Background/Aims: Clinical studies have shown that hyperuricaemia is strongly associated with cardiovascular disease. However, the molecular mechanisms of high uric acid (HUA) associated with cardiovascular disease remain poorly understood. In this study, we investigated the effect of HUA on cardiomyocytes. Methods: We exposed H9c2 cardiomyocytes to HUA, then cell viability was determined by MTT assay, and reactive oxygen species’ (ROS) production was detected by a fluorescence assay. Western blot analysis was used to examine phosphorylation of extracellular signal-regulated kinase (ERK), p38, phosphatidylinositol 3-kinase (PI3K) and Akt. We monitored the impact of HUA on phospho-ERK and phospho-p38 levels in myocardial tissue from an acute hyperuricaemia mouse model established by potassium oxonate treatment. Results: HUA decreased cardiomyocyte viability and increased ROS production in cardiomyocytes; pre-treatment with N-acetyl-L-cysteine, a ROS scavenger, and PD98059, an ERK inhibitor, reversed HUA-inhibited viability of cardiomyocytes. Further examination of signal transduction pathways revealed HUA-induced ROS involved in activating ERK/P38 and inhibiting PI3K/Akt in cardiomyocytes. Furthermore, the acute hyperuricaemic mouse model showed an increased phospho-ERK/p38 level in myocardial tissues. Conclusion: HUA induced oxidative damage and inhibited the viability of cardiomyocytes by activating ERK/p38 signalling, for a novel potential mechanism of hyperuricaemic-related cardiovascular disease.

Hyperuricaemia has been identified as a potential risk factor for gout, dyslipidaemia and hypertension in the clinic but is also strongly associated with cardiovascular diseases, including atrial fibrillation, ischaemic heart disease and heart failure [1‒6]. Moreover, increased uric acid (UA) concentration may reflect increased activity of the xanthine oxidase pathway, an important source of oxygen free radicals, which are related to various detrimental processes, such as increased cytokine production, cell apoptosis and endothelial dysfunction and may contribute to abnormal energy metabolism in human cardiomyopathy [7‒10]. However, the molecular mechanisms of high UA (HUA) level associated with cardiovascular disease remain poorly understood.

Reactive oxygen species (ROS) are generated by a variety of endogenous and exogenous processes through several pathways; mitochondria are the major source of intracellular ROS [11‒12]. ROS are generally believed to be harmful to cells and tissues [13]. HUA levels increase oxidative stress in multiple cells [14‒18] and cause damaged endothelia, renal injury, insulin resistance and inflammation. The three members of the mitogen-activated protein kinase (MAPK) family, extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK) and p38, regulate a variety of physiological processes, such as cell growth, metabolism, differentiation and cell death. Several signalling cascades, including those involving the MAPKs p38 and ERK, as well as phosphoinositide 3-kinase (PI3K)/Akt and JNK, regulate the dedifferentiation of cells by modulating the generation of ROS [19‒20]. Although ROS may inhibit cell differentiation, the mechanisms involved have not been fully elucidated. A previous study of rabbit chondrocyte cartilage degradation suggested that ROS modulates the ERK/p38 signalling cascades, thereby leading to dedifferentiation and inflammation. However, whether HUA interferes with ERK/p38 signalling in cardiomyocytes remains unclear.

In the present study, we investigated the effect of HUA on oxidative stress, ERK/p38 and PI3K/Akt signalling, as manifested by changes in ROS production, cardiomyocyte viability and phospho-ERK/p38 and phospho-PI3K/Akt activity in cardiomyocytes and cardiac tissue from an acute hyperuricaemia mouse model. We examined whether the antioxidant, N-acetyl-L-cysteine (NAC), and PD98059, an ERK inhibitor, could prevent HUA-inhibited cardiomyocyte viability, to examine the potential role of oxidative stress and ERK/p38 signalling in the process.

Reagents

3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT), 2, 7,-dichlorodihydrofluorescein diacetate (DCFH-DA), UA, PD98059 and insulin were from Sigma (St. Louis, MO, USA). Anti-phospho/total-ERK, anti-phospho-Akt and anti-Akt antibodies were from Bioworld (St. Louis Park, MN, USA). Anti-phospho/total-p38 antibodies were from Cell Signaling (Danvers, MA, USA). Anti-phospho-PI3K and anti-Pi3K antibodies, rabbit GAPDH and SB203580 were from Abcam. NAC was from ENZO Life Sciences (Farmingdale, NY, USA). All chemical reagents were of analytical grade. For the primary buffer, a UA (final concentration 15 mg/dl) stock solution was prepared at 15 mg/ml in 0.5 M NaOH. An NAC stock solution was prepared at 500 mM in ultrapure water. In experiments involving reagents, cells were pre-treated with antioxidant (NAC, 5 mM), ERK inhibitor (PD98059, 20 mM) or insulin (100 nM) for 30 min before adding UA.

Cell culture and treatment

Cellular studies were conducted with H9c2 rat heart–derived embryonic myocytes (CRL-1446; American Type Culture Collection, Manassas, VA) incubated with DMEM or low-glucose MEM supplemented with 10% foetal bovine serum (FBS), 100 U/ml penicillin G, 100 mg/ml streptomycin, and 2 mM L-glutamine. Primary cardiomyocytes were obtained from neonatal C57BL/6 mice, as previously reported [21]. Cells were incubated at 37°C with 5% CO2 and 95% air. For all experiments, cells were plated in 6-well plates at 2.0×105 cells/ml. For HUA treatment, cells were incubated with 15 mg/dl HUA in fresh cell-culture medium for 24 h or the indicated times, then harvested for biochemical or molecular assays. All experiments were repeated at least 3 times.

Cell viability

The viability of H9c2 cardiomyocytes was determined by MTT assay. Cells were inoculated at 4000 cells/well into 96-well plates and treated with UA at various concentrations (0–15 mg/dl). At 12, 24, 48, and 72 h after exposure to UA, the culture medium was discarded and 150 µl of MTT solution was added to each well (0.5 mg/ml, final concentration) and incubated at 37°C for 4 h. Then, MTT solution was removed and 150 µl of DMSO was added to dissolve the formazan crystals. The absorbance was measured at 492 nm on a microplate reader. The optical density of the treated cells was normalized to that of control cells (0 mg/dl).

Measurement of intracellular ROS levels

Cells were subcultured in 6-well plates (2.0×105 cells/well), allowed to attach for 24 h, exposed to HUA (15 mg/dl) for 24 h, and stained with 10 mM DCFH-DA for 30 min at 37°C as described [22]. Stained cells were imaged by fluorescence microscopy and analysed by flow cytometry with excitation and emission at 530 and 480 nm, respectively.

Animals

This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the U.S. National Institutes of Health. The protocol was approved by the Committee on the Ethics of Animal Experiments of the University of Shantou (Permit no. SYXK2007-0079). Eight-week-old male C57BL/6J mice (20±2 g) were from Vital River Laboratories (Beijing) and housed in the Laboratory Animal Center of Shantou University Medical College. All surgery was performed under sodium pentobarbital anaesthesia, and all efforts were made to minimize suffering. Mice were fed a standard diet and maintained in individual cages with routine light–dark cycles and allowed to adapt to the laboratory environment for 1 week. Animals were anaesthetized by injecting sodium pentobarbital (50 mg/kg intraperitoneally). Single ventricular myocytes were isolated as described [23].

Ten-week-old male C57BL/6J mice were randomly assigned to 2 groups (n = 4 each) for treatment: control and HUA. For HUA treatment, following an 18-h overnight fast, mice received an intraperitoneal (i.p.) injection of potassium oxonate (300 mg/kg) and intragastric administration of hypoxanthine (500 mg/kg) for 1–2 h to create the acute hyperuricaemia model. The volume of the drug was based on body weight measured immediately before each dose. Then, the serum UA level was measured at different times by the phosphotungstic acid method [24]. For control and HUA mice, left-ventricle cardiac muscle tissue was excised. All tissue samples were immediately stored in liquid nitrogen.

Western blot analysis

Cells were lysed, sonicated and homogenized in radioimmunoprecipitation assay (RIPA) buffer, supplemented with protease inhibitors (1 mmol/l phenylmethanesulfonyl fluoride, PMSF) and phosphatase inhibitors (phosphatase inhibitor mixture I). The supernatant protein concentration was determined by use of the BCA Protein Assay Kit (Pierce, IL, USA), then equal amounts of total protein underwent 10% SDS-PAGE and were transferred to polyvinylidene difluoride membranes (Millipore, Shanghai), which were blocked with 5% non-fat milk and incubated with primary antibodies for phosphorylated and total proteins (1: 1000 dilution), then horseradish peroxidase-conjugated secondary antibody (1: 10, 000 dilution). An enhanced chemiluminescence kit (Pierce, IL, USA) was used for signal detection. Images of blots were acquired by using a digital image processing system (Universal Hood II76S/0608, Bio-Rad, Hercules, CA) and quantified by use of Quantity One (Bio-Rad).

Statistical Analysis

Data are described with the mean ± SD and were analysed by using SPSS17.0 (SPSS Inc., Chicago, IL) with unpaired Student t test or one-way ANOVA. Significant differences were determined by Duncan multiple range tests. Data were considered significant at P < 0.05.

HUA inhibits viability of H9c2 cardiomyocytes

To examine the effect of UA on cell viability, H9c2 cardiomyocytes were treated with different concentrations of UA (0–15 mg/dl) for various times, and cell viability was evaluated by MTT assay. UA time- and dose-dependently inhibited the viability of H9c2 cardiomyocytes (Fig. 1A). Pre-incubation with HUA (15 mg/dl) decreased cell viability by 21% and 32% at 24 h and 48 h, respectively; whereas, with a lower concentration of UA, a longer time was needed for a similar level of growth inhibition. We used 15 mg/dl for subsequent experiments.

Fig. 1.

(A-B) Effect of uric acid (UA) on the proliferation of H9c2 cardiomyocytes. H9c2 cardiomyocytes were treated with different concentrations of UA (0–15 mg/dl) for 12, 24, 48, and 72 h (A); *P<0.01 vs. 12 h; #P<0.05 vs. 10 mg/dl. Pre-treatment with N-acetyl-L-cysteine (NAC) reversed high UA (HUA)-inhibited cardiomyocyte viability as measured by MTT assay (B); *P<0.01 vs. control; **P<0.01 vs. HUA; #P<0.05 vs. HUA. Data are the mean ± SEM from 5 independent experiments. (C-H) Effect of HUA on ROS generation in H9c2 cardiomyocytes (C-E) and primary cardiomyocytes (F-H). Cells were co-incubated with HUA and stained with DCFH-DA for fluorescence microscopy (C, F) and analysed by flow cytometry (E, H); (F) *P<0.01 vs. control, #P<0.01 vs. HUA and N-acetyl-L-cysteine (NAC).

Fig. 1.

(A-B) Effect of uric acid (UA) on the proliferation of H9c2 cardiomyocytes. H9c2 cardiomyocytes were treated with different concentrations of UA (0–15 mg/dl) for 12, 24, 48, and 72 h (A); *P<0.01 vs. 12 h; #P<0.05 vs. 10 mg/dl. Pre-treatment with N-acetyl-L-cysteine (NAC) reversed high UA (HUA)-inhibited cardiomyocyte viability as measured by MTT assay (B); *P<0.01 vs. control; **P<0.01 vs. HUA; #P<0.05 vs. HUA. Data are the mean ± SEM from 5 independent experiments. (C-H) Effect of HUA on ROS generation in H9c2 cardiomyocytes (C-E) and primary cardiomyocytes (F-H). Cells were co-incubated with HUA and stained with DCFH-DA for fluorescence microscopy (C, F) and analysed by flow cytometry (E, H); (F) *P<0.01 vs. control, #P<0.01 vs. HUA and N-acetyl-L-cysteine (NAC).

Close modal

HUA inhibited cardiomyocyte viability via oxidative stress

The ROS level was higher in H9c2 and primary cardiomyocytes with, rather than without, HUA treatment (Fig. 1C–1H). Pre-treatment with the antioxidant NAC partially reversed the HUA-generated ROS (Fig. 1C–1H), which suggests that HUA directly caused oxidative stress in cardiomyocytes. To test whether HUA treatment inhibited cardiomyocyte viability via oxidative stress, pre-treatment with NAC reversed the HUA-inhibited viability of H9c2 cardio-myocytes (Fig. 1B, 1C-1H), so oxidative stress may play a key role in HUA-inhibited viability in H9c2 cardiomyocytes.

HUA-inhibited cardiomyocyte viability was mediated by an increased phospho-ERK/p38 level via oxidative stress

ERK and p38 regulate a variety of physiological processes such as cell growth, metabolism, differentiation and cell death. We examined whether HUA-inhibited cardiomyocyte viability was mediated by ERK and p38. HUA exposure increased phospho-ERK and phospho-p38 levels in H9c2 and primary cardiomyocytes (Fig. 2A-2B). Furthermore, NAC plus PD98059, an ERK inhibitor, reversed the HUA-activated level of phospho-ERK/p38 in the cardiomyocytes (Fig. 2A-2B). Moreover, pre-treatment with NAC, PD98059 and SB203580 reversed the HUA-inhibited viability of H9c2 cardiomyocytes (Fig. 3C, Fig. 4A). Additionally, SB203580, a p38 inhibitor, reversed HUA-activated phospho-p38 and phospho-ERK levels in H9c2 cardiomyocytes (Fig. 3A-3B). Hence, HUA inhibited cardiomyocyte viability through the ERK/p38 pathway (p38 regulated by ERK) via oxidative stress.

Fig. 2.

Effect of HUA on phospho-ERK (A) and phospho-p38 (B) levels in H9c2 and primary cardiomyocytes. Western blot analysis of cells pre-treated with HUA with or without NAC and PD98059, an ERK inhibitor. Data are the mean ± SD from 3 independent experiments; *P<0.01 vs. control; #P<0.01 vs. HUA.

Fig. 2.

Effect of HUA on phospho-ERK (A) and phospho-p38 (B) levels in H9c2 and primary cardiomyocytes. Western blot analysis of cells pre-treated with HUA with or without NAC and PD98059, an ERK inhibitor. Data are the mean ± SD from 3 independent experiments; *P<0.01 vs. control; #P<0.01 vs. HUA.

Close modal
Fig. 3.

Effect of SB203580 on HUA-induced phospho-ERK (A) and phospho-p38 (B) levels in H9c2 cardiomyocytes and cardiomyocyte viability (C). Western blot analysis of cells pre-treated with HUA with or without SB203580, a p38 inhibitor. Data are the mean ± SD from 3 independent experiments; *P<0.01 vs. control; #P<0.01 vs. HUA.

Fig. 3.

Effect of SB203580 on HUA-induced phospho-ERK (A) and phospho-p38 (B) levels in H9c2 cardiomyocytes and cardiomyocyte viability (C). Western blot analysis of cells pre-treated with HUA with or without SB203580, a p38 inhibitor. Data are the mean ± SD from 3 independent experiments; *P<0.01 vs. control; #P<0.01 vs. HUA.

Close modal
Fig. 4.

(A) Pre-treatment with NAC and PD98059 reversed HUA-inhibited cardiomyocyte viability; *P<0.01 vs. control; #P<0.05 vs. HUA. Viable cells were measured by MTT assay. (B-D) HUA inhibits phospho-PI3K/ Akt expression (B-C), but insulin, an activator of PI3K/Akt, did not affect HUA-inhibited cardiomyocyte viability (D). Western blot analysis of cells pre-treated with HUA with or without insulin (B-C); *P<0.01 vs. control; #P<0.05 vs. HUA; (D) *P<0.01 vs. control; #P<0.05 vs. insulin (E-F) Western blot analysis of phospho-ERK (E) and phospho-p38 (F) levels in cardiac tissues of hyperuricaemic mice; *P<0.01 vs. control. Data are the mean ± SEM from 5 independent experiments.

Fig. 4.

(A) Pre-treatment with NAC and PD98059 reversed HUA-inhibited cardiomyocyte viability; *P<0.01 vs. control; #P<0.05 vs. HUA. Viable cells were measured by MTT assay. (B-D) HUA inhibits phospho-PI3K/ Akt expression (B-C), but insulin, an activator of PI3K/Akt, did not affect HUA-inhibited cardiomyocyte viability (D). Western blot analysis of cells pre-treated with HUA with or without insulin (B-C); *P<0.01 vs. control; #P<0.05 vs. HUA; (D) *P<0.01 vs. control; #P<0.05 vs. insulin (E-F) Western blot analysis of phospho-ERK (E) and phospho-p38 (F) levels in cardiac tissues of hyperuricaemic mice; *P<0.01 vs. control. Data are the mean ± SEM from 5 independent experiments.

Close modal

HUA inhibited phospho-PI3K/Akt expression, but insulin, a PI3K/Akt activator, did not affect HUA-inhibited cardiomyocyte viability

The Akt family of intracellular Ser/Thr kinases regulates both cardiac growth and metabolism [25, 26]. Akt regulates insulin-stimulated glucose uptake downstream of PI3K in cardiomyocytes [26]. We determined whether HUA-inhibited cardiomyocyte viability was mediated by PI3K/Akt. HUA inhibited and insulin increased phospho-PI3K/Akt expression (Fig. 4B-4C). However, insulin did not affect HUA-inhibited cardiomyocyte viability (Fig. 4D). Therefore, the PI3K/Akt signal pathway may not be involved in mechanisms of HUA-inhibited viability of H9c2 cardiomyocytes.

Hyperuricaemia increased the phospho-ERK/p38 level in mouse cardiac tissue

As expected, serum UA levels in our mouse model were higher after, rather than before, hyperuricaemia induction (113.81±17.98 vs 38.03±2.68 mg/l), which agreed with levels in patients with primary hyperuricaemia [27]. We examined ERK/p38 signalling in cardiac tissues of the acute hyperuricaemic mouse model. Hyperuricaemia increased phospho-ERK/ p38 level in mouse cardiac tissues (Fig. 4E-4F), which demonstrates a profound effect of hyperuricaemia on hyperuricaemic-related cardiac damage in vivo.

Oxidative stress is caused by abnormal cell metabolism exceeding the physiological buffering capacity. Oxidative stress has been described to increase cellular ageing, thus weakening organ function [28]. Previous studies have demonstrated that HUA increases oxidative stress in a variety of cell types, such as pancreatic ß cells, adipocytes and mesangial cells. ROS are second messengers that mediate gene transcription, cell proliferation, necrosis, apoptosis and differentiation in a variety of cell types [29]. In this study, we investigated the effect of HUA on cardiomyocytes and myocardial tissues from an acute hyperuricaemic mice model. HUA decreased H9c2 cardiomyocyte viability and increased ROS production in H9c2 cardiomyocytes; pre-treatment with NAC and PD98059 reversed HUA-inhibited viability of H9c2 cardiomyocytes. Thus, HUA inhibits cardiomyocyte viability through the ERK/p38 pathway via oxidative stress. Furthermore, the acute hyperuricaemic mice model showed an increased phospho-ERK/p38 level and inhibited phospho-PI3K/Akt level in myocardial tissues.

Serum HUA is strongly associated with many cardiovascular diseases, such as heart failure [2, 3], hypertension [5], and coronary artery disease [4, 6], but a pathological mechanism to explain this association has been missing. In previous studies [30], doxorubicin significantly induced ROS production and impaired cardiomyocyte viability. Indeed, reduced ROS production prompted survival of a variety of cell types [31]. In the present study, HUA stimulated ROS formation, thereby impairing H9c2 cardiomyocyte viability.

Dedifferentiation and inflammation are supported by an intracellular signalling network involving the MAPK and PI3K/Akt pathways [32]. MAPKs are a family of proteins promoting a phosphorylative signalling cascade, leading to the activation of transcription factors involved in cellular dedifferentiation or inflammation [20]. Phosphorylated Akt translocates to the nucleus and phosphorylates numerous target molecules to mediate signals [19]. ROS induces dedifferentiation, inflammation and proliferation in a variety of cell types via the temporal activation of the MAPK and PI3K pathways [19, 20]. We explored several cellular signalling pathways suggested to be stimulated by ROS. MAPKs, mainly including ERK, p38 and JNK, are also key regulators of cell survival [32]. We chose ERK/p38 as a representative signalling effecter in the MAPK signalling pathway. Phosphorylation of ERK/ p38 was significantly enhanced with HUA, which indicated an involvement in the impaired survival of H9c2 cardiomyocytes. We also tested cell viability with MTT assay and found that HUA time- and dose-dependently impaired cardiomyocyte viability. Therefore, HUA reduced cardiomyocyte viability via the ROS–ERK/p38 pathway. This result is consistent with other studies showing the stress response effect of abnormal stimulation on cardiomyocytes [34, 35].

Moreover, we determined whether HUA-inhibited cardiomyocyte viability was mediated by PI3K/Akt. HUA inhibited but insulin promoted phospho-PI3K/Akt expression. However, insulin did not affect cardiomyocyte viability inhibited by HUA. Thus, the PI3K/Akt signal pathway may not be involved in mechanisms of HUA inhibiting cardiomyocyte viability.

In summary, HUA inhibited cardiomyocyte viability through ERK/p38 pathway via oxidative stress. We provide a novel potential mechanism for hyperuricaemia associated with cardiovascular disease. However, further study is needed to confirm how HUA leads to myocardial injury and its significance in heart disease.

Limitations

Our study contains some limitations. First, we studied only the effect of HUA on H9c2 cardiomyocyte viability and did not assess its effect on cardiomyocyte apoptosis. We did not compare the contribution of cardiomyocyte viability in heart function loss. Second, the ERK/p38 signal pathway is not the only regulator of cell viability. We did not study the other signal pathways.

The authors declare no Disclosure Statements regarding the publication of this article.

1.
Zhang CH, Huang DS, Shen D, Zhang LW, Ma YJ, Wang YM, Sun HY: Association Between Serum Uric Acid Levels and Atrial Fibrillation Risk. Cell Physiol Biochem 2016; 38: 1589-1595.
[PubMed]
2.
Anker SD, Doehner W, Rauchhaus M, Sharma R, Francis D, Knosalla C, Davos CH, Cicoira M, Shamim W, Kemp M, Segal R, Osterziel KJ, Leyva F, Hetzer R, Ponikowski P, Coats AJ: Uric acid and survival in chronic heart failure: Validation and application in metabolic, functional, and hemodynamic staging. Circulation 2003; 107: 1991–1997.
[PubMed]
3.
Leyva F, Anker S, Swan JW, Godsland IF, Wingrove CS, Chua TP, Stevenson JC, Coats AJ: Serum uric acid as an index of impaired oxidative metabolism in chronic heart failure. Eur Heart J 1997; 18: 858–865.
[PubMed]
4.
Krishnan E, Baker JF, Furst DE, Schumacher HR: Gout and the risk of acute myocardial infarction. Arthritis Rheum 2006; 54: 2688–2696.
[PubMed]
5.
Verdecchia P, Schillaci G, Reboldi G, Porcellati C, Brunetti P: Relation between serum uric acid and risk of cardiovascular disease in essential hypertension: the PIUMA study. Hypertension 2000; 36: 1072–1078.
[PubMed]
6.
Bos MJ, Koudstaal PJ, Hofman A, Witteman JC, Breteler MM: Uric acid is a risk factor for myocardial infarction and stroke: the Rotterdam Study. Stroke 2006; 37: 1503–1507.
[PubMed]
7.
Hare JM, Johnson RJ: Uric acid predicts clinical outcomes in heart failure: Insights regarding the role of xanthine oxidase and uric acid in disease pathophysiology. Circulation 2003; 107: 1951-1953.
[PubMed]
8.
Cappola TP, Kass DA, Nelson GS, Berger RD, Rosas GO, Kobeissi ZA, Marbán E, Hare JM: Allopurinol improves myocardial efficiency in patients with idiopathic dilated cardiomyopathy. Circulation 2001; 104: 2407-2411.
[PubMed]
9.
Anker SD, Doehner W, Rauchhaus M, Sharma R, Francis D, Knosalla C, Davos CH, Cicoira M, Shamim W, Kemp M, Segal R, Osterziel KJ, Leyva F, Hetzer R, Ponikowski P, Coats AJ: Uric acid and survival in chronic heart failure: Validation and application in metabolic, functional, and hemodynamic staging. Circulation 2003; 107: 1991-1997.
[PubMed]
10.
Leyva F, Anker S, Swan JW, Godsland IF, Wingrove CS, Chua TP, Stevenson JC, Coats AJ: Serum uric acid as an index of impaired oxidative metabolism in chronic heart failure. Eur Heart J 1997; 18: 858-865.
[PubMed]
11.
Tormos KV, Anso E, Hamanaka RB, Eisenbart J, Joseph J, Kalyanaraman B, Chandel NS: Mitochondrial complex III ROS regulate adipocyte differentiation. Cell Metab 2011; 14: 537-544.
[PubMed]
12.
Nathan C, Cunningham-Bussel A: Beyond oxidative stress: an immunologist’s guide to reactive oxygen species. Nat Rev Immunol 2013; 13: 349-361.
[PubMed]
13.
Droge W: Free radicals in the physiological control of cell function. Physiol Rev 2002; 82: 47-95.
[PubMed]
14.
Zhu Y, Hu Y, Huang T, Zhang Y, Li Z, Luo C, Luo Y, Yuan H, Hisatome I, Yamamoto T, Cheng J: High uric acid directly inhibits insulin signalling and induces insulin resistance. Biochem Biophys Res Commun 2014; 447: 707–714.
[PubMed]
15.
Zhang YN, Yamamoto T, Hisatome I, Li Y, Cheng W, Sun N, Cai B, Huang T, Zhu Y, Li Z, Jing X, Zhou R, Cheng J: Uric acid induces oxidative stress and growth inhibition by activating adenosine monophosphate-activated protein kinase and extracellular signal-regulated kinase signal pathways in pancreatic β cells. Mol Cell Endocrinol 2013; 375: 89–96.
[PubMed]
16.
Zhang JX, Zhang YP, Wu QN, Chen B: Uric acid induces oxidative stress via an activation of the reninangiotensin system in 3T3-L1 adipocytes. Endocrine 2015; 48: 135–142.
[PubMed]
17.
Zhuang Y, Feng Q, Ding G, Zhao M, Che R, Bai M, Bao H, Zhang A, Huang S: Activation of ERK1/2 by NADPH oxidase-originated reactive oxygen species mediates uric acid-induced mesangial cell proliferation. Am J Physiol Renal Physiol 2014; 307:F396–406.
[PubMed]
18.
Luo C, Lian X, Hong L, Zou J, Li Z, Zhu Y, Huang T, Zhang Y, Hu Y, Yuan H, Wen T, Zhuang W, Cai B, Zhang X, Hisatome I, Yamamoto T, Huang J, Cheng J: High Uric Acid Activates the ROS-AMPK Pathway, Impairs CD68 Expression and Inhibits OxLDL-Induced Foam-Cell Formation in a Human Monocytic Cell Line, THP-1 Cell Physiol Biochem 2016; 40: 538-548.
[PubMed]
19.
Wang X, Liu JZ, Hu JX, Wu H, Li YL, Chen HL, Bai H, Hai CX: ROS-activated p38 MAPK/ERK-Akt cascade plays a central role in palmitic acid-stimulated hepatocyte proliferation. Free Radic Biol Med 2011; 51: 539-551.
[PubMed]
20.
Lin CF, Young KC, Bai CH, Yu BC, Ma CT, Chien YC, Su HC, Wang HY, Liao CS, Lai HW, Tsao CW: Blockade of reactive oxygen species and Akt activation is critical for anti-inflammation and growth inhibition of metformin in phosphatase and tensin homolog-deficient RAW264.7 cells. Immunopharmacol Immunotoxicol 2013; 35: 669-677.
[PubMed]
21.
Yue R, Hu H, Yiu KH, Luo T, Zhou Z, Xu L, Zhang S, Li K, Yu Z: Lycopene Protects against Hypoxia/Reoxygenation-Induced Apoptosis by Preventing Mitochondrial Dysfunction in Primary Neonatal Mouse Cardiomyocytes. PLoS ONE 2012; 7:e50778.
[PubMed]
22.
Barbu A, Welsh N, Saldeen J: Cytokine-induced apoptosis and necrosis are preceded by disruption of the mitochondrial membrane potential (Deltapsi(m)) in pancreatic RINm5F cells: prevention by Bcl-2 Mol Cell Endocrinol 2002; 190: 75–82.
[PubMed]
23.
Chen W, Nan L, Luan R, Yan Li, Wang D, Zou W, Xing Y, Tao L, Cao F, Wang H: Apelin protects sarcoplasmic reticulum function and cardiac performance in ischaemia-reperfusion by attenuating oxidation of sarcoplasmic reticulum Ca2+-ATPase and ryanodine receptor. Cardiovascular Research 2013; 100: 114–124.
[PubMed]
24.
Li JM, Zhang X, Wang X, Xie YC, Kong LD: Protective effects of cortex fraxini coumarines against oxonate-induced hyperuricemia and renal dysfunction in mice. Eur J Pharmacol 2011; 666: 196–204.
[PubMed]
25.
Matsui T, Rosenzweig A: Convergent signal transduction pathways controlling cardiomyocyte survival and function: the role of PI 3-kinase and Akt. J Mol Cell Cardiol 2005; 38: 63–71.
[PubMed]
26.
Yang Jl, Holman GD: Long-Term Metformin Treatment Stimulates Cardiomyocyte Glucose Transport through an AMPActivated Protein Kinase-Dependent Reduction in GLUT4 Endocytosis. Endocrinology 2006; 147: 2728–2736.
[PubMed]
27.
Nakagawa T, Hu HB, Zharikov S, Tuttle KR, Short RA, Glushakova O, Ouyang X, Feig DI, Block ER, Herrera-Acosta J, Patel JM, Johnson RJ: A causal role for uric acid in fructose-induced metabolic syndrome. Am J Physiol-Renal 2006; 290: F625–F631.
[PubMed]
28.
Brouilette S, Singh RK, Thompson JR, Goodall AH, Samani NJ: White cell telomere length and risk of premature myocardial infarction. Arterioscler Thromb Vasc Biol 2003; 23: 842-846.
[PubMed]
29.
Korbecki J, Baranowska-Bosiacka I, Gutowska I, Chlubek D: The effect of reactive oxygen species on the synthesis of pros-tanoids from arachidonic acid. J Physiol Pharmacol 2013; 64: 409-421.
[PubMed]
30.
Zhou LL, Chen LP, Wang J, Deng YJ: Astragalus polysaccharide improves cardiac function in doxorubicin-induced cardiomyopathy through ROS-p38 signaling. Int J Clin Exp Med 2015; 8: 21839-21848.
[PubMed]
31.
Muzi-Filho H, Bezerra CG, Souza AM, Boldrini LC, Takiya CM, Oliveira FL, Nesi RT, Valença SS, Einicker-Lamas M, Vieyra A, Lara LS, Cunha VM: Undernutrition affects cell survival, oxidative stress, ca2+ handling and signaling pathways in vas deferens, crippling reproductive capacity. PLoS One 2013; 8:e69682.
[PubMed]
32.
Torres M, Forman HJ: Redox signaling and the MAP kinase pathways. Biofactors 2003; 17: 287-296.
[PubMed]
33.
CKim EK, Choi EJ: Pathological roles of mapk signaling pathways in human diseases. Biochim Biophys Acta 2010; 1802: 396-405.
[PubMed]
34.
Fang SJ, Wu XS, Han ZH, Zhang XX, Wang CM, Li XY, Lu LQ, Zhang JL: Neuregulin-1 preconditioning protects the heart against ischemia/reperfusion injury through a pi3k/akt-dependent mechanism. Chin Med J (Engl) 2010; 123: 3597-3604.
[PubMed]
35.
Ravingerova T, Matejikova J, Neckar J, Andelova E, Kolar F: Differential role of pi3k/akt pathway in the infarct size limitation and antiarrhythmic protection in the rat heart. Mol Cell Biochem 2007; 297: 111-120.
[PubMed]