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

This cell-based study showed that extracts of magic mushrooms (containing psilocybin) didn’t induce cell injury and possibly could even have a protective effect. This study provides some evidence for their safety in use for those with cardiovascular disease conditions.

Abstract

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.

Authors: Sanah M. Nkadimeng, Christiaan M. L. Steinmann & Jacobus N. Eloff

Summary

Heart failure is a complex clinical syndrome that may result from any structural and/or functional cardiac disorder. Depression in heart failure patients may lead to a two-fold increase in mortality.

Psilocybin-containing mushrooms have been used since ancient times for their mind healing properties. Psilocybin is also known to cause temporary increase in heart rate and blood pressure, which may present a risk to users suffering from cardiovascular illnesses such as heart failure. The study investigates the effects of Psilocybe cubensis and Panaeolus cyanescens magic mushrooms on pathological hypertrophy conditions in which endothelin-1 (ET1-1) disorder is a contributor to pathogenesis. Endothelin-1 is a potent vasoconstrictor that plays a critically important role in the induction of myocyte hypertrophy.

This study evaluates the effects of Psilocybe cubensis and Panaeolus cyanescens magic mushrooms water extracts on ET-1-induced hypertrophy and tumor necrosis factor-induced cell injury and death on rat embryonic ventricular H9C2 cardiomyocytes.

Endothelin-1 stimulation increased cell sizes and the positive control ambrisentan reversed the cell sizes. The hot-water and cold-water extracts of P. cubensis and Pan cyanescens mushrooms significantly reduced the sizes of the treated cells when compared with ET-1 control cells.

The control ET-1 induced cells significantly increased the levels of BNP compared to the NO-ET1 non-induced cells, Fig. 2. The positive control ambrisentan reduced the ET-1 effect significantly compared to the ET-1 control.

Treatment with ET-1 reduced mitochondrial activity of the cells significantly, and the water extracts of P. cubensis and Pan cyanescens increased cell viability.

The ET-1 stimulation increased the TNF- level of the cells compared to NO-ET1 cells. The hot-water (GH) and cold-water (GC) extracts of P. cubensis and hot-water (PH) and cold-water (PC) extracts of Pan cyanescens reduced the TNF- levels significantly.

Figure 3 shows the effects of hot-water and cold-water extracts of P. cubensis, Pan cyanescens and ambrisentan on cell viability.

ET-1 increased the intracellular ROS productions, which were reduced by the positive control ambrisentan. The water extracts of Pan cyanescens and P. cubensis significantly decreased the ROS productions, while the water extract of P. cubensis increased the ROS production very close to the positive control.

ET-1 stimulation and extract treatment increased cell growth, whereas ET-1 stimulated cells lowered cell growth when compared to NO-ET1 non-stimulated cells. The two water extracts of Pan cyanescens induced lower rate of cell growth when compared to NO-ET1 cells.

The growth rate of the cells increased after 12 hours with the highest growth rate achieved with cold water extracts of Pan cyanescens mushrooms.

Stimulation with TNF-induced cell injury and death. P. cubensis and Pan cyanescens mushrooms increased cell viability above 100%.

Discussion

Heart failure is a public health problem that significantly impacts daily management and the quality of life of many affected persons. Our study evaluated the effects of 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.

The hot-water and cold-water extracts of P. cubensis and Pan cyanescens mushrooms reduced significantly the ET-1 induced cell size measurements of treated cells and also reduced the ET-1 induced concentrations of BNP. The four extracts also improved mitochondrial activity of the cells in a dose-dependent manner.

Figure 6 shows the effects of hot-water, cold-water, and ambrisentan extracts on intracellular ROS production and cell viability.

The hot-water and cold-water extracts of P. cubensis and hot-water and cold-water extracts of Pan cyanescens mushrooms reduced the TNF- concentration in the treated cells compared to ET-1-induced control cells and demonstrated potential safety in heart failure conditions.

ROS, especially superoxide and hydroxyl radicals, play an important role in the progression of cardiovascular diseases such as heart failure by inducing oxidative stress. The four water mushroom extracts reversed the ET-1-induced ROS levels significantly.

The decrease in ROS levels observed with the extracts was not due to toxicity, as the cell growth rate continued to increase after 1 h treatment. However, the water extract of Pan cyanescens contained other compounds that lowered cell growth rate. Pan cyanescens mushrooms have high levels of urea, which is known to induce cell cycle delay and promote a slow rate of increase of cells in log phase of growth. However, the growth rate of cells improved after 12 hours.

The results showed that the four extracts of the two magic mushrooms reversed the TNF-induced cell injury and death in a dose dependent manner, which is beneficial in a pathological hypertrophy condition.

The cold-water extract of Pan cyanescens mushroom protected against ET-1 induced cell death by increasing cell viability even higher than the positive control and non-induced cells at the concentrations used in the study. This effect suggests that the extract may have compounds that blocked the induced cell death cascades.

Sphingosine, a well-known effective inducer of apoptosis on cardiomyocytes, may also inhibit protein kinase C (PKC), a protein that protects cells from apoptotic cell death. The cold-water extraction of Pan cyanescens mushroom may contain compounds that may promote or activate overexpression and/or phosphorylation of Blc-2 proteins pathways.

The two-water extract of P. cubensis and the hot-water extract of Pan cyanescens suppressed ET-induced TNF- levels and the expression of several pro-inflammatory cytokines and the genes associated with apoptosis in vitro.

Pan cyanescens and P. cubensis mushrooms contain mycochemical compounds with known biological activities, including alkaloids, saponins, flavonoids and tannins, which may have contributed to the cardioprotective effects exhibited by the water extracts of the two mushrooms in the study.

The study demonstrated that Panaeolus cyanescens and Psilocybe cubensis mushrooms significantly reduced ET-1-induced hypertrophy, BNP, TNF- and ROS levels and decreased mitochondrial activity in stimulated cardiomyocyte cells. The two mushrooms also had cardioprotective potential properties and alleviated against TNF-induced cell injury and death.

This study was approved by the University of Pretoria research committee and the Medical Control Council committee of the South African Health Department.

Growing mushrooms and making extracts. Psilocybe cubensis and Panaeolus cyanescens mushrooms were grown in a sterilised monotub with monitored temperature and humidity under sterile conditions and extracts were made with hot boiling and cold water solvent according to Nkadimeng et al.31 method.

Materials and methods

Rat H9C2 cardiomyoblast cells were cultured in DMEM supplemented with 10% FBS and 1% penicillin/streptomycin in 75 cm2 tissue culture treated flasks at 37 °C in 5% CO2 balanced air.

H9C2 cardiomyoblast cells were cultured according to the method of18,32 with modification, and were treated with endothelin-1 (ET-1) 1160/100U (R&D, Whitehead scientific) for 45 min before being treated with the four water extracts and positive control ambrisentan (SML2104, Sigma-aldrich) over 48 h.

To test for mitochondrial activity, cells were deprived of serum, treated with four extracts and positive controls, and then cultured for 48 h. Viability was calculated using the Resazurin assay kit AR002 (R & D, Whitehead scientific).

After 48 h treatment, cell surface area measurements were taken with a light microscopy Olympus BX63 using 20 m lenses. The results showed represented analysis from three independent experiments.

Brain natriuretic peptide (BNP) concentrations were measured using an enzyme immunoassay after 48 h treatment with the extracts.

After 48 h treatment, TNF levels were measured using an ELISA kit. The concentrations were calculated from the standard curve.

Intracellular ROS measurements were performed on ET-1-induced cardiomyocytes and treated with four water mushroom extracts and ambrisentan for 1 h. A fluorometric intracellular ROS assay kit was used to detect intracellular ROS in live cells.

The rate of cell growth was determined by measuring the absorbance in each well before the cells were stimulated ET-1, after 2 h ET-1 stimulation and 1 h treatment with the extracts and positive control ambrisentan. The percentage of cell survival and growth was calculated using the formula.

Four water extracts were tested for their effect on TNF-induced cardiomyocyte injury and cell death. The effect of the extracts was determined by using method of33 with modifications.

Statistical analysis was performed using one way ANOVA analysis of variance, normality test, Shapiro-Wilk and equal variance test of Brown-Forsythe. A p value of 0.050 was considered statistically significant.

Additional information

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