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Qian Yang Yu Yin Granule Improves Renal Injury of Hypertension by Regulating Metabolic Reprogramming Mediated by HIF-1Α/PKM2 Positive Feedback Loop

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Qian Yang Yu Yin Granule Improves Renal Injury of Hypertension by Regulating Metabolic Reprogramming Mediated by HIF-1Α/PKM2 Positive Feedback Loop ORIGINAL RESEARCH published: 07 June 2021 doi: 10.3389/fphar.2021.667433 Qian Yang Yu Yin Granule Improves Renal Injury of Hypertension by Regulating Metabolic Reprogramming Mediated by HIF-1α/PKM2 Positive Feedback Loop Lichao Qian†, Shuai Ren†, Zhongchi Xu, Yawei Zheng, Lihua Wu, Ying Yang, Yixuan Wang, Jie Li, Shihai Yan* and Zhuyuan Fang* Jiangsu Province Hospital of Chinese Medicine, Affiliated Hospital of Nanjing University of Chinese Medicine, Nanjing, China Protection against hypoxia injury is an important therapeutic strategy for treating Edited by: Takashi Sato, hypertensive nephropathy. In this study, the effects of Qian Yang Yu Yin granule Tokyo University of Pharmacy and Life (QYYY) on spontaneously hypertensive rats fed with high salt diet and HEK293T cells Sciences, Japan exposed to hypoxia were investigated. After eight weeks’ treatment of QYYY, blood Reviewed by: pressure, serum creatinine, serum cystatin C, blood urea nitrogen, urinary β2- Omar Estrada, Instituto Venezolano de microglobulin, urinary N-acetyl-β-glucosaminidase, and urinary microalbumin were Investigaciones Científicas, Venezuela assessed. The changes of hypoxia-inducible factor-1α (HIF-1α), pyruvate kinase M2 Guang-Bo Ge, Shanghai University of Traditional (PKM2), glucose transport 1 (GLUT1), lactate dehydrogenase A (LDH-A), connective Chinese Medicine, China tissue growth factor (CTGF), transforming growth factor-β1 (TGF-β1), ATP, lactate, *Correspondence: pyruvate, and pathology were also assessed in vivo. HEK293T cells pre-treated with Shihai Yan QYYY and/or HIF-1α over expressing cells were cultured in a three gas hypoxic incubator [email protected] Zhuyuan Fang chamber (5% CO2,1%O2, 94% N2) for 12 h and then the expressions of HIF-1α, PKM2, [email protected] GLUT1, LDH-A, CTGF, TGF-β1, ATP, lactate, and pyruvate were detected. Our results † These authors have contributed showed that QYYY promoted the indicators of renal inflammation and fibrosis mediated by equally for this work HIF-1α/PKM2 positive feedback loop in vivo and vitro. Our findings indicated that QYYY Specialty section: treated hypertensive nephropathy by regulating metabolic reprogramming mediated by This article was submitted to HIF-1α/PKM2 positive feedback loop. Ethnopharmacology, a section of the journal Keywords: hypertensive nephropathy, metabolic reprogramming, HIF-1α, PKM2, Qian Yang Yu Yin granule Frontiers in Pharmacology Received: 13 February 2021 Accepted: 21 May 2021 INTRODUCTION Published: 07 June 2021 Citation: Hypertension is a commonly observed cardiovascular disease and kidney damage is one of the major Qian L, Ren S, Xu Z, Zheng Y, Wu L, complications found in patients with hypertension. Hypertension is one of the important causes of Yang Y, Wang Y, Li J, Yan S and Fang Z chronic kidney disease. Statistics from the American Nephrology Association in 2009 showed that (2021) Qian Yang Yu Yin Granule about 28% of patients with end-stage kidney disease were induced by hypertension (Mahmoodi et al., Improves Renal Injury of Hypertension 2012). Traditional Chinese medicine (TCM) has its unique curative effects in the treatment of by Regulating Metabolic α hypertensive renal damage, but the mechanism of TCM in the treatment of hypertensive renal Reprogramming Mediated by HIF-1 / fi “ ” PKM2 Positive Feedback Loop. damage is insuf cient (Guo et al., 2019). Under the guidance of the theory of blood stasis and heat Front. Pharmacol. 12:667433. in TCM, Qian Yang Yu Yin granule (QYYY) was created and applied as hospital preparation and has doi: 10.3389/fphar.2021.667433 been widely used in clinical practice for more than 20 years in Jiangsu Province Hospital of Chinese Frontiers in Pharmacology | www.frontiersin.org 1 June 2021 | Volume 12 | Article 667433 Qian et al. QYYY and Hypertensive Nephropathy Medicine. It is safe and effective in treating hypertensive kidney A large number of studies have confirmed that HIF-1α is injury. Previous studies of our project group showed that QYYY closely related to PKM2 (Luo et al., 2011). For instance, Hasan D had antioxidant, anti-inflammatory and anti fibrosis effects found that the antisense chain of the first intron of PKM gene (Chen et al., 2013; Wang et al., 2014; Yan D et al., 2014; contained hypoxia response element (HRE), and proved that Ding et al., 2015; Yan et al., 2018; Liu et al., 2019; Zhang HIF-1α significantly promoted the expression of PKM2, which et al., 2020). also formed a positive feedback mechanism at the gene level and Metabolic reprogramming refers to the change of cell played a key role in cell metabolic reprogramming (Hasan et al., metabolic mode. In normal cells, when oxygen is sufficient, 2018). In another study, Chai Xin Xin used shikonin, a specific cells primarily obtain energy through the process of oxidative inhibitor of PKM2, to down-regulate the expression of PKM2. phosphorylation. However, under hypoxia, cells obtain energy They found that the down-regulation of PKM2 in breast cancer through glycolysis, pentose phosphate and other pathways. This cells caused the down-regulation of HIF-1α (Chai et al., 2019). switch of cell energy acquisition mode has been termed as Therefore, we speculated that HIF-1α/PKM2 positive feedback is metabolic reprogramming (Vander et al., 2009; Krisher and the key pathway of metabolic reprogramming of renal cell in Prather, 2012). Kidney is an organ that requires high-energy hypertension. consumption. Therefore, the normal and optimal energy metabolism system serves as an important biochemical basis to maintain the specific structure and physiological functions MATERIALS AND METHODS of the kidney. A number of previous studies have reported that alteration in renal cell metabolism from oxidative Preparation of Qian Yang Yu Yin phosphorylation to glycolysis is the main feature of cell QYYY (Batch No. Z20100007) was obtained from Jiangsu activation during renal fibrosis and inhibition of renal cell Province of Chinese Medicine. It is made up of Cyathula glycolysis can significantly reduce renal fibrosis (Ding et al., officinalis K. C. Kuan [Amaranthaceae; cyathulae radix], 2017). Moreover, gene and protein analyses have showed that Scrophularia ningpoensis Hemsl [Scrophulariaceae; the expression of glycolytic enzyme was significantly up-regulated scrophulariae radix], Bidens pilosa L. [Asteraceae; Bidens in renal interstitial fibroblasts treated with unilateral ureteral bipinnata L.], Cornus officinalis Siebold and Zucc [Cornaceae; obstruction (UUO) or transforming growth factor-β1 (TGF- corni fructus], Alisma plantago-aquatica subsp. orientale (Sam.) β1). In nephropathy caused by UUO or renal interstitial Sam. [Alismataceae; alismatis rhizoma] and Reynoutria fibroblasts treated with TGF-β1, the aerobic glycolysis flux was multiflora (Thunb.) Moldenke [Polygonaceae; polygoni increased with glucose uptake and lactate production and it was multiflori radix] (Rivera et al., 2014). Detailed information on reported to be positively correlated with the fibrosis process. the components of QYYY is listed in Table 1. The preparing Glycolysis inhibitors can be used as a potential anti fibrosis process of QYYY was as below: six ingredients were mixed strategy. together. Then the mixture was decocted with 12 times of the Protection against hypoxia injury is an important therapeutic amount of water twice, for 1 h each time. The supernatant strategy for treating hypertensive nephropathy (Watson et al., obtained by high-speed centrifugation was concentrated to a 2019). Hypoxia inducible factor-1α (HIF-1α) is a special protein relative density of 1.2 g/ml at 60°C. After stevioside was added distributed in mammalian cells. Knockout or inhibition of HIF- to a concentration of 1% (weight/volume), the concentrated 1α effectively improved hypertensive renal injury (Huang et al., supernatant was mixed with dextrin and granulated in a 2019). In renal injury model, HIF-1α mediated gene expression in fluidized bed granulator. The obtained particles were dried at renal medulla such as glucose transporter 1 (GLUT1), pyruvate 60°C for 1 h and then stored at 4°C(Ding et al., 2015). High kinase M2 (PKM2), connective tissue growth factor (CTGF), performance liquid chromatogram (HPLC) was established to TGF-β1, and other related genes were increased. A related study determine the active components in QYYY and 2,3,5,4′-tetra- showed that long-term over-expression of HIF-1α was a hydroxystilbene-2-O-β-Dglucopyranoside (2,3,5,4-TDG), pathogenic factor leading to chronic kidney injury and morroniside, harpgide, ecdysterone, and hyperin were stimulating the expression of HIF-1α in cells induced renal detected. The quality evaluation of QYYY is shown in Table 2 injury, hypertension and disease progression (Armutcu et al., (Zhang et al., 2020). 2019). Moreover, a related study found that the level of serum HIF-1α may reflect the degree of damage of chronic Animals and Treatment glomerulonephritis and actively participate in the occurrence 50 spontaneously hypertensive rats (SHR), 10 Wistar-Kyoto rats and development of chronic glomerulonephritis (Dallatu et al., (WKY), 8 weeks old, male, weighing 200 ± 30 g, were purchased 2014). It has been reported that an increase of HIF-1α level could from Beijing Weitong Lihua Experimental Animal Technology participate in the formation of proteinuria, promote metabolic Co., Ltd. After four weeks of adaptive feeding, the rats were reprogramming and renal fibrosis, and thereby aggravate the weighed and administrated. SHR were randomly divided into five progressive deterioration of renal function.
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