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, Southwest Minzu University Chengdu 610041,China.
Zhongguo Zhong Yao Za Zhi. 2022 Sep;47(17):4715-4722. doi: 10.19540/j.cnki.cjcmm.20220207.704.
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.
本研究旨在探讨藏药邦那（铁棒锤和黄草乌）中的生物碱对骨关节炎（OA）大鼠的体内外治疗作用及其潜在机制。从2 - 3周龄雄性SD大鼠分离软骨细胞，体外使用脂多糖（LPS）诱导软骨细胞发生骨关节炎。采用甲基噻唑基四氮唑（MTT）法研究7种生物碱（12 - 表那碎因、松果灵、苯甲酰乌头原碱、乌头碱、3 - 乙酰乌头碱、新乌头碱和苯甲酰新乌头碱）对软骨细胞的毒性。软骨细胞分为对照组、模型组（用5 μg·mL⁻¹ LPS诱导12 h）和给药组（用5 μg·mL⁻¹ LPS诱导12 h并孵育24 h）。通过蛋白质免疫印迹法检测各组炎症因子环氧化酶 - 2（COX - 2）、诱导型一氧化氮合酶（iNOS）、肿瘤坏死因子 - α（TNF - α）和白细胞介素 - 1β（IL - 1β）的蛋白表达，通过免疫荧光染色检测基质金属蛋白酶 - 13（MMP - 13）、聚集蛋白聚糖、Ⅱ型胶原、成纤维细胞生长因子2（FGF2）的蛋白表达。对于体内实验，用碘乙酸钠诱导大鼠发生骨关节炎，通过免疫组织化学检测各组大鼠软骨组织中MMP - 13、TNF - α和FGF2的表达。结果表明，在一定剂量范围内，7种生物碱处理下软骨细胞活力可达9０%以上。与模型组相比，乌头碱、12 - 表那碎因、松果灵、3 - 乙酰乌头碱和新乌头碱可降低炎症因子COX - 2、iNOS、TNF - α和IL - 1β的蛋白表达。此外，12 - 表那碎因、乌头碱和新乌头碱可下调MMP - 13的表达，上调聚集蛋白聚糖和Ⅱ型胶原的表达。另外，与模型组和其他邦那生物碱相比，12 - 表那碎因显著上调FGF2的表达。因此，选择12 - 表那碎因进行体内动物实验。免疫组织化学结果显示，与模型组相比，12 - 表那碎因可显著降低软骨组织中MMP - 13和TNF - α的表达，并上调FGF2的表达。综上所述，在7种邦那生物碱中，12 - 表那碎因可通过下调MMP - 13和TNF - α的表达以及上调FGF2的表达来促进大鼠骨关节炎的修复。
