活血通络胶囊对激素性股骨头坏死囊性变成血管修复的影响

Effects of Huoxue Tongluo Jiaonang(活血通络胶囊)on angiogenesis repair of cystic lesions of steroid-induced osteonecrosis of the femoral head

目的:探讨活血通络胶囊对激素性股骨头坏死(steroid-induced osteonecrosis of the femoral head,SONFH)囊性变成血管修复的影响。方法:(1)将从全髋关节置换术中收集的一部分SONFH囊性变组织制备成单细胞悬液,采用单细胞转录组学分析囊性变组织中的细胞组分。将另一部分SONFH囊性变组织制成病理切片,采用苏木素-伊红染色法观察囊性变中血管形态,并采用免疫荧光法观察囊性变中血管内皮细胞特异性蛋白的表达情况。(2)分别按照每天20 mg·kg^(-1)、40 mg·kg^(-1)、80 mg·kg^(-1)体质量以浓度1 mg·mL^(-1)、2 mg·mL^(-1)、4 mg·mL^(-1)的活血通络胶囊溶液和20 mL·kg^(-1)生理盐水给大鼠灌胃,获取低、中、高剂量活血通络胶囊含药血清及空白血清。(3)取生长良好的人脐静脉内皮细胞(human umbilical vein endothelial cells,HUVECs),分为空白血清组和低、中、高剂量含药血清组,空白血清组细胞常规培养,低、中、高剂量含药血清组细胞分别采用低、中、高剂量活血通络胶囊含药血清干预。干预48 h后,通过细胞计数试剂盒-8法测定活血通络胶囊含药血清对HUVECs增殖能力的影响。(4)将HUVECs分为对照组、地塞米松组和活血通络组,对照组细胞常规培养,地塞米松组细胞采用地塞米松干预,活血通络组细胞采用地塞米松和中剂量活血通络胶囊含药血清干预。干预至细胞长满后进行细胞划痕实验,观察活血通络胶囊含药血清对HUVECs迁移能力的影响。(5)HUVECs分组与干预方法同(4),干预7 d后进行成血管实验,观察活血通络胶囊含药血清对HUVECs成血管能力的影响。结果:(1)SONFH囊性变组织中细胞组分分析结果。SONFH囊性变组织中存在内皮细胞及具有血管生成功能的内皮细胞亚群。(2)SONFH囊性变组织病理学观察结果。苏木素-伊红染色结果显示,SONFH囊性变组织中存在椭圆形的血管腔,血管内膜稍增厚、弹力纤维增生分层。免疫荧光染色结果显示,SONFH囊性变组织内存在血管内皮细胞特异性蛋白(CD31蛋白和血管内皮生长因子蛋白),且可见明显的管形。(3)HUVECs相对增殖率测定结果。各组HUVECs相对增殖率比较,差异有统计学意义[0%,(0.15±0.83)%,(18.70±6.10)%,(24.9±15.10)%,F=7.541,P=0.001];低剂量含药血清组HUVECs相对增殖率与空白血清组的差异无统计学意义(P=0.999),中剂量含药血清组和高剂量含药血清组HUVECs相对增殖率均高于空白血清组(P=0.046,P=0.006),中剂量含药血清组HUVECs相对增殖率与高剂量含药血清组的差异无统计学意义(P=0.782)。(4)HUVECs迁移率测算结果。地塞米松组HUVECs迁移率(10%)明显低于对照组(26%),活血通络组HUVECs迁移率(24%)明显高于地塞米松组(10%)。(5)HUVECs形成的管形分支数测算结果。每个放大40倍视野下,各组HUVECs形成的管形分支数比较,差异有统计学意义[(112.20±7.96)个,(83.40±14.02)个,(122.40±14.59)个,F=2.980,P=0.001];地塞米松组HUVECs形成的管形分支数少于对照组和活血通络组(P=0.006,P=0.001),对照组HUVECs形成的管形分支数与活血通络组的差异无统计学意义(P=0.292)。结论:活血通络胶囊可通过提高血管内皮细胞的增殖、迁移和成血管能力,促进SONFH囊性变的成血管修复。

Objective:To explore the effects of Huoxue Tongluo Jiaonang(活血通络胶囊,HXTLJN)on angiogenesis repair of cystic lesions of steroid-induced osteonecrosis of femoral head(SONFH).Methods:①The tissues collected from the site of cystic lesions during the total hip arthroplasty in patients with SONFH were acquired,one part was made into single-cell suspension to analyze the cellular components in cystic lesions tissues based on the single cell transcriptomics,while,the other part was made into pathological sections to observe the vascular morphology and detect the expression of vascular endothelial cells(VECs)-specific proteins in cystic lesions tissues by using hematoxylin-eosin(HE)staining and immunofluorescence method,respectively.②Twelve rats were selected and ran domized into 4 groups.The rats in any three groups were intragastric administrated with HXTLJN solution at concentration of 1,2 and 4 mg/mL in daily dosage of 20,40 and 80 mg/kg,respectively,while,the ones in the last group with normal saline in daily dosage of 20 mL/kg,for making low-,medium-,and high-dose HXTLJN medicated serum and blank serum,respectively.③The well-growned human umbilical vein endothelial cells(HUVECs)were fetched and divided into blank serum group,and low-,medium-,and high-dose medicated serum groups.The HUVECs in blank serum group were cultured in the conventional Dulbecco’s Modified Eagle’s Medium(DMEM),while the ones in low-,medium-,and high-dose medicated serum groups were intervened with low-,medium-,and high-dose HXTLJN medicated serum,respectively.After 48-hour intervention,the effect of HXTLJN medicated serum on the proliferation ability of HUVECs was determined by using the cell counting kit-8(CCK8)assay.④The HUVECs were further divided into control group,dexamethasone(DEX)group and Huoxue Tongluo(活血通络,HXTL)group.The HUVECs in control group were cultured in the conventional DMEM,while the ones in DEX group were intervened with DEX,and the ones in HXTL group with DEX and medium-dose HXTLJN medicated serum.After the cells reached 100%confluence,the effect of HXTLJN medicated serum on the migration ability of HUVECs was observed by employing a cell scratch assay.⑤The grouping and intervention methods for HUVECs were the same as those in item④.After 7-day intervention,the effect of HXTLJN medicated serum on the angiogenesis ability of HUVECs was observed by a tube formation assay.Results:①The results of analysis on the cellular components in the SONFH-associated cystic lesions tissues.The endothelial cells and endothelial cell subsets with angiogenesis function existed in the SONFH-associated cystic lesions tissues.②The results of observation on histopathological changes in SONFH-associated cystic lesions tissues.The results of HE staining showed that the oval-shaped vascular lumens,with slightly thickened vascular intima and hyperplastic,layered elastic fibers,were presented in the SONFH-associated cystic lesions tissues.The results of immunofluorescence staining revealed that the VEC-specific proteins(CD31 and vascular endothelial growth factor)existed in the SONFH-associated cystic lesions tissues,and the obvious tubular structures were observed.③The results of detection on the relative proliferation rate of HUVECs.The difference was statistically significant among the groups in the relative proliferation rate of HUVECs(0%,0.15±0.83,18.70±6.10,24.9±15.10%,F=7.541,P=0.001).The relative proliferation rate of HUVECs were higher in medium-and high-dose medicated serum groups compared to blank serum group(P=0.046,P=0.006),while,the comparisons between low-dose medicated serum group and blank serum group,as well as between medium-dose medicated serum group and high-dose medicated serum group revealed no significant differences(P=0.999;P=0.782).④The results of calculation on migration rate of HUVECs.The migration rate of HUVECs was significantly lower in DEX group(10%)compared to control group(26%),while,it was obviously higher in HXTL group(24%)compared to DEX group(10%).⑤The results of calculation on the number of tube-like branches formed by HUVECs.Under the 40-fold magnification fields,the difference was statistically significant among the groups in the number of tube-like branches formed by HUVECs(112.20±7.96,83.40±14.02,122.40±14.59 branches,F=2.980,P=0.001).The tube-like branches formed by HUVECs were less in DEX group compared to control group and HXTL group(P=0.006,P=0.001),while,the comparison between control group and HXTL group revealed no significant difference(P=0.292).Conclusion:The HXTLJN can promote the angiogenesis repair in SONFH-associated cystic lesions by enhancing the proliferation,migration,and angiogenesis capabilities of VECs.