[Effect of salvia miltiorrhiza combined with roxadustat on wound healing of full-thickness skin defects in diabetic rats and its mechanism].

Objective: To explore the effect of salvia miltiorrhiza combined with roxadustat on wound healing of full-thickness skin defects in diabetic rats and its mechanism. Methods: This study was an experimental study. Twenty male 8-week-old Sprague-Dawley rats were used to successfully establish diabetic model, then full-thickness skin defect wounds on their backs were made. The rats were divided into normal saline group, roxadustat alone group, salvia miltiorrhiza alone group, and roxadustat+salvia miltiorrhiza group according to the random number table, with 5 rats in each group. Immediately after injury, the rats in normal saline group were given 5 mL normal saline by gavage, the rats in roxadustat alone group were given 1.5 mg/mL roxadustat suspension by gavage at 25 mg/kg, the rats in salvia miltiorrhiza alone group were given 18 mg/mL salvia miltiorrhiza suspension by gavage at 300 mg/kg, and the rats in roxadustat+salvia miltiorrhiza group were given 19.5 mg/mL roxadustat and salvia miltiorrhiza suspension at roxadustat 25 mg/kg and salvia miltiorrhiza 300 mg/kg. All were administered once a day for 2 weeks. The wounds at 0 (immediately), 4, 8, and 12 d after injury were observed, and the wound healing rates at 4, 8, and 12 d after injury were calculated (n=5). At 14 d after injury, abdominal aortic blood was collected, and hemoglobin, red cell count, and white blood cell count were detected (n=5). The wound tissue was collected for hematoxylin-eosin staining to observe inflammatory infiltration, skin tissue structure, and neovascularization, for Masson staining to observe the proportion of collagen fiber (n=3), for Western blotting to detect the protein expression levels of vascular endothelial growth factor (VEGF), CD31, interleukin 6 (IL-6), tumor necrosis factor α (TNF-α), and IL-1β (n=3), and for immunohistochemical staining to determine the protein expression levels of epidermal growth factor receptor (EGFR), hypoxia-inducible factor 1α (HIF-1α), and proliferating cell nuclear antigen (PCNA), with sample number of 3. Results: From 0 to 12 d after injury, the wound areas of rats in 4 groups were gradually decreased. At 4 d after injury, the wound healing rates of rats in salvia miltiorrhiza alone group and roxadustat+salvia miltiorrhiza group were significantly higher than those in normal saline group and roxadustat alone group (P<0.05). At 8 d after injury, the wound healing rates of rats in roxadustat alone group and salvia miltiorrhiza alone group were significantly higher than the rate in normal saline group (P<0.05), and the wound healing rate of rats in roxadustat+salvia miltiorrhiza group was significantly higher than the rates in the other 3 groups (with P values all <0.05). At 12 d after injury, the wound healing rates of rats in roxadustat alone group, salvia miltiorrhiza alone group, and roxadustat+salvia miltiorrhiza group were significantly higher than the rate in normal saline group (P<0.05). At 14 d after injury, there were no statistically significant differences in the hemoglobin or red blood cell count of rats in 4 groups (P<0.05). The white blood cell count of rats in roxadustat alone group, salvia miltiorrhiza alone group, and roxadustat+salvia miltiorrhiza group were respectively (24.3±1.2)×109/L, (26.3±2.4)×109/L, and (15.0±0.7)×109/L, which were significantly lower than (33.8±2.7)×109/L in normal saline group (P<0.05); the white blood cell count of rats in roxadustat+salvia miltiorrhiza group was significantly lower than that in roxadustat alone group and salvia miltiorrhiza alone group (with P values both <0.05). At 14 d after injury, a large number of inflammatory cell infiltration, disordered skin tissue structure, and few new blood vessels were observed in the wounds of rats in normal saline group; while a small amount of inflammatory cell infiltration, tight skin tissue structure, and rich neovascularization were observed in the wounds of rats in the other 3 groups. There were no statistically significant differences in the proportion of collagen fiber of wounds in rats among the 4 groups (P>0.05). At 14 d after injury, the protein expression levels of VEGF and CD31 in the wound tissue of rats in roxadustat alone group, salvia miltiorrhiza alone group, and roxadustat+salvia miltiorrhiza group were significantly higher than those in normal saline group (P<0.05), the protein expression level of CD31 in the wound tissue of rats in roxadustat+salvia miltiorrhiza group was significantly higher than the levels in roxadustat alone group and salvia miltiorrhiza alone group (with P values both <0.05). At 14 d after injury, the protein expression levels of IL-6, TNF-α, and IL-1β in the wound tissue of rats in roxadustat alone group, salvia miltiorrhiza alone group, and roxadustat+salvia miltiorrhiza group were significantly lower than those in normal saline group (P<0.05); the protein expression levels of IL-6 and IL-1β in the wound tissue of rats in roxadustat+salvia miltiorrhiza group were significantly lower than those in roxadustat alone group and salvia miltiorrhiza alone group (P<0.05); the protein expression level of TNF-α in the wound tissue of rats in roxadustat+salvia miltiorrhiza group was significantly lower than that in salvia miltiorrhiza alone group (P<0.05). At 14 d after injury, the protein expression level of EGFR in the wound tissue of rats in roxadustat+salvia miltiorrhiza group was significantly higher than the levels in the other 3 groups (with P values all <0.05); the protein expression levels of HIF-1α in the wound tissue of rats in roxadustat alone group and roxadustat+salvia miltiorrhiza group were significantly higher than the level in normal saline group (P<0.05), and the protein expression level of HIF-1α in the wound tissue of rats in roxadustat+salvia miltiorrhiza group was significantly higher than that in salvia miltiorrhiza alone group (P<0.05); there were no statistically significant differences in the protein expression level of PCNA in the wound tissue of rats in 4 groups (P>0.05). Conclusions: Roxadustat combined with salvia miltiorrhiza can promote the wound healing of full-thickness skin defects in diabetic rats by promoting blood vessel regeneration and reducing inflammatory response.目的： 探究丹参联合罗沙司他对糖尿病大鼠全层皮肤缺损创面愈合的影响及其机制。 方法： 该研究为实验研究。将20只8周龄雄性SD大鼠成功制成糖尿病模型后，在其背部制作全层皮肤缺损创面，之后将大鼠按照随机数字表法分为生理盐水组、单纯罗沙司他组、单纯丹参组、罗沙司他+丹参组，每组5只。伤后即刻，对生理盐水组大鼠予5 mL生理盐水灌胃，对单纯罗沙司他组大鼠予1.5 mg/mL的罗沙司他混悬液按照25 mg/kg灌胃，对单纯丹参组大鼠予18 mg/mL的丹参混悬液按照300 mg/kg灌胃，对罗沙司他+丹参组的大鼠予19.5 mg/mL的罗沙司他和丹参混悬液按照罗沙司他25 mg/kg、丹参300 mg/kg灌胃，均持续给药2周，1次/d。观察伤后0（即刻）、4、8、12 d创面情况，计算伤后4、8、12 d的创面愈合率（样本数为5）。伤后14 d，取腹主动脉血行血红蛋白、红细胞计数、白细胞计数检测（样本数为5）；取创面组织，行苏木精-伊红染色后观察炎性浸润、皮肤组织结构及新生血管生成情况，行Masson染色后观测胶原纤维占比（样本数为3），行蛋白质印迹法检测血管内皮生长因子（VEGF）、CD31、白细胞介素6（IL-6）、肿瘤坏死因子α（TNF-α）、IL-1β的蛋白表达水平（样本数为3），行免疫组织化学染色检测表皮生长因子受体（EGFR）、缺氧诱导因子1α（HIF-1α）、增殖细胞核抗原（PCNA）的蛋白表达水平（样本数为3）。 结果： 伤后0~12 d，4组大鼠创面面积均逐渐缩小。伤后4 d，单纯丹参组、罗沙司他+丹参组大鼠创面愈合率均明显高于生理盐水组和单纯罗沙司他组（P<0.05）；伤后8 d，单纯罗沙司他组和单纯丹参组大鼠创面愈合率均明显高于生理盐水组（P<0.05），罗沙司他+丹参组大鼠创面愈合率明显高于其余3组（P值均<0.05）；伤后12 d，单纯罗沙司他组、单纯丹参组、罗沙司他+丹参组大鼠创面愈合率均明显高于生理盐水组（P<0.05）。伤后14 d，4组大鼠的血红蛋白和红细胞计数比较，差异均无统计学意义（P>0.05）；单纯罗沙司他组、单纯丹参组、罗沙司他+丹参组大鼠白细胞计数分别为（24.3±1.2）×109/L、（26.3±2.4）×109/L、（15.0±0.7）×109/L，均明显低于生理盐水组的（33.8±2.7）×109/L（P<0.05）；罗沙司他+丹参组大鼠白细胞计数明显低于单纯罗沙司他组和单纯丹参组（P值均<0.05）。伤后14 d，生理盐水组大鼠创面可见大量炎症细胞浸润，皮肤组织结构紊乱，新生血管少；其余3组大鼠创面可见少量炎症细胞浸润，皮肤组织结构紧密，新生血管丰富。4组大鼠创面的胶原纤维占比组间总体比较，差异无统计学意义（P>0.05）。伤后14 d，单纯罗沙司他组、单纯丹参组、罗沙司他+丹参组大鼠创面组织中VEGF、CD31的蛋白表达水平均明显高于生理盐水组（P<0.05），罗沙司他+丹参组大鼠创面组织中CD31的蛋白表达水平明显高于单纯罗沙司他组和单纯丹参组（P值均<0.05）。伤后14 d，单纯罗沙司他组、单纯丹参组、罗沙司他+丹参组大鼠创面组织中IL-6、TNF-α、IL-1β的蛋白表达水平均明显低于生理盐水组（P<0.05），罗沙司他+丹参组大鼠创面组织中IL-6、IL-1β的蛋白表达水平均明显低于单纯罗沙司他组和单纯丹参组（P<0.05），罗沙司他+丹参组大鼠创面组织中TNF-α的蛋白表达水平明显低于单纯丹参组（P<0.05）。伤后14 d，罗沙司他+丹参组大鼠创面组织中EGFR的蛋白表达水平明显高于其余3组（P值均<0.05）；单纯罗沙司他组和罗沙司他+丹参组大鼠创面组织中HIF-1α的蛋白表达水平均明显高于生理盐水组（P<0.05），罗沙司他+丹参组大鼠创面组织中HIF-1α的蛋白表达水平明显高于单纯丹参组（P<0.05）；4组大鼠创面组织中PCNA的蛋白表达水平比较，差异无统计学意义（P>0.05）。 结论： 罗沙司他联合丹参可以通过促进血管再生、降低炎症反应，进而促进糖尿病大鼠全层皮肤缺损创面愈合。.