摘要
[目的]探究花青素缓解小鼠骨关节炎(OA)疼痛的内在机制。[方法]C57/BL6小鼠双膝关节注射碘乙酸构建OA模型,用花青素、胰岛素生长因子结合蛋白4(IGFBP-4)过表达和敲减腺病毒处理OA模型。随机分为8组,每组10只:正常(NC)组、对照(Sham)组、OA组、花青素治疗(AC)组,IGFBP-4过表达(BP4+OA)组、IGFBP-4过表达空载(CON267+OA)组、IGFBP-4敲减(BP4-OA)组、IGFBP-4敲减空载(CON098+OA)组。小动物活体成像观察腺病毒关节腔内注射是否成功;苏木精-伊红(HE)染色评估模型是否构建成功;Von Frey、热板实验评估机械痛阈及热痛阈变化;酶联免疫吸附实验(ELISA)检测血清P物质含量;尼氏染色评价脊髓背根神经节(DRG)形态变化;免疫组化检测DRG瞬时受体电位香草酸受体1(TRPV1)表达;蛋白免疫印迹(Western blot)检测DRG中TRPV1、滑膜肿瘤坏死因子-α(TNF-α)、白细胞介素-6(IL-6)、IGFBP-4、Wnt3a、β-catenin表达。[结果]BP4+OA组、CON267+OA组、BP4-OA组、CON098+OA组膝关节处观察到荧光信号;BP4+OA组、BP4-OA组荧光信号辐射效率高于各自空载组(P<0.05)。OA组、CON267+OA组、CON098+OA组滑膜炎症细胞浸润、结缔组织增生、软骨表面侵蚀缺损;AC组和BP4+OA组滑膜和软骨病理变化减轻;BP4-OA组滑膜和软骨病理损伤加重。与Sham组比较,OA组、CON267+OA组、CON098+OA组PWMT和PWTL降低;与OA组比较,AC组、BP4+OA组PWMT和PWTL延长;与OA组比较,BP4-OA组PWMT和PWTL缩短(P<0.05)。OA组、CON267+OA组、CON098+OA组部分神经元核仁消失,胞浆呈空泡状改变,尼氏染色变浅;AC组和BP4+OA组DRG形态损伤减轻;BP4-OA组DRG形态损伤加重。OA组P物质含量、TRPV1表达高于Sham组;AC组P物质含量、TRPV1表达低于OA组;BP4+OA组P物质含量、TRPV1表达低于其空载组和OA组;BP4-OA组P物质含量、TRPV1表达高于其空载组和OA组,明显高于BP4+OA组和AC组(P<0.05)。OA组滑膜TNF-α、IL-6、Wnt3a、β-catenin表达高于Sham组;AC组滑膜TNF-α、IL-6、Wnt3a、β-catenin表达低于OA组;BP4+OA组滑膜TNF-α、IL-6、Wnt3a、β-catenin表达低于其空载组和OA组;BP4-OA组滑膜TNF-α、IL-6、Wnt3a、β-catenin表达高于其空载组和OA组,明显高于BP4+OA组和AC组(P<0.05)。OA组滑膜IGFBP-4表达高于Sham组;AC组IGFBP-4表达高于OA组,且明显高于Sham组;BP4+OA组滑膜IGFBP-4表达高于其空载组;BP4-OA组滑膜IGFBP-4表达低于其空载组,明显低于BP4+OA组和AC组(P<0.05)。[结论]花青素可能通过上调滑膜IGFBP-4抑制Wnt/β-catenin通路,减轻炎症反应、缓解KOA小鼠外周敏化及疼痛。
[Objective]To explore the mechanism of anthocyanins in relieving the pain of osteoarthritis mice.[Methods]The OA model were constructed in C57/BL6 mice by injecting iodoacetic acid.The OA model was treated with over expression of anthocyanin and insulin growth factor binding protein 4(IGFBP-4)and knockdown adenovirus.Randomly divided into 8 groups with 10 animals in each group:normal group,sham group,OA group,anthocyanins treatment(AC)group,IGFBP-4 overexpression(BP4+OA)group,IGFBP-4 overexpression control(CON267+OA)group,IGFBP-4 knockdown(BP4-OA)group,IGFBP-4 knockdown control(CON098+OA)group.The successful construction of IGFBP-4 adenovirus model was observed with small animal in imaging system.HE staining evaluated whether the model was successfully constructed.The changes of the mechanical pain threshold and thermal pain threshold were evaluated by Von Frey and hot plate experiments.Substance P content in serum was detected by ELISA.The morphological changes of spinal dorsal root ganglion(DRG)were evaluated by Nissl’s staining.Immunohistochemical staining was used to detect the expression of transient receptor potential vanillate receptor 1(TRPV1)in DRG.Western blot was used to detect TRPV1 protein expression in DRG and IGFBP-4,Wnt3a,β-catenin,TNF-αand IL-6 in synovial.[Results]Fluorescence signals were observed in the knee joint of BP4+OA,CON267+OA,BP4-OA and CON098+OA groups;the radiation efficiency of BP4+OA group and BP4-OA group were higher than that of no-load control group(P<0.05).In OA group,CON267+OA group and CON098+OA group,there were synovial inflammatory cell infiltration,connective tissue hyperplasia,and cartilage surface erosion defect;the pathological changes of synovial membrane and cartilage were improved in AC group and BP4+OA group.The pathological injury of synovium and cartilage in BP4-OA group was further aggravated.Compare with Sham group,OA group,CON267+OA group and CON098+OA group of PWMT and PWTL were reduced.Compare with OA group,PWMT and PWTL in AC group and BP4+OA group were prolonged than OA group;PWMT and PWTL were shortened in BP4-OA group(P<0.05).Some of the neurons disappeared,the cytoplasm was vacuolar and Nil staining became shallow in OA group,CON267+OA group and CON098+OA group;DRG morphological change was reduced in AC group and BP4+OA group;DRG morphological damage of in BP4-OA group was further aggravated.Substance P content and TRPV1 in DRG in OA group were higher than that in Sham group;substance P content and TRPV1 in DRG in AC group were lower than that in OA group.Substance P content and TRPV1 in DRG in BP4+OA group were lower than that in CON267+OA group and OA group.Substance P content and TRPV1 in DRG in BP4-OA group were higher than that in CON098+OA and OA groups,and significantly higher than that in BP4+OA and AC groups(P<0.05).TNF-α,IL-6,Wnt3a,β-catenin in synovial in OA group were higher than that in Sham group.TNF-α,IL-6,Wnt3a,β-catenin in synovial in AC group were lower than that in OA group.TNF-α,IL-6,Wnt3a,β-catenin in synovial in BP4+OA group were lower than that in CON267+OA group and OA group.TNF-α,IL-6,Wnt3a,β-catenin in synovial in BP4-OA group were higher than that in CON098+OA and OA groups,and significantly higher than that in BP4+OA and AC groups(P<0.05).IGFBP-4 in synovial in OA group was higher than that in sham group.IGFBP-4 in synovial in AC group was higher than those in OA group,and significantly higher than that in Sham group.IGFBP-4 in synovial in BP4+OA group was higher than that in CON267+OA group.IGFBP-4 in synovial in BP4-OA group was significantly lower than that in CON098+OA,and significantly lower than that in BP4+OA and AC groups(P<0.05).[Conclusion]Anthocyanins may inhibit Wnt/β-catenin pathway by up-regulating the expression of synovial IGFBP-4,reduce inflammatory response,and relieve peripheral sensitization and pain in KOA mice.
作者
王睿
赵雪梅
李达
乔玉雪
江小华
WANG Rui;ZHAO Xuemei;LI Da;QIAO Yuxue;JIANG Xiaohua(North China University of Science and Technology,Tangshan 063210,China;Hebei Key Laboratory for Chronic Diseases,Tangshan 063210,China)
出处
《天津中医药》
2025年第3期343-352,共10页
Tianjin Journal of Traditional Chinese Medicine
基金
国家重点实验室开放课题项目(MDK2012023)。
