Yimin Y M, Huang X, Meng X C, Gu S C, Zhang Z W, Liu Y H, Luo S Y, Zan T
Department of Plastic and Reconstructive Surgery, Shanghai Ninth People's Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200011, China.
Department of Plastic and Cosmetic Surgery, the First Affiliated Hospital of Guangxi Medical University, Nanning 530021, China.
Zhonghua Shao Shang Yu Chuang Mian Xiu Fu Za Zhi. 2022 May 20;38(5):434-446. doi: 10.3760/cma.j.cn501120-20210419-00138.
To analyze the effects of transient receptor potential vanilloid type 4 (TRPV4) activation on the function and endothelial-to-mesenchymal transition (EndMT) of human umbilical vein endothelial cells (HUVECs), as well as to explore the effects of TRPV4 activation on blood perfusion and survival of rat perforator flap and the mechanism. The experimental research methods were used. The 3 to 6 passages of HUVECs were used for experiments and divided into 0.5 μmol/L 4α-phorbol 12, 13-didecanoate (4αPDD) group, 1.0 μmol/L 4αPDD group, 3.0 μmol/L 4αPDD group, 10.0 μmol/L 4αPDD group, and phosphate buffer solution (PBS) group, which were cultivated in corresponding final molarity of 4αPDD and PBS, respectively. The cell proliferation activity at 6 and 12 h of culture was detected using cell counting kit-8 (CCK-8). Another batch of cells was acquired and divided into PBS group, 1 μmol/L 4αPDD group, and 3 μmol/L 4αPDD group, which were treated similarly as described before and then detected for cell proliferation activity at 6, 12, 24, and 48 h of culture. The residual scratch area of cells at post scratch hour (PSH) 12, 24, and 48 was detected by scratch test, and the percentage of the residual scratch area was calculated. The number of migrated cells at 24 and 48 h of culture was detected by Transwell experiment. The tube-formation assay was used to measure the number of tubular structures at 4 and 8 h of culture. The protein expressions of E-cadherin, N-cadherin, Slug, and Snail at 24 h of culture were detected by Western blotting. All the sample numbers in each group at each time point in vitro experiments were 3. A total of 36 male Sprague-Dawley rats aged 8 to 10 weeks were divided into delayed flap group, 4αPDD group, and normal saline group according to the random number table, with 12 rats in each group, and iliolumbar artery perforator flap models on the back were constructed. The flap surgical delay procedure was only performed in the rats in delayed flap group one week before the flap transfer surgery. Neither rats in 4αPDD group nor normal saline group had flap surgical delay; instead, they were intraperitoneally injected with 4αPDD and an equivalent mass of normal saline, respectively, at 10 min before, 24 h after, and 48 h after the surgery. The general state of flap was observed on post surgery day (PSD) 0 (immediately), 1, 4, and 7. The flap survival rates were assessed on PSD 7. The flap blood perfusion was detected by laser speckle contrast imaging technique on PSD 1, 4, and 7. The microvascular density in the flap's choke vessel zone was detected by immunohistochemical staining. All the sample numbers in each group at each time point in vivo experiments were 12. Data were statistically analyzed with analysis of variance for factorial design, analysis of variance for repeated measurement, one-way analysis of variance, least significant difference test, and Bonferroni correction. At 6 and 12 h of culture, there were no statistically significant differences in cell proliferation activity in the overall comparison among PBS group, 0.5 μmol/L 4αPDD group, 1.0 μmol/L 4αPDD group, 3.0 μmol/L 4αPDD group, and 10.0 μmol/L 4αPDD group (>0.05). At 6, 12, 24, and 48 h of culture, there were no statistically significant differences in cell proliferation activity in the overall comparison among PBS group, 1 μmol/L 4αPDD group, and 3 μmol/L 4αPDD group (>0.05). At PSH 12, the percentages of the residual scratch area of cells in 1 μmol/L 4αPDD group and 3 μmol/L 4αPDD group were close to that in PBS group (>0.05). At PSH 24 and 48, compared with those in PBS group, the percentages of the residual scratch area of cells in 3 μmol/L 4αPDD group were significantly decreased (with values of 2.83 and 2.79, respectively, <0.05), while the percentages of the residual scratch area of cells in 1 μmol/L 4αPDD group showed no significant differences (>0.05). At 24 h of culture, the number of migrated cells in 1 μmol/L 4αPDD group and 3 μmol/L 4αPDD group were close to that in PBS group (>0.05). At 48 h of culture, the number of migrated cells in 1 μmol/L 4αPDD group and 3 μmol/L 4αPDD groups were significantly greater than that in PBS group (with values of 6.20 and 9.59, respectively, <0.01). At 4 h of culture, the numbers of tubular structures of cells in 1 μmol/L 4αPDD group and 3 μmol/L 4αPDD group were significantly greater than that in PBS group (with values of 4.68 and 4.95, respectively, <0.05 or <0.01). At 8 h of culture, the numbers of tubular structures of cells in 1 μmol/L 4αPDD and 3 μmol/L 4αPDD groups were similar to that in PBS group (>0.05). At 24 h of culture, compared with those in PBS group, the protein expression level of E-cadherin of cells in 3 μmol/L 4αPDD group was significantly decreased (=5.13, <0.01), whereas there was no statistically significant difference in the protein expression level of E-cadherin of cells in 1 μmol/L 4αPDD group (>0.05); the protein expression level of N-cadherin of cells in 3 μmol/L 4αPDD group was significantly increased (=4.93, <0.01), whereas there was no statistically significant difference in the protein expression level of N-cadherin of cells in 1 μmol/L 4αPDD group (>0.05); the protein expression levels of Slug of cells in 1 μmol/L 4αPDD group and 3 μmol/L 4αPDD group were significantly increased (with values of 3.85 and 6.52, respectively, <0.05 or <0.01); and the protein expression level of Snail of cells in 3 μmol/L 4αPDD group was significantly increased (=4.08, <0.05), whereas there was no statistically significant difference in the protein expression level of Snail of cells in 1 μmol/L 4αPDD group (>0.05). There were no statistically significant differences in the protein expression levels of E-cadherin, N-cadherin, Slug, or Snail of cells between 1 μmol/L 4αPDD group and 3 μmol/L 4αPDD group (>0.05). The general condition of flaps of rats in the three groups was good on PSD 0. On PSD 1, the flaps of rats in the three groups were basically similar, with bruising and swelling at the distal end. On PSD 4, the swelling of flaps of rats in the three groups subsided, and the distal end turned dark brown and necrosis occurred, with the area of necrosis in flaps of rats in normal saline group being larger than the areas in 4αPDD group and delayed flap group. On PSD 7, the necrotic areas of flaps of rats in the 3 groups were fairly stable, with the area of necrosis at the distal end of flap of rats in delayed flap group being the smallest. On PSD 7, the flap survival rates of rats in 4αPDD group ((80±13)%) and delayed flap group ((87±9)%) were similar (>0.05), and both were significantly higher than (70±11)% in normal saline group (with values of 2.24 and 3.65, respectively, <0.05 or <0.01). On PSD 1, the overall blood perfusion signals of rats in the 3 groups were basically the same, and the blood perfusion signals in the choke vessel zone were relatively strong, with a certain degree of underperfusion at the distal end. On PSD 4, the boundary between the surviving and necrotic areas of flaps of rats in the 3 groups became evident, and the blood perfusion signals in the choke vessel zone were improved, with the normal saline group's distal hypoperfused area of flap being larger than the areas in delayed flap group and 4αPDD group. On PSD 7, the blood perfusion signals of overall flap of rats had generally stabilized in the 3 groups, with the intensity of blood perfusion signal in the choke vessel zone and overall flap of rats in delayed flap group and 4αPDD group being significantly greater than that in normal saline group. On PSD 7, the microvascular density in the choke vessel zone of flap of rats in 4αPDD group and delayed flap group were similar (>0.05), and both were significantly higher than that in normal saline group (with values of 4.11 and 5.38, respectively, <0.01). After activation, TRPV4 may promote the migration and tubular formation of human vascular endothelial cells via the EndMT pathway, leading to the enhanced blood perfusion of perforator flap and microvascular density in the choke vessel zone, and therefore increase the flap survival rate.
分析瞬时受体电位香草酸亚型4（TRPV4）激活对人脐静脉内皮细胞（HUVECs）功能及内皮-间质转化（EndMT）的影响，探讨TRPV4激活对大鼠穿支皮瓣血运及存活的影响及其机制。采用实验研究方法。取3至6代HUVECs进行实验，分为0.5 μmol/L 4α-佛波醇12,13-十四酸酯（4αPDD）组、1.0 μmol/L 4αPDD组、3.0 μmol/L 4αPDD组、10.0 μmol/L 4αPDD组和磷酸盐缓冲液（PBS）组，分别用相应终浓度的4αPDD和PBS培养。采用细胞计数试剂盒-8（CCK-8）检测培养6 h和12 h时的细胞增殖活性。另取一批细胞，分为PBS组、1 μmol/L 4αPDD组和3 μmol/L 4αPDD组，按上述方法处理后，检测培养6、12、24和48 h时的细胞增殖活性。采用划痕试验检测划痕后12、24和48 h细胞的残余划痕面积，计算残余划痕面积百分比。采用Transwell实验检测培养24 h和48 h时的迁移细胞数。采用管腔形成实验检测培养4 h和8 h时的管状结构数量。采用蛋白质印迹法检测培养24 h时E-钙黏蛋白、N-钙黏蛋白、Slug和Snail的蛋白表达。体外实验中每组各时间点的样本数均为3。选取36只8至10周龄的雄性Sprague-Dawley大鼠，按随机数字表法分为延迟皮瓣组、4αPDD组和生理盐水组，每组12只，建立背部髂腰动脉穿支皮瓣模型。仅在延迟皮瓣组大鼠皮瓣转移手术前1周进行皮瓣延迟手术。4αPDD组和生理盐水组大鼠均未进行皮瓣延迟手术，而是分别在术后10 min、24 h和48 h腹腔注射4αPDD和等量生理盐水。于术后第0天（即刻）、1、4和7天观察皮瓣一般情况。于术后第7天评估皮瓣存活率。采用激光散斑对比成像技术检测术后第1、4和7天皮瓣血运情况。采用免疫组织化学染色检测皮瓣缺血区微血管密度。体内实验中每组各时间点的样本数均为12。数据采用析因设计方差分析、重复测量方差分析、单因素方差分析、最小显著差法检验和Bonferroni校正进行统计学分析。培养6 h和12 h时，PBS组、0.5 μmol/L 4αPDD组、1.0 μmol/L 4αPDD组、3.0 μmol/L 4αPDD组和10.0 μmol/L 4αPDD组细胞增殖活性总体比较差异无统计学意义（>0.05）。培养6、12、24和48 h时，PBS组、1 μmol/L 4αPDD组和3 μmol/L 4αPDD组细胞增殖活性总体比较差异无统计学意义（>0.05）。划痕后12 h，1 μmol/L 4αPDD组和3 μmol/L 4αPDD组细胞残余划痕面积百分比与PBS组接近（>0.05）。划痕后24 h和48 h，与PBS组相比，3 μmol/L 4αPDD组细胞残余划痕面积百分比显著降低（分别为2.83和2.79，<0.05），而1 μmol/L 4αPDD组细胞残余划痕面积百分比差异无统计学意义（>0.05）。培养24 h时，1 μmol/L 4αPDD组和3 μmol/L 4αPDD组迁移细胞数与PBS组接近（>0.05）。培养48 h时，1 μmol/L 4αPDD组和3 μmol/L 4αPDD组迁移细胞数显著多于PBS组（分别为6.20和9.59，<0.01）。培养4 h时，1 μmol/L 4αPDD组和3 μmol/L 4αPDD组细胞管状结构数量显著多于PBS组（分别为4.68和4.95，<0.05或<0.01）。培养8 h时，1 μmol/L 4αPDD组和3 μmol/L 4αPDD组细胞管状结构数量与PBS组相似（>0.05）。培养24 h时，与PBS组相比，3 μmol/L 4αPDD组细胞E-钙黏蛋白表达水平显著降低（F = 5.13，<0.01），而1 μmol/L 4αPDD组细胞E-钙黏蛋白表达水平差异无统计学意义（>0.05）；3 μmol/L 4αPDD组细胞N-钙黏蛋白表达水平显著升高（F = 4.93，<0.01），而1 μmol/L 4αPDD组细胞N-钙黏蛋白表达水平差异无统计学意义（>0.05）；1 μmol/L 4αPDD组和3 μmol/L 4αPDD组细胞Slug表达水平显著升高（分别为3.85和6.52，<0.05或<0.01）；3 μmol/L 4αPDD组细胞Snail表达水平显著升高（F = 4.08，<0.05），而1 μmol/L 4αPDD组细胞Snail表达水平差异无统计学意义（>0.05）。1 μmol/L 4αPDD组和3 μmol/L 4αPDD组细胞E-钙黏蛋白、N-钙黏蛋白、Slug或Snail蛋白表达水平差异无统计学意义（>0.05）。术后第0天，三组大鼠皮瓣一般情况良好。术后第1天，三组大鼠皮瓣基本相似，远端出现瘀斑和肿胀。术后第4天，三组大鼠皮瓣肿胀消退，远端变为深褐色并出现坏死，生理盐水组大鼠皮瓣坏死面积大于4αPDD组和延迟皮瓣组。术后第7天，三组大鼠皮瓣坏死面积基本稳定，延迟皮瓣组大鼠皮瓣远端坏死面积最小。术后第7天，4αPDD组（（80±13）%）和延迟皮瓣组（（87±9）%）大鼠皮瓣存活率相似（>0.05），且均显著高于生理盐水组（（70±11）%）（分别为2.24和3.65，<0.05或<0.01）。术后第1天，三组大鼠皮瓣总体血运信号基本相同，缺血区血运信号较强，远端有一定程度的灌注不足。术后第4天，三组大鼠皮瓣存活区与坏死区边界清晰，缺血区血运信号改善，生理盐水组大鼠皮瓣远端灌注不足面积大于延迟皮瓣组和4αPDD组。术后第7天，三组大鼠皮瓣总体血运信号基本稳定，延迟皮瓣组和4αPDD组大鼠皮瓣缺血区及总体血运信号强度显著高于生理盐水组。术后第7天，4αPDD组和延迟皮瓣组大鼠皮瓣缺血区微血管密度相似（>0.05），且均显著高于生理盐水组（分别为4.11和5.38，<0.01）。TRPV4激活后可能通过EndMT途径促进人血管内皮细胞迁移和管腔形成，从而增强穿支皮瓣血运及缺血区微血管密度，提高皮瓣存活率。
