MMP-9, PDGF, TGF-β 1, VEGF, and CD31 in wound tissue of mice in diabetes+high-concentration berberine group were significantly increased ( P<0.05), the mRNA level of caspase-3 was significantly decreased ( P<0.05). Conclusions Berberine can promote the proliferation and migration of MDF and the healing of full-thickness skin defect wounds of mice in diabetic mice by up-regulating the expression of biofactors including MMP-9, PDGF, TGF-β 1, and VEGF, and down-regulating the expression of caspase-3, a pro-apoptotic factor in wound tissue of mice. -
Key words:
- Berberine /
- Diabetes mellitus, experimental /
- Fibroblasts /
- Cytokines /
- Wound repair
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参考文献
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1 培养48 h后划痕试验检测3组小鼠真皮成纤维细胞24 h迁移情况。1A、1B、1C.分别为正常对照组、单纯高糖组和高糖+小檗碱组划痕0 h（即刻）细胞情况，其划痕情况近似；1D、1E、1F.分别为正常对照组、单纯高糖组和高糖+小檗碱组细胞划痕24 h细胞迁移情况，图1E中划痕剩余面积最大
注：图中竖线为细胞迁移的边缘线；高糖指葡萄糖物质的量浓度为30 mmol/L，小檗碱质量浓度为20.00 μg/mL
2 培养48 h后Transwell实验检测3组小鼠真皮成纤维细胞24 h迁移情况 结晶紫×200。2A、2B、2C.分别为正常对照组、单纯高糖组和高糖+小檗碱组Transwell下室细胞迁移数量，图2B中迁移的细胞数最少
注：高糖指葡萄糖物质的量浓度为30 mmol/L，小檗碱质量浓度为20.00 μg/mL
3 蛋白质印迹法检测3组小鼠真皮成纤维细胞培养48 h后多种细胞因子蛋白表达水平
注：高糖指葡萄糖物质的量浓度为30 mmol/L，小檗碱质量浓度为20.00 μg/mL；条带上方1、2、3分别指示正常对照组、单纯高糖组和高糖+小檗碱组；PDGF为血小板源性生长因子，VEGF为血管内皮生长因子，TGF-β1为转化生长因子β1，MMP-9为基质金属蛋白酶9，caspase-3为胱天蛋白酶3
4 3组糖尿病小鼠全层皮肤缺损创面伤后各时间点创面面积。4A、4B、4C、4D、4E.分别为单纯糖尿病组小鼠伤后0（即刻）、3、7、14、21 d创面情况；4F、4G、4H、4I、4J.分别为糖尿病+低浓度（25 μg/mL）小檗碱组小鼠伤后0、3、7、14、21 d创面情况，图4I、4J创面面积较图4D、4E明显缩小；4K、4L、4M、4N、4O.分别为糖尿病+高浓度（75 μg/mL）小檗碱组小鼠伤后0、3、7、14、21 d创面情况，3组小鼠创面均随时间延长趋向闭合，图4L、4M、4N、4O创面面积分别较图4B、4C、4D、4E和图4G、4H、4I、4J明显缩小
5 3组糖尿病小鼠全层皮肤缺损创面伤后21 d病理变化及胶原分布。5A、5B、5C.分别为单纯糖尿病组、糖尿病+低浓度（25 μg/mL）小檗碱组、糖尿病+高浓度（75 μg/mL）小檗碱组小鼠创面组织病理情况，其中图5C中的创面上皮化形成情况最好，真皮层有少量毛囊和少量炎症细胞 苏木精-伊红×200；5D、5E、5F.分别为单纯糖尿病组、糖尿病+低浓度小檗碱组、糖尿病+高浓度小檗碱组小鼠创面组织胶原分布情况，其中图5F的创面中胶原分布最密集 Masson×200
表1 实时荧光定量反转录PCR检测的小鼠真皮成纤维细胞中多种细胞因子的引物序列及产物大小
表1. The primer sequences and product sizes of polycytokines in mouse dermal fibroblasts detected by real‑time fluorescence quantitative reverse transcription polymerase chain reaction
引物名称 引物序列（5'→3'） 产物大小（bp） β肌动蛋白 上游引物： CTGAGAGGGAAACGTGCGT 208 下游引物： CCACAGGATTCCATACCCAAGA PDGF 上游引物： CAGTGCTGGAAGTGGTTAACG 183 下游引物： CATCCTCTTCCACGATGACTAAG VEGF 上游引物： GCTACTGCCGTCCGATTGAG 268 下游引物： GGCTTTGTTCTGTCTTTCTTTGGT TGF-β 1 上游引物： AGAGCCCTGGATACCAACTATTG 286 下游引物： TGCGACCCACGTAGTAGACG MMP-9 上游引物： GTGTCTGGAGATTCGACTTGAAG 130 下游引物： CAGAAATAGGCTTTGTCTTGGTACT caspase-3 上游引物： GTGGGACTGATGAGGAGATGGC 137 下游引物： AAGGGACTGGATGAACCACGAC 注：PDGF为血小板源性生长因子，VEGF为血管内皮生长因子，TGF-β 1为转化生长因子β 1，MMP-9为基质金属蛋白酶9，caspase-3为胱天蛋白酶3 
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表2 含不同质量浓度小檗碱的培养基培养48 h后小鼠真皮成纤维细胞吸光度值比较（
表2. Comparison of absorbance value of mouse dermal fibroblasts cultured in medium with different concentrations of berberine for 48 h
小檗碱质量浓度（μg/mL） 样本数 吸光度值 t值 P值 0 6 0.72±0.05 — — 1.25 6 0.77±0.05 0.42 0.680 2.50 6 0.82±0.05 2.00 0.060 5.00 6 0.92±0.05 3.27 0.004 10.00 6 0.95±0.05 3.74 0.002 20.00 6 0.98±0.07 4.00 0.001 40.00 6 0.97±0.07 3.92 0.002 80.00 6 0.90±0.06 1.83 0.085 160.00 6 0.80±0.04 <0.01 1.000 注：“—”表示无此项；细胞吸光度值总体比较， F=28.74， P<0.001； t值、 P值为其他质量浓度与0 μg/mL比较所得
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表3 3组小鼠真皮成纤维细胞培养48 h后多种细胞因子mRNA和蛋白表达水平的比较（
表3. Comparison of mRNA and protein expression levels of polycytokines of mouse dermal fibroblasts in the three groups after 48 hours of culture
组别 样本数 mRNA 蛋白 PDGF VEGF TGF-β 1 MMP-9 caspase-3 PDGF VEGF TGF-β 1 MMP-9 caspase-3 正常对照组 3 1.063± 0.947± 1.090± 1.053± 0.94± 0.694± 0.769± 0.673± 0.743± 0.317± 0.058 0.041 0.067 0.039 0.04 0.040 0.040 0.052 0.105 0.026 单纯高糖组 3 0.203± 0.260± 0.163± 0.287± 2.29± 0.210± 0.060± 0.150± 0.057± 0.614± 0.021 a 0.014 a 0.005 a 0.024 a 0.22 a 0.029 a 0.010 a 0.047 a 0.008 a 0.024 a 高糖+小檗碱组 3 0.477± 0.533± 0.400± 0.593± 1.75± 0.350± 0.238± 0.300± 0.224± 0.399± 0.031 ab 0.025 ab 0.043 ab 0.039 ab 0.08 ab 0.043 ab 0.035 ab 0.016 ab 0.015 ab 0.022 ab F值 328.67 157.33 242.67 178.00 3 562.33 29.33 212.00 16.00 84.33 72.33 P值 <0.001 <0.001 <0.001 <0.001 <0.001 0.002 <0.001 0.006 <0.001 <0.001 注：高糖指葡萄糖物质的量浓度为30 mmol/L，小檗碱质量浓度为20.00 μg/mL；PDGF为血小板源性生长因子，VEGF为血管内皮生长因子，TGF-β 1为转化生长因子β 1，MMP-9为基质金属蛋白酶9，caspase-3为胱天蛋白酶3；与正常对照组比较， a P<0.05；与单纯高糖组比较， b P<0.05
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表4 3组糖尿病小鼠全层皮肤缺损创面伤后各时间点创面面积比较（cm 2，
表4. Comparison of wound area of full-thickness skin defect wounds in diabetic mice in the three groups at each time point after injury
组别 样本数 0 d（即刻） 3 d 7 d 14 d 21 d 单纯糖尿病组 5 0.621±0.041 0.521±0.035 0.431±0.015 0.342±0.020 0.112±0.008 糖尿病+低浓度小檗碱组 5 0.623±0.018 0.516±0.013 0.412±0.016 0.238±0.018 a 0.088±0.007 a 糖尿病+高浓度小檗碱组 5 0.626±0.018 0.425±0.019 ab 0.279±0.019 ab 0.155±0.017 ab 0.022±0.004 ab F值 0.05 15.08 61.55 56.62 92.77 P值 0.950 <0.001 <0.001 <0.001 <0.001 注：小檗碱低质量浓度、高质量浓度分别为25 μg/mL、75 μg/mL；处理因素主效应， F=231.60， P<0.001；时间因素主效应， F=879.40， P<0.001；两者交互作用， F=115.50， P<0.001；与单纯糖尿病组比较， a P<0.05；与糖尿病+低浓度小檗碱组比较， b P<0.05
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表5 伤后21 d 3组糖尿病小鼠全层皮肤缺损创面组织中各细胞因子的蛋白表达和mRNA水平比较（
表5. Comparison of protein expression and mRNA levels of various cytokines in the wound tissue of full-thickness skin defects of mice in the three groups on post injury day 21
组别 样本数 蛋白表达水平 基质金属蛋白酶9 血小板源性生长因子 转化生长因子β 1 血管内皮生长因子 CD31 胱天蛋白酶3 单纯糖尿病组 5 419±44 470±76 201±9 827±71 293±40 5 347±686 糖尿病+低浓度小檗碱组 5 1 872±242 a 1 116±158 a 1 616±87 a 1 811±254 a 1 498±215 a 3 266±461 a 糖尿病+高浓度小檗碱组 5 3 089±342 ab 2 184±286 ab 2 856±447 ab 3 311±495 ab 3 236±446 ab 2 196±290 ab F值 49.93 41.00 60.38 145.50 108.72 60.39 P值 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 注：小檗碱低质量浓度、高质量浓度分别为25 μg/mL、75 μg/mL；与单纯糖尿病组比较， a P<0.05；与糖尿病+低浓度小檗碱组比较， b P<0.05
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