兰州大学机构库 >第一临床医学院
SGLT2抑制剂—达格列净抗动脉粥样硬化作用的机制研究
Alternative TitleThe Mechanism of Anti-atherosclerosis of Dapagliflozin, an SGLT2 Inhibitor
黄亚晓
Subtype硕士
Thesis Advisor张锦
2023-05-23
Degree Grantor兰州大学
Place of Conferral兰州
Degree Name医学硕士
Degree Discipline内科学
Keyword钠-葡萄糖协同转运蛋白2抑制剂 sodium-glucose cotransporter 2 inhibitor 达格列净 dapagliflozin 动脉粥样硬化 atherosclerosis 自噬 autophagy ROS-NLRP3 ROS-NLRP3
Abstract

背景和目的  动脉粥样硬化(AS)是冠心病典型的病理基础。炎症和血脂异常是AS的病理基础。临床研究已经证实,即使低密度脂蛋白(LDL)显著降低后,仍存在动脉粥样硬化性心血管疾病残余风险。这种残余风险的一部分可以用炎症来解释,因为抗炎治疗可以有效提高LDL抑制剂治疗的疗效。核苷酸结合寡聚化结构域样受体蛋白3(NLRP3)炎症小体被认为是脂质代谢和炎症之间的联系,因胆固醇结晶和氧化低密度脂蛋白(ox-LDL)可激活NLRP3炎性小体。还有研究发现,活性氧(ROS)可促使低密度脂蛋白(LDL)转化为ox-LDL,进一步导致ROS的分泌,并激活NLRP3炎症小体及下游细胞因子释放。细胞自噬可通过清除ROS的堆积,抑制NLRP3及后续因子的激活。本研究通过构建AS大鼠模型,探讨细胞自噬抑制ROS-NLRP3通路改善AS及达格列净是否通过激活自噬并抑制ROS-NLRP3通路的来抑制AS的发展,从动物水平探究达格列净改善AS的可能机制。

方法  将40只雄性SD大鼠分为空白对照组(n=10)、AS模型构建组(n=30)AS模型构建组给予维生素D3灌胃(前3天)+高脂饲料喂养16周诱导AS病变形成; AS模型成功后分为模型组、雷帕霉素组、达格列净组。雷帕霉素组、达格列净组分别予以雷帕霉素(1 mg·kg-1)、达格列净(3 mg·kg-1)灌胃4周,空白对照组及模型组则灌胃等量的0.9%NaCl 4周。每周监测体重。药物干预结束后,取大鼠主动脉行油红O染色并观察动脉粥样硬化病变面积;行苏木精-伊红染色(HE),观察泡沫细胞;采用比色法测试大鼠血清血糖、血脂水平。用酶联免疫吸附实验法检测大鼠血清ox-LDL、ROS和IL-1β的含量,用Western blot检测腹主动脉的微管相关蛋白轻链3(LC3)、选择性自噬接头蛋白(p62)和NLRP3蛋白的表达水平。

结果  

⑴体重  与AS模型构建成功时相比,药物干预4周后两组(雷帕霉素组和达格列净组)的体重明显减轻,差异有统计学意义(均P<0.05)。

⑵血清指标  空白对照组、模型组、雷帕霉素组和达格列净组血清LDL水平分别为(2.67 ± 1.94),(19.75 ± 4.86),(9.18 ± 5.16),(9.19 ± 1.46)mmol·L-1;ox-LDL水平分别为(375.06 ± 115.43)(586.39 ± 223.70),(358.17 ± 107.32)和(400.45 ± 129.21)pg·mL-1;ROS水平分别为(7.89 ± 1.07),(15.47 ± 2.65)(8.45 ± 2.20)和(8.17 ± 1.52)ng·mL-1;IL-1β水平分别为(62.97 ± 18.21),(171.22 ± 80.2),(65.50 ± 18.55)和(85.70 ± 38.82)pg·mL-1;模型组的上述指标与空白对照组、雷帕霉素组、达格列净组相比,差异均有统计学意义(均P<0.05)。

⑶组织形态学  药物干预结束后大鼠主动脉大体油红染色显示,模型组与空白对照组相比,其斑块面积明显增多。雷帕霉素及达格列净组与模型组相比,斑块面积明显减少,且两组之间无明显差异。HE染色显示,空白对照组的主动脉弓组织结构清楚,形态正常,模型组主动脉组织内膜增厚,可见泡沫细胞,用药组则明显减轻病变。

⑷组织蛋白表达  Western blot法检测大鼠腹主动脉NLRP3蛋白相对表达水平分别为0.52 ± 0.01,1.58 ± 0.03,0.80 ± 0.01和0.84 ± 0.05;LC3-Ⅱ/ LC3-Ⅰ蛋白相对表达水平分别为2.69 ± 0.19,0.55 ± 0.25,0.74 ± 0.25和0.99 ± 0.17,p62蛋白相对表达水平分别为0.72 ± 0.14,1.41 ± 0.14,0.91 ± 0.01和0.79 ± 0.01。模型组的上述指标与空白对照组、雷帕霉素组、达格列净组相比,差异均有统计学意义(均P<0.05)。

结论  SGLT2抑制剂—达格列净可能通过激活自噬,抑制ROS-NLRP3通路发挥抗动脉粥样硬化的作用。

Other Abstract

Background and Objective  Atherosclerosis (AS) is the typical pathological basis of coronary heart disease. Inflammation and dyslipidemia underlie the pathological basis of AS. Clinical studies have confirmed that there is still residual risk of atherosclerotic cardiovascular diseases (ASCVD) even after intense reduction of LDL. Some of this residual risk can be explained by inflammation as anti-inflammatory therapy is effective in improving the efficacy of LDL inhibitor therapy. Nod-like receptor pyrin domain-containing protein 3 (NLRP3) inflammasome is believed to be a link between lipid metabolism and inflammation, as cholesterol crystallization and oxidized low-density lipoprotein (ox-LDL) can activate NLRP3 inflammasome. Research has also found that reactive oxygen species (ROS) can promote the conversion of low-density lipoprotein (LDL) into ox-LDL, further leading to ROS secretion and activating NLRP3 inflammasomes and downstream cytokine release. Autophagy can inhibit the activation of NLRP3 and subsequent cytokines by clearing the accumulation of ROS. In this study, we constructed an AS rat model to investigate whether autophagy inhibits ROS-NLRP3 pathway to improve AS and whether dapagliflozin inhibits the development of AS by activating autophagy and inhibiting ROS-NLRP3 pathway, and explore the possible mechanism of daggligin to improve AS at the animal level.

Methods  Forty male SD rats were divided into a blank control group (n=10) and an AS model construction group (n=30). The AS model construction group was given vitamin D3 by gavage (the first 3 days) and fed with high-fat feed for 16 weeks to induce AS the formation of atheroscloma lesions .After successful modeling of AS , it was divided into a model group, a rapamycin group, and daggliflozin group. The rapamycin group and daggliflozin group were intragastrically administered  by rapamycin (1 mg·kg-1)and daggliflozin(3 mg·kg-1) for 4 weeks respectively, while the blank control group and model group were given the same amount of 0.9%NaCl for 4 weeks. At the end of drug intervention, aortic oil red O staining was observed the area of AS lesions; Hematoxylin eosin staining (HE) was used to observe foam cells; The serum levels of rat in GLU、TC、TG、HDL-C、LDL-C were determined by colorimetric method. The serum levels of rat in oxidized low density lipoprotein (ox-LDL), ROS and IL-1β in rats  were determined by enzyme-linked immunosorbent assay. Western blot was used to detect the expression level of microtubule-associated protein light chain 3 (LC3), selective autophagy adaptor protein(p62), NLRP3 proteins in rat abdominal aorta.

Results  

⑴Weight  Compared with the successful construction of the AS model, after 4 weeks of drug intervention, the weight of the two groups (rapamycin group and daggliflozin group) significantly reduced, with a statistically significant difference(all P<0.05).

⑵Serum indicators  The levels of the LDL-C in the plasma of the blank normal control group,model group ,rapamycin group and dapagliflozin group were (2.67 ± 1.94), (19.75 ± 4.86), (9.18 ± 5.16)and(9.19 ± 1.46)mmol·L-1; the levels of the oxLDL in the plasma of the blank normal control group,model group ,rapamycin group, and dapagliflozin group were(375.06 ± 115.43)(586.39 ± 223.70), (358.17 ± 107.32)and(400.45 ± 129.21)pg·mL-1; the levels of the ROS in the plasma of the blank normal control group,model group ,rapamycin group, and dapagliflozin group were (7.89 ± 1.07),(15.47 ± 2.65), (8.45 ± 2.20)and(8.17 ± 1.52)ng·mL-1; the levels of the IL-1β in the plasma of the blank normal control group, model group ,rapamycin group, and dapagliflozin group were (62.97 ± 18.21), (171.22 ± 80.2), (65.50 ± 18.55)和(85.70 ± 38.82)pg·mL-1; There were significant differences of the aboves between model group and blank normal control group and rapamycin group and dapagliflozin group(all P<0.05).

⑶Histomorphology   After the end of drug intervention, gross oil red staining of the aorta of rats showed that the plaque area of the model group was significantly increased , compared to the blank control group. Compared with the model group, the plaque area in the rapamycin and daggliflozin groups was significantly reduced, and there was no significant difference between the two groups. HE staining showed that the structure of aortic arch in the blank control group was clear and the morphology was normal. The intima of aortic tissue in the model group was thickened, and foam cells were visible. The drug group significantly alleviated the lesion.

⑷Tissue protein expression  The relative expressions levels of NLRP3 protein were 0.52 ± 0.01,1.58 ± 0.03,0.80 ± 0.01 and 0.84 ± 0.05; the relative expressions levels of LC3-Ⅱ/ LC3-Ⅰ protein were 2.69 ± 0.19, 0.55 ± 0.25, 0.74 ± 0.25 and 0.99 ± 0.17;  the relative expression levels of p62 protein were 0.72 ± 0.14, 1.41 ± 0.14, 0.91 ± 0.01 and 0.79 ± 0.01. There were significant differences of the aboves between model group and blank normal control group and rapamycin group and dapagliflozin group(all P<0.05).

Conclusion  Sodium-glucose cotransporter 2 inhibitor dapagliflozin may play an anti-atherosclerosis by activating autophagy and inhibiting ROS-NLRP3 pathway.

MOST Discipline Catalogue医学 - 临床医学 - 内科学(含:心血管病、血液病、呼吸系病、消化系病、内分泌与代谢病、肾病、风湿病、传染病)
URL查看原文
Language中文
Other Code262010_220200906040
Document Type学位论文
Identifierhttps://ir.lzu.edu.cn/handle/262010/539602
Collection第一临床医学院
Affiliation
兰州大学第一临床医学院
Recommended Citation
GB/T 7714
黄亚晓. SGLT2抑制剂—达格列净抗动脉粥样硬化作用的机制研究[D]. 兰州. 兰州大学,2023.
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