In H9C2 cardiomyocytes, water extracts of Psilocybe cubensis and Panaeolus cyanescens did not exacerbate endothelin‑1‑induced hypertrophy and, at the tested concentrations, protected against TNF‑α‑induced injury and cell death. These results support the potential safety of controlled medicinal use of these psilocybin‑containing mushrooms in this pathological context, with caution advised at higher concentrations.
Prevalence of major depression in people with chronic heart failure is higher than in normal populations. Depression in heart failure has become a major issue. Psilocybin-containing mushrooms commonly known as magic mushrooms, have been used since ancient times for their mind healing properties. Their safety in cardiovascular disease conditions is not fully known and may pose as a risk for users suffering from these illnesses. Study investigates the effects and safety of Psilocybe cubensis and Panaeolus cyanescens magic mushrooms use from genus Psilocybe and Panaeolus respectively, in a pathological hypertrophy conditions in which endothelin-1 disorder is a contributor to pathogenesis. We examined the effects of the mushrooms extracts on endothelin-1-induced hypertrophy and tumor necrosis factor-α (TNF- α)-induced cell injury in H9C2 cardiomyocytes. Mushrooms were oven dried and extracted with cold and boiling-hot water. H9C2 cardiomyocytes were induced with endothelin-1 prior to treatment with extracts over 48 h. Cell injury was stimulated with TNF-α. Results proposed that the water extracts of Panaeolus cyanescens and Psilocybe cubensis did not aggravate the pathological hypertrophy induced by endothelin-1 and also protected against the TNF-α-induced injury and cell death in concentrations used. Results support medicinal safe use of mushrooms under controlled conditions and cautioned use of higher concentrations.
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.
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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.
Ethical and protocol clearances. The protocol for this study was submitted to the University of Pretoria research committee (UPREC) and approved with the number REC045-18. The protocol was also submitted and approved by the Medical Control Council (MCC) committee of South African Health Department and a permit (POS 223/2019/2020) as psilocybin-containing mushrooms are schedule 7 substances in South Africa.
Effects on cell width measurements and BNP concentrations. Morphological Effects on mitochondrial activity. Treatment with ET-1 reduced mitochondrial activity of the cells significantly (p < 0.0001 indicated by % cell viability below 80% in comparison to the NO-ET1 cells, Fig.. The water extracts of P. cubensis and Pan cyanescens increased the viability of cells above 80% in safe margins in a dose dependant manner same as ambrisentan, positive control, Fig..
Heart failure is a public health problem that significantly impacts daily management and the quality of life of many affected persons 2 . Major depression in chronic heart failure and its increasing role in heart failure mortality is an additional problem. Although magic mushrooms have been used in ancient and recent times for mind healing and are known to improve the quality of life, their safety in cardiovascular diseases such as heart failure is not known. Our study investigated for the first time, the effects of the hot-water and cold-water extracts of Panaeolus cyanescens and Psilocybe cubensis magic mushrooms on ET-1, a major physiological inducer of hypertrophic changes in vitro on rat H9C2 cardiomyocytes where we evaluate the safety or ability of the extracts to exacerbate these effects. The in vitro H9C2 cardiomyoblast cells protocol model used in the study was chosen based on their established and proven capacity to exhibit physiological responses useful in drug discovery for cardiovascular medicine. The results from the study demonstrated that treatment with ET-1 increased significantly the cell measurements sizes, BNP levels of the stimulated cells and decreased mitochondrial activity significantly as indicated by cell viability when compared to the non-induced NO-ET1 cells. These effects were in agreement with previous studies indicating successful cellular ET-1-induced hypertrophy in our study. manner same as positive control, ambrisentan indicating safety at the concentrations investigated in the study. Furthermore the two extracts of P. cubensis and hot-water extract of Pan cyanescens mushrooms reduced the TNF-α concentration in the treated cells compared to ET-1-induced control cells while the cold-water of Pan cyanescens slightly increased it non-significantly. TNF-α is a key pro-inflammatory cytokine that is known to promote cardiac dysfunctions and contributes to heart failure. By reducing TNF-α, the three extracts demonstrated potential safety in heart failure conditions. Many studies have established that ROS plays a very important role in the progression of cardiovascular diseases such as heart failure by inducing oxidative stress which in turn leads to cell and tissue injury. Superoxide and hydroxyl radicals are among the most prominent ROS causing toxic insults to the human body. In our study we measured ROS levels especially superoxide and hydroxyl radicals over 1 h of treatment after 2 h ET-1 stimulation and the results showed that the four water mushroom extracts reversed the ET-1-induced ROS levels significantly same as the positive control when compared to ET-1-induced control cells. By decreasing ROS levels, the extracts demonstrated safety and protective effect against ET-1 induced oxidative stress that will be beneficial in heart failure. Furthermore, the decrease in ROS observed with the extracts was not due to toxicity based on the positive increase in cell growth rate (Fig.) where % viability of cells continued to increase after 1 h treatment. However, it was also quite interesting to perceive the differences between the water extracts of the two mushrooms on cell growth rate analysis in comparison to the NO-ET-1 cells. The cells treated with P. cubensis extract after 1 h continued to grow at the rate close to positive control, ambrisentan and NO-ET1 cells while the water extract of Pan cyanescens lead to a reduction in rate of cell growth even slower than that observed with ET-1-induced control cells. This effect showed that although Pan cyanescens water extracts reduced ROS levels, they also contain other compounds that lowered rate of cell growth. However, it is known that Pan cyanescens mushrooms are unique in that they possess very high levels of urea in addition to psilocybin, psilocin, baeocystin, and other compounds generally known to be present in magic mushrooms. Urea is also known to induce cell cycle delay and promote a slow rate of increase of cells in log phase of growth. This could be the reason behind such a reduction in rate of cell growth observed with Pan cyanescens water extracts treatment compared to the other samples. However, we also observed an improvement change from 12 h in the growth rate of the two water extracts of Pan cyanescence such that the cold water exhibited the highest rate of growth by the 48th hour treatment. Caution is however needed with higher concentrations of Pan cyanescens mushroom water extracts as they may have potential to induce cell cycle delay in the first hour after consumption. To further investigate safety of the extracts on cell injury, the results showed that the cardiomyocytes induced with TNF-α stimulated a significant cell death indicated by reduction in the viability of induced cells below 80% compared to normal non-induced cells. The four extracts of the two magic mushrooms reversed the TNF-αinduced cell injury and death signified by increasing % cell viability of the treated cells same as positive control quercetin in a dose dependent manner. This effect demonstrated the protective effect of the mushroom extracts against cardiomyocyte injury that will be beneficial in a pathological hypertrophy condition. Moreover, it was also interesting to observe that although the cold-water extract of Pan cyanescens mushroom did not inhibit production of TNF-α concentration in the ET-1 stimulated cells after 48 h, the extract still protected against ET-1 induced cell death by increasing % cell viability of cells even higher than the positive control and non-induced cells at the concentrations used in the study as shown in Fig.. This effect combine with the protective effect of the cold-water extract on TNF-α-induced cell injury above demonstrate that the extract may have compounds that blocked the induced-cardiomyocyte apoptosis cascades probably by activating or promoting expressions of the cell death repressors. Studies have shown that the apoptosis effects of TNF-α in the heart is depending on the type of its receptor whereby it exhibits its cardiotoxic effect through its receptor TNFR1 (tumor necrosis factor receptor1). After binding to its TNFR1 receptor, TNF-α can stimulates apoptosis in cardiomyocytes by activating sphingomyelin signal transduction pathway leading to production of the intracellular signalling molecule, sphingosine. Sphingosine is a well-known effective inducer of apoptosis on cardiomyocytes and it induces its effect by down-regulating the expressions of cell death repressors, Bcl-2 (B Cell Lymphoma-2) family protein in the same manner as it does in other cell types. Furthermore, sphingosine is also a potent inhibitor of protein kinase C (PKC), which has been found to protect cells from apoptotic cell death; consequently, sphingosine may promote apoptosis through PKC inhibition by changing the level of Blc-2 phosphorylation. Moreover, many studies have also found that the beta-adrenergic receptor1 blocker (β 1 -blocker) enhances the resistance of cardiomyocytes to cell death by expanding the survival range of the switching response of Bcl-2. Beta-adrenergic receptor1 is one of the β-adrenergic receptors known to transduce the cell death signal via cyclic 3′,5′-adenosine monophosphate (cAMP)-dependent signalling pathways of cardiomyocytes which may result in the reduction of cardiac contractility related to the pathophysiology of heart failure. We propose possibility that the water extracts of Pan cyanescens and P. cubensis mushrooms may possess compounds with potential ability to promote or activate overexpression and/or phosphorylation of Blc-2 proteins pathways thereby inhibiting the induced-apoptosis and preserving mitochondrial membrane integrity of the treated cells. And this compound/s may be more pronounced in the cold-water extraction of Pan cyanescens mushroom. Moreover, the suppressive effects of the two-water extract of P. cubensis and the hot-water extract of Pan cyanescens on ET-induced TNF-α levels of treated cells also indicated that these extracts may also have activity on the nuclear factor (NF)-κB signalling, a transcription factor that regulates the expression of many proinflammatory cytokines including TNF-α and the genes associated with apoptosis. Studies have proposed that the inflammation-related NF-κB signaling and its correlation with apoptosis is the underlying mechanism in the pathogenesis of heart failure. Furthermore, oxidative stress may also activate NF-κB and initiate the transcription of numerous pro-apoptotic genes, which includes Bax, Fas and Fas ligand, inducing myocardial cell apoptosis and further promoting heart failure condition. A further study into the mechanisms of action in vitro and in vivo is therefore recommended. Furthermore, in previous studies, mycochemical compounds were verified to be present in both Pan cyanescens and P. cubensis mushrooms such as alkaloids, with known biological activities including toxicity against cells of foreign organisms. Saponins which are known as potent antioxidant that neutralises free radicals and flavonoids with antioxidant, anti-inflammatory and anti-carcinorgenic activities. Finally, tannins with antioxidant properties related to their scavenging activities reported to have been used against heart diseases were also detected in the two mushrooms. Presence of these compounds could have also played a role in the protective activities exhibited by the water extracts of Pan cyanescens and P. cubensis mushrooms in the study. The study also showed that in general the cardioprotective effects were more pronounced with the hot-water extracts of the two mushrooms compared to the cold-water extractions suggesting more benefit with users of the mushrooms that consume the mushrooms with tea. In conclusion, the study demonstrated that ET-1 significantly increased cell size measurements, BNP, TNF-α and ROS levels and decreased mitochondrial activity of the stimulated cardiomyocyte cells. The results indicated that the water extracts of P. cubensis and Pan cyanescens mushrooms significantly reversed the cell size and BNP levels which are two indices of hypertrophy and increased viability of cells. The two water extracts of P. cubensis and hot-water extract of Pan cyanescens mushrooms also significantly reduced the ET-1-induced TNF-α, a pro-inflammatory cytokine involved in the progression of pathological hypertrophy and heart failure. The four extracts also inhibited the ET-1 induced intracellular ROS levels significantly indicating potential safety in these conditions. Furthermore, the extracts exhibited protective properties against TNF-α-induced cell injury and death in the concentrations investigated in the study. Finally, the study proposed that the water extracts of Panaeolus cyanescens and Psilocybe cubensis mushrooms did not increase the ET-1-induced hypertrophic changes, instead the two mushrooms had cardioprotective potential properties and also alleviated against TNF-α-induced cell injury and death in the concentrations investigated. The study indicated for the first time the safety and potential beneficial properties of Panaeolus cyanescens and Psilocybe cubensis mushrooms usage in heart failure conditions where ET-1 is the course of pathological hypertrophic changes. However, cautioned with higher concentrations. Further investigation is required to establish the underlying mechanisms of action.
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