The cardioprotective effect of S. africana caerulea/Blue Sage in ischaemia and reperfusion induced oxidative stress

Background: Since antiquity, alternative herbal remedies, such as S. africana caerulea/Blue Sage (BLS) water infusion extract (WIE) has been used by traditional healers, for the effective treatment of various chronic inflammatory disorders associated with reduced cellular antioxidant defense mechanisms and free radical cellular damage. In the heart, ischaemia—reperfusion (I/R) induced oxidative stress becomes an early crucial event in the pathogenesis of ischaemia—reperfusion injury (I/RI) and subsequent heart failure.Purpose/Aim: To investigate whether BLS WIE treatment during ischaemia and/or reperfusion may be cardioprotective.Study design: Isolated perfused rat hearts were exposed to 35 min regional ischaemia (RI) and 60 min reperfusion. The BLS WIE was applied: i) for the last 10 min of RI (PerT) or ii) from onset of reperfusion (PostT) or iii) both (PerT) + (PostT). Methods: Endpoints were functional recovery and infarct size (IS). In another set of experiments, left ventricles were freeze-clamped after RI and 10 min reperfusion for detection of total and phosphorylated p-ERK p44/p42, p-Akt, p-p38-MAPK, p-JNK, Nrf-2, NF-kB, Bax, Bcl-2, Caspase-3, and PGC-1α by Western blot analysis.Results: BLS (PostT) significantly increased ERK p44, p-Akt, Nrf-2, and Bcl-2 levels; significantly decreased p-p38-MAPK as well as p-JNK p46 phosphorylation; did not affect Bax levels and significantly decreased Bax/Bcl-2 ratios. This was associated with significantly reduced Caspase-3 levels and increased PGC-1α phosphorylation, particlarly when BLS WIE was administered as PostT.Conclusion: The administration of polyphenol-rich BLS WIE at different stages of ischaemia and/or reperfusion, activate/inhibit several signaling events simultaneously and mediate cardioprotection in a multitarget manner

mRNA expression of C3A cell-lysates normalized to <i>β-actin</i>, showing the effects of steatosis induction [1 mM oleic acid] and treatment effects of pioglitazone and Afriplex GRT^TM.

Data represents mean ± SEM (n = 3), where *P #P ##P a) ChREBP (b) FASN, (c) SREBF1, (d) IRS-1 & (e) SOD2 respectively. ChREBP—carbohydrate response element binding protein, FASN—fatty acid synthase, SREBF1—sterol regulatory element-binding transcription factor 1, IRS-1—insulin receptor substrate 1, SOD2—superoxide dismutase 2.</p

List of measurements, list of abbreviations and table of chemicals/kits.

List of measurements, list of abbreviations and table of chemicals/kits.</p

List of antibodies.

BackgroundCurrently, 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.MethodsOleic 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.ResultsKey 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.ConclusionAfriplex 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.</div

Afriplex^TM GRT product safety data sheet with physical and chemical properties.

AfriplexTM GRT product safety data sheet with physical and chemical properties.</p

Protein expression of C3A cell-lysates normalized to β-actin, showing the effects of steatosis induction [1 mM oleic acid] and treatment effects of pioglitazone and Afriplex GRT^TM.

Data represents mean ± SEM (n = 4), where *P #P ##P ####P a) Caspase-3, (b) IRS-1, (c) GSTZ1, (d) TNF-α, (e) FASN & (f) NFκB respectively. IRS-1—insulin receptor substrate 1, GSTZ-1—glutathione S-transferase zeta 1, TNF-α - tumour necrosis factor-alpha, FASN—fatty acid synthase, NFκB—nuclear factor kappa B.</p

MTT assay, showing the effects of steatosis induction [1 mM oleic acid] and treatment effects of pioglitazone and Afriplex GRT^TM (GRT), on mitochondrial activity.

Data represents mean ± SEM (n = 3), where *P < 0.05, **P < 0.01 and ***P < 0.001 when compared to the control.</p

Full Western blot images.

BackgroundCurrently, 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.MethodsOleic 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.ResultsKey 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.ConclusionAfriplex 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.</div

Gene expression TaqMan probes and qRT-PCR conditions.

Gene expression TaqMan probes and qRT-PCR conditions.</p

Oil Red O assay, showing the effects of steatosis induction [1 mM oleic acid] and treatment effects of pioglitazone and Afriplex GRT^TM (GRT), on intracellular lipid content.

Images are a representation of one experiment, captured at 200x magnification using a Nikon Eclipse Ti/S inverted light microscope with a digital camera (scale represents: 100 μm). Histogram illustrates spectrophotometric data of three independent repeats of the ORO assay. Data represents mean ± SEM (n = 3), where **P #P < 0.05, when compared to the oleic acid-induced cells respectively.</p