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.

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.

The Implication of Low Dose Dimethyl Sulfoxide on Mitochondrial Function and Oxidative Damage in Cultured Cardiac and Cancer Cells.

Although numerous studies have demonstrated the biological and multifaceted nature of dimethyl sulfoxide (DMSO) across different in vitro models, the direct effect of "non-toxic" low DMSO doses on cardiac and cancer cells has not been clearly explored. In the present study, H9c2 cardiomyoblasts and MCF-7 breast cancer cells were treated with varying concentrations of DMSO (0.001-3.7%) for 6 days. Here, DMSO doses < 0.5% enhanced the cardiomyoblasts respiratory control ratio and cellular viability relative to the control cells. However, 3.7% DMSO exposure enhanced the rate of apoptosis, which was driven by mitochondrial dysfunction and oxidative stress in the cardiomyoblasts. Additionally, in the cancer cells, DMSO (≥0.009) led to a reduction in the cell's maximal respiratory capacity and ATP-linked respiration and turnover. As a result, the reduced bioenergetics accelerated ROS production whilst increasing early and late apoptosis in these cells. Surprisingly, 0.001% DMSO exposure led to a significant increase in the cancer cells proliferative activity. The latter, therefore, suggests that the use of DMSO, as a solvent or therapeutic compound, should be applied with caution in the cancer cells. Paradoxically, in the cardiomyoblasts, the application of DMSO (≤0.5%) demonstrated no cytotoxic or overt therapeutic benefits.

The triterpene, methyl-3ß-hydroxylanosta-9,24-dien-21-oate (RA3), attenuates high glucose-induced oxidative damage and apoptosis by improving energy metabolism.

BACKGROUND: Hyperglycemia-induced cardiovascular dysfunction has been linked to oxidative stress and accelerated apoptosis in the diabetic myocardium. While there is currently no treatment for diabetic cardiomyopathy (DCM), studies suggest that the combinational use of anti-hyperglycemic agents and triterpenes could be effective in alleviating DCM. HYPOTHESIS: To investigate the therapeutic effect of methyl-3ß-hydroxylanosta-9,24-dien-21-oate (RA3), in the absence or presence of the anti-diabetic drug, metformin (MET), against hyperglycemia-induced cardiac injury using an in vitro H9c2 cell model. METHODS: To mimic a hyperglycemic state, H9c2 cells were exposed to high glucose (HG, 33 mM) for 24 h. Thereafter, the cells were treated with RA3 (1 µM), MET (1 µM) and the combination of MET (1 µM) plus RA3 (1 µM) for 24 h, to assess the treatments therapeutic effect. RESULTS: Biochemical analysis revealed that RA3, with or without MET, improves glucose uptake via insulin-dependent (IRS-1/PI3K/Akt signaling) and independent (AMPK) pathways whilst ameliorating the activity of antioxidant enzymes in the H9c2 cells. Mechanistically, RA3 was able to alleviate HG-stimulated oxidative stress through the inhibition of reactive oxygen species (ROS) and lipid peroxidation as well as the reduced expression of the PKC/NF-кB cascade through decreased intracellular lipid content. Subsequently, RA3 was able to mitigate HG-induced apoptosis by decreasing the activity of caspase 3/7 and DNA fragmentation in the cardiomyoblasts. CONCLUSION: RA3, in the absence or presence of MET, demonstrated potent therapeutic properties against hyperglycemia-mediated cardiac damage and could be a suitable candidate in the prevention of DCM.

The Prophylactic Effect of Pinocembrin Against Doxorubicin-Induced Cardiotoxicity in an In Vitro H9c2 Cell Model.

BACKGROUND: The clinical use of Doxorubicin (Dox) is significantly limited by its dose-dependent cardiotoxic side effect. Accumulative evidence suggests that the use of flavonoids, such as the antioxidative Pinocembrin (Pin), could be effective in the prevention of Dox-induced cardiotoxicity. Accordingly, we investigated the ability of pinocembrin (Pin) to attenuate Dox-induced cardiotoxicity in an in vitro H9c2 cardiomyoblast model. METHODOLOGY: The cardioprotective potential of Pin was established in H9c2 cells. Here, cells were treated with Dox (2µM), Dox (2µM) + Pin (1µM), and Dox (2µM) + Dexrazoxane (20µM) for 6 days. Thereafter, the safe co-administration of Pin with Dox, in a cancer environment, was investigated in MCF-7 breast cancer cells subjected to the same experimental conditions. Untreated cells served as the control. Subsequently, Pin's ability to attenuate Dox-mediated oxidative stress, impaired mitochondrial bioenergetics and potential, as well as aggravated apoptosis was quantified using biochemical assays. RESULTS: The results demonstrated that co-treatment with Pin mitigates Dox-induced oxidative stress by alleviating the antioxidant enzyme activity of the H9c2 cells. Pin further reduced the rate of apoptosis and necrosis inferred by Dox by improving mitochondrial bioenergetics. Interestingly, Pin did not decrease the efficacy of Dox but, rather increased the rate of apoptosis and necrosis in Dox-treated MCF-7 cells. CONCLUSION: The findings presented in this study showed, for the first time, that Pin attenuates Dox-induced cardiotoxicity without reducing its chemotherapeutic effect. We propose that additional studies, using in vivo models, should be conducted to further investigate Pin as a suitable candidate in the prevention of the cardiovascular dysfunction inferred by Dox administration.

In Utero One-Carbon Metabolism Interplay and Metabolic Syndrome in Cardiovascular Disease Risk Reduction.

The maternal obesogenic environment plays a role in programing the susceptibility of the fetus to postnatal non-alcoholic fatty liver disease (NAFLD), a risk factor for cardiovascular disease (CVD). NAFLD is a multisystem disease that is characterized by hepatic fat accumulation due in part to dysregulated energy metabolism network through epigenetic mechanisms such as DNA methylation. DNA methylation affects fetal programing and disease risk via regulation of gene transcription; it is affected by methyl donor nutrients such as vitamin B12 , methionine, folic acid, vitamin B6 , and choline. Although several studies have documented the role of several maternal methyl donor nutrients on obesity-induced NAFLD in offspring, currently, data are lacking on its impact on CVD risk as an endpoint. The aim of this paper is to use current knowledge to construct a postulation for the potential role of a comprehensive gestational methyl donor nutrients supplementary approach on the susceptibility of offspring to developing metabolic-syndrome-related cardiovascular complications.

An In Vitro Study on the Combination Effect of Metformin and N-Acetyl Cysteine against Hyperglycaemia-Induced Cardiac Damage.

Chronic hyperglycaemia is a major risk factor for diabetes-induced cardiovascular dysfunction. In a hyperglycaemic state, excess production of reactive oxygen species (ROS), coupled with decreased levels of glutathione, contribute to increased lipid peroxidation and subsequent myocardial apoptosis. N-acetylcysteine (NAC) is a thiol-containing antioxidant known to protect against hyperglycaemic-induced oxidative stress by promoting the production of glutathione. While the role of NAC against oxidative stress-related cardiac dysfunction has been documented, to date data is lacking on its beneficial effect when used with glucose lowering therapies, such as metformin (MET). Thus, the aim of the study was to better understand the cardioprotective effect of NAC plus MET against hyperglycaemia-induced cardiac damage in an H9c2 cardiomyoblast model. H9c2 cardiomyoblasts were exposed to chronic high glucose concentrations for 24 h. Thereafter, cells were treated with MET, NAC or a combination of MET and NAC for an additional 24 h. The combination treatment mitigated high glucose-induced oxidative stress by improving metabolic activity i.e. ATP activity, glucose uptake (GU) and reducing lipid accumulation. The combination treatment was as effective as MET in diminishing oxidative stress, lipid peroxidation and apoptosis. We observed that the combination treatment prevented hyperglycaemic-induced cardiac damage by increasing GLUT4 expression and mitigating lipid accumulation via phosphorylation of both AMPK and AKT, while decreasing nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kB), as well as protein kinase C (PKC), a known activator of insulin receptor substrate-1 (IRS-1), via phosphorylation at Ser307. On this basis, the current results support the notion that the combination of NAC and MET can shield the diabetic heart against impaired glucose utilization and therefore its long-term protective effect warrants further investigation.

Lanosteryl triterpenes from Protorhus longifolia as a cardioprotective agent: a mini review.

The epidemic of cardiovascular diseases is a global phenomenon that is exaggerated by the growing prevalence of diabetes mellitus. Coronary artery disease and diabetic cardiomyopathy are the major cardiovascular complications responsible for exacerbated myocardial infarction in diabetic individuals. Increasing research has identified hyperglycemia and hyperlipidemia as key factors driving the augmentation of oxidative stress and a pro-inflammatory response that usually results in increased fibrosis and reduced cardiac efficiency. While current antidiabetic agents remain active in attenuating diabetes-associated complications, overtime, their efficacy proves limited in protecting the hearts of diabetic individuals. This has led to a considerable increase in the number of natural products that are screened for their antidiabetic and cardioprotective properties. These natural products may present essential ameliorative properties relevant to their use as a monotherapy or as an adjunct to current drug agents in combating diabetes and its associated cardiovascular complications. Recent findings have suggested that triterpenes isolated from Protorhus longifolia (Benrh.) Engl., a plant species endemic to Southern Africa, display strong antioxidant and antidiabetic properties that may potentially protect against diabetes-induced cardiovascular complications. Thus, in addition to discussing the pathophysiology associated with diabetes-induced cardiovascular injury, available evidence pertaining to the cardiovascular protective potential of lanosteryl triterpenes from Protorhus longifolia will be discussed.