利格列汀对糖尿病Wistar大鼠心脏保护作用的研究

doi:10.5246/jcps.2021.04.027

摘要/Abstract

摘要：

糖尿病性心肌损害(DCM)是糖尿病的一个重要的心血管并发症, 然而其发病机制未完全阐明。本文目的研究利格列汀对糖尿病大鼠心肌细胞的影响及其作用机制。用二维超声评价各组大鼠不同时间点的心肌功能; HE染色观察心肌损伤及炎症情况; 采用天狼星红染色, 光镜下观察心肌纤维化程度; TUNEL染色观察心肌细胞凋亡程度; 采用逆转录聚合酶链反应(RT-PCR)和Western blot法分别检测各组相关细胞Bcl-2、Bax、TNF-α、PAI-1、CTGF和TGF-β1的mRNA表达。用RT-PCR、Western blot和免疫荧光技术研究Nrf2相关因子在心肌中的表达及转录功能。结果表明, 糖尿病 + 利格列汀组大鼠的左室体积(LV)、左室厚度(LT)、空腹血糖水平和心重/体重(HW/BW)均显著低于糖尿病组(P < 0.05), 而射血分数(EF)高于糖尿病组(P < 0.05)。从HE染色图像来看, 利格列汀的治疗使心肌纤维排列更加规则, 心肌细胞条纹清晰。Sirus-red染色显示糖尿病组大鼠有明显的胶原聚集, 心肌纤维化明显, 而糖尿病+利格列汀组症状减轻。TUNEL染色显示, 4周时糖尿病+利格列汀组心肌细胞凋亡程度明显低于糖尿病组(P < 0.01)。利格列汀能使糖尿病大鼠上调的cleaved-caspase-3、TNF-α、PAI-1、CTGF、TGF-β1等蛋白的表达水平下降, 具有统计学意义(cleaved-caspase-3: P < 0.01; TNF-α: P < 0.01; PAI-1: P < 0.05; CTGF: P < 0.05; TGF-β1: P < 0.05); 在糖尿病+利格列汀组, Bcl-2/Bax比值较糖尿病组增加, 具有统计学意义(Bcl-2/Bax: P < 0.05)。通过RT-PCR、western-blot和免疫荧光法检测Nrf2在细胞核和胞浆中的表达, 发现利格列汀促进心肌组织细胞Nrf2核移位。并且糖尿病大鼠心脏Nrf2表达明显下调(P < 0.01), 而糖尿病+利格列汀组Nrf2下降趋势明显改善。本文研究了利格列汀对糖尿病心肌损伤的影响, 发现其保护机制可能与抗氧化应激Nrf2信号通路有关, 这为糖尿病性心肌损害的防治提供了新的思路和治疗靶点。

关键词: 糖尿病性心肌病, 利格列汀, Nrf2, 氧化应激反应

Abstract:

Diabetic cardiomyopathy (DCM) is an important cardiovascular complication of diabetes mellitus, while the pathogenesis of DCM has not been fully elucidated. In the present study, we aimed to investigate the effect of Linagliptin on cardiomyocytes of diabetic rats and its mechanism. Cardiac function was evaluated by two-dimensional ultrasound at different time points for each group. HE staining was used to evaluate myocardial injury and inflammatory condition. Sirius-red staining was used to observe the degree of myocardial fibrosis under optical microscope. TUNEL staining was used to investigate the degree of cardiomyocyte apoptosis in four groups. The expressions of mRNAs in relevant cells, including Bcl-2, Bax, TNF-α, PAI-1, CTGF and TGF-β1, were measured by reverse transcription polymerase chain reaction (RT-PCR) and Western blotting analysis in different groups. The expression and transcriptional function of Nrf2 in myocardium activated by Lingliptin were determined using RT-PCR, Western blotting analysis and immunofluorescence. The results showed that the left ventricular volume (LV), left ventricular thickness (LT), fasting blood glucose (FBG) and heart weight/body weight (HW/BW) in diabetes and Linagliptin CO treatment group were significantly lower compared with diabetic group (P < 0.05), while the ejection fraction (EF) was higher compared with diabetic group (P < 0.05). From HE staining, the treatment of Linagliptin made the arrangement of myocardial fibers more regular, and the striation of myocardial cells became clearer. The Sirus-red staining showed that there was significant accumulation of collagen in the diabetic group rats, indicating that the rats in diabetic group had cardiac fibrosis. The phenomenon in diabetes and Linagliptin CO treatment group was alleviated. TUNEL staining showed that at time point of 4 weeks, the degree of cardiomyocyte apoptosis in diabetes and Linagliptin CO treatment group was lower compared with diabetic group (P < 0.01). The expressions of cleaved-caspase-3, TNF-α, PAI-1, CTGF and TGF-β1 proteins in diabetic rats were significantly decreased by Linagliptin, and the difference was statistically significant (cleaved-caspase-3: P < 0.01; TNF-α: P < 0.01; PAI-1: P < 0.05; CTGF: P < 0.05; TGF-β1: P < 0.05). Compared with the diabetic group, the ratio of Bcl-2/Bax was inecreased in diabetes and Linagliptin CO treatment group, and the difference was statistically significant (P < 0.05). From Nrf2 expression in the nucleus and cytosol by RT-PCR, Western blotting analysis and immunofluorescence test, the results showed that Linagliptin promoted the Nrf2 nuclear translocation in myocardial tissue cells. The expression of Nrf2 was significantly down-regulated in the heart of diabetic rats (P < 0.01), while this phenomenon in diabetes and Linagliptin CO treatment group was greatly ameliorated. This paper studied the effect of Linagliptin on diabetic myocardial injur and found that the protective mechanism might be related to Nrf2 signaling pathway of antioxidant stress. Collectively, our finding provided new ideas and therapeutic targets for the prevention and treatment of DCM.

Key words: Diabetic cardiomyopathy, Linagliptin, Nrf2, Oxidative stress

Supporting:

李荣, 宋利华, 刘杰, 白杨, 杜玉茗, 林春华, 苏秀媛, 于宗学. 利格列汀对糖尿病Wistar大鼠心脏保护作用的研究[J]. 中国药学(英文版), 2021, 30(4): 334-346.

Rong Li, Lihua Song, Jie Liu, Yang Bai, Yuming Du, Chunhua Lin, Xiuyuan Su, Zongxue Yu. Cardioprotective effect of Linagliptin on diabetic Wistar rats[J]. Journal of Chinese Pharmaceutical Sciences, 2021, 30(4): 334-346.

图/表 14

Table 1. Feeding and drug treatment of rats in each group (lasting for 4 weeks).

Table 2. Primer sequences of target genes.

Table 3. Comparisons of myocardial function parameters at different time points for each group (n = 5).

Figure 1. Hematoxylin-eosin inflammation staining results of myocardial tissue at different time points in each group (× 200). (A) Control group; (B) Linagliptin treatment group; (C) Diabetes group; (D) Diabetes and Linagliptin CO treatment group.

Figure 2. Sirius red fibrosis staining results of myocardial tissue at different time points in each group (× 200). (A) Control group; (B) Linagliptin treatment group; (C) Diabetes group; (D) Diabetes and Linagliptin CO treatment group.

Figure 3. TUNEL apoptosis staining results of myocardial tissue at different time points in each group (× 400). (A) Control group; (B) Linagliptin treatment group; (C) Diabetes group; (D) Diabetes and Linagliptin CO treatment group.

Figure 4. Apoptotic rates of cardiomyocytes at different time points in each group (n = 5). **P < 0.01, compared with group C; ##P < 0.01, compared with group A and group B.

Table 4. The ratio of Bcl-2/Bax at different time points in each group (n = 5).

Figure 5. mRNA expressions of cleaved caspase-3, TNF-α, PAI-1, CTGF and TGF-β1 in myocardial tissue at different time points in each group (n = 5). *P < 0.05, compared with group C; **P < 0.01, compared with group C.

Figure 6. Immunoblotting of Bcl-2, Bax, TNF-α, PAI-1, CTGF, TGF-β1 and cleaved caspase-3 at different time points in each group (n = 5).

Figure 7. Nrf2 protein expression at different time points in each group (n = 5). (A) Cytosol; (B) Nucleus.

Figure 8. Nrf2 mRNA expression of cytosol at different time points in each group (n = 5).

Figure 9. Nrf2 mRNA expression of nucleus at different time points in each group (n = 5).

Figure 10. The nuclear expression of Nrf2 at different time points in each group (n = 5). (A) Control group; (B) Linagliptin treatment group; (C) Diabetes group; (D) Diabetes and Linagliptin CO treatment group.

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