飞龙掌血对胶原诱发关节炎大鼠心血管损害的干预作用及机制

Intervention effects and mechanism of Toddalia asiatica on cardiovascular damage in rats with collageninduced arthritis

目的基于维生素D(VD)和中性粒细胞胞外诱捕网(NETs)探讨苗药飞龙掌血对胶原诱发关节炎(CIA)大鼠心血管损害的干预作用及潜在机制。方法将SD大鼠随机分为正常组(9只)和造模组(61只)。造模组大鼠以多点注射牛Ⅱ型胶原+弗氏不完全佐剂的方式制备CIA模型。将造模成功的大鼠分为模型组、甲氨蝶呤组(阳性对照,1.5 mg/kg,每周2次)、VD组[通路验证,1000单位/(kg·d),每天1次]和飞龙掌血低、中、高剂量组[0.54、1.08、2.16 g/(kg·d),以生药量计,每天1次],每组9只,灌胃给药4周。于造模后、给药前进行关节炎指数评分,分别于给药前和末次给药后测量足跖厚度,观察大鼠踝关节、心脏和腹主动脉组织病理学变化;检测血清中髓过氧化物酶(MPO)、白细胞介素6(IL-6)、25-羟基维生素D_(3)[25(OH)D_(3)]含量;检测心脏组织中肽酰基精氨酸脱亚氨酶4(PAD4)、NETs标志物[瓜氨酸化组蛋白H3(CitH3)、MPO]、VD相关指标[维生素D受体(VDR)、25羟基维生素D-1α羟化酶(CYP27B1)]、IL-6蛋白的表达情况。结果与正常组比较,模型组大鼠关节炎指数和足跖厚度均显著增加(P<0.01),踝关节组织可见明显的炎症细胞浸润和纤维组织增生,心脏组织可见明显的组织空泡和部分周围血管壁增厚,腹主动脉组织可见内皮脱落;血清MPO、IL-6含量显著增高(P<0.01),25(OH)D_(3)水平显著下降(P<0.01);心肌组织中PAD4、CitH3、MPO、IL-6蛋白的表达均显著上调(P<0.01);VDR、CYP27B1蛋白的表达虽有改变,但差异均无统计学意义(P>0.05)。与模型组比较,各药物组大鼠踝关节、心脏组织的病理改变均明显改善,上述各指标均普遍逆转(P<0.05或P<0.01)。结论飞龙掌血可通过抑制NETs的形成及炎症反应的发生来改善类风湿性关节炎症状及相关心血管损害,其机制可能与调节VD水平有关。

OBJECTIVE To investigate the intervention effects and potential mechanism of Miao medicine Toddalia asiatica on cardiovascular damage in rats with collagen-induced arthritis(CIA)based on vitamin D(VD)and neutrophil extracellular traps(NETs).METHODS SD rats were randomly divided into the normal group(9 rats)and the modeling group(61 rats).CIA model was prepared by multi-point injection of typeⅡbovine collagen+Fisher’s incomplete adjuvant;the model rats were randomly divided into the model group,methotrexate group(positive control,1.5 mg/kg,twice a week),vitamin D group[pathway validation,1000 IU/(kg·d),once a day],T.asiatica low-dose,medium-dose and high-dose groups[0.54,1.08,2.16 g/(kg·d),calculated by crude drug,once a day],with 9 rats in each group;they were given relevant medicine intragastrically for 4 consecutive weeks.Arthritis index scoring was performed after modeling and before administration,and plantar thickness was measured before and after the last administration;the histopathological changes of ankle joint,heart and abdominal aorta were observed in rats;the serum contents of myeloperoxidase(MPO),interleukin-6(IL-6)and 25-hydroxyvitamin D_(3)[25(OH)D_(3)]were detected;the expressions of peptidylarginine deiminase 4(PAD4),NETs markers[citrullinated histone H3(CitH3),MPO],VD-related indicators[vitamin D receptor(VDR),25-hydroxyvitamin D-1α-hydroxylase(CYP27B1)]and IL-6 were determined in cardiac tissue.RESULTS Compared with the normal group,the plantar thickness of the arthritis index increased significantly in the model group(P<0.01).The obvious inflammatory cell infiltration and fibrous tissue hyperplasia were found in the ankle joint,the obvious myocardial fiber vacuoles and thickening of some surrounding blood vessel walls were found in the heart tissue,and the endothelial detachment was found in the abdominal aorta.The contents of MPO and IL-6 in serum increased significantly(P<0.01),while the level of 25(OH)D_(3) decreased significantly(P<0.01);the protein expressions of PAD4,CitH3,MPO and IL-6 in myocardial tissue up-regulated significantly(P<0.01),while protein expression of VDR and CYP27B1 changed to acertain extent without significance(P>0.05).Compared with the model group,the pathological changes of ankle joints and cardiac tissue in rats were significantly improved in administration groups,and the above indicators were generally reversed(P<0.05 or P<0.01).CONCLUSIONS T.asiatica can improve rheumatoid arthritis symptoms and cardiovascular damage by inhibiting the formation of NETs and inflammatory response,the mechanism of which may be associated with the regulation of VD expression.