[1]杨晓强,肖欢,田佳庆,等.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[J].中医正骨,2024,36(09):40-48.
　YANG Xiaoqiang,XIAO Huan,TIAN Jiaqing,et al.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[J].The Journal of Traditional Chinese Orthopedics and Traumatology,2024,36(09):40-48.
点击复制

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()
分享到：

《中医正骨》[ISSN:1001-6015/CN:41-1162/R]

卷:
第36卷
期数:
2024年09期
页码:
40-48
栏目:
技术研究
出版日期:
2024-09-20

文章信息/Info

Title:
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
作者:
杨晓强1; 肖欢2; 田佳庆1; 方伟华1; 彭鹏1; 肖方骏1; 何伟3; 何敏聪3; 魏秋实3
(1.广州中医药大学第三临床医学院,广东 广州 510006; 2.毕节市中医医院,贵州 毕节 551700; 3.广州中医药大学第三附属医院,广东 广州 510378)
Author(s):
YANG Xiaoqiang1; XIAO Huan2; TIAN Jiaqing1; FANG Weihua1; PENG Peng1; XIAO Fangjun1; HE Wei3; HE Mincong3; WEI Qiushi3
1.The Third Clinical Medical College of Guangzhou University of Chinese Medicine,Guangzhou 510006,Guangdong,China 2.Bijie Traditional Chinese Medicine Hospital,Bijie 551700,Guizhou,China 3.The Third Affiliated Hospital of Guangzhou University of Chinese Medicine,Guangzhou 510378,Guangdong,China
关键词:
股骨头坏死;  囊性变;  成血管细胞;  人脐静脉内皮细胞;  活血通络;  成血管修复
Keywords:
femur head necrosis;  cystic lesions;  hemangioblasts;  human umbilical vein endothelial cells;  activating blood dredging collaterals;  angiogenesis repair
摘要:
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。⑤HUVECs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:①SONFH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。②SONFH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。③HUVECs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)。④HUVECs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%)。⑤HUVECs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。
Abstract:
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 randomized 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 item ④.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.

参考文献/References:

[1] MOTTA F,TIMILSINA S,GERSHWIN M E,et al.Steroid-induced osteonecrosis[J].J Transl Autoimmun,2022,5:100168.
[2] 何晓铭,庞凤祥,李子祺,等.基于“态靶结合”理论探讨股骨头坏死囊性变的辨治思路[J].辽宁中医杂志,2022,49(2):44-47.
[3] 魏秋实,何伟,张庆文,等.围塌陷期股骨头坏死不同影像学表现研究[J].中国修复重建外科杂志,2021,35(9):1105-1110.
[4] GAO F,HAN J,HE Z,et al.Radiological analysis of cystic lesion in osteonecrosis of the femoral head[J].Int Orthop,2018,42(7):1615-1621.
[5] LIU G B,LI R,LU Q,et al.Three-dimensional distribution of cystic lesions in osteonecrosis of the femoral head[J].J Orthop Translat,2019,22:109-115.
[6] LAKHOTIA D,SWAMINATHAN S,SHON W Y,et al.Healing process of osteonecrotic lesions of the femoral head following transtrochanteric rotational osteotomy:a computed tomography-based study[J].Clin Orthop Surg,2017,9(1):29-36.
[7] TUCKERMANN J,ADAMS R H.The endothelium-bone axis in development,homeostasis and bone and joint disease[J].Nat Rev Rheumatol,2021,17(10):608-620.
[8] WEI Q S,HONG G J,YUAN Y J,et al.Huo Xue Tong Luo capsule,a vasoactive herbal formula prevents progression of asymptomatic osteonecrosis of femoral head:a prospective study[J].J Orthop Translat,2018,18:65-73.
[9] 沈莹姗,陈镇秋,何伟,等.基于“活血通络法”非手术保髋疗法在全身性多发骨坏死患者的运用[J].时珍国医国药,2020,31(6):1389-1392.
[10] HE X M,HE M C,YANG P,et al.The therapeutic effect of Huo Xue Tong Luo capsules in Association Research Circulation Osseous(ARCO)stage Ⅱ osteonecrosis of the femoral head:a clinical study with an average follow-up period of 7.95 years[J].Front Pharmacol,2021,12:773758.
[11] 魏伟,吴希美,李元健.药理实验方法学[M].4版.北京:人民卫生出版社,2010:71-72.
[12] CUI L,ZHUANG Q,LIN J,et al.Multicentric epidemiologic study on six thousand three hundred and ninety five cases of femoral head osteonecrosis in China[J].Int Orthop,2016,40(2):267-276.
[13] TAN B,LI W,ZENG P,et al.Epidemiological study based on china osteonecrosis of the femoral head database[J].Orthop Surg,2021,13(1):153-160.
[14] FUKUSHIMA W,FUJIOKA M,KUBO T,et al.Nationwide epidemiologic survey of idiopathic osteonecrosis of the femoral head[J].Clin Orthop Relat Res,2010,468(10):2715-2724.
[15] KUBO T,UESHIMA K,SAITO M,et al.Clinical and basic research on steroid-induced osteonecrosis of the femoral head in Japan[J].J Orthop Sci,2016,21(4):407-413.
[16] BIRLA V,VAISH A,VAISHYA R.Risk factors and pathogenesis of steroid-induced osteonecrosis of femoral head—a scoping review[J].J Clin Orthop Trauma,2021,23:101643.
[17] 张程,何海军.中医药防治股骨头坏死的优势与面临的挑战[J].中医正骨,2023,35(11):60-63.
[18] 沈莹姗,何晓铭,赵雄,等.关于“血瘀证是股骨头坏死主要证候”的调查研究[J].中医正骨,2022,34(4):25-29.
[19] 乌日莎娜,沈莹姗,袁颖嘉,等.基于“瘀去、新生、骨合”理论从细胞层面探讨祛瘀、活血及补肾中药治疗股骨头坏死的作用机制[J].中医正骨,2022,34(4):61-63.
[20] 郭雪峰,于睿,任艳玲.基于“成骨-成血管耦联”理论探讨中医药防治激素性股骨头坏死的科学内涵[J/OL].中华中医药学刊,2023[2024-01-13]. https://link.cnki.net/urlid/21.1546.R.20230915.1547.058.
[21] 何宪顺,韦雨柔,何敏聪,等.活血通络法对激素性股骨头坏死囊性变患者血清骨吸收/成血管/成骨蛋白调节作用的研究[J].中国全科医学,2024,27(12):1504-1510.
[22] 田佳庆,何敏聪,韦雨柔,等.激素性股骨头坏死囊性变分布规律及病理特点[J].中国组织工程研究,2023,27(31):4996-5001.
[23] CHANG C,GREENSPAN A,GERSHWIN M E.The pathogenesis,diagnosis and clinical manifestations of steroid-induced osteonecrosis[J].J Autoimmun,2020,110:102460.
[24] HUANG C,WEN Z,NIU J,et al.Steroid-induced osteonecrosis of the femoral head: novel insight into the roles of bone endothelial cells in pathogenesis and treatment[J].Front Cell Dev Biol,2021,9:777697.
[25] YU H,YUE J,WANG W,et al.Icariin promotes angiogenesis in glucocorticoid-induced osteonecrosis of femoral heads:in vitro and in vivo studies[J].J Cell Mol Med,2019,23(11):7320-7330.
[26] NOWAK-SLIWINSKA P,ALITALO K,ALLEN E,et al.Consensus guidelines for the use and interpretation of angiogenesis assays[J].Angiogenesis,2018,21(3):425-532.
[27] BELOGLAZOVA I,ZUBKOVA E,DERGILEV K,et al.New insight on 2D in vitro angiogenesis models:all that stretches is not a tube[J].Cells,2022,11(20):3278.

相似文献/References:

[1]陈雷雷,张颖.何伟教授采用中医药疗法治疗股骨头坏死的经验[J].中医正骨,2015,27(10):74.
[2]徐西林,赵永兰,张晓峰,等.活骨注射液髋关节腔灌注对兔股骨头坏死模型 血管内皮生长因子表达的动态影响[J].中医正骨,2015,27(08):1.
　XU Xilin,ZHAO Yonglan,ZHANG Xiaofeng,et al.Dynamic effect of intra-articular hip injection of Huogu injection on the expression of vascular endothelial growth factor in rabbit models with femur head necrosis[J].The Journal of Traditional Chinese Orthopedics and Traumatology,2015,27(09):1.
[3]周勇,任菲菲,丰凡翔,等.血管内皮生长因子和骨形态发生蛋白2 在非创伤性股骨头坏死不同区域的表达[J].中医正骨,2015,27(08):7.
　ZHOU Yong,REN Feifei,FENG Fanxiang,et al.Expressions of vascular endothelial growth factor and bone morphogenetic protein 2 in different zones of femoral head with non-traumatic osteonecrosis[J].The Journal of Traditional Chinese Orthopedics and Traumatology,2015,27(09):7.
[4]鲍荣华,王国平,夏晓斌,等.钽棒植入治疗非创伤性股骨头坏死的疗效观察[J].中医正骨,2015,27(02):28.
[5]张兵,马凰富,刘波,等.非创伤性股骨头坏死的舌象定量研究[J].中医正骨,2015,27(04):8.
　ZHANG Bing,MA Huangfu,LIU Bo,et al.Quantitative study on tongue manifestation of patients with nontraumatic osteonecrosis of femoral head[J].The Journal of Traditional Chinese Orthopedics and Traumatology,2015,27(09):8.
[6]李刚,程春生.股骨头坏死血瘀证症状与平乐郭氏正骨方药药物组成 最大频繁关联模式挖掘[J].中医正骨,2015,27(04):21.
　LI Gang,CHENG Chunsheng.Maximal frequent association pattern mining for studying the relationship between BLOOD STASIS syndrome in patients with osteonecrosis of femoral head and drug components of Pingle Guo's orthopedics prescription[J].The Journal of Traditional Chinese Orthopedics and Traumatology,2015,27(09):21.
[7]李文龙,梅沉成,杜贵强,等.微创减压植骨多孔钽棒植入治疗 ARCOⅡ期非创伤性缺血性股骨头坏死[J].中医正骨,2016,28(02):43.
[8]张磊,金红婷,童培建.骨健口服液早期干预非创伤性股骨头坏死的临床研究[J].中医正骨,2016,28(03):14.
　ZHANG Lei,JIN Hongting,TONG Peijian.Clinical study on Gujian Koufuye(骨健口服液)for early intervention of nontraumatic osteonecrosis of femoral head[J].The Journal of Traditional Chinese Orthopedics and Traumatology,2016,28(09):14.
[9]鲍荣华,王国平,夏晓斌,等.直接前入路微创全髋关节置换术治疗晚期股骨头坏死[J].中医正骨,2016,28(03):61.
[10]张超,姚晨,沈计荣.微创髓芯减压打压支撑植骨术结合补肾活血汤口服治疗ARCOⅡ、Ⅲ期股骨头坏死的近期疗效观察[J].中医正骨,2016,28(03):20.
　ZHANG Chao,YAO Chen,SHEN Jirong.[J].The Journal of Traditional Chinese Orthopedics and Traumatology,2016,28(09):20.
[11]彭鹏,肖欢,方伟华,等.激素性股骨头坏死囊性变组织骨修复机制分析与活血通络胶囊含药血清对肥大软骨细胞成骨分化影响的实验研究[J].中医正骨,2024,36(09):19.
　PENG Peng,XIAO Huan,FANG Weihua,et al.[J].The Journal of Traditional Chinese Orthopedics and Traumatology,2024,36(09):19.

备注/Memo

备注/Memo:
基金项目:国家自然科学基金项目(8227150353,82004392); 广东省基础与应用基础研究基金项目(2023A1515010551); 广东省中医骨伤研究院开放课题重点项目(GYH202101-01,GYH202101-04); 毕节市科学技术局2022年度“揭榜挂帅”项目(毕科合重大专项〔2022〕1号); 中国博士后科学基金第75批面上资助项目(2024M750819); 中医证候全国重点实验室研究生项目(SKLKY2024A0003)
通讯作者:魏秋实 E-mail:weiqshi@126.com
更新日期/Last Update: 1900-01-01