[1]张若谷,葛钦文,邹凯奥,等.补肾活血方对腰椎间盘退变小鼠腰椎终板软骨的影响及其作用机制[J].中医正骨,2025,37(03):5-13.
　ZHANG Ruogu,GE Qinwen,ZOU Kaiao,et al.Effects and mechanism of Bushen Huoxue Fang(补肾活血方)on lumbar endplate cartilage in mice with intervertebral disc degeneration:an experimental study[J].The Journal of Traditional Chinese Orthopedics and Traumatology,2025,37(03):5-13.
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补肾活血方对腰椎间盘退变小鼠腰椎终板软骨的影响及其作用机制()

《中医正骨》[ISSN:1001-6015/CN:41-1162/R]

卷:
第37卷
期数:
2025年03期
页码:
5-13
栏目:
基础研究
出版日期:
2025-03-20

文章信息/Info

Title:
Effects and mechanism of Bushen Huoxue Fang(补肾活血方)on lumbar endplate cartilage in mice with intervertebral disc degeneration:an experimental study
作者:
张若谷; 葛钦文; 邹凯奥; 厉驹; 金红婷; 童培建; 王萍儿
浙江中医药大学第一临床医学院,浙江 杭州 310053
Author(s):
ZHANG Ruogu; GE Qinwen; ZOU Kaiao; LI Ju; JIN Hongting; TONG Peijian; WANG Pinger
The First Clinical Medical College of Zhejiang Chinese Medical University,Hangzhou 310053,Zhejiang,China
关键词:
椎间盘退化;  腰椎;  补肾活血方;  终板软骨;  小鼠;  动物实验
Keywords:
intervertebral disc degeneration;  lumbar vertebrae;  Bushen Huoxue Fang;  endplate cartilage;  mice;  animal experimentation
摘要:
目的:探讨补肾活血方对腰椎间盘退变小鼠腰椎终板软骨的影响及其作用机制。方法:将18只10周龄雄性SPF级C57BL/6J小鼠随机分为空白对照组、模型组、补肾活血方组,每组6只。模型组和补肾活血方组采用腰椎失稳术构建腰椎间盘退变模型。造模后第2天,补肾活血方组小鼠用补肾活血方中药提取液灌胃,空白对照组和模型组小鼠用生理盐水灌胃,各组小鼠均每天灌胃1次,连续灌胃8周。灌胃结束后,脱颈处死各组小鼠,切取小鼠L4和L5椎体,进行Micro-CT分析,并计算腰椎间盘相对高度。常规进行小鼠腰椎终板软骨组织病理学观察,计算小鼠的腰椎终板软骨组织形态学评分,测定小鼠腰椎终板软骨组织中Ⅱ型胶原蛋白α1链(collagen typeⅡalpha 1 chain,Col2α1)、基质金属蛋白酶-13(matrix metalloproteinase-13,MMP-13)和pSmad2的表达情况。结果:①小鼠腰椎Micro-CT分析结果。空白对照组和补肾活血方组小鼠腰椎终板软骨骨化区域的骨体积分数均低于模型组(P=0.000,P=0.022),骨小梁数量均少于模型组(P=0.002,P=0.001),腰椎间盘相对高度均高于模型组(P=0.000,P=0.000)。补肾活血方组小鼠腰椎终板软骨骨化区域的骨体积分数高于空白对照组(P=0.003),骨小梁数量、腰椎间盘相对高度与空白对照组的差异均无统计学意义(P=0.882,P=0.794)。②小鼠腰椎终板软骨组织病理学观察结果。空白对照组小鼠的腰椎终板软骨细胞异质性较小,软骨厚度正常,骨化面积较小。与空白对照组相比,模型组小鼠的腰椎终板软骨细胞异质性显著增大,软骨明显变薄,骨化面积增大。与模型组相比,补肾活血方组小鼠的腰椎终板软骨细胞异质性较小,软骨厚度有所增加,骨化面积有所减小,但均未达到空白对照组的水平。空白对照组和补肾活血方组小鼠的腰椎终板软骨组织形态学评分均低于模型组(P=0.000,P=0.000),空白对照组与补肾活血方组小鼠的腰椎终板软骨组织形态学评分差异无统计学意义(P=0.591)。③小鼠腰椎终板软骨组织中MMP-13和Col2α1表达水平测定结果。空白对照组和补肾活血方组小鼠腰椎终板软骨组织中MMP-13阳性细胞数占比均低于模型组(P=0.002,P=0.010),Col2α1阳性表达面积比均高于模型组(P=0.000,P=0.026)。空白对照组与补肾活血方组小鼠腰椎终板软骨组织中MMP-13阳性细胞数占比的差异无统计学意义(P=0.226)。空白对照组小鼠腰椎终板软骨组织中Col2α1阳性表达面积比高于补肾活血方组(P=0.000)。④小鼠腰椎终板软骨组织中pSmad2表达水平测定结果。空白对照组小鼠腰椎终板软骨组织中可见大量pSmad2阳性细胞。空白对照组和补肾活血方组小鼠腰椎终板软骨组织中pSmad2阳性细胞数占比均高于模型组(P=0.014,P=0.040)。空白对照组与补肾活血方组小鼠腰椎终板软骨组织中pSmad2阳性细胞数占比的差异无统计学意义(P=0.444)。结论:补肾活血方可延缓小鼠腰椎间盘退变的病理进程,其作用机制可能与补肾活血方能够通过促进腰椎终板软骨细胞中Smad2的磷酸化,下调MMP-13的表达并上调Col2α1的表达有关。
Abstract:
Objective:To observe the effects of Bushen Huoxue Fang(补肾活血方,BSHXF)on lumbar endplate cartilage in mice with intervertebral disc degeneration(IVDD),and to explore its underlying mechanism.Methods:Eighteen 10-week-old SPF-grade male C57BL/6J mice were randomized into blank control group,model group,and BSHXF group,6 cases in each group.All mice but the ones in blank control group were modeled by lumbar spine instability(LSI)surgery for inducing IVDD.The next day after successful modeling,the mice in BSHXF group were intervened by intragastric administration with BSHXF extract,while the ones in blank control group and model group with the same dose of normal saline,once a day for consecutive 8 weeks.After the end of the last intervention,all mice were sacrificed by cervical dislocation,and their L4 and L5 vertebrae were harvested for Micro-CT examination,and the relative height of the L4-5 intervertebral disc was calculated.Furthermore,the pathological changes of the lumbar endplate cartilage tissues were observed,and the morphological score of lumbar endplate cartilage tissues was calculated,meanwhile,the expression levels of collagen typeⅡalpha 1 chain(Col2α1),matrix metalloproteinase-13(MMP-13),and pSmad2 in the lumbar endplate cartilage tissues were detected.Results:①The Micro-CT analysis results of the lumbar vertebrae.The blank control group and BSHXF group presented lower bone volume fraction(BVF)and fewer trabecular number(Tb.N)in the ossification area of lumbar endplate cartilage,and higher relative height of intervertebral disc compared to model group(P=0.000,P=0.022; P=0.002,P=0.001; P=0.000,P=0.000).Moreover,the BSHXF group exhibited higher BVF compared to blank control group(P=0.003),while,there was no significant differences between the 2 groups in the Tb.N and relative height of intervertebral disc(P=0.882,P=0.794).②The pathological observation of the lumbar endplate cartilage tissues.In blank control group,the lumbar endplate chondrocytes presented as low heterogeneity,normal cartilage thickness,and a small ossified area.Compared with that of blank control group,the changes in the lumbar endplate chondrocytes,manifesting as significantly increased heterogeneity,thinned cartilage layer,and an enlarged ossified area,were observed in mice of model group; while,compared with that of model group,the changes in the lumbar endplate chondrocytes,manifesting as reduced heterogeneity,increased cartilage thickness,and a decreased ossified area,were observed in mice of BSHXF group,but all these did not reach the levels of blank control group.Besides,the morphological score of lumbar endplate cartilage tissues was lower in blank control group and BSHXF group compared to model group(P=0.000,P=0.000),with no significant difference between blank control group and BSHXF group(P=0.591).③The expression levels of MMP-13 and Col2α1 in the lumbar endplate cartilage tissues.The proportion of MMP-13-positive cells was lower,while the ratio of Col2α1-positive expression area was higher in blank control group and BSHXF group compared to model group(P=0.002,P=0.010; P=0.000,P=0.026).Furthermore,the ratio of Col2α1-positive expression area was higher in blank control group compared to BSHXF group(P=0.000),while there was no significant difference in the proportion of MMP-13-positive cells between the 2 groups(P=0.226).④The expression level of pSmad2 in the lumbar endplate cartilage tissues.A high percentage of pSmad2-positive cells were observed in the lumbar endplate cartilage tissues of blank control group.The proportion of pSmad2-positive cells in the lumbar endplate cartilage tissues was higher in blank control group and BSHXF group compared to model group(P=0.014,P=0.040),with no significant difference between blank control group and BSHXF group(P=0.444).Conclusion:BSHXF can delay the pathological progression of lumbar IVDD in mice.It may work by down-regulating the expression of MMP-13,and up-regulating the expression of Col2α1 through promoting the phosphorylation of Smad2 in lumbar endplate chondrocytes.

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备注/Memo

备注/Memo:
基金项目:浙江省自然科学基金探索项目(LY23H270005)
通讯作者:王萍儿 E-mail:apple63209321@126.com
更新日期/Last Update: 1900-01-01