Canagliflozin and Dapagliflozin Attenuate Glucolipotoxicity-Induced Oxidative Stress and Apoptosis in Cardiomyocytes via Inhibition of Sodium-Glucose Cotransporter-1.

Sodium-dependent glucose cotransporter 2 inhibitors (SGLT2) are recently approved drugs for the treatment of diabetes that regulate blood glucose levels by inhibiting reabsorption of glucose and sodium in the proximal tubules of the kidney. SGLT2 inhibitors have also shown cardiovascular (CV) benefits in diabetic patients. However, the therapeutic efficacy of SGLT2 inhibitors with respect to CV disease needs further investigation. Thus, the aim of the present study was to examine the effects of SGLT2 inhibitors, canagliflozin (CANA) and dapagliflozin (DAPA) in vitro under glucolipotoxic condition by treating cultured cardiomyocytes (H9C2) with high glucose (HG) and high lipid, palmitic acid (PA), to investigate whether inhibition of sodium glucose cotransporter could prevent any harmful effects of glucolipotoxicity in these cells. SGLT1 expression was measured by immunofluorescence staining and quantitative polymerase chain reaction. Oxidative stress and apoptosis were measured by flow cytometry. Hypertrophy was measured by hematoxylin and eosin (H&E) and crystal violet staining. A significant increase in SGLT1 expression was observed in HG- and PA-treated cardiomyocytes. Also, a significant increase in reactive oxygen species generation and apoptosis was observed in HG+PA-treated cultured cardiomyocytes. HG- and PA-treated cardiomyocytes developed significant structural alterations. All these effects of HG and PA were attenuated by CANA and DAPA. In conclusion, our study demonstrates upregulation of SGLT1 induces oxidative stress and apoptosis in cultured cardiomyocytes. Thus, inhibition of SGLT1 may be used as a possible approach for the treatment of CVD in diabetic patients.

Syzygium cumini (jamun) fruit-extracted phytochemicals exert anti-proliferative effect on ovarian cancer cells.

BACKGROUND: The medicinal properties of Syzygium sp., especially the antidiabetic property, date back to the ancient times. However, in the recent past, extracts from different parts of the Syzygium sp. have demonstrated promising anticancer activities in diverse cancer types, and now, attempts are being made to identify the active phytochemicals. AIMS AND OBJECTIVES: In this study, we intended to test the anticancer properties of phytochemicals extracted from the fruit of Syzygium cumini plant in ovarian cancer cells. MATERIALS AND METHODS: A total of nine phytochemicals extracted from the S. cumini fruits using chloroform were tested for their anticancer activity in the ovarian cancer cell line PA-1. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide tetrazolium assay was performed to calculate the 50% inhibition (IC50) concentration and cell cytotoxicity values. Cell scratch assay was performed to assess the proliferation inhibition activity of the phytochemicals. Cisplatin was used as positive control. RESULTS: Out of the nine phytochemicals tested, quercetin (QC), gallic acid (GA), and oleanolic acid (OA) were found active. QC and GA were most effective with more than 90% cell cytotoxicity at 2.5 µ g/ml and above concentrations and OA moderately effective up to 5 µg/ml serial concentrations. Cell proliferation was significantly inhibited by QC and GA and moderately but significantly by OA. CONCLUSION: Our data demonstrate the anticancer activity of QC, GA, and OA phytochemicals, which is consistent with the previous reports. However, this is the first report showing the anticancer activity of these phytochemicals derived from S. cumini in the ovarian cancer cells. These data suggest that there is a potential to develop these phytochemicals as anticancer therapeutic agents either as monotherapeutic agents or in combination with commonly used chemotherapeutic agents, which needs to be explored.

Inhibition of double stranded RNA dependent protein kinase (PKR) abrogates isoproterenol induced myocardial ischemia in vitro in cultured cardiomyocytes and in vivo in wistar rats.

Protein kinase R (PKR) plays a main role in inflammation, insulin resistance, and glucose balance. It is activated by various stress signals and is key mediators of diabetes and associated complications. In the present study, we investigated the effect of PKR inhibition on myocardial dysfunction, inflammatory, cell death and interrelated signalling pathways in isoproterenol induced myocardial ischemia in vivo in wistar rats and in vitro in cultured cardiomyocytes. H9C2 rat cardiomyocytes were treated with 10 µM Isoproterenol (ISO). For in vivo studies, rats were divided into 4 groups: control, ischemic group (ISO), preventive group, curative group and each group consist of 8 rats. Myocardial Ischemia (MI) was induced with two subsequent doses of ISO (100 mg/kg, s.c.). The rats were treated with PKR inhibitor, C16 (166.5 µg/kg, i.p.) for 14 days. Heart rate, systolic, diastolic and mean arterial pressures were measured by non-invasive BP apparatus. Cardiac biomarkers were measured by commercial kits. Ischemic Zone, Morphological abnormalities and fibrosis of heart was detected by TTC, haematoxylin & eosin staining, Masson's and Sirius red staining respectively. Protein expression was done by western blotting and immune histochemistry. mRNA expression was done by RT-PCR. MI was characterized by declined myocardial performance along with elevation of cardiac biomarkers and associated with increased expression of PKR, oxidative-nitrosative stress, activated various inflammatory pathways (nuclear factor kappa light chain enhancer of activated B cells -NF-κB); Mitogen-activated protein kinases-MAPK; c-Jun N-terminal kinase-JNK), increased expression of inflammatory markers (Tumour necrosis factor alpha-TNF-α), markers of fibrosis (Alpha smooth muscle actin -α-SMA; Transforming growth factor beta-TGF-ß), enhanced cell death (Ischemic zone) and increased expression of extracellular regulated-kinases (ERK-1/2) and advanced glycation end products (AGE's). Interestingly, inhibition of PKR attenuated myocardial dysfunction, cardiac fibrosis, oxidative/nitrosative stress, inflammation, cell death, and inter-related signalling pathways. Our findings report that inhibition of PKR improves the ischemic mediated inflammation, apoptosis, cardiac hypertrophy and fibrosis in MI induced rats. Hence, inhibition of PKR might be one of intervention therapy for the treatment of myocardial ischemia.

High fructose and streptozotocin induced diabetic impairments are mitigated by Indirubin-3-hydrazone via downregulation of PKR pathway in Wistar rats.

Metabolic disorders are becoming more common in young population due to increased consumption of carbohydrate rich diet, lack of physical activity and stress. Fructose is used as a sweetener in many carbonated beverages and is a known inducer of oxidative stress and hypertension. Up-regulation of the double-stranded RNA-dependent protein kinase (PKR) causes impairment in insulin signaling pathway and metabolic dysfunctions in type 2 diabetes mellitus. In the present study we investigated the role of PKR and associated pathways in high fructose (HF) and streptozotocin (STZ) induced diabetes and whether indirubin-3-hydrazone (IHZ), a novel PKR inhibitor can reverse the HF and STZ induced diabetic impairments in Wistar rats. Diabetes was induced by feeding rats 20% high fructose in drinking water for 6 weeks and by giving a single dose of STZ (35 mg/kg., i.p) at the end of week 5. Glucose and lipid levels were measured by using assay kits. Expression of PKR and its downstream genes were determined by immunohistochemistry, qRT-PCR and western blotting techniques. Histo-pathological studies were performed using H&E staining. Fibrosis was detected in insulin sensitive tissues and organs using Sirius red and Masson's trichrome staining and apoptosis by TUNEL assay. HF and STZ induced hyperglycemia, fibrosis, oxidative stress, and inflammation in liver, pancreas, skeletal muscle and adipose tissue are mediated via PKR pathway and its downstream effectors, and these effects were attenuated by PKR inhibitor IHZ. Thus, inhibition of PKR can protect insulin sensitive organs and tissues from HF induced diabetic impairments via the inhibition of c-Jun N-terminal kinase (JNK) pathway.

PKR inhibitor imoxin prevents hypertension, endothelial dysfunction and cardiac and vascular remodelling in L-NAME-treated rats.

AIMS: Hypertension is one of the leading causes of cardiovascular mortality and morbidity. It is associated with severe cardiac and vascular dysfunction. Double-stranded RNA-dependent protein kinase (PKR), is a known inducer of inflammation and apoptosis. However, no research has been done to elucidate the role of the PKR in an experimental model of hypertension, and related cardiovascular complications. MAIN METHODS: L-NAME (NG-Nitro-L-arginine-methyl ester) was used to induce the hypertension. Imoxin treatment was given to Wistar rats for the four weeks along with the L-NAME, to investigate the influence on the hypertension. Changes in physiological parameter were assessed by recording non-invasive blood pressure. Expression of PKR and downstream markers for inflammation, fibrosis, and vascular damage in rat heart and aorta was determined by western blot and immunohistochemistry. Histological examination and fibrosis assessment were done by using assay kits. Vascular reactivity was determined by ex-vivo isometric tension studies on rat aortic rings. KEY FINDINGS: L-NAME-treated rats showed a significant increase in PKR expression followed by cardiac damage and vascular alterations compared to that of control animals. Results of western blot and immunohistochemistry indicate a significant increase in the inflammatory markers downstream to PKR. Endothelium-dependent vascular relaxation was significantly impaired in L-NAME administered rats. All effects of the L-NAME were attenuated by selective inhibition of PKR by imoxin. SIGNIFICANCE: Alterations in the heart and vasculature could be mediated in part by activation of the PKR pathway. Hence selective inhibition of PKR has therapeutic potential for combating hypertension and associated cardiovascular complications.

Upregulation of PKR pathway mediates glucolipotoxicity induced diabetic cardiomyopathy in vivo in wistar rats and in vitro in cultured cardiomyocytes.

AIMS: Protein Kinase R (PKR) plays a key role in inflammation and insulin resistance. Cytokines, high fat diet, infection and various stress signals can activate PKR. However, the functional significance of PKR in diabetic cardiomyopathy (DCM) is not explored so far. Thus the aim of the present study was to investigate the role of PKR in DCM in vivo in a rat model of DCM and underlying molecular mechanism. METHODS AND RESULTS: DCM was induced in Wistar rats by recipe of high fat diet and single injection of streptozotocin. Vital parameters were measured by non-invasive BP apparatus. Morphology, fibrosis and protein expression in heart was done by haematoxylin & eosin staining, masson's trichome/sirius red staining and western blotting respectively. For molecular mechanism studies, PKR gene silencing was done in cultured H9C2 cardiomyocytes and effect was observed in the presence of high glucose and high fat. Significant upregulation of PKR along with increase in cardiac biomarkers, decreased systolic and diastolic cardiac functions, oxidative stress, inflammatory markers, markers of fibrosis, enhanced cell death and AGEs' was observed in DCM disease model. Moreover, selective inhibition of PKR alleviated cardiac dysfunction, fibrosis, oxidative stress, inflammation and cell death. Additionally knockdown of PKR attenuated glucolipotoxicty-induced markers of inflammation, oxidative stress and apoptosis in cultured H9C2 cardiomyocytes. CONCLUSION: Our present study reports for the first time that inhibition of PKR may have great therapeutic potential in the treatment of DCM by attenuating inflammation, oxidative stress, apoptosis and fibrosis.

Selective inhibition of PKR improves vascular inflammation and remodelling in high fructose treated primary vascular smooth muscle cells.

BACKGROUND AND OBJECTIVE: Double-stranded RNA dependent protein kinase (PKR) is reported to play a critical role in the pathogenesis of diabetes and associated vascular complications. Increased PKR activity is observed in metabolic disorders. Increased PKR activity is reported to induce inflammation and oxidative stress. Inflammation and oxidative stress are implicated in the pathogenesis of vascular disease. There are no studies done so far about the role of PKR in vascular smooth muscle cells (VSMCs) and the underlying molecular mechanism. Thus the aim of the present study is to investigate the role of PKR in high fructose treated VSMCs. Moreover, a selective PKR inhibitor, imoxin (C16) was used to investigate the underlying molecular mechanism. METHODS: VSMCs were isolated by enzymatic digestion method from thoracic aorta of rats and incubated with high fructose (HF) and PKR inhibitor. Immunocytochemistry and Western blotting were performed for PKR and its downstream markers of inflammation, apoptosis and phenotypic transition (AGEs, MMP-9, and ERK1/2). Oxidative stress was measured using flow cytometry. Cellular hypertrophy and proliferative index were determined by haematoxylin and eosin staining, MTT assay, BrdU labelling assay and agarose gel electrophoresis. Scratch test was done for migratory behaviour. Alizarin red staining was performed for assessing vascular calcification. Mitochondrial membrane potential and chromatin condensation was determined by rhodamine 6G and DAPI staining. RESULTS: PKR expression was significantly increased in HF treated VSMCs which was accompanied by increase in levels of gene markers of inflammation, oxidative stress and apoptosis. Moreover, increase in cellular proliferation, phenotypic switch and decrease in membrane potential was observed in HF treated VSMCs. All these effects of HF were attenuated by selective PKR inhibitor, imoxin (C16). CONCLUSION: In conclusion PKR activation plays an important role in the pathogenesis of vascular inflammation and remodelling, and therapeutically targeting PKR could be an effective approach to treat the abnormalities associated with vascular complications.

Inhibition of protein kinase R protects against palmitic acid-induced inflammation, oxidative stress, and apoptosis through the JNK/NF-kB/NLRP3 pathway in cultured H9C2 cardiomyocytes.

BACKGROUND AND PURPOSE: Double-stranded RNA-dependent protein kinase (PKR) is a critical regulator of apoptosis, oxidative stress, and inflammation under hyperlipidemic and insulin resistance conditions. Saturated free fatty acids, such as palmitic acid (PA), are known inducers of apoptosis in numerous cell types. However, the underlying molecular mechanism is not fully understood. The aim of the present study was to examine the effect of PA on cultured rat H9C2 cardiac myocytes cells and to investigate the PKR mediated harmful effects of PA in vitro in cultured cardiomyocytes. EXPERIMENTAL APPROACH: PKR expression was determined by immunofluorescence and immunoblotting. Oxidative stress and apoptosis were determined by flow cytometry and assay kits. The expression of different gene markers of apoptosis, oxidative stress, and inflammation were measured by Western blot analysis and reverse transcription polymerase chain reaction. KEY RESULTS: PKR expression, reactive oxygen species levels as well as apoptosis were increased in PA-treated cultured H9C2 cardiomyocytes. The harmful effects of PA were attenuated by a selective PKR inhibitor, C16. Moreover, we observed that upregulation of c-Jun N-terminal kinase (JNK), nuclear factor-kB (NF-kB) and NACHT, LRR and PYD domains-containing protein 3 (NLRP3) pathways is associated with increased expression of interleukin 6 and tumor necrosis factor-α in PA-treated cardiomyocytes and attenuation by a selective PKR inhibitor. CONCLUSION AND IMPLICATIONS: Our study reports, for the first time, that PKR-mediated harmful effects of PA in cultured cardiomyocytes via activation of JNK, NF-kB, and NLRP3 pathways. Inhibition of PKR is one of the possible mechanistic approaches to inhibit inflammation, oxidative stress, and apoptosis in lipotoxicity-induced cardiomyocyte damage.

Pharmacological evaluation of novel PKR inhibitor indirubin-3-hydrazone in-vitro in cardiac myocytes and in-vivo in wistar rats.

AIMS: Double stranded protein kinase R cellular response is associated with various stress signals such as nutrients, endoplasmic stress, cytokines and mechanical stress. Increased PKR activity has been observed under diabetic and cardiovascular disease conditions. Most of the currently available PKR inhibitors are non-specific and have other effects as well. Thus, the aim of the present study was to examine the effect of novel PKR inhibitor indirubin-3-hydrazone (IHZ) in cultured rat H9C2 cardiomyocytes and wistar rats. MATERIALS AND METHODS: PKR expression was determined by Q-PCR, immunofluorescence and immunoblotting. The expression of different gene markers for apoptosis was measured by RT-PCR. Apoptosis and oxidative stress were determined by flow cytometry. KEY FINDINGS: High glucose (HG) treated H9C2 cardiomyocytes and high fructose (HF) treated wistar rats developed a significant increase in PKR expression. A significant increase in apoptosis and generation of reactive oxygen species was also observed in HG treated H9C2 cells and HF treated rats. Reduced vacuole formation and prominent nuclei were also observed in high glucose treated cells. Cardiac hypertrophy and increased fibrosis were observed in HF treated rats. All these effects of HG and HF were attenuated by novel PKR inhibitor, indirubin-3-hydrazone. SIGNIFICANCE: Our results indicate IHZ as an effective inhibitor of PKR in vitro and in-vivo, thus it may prove very useful in blocking the multiple harmful effects of PKR.