马钱子水提物改善Ⅱ型胶原诱导型类风湿关节炎大鼠骨破坏的作用及机制研究

Effect and mechanism of aqueous extract of Strychni Semen on bone destruction in rats with typeⅡcollagen⁃induced rheumatoid arthritis

考察马钱子水提物(aqueous extract of Strychni Semen,SA)对Ⅱ型胶原诱导型关节炎(typeⅡcollagen⁃induced arthri⁃tis,CIA)大鼠骨破坏的作用机制。将SD大鼠随机分为正常组,模型组,SA低、中、高剂量组(2.85、5.70、11.40 mg·kg^(-1)),甲氨蝶呤组。除正常组外,其他各组均制备CIA模型。二次免疫后,SA低、中、高剂量组给予不同剂量的SA,每日1次,甲氨蝶呤组每3 d给药1次,正常组和模型组给予0.3%羟甲基纤维素钠(CMC⁃Na),连续28 d。每3 d进行关节炎临床评分。采用微计算机断层扫描技术(micro computed tomography,Micro⁃CT)评价骨破坏。通过苏木素⁃伊红(hematoxylin⁃eosin,HE)染色以及抗酒石酸酸性磷酸酶(tartrate⁃resistant acid phosphatase,TRAP)染色评价CIA大鼠关节组织病理学变化及破骨细胞数量。免疫组织化学法检测大鼠关节组织中白细胞介素⁃1β(interleukin⁃1β,IL⁃1β)的表达。免疫蛋白印记法检测大鼠关节组织中丝裂原活化蛋白激酶(mitogen⁃activated protein kinase,MAPK)、磷脂酰肌醇3⁃激酶(phosphatidylinositol 3⁃kinase,PI3K)/蛋白激酶B(pro⁃tein kinase B,Akt)信号通路关键蛋白的表达。结果显示,SA各剂量均能不同程度地改善CIA大鼠炎症关节的红、肿和关节畸形,降低临床评分,抑制滑膜炎症、血管翳、软骨侵蚀和骨破坏,并减少骨组织中TRAP阳性细胞数;Micro⁃CT结果显示SA能够升高骨密度、骨体积分数、骨小梁厚度、骨小梁数量,并降低骨表面体积比和骨小梁分离度。SA各剂量均能不同程度下调IL⁃1β、p⁃JNK、p⁃ERK、p⁃p38、PI3K、p⁃Akt蛋白表达水平。综上,SA能够改善CIA大鼠的疾病严重程度,减轻关节组织病理学和影像学改变,具有骨保护作用,其作用机制可能与抑制MAPK、PI3K/Akt信号通路过度活化有关。

To investigate the mechanism of action of aqueous extract of Strychni Semen(SA)on bone destruction in rats with typeⅡcollagen⁃induced arthritis(CIA),the SD rats were randomly divided into normal group,model group,low,medium,and high dose(2.85,5.70,and 11.40 mg·kg^(-1))groups of SA,and methotrexate group.Except for the normal group,the CIA model was prepared for the other groups.After the second immunization,different doses of SA were given to the low,medium,and high dose groups of SA once a day,and the methotrexate group was given once every three days.0.3%sodium hydroxymethylcellulose(CMC⁃Na)was given once a day to the normal and model groups for 28 d.The clinical score of arthritis was evaluated every three days.Micro computed tomography(Micro⁃CT)method was used to evaluate the degree of bone destruction.Histopathological changes in the joint tissue and the number of osteoclasts in CIA rats were evaluated by hematoxylin⁃eosin(HE)staining and tartrate⁃resistant acid phosphatase(TRAP)staining.The expression of interleukin⁃1β(IL⁃1β)in the joint tissue of rats was detected by immunohistochemistry.Western blot was used to detect key protein expression in mitogen⁃activated protein kinase(MAPK)and phosphatidylinositol 3⁃kinase/protein kinase B(PI3K/Akt)signaling pathways in the joint tissue of rats.The results showed that different doses of SA were able to improve the red and swollen inflammatory joint and joint deformity in CIA rats to varying degrees,reduce the clinical score,inhibit synovial in⁃flammation,vascular opacification,cartilage erosion,and bone destruction,and reduce the number of TRAP⁃positive cells in bone tis⁃sue.Micro⁃CT results showed that the SA was able to increase bone mineral density,bone volume fraction,trabecular reduce,and tra⁃becular number and reduce bone surface/bone volume and trabecular separation/spacing.Different doses of SA could down⁃regulate the protein expression of IL⁃1β,p⁃JNK,p⁃ERK,p⁃p38,PI3K,and p⁃Akt to varying degrees.In conclusion,SA can improve disease se⁃verity,attenuate histopathological and imaging changes in joints,and have osteoprotective effects in CIA rats,and its mechanism of ac⁃tion may be related to the inhibition of the overactivation of MAPK and PI3K/Akt signaling pathways.