Tetramethylpyrazine attenuates chronic intermittent hypoxia-exacerbated diabetic atherosclerosis: a mechanistic study of the IRE1α-XBP1 signaling pathway

Abstract

Objective: A complex relationship exists between obstructive sleep apnea syndrome and diabetes mellitus (DM). Chronic intermittent hypoxia (CIH), which is a core pathological feature of obstructive sleep apnea syndrome, may play an important role in the onset and development of DM-related atherosclerosis (DM-AS). This study aimed to investigate the mechanism of action of tetramethylpyrazine (TMP) in CIH-associated DM-AS.

Methods: In vivo, a DM-AS mouse model was established by intraperitoneal injection of streptozotocin combined with a high-fat diet. They were exposed to CIH or normoxic conditions for 8 weeks and received different doses of TMP, rosuvastatin, toyocamycin, and purified water. Glycolipid metabolism, inflammation levels, degree of aortic AS, and expression levels of endoplasmic reticulum stress (ERS) and autophagy proteins were examined in mice. In vitro, human umbilical vein endothelial cells (HUVECs) were treated with high glucose and fat in combination with insulin to establish an insulin-resistant cell model (HUVEC-IR). After pretreatment with 4μ8C (IRE1 inhibitor) and different doses of TMP, intermittent hypoxic intervention was performed. Changes in cell morphology, proliferative activity, glucose consumption, and ability to migrate were observed, and the expression levels of ERS and autophagy proteins were detected.

Results: In vivo experiments showed that CIH significantly increased blood glucose levels and Homeostasis Model Assessment of Insulin Resistance (HOMA-IR) (P < 0.001 or P < 0.05), low-density lipoprotein cholesterol (LDL-C) content (P < 0.001), and the levels of three inflammatory factors [interleukin-1 beta (IL-1β), IL-6, and tumor necrosis factor-alpha (TNF-α)] in the mice (P < 0.001) compared with those of the mice with DM alone; moreover, the aortic atherosclerotic (AS) plaque area in CIH mice was significantly enlarged (P < 0.001). Western blotting results showed that the expressions of aortic IRE1α, XBP1s, Beclin1, and LC3A proteins were significantly increased in CIH mice compared with DM mice (P < 0.05). After treatment with different doses of TMP, rosuvastatin calcium, and toyocamycin, serum inflammation and lipid levels and plaque area were significantly reduced in mice (P < 0.001 and P < 0.001, respectively). The expression levels of aortic XBP1s, Beclin1, and LC3A were reduced in TMP- and toyocamycin-treated mice (P < 0.05). In the in vitro experiments, compared with normoxic cells, the cells treated with intermittent hypoxia (IH) showed a significant decrease in cell migration distance (P < 0.05), a significant increase in apoptosis rate (P < 0.001), a substantial increase in proliferation inhibition rate (P < 0.001), a significant increase in the levels of XBP1s and LC3A proteins (P < 0.05), and an increase in the number of autophagic vesicles/lysosomes, as observed under transmission electron microscopy. After treatment with different doses of TMP and 4μ8C, cell morphology was significantly restored, apoptosis rate significantly reduced (P < 0.001), and XBP1s, Beclin1, and LC3A expressions significantly inhibited (P < 0.05).

Conclusion: CIH promoted the onset of DM-AS, whereas TMP attenuated ERS and excessive autophagy by modulating the IRE1α-XBP1 signaling pathway and inhibiting XBP1 splicing, thereby ameliorating DM-AS exacerbated by CIH.

Keywords

Arteriosclerosis / Diabetes / Intermittent hypoxia / IRE1α-XBP1 / TMP

Conflict of interest statement

The authors declare no conflict of interest.

Funding

The research fund of this project comes from the National Natural Science Foundation of China (82074264).

Author contributions

Yue Liu designed the manuscript. Binyu Luo and Wenting Wang did the literature search, wrote the manuscript, and drafted figures. Yanfei Liu, Yiwen Li, Binyu Luo, and Wenting Wang revised the manuscript. All authors listed have made a substantial contribution to the work. All authors have read and approved the article.

Ethical approval of studies and informed consent

This animal study was approved by the Animal Ethics Committee of Xiyuan Hospital, China Academy of Chinese Medical Sciences (2021XLC046-1).

Acknowledgments

None.

Data availability

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

References

[1] Marin JM, Carrizo SJ, Vicente E, et al. Long-term cardiovascular outcomes in men with obstructive sleep apnoea-hypopnoea with or without treatment with continuous positive airway pressure: an observational study. Lancet (London) 2005; 365(9464): 1046-1053.

[2] Yaggi HK, Concato J, Kernan WN, et al. Obstructive sleep apnea as a risk factor for stroke and death. N Engl J Med 2005; 353(19):2034-2041.

[3] De Vega Sánchez B, Juarros Martínez SA, Del Olmo Chiches M, et al. Obstructive sleep apnoea, type 2 diabetes and cardiovascular risk factors. Eur J Intern Med 2017;39:e16-e17.

[4] Huang T, Lin BM, Stampfer MJ, et al. A population-based study of the bidirectional association between obstructive sleep apnea and type 2 diabetes in three prospective U.S. cohorts. Diabetes Care 2018; 41(10):2111-2119.

[5] Muraki I, Wada H, Tanigawa T. Sleep apnea and type 2 diabetes. J Diabetes Investig 2018; 9(5):991-997.

[6] Trzepizur W, Le Vaillant M, Meslier N, et al. Independent association between nocturnal intermittent hypoxemia and metabolic dyslipidemia. Chest 2013; 143(6):1584-1589.

[7] Ma L, Zhang J, Qiao Y, et al. Intermittent hypoxia composite abnormal glucose metabolism-mediated atherosclerosis in vitro and in vivo: the role of SREBP-1. Oxid Med Cell Longev 2019;2019:4862760.

[8] Tian J, Yue L, Lizhi L, et al. Endoplasmic reticulum stress and autophagy: a potential therapeutic target for atherosclerosis. Life Sci 2017; 29(4):356-363.

[9] Tufanli O, Telkoparan Akillilar P, Acosta-Alvear D, et al. Targeting IRE1 with small molecules counteracts progression of atherosclerosis. Proc Natl Acad Sci USA 2017; 114(8):E1395-E1404.

[10] Huang S, Xing Y, Liu Y. Emerging roles for the ERS sensor IRE1α in metabolic regulation and disease. J Biol Chem 2019; 294(49):18726-18741.

[11] Margariti A, Li H, Chen T, et al. XBP1 mRNA splicing triggers an autophagic response in endothelial cells through BECLIN-1 transcriptional activation. J Biol Chem 2013; 288(2):859-872.

[12] Tian PG, Jiang ZX, Li JH, et al. Spliced XBP1 promotes macrophage survival and autophagy by interacting with Beclin-1. Biochem Biophys Res Commun 2015; 463(4):518-523.

[13] Chen HR. Cluster Analysis of Traditional Chinese Medicine Syndrome of Obstructive Sleep Apnea Hypopnea Syndrome Combined with Type 2 Diabetes Mellitus and its Correlation with Insulin Resistance. Fujian University of Chinese Medicine, 2019.

[14] Xudong Z, Lu H. Value of thrombologram in type 2 diabetes mellitus patients with obstructive sleep apnea hypopnea syndrome. J Clin Med Lit Electron 2018; 5(6):6-8.

[15] Guo M, Liu Y, Shi D. Cardiovascular actions and therapeutic potential of tetramethylpyrazine (active component isolated from Rhizoma Chuanxiong): roles and mechanisms. Biomed Res Int 2016;2016:2430329.

[16] Zhao Y, Liu Y, Chen K. Mechanisms and clinical application of tetramethylpyrazine (an Interesting Natural Compound Isolated from Ligusticum Wallichii): current status and perspective. Oxid Med Cell Longevity 2016;2016:2124638.

[17] Ma L, Zhang J, Liu Y, et al. Intervention effects of qi-benefiting and blood-activating traditional Chinese medicine combinations on Sterol regulatory element-binding protein 1c/Fatty acid synthase (SREBP-1c/FAS) signaling pathway in an intermittent hypoxia-complex insulin resistance cell model. World Sci Technol Modernization Tradit Chin Med 2019; 21(11):2516-2525.

[18] Sun WT, Wang XC, Novakovic A, et al. Protection of dilator function of coronary arteries from homocysteine by tetramethylpyrazine: role of ERS in modulation of BKCa channels. Vascul Pharmacol 2019;113:27-37.

[19] Jiang K, Xu Y, Wang D, et al. Cardioprotective mechanism of SGLT2 inhibitor against myocardial infarction is through reduction of autosis. Protein Cell 2022; 13(5):336-359.

[20] Asaf R, Blum S, Roguin A, et al. Haptoglobin genotype is a determinant of survival and cardiac remodeling after myocardial infarction in diabetic mice. Cardiovasc Diabetol 2009;8:29.

[21] Andrés-Manzano MJ, Andrés V, Dorado B. Oil Red O and hematoxylin and eosin staining for quantification of atherosclerosis burden in mouse aorta and aortic root. Methods Mol Biol 2015;1339:85-99.

[22] Zhu J, Liu B, Wang Z, et al. Exosomes from nicotine-stimulated macrophages accelerate atherosclerosis through miR-21-3p/ PTEN-mediated VSMC migration and proliferation. Theranostics 2019; 9(23):6901-6919.

[23] Wang W, Wang S, Li Y, et al. Network pharmacology, molecular docking, and in vitro experimental verification of the mechanism of Guanxining in treating diabetic atherosclerosis. J Ethnopharmacol 2024;324:117792.

[24] Zhang Y, Liu D, Hu H, et al. HIF-1α/BNIP3 signaling pathway-induced-autophagy plays protective role during myocardial ischemia-reperfusion injury. Biomed Pharmacother 2019;120: 109464.

[25] Dong Y, Fernandes C, Liu Y, et al. Role of endoplasmic reticulum stress signalling in diabetic endothelial dysfunction and atherosclerosis. Diab Vasc Dis Res 2017; 14(1):14-23.

[26] Galán M, Kassan M, Kadowitz PJ, et al. Mechanism of endoplasmic reticulum stress-induced vascular endothelial dysfunction. Biochim Biophys Acta 2014; 1843(6):1063-1075.

[27] Yao ST, Qin SC. The relationship of autophagy with endoplasmic reticulum stress and its role in pathogenesis, prevention and therapy of atherosclerosis. Sheng Li Xue Bao 2017; 69(4):515-521.

[28] Poznyak A, Grechko AV, Poggio P, et al. The diabetes mellitus-atherosclerosis connection: the role of lipid and glucose metabolism and chronic inflammation. Int J Mol Sci 2020; 21(5):1835.

[29] Nagayoshi M, Punjabi NM, Selvin E, et al. Obstructive sleep apnea and incident type 2 diabetes. Sleep Med 2016;25:156-161.

[30] Ota H, Fujita Y, Yamauchi M, et al. Relationship between intermittent hypoxia and type 2 diabetes in sleep apnea syndrome. Int J Mol Sci 2019; 20(19):4756.

[31] Zheng L, Zhuo W, Yanzhen G, et al. Study on the protective effect of ligustrazine on intermittent hypoxia-induced pulmonary artery endothelial injury in rats. World Clin Med 2016; 10(9):1-2.

[32] Metcalf MG, Higuchi-Sanabria R, Garcia G, et al. Beyond the cell factory: homeostatic regulation of and by the UPR(ER). Sci Adv 2020; 6(29):eabb9614.

[33] Hetz C, Zhang K, Kaufman RJ. Mechanisms, regulation and functions of the unfolded protein response. Nat Rev Mol Cell Biol 2020; 21(8):421-438.

[34] Wu R, Zhang QH, Lu YJ, et al. Involvement of the IRE1α-XBP1 pathway and XBP1s-dependent transcriptional reprogramming in metabolic diseases. DNA Cell Biol 2015; 34(1):6-18.

[35] Sha H, He Y, Yang L, et al. Stressed out about obesity: IRE1α- XBP1 in metabolic disorders. Trends Endocrinol Metab 2011; 22(9):374-381.

[36] Chen Y, Wu Z, Huang S, et al. Adipocyte IRE1α promotes PGC1α mRNA decay and restrains adaptive thermogenesis. Nat Metab 2022; 4(9):1166-1184.

[37] Zeng L, Zampetaki A, Margariti A, et al. Sustained activation of XBP1 splicing leads to endothelial apoptosis and atherosclerosis development in response to disturbed flow. Proc Natl Acad Sci U S A 2009; 106(20):8326-8331.

[38] Jinfan T, Yue L, Zhi L, et al. Endoplasmic reticulum stress, autophagy and their interaction: a potential new target for the treatment of atherosclerosis. Life sci 2017; 29(4):356-363.