Overexpression of G Protein-Coupled Receptor 40 Protects Obesity-Induced Cardiomyopathy Through the SIRT1/LKB1/AMPK Pathway.

Obesity has become a serious global public health problem, and cardiomyopathy caused by obesity has recently gained attention. As an important protein involved in glucose and lipid metabolism, G protein-coupled receptor 40 (GPR40) exerts cardioprotective effects in some disease models. This study aimed to explore whether GPR40 plays a protective role in obesity-induced cardiomyopathy. We established an obesity model by feeding rats with a high-fat diet, and H9c2 cells were stimulated with palmitic acid to mimic high fat stimulation. Overexpression of GPR40 was achieved by infection with lentivirus or cDNA plasmids. Obesity-induced cardiac injury models exhibit cardiac dysfunction, myocardial hypertrophy, and collagen accumulation, which are accompanied by increased inflammation, oxidative stress, and apoptosis. However, GPR40 overexpression attenuated these alterations. The anti-inflammatory effect of GPR40 may be by inhibiting the nuclear factor-κB pathway, and the antioxidative stress may occur as a result of nuclear transcription factor erythroid 2-related factor 2 pathway activation. In terms of the mechanisms of GPR40 against obese cardiomyopathy, GPR40 overexpression not only activated the sirtuin 1 (SIRT1)-liver kinase B1 (LKB1)-AMP-activated protein kinase (AMPK) pathway but also enhanced the binding of SIRT1 to LKB1. The antifibrotic, anti-inflammatory, antioxidative stress, and antiapoptotic effects of GPR40 overexpression were inhibited by SIRT1 small interfering RNA. In conclusion, GPR40 overexpression protects against obesity-induced cardiac injury in rats, possibly through the SIRT1-LKB1-AMPK pathway.

Obesity increases neuropathic pain via the AMPK-ERK-NOX4 pathway in rats.

This study focused on the relationship between extracellular-regulated kinase (ERK) and obesity-induced increases in neuropathic pain. We fed rats a high-fat diet to establish the obesity model, and rats were given surgery to establish the chronic compression of the dorsal root ganglia (CCD) model. U0126 was applied to inhibit ERK, and metformin or 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR) was applied to cause AMP-activated protein kinase (AMPK) activation. Paw withdrawal mechanical threshold (PWMT) were calculated to indicate the level of neuropathic pain. The data indicated that compared with normal CCD rats, the PWMT of obese CCD rats were decreased, accompanied with an increase of ERK phosphorylation, NAD(P)H oxidase 4 (NOX4) protein expression, oxidative stress and inflammatory level in the L4 to L5 spinal cord and dorsal root ganglia (DRG). Administration of U0126 could partially elevate the PWMT and reduce the protein expression of NOX4 and the above pathological changes in obese CCD rats. In vitro, ERK phosphorylation, NOX4 protein expression increased significantly in DRG neurons under the stimulation of palmitic acid (PA), accompanied with increased secretion of inflammatory factors, oxidative stress and apoptosis level, while U0126 partially attenuated the PA-induced upregulation of NOX4 and other pathological changes. In the rescue experiment, overexpression of NOX4 abolished the above protective effect of U0126 on DRG neurons in high-fat environment. Next, we explore upstream mechanisms. Metformin gavage significantly reduced neuropathic pain in obese CCD rats. For the mechanisms, activating AMPK with metformin (obese CCD rats) or AICAR (DRG neurons in a high-fat environment) not only inhibited the ERK-NOX4 pathway, but also improved oxidative stress and inflammation caused by high-fat. In conclusion, the AMPK-ERK-NOX4 pathway may has a pivotal role in mediating obesity-induced increases in neuropathic pain.

Yes-Associated Protein is Involved in Myocardial Fibrosis in Rats with Diabetic Cardiomyopathy.

INTRODUCTION: Recent studies have shown that YAP is closely related to the pathological process of cardiovascular diseases. But the role of YAP in cardiac injury of diabetic cardiomyopathy (DCM) is still unclear. METHODS: Diabetic cardiomyopathy rat model was established and divided into control group, DCM group, LV-SC-shRNA group and LV-YAP-shRNA group. LV-SC-shRNA group and LV-YAP-shRNA group were injected with lentivirus expressing SC-shRNA and YAP-shRNA via tail vein, respectively. Primary rat cardiac fibroblasts (CFs) were stimulated with high concentration of glucose and treated with recombinant lentivirus expressing either SC-shRNA or YAP-shRNA to observe the expression of CTGF and fibronectin, so as to observe the effect of inhibiting YAP on the pathogenesis of DCM. RESULTS: Compared with control group, high glucose markedly increased YAP mRNA and protein expression in DCM and CFs. Inhibition of YAP decreased myocardial fibrosis and improved cardiac function in the DCM model and decreased the expression of CTGF and fibronectin in CFs. The result suggested that YAP plays a key role in the pathological progression of DCM, and the underlying mechanisms may be associated with TEAD and CTGF. DISCUSSION: We found that the expression of YAP was increased both in vivo and in vitro, suggesting that YAP is closely related to DCM, and YAP knockdown can reduce myocardial fibrosis in rat model of DCM by reducing the expression of PAI-1, collagen I, collagen III, CTGF and profilin, as well as the expression of CTGF and fibronectin in CFs. This study revealed that YAP plays an important role in the pathological process of diabetic cardiomyopathy, and down-regulation of YAP expression may provide a new therapeutic target for DCM.

Excessive expression of miR-1a by statin causes skeletal injury through targeting mitogen-activated protein kinase kinase kinase 1.

BACKGROUNDS: A major side effect of statin, a widely used drug to treat hyperlipidemia, is skeletal myopathy through cell apoptosis. The aim of this study is to investigate the roles of microRNA in statin-induced injury. METHODS: Apolipoprotein E knockout (ApoE-/-) mice were administered with simvastatin (20 mg/kg/day) for 8 weeks. Exercise capacity was evaluated by hanging grid test, forelimb grip strength, and running tolerance test. RESULTS: In cultured skeletal muscle cells, statin increased the levels of miR-1a but decreased the levels of mitogen-activated protein kinase kinase kinase 1 (MAP3K1) in a time or dose dependent manner. Both computational target-scan analysis and luciferase gene reporter assay indicated that MAP3K1 is the target gene of miR-1a. Statin induced cell apoptosis of skeletal muscle cells, but abolished by downregulating of miR-1a or upregulation of MAP3K1. Further, the effects of miR-1a inhibition on statin-induced cell apoptosis were ablated by MAP3K1 siRNA. In ApoE-/- mice, statin induced cell apoptosis of skeletal muscle cells and decreased exercise capacity in mice infected with vector, but not in mice with lentivirus-mediated miR-1a gene silence. CONCLUSION: Statin causes skeletal injury through induction of miR-1a excessive expression to decrease MAP3K1 gene expression.

Vitamin B6 Alleviates Lipopolysaccharide-induced Myocardial Injury by Ferroptosis and Apoptosis Regulation.

Vitamin B6 (VitB6) is a water-soluble vitamin and includes pyridoxine, pyridoxal, pyridoxamine, and their phosphorylated forms. In the current study, we demonstrated that VitB6 could improve lipopolysaccharide (LPS)-induced myocardial injury. We demonstrated that VitB6 can suppress LPS-induced oxidative stress and lipid peroxidation that lead to ferroptosis and apoptosis in vivo and in vitro. Moreover, we found that VitB6 can regulate the expression of iron regulatory proteins, maintaining intracellular iron homeostasis. To confirm that VitB6 could inhibit LPS-induced ferroptosis and apoptosis, we pretreated mice with ferrostatin-1 (Fer-1) and emricasan that efficiently mimicked VitB6 pharmacological effects. This improved the survival rate of mice challenged with a high LPS dose. In addition, VitB6 regulated the expression of LPS-induced apoptosis-related proteins and iron regulatory proteins. It mediated the expression of Nrf2, transcription factor NF-E2-related factor 2, which promoted the expression of antioxidant enzymes and restrained LPS-induced ferroptosis and apoptosis. Overall, our results indicated that VitB6 can be used on novel therapies to relieve LPS-induced myocardial injury.

Vitamin B6 inhibits macrophage activation to prevent lipopolysaccharide-induced acute pneumonia in mice.

Macrophage activation participates in the pathogenesis of pulmonary inflammation. As a coenzyme, vitamin B6 (VitB6) is mainly involved in the metabolism of amino acids, nucleic acids, glycogen and lipids. We have previously reported that activation of AMP-activated protein kinase (AMPK) produces anti-inflammatory effects both in vitro and in vivo. Whether VitB6 via AMPK activation prevents pulmonary inflammation remains unknown. The model of acute pneumonia was induced by injecting mice with lipopolysaccharide (LPS). The inflammation was determined by measuring the levels of interleukin-1 beta (IL-1ß), IL-6 and tumour necrosis factor alpha (TNF-α) using real time PCR, ELISA and immunohistochemistry. Exposure of cultured primary macrophages to VitB6 increased AMP-activated protein kinase (AMPK) Thr172 phosphorylation in a time/dose-dependent manner, which was inhibited by compound C. VitB6 downregulated the inflammatory gene expressions including IL-1ß, IL-6 and TNF-α in macrophages challenged with LPS. These effects of VitB6 were mirrored by AMPK activator 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR). However, VitB6 was unable to inhibit LPS-induced macrophage activation if AMPK was in deficient through siRNA-mediated approaches. Further, the anti-inflammatory effects produced by VitB6 or AICAR in LPS-treated macrophages were abolished in DOK3 gene knockout (DOK3-/- ) macrophages, but were enhanced in macrophages if DOK3 was overexpressed. In vivo studies indicated that administration of VitB6 remarkably inhibited LPS-induced both systemic inflammation and acute pneumonia in wild-type mice, but not in DOK3-/- mice. VitB6 prevents LPS-induced acute pulmonary inflammation in mice via the inhibition of macrophage activation.

(Pro)renin Receptor is Involved in Myocardial Damage in Alcoholic Cardiomyopathy.

BACKGROUND: (Pro)renin receptor (PRR), a novel member of the renin-angiotensin system, participates in various cardiovascular diseases. However, the role of PRR in alcoholic cardiomyopathy (ACM), which is caused by alcohol intake and manifests as myocardial damage and cardiac dysfunction, remains unclear. METHODS: PRR gene silencing was achieved by transfecting recombinant adenovirus expressing anti-PRR short hairpin RNA (PRR-shRNA). In vitro, primary rat cardiac fibroblasts (CFs) were cultured with the stimulation of alcohol (200 mM), with or without PRR-shRNA and PD98059. Immunofluorescence, RT-PCR, and Western blot were used to measure the protein and messenger (mRNA) expression of PRR, fibrotic factors, and members of related signaling pathways. In vivo, Wistar rats were fed a diet containing 9% (v/v) alcohol or a normal diet for 3 months, with or without PRR-shRNA. Sirius Red staining, immunohistochemical staining, and toluidine blue staining were used to evaluate myocardial fibrosis, oxidative stress, and inflammation response. RESULTS: Alcohol markedly increased PRR mRNA and protein expression in a time- and concentration-dependent manner in CFs. The increased expression of fibrotic factors induced by alcohol was prevented by PRR-shRNA and PD98059. Moreover, PRR-shRNA decreased the phosphorylation of extracellular regulated protein kinases (ERK) 1/2 in CFs. Furthermore, PRR-shRNA decreased cardiac fibrosis, reduced oxidative stress, and alleviated inflammation response in the myocardial tissue. CONCLUSIONS: Our results show that PRR-ERK1/2 signaling was involved in the development of ACM and that PRR could be a new target for the treatment of ACM.