丹蒌片抗痰瘀互结型动脉粥样硬化大鼠炎症反应及机制

Danlou Tablet Fought against Inflammatory Reaction in Atherosclerosis Rats with Intermingled Phlegm and Blood Stasis Syndrome and Its Mechanism Study

目的观察丹蒌片对痰瘀互结型动脉粥样硬化(atherosclerosis,AS)大鼠炎症因子以及脂蛋白相关磷脂酶A2(lipoprotein-associated phospholipase A2,LP-PLA2)和分泌型磷脂酶A2(secretory phospholipase A2,s PLA2)表达的影响,分析其可能机制。方法 40只雄性Wistar大鼠随机分为正常组、模型组、西药组、丹蒌低剂量组(低剂量组)、丹蒌高剂量组(高剂量组),每组8只。正常组大鼠喂食基础饲料12周,余4组均采用高糖高脂饮食联合维生素D3腹腔注射复制大鼠痰瘀互结型AS模型。模型组、西药组、低剂量组、高剂量组分别给予生理盐水、阿托伐他汀钙混悬液[1.8 mg/(kg·d)]、丹蒌片低剂量[450 mg/(kg·d)]、高剂量混悬液[900 mg/(kg·d)]灌胃干预8周。观察大鼠一般情况。灌胃结束后,处死大鼠取血清,检测大鼠血清中TC、TG、HDL-C、LDL-C、IL-6、TNF-α、单核细胞趋化蛋白-1(monocyte chemoattractant protein,MCP-1)、氧化低密度脂蛋白(oxidized low-density lipoprotein,ox-LDL)、LP-PLA2、s PLA2,HE染色观察大鼠胸主动脉病理学改变,Western blot和荧光定量PCR法分别检测大鼠胸主动脉内LP-PLA2、s PLA2蛋白及mRNA表达。结果与正常组比较,模型组大鼠精神萎靡,反应迟钝,胸主动脉可见典型的AS斑块,血清TC、TG、LDL-C、IL-6、TNF-α、MCP-1、ox-LDL、LP-PLA2、s PLA2水平升高(P<0.05),胸主动脉LP-PLA2、s PLA2蛋白及mRNA表达升高(P<0.05)。干预8周后,3个给药组大鼠均变活跃,胸主动脉HE染色见斑块消退。与模型组比较,3个给药组TC、TG、LDL-C、IL-6、TNF-α、MCP-1、ox-LDL、LP-PLA2水平降低(P<0.01,P<0.05),西药组血清s PLA2水平降低,大鼠胸主动脉LP-PLA2、s PLA2蛋白及mRNA表达降低(P<0.01,P<0.05);低剂量组胸主动脉LPPLA2蛋白及mRNA表达降低(P<0.01,P<0.05);高剂量组胸主动脉LP-PLA2、s PLA2蛋白及mRNA表达降低(P<0.01,P<0.05)。结论丹蒌片可能通过抑制LP-PLA2的表达,减少ox-LDL生成,从而发挥其抗炎、抗AS的作用。

Objective To observe the effects of Danlou Tablet（DT） on inflammatory reaction,and expressions of lipoprotein-associated phospholipase A2（LP-PLA2）,secretory phospholipase A2（sPLA2）,and to analyze potential mechanisms.Methods Forty male Wistar rats were randomly and equally divided into five groups,i.e.,the normal control group,the model group,the Western medicine（WM） group,the low dose DT group,the high dose DT group,8 in each group.Rats in the normal control group were fed with basic forage for 1 2 successive weeks,while AS rat model was established in rats of the other four groups by feeding high fat and sugar forage plus intraperitoneal injection of vitamin D_3.Normal saline,atorvastatin calcium suspension（at the daily dose of 1.8 mg/kg）,low dose DT suspension（at the daily dose of 450 mg/kg）,and high dose DT suspension（at the daily dose of 9 0 0 mg/kg） were administered to rats in the model group,the WM group,the low dose DT group,the high dose DT group respectively by gastragavage for 8 successive weeks.The general condition of all rats was observed.Rats were sacrificed after gastric administration and their serum collected.Serum levels of lipids（TC,TG,HDL-C,LDL-C） and inflammatory factors[IL-6,TNF-ct,monocyte chemoattractant protein 1（MCP-1）,oxidized low-density lipoprotein（ox-LDL）,lipoprotein-associated phospholipase A2（LP-PLA2）,secretory phospholipase A2（sPLA2）]were detected.Pathological changes of thoracic aorta were observed by HE staining.Protein and gene expressions of LP-PLA2 and sPLA2 in thoracic aorta were measured by Western blot and real-time fluorescent quantitative PCR respectively.Results Compared with the normal control group,rats in the model group were in low spirits and responded poorly.Typical atherosclerotic plaque could be seen in thoracic aorta of rats in the model group.Serum levels of TC,TG,LDL-C,IL-6,TNF-α,MCP-1,ox-LDL,LP-PLA2,and sPLA2 significantly increased（P 〈0.05）;protein and gene expressions of LP-PLA2 and sPLA2 in rat thoracic aorta increased（P 〈0.05） in the model group.After 8 weeks of intervention,rats in 3 medication groups appeared active,and HE staining showed subsidence of plaque in rat thoracic aorta.Compared with the model group,serum levels of TC,TG,LDL-C,IL-6,TNF-α,MCP-1,ox-LDL,and LP-PLA2 decreased in 3 medication groups（P 〈0.01,P 〈0.05）;serum sPLA2 level decreased,protein and mRNA expressions of LP-PLA2 and sPLA2 in rat thoracic aorta decreased in the WM group（P0.01,P0.05）;protein and mRNA expressions of LP-PLA2 in rat thoracic aorta significantly decreased in the low dose DT group（P0.01,P 〈0.05）,and those of LP-PLA2 and sPLA2 decreased in the high dose DT group（P 〈0.01,P 〈0.05）.Conclusion DT could fight against inflammatory reaction and AS possibly through inhibiting LP-PLA2 expression and reducing oxLDL production.