来源：风湿病与关节炎,2024,13（5）:1-8.

脉冲电磁场对老年大鼠关节软骨及软骨下骨凋亡蛋白的影响（基础研究）

李萌萌¹，尹林伟¹，牛尚博¹，刘　静¹，黄夏荣¹，王金玲¹，孙光华¹，钟培瑞¹，彭　婷¹，曾国锋¹，周　君^1,2

　　【摘　要】目的：探讨脉冲电磁场对老年大鼠关节软骨及软骨下骨凋亡蛋白的影响。方法：采用随机抽样法将16只24月龄SD自然衰老雄性大鼠随机等分为老年组和脉冲电磁组，8只6月龄SD青年雄性大鼠作为青年组。脉冲电磁组予脉冲电磁场干预，频率为8 Hz，强度为3.82 mT，每日40 min，每周5 d，持续8周。青年组和老年组固定于脉冲电磁场下，仪器不通电。8周后取材，ELISA法检测血清Ⅱ型胶原C端肽（CTX-Ⅱ）含量；显微CT检测左侧胫骨软骨下骨微结构并分析骨组织形态量化指标；番红O-固绿染色观察大鼠左侧胫骨平台软骨组织形态结构，改良Mankin's评分评估大鼠膝关节软骨组织退行性变化程度。RT-qPCR、Western blot检测半胱氨酸天冬氨酸蛋白酶-3（Caspase-3）、半胱氨酸天冬氨酸蛋白酶-8（Caspase-8）、血小板反应蛋白解整合素金属蛋白酶-4（ADAMTS-4），以及血小板反应蛋白解整合素金属蛋白酶-5（ADAMTS-5）的mRNA与蛋白表达水平。结果：①与青年组比较，老年组大鼠软骨胶原分解产物CTX-Ⅱ水平升高（P < 0.05）；骨密度、骨体积分数、骨小梁数量均降低（P < 0.05），骨小梁厚度、骨小梁间距均增大（P < 0.05）；番红O-固绿染色显示，大鼠软骨表面粗糙有裂隙，软骨与软骨下骨界限模糊不清，且有潮线，低细胞化，细胞形态结构不规则，蛋白多糖丢失且红染变淡；改良Mankin's评分增加（P < 0.05）；Caspase-3、Caspase-8、ADAMTS-4、ADAMTS-5的mRNA及蛋白表达升高（P < 0.05）。②与老年组比较，脉冲电磁组大鼠软骨胶原分解产物CTX-Ⅱ水平降低（P < 0.05）；左侧胫骨软骨下骨的骨密度、骨体积分数升高，骨小梁数量增加（P < 0.05），骨小梁间距减小（P < 0.05），骨小梁厚度有降低趋势（P > 0.05）；番红O-固绿染色显示，大鼠软骨表面较为平整，软骨与软骨下骨界限分明，细胞形态较规则、数量较多，蛋白多糖丰富且染色均匀；改良Mankin's评分较小（P < 0.05）；Caspase-3、Caspase-8、ADAMTS-4、ADAMTS-5的mRNA及蛋白表达降低（P < 0.05）。结论：脉冲电磁场可能通过降低凋亡相关蛋白Caspase-3、Caspase-8的表达及金属蛋白酶ADAMTS-4、ADAMTS-5的表达，延缓老年大鼠关节软骨退变与细胞外基质降解，抑制局部骨质疏松，发挥抗骨关节炎作用。

　　【关键词】　膝骨关节炎；脉冲电磁场；关节软骨；软骨下骨；凋亡；大鼠

The Effect of Pulsed Electromagnetic Field on Apoptosis Proteins in Articular Cartilage and Subchondral Bone of Elderly Rats

LI Meng-meng,YIN Lin-wei,NIU Shang-bo,LIU Jing,HUANG Xia-rong,WANG Jin-ling,SUN Guang-hua,ZHONG Pei-rui,PENG Ting,ZENG Guo-feng,ZHOU Jun

　　【ABSTRACT】Objective:To explore

the effects of pulsed electromagnetic fields on apoptosis proteins in articular cartilage and subchondral bone of elderly rats.Methods:Sixteen 24-month-old SD naturally aging male rats were randomly divided into an elderly group and a pulsed electromagnetic group using random sampling method.Eight 6-month-old SD young male rats were selected as the youth group.The pulsed electromagnetic group received pulsed electromagnetic field intervention at a frequency of 8 Hz and an intensity of 3.82 mT,with a duration of 40 minutes per day and 5 days per week for 8 weeks.The youth and elderly groups were fixed under pulsed electromagnetic fields,and the instruments were not powered on.After 8 weeks,samples were taken and ELISA was used to detect the content of C-telopeptide of typeⅡcollagen（CTX-Ⅱ）in serum;Micro CT（Micro-CT）was used to detect the microstructure of the left tibial subchondral bone and analyze quantitative indicators of bone tissue morphology;the morphological structure of the cartilage tissue on the left tibial plateau of rats was observed using safranin O-solid green staining,and the degree of degenerative changes in the knee joint cartilage tissue of rats was evaluated using the improved Mankin's score.RT-qPCR and Western blot were used to detect the mRNA and protein expression levels of Caspase-3,Caspase-8,ADAMTS-4 and ADAMTS-5.Results:①Compared with the young group,the CTX - Ⅱ level of cartilage collagen decomposition product in the elderly group rats increased（P < 0.05）;bone density,bone volume fraction,and number of trabeculae all decreased（P < 0.05）,while bone trabecular thickness and spacing increased（P < 0.05）;safranin O-solid green staining showed that the surface of rat cartilage was rough with cracks,the boundary between cartilage and subchondral bone was unclear,and there was a tidal line,low cell count,irregular cell morphology and structure,loss of proteoglycans,and pale red staining;improved Mankin's score increased（P < 0.05）;the mRNA and protein expression of Caspase-3,Caspase-8,ADAMTS-4,and ADAMTS-5 increased（P < 0.05）.②Compared with the elderly group,the CTX -Ⅱ level of cartilage collagen decomposition products in the pulsed electromagnetic group rats decreased

（P < 0.05）;the bone density and bone volume fraction of the subchondral bone of the left tibia increased,the number of trabeculae increased（P < 0.05）,the distance between trabeculae decreased（P < 0.05）,and the thickness of trabeculae showed a decreasing trend（P > 0.05）;safranin O-solid green staining showed that the surface of rat cartilage was relatively flat,with clear boundaries between cartilage and subchondral bone,the cell morphology was more regular and numerous,and protein polysaccharides were abundant and evenly stained;the improved Mankin's score was relatively small（P < 0.05）;the mRNA and protein expression of Caspase-3,Caspase-8,ADAMTS-4,and ADAMTS-5 decreased（P < 0.05）.Conclusion:Pulsed electromagnetic fields may delay articular cartilage degeneration and extracellular matrix degradation in elderly rats,inhibit local osteoporosis,and exert anti osteoarthritis effects by reducing the expression of apoptosis related proteins Caspase-3 and Caspase-8,as well as the expression of metalloproteinases ADAMTS-4 and ADAMTS-5.

　　【Keywords】 knee osteoarthritis;pulsed electromagnetic field;joint cartilage;subchondral bone;apoptosis;rats