Afriplex GRTTM extract attenuates hepatic steatosis in an in vitro model of NAFLD.

BACKGROUND: Currently, it is acknowledged that vitamin E, insulin sensitizers and anti-diabetic drugs are used to manage non-alcoholic fatty liver disease (NAFLD), however, these therapeutic interventions harbour adverse side effects. Pioglitazone, an anti-diabetic drug, is currently the most effective therapy to manage NAFLD. The use of natural medicines is widely embraced due to the lack of evidence of their negative side effects. Rooibos has been previously shown to decrease inflammation and oxidative stress in experimental models of diabetes, however, this is yet to be explored in a setting of NAFLD. This study was aimed at investigating the effects of an aspalathin-rich green rooibos extract (Afriplex GRTTM) against markers of hepatic oxidative stress, inflammation and apoptosis in an in vitro model of NAFLD. METHODS: Oleic acid [1 mM] was used to induce hepatic steatosis in C3A liver cells. Thereafter, the therapeutic effect of Afriplex GRTTM, with or without pioglitazone, was determined by assessing its impact on cell viability, changes in mitochondrial membrane potential, intracellular lipid accumulation and the expression of genes and proteins (ChREBP, SREBF1, FASN, IRS1, SOD2, Caspase-3, GSTZ1, IRS1 and TNF-α) that are associated with the development of NAFLD. RESULTS: Key findings showed that Afriplex GRTTM added to the medium alone or combined with pioglitazone, could effectively block hepatic lipid accumulation without inducing cytotoxicity in C3A liver cells exposed oleic acid. This positive outcome was consistent with effective regulation of genes involved in insulin signaling, as well as carbohydrate and lipid metabolism (IRS1, SREBF1 and ChREBP). Interestingly, in addition to reducing protein levels of an inflammatory marker (TNF-α), the Afriplex GRTTM could ameliorate oleic acid-induced hepatic steatotic damage by decreasing the protein expression of oxidative stress and apoptosis related markers such as GSTZ1 and caspase-3. CONCLUSION: Afriplex GRTTM reduced hepatic steatosis in oleic acid induced C3A liver cells by modulating SREBF1, ChREBP and IRS-1 gene expression. The extract may also play a role in alleviating inflammation by reducing TNF-α expression, suggesting that additional experiments are required for its development as a suitable therapeutic option against NAFLD. Importantly, further research is needed to explore its antioxidant role in this model.

Doxorubicin-Induced Cardiomyopathy: A Preliminary Study on the Cardioprotective Benefits of 7-Hydroxyflavanone.

The therapeutic properties of flavonoids are reported to offer cardioprotective benefits against doxorubicin (Dox)-induced cardiotoxicity (DIC). In the current study, we aimed to investigate the prophylactic properties of 7-hydroxyflavanone (7H), a flavonoid with antioxidative properties, against DIC. An in vitro model of DIC was established by exposing H9c2 cardiomyoblasts to Dox for 6 days. Similarly, cells were also co-treated with 7H to assess its ability to mitigate DIC. The data obtained indicate that 7H, as a co-treatment, alleviates Dox-induced oxidative stress by enhancing total glutathione content (p ≤ 0.001) and superoxide dismutase activity (p ≤ 0.001) whilst decreasing ROS (p ≤ 0.001), malondialdehyde production (p ≤ 0.001) and the secretion of interleukin-6 (p ≤ 0.001). The data also showed an improvement in mitochondrial function as shown via enhanced bioenergetics, mitochondrial membrane potential, and PGC1-alpha (p ≤ 0.05) and pAMPK (p ≤ 0.001) expression. The cardioprotective potential of 7H was further highlighted by its ability attenuate Dox-induced caspase 3/7 activity (p ≤ 0.001), apoptosis (p ≤ 0.001) and necrosis (p ≤ 0.05). In conclusion, our findings demonstrated the cardioprotective benefits of 7H and thus suggests that it could be a suitable candidate cardioprotective agent against DIC.

Cyclopia intermedia (Honeybush) Induces Uncoupling Protein 1 and Peroxisome Proliferator-Activated Receptor Alpha Expression in Obese Diabetic Female db/db Mice.

Previously, we reported that a crude polyphenol-enriched fraction of Cyclopia intermedia (CPEF), a plant consumed as the herbal tea, commonly known as honeybush, reduced lipid content in 3T3-L1 adipocytes and inhibited body weight gain in obese, diabetic female leptin receptor-deficient (db/db) mice. In the current study, the mechanisms underlying decreased body weight gain in db/db mice were further elucidated using western blot analysis and in silico approaches. CPEF induced uncoupling protein 1 (UCP1, 3.4-fold, p < 0.05) and peroxisome proliferator-activated receptor alpha (PPARα, 2.6-fold, p < 0.05) expression in brown adipose tissue. In the liver, CPEF induced PPARα expression (2.2-fold, p < 0.05), which was accompanied by a 31.9% decrease in fat droplets in Hematoxylin and Eosin (H&E)-stained liver sections (p < 0.001). Molecular docking analysis revealed that the CPEF compounds, hesperidin and neoponcirin, had the highest binding affinities for UCP1 and PPARα, respectively. This was validated with stabilising intermolecular interactions within the active sites of UCP1 and PPARα when complexed with these compounds. This study suggests that CPEF may exert its anti-obesity effects by promoting thermogenesis and fatty acid oxidation via inducing UCP1 and PPARα expression, and that hesperidin and neoponcirin may be responsible for these effects. Findings from this study could pave the way for designing target-specific anti-obesity therapeutics from C. intermedia.

Prevention of Anthracycline-Induced Cardiotoxicity: The Good and Bad of Current and Alternative Therapies.

Doxorubicin (Dox)-induced cardiotoxicity (DIC) remains a serious health burden, especially in developing countries. Unfortunately, the high cost of current preventative strategies has marginalized numerous cancer patients because of socio-economic factors. In addition, the efficacy of these strategies, without reducing the chemotherapeutic properties of Dox, is frequently questioned. These limitations have widened the gap and necessity for alternative medicines, like flavonoids, to be investigated. However, new therapeutics may also present their own shortcomings, ruling out the idea of "natural is safe". The U.S. Food and Drug Administration (FDA) has stipulated that the concept of drug-safety be considered in all pre-clinical and clinical studies, to explore the pharmacokinetics and potential interactions of the drugs being investigated. As such our studies on flavonoids, as cardio-protectants against DIC, have been centered around cardiac and cancer models, to ensure that the efficacy of Dox is preserved. Our findings thus far suggest that flavonoids of Galenia africana could be suitable candidates for the prevention of DIC. However, this still requires further investigation, which would focus on drug-interactions as well as in vivo experimental models to determine the extent of cardioprotection.

Sclerocarya birrea (Marula) Extract Inhibits Hepatic Steatosis in db/db Mice.

Non-alcoholic fatty liver disease (NAFLD) is a spectrum of hepatic metabolic perturbations ranging from simple steatosis to steatohepatitis, cirrhosis and hepatocellular carcinoma. Currently, lifestyle modifications to reduce weight gain are considered the most effective means of preventing and treating the disease. The aim of the present study was to determine the therapeutic benefit of Sclerocarya birrea (Marula leaf extract, MLE) on hepatic steatosis. Obese db/db mice were randomly stratified into the obese control, metformin (MET) or MLE-treated groups. Mice were treated daily for 29 days, at which point all mice were euthanized and liver samples were collected. Hematoxylin and eosin staining was used for histological assessment of the liver sections, while qRT-PCR and Western blot were used to determine hepatic mRNA and protein expression, respectively. Thereafter, the association between methylenetetrahydrofolate reductase (Mthfr a key enzyme in one-carbon metabolism and DNA-methylation-induced regulation of gene transcription) and lipogenic genes was evaluated using Pearson's correlation coefficient. Mice treated with MLE presented with significantly lower body and liver weights as compared with the obese control and MET-treated mice (p ≤ 0.05). Further, MLE treatment significantly inhibited hepatic steatosis as compared with the obese control and MET-treated mice (p ≤ 0.05). The reduced lipid accumulation was associated with low expression of fatty acid synthase (Cpt1; p ≤ 0.05) and an upregulation of the fatty acid oxidation gene, carnitine palmitoyltransferase (Cpt1; p ≤ 0.01), as compared with the obese control mice. Interestingly, MLE treatment improved the correlation between Mthfr and Cpt1 mRNA expression (r = 0.72, p ≤ 0.01). Taken together, the results suggest that Marula leaf extracts may inhibit hepatic steatosis by influencing the association between Mthfr and genes involved in hepatic lipid metabolism. Further studies are warranted to assess DNA methylation changes in lipid metabolism genes.

Molecular insights into the pathophysiology of doxorubicin-induced cardiotoxicity: a graphical representation.

A breakthrough in oncology research was the discovery of doxorubicin (Dox) in the 1960's. Unlike other chemotherapy drugs, Dox was determined to have a greater therapeutic index. Since its discovery, Dox has, in part, contributed to the 5-10-year survival increase in cancer patient outcomes. Unfortunately, despite its efficacy, both in adult and pediatric cancers, the clinical significance of Dox is tainted by its adverse side effects, which usually manifest as cardiotoxicity. The issue stems from Dox's lack of specificity which prevents it from accurately distinguishing between cancer cells and healthy cell lines, like cardiomyocytes. In addition, the high binding affinity of Dox to topoisomerases, which are abundantly found in cancer and cardiac cells in different isoforms, potentiates DNA damage. In both cell lines, Dox induces cytotoxicity by stimulating the production of pro-oxidants whilst inhibiting antioxidant enzymatic activity. Given that the cardiac muscle has an inherently low antioxidant capacity makes it susceptible to oxidative damage thereby, allowing the accumulation of Dox within the myocardium. Subsequently, Dox drives the activation of cell death pathways, such as ferroptosis, necroptosis and apoptosis by triggering numerous cellular responses that have been implicated in diseases. To date, the exact mechanism by which Dox induces the cardiotoxicity remains an aspect of much interest in cardio-oncology research. Hence, the current review summarizes the proposed mechanisms that are associated with the onset and progression of DIC.

Metformin and heart failure-related outcomes in patients with or without diabetes: a systematic review of randomized controlled trials.

Metformin is considered a safe anti-hyperglycemic drug for patients with type 2 diabetes (T2D); however, information on its impact on heart failure-related outcomes remains inconclusive. The current systematic review explored evidence from randomized clinical trials (RCTs) reporting on the impact of metformin in modulating heart failure-related markers in patients with or without T2D. Electronic databases such as MEDLINE, Cochrane Library, and EMBASE were searched for eligible studies. Included studies were those assessing the use of metformin as an intervention, and also containing the comparison group on placebo, and all articles had to report on measurable heart failure-related indices in individuals with or without T2D. The modified Downs and Black checklist was used to evaluate the risk of bias. Overall, nine studies met the inclusion criteria, enrolling a total of 2486 patients. Although summarized evidence showed that metformin did not affect left ventricular function, this antidiabetic drug could improve myocardial oxygen consumption concomitant to reducing prominent markers of heart failure such as n-terminal pro-brain natriuretic peptide and low-density lipoprotein levels, inconsistently between diabetic and nondiabetic patients. Effective modulation of some heart failure-related outcomes with metformin treatment was related to its beneficial effects in ameliorating insulin resistance and blocking pro-inflammatory markers such as the aging-associated cytokine CCL11 (C-C motif chemokine ligand 11). Overall, although such beneficial effects were observed with metformin treatment, additional RCTs are necessary to improve our understanding on its modulatory effects on heart failure-related outcomes especially in diabetic patients.

Exploring the Comparative Efficacy of Metformin and Resveratrol in the Management of Diabetes-associated Complications: A Systematic Review of Preclinical Studies.

Food-derived bioactive compounds such as resveratrol are increasingly explored for their protective effects against metabolic complications. Evidence supports the strong antioxidant properties and therapeutic effects of resveratrol in managing diabetes and its associated complications. However, evidence informing on the comparative or combination effects of this natural compound with an accomplished and well-characterized antidiabetic agent like metformin has not been revised. Thus, we conducted a comprehensive systematic search of the major electronic databases which included MEDLINE, Cochrane Library, and EMBASE. The cumulative evidence strongly supports the comparative effects of metformin and resveratrol in ameliorating diabetes-associated complications in preclinical settings. In particular, both compounds showed strong ameliorative effects against hyperglycemia, dyslipidemia, insulin resistance, a pro-inflammatory response, and lipid peroxidation in various experimental models of diabetes. Enhancing intracellular antioxidant capacity in addition to activating NAD-dependent deacetylase sirtuin-1 (SIRT1) and AMP-activated protein kinase (AMPK) are the prime mechanisms involved in the therapeutic effects of these compounds. Of interest, preclinical evidence also demonstrates that the combination treatment with these compounds may have a greater efficacy in protecting against diabetes. Thus, confirmation of such evidence in well-organized clinical trials remains crucial to uncover novel therapeutic strategies to manage diabetes and its linked complications.

Age-dependent development of left ventricular wall thickness in type 2 diabetic (db/db) mice is associated with elevated low-density lipoprotein and triglyceride serum levels.

Diabetic cardiomyopathy (DCM) is a disease of heart muscle that remains one of the leading causes of death in diabetic individuals. Shifts in substrate preference resulting in aberrant serum lipid content and enlarged left ventricular wall thickness are well-established characteristics associated with the development of DCM. As underlying mechanisms driving the onset of the DCM remain relatively unclear, this study sought to characterize age-dependent development of left ventricular (LV) wall thickness in diabetic (db/db) mice. Such data were compared with low-density lipoprotein (LDL) and triglyceride serum levels to assess whether any correlation exists between the parameters here investigated. For methods, db/db mice together with nondiabetic controls (n = six per group) were monitored from the age of 6-16 weeks. Mice were terminated each week to measure body weights, heart weights, liver weights, tibia length, and fasting plasma glucose levels. Heart tissues were stained with haematoxylin and eosin to measure LV wall and interventricular septum thickness together with an assessment of myocardial remodeling. Serum was collected weekly and used to measure LDL and triglyceride levels. Results showed that db/db mice presented significantly increased body weights, liver/body weight, and fasting plasma glucose levels from the age of 6-16 weeks. They further displayed a marked enlargement of LV wall and interventricular septum thickness from the age of 11 weeks, while increased heart weight/tibia length was recorded only from week 16. From week 11, the LV wall and interventricular septum thickness results corresponded with cardiac remodeling and raised LDL and triglyceride serum levels. In summary, age-dependent development of LV wall thickness in db/db mice is partially associated with increased LDL and triglyceride levels, elucidating a potential pathophysiological mechanism.

Aspalathin Protects the Heart against Hyperglycemia-Induced Oxidative Damage by Up-Regulating Nrf2 Expression.

Aspalathin (ASP) can protect H9c2 cardiomyocytes against high glucose (HG)-induced shifts in myocardial substrate preference, oxidative stress, and apoptosis. The protective mechanism of ASP remains unknown. However, as one of possible, it is well known that phytochemical flavonoids reduce oxidative stress via nuclear factor (erythroid-derived 2)-like 2 (Nrf2) activation resulting in up-regulation of antioxidant genes and enzymes. Therefore, we hypothesized that ASP protects the myocardium against HG- and hyperglycemia-induced oxidative damage by up-regulating Nrf2 expression in H9c2 cardiomyocytes and diabetic (db/db) mice, respectively. Using an oxidative stress RT² Profiler PCR array, ASP at a dose of 1 µM was demonstrated to protect H9c2 cardiomyocytes against HG-induced oxidative stress, but silencing of Nrf2 abolished this protective response of ASP and exacerbated cardiomyocyte apoptosis. Db/db mice and their non-diabetic (db/+) littermate controls were subsequently treated daily for six weeks with either a low (13 mg/kg) or high (130 mg/kg) ASP dose. Compared to nondiabetic mice the db/db mice presented increased cardiac remodeling and enlarged left ventricular wall that occurred concomitant to enhanced oxidative stress. Daily treatment of mice with ASP at a dose of 130 mg/kg for six weeks was more effective at reversing complications than both a low dose ASP or metformin, eliciting enhanced expression of Nrf2 and its downstream antioxidant genes. These results indicate that ASP maintains cellular homeostasis and protects the myocardium against hyperglycemia-induced oxidative stress through activation of Nrf2 and its downstream target genes.

Phenylpyruvic Acid-2-O-ß-D-Glucoside Attenuates High Glucose-Induced Apoptosis in H9c2 Cardiomyocytes.

Chronic hyperglycemia is closely associated with impaired substrate metabolism, dysregulated mitochondrial membrane potential, and apoptosis in the diabetic heart. As adult cardiomyocytes display a limited capacity to regenerate following an insult, it is essential to protect the myocardium against the detrimental effects of chronic hyperglycemia. This study therefore investigated whether phenylpyruvic acid-2-O-ß-D-glucoside, present in Aspalathus linearis (rooibos), is able to attenuate hyperglycemia-induced damage in H9c2 cardiomyocytes. H9c2 cardiomyocytes were exposed to a high glucose concentration (33 mM) prior to treatment with phenylpyruvic acid-2-O-ß-D-glucoside (1 µM), metformin (1 µM), or a combination of phenylpyruvic acid-2-O-ß-D-glucoside and metformin (both at 1 µM). Our data revealed that high glucose exposure increased cardiac free fatty acid uptake and oxidation, mitochondrial membrane potential, and apoptosis (caspase 3/7 activity and TUNEL), and decreased the Bcl2/Bax protein expression ratio. Phenylpyruvic acid-2-O-ß-D-glucoside treatment, alone or in combination with metformin, attenuated these glucose-induced perturbations, confirming its protective effect in H9c2 cardiomyocytes exposed to chronic hyperglycemia.