2017 Volume 40 Issue 10 Pages 1779-1783
Persistent inhalation of mitragynine (MG), a major alkaloid in the leaves of Mitragyna speciosa, causes various systemic adverse effects such as seizure, diarrhea and arthralgias, but its toxicity to endothelial cells and effects on barrier function of the cells are poorly understood. In this study, we compared toxicities of MG and mitraphylline, another constituent of the leaves, against human aortic endothelial (HAE), bronchial BEAS-2B, neuronal SK-N-SH, hepatic HepG2, kidney HEK293, gastric MKN45, colon DLD1, lung A549, breast MCF7 and prostate LNCaP cells, and found that MG, but not mitraphylline, shows higher toxicity to HAE cells compared to the other cells. Forty-eight-hours incubation of HAE cells with a high concentration of MG (60 µM) provoked apoptotic cell death, which was probably due to signaling through enhanced reactive oxygen species (ROS) generation and resultant caspase activation. Treatment of the cells with MG at sublethal concentrations less than 20 µM significantly lowered transendothelial electrical resistance and elevated paracellular permeability, without affecting the cell viability. In addition, the MG-elicited lowering of the resistance was abolished by a ROS inhibitor N-acetyl-L-cysteine and augmented by H2O2 and 9,10-phenanthrenequinone, which generates ROS through its redox cycle. These results suggest the contribution of ROS generation to the increase in endothelial barrier permeability.
A Southeast Asian tree Mitragyna speciosa KORTH, also designated as Kratom, is traditionally used for minimizing physical and mental fatigues.1–4) Kratom possesses diverse pharmacological activities, showing a stimulant-like activity at low doses and opioid-like activity at higher doses. It also has various beneficial activities such as anti-inflammatory, anti-diarrheal and hypophagic activities, as well as narcotic and analgesic action derived from activation of opioid receptors-dependent signaling cascade.1,2) The narcotic effects are considered to be mainly attributable to mitragynine (MG), the most abundant alkaloid included in the Kratom leaves (Fig. 1). MG is also known to exhibit almost all of the above mentioned activities of Kratom.1–3) The leaf preparations contain lots of other indole-based alkaloids such as mitraphylline, speciogynine, speciociliatine and a MG metabolite 7-hydroxy-MG, whose structures closely resemble that of MG. Based on the facts that the phamacological activities of MG and its 7-hydroxy metabolite are much more potent than that of morphine5,6) and that oral administration of MG results in a long elimination half time (3.9–6.6 h),3,7,8) MG is thought to play a crucial role in clinical symptoms in patients who received an overdose of Kratom. Previous animal experiments concluded that a fatal risk of MG is relatively low.9) However, a recent acute toxicity test of Kratom extract has reported occurrence of hypertension, hepatotoxicity and nephrotoxicity in rodents.10) In humans, a variety of adverse effects of MG such as seizures, coma, hallucinations, diarrhea, arthralgias myalgias and respiratory depression are reported.11) Additionally, recent cell-based experiments revealed that a low concentration (10 µM) of MG provokes toxicity against human cardiomyocytes.4) Thus, there has been increased documentation reporting that overuse of MG would cause systemic health hazards through exerting the drug toxicity. However, little is known about effect of MG on cardiovascular system and endothelial barrier. In this study, we examined toxicities against human aortic endothelial (HAE), bronchial epithelial, neuronal, hepatic, kidney, gastric, colon, lung, breast and prostate cells of MG, compared with those of mitraphylline, that was reported to exert cancer cell toxicities.12) Because MG was shown to exert toxicity most potently against HAE cells, we explored its toxic mechanism by monitoring production of reactive oxygen species (ROS), caspase-3 activation and DNA fragmentation in the cells. In addition, it was investigated whether MG alters permeability of the endothelial cell barrier by treatment at its sublethal concentrations or not.
MG and mitraphylline were purchased from Cayman Chemicals (Ann Arbor, MI, U.S.A.), and N-acetyl-L-cysteine (NAC), N-acetyl aspartic acid (Asp)-glutamic acid (Glu)-valine (Val)-Asp-7-amido-4-methylcoumarin and fluorescein isothiocyanate (FITC)-dextran were from Sigma-Aldrich (St. Louis, MO, U.S.A.). 6-Carboxy-2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA) and bicinchoninic acid protein assay reagent were obtained from Molecular Probes (Rockland, ME, U.S.A.) and Pierce (Rockford, IL, U.S.A.), respectively. Polyethylene glycol-conjugated catalase (PEG-cat) was generously gifted from Dr. Yoshito Kumagai (University of Tsukuba, Japan). All other chemicals were of the highest grade that could be obtained commercially.
Cell Culture and Cytotoxicity AssayHAE cells were obtained from Clonetics (Walkersville, MD, U.S.A.), and cultured in EBM2 medium in type-I collagen-coated dishes at 37°C in a humidified incubator containing 5% CO2. In the majority of experiments, the cells were used at passage 4–8, and the endothelial cobblestone morphology was confirmed microscopically before use. For cytotoxicity assay, the cells were seeded at densities of 2×104 cells/well into a type-I collagen-coated 96-well microplate and, after reaching a 90% confluence of the cells, the medium was replaced with EBM medium supplemented with 0.5% fetal bovine serum and antibiotics alone 2 h before treatment of agent. The culture and treatment of bronchial BEAS-2B (American Type Culture Collection, Manassas, VA, U.S.A.), neuronal SK-N-SH cells (RIKEN Cell Bank, Tsukuba, Japan), hepatic HepG2 (American Type Culture Collection) and kidney HEK293 cells (American Type Culture Collection), gastric MKN45 (Health Science Research Resources Bank, Osaka, Japan), colon DLD1 (American Type Culture Collection), lung A549 (American Type Culture Collection), breast MCF7 (American Type Culture Collection) and prostate LNCaP cells (American Type Culture Collection) were similarly performed, except that Dulbecco’s modified Eagle’s medium (DMEM)/F-12, Minimum Essential Medium alpha media and DMEM supplemented with the serum (10% for culture and 2% for assay) and antibiotics were used as culture media of BEAS-2B, SK-N-SH and other seven cells, respectively. Viable cell number was evaluated by a tetrazolium dye-based cytotoxicity assay using 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium monosodium salt (Wako Pure Chemical Industries, Ltd., Osaka, Japan).
Measurement of ROS, Caspase-3 Activity and DNA FragmentationLevel of intracellular H2O2 was monitored using the fluorogenic probe, DCFH-DA, as previously reported.13) After washing with Dulbecco’s phosphate-buffered saline (DPBS), the cells were incubated for 20 min in fresh serum-free medium containing 20 µM the fluorogenic probe, and then sufficiently washed with DPBS. DCF fluorescence-positive cells were counted using a Becton Dickinson FACSVerse flow cytometer equipped with BD FACSuite software. The activity of caspase-3 in cell extracts was measured using acetyl Asp-Glu-Val-Asp-7-amido-4-methylcoumarin as the fluorogenic substrates.14) DNA fragmentation analysis was performed according to the method reported previously.13)
Measurement of trans-Endothelial Electrical Resistance (TEER) and Paracellular PermeabilityCells were seeded at a density of 5×104 cells/well into a type-I collagen-coated Millicell Hanging Cell Culture Insert (8-µm pore) and then cultured for 5 d before treatment of the cells with MG and/or agents. TEER between apical and basal components of the insert was measured using a Millipore volt ohmmeter. To estimate the paracellular permeability, FITC-dextran (average molecular weight: 150000) was added into the insert and the fluorescence intensity in the basal side was measured 4 h after the addition.
Statistical AnalysisData are expressed as the means±standard deviation (S.D.) of at least three independent experiments. Statistical evaluation of the data was performed by using Student’s t-test for comparison between two groups and one-way ANOVA followed by Dunnett’s test for multiple comparisons.
When viability of HAE cells after 48-h treatment of MG was measured, it was remarkably lowered at MG concentrations higher than 40 µM (Fig. 2). As the treatment induced death of HAE, BEAS-2B and SK-N-SH cells with the 50% lethal concentration values of 43.1±6.7, 67.2±7.1 and 50.9±4.6 µM, respectively, the sensitivity to the drug toxicity of HAE cells is presumed to be higher than those of the bronchial and neuronal cells. It should be noted that no considerable alteration in viability by treatment of 60 µM MG was observed for other seven cells (92.3% in HepG2, 86.7% in HEK293, 90.7% in MKN45, 85.9% in DLD1, 94.7% in A549, 84.4% in MCF7 and 87.1% in LNCaP). In contrast to MG, mitraphylline, an oxindole alkaloid of Kratom, at concentrations up to 60 µM did not affect viability of all the cells. The results suggest that exposure to MG exerts toxicity more potently against endothelial cells than against other cells.
HAE, BEAS-2B and SK-N-SH cells were treated for 48 h with the indicated concentrations of mitraphylline (○) or MG (●). The viability values were estimated by the formazan dye-based cytotoxicity assay and are expressed as percentages of that in the control cells treated with the vehicle dimethyl sulfoxide (DMSO) at the final concentration of 0.5%. Significant difference from the group treated with DMSO alone (shown as 0 µM), * p<0.05 and ** p<0.01.
To investigate whether production of ROS participates in the apoptotic mechanism triggered by MG, we measured effects of antioxidants, NAC and PEG-cat, on HAE cell toxicity elicited by a high concentration (60 µM) of the drug. Pretreatment with the antioxidants resulted in significant protection from the cell damage provoked by MG treatment (Fig. 3A). Additionally, flow cytometric analysis using the fluorogenic probe DCFH-DA revealed the elevation of intracellular concentration of H2O2 by MG treatment (Fig. 3B). Furthermore, the treatment showed typical apoptotic features, caspase-3 activation (Fig. 3C) and DNA fragmentation (Fig. 3D), which were almost completely abrogated by preincubation with NAC. These results clearly demonstrate that the treatment with MG elicits apoptosis of HAE cells through ROS production and resultant caspase-3 activation. We provide the first evidence that ROS production mediates the endothelial cell apoptosis elicited by MG. Because the MG-triggered cellular processes involved in ROS production are still unknown, biochemical and cell-based experiments to identify them are now ongoing in our laboratory.
(A) Effects of antioxidants on HAE cell damage by MG. The cells were pretreated for 2 h with the indicated concentrations of NAC or PEG-cat, and then treated for 48 h with 60 µM MG. (B) ROS generation. After 24-h treatment with 0 (1) or 60 µM MG (2), the intracellular ROS levels were estimated by the oxidation of DCFH-DA, and are expressed as percentages of the DCF fluorescence-positive cells to total cells. (C) Caspase-3 activation. The cells were treated for 48 h with 0, 10, 20, 40 or 60 µM MG. In the NAC group, the cells were pretreated for 2 h with 2 mM NAC prior to the MG treatment. The caspase-3 activity in the cell extract was measured and is expressed as the percentage of that of the control cells treated with DMSO alone (shown as 0 µM). (D) DNA fragmentation analysis. HAE cells were treated for 48 h with 60 µM MG in the absence or presence of NAC pretreatment. DNA was isolated from the cells and then followed by agarose gel electrophoresis and ethidium bromide staining. Significant difference from the group treated with DMSO alone, * p<0.05 and ** p<0.01. Significant difference from the group treated with 60 µM MG alone, # p<0.05 and ## p<0.01.
Postmortem screening of MG-related cases has shown that the drug concentrations in peripheral bloods were 0.23–1.06 mg/L (corresponding to 0.6–2.7 µM),15,16) which are much less than the 50% lethal concentration values mentioned above (43.1 µM for HAE cells). Considering that diverse effects such as seizure were observed even at the lower MG concentrations,17) it is assumed that at sublethal concentrations MG is capable of transmigrating itself across endothelial cell monolayer to extravascular tissues such as brain and muscle. To test this assumption, we monitored value of TEER between apical and basal compartments in the insert, where HAE cells grown to confluence were treated with MG. Intriguingly, at the low concentrations (less than 20 µM) that hardly affected the HAE cell viability as indicated in Fig. 4A, the TEER value was decreased in a manner dependent on concentration of the drug (Fig. 4B). By synchronizing decrease in TEER, it was evident that the endothelial monolayer permeability was increased, as the results of the paracellular permeability assay using the FITC-dextran are shown in Fig. 4C. Furthermore, the MG-decreased TEER was recovered and deteriorated by pretreating with NAC and ROS (H2O2 and 9,10-phenanethrenequinone that generates ROS via its redox cycling), respectively (Fig. 4B). These results strongly suggest the participation of ROS formation in the mechanism controlling endothelial barrier permeability. To our knowledge, this is the first report that MG elevates permeability of endothelial barrier via ROS production. The involvement of ROS in elevation of endothelial barrier permeability is consistent with previous reports.18–20) As evident from the TEER value in Fig. 4A, the endothelial permeability was significantly promoted by long-term (5 d) incubation with MG at its sublethal concentration (5 µM), which is relatively close to those in the postmortem screening, inferring the critical contribution of endothelial barrior dysfunction to the MG-elicited adverse effects. The permeability between endothelial cells is regulated by tight junction proteins, claudins and occludins, of which claudin-5 plays a key role in endothelial barrier permeability. Therefore, it would be of interest to elucidate alteration in expression, localization and function of claudin-5 as a mechanism underlying the endothelial barrier disruption by ROS.
After 5-d culture after seeding into the inserts, HAE cells were pretreated for 2 h with 50 µM H2O2, 0.5 µM 9,10-phenanthrenequinone (PQ) or 2 mM NAC, and then treated for 48 h with 0, 5, 10 or 20 µM MG. In group of 5 d treatment (5 d), the cells were treated for the period by exchanging the assay medium supplemented with 5 µM MG every other day. (A) Cell viability. (B) TEER. (C) Paracellular permeability. The values are expressed as the percentage of those in the control cells treated with DMSO alone. * Significant difference from the group treated with DMSO alone, p<0.05. # Significant difference from the group treated with MG alone, p<0.05.
We thank Dr. Akira Hara for insightful discussion and critical reading of the manuscript.
The authors declare no conflict of interest.
