电针对前交叉韧带断裂大鼠炎症反应及cGAS/STING信号通路的影响

Effects of Electroacupuncture on Inflammatory Response and cGAS/STING Signaling Pathway in Rats with Anterior Cruciate Ligament Rupture

目的:探究电针对前交叉韧带断裂后创伤性骨关节炎早期炎症反应与环状GMP-AMP合成酶(cGAS)/干扰素基因刺激因子(STING)信号通路的影响。方法:24只SD大鼠随机分为假手术组、前交叉韧带损伤(ACLT)组和电针组,每组8只。其中ACLT组和电针组实施前交叉韧带切断术。电针组大鼠予双侧“阳陵泉”“血海”“足三里”“太溪”电针刺激,1次/d,每周干预5 d,连续2周,其余两组不做处理。2周后ELISA法检测大鼠血清白介素-1β(IL-1β)和肿瘤坏死因子-α(TNF-α)水平;取大鼠左膝关节软骨组织切片分别进行番红O-固绿染色和苏木精-伊红(HE)染色,镜下观察软骨损伤程度并评分(Mankin’s评分);RT-qPCR检测大鼠右膝关节软骨基质金属蛋白酶-13(MMP-13)mRNA、cGAS mRNA、STING mRNA表达水平。Western blotting检测大鼠右膝关节软骨MMP-13、cGAS、STING蛋白表达水平。结果:ACLT组大鼠血清IL-1β、TNF-α水平均高于假手术组(P<0.05);电针组大鼠血清IL-1β、TNF-α水平均低于ACLT组(P<0.05)。番红O-固绿染色显示假手术组大鼠软骨表面光滑;ACLT组大鼠软骨表面出现轻微缺损和细小裂层;电针组大鼠软骨表面较平滑,无明显裂层。HE染色显示假手术组软骨表面平整且光滑,软骨细胞均排列规整;ACLT组大鼠软骨层表面变薄;电针组大鼠软骨表面略粗糙。ACLT组大鼠Mankin’s评分高于假手术组(P<0.05);电针组大鼠Mankin’s评分低于ACLT组(P<0.05)。ACLT组大鼠MMP-13 mRNA、cGAS mRNA、STING mRNA相对表达量均高于假手术组(P<0.05);电针组大鼠MMP-13 mRNA、cGAS mRNA、STING mRNA相对表达量均低于ACLT组(P<0.05)。ACLT组大鼠MMP-13、cGAS、STING蛋白相对表达量均高于假手术组(P<0.01);电针组大鼠MMP-13、cGAS、STING蛋白相对表达量均低于ACLT组(P<0.01)。结论:电针可能通过下调cGAS/STING信号通路而减轻ACLT大鼠炎症反应,延缓关节软骨退行性改变。

Objective:To investigate the progression of traumatic osteoarthritis after anterior cruciate ligament rupture(ACLT)and the effect of electroacupuncture on its early inflammatory response and cyclic GMP-AMP synthase(cGAS)/interferon gene stimulating factor(STING)signaling pathway.Methods:A total of 24 SD rats were randomly divided into sham operation group,anterior cruciate ligament injury(ACLT)group and electroacupuncture group,with 8 rats in each group.Among them,ACLT group and electroacupuncture group were subjected to ACL amputation.In the electroacupuncture group,rats were given bilateral electroacupuncture stimulation of"Yanglingquan"(GB 34),"Xuehai"(SP 10),"Zusanli"(ST 36)and"Taixi"(KI 3)once a day,and the intervention was carried out for 5 days per week.After 2 weeks,the rats'serum interleukin-1β(IL-1β)and tumor necrosis factor-α(TNF-α)levels were detected by ELISA;The cartilage tissue sections of the left knee joint were stained with sapphire red O-green and hematoxylin-eosin(HE),and the extent of the cartilage damage was observed and scored under the microscope(Mankin's score);RT-qPCR was performed to detect the expression levels of matrix metalloproteinase-13(MMP-13)mRNA,cGAS mRNA and STING mRNA in the cartilage of the right knee joint of rats.Western blotting was performed to detect the expression levels of MMP-13,cGAS and STING proteins in the cartilage of the right knee joint of rats.Results:The ACLT group showed higher serum IL-1βand TNF-αlevels than sham operation group(P<0.05).The electroacupuncture group showed lower serum IL-1βand TNF-αlevels than ACLT group(P<0.05).The surface of cartilage of rats in the sham operation group was smooth;The surface of cartilage of rats in the ACLT group showed slight defects and small cracks;The surface of cartilage of rats in the electro-acupuncture group was smooth without obvious cracks.HE staining showed that the surface of cartilage of rats in the sham operation group was smooth and even,and the chondrocytes were arranged in a regular way;The surface of cartilage of rats in the ACLT group was thinned;The surface of cartilage of rats in the electroacupuncture group was a little bit rough;The ACLT group showed higher Mankin's score than sham operation group(P<0.05);The electroacupuncture group showed lower Mankin's score than ACLT group(P<0.05).The ACLT group showed higher relative expression of MMP-13 mRNA,cGAS mRNA and STING mRNA than sham operation group(P<0.05);The electroacupuncture group showed lower relative expression of MMP-13 mRNA,cGAS mRNA and STING mRNA than ACLT group(P<0.05).The ACLT group showed higher relative expression of MMP-13,cGAS and STING proteins than sham-operated group(P<0.01);The electroacupuncture group showed lower relative expression of MMP-13,cGAS and STING proteins than ACLT group(P<0.01).Conclusion:Electroacupuncture may reduce inflammatory response and delay articular cartilage degeneration in ACLT rats by down-regulating cGAS/STING signaling pathway.