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The Disappearance of IPO in Myocardium of Diabetes Mellitus Rats Is Associated with the Increase of Succinate Dehydrogenase-Flav

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The Disappearance of IPO in Myocardium of Diabetes Mellitus Rats Is Associated with the Increase of Succinate Dehydrogenase-Flav Deng et al. BMC Cardiovasc Disord (2021) 21:142 https://doi.org/10.1186/s12872-021-01949-z RESEARCH ARTICLE Open Access The disappearance of IPO in myocardium of diabetes mellitus rats is associated with the increase of succinate dehydrogenase-favin protein Mengyuan Deng1†, Wei Chen1†, Haiying Wang1*, Yan Wang1, Wenjing Zhou2 and Tian Yu3 Abstract Background: The aim of the present study was to investigate whether the disappearance of ischemic post-process- ing (IPO) in the myocardium of diabetes mellitus (DM) is associated with the increase of succinate dehydrogenase- favin protein (SDHA). Methods: A total of 50 Sprague Dawley rats, weighing 300–400 g, were divided into 5 groups according to the ran- dom number table method, each with 10 rats. After DM rats were fed a high-fat and -sugar diet for 4 weeks, they were injected with Streptozotocin to establish the diabetic rat model. Normal rats were fed the same regular diet for the same number of weeks. Next, the above rats were taken to establish a cardiopulmonary bypass (CPB) model. Intraperi- toneal glucose tolerance test (IPGTT) and oral glucose tolerance test (OGTT) were used to detect whether the DM rat model was established successfully. Taking blood from the femoral artery to collect the blood-gas analysis indicators, and judged whether the CPB model is established. After perfusion was performed according to the experimental strategy, the area of myocardial infarction (MI), and serum creatine kinase isoenzyme (CK-MB) and cardiac troponin (CTnI) levels were measured. Finally, the relative mRNA and protein expression of SDHA was detected. Results: The OGTT and IPGTT suggested that the DM rat model was successfully established. The arterial blood gas analysis indicated that the CPB model was successfully established. As compared with the N group, the heart func- tion of the IR group was signifcantly reduced, the levels of myocardial enzyme markers, the area of MI, as well as the relative mRNA and protein expression of SDHA, were all increased. As compared with the IR group, the CK-MB and CTnI levels in the IPO group, the MI area, relative mRNA and protein expression of SDHA decreased. As compared with the IPO group, the myocardial enzyme content in the DM IPO group, the MI area and the relative mRNA and protein expression of SDHA increased. As compared with the DM + IPO group, in the DM IPO dme group, the myocardial enzyme content, area of MI and relative mRNA and protein+ expression were all decreased.+ + *Correspondence: [email protected] †Mengyuan Deng and Wei Chen have contributed equally to this work 1 Department of Anesthesiology, Afliated Hospital of Zunyi Medical University, 149 Dalian Road, Huichuan District, Zunyi 563003, Guizhou, People’s Republic of China Full list of author information is available at the end of the article © The Author(s) 2021. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creat iveco mmons .org/licen ses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creat iveco mmons .org/publi cdoma in/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Deng et al. BMC Cardiovasc Disord (2021) 21:142 Page 2 of 11 Conclusion: IPO can inhibit the expression of SDHA, reduce MIRI and exert a cardioprotective efect in the normal rats. However, the protective efect of IPO disappears in the diabetic rats. The inhibitor dme combined with IPO can increase the expression of SDHA and restore the protective efect of IPO in DM myocardia. Introduction expression of SDHA increases and is two times higher Myocardial ischemia–reperfusion injury (MIRI) is the than that of the control group, and that IPO can reduce pathological core of several cardiovascular diseases, the expression of SDHA and reduce MIRI [19]. Based such as stroke and myocardial infarction (MI) [1]. Its on the results of this study, we speculated that SDHA factors are multifaceted, such as the production of overexpression may be involved in MIRI, and the car- reactive oxygen species (ROS) during reperfusion [2], dioprotective efect of IPO may be associated with the and the opening of the permeability transition pore [3] inhibition of SDH. Te increased oxidative damage and activation of nuclear factor erythroid 2-related fac- response or energy metabolism disorder caused by the tor 2 [4]. Ischemia post-processing (IPO) can induce increased expression of SDHA may be the main rea- the miR-21 regulation axis through hypoxia-inducible son for the reduced anti-MIRI efect of IPO in the DM factor 1-α, downregulate the infammatory media- myocardium, and dme may restore the myocardial pro- tors programmed cell death protein 4 and Fas ligand, tective efect of IPO in the DM myocardium. inhibit infammatory response and exert myocardial protection [5]. In the diabetes mellitus (DM) myocar- Materials and methods dium, the anti-MIRI efect of IPO is weakened or dis- Animal experiment group appears [6, 7]—which may be due to the decreased A total of 50 rats were divided into fve groups accord- expression modulation of cystathionine γ-lyase [8]— ing to the random number table method (ten rats in phosphorylate extracellular signal related kinase fails each group): Te normal (N), ischemia reperfusion [9], phosphoinositide-3-kinase (PI3K) is activated (IR), ischemic post-treatment (IPO), diabetic mellitus [10], the mechanistic target of rapamycin is upregu- ischemia postconditioning (DM + IPO) and ischemic lated [11] and autophagy is reduced. But the specifc postconditioning of diabetic rats and combined of inhibi- mechanism remains unclear. SDH, also known as suc- tor group (dme is an inhibitor of SDHA), which is abbre- cinate ubiquinone oxidoreductase or mitochondrial viated as (DM + IPO + dme) groups (Fig. 1). complex II, is the only multi-subunit enzyme integrated in the inner mitochondrial membrane of the tricarbo- Materials xylic acid cycle (TCA). It is composed of two water- Dimethyl malonate (dme), streptozocin, citric acid and soluble heterodimers of dehydrogenase-favin protein sodium citrate were purchased from Merck KGaA. (SDHA) and succinate dehydrogenase-iron-sulfur pro- tein (SDHB), combined with two hydrophobic subunits SDHC (CybL) and SDHD (Cybs). However, as the only active group in the SDH structure, SDHA is a key fac- tor in coupling mitochondrial electron transport and respiratory function, while SDHB, C, D, etc. are inac- tive groups that only play a role in maintaining struc- tural stability [12]. Terefore, this experiment focused on SDHA. When SDH is mutated, it causes massive accumulation of succinic and fumaric acid [13], ade- nine nucleotide decomposition [14], partial reversal of the malic acid/aspartic acid shuttle process and the opening of the hypoxic signal pathway. When the mito- chondrial self-storage breathing capacity disappears completely, a large amount of ROS is generated, result- Fig. 1 Grouping and perfusion strategy in rats. A total of 50 standard ing in myocardial cells death [15]. male Sprague–Dawley rats were randomly divided into 5 groups (n 10, each group): The N, IR, IPO, DM IPO and DM IPO dme SDH competitive inhibitor-dimethyl malonate (dme) = + + + can reduce the accumulation of succinic acid and groups. DM, diabetes mellitus; N, normal; IR, ischemia–reperfusion; IPO ischemia post-conditioning, DM IPO DM ischemia + reduce oxidation, and fnally decrease MIRI [16–18]. post-conditioning, DM IPO dme DM ischemia post-conditioning + + Our previous study found that when MIRI occurs, the with inhibitor Deng et al. BMC Cardiovasc Disord (2021) 21:142 Page 3 of 11 Rabbit SDHA antibody (cat. no. EPR9043) was pur- easiest and smoothest here). After the tube had been chased from Abcam. Rabbit GAPDH antibody (cat. no. placed, the skin was cut longitudinally along the sternum, EPR16891) was purchased from Abcam. HRP Goat anti- the skin, was cut along the middle (parallel to the level of rabbit secondary antibody was purchased from Abcam the third rib) to expose and aortic root. Heparin (4 mg/ (cat. no. ab205718). Reverse transcription kit SYBG- kg) was injected through the tail vein. When ACT was QPCR kit was purchased from Vazyme. Te primers at > 480 s, the right jugular vein was opened for perfu- were purchased from Jiangsu Synthgene Biotechnology sion, and the target fow was adjusted to 100 ml/kg·min. Co, Ltd.. Succinate acid, rotenone and adenosine diphos- At this time, the CPB model was considered successful. A phate was purchased from Beijing Solarbio Science & mean arterial pressure (MAP) of > 60 mmHg was main- Technology Co., Ltd.. tained during the experiment. In the N group, perfusion was maintained at 100 ml/ Establishment of DM rat model kg·min for 100 min and then the experiment was ended. Adult male Sprague Dawley (SD) rats (age, 16–20 weeks; In the IR group, the aforementioned fow perfusion was weight, ~ 300 g) were purchased from Changsha Tianqin maintained for 10 min, the overhead light and insulation Biotechnology Co., Ltd. [license no., SCXK (Xiang) 2014- blanket were turned of, the aortic root was clamped so 0011].
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