Synthesis and evaluation of the effects of solid lipid nanoparticles of ivermectin and ivermectin on cuprizone-induced demyelination via targeting the TRPA1/NF-kB/GFAP signaling pathway.

Objectives: Multiple sclerosis (MS) is a chronic disease of the central nervous system (CNS) and its cause is unknown. Several environmental and genetic factors may have roles in the pathogenesis of MS. The synthesis of solid lipid nanoparticles (SLNs) for ivermectin (IVM) loading was performed to increase its efficiency and bioavailability and evaluate its ability in improving the behavioral and histopathological changes induced by cuprizone (CPZ) in the male C57BL/6 mice. Materials and Methods: Four groups of 7 adult C57BL/6 mice including control (normal diet), CPZ, IVM, and nano-IVM groups were chosen. After synthesis of nano-ivermectin, demyelination was induced by adding 0.2% CPZ to animal feed for 6 weeks. IVM and nano-IVM (1 mg/kg/day, IP) were given for the final 14 days of the study. At last, behavioral tests, histochemical assays, and immunohistochemistry of TRPA1, NF-kB p65, and GFAP were done. Results: The time of immobility of mice in the IVM and nano-IVM groups was reduced compared to the CPZ group. Histopathological examination revealed demyelination in the CPZ group, which was ameliorated by IVM and nano-IVM administration. In IVM and nano-IVM groups corpus callosum levels of TRPA1, NF-kB p65, and GFAP were decreased compared to the CPZ group. In the IVM and nano-IVM groups, the levels of MBP were significantly higher than in the CPZ group. Conclusion: The results evidenced that IVM and nano-IVM administration is capable of reducing demyelination in mice.

Lithium reverses the effect of opioids on eNOS/nitric oxide pathway in human umbilical vein endothelial cells.

The main challenge of pain management with opioids is development of acute and chronic analgesic tolerance. Several studies on neuronal cells have focused on the molecular mechanisms involved in tolerance such as cyclic AMP (cAMP) activation, and nitric oxide (NO) pathway. However, the effects of opioids on non-neuronal cells and tolerance development have been poorly investigated. Lithium chloride is a glycogen synthase kinase 3ß (GSK-3ß) inhibitor and exert its effects through modulation of nitric oxide pathway. In this study we examined the effect of lithium on acute/chronic morphine and methadone administration in endothelial cells which express mu opioid receptors. Human umbilical vein endothelial cells (HUVECs) were treated with different doses of morphine, methadone, and lithium for six and 48 h. Then we evaluated cell viability, nitrite and cyclic AMP levels, as well as the expression of endothelial nitric oxide synthase (eNOS) protein using Immunocytochemistry (ICC) assay and phosphorylated GSK-3ß enzyme by western blot analysis in cells. Both chronic morphine and methadone treatment increased NO level and eNOS expression in HUVECs. Morphine induced cAMP overproduction after 48 h exposure with cells. Lithium pretreatment (10 mM) in both morphine and methadone received groups significantly reduced nitrite and cAMP levels as well as eNOS expression as compared to the control. The decreased amount of phospho GSK-3ß due to the opioid exposure was increased following lithium treatment. Tolerance like pattern may occur in non-neuronal cells with opioid receptors and this study clearly revealed the attenuation of morphine and methadone tolerance like behavior by lithium treatment in HUVECs.

Evaluation of the effects of metformin administration on morphine tolerance in mice.

Opioids are used in clinical practice to relieve moderate to severe pain. Prolonged use of opioids can lead to a situation of analgesic tolerance and dependence. Several mechanisms are involved in the tolerance to analgesic opioids, including desensitization or internalization of the opioid receptor, elevation of cAMP levels, microglial activation and neuroinflammation, elevation of spinal mTOR activity and change in the expression of some proteins involved in tolerance, such as nNOS. Activation of the AMPK pathway inhibits mTOR and p38 MAPK ameliorating neuroinflammation and tolerance induced by morphine. Metformin, a potent antidiabetic agent, can also activate AMPK. Morphine tolerance was induced in mice by intraperitoneal administration three times daily at 08:00, 11.00 and 16.00 h of 50, 50 and 75 mg/kg morphine, respectively during four days. On the fifth day mice received a single injection of morphine 50 mg/kg. To evaluate the effects of metformin in development of morphine-induced analgesic tolerance a group of mice received metformin (10 mg/kg) 45 min before each morphine administration. Tail flick and hot plate tests were performed to estimate analgesic latency on days 1, 3 and 5. At five days, the animals were sacrificed, the brain dissected and nitrite levels determined. Chronic metformin administration significantly increased the analgesic latency on days 3 and 5 compared to the morphine group in hot plate test and in tail flick test. Chronic and acute metformin administration significantly decreased nitric oxide level compare to morphine group. The present results revealed that metformin attenuated analgesic tolerance induced by repeated intraperitoneal injections of morphine in mice.

In vitro evaluation of effects of metformin on morphine and methadone tolerance through mammalian target of rapamycin signaling pathway.

The chronic use of opioids leads to tolerance, psychological, and physical dependence that limits their use as an effective long-term pain control. Several studies have shown that mammalian target of rapamycin (mTOR) plays a crucial role in the development of opioid tolerance. Metformin activates 5' adenosine monophosphate-activated protein kinase (AMPK) which directly suppresses the mTOR complex 1 signaling pathway. On the other hand, metformin can also inhibit mTOR directly and in an AMPK-independent manner. Thus, in the current study, we aimed to investigate the effects of metformin on the development of morphine and/or methadone-induced tolerance in human glioblastoma (T98G) cell line. We examined the effects of chronic treatment of morphine and/or methadone in the presence or absence of metformin with or without AMPK inhibitor (dorsomorphin hydrochloride) on levels of nitric oxide and cyclic adenosine monophosphate (cAMP), phosphorylated and dephosphorylated ribosomal protein S6 kinase ß-1 (S6K1) and 4E-binding protein 1 (4E-BP1) in T98G cells. Pretreatment of cells with metformin (40 µM) with or without AMPK inhibitor (dorsomorphin hydrochloride; 1 µM) before adding of morphine (2.5 µM) or methadone (1 µM) revealed a protective effects on the development of opioid tolerance. Prior administration of metformin reversed the elevation of nitric oxide levels induced by morphine (p < 0.001) and methadone (p < 0.001) and also prevented the raise of cAMP levels induced by morphine in T98G cells (p < 0.05). Contribution of mTOR signaling pathway in metformin-induced effect was shown by the inhibition of phosphorylation of S6K1 and 4E-BP1, the downstream targets of mTOR. mTOR activation suppresses opioid-induced antinociception, and its activity has also been increased during opioid tolerance.

Cyclosporin A attenuating morphine tolerance through inhibiting NO/ERK signaling pathway in human glioblastoma cell line: the involvement of calcineurin.

Cyclosporin A (CsA) is known to have an immunosuppressive action. However, it is also attracting attention due to its effects on the nervous system, such as inhibiting the development and expression of morphine-induced tolerance and dependence through unknown mechanisms. It has been shown that CsA modulates the nitric oxide (NO) synthesis and extracellular signal-regulated kinases (ERK) activation, which are potentially involved in signaling pathways in morphine-induced tolerance in cellular models. Therefore, the current study was designed to evaluate the modulatory role of CsA on the MOR tolerance, by targeting the downstream signaling pathway of NO and ERK using an in vitro model. For this purpose, T98G cells were pretreated with CsA, calcineurin autoinhibitory peptide (CAIP), and NG-nitro-l-arginine methyl ester (L-NAME) 30 min before 18 h exposure to MOR. Then, we analyzed the intracellular cyclic adenosine monophosphate (cAMP) levels and also the expression of phosphorylated ERK and nitric oxide synthase (nNOS) proteins. Our results showed that CsA (1 nM, 10 nM, and 100 nM) and CAIP (50 µM) have significantly reduced cAMP and nitrite levels as compared to MOR-treated (2.5 µM) T98G cells. This clearly revealed the attenuation of MOR tolerance by CsA. The expression of nNOS and p-ERK proteins were down-regulated when the T98G cells were pretreated with CsA (1 nM, 10 nM, and 100 nM), CAIP (50 µM), and L-NAME (0.1 mM) as compared to MOR. In conclusion, the CsA pretreatment had a modulatory role in MOR-induced tolerance, which was possibly mediated through NO/ERK signaling pathway.

The anticonvulsant activity and cerebral protection of chronic lithium chloride via NMDA receptor/nitric oxide and phospho-ERK.

The underlying mechanisms for the neuroprotective effects of lithium chloride in neurodegenerative diseases such as seizures remain unknown. In present study the downstream signaling pathway of phospho-ERK/NMDA receptors/nitric oxide has been studied. For this purpose, acute and chronic effect of lithium in seizure animal model and the interaction of NMDA receptor antagonist (MK-801) and neuronal nitric oxide synthase (nNOS) inhibitor (7-NI) with these neuroprotection has been studied. Acute lithium administration showed pro-convulsive properties in pentylenetetrazole (PTZ)-induced seizure model while chronic treatment increased the seizure threshold significantly. The serum level of lithium in treated mice were 0.48 mEq/L corresponding the therapeutic range. Administration of 7-NI (30mg/kg, i.p.) and MK-801 (0.001mg/kg, i.p.) had no effect on seizure threshold, while co-administration of them before the sub-effective dose of lithium (4mg/kg, i.p.) increased the anticonvulsant effect of lithium significantly. Furthermore, acute injection of MK-801 (0.05mg/kg) or 7-NI (60mg/kg) and co-administration of them significantly suppressed the anticonvulsant effect of effective dose of lithium (10mg/kg). This data demonstrated involvement of NMDA receptors/nitric oxide pathway in anticonvulsant effect of lithium. In cerebellar granule neurons (CGNs) culture studies on glutamate excitotoxicity western blot analysis, nitrite assay by Griess reaction, cell viability and microscopic morphology evaluation has been carried out to find the role of NMDA receptor/nitric oxide and phospho-ERK signaling in lithium neuroprotection. Using MTT assay and morphologic examinations, chronic lithium treatment showed protective effects against glutamate toxicity in primary cerebellar culture neurons. The level of nitric oxide was significantly reduced in co-administration of lithium and glutamate while glutamate significantly increased levels of nitric oxide. The involvement of NMDA receptors/nitric oxide and phospho-ERK pathway in the effects of lithium on cerebellar neurons has been shown. Inhibition of ERK signaling may be reconsidered as a pharmacological approach for seizure control.

Therapeutic Effect of Scrophularia striata Ethanolic Extract against Localized Cutaneous Leishmaniasis Caused by Leishmania major (MRHO/IR/75/ER).

BACKGROUND: We evaluated the effect of the ethanolic extract of Scrophularia striata on the Iranian strain of Leishmania major (MRHO/IR/75/ER) both in vitro and in vivo conditions. METHODS: The effective dose (ED) of ethanolic extract of S. striata were determined using MTT assay on the growth of promastigote forms of L. major in axenic culture media. Then, the ED50 of S. striata on mice peritoneal macrophages was determined using calculation of amastigote forms on mice peritoneal macrophages. For in vivo experiments, the therapeutic effects of various concentrations of S. striata on infected BALB/c mice was studied. A total of 75 infected mice were randomly divided into five groups: two groups (10% and 50% of S. striata) as experimental and three as control (ethanol 50%, Glucantime® and no treatment). The efficacy were determined by comparing the diameters of lesions and the microscopically examinations. RESULTS: The effect of S. striata extract (0/625%, 1/25%, 2/5%, 5%, 10%, 20% and 50%) on peritoneal macrophages of Balb/c mice infected with L.major in tissue-culture slides was assessed. S. striata extract (10%) removed the L.major amastigotes-infected macrophages significantly after 24 h (P < 0.05). The higher concentrations of S. striata ethanolic extract (20%, and 50%) had highly toxic effects on macrophages, resulted in the disintegration of the cytoplasm of macrophages after 48 and 72 h. In concentration 10% of S. striata, more than 85% of L. major amastigotes-infected macrophages were damaged without cytotoxicity effects on macrophages. The higher concentrations had toxic effects on cultured macrophages. CONCLUSION: S. striata ethanolic extract 10% had anti leishmanial effects in both in vivo and in vitro.

Lithium chloride protects PC12 pheochromocytoma cell line from morphine-induced apoptosis.

BACKGROUND: Opioid drugs are considered as important members of drugs of abuse. Opioid abusers are more likely to be infected which may be due to apoptotic effects of the drugs on immune cells. Furthermore, there are some reports on the apoptotic effect of morphine on neural cells. In the present study, the effect of morphine and lithium on apoptosis in PC12 cell line (as a model of neural cells) was examined. METHODS: We used 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, annexin V-fluorescein isothiocyanate test and quantitative real time RT-polymerase chain reaction for detection of necrosis and apoptosis (programmed cell death). RESULTS: PC12 cells were exposed to different concentrations of morphine for six, 12, 24, 48, and 96 hours. Quantitative real-time RT-polymerase chain reaction revealed that mRNA expression of BAX (proapoptotic element) increased while a decrement in the mRNA expression of BCL-2 (protective element) was observed after six hours (but not after 12 or 24 hours) exposure to morphine. Furthermore, the results of MTT assay and annexin V-fluorescein isothiocyanate test indicated that morphine exposure causes an increase in the percentage of apoptotic and necrotic cells, respectively. Interestingly, the results of MTT assay and annexin V-fluorescein isothiocyanate test were observed 12 and 24 hours after morphine exposure. Thus, it can be concluded that alteration in mRNA expression is an early event rather than as a consequence of apoptosis or necrosis. On the other hand, lower concentrations of lithium elicit protective effect against apoptosis in some of mammalian cells while the higher concentrations are toxic. Despite large body of evidences on the protective effect of lithium, elucidation of downstream events are still unknown. In the present study, 72-hour preincubation of PC12 cells with 1.2 mM lithium chloride reversed the effects of morphine on the mRNA expression of BAX and BCL-2. Furthermore, the results of real time RT-polymerase chain reaction were supported by annexin V-fluorescein isothiocyanate test and MTT assay. CONCLUSION: The protective effect of lithium on the morphine-induced cytotoxicity is mediated via down-regulation of BAX and up-regulation of BCL-2 mRNA expression.

Comparative Measurement of In Vitro Paraquat and Aflatoxin B1 Cytotoxicity Using Three Different Cytotoxicity Assays in Pheochromocytoma Cells (PC-12).

ABSTRACT Among the herbicides and mycotoxins, paraquat (PQ) and aflatoxin B1 (AFB1) are highly cytotoxic. In this study the toxicity of PQ and AFB1 in the cultured cell were determined using MTT [3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide], JG-B (Janus green B), and NR (neutral red) assay by multiwell scanning spectophotometry. JG-B was used not only for the vital staining of mitochondria but also for viability assay and was compared to MTT and NR assay. Various concentrations of paraquat (0.1 mM to 100 mM) and AFB1 (0.001 nM to 10 nM) on the PC-12 cells were investigated. The 50% lethal concentration of toxins (LC50) were determined for PQ (7.70 +/- 2.50, 3.67 +/- 1.53, 4.85 +/- 2.44) and AFB1 (0.16 +/- 0.01, 0.13 +/- 0.04, 0.14 +/- 0.02) as determined by these methods (JG-B, NR, and MTT, respectively). A significant correlation was found among the JG-B and MTT using PQ (r(2) = 0.99, p < 0.05) and significant correlation was also found among the three methods (r(2) = 0.95, 0.93, and 0.92, p < 0.05) using AFB1. No significant correlation was found between JG-B and MTT with NR (r(2) = 0.34 and 0.35, p < 0.05, respectively) using PQ. These results suggest that both methods (MTT assay and JG-B assay) are reliable and are comparable for determining the cytotoxicity. It is concluded that the JG-B assay may be preferable to MTT assay methods because of its simplicity, low cost, sensitivity, and objectivity; in addition, this method takes little time to be done.