摘要
目的:分析青蒿素通过调节Cav1.2/钙调蛋白(calmodulin,CaM)/2型钙调蛋白激酶(Ca^2+/calmodulin-dependent protein kinaseⅡ,CaMKⅡ)通路对心房纤颤(atrial fibrillation,AF)大鼠模型的治疗作用。方法:从60只SD大鼠中随机挑选出50只尾静脉注射乙酰胆碱(acetyl choline,Ach)和氯化钙(CaCl 2)混合液建立大鼠AF模型。建模成功后随机分为模型组、阳性对照组、青蒿素高剂量组、青蒿素中剂量组、青蒿素低剂量组,剩余10只为空白组。阳性对照组以维拉帕米6.36 mg溶于2 mL生理盐水后灌胃,每天1次;青蒿素高剂量组、青蒿素中剂量组和青蒿素低剂量组分别以青蒿素8 mg、4 mg和2 mg溶于2 mL生理盐水后灌胃,每天1次;模型组和空白组给予2 mL生理盐水灌胃。每天1次,连续给药28 d。大鼠给药期间继续尾静脉注射Ach-CaCl 2。给药后使用生物机能系统记录大鼠肢体Ⅱ导联心电图测AF持续时间;给药后使用Landendrff设备检测大鼠心房肌有效不应期;HE染色观察大鼠心房肌组织病理变化;RT-qPCR检测Cav1.2、CaM、CaMKⅡ、2型兰尼碱受体(ryanodine recepyor 2,RyR2)mRNA表达;通过蛋白免疫印迹法检测Cav1.2、CaM、CaMKⅡ、RyR2蛋白表达及2型兰尼碱受体磷酸化(phosphorylated-ryanodine recepyor 2,p-RyR2)水平。结果:AF持续时间比较:模型组大鼠AF持续时间显著长于空白组(P<0.05),阳性对照组和青蒿素各剂量组短于模型组且长于空白组(P<0.05),青蒿素高剂量组、青蒿素中剂量组短于阳性对照组、青蒿素低剂量组(P<0.05),青蒿素高剂量组短于模型组(P<0.05)。有效不应期比较:模型组显著短于空白组(P<0.05),阳性对照组、青蒿素各剂量组均长于模型组且短于空白组(P<0.05),其中青蒿素高剂量组、青蒿素中剂量组长于阳性对照组、青蒿素低剂量组(P<0.05),青蒿素高剂量组长于模型组(P<0.05)。HE染色结果:模型组心肌细胞病变严重,阳性对照组、青蒿素各剂量组病变程度均有所减轻,青蒿素高剂量组与空白组结果相近。Cav1.2 mRNA和蛋白表达比较:模型组Cav1.2 mRNA和蛋白表达显著低于空白组(P<0.05),阳性对照组、青蒿素各剂量组均高于模型组且低于空白组(P<0.05),青蒿素高剂量组、青蒿素中剂量组高于阳性对照组、青蒿素低剂量组(P<0.05),青蒿素高剂量组高于模型组(P<0.05)。CaM、CaMKⅡmRNA和蛋白表达及p-RyR2水平比较:模型组CaM、CaMKⅡmRNA和蛋白表达及p-RyR2水平高于空白组(P<0.05),阳性对照组、青蒿素各剂量组均低于模型组且高于空白组(P<0.05),青蒿素高剂量组、青蒿素中剂量组低于阳性对照组、青蒿素低剂量组(P<0.05),青蒿素高剂量组低于模型组(P<0.05)。结论:青蒿素能显著延长心房有效不应期,缩短大鼠AF持续时间,推测青蒿素通过上调Cav1.2钙离子通道表达水平,下调CaM和CaMKⅡ的表达水平,导致p-RyR2水平被抑制,从而对大鼠AF起到治疗效果。
Objective: To analyze the effects of artemisinin on atrial fibrillation by regulating the Cav1. 2/calmodulin(CaM)/2 type calmodulin kinase(Ca2 +/calmodulin-dependent protein kinase Ⅱ,CaMK Ⅱ) pathway,AF) Therapeutic effect of rat model.Method: 50 SD rats were randomly selected from 60 SD rats by tail vein injection of acetyl choline(Ach) and calcium chloride(CaCl2) mixture to establish rat AF models. After successful modeling,they were randomly divided into 5 groups. Group,positive control group,high-dose artemisinin group,medium-dose artemisinin group,low-dose artemisinin group,and the remaining 10 were blank control groups. The positive control group was given 6. 36 mg of verapamil dissolved in 2 m L of normal saline and gavage once a day;the artemisinin high-dose group,artemisinin medium-dose group and artemisinin low-dose group were given artemisinin respectively 8 mg,4 mg and 2 mg were dissolved in 2 m L of normal saline and then gavage once a day;the model group and blank control group were gavage with 2 m L of normal saline. It was administered once a day for 28 days. During the administration period,the rats continued to inject Ach-CaCl2 into the tail vein. After administration,the biological function system was used to record the duration of AF in the Ⅱ lead electrocardiogram of rat limbs;Landendrff equipment was used to detect the effective refractory period of rat atrial muscle after administration;HE staining was used to observe the pathological changes of rat atrial muscle tissue;RT-qPCR The m RNA expression of Cav1. 2,CaM,CaMKⅡ,and type 2 ryanodine receptor(Ry R2) was detected;the protein expression of Cav1. 2,CaM,CaMKⅡ,Ry R2 and the level of p-RyR2 were detected by Western blotting(WB). Results: Comparison of AF duration: The duration of AF in the model group was significantly longer than that of the blank control group(P < 0. 05),and the positive control group and artemisinin dose groups were shorter than the model group and longer than the blank control group(P < 0. 05). The artemisinin high-dose group and the artemisinin medium-dose group were shorter than the positive control group and the artemisinin low-dose group(P < 0. 05),and the artemisinin high-dose group was shorter than the model group(P <0. 05). Comparison of effective refractory period: the model group was significantly shorter than the blank control group(P <0. 05),the positive control group and artemisinin dose groups were longer than the model group and shorter than the blank control group(P < 0. 05),of which artemisinin The high dose group and the artemisinin medium dose group were longer than the positive control group,the artemisinin low dose group(P < 0. 05),and the artemisinin high dose group was longer than the model group(P < 0. 05). HE staining results: the cardiomyocyte pathological changes in the model group were severe. The pathological changes of the positive control group and the artemisinin dose groups were all reduced. The results of the high-dose artemisinin group and the blank control group were similar. Cav1. 2 m RNA and protein expression comparison: The Cav1. 2 m RNA and protein expression of the model group was significantly lower than that of the blank control group(P < 0. 05),and the positive control group and artemisinin dose groups were higher than the model group and lower than the blank control group Artemisinin high-dose group and artemisinin medium-dose group were higher than positive control group,artemisinin low-dose group(P < 0. 05),artemisinin highdose group was higher than model group(P < 0. 05). Comparison of CaM,CaMKⅡ m RNA and protein expression and p-RyR2 level: Model group CaM,CaMKⅡ m RNA and protein expression and p-RyR2 level were higher than that of blank control group(P < 0. 05),positive control group,artemisinin each dose The two groups were lower than the model group and higher than the blank control group(P < 0. 05). The artemisinin high-dose group and the artemisinin medium-dose group were lower than the positive control group and the artemisinin low-dose group(P < 0. 05). The artemisinin high-dose group was lower than the model group(P < 0. 05). Conclusion: Artemisinin can significantly prolong the effective atrial refractory period and shorten the duration of AF in rats. It is speculated that artemisinin can up-regulate the expression level of Cav1. 2 calcium channels and down-regulate the expression levels of CaM and CaMKⅡ,leading to p-RyR2 levels It is inhibited and has a therapeutic effect on rat AF.
作者
王宇
裴斐
WANG Yu;PEI Fei(The Second Affiliated Hospital of Xi′an Jiaotong University,Xi′an Shaanxi China 710004)
出处
《中医学报》
CAS
2020年第10期2182-2189,共8页
Acta Chinese Medicine
基金
国家自然科学基金面上项目(81672300)。
