Effects and safety of Psilocybe cubensis and Panaeolus cyanescens magic mushroom extracts on endothelin-1-induced hypertrophy and cell injury in cardiomyocytes

Heart failure is a major public health problem associated with reduced quality of life and a substantially elevated prevalence of major depression compared with the general population. Previous research has shown that psilocybin, the principal psychoactive compound in so-called magic mushrooms, has antidepressant effects, but the cardiovascular safety of psilocybin-containing mushrooms in conditions such as heart failure is not well established. Endothelin-1 (ET-1) is a potent vasoconstrictor and a recognised inducer of pathological cardiomyocyte hypertrophy; tumour necrosis factor-α (TNF-α) contributes to cardiac injury and apoptosis. These pathways are relevant to heart failure and to comorbid depression in affected patients, creating a need to evaluate whether mushroom preparations might aggravate or ameliorate cardiac pathology. Nkadimeng and colleagues set out to test whether water extracts of two species commonly used as magic mushrooms, Psilocybe cubensis and Panaeolus cyanescens, affect ET-1-induced hypertrophy and TNF-α-induced cell injury in vitro. The study employed hot-water and cold-water extracts—methods reflecting common routes of human consumption—and used the rat H9C2 cardiomyoblast cell line to assess morphological, biochemical and viability endpoints. The work is presented as the first in vitro evaluation of these species in an ET-1-induced hypertrophy model and aims to provide preliminary safety data relevant to use in patients with heart failure-related depression.

This was an in vitro experimental study using rat H9C2 cardiomyoblasts. Nkadimeng and colleagues grew Psilocybe cubensis and Panaeolus cyanescens from purchased spore syringes under sterile conditions, prepared hot-boiling and cold-water extracts of each species, and labelled them GH (P. cubensis hot), GC (P. cubensis cold), PH (P. cyanescens hot) and PC (P. cyanescens cold). Ethics and regulatory approvals from the University of Pretoria and the South African Health Department were obtained because psilocybin-containing mushrooms are controlled substances in South Africa. Cell culture procedures included seeding at specified densities depending on assay: 1 × 10^6 cells on cover slips in six-well plates for morphology, 1 × 10^4 cells per well in 96-well plates for mitochondrial activity and ROS assays, and 1 × 10^5 cells per well for TNF-α apoptosis assays. Cells were serum-deprived for 18 h prior to induction. Hypertrophy was induced with 100 nM ET-1 for 45 min (or 2 h for ROS measurements) before treatment with mushroom extracts at 50 µg/mL; ambrisentan (25 µg/mL), an ET-A receptor antagonist, served as a positive control for ET-1 experiments. For TNF-α injury assays, cells were exposed to 250 pg/mL TNF-α for 2 h and then treated with extracts at 25 and 50 µg/mL; quercetin (12.5 and 25 µg/mL) was used as a positive control for protection against apoptosis. Outcome measures included mitochondrial activity (resazurin assay, reported as % viability), cell surface area measured by haematoxylin & eosin staining and image analysis (60–80 cells per group), BNP concentration in culture medium (EIA), TNF-α concentration in medium (ELISA), intracellular reactive oxygen species (ROS) measured fluorometrically with a kit targeting superoxide and hydroxyl radicals, and a spectrophotometric-derived rate-of-growth assay based on absorbance differences before and after stimulation. Experiments were performed in triplicate and repeated three times. Statistical analysis used one-way ANOVA with normality (Shapiro–Wilk) and equal variance (Brown–Forsythe) tests; results are presented as mean ± SD and p ≤ 0.050 was considered significant.

ET-1 produced the expected hypertrophic and injurious phenotype in H9C2 cells: cell size measurements and BNP concentrations increased after ET-1 exposure, while mitochondrial activity decreased significantly to under 80% viability versus non-induced controls (NO-ET1). ET-1 also raised extracellular TNF-α (p = 0.006) and intracellular ROS (p < 0.0001). When applied at the concentrations used, the water extracts of both species ameliorated several ET-1-induced changes. All four extracts increased cell viability above 80% in a dose-dependent manner, comparable to ambrisentan. Regarding TNF-α levels in ET-1-stimulated cultures, both hot- and cold-water extracts of P. cubensis reduced TNF-α significantly (p = 0.047 and p = 0.024 respectively). For P. cyanescens, the hot-water extract decreased TNF-α significantly (p = 0.002) while the cold-water extract produced a non-significant increase compared with ET-1 controls. Ambrisentan reduced TNF-α non-significantly. All four water extracts produced marked reductions in ET-1-induced intracellular ROS: ET-1 increased ROS (p < 0.0001) and ambrisentan significantly decreased ROS (p < 0.0001); both P. cubensis extracts (GH and GC) and both P. cyanescens extracts (PH and PC) significantly reduced ROS production (all p < 0.0001 compared with ET-1 control). Cell growth-rate assays showed that ET-1 lowered growth versus NO-ET1; GC (P. cubensis cold) increased growth similar to ambrisentan and GH showed the highest growth rate relative to NO-ET1 at early time points. In contrast, the two P. cyanescens extracts initially induced lower growth rates than NO-ET1, even lower than ET-1 control, though the cold extract’s growth rate improved over time and by 48 h was close to ambrisentan. In TNF-α-induced injury assays, TNF-α caused a significant reduction in viability below 80% (p < 0.0001). Both hot and cold extracts of P. cubensis and P. cyanescens increased % viability of TNF-α-treated cells above 100%, comparable to the positive control quercetin; this protective effect was reported as dose dependent. Notably, the cold P. cyanescens extract did not lower ET-1-induced TNF-α at 48 h but nevertheless increased cell viability beyond control and positive-control levels at the concentrations tested.

Nkadimeng and colleagues interpret their results as indicating that water extracts of Psilocybe cubensis and Panaeolus cyanescens do not exacerbate ET-1-induced hypertrophic changes in H9C2 cardiomyoblasts and, at the concentrations studied, display protective properties. The extracts reversed ET-1-induced increases in cell size and BNP, reduced ROS generation, increased mitochondrial activity/viability, and protected against TNF-α-induced cell injury and death. The authors note that three of the extracts (both P. cubensis extracts and the hot P. cyanescens extract) also reduced ET-1-induced TNF-α concentrations, which they regard as favourable given TNF-α’s role in cardiac dysfunction and heart failure progression. Possible mechanisms proposed by the investigators include antioxidant activity and modulation of apoptosis-regulatory pathways. They suggest the extracts might influence NF-κB signalling and/or promote Bcl-2 family cell-survival pathways, thereby limiting apoptosis and preserving mitochondrial integrity; these hypotheses are offered to explain why the cold P. cyanescens extract protected viability despite not lowering TNF-α. The discussion also highlights known mycochemical constituents detected in these species—alkaloids, saponins, flavonoids and tannins—which have documented antioxidant and anti-inflammatory properties and therefore might account for the observed effects. The authors observed a difference between hot- and cold-water extracts: cardioprotective effects were generally more pronounced with hot-water extracts, prompting the suggestion that preparations such as mushroom tea could yield greater benefit. They also caution that P. cyanescens extracts may transiently slow cell growth early after exposure, a phenomenon the authors attribute to the species’ reportedly high urea content and its known effects on cell-cycle dynamics. Overall, the investigators recommend caution at higher concentrations, acknowledge that mechanistic pathways remain to be established, and call for further in vitro and in vivo studies to clarify the underlying mechanisms and to extend safety evaluation in relevant models.