摘要
该研究主要探讨了藏族药榜那生物碱成分体外和体内对骨关节炎(OA)大鼠的治疗作用及其相关机制。从2~3周SD雄性大鼠中分离软骨细胞,通过脂多糖(LPS)诱导软骨细胞构建体外关节炎损伤模型,采用MTT实验考察榜那7种生物碱(12-表-欧乌头碱、宋果灵、苯甲酰乌头原碱、乌头碱、3-乙酰乌头碱、新乌头碱、苯甲酰新乌头碱)对软骨细胞的毒性影响,后将实验分为对照组、模型组(LPS 5μg·mL^(-1)诱导12 h)和给药组(LPS 5μg·mL^(-1)诱导12 h后给药孵育24 h)。通过Western blot分别检测各组细胞中炎症因子环氧合酶2 (COX-2)、一氧化氮合酶(iNOS)、肿瘤坏死因子α(TNF-α)、白细胞介素-1β(IL-1β)的蛋白表达情况,采用免疫荧光染色分别检测各组细胞中基质金属蛋白酶13 (MMP-13)、聚集蛋白聚糖(aggrecan)、Ⅱ型胶原(collagenⅡ)、成纤维细胞生长因子2 (FGF2)的蛋白表达情况。体内实验利用碘乙酸钠制备了大鼠骨关节炎模型,通过免疫组化检测各组大鼠软骨组织中MMP-13、TNF-α及FGF2的表达情况。结果显示,榜那中的7种生物碱成分在一定剂量范围内可使软骨细胞的存活率达到90%以上;Western blot结果显示,与模型组相比,乌头碱、12-表-欧乌头碱、宋果灵、3-乙酰乌头碱、新乌头碱可降低炎症因子COX-2、iNOS、TNF-α、IL-1β的蛋白表达;免疫荧光结果显示乌头碱、12-表-欧乌头碱、新乌头碱可抑制MMP-13的表达,上调aggrecan、collagenⅡ的表达;此外,与模型组及其他榜那生物碱成分组相比,唯有12-表-欧乌头碱可显著上调FGF2的蛋白表达。因此,选用12-表-欧乌头碱进行体内动物实验,免疫组化结果显示,与模型组相比,12-表-欧乌头碱可明显降低软骨组织中MMP-13和TNF-α的表达,上调FGF2的表达。该研究结果表明,在7种榜那生物碱成分中,12-表-欧乌头碱可通过下调MMP-13和TNF-α的表达,并上调FGF2的表达促进了大鼠骨关节炎损伤的修复。
This study aims to investigate the therapeutic effects of alkaloids in Tibetan medicine Bangna(Aconiti Penduli et Aconiti Flavi Radix) on osteoarthritis(OA) rats in vitro and in vivo and the underlying mechanisms. Chondrocytes were isolated from 2-3 week-old male SD rats and lipopolysaccharide(LPS) was used to induce OA in chondrocytes in vitro. Methyl thiazolyl tetrazolium(MTT) assay was used to investigate the toxicity of seven alkaloids(12-epi-napelline, songorine, benzoylaconine, aconitine, 3-acetylaconitine, mesaconitine, and benzoylmesaconine) to chondrocytes. Chondrocytes were classified into the control group, model group(induced by LPS 5 μg·mL^(-1)for 12 h), and administration groups(induced by LPS 5 μg·mL^(-1)for 12 h and incubated for 24 h). The protein expression of inflammatory factors cyclooxygenase-2(COX-2), inducible nitric oxide synthetase(iNOS), tumor necrosis factor-α(TNF-α), and interleukin-1β(IL-1β) in each group were detected by Western blot, and the protein expression of matrix metalloprotease-13(MMP-13), aggrecan, collagen Ⅱ, fibroblast growth factor 2(FGF2) by immunofluorescence staining. For the in vivo experiment, sodium iodoacetate was used to induce OA in rats, and the expression of MMP-13, TNF-α, and FGF2 in cartilage tissues of rats in each group was detected by immunohistochemistry. The results showed that the viability of chondrocytes could reach more than 90% under the treatment of the seven alkaloids in a certain dose range. Aconitine, 12-epi-napelline, songorine, 3-acetylaconitine, and mesaconitine could decrease the protein expression of inflammatory factors COX-2, iNOS, TNF-α and IL-1β compared with the model group. Moreover, 12-epi-napelline, aconitine, and mesaconitine could down-regulate the expression of MMP-13 and up-regulate the expression of aggrecan and collagen Ⅱ. In addition, compared with the model group and other Bangna alkaloids, 12-epi-napelline significantly up-regulated the expression of FGF2. Therefore, 12-epi-napelline was selected for the animal experiment in vivo. Immunohistochemistry results showed that 12-epi-napelline could significantly reduce the expression of MMP-13 and TNF-α in cartilage tissues, and up-regulate the expression of FGF2 compared with the model group. In conclusion, among the seven Bangna alkaloids, 12-epi-napelline can promote the repair of OA in rats by down-regulating the expression of MMP-13 and TNF-α and up-regulating the expression of FGF2.
作者
王琦
彭静
刘洋
田洋
李洁
任瑶瑶
顾健
谭睿
WANG Qi;PENG Jing;LIU Yang;TIAN Yang;LI Jie;REN Yao-yao;GU Jian;TAN Rui(School of Life Science and Engineering,Southwest Jiaotong University,Chengdu 610031,China;College of Pharmacy,Southuest Minzu University,Chengdu 610041,China)
出处
《中国中药杂志》
CAS
CSCD
北大核心
2022年第17期4715-4722,共8页
China Journal of Chinese Materia Medica
基金
国家自然科学基金委面上项目(82074003,82174083)
四川省重点研发计划项目(20ZDYF3291)
四川省应急重大项目(2021XYCZ008)。