Protective effect of betulinic acid on Freund's complete adjuvant‐induced arthritis in rats

The purpose of the experiment was to study the effects of betulinic acid (BA) on adjuvant‐induced arthritis in rats. The rat model of rheumatoid arthritis (AA) was established by Freund's complete adjuvant. Arthritis index, joint pathology, toe swelling, hemorheology, related cytokines and ROCK/NF‐κB signaling pathway were measured in rats. BA can significantly inhibit the arthritis index, improve joint pathology, reduce toe swelling, improve blood rheology, improve synovial cell apoptosis, and restore related cytokine negative regulation of ROCK/NF‐κB signaling pathways. BA has an obvious therapeutic effect on joint inflammation of toes in AA model rats, which may be due to the regulation of ROCK/NF‐κB signaling pathway.     

Activation and inhibition of the proteasome by betulinic acid and its derivatives

This study discovered that betulinic acid (BA) is a potent proteasome activator that preferentially activates the chymotrypsin-like activity of the proteasome. Chemical modifications can transform BA into proteasome inhibitors. Chemical modifications at the C-3 position of BA resulted in compounds, such as dimethylsuccinyl BA (DSB), with various inhibitory activities against the human 20S proteasome. Interestingly, the proteasomal activation by BA and the inhibitory activity of DSB could be abrogated by introducing a side chain at the C-28 position. In summary, this study discovered a class of small molecules that can either activate or inhibit human proteasome activity depending on side chain modifications.     

Mephedrone does not damage dopamine nerve endings of the striatum,but enhances the neurotoxicity of methamphetamine,amphetamine, and MDMA

Mephedrone (4‐methylmethcathinone) is a β‐ketoamphetamine stimulant drug of abuse with close structural and mechanistic similarities to methamphetamine. One of the most powerful actions associated with mephedrone is the ability to stimulate dopamine (DA) release and block its re‐uptake through its interaction with the dopamine transporter (DAT). Although mephedrone does not cause toxicity to DA nerve endings, its ability to serve as a DAT blocker could provide protection against methamphetamine‐induced neurotoxicity like other DAT inhibitors. To test this possibility, mice were treated with mephedrone (10, 20, or 40 mg/kg) prior to each injection of a neurotoxic regimen of methamphetamine (four injections of 2.5 or 5.0 mg/kg at 2 h intervals). The integrity of DA nerve endings of the striatum was assessed through measures of DA, DAT, and tyrosine hydroxylase levels. The moderate to severe DA toxicity associated with the different doses of methamphetamine was not prevented by any dose of mephedrone but was, in fact, significantly enhanced. The hyperthermia caused by combined treatment with mephedrone and methamphetamine was the same as seen after either drug alone. Mephedrone also enhanced the neurotoxic effects of amphetamine and 3,4‐methylenedioxymethamphetamine on DA nerve endings. In contrast, nomifensine protected against methamphetamine‐induced neurotoxicity. As mephedrone increases methamphetamine neurotoxicity, the present results suggest that it interacts with the DAT in a manner unlike that of other typical DAT inhibitors. The relatively innocuous effects of mephedrone alone on DA nerve endings mask a potentially dangerous interaction with drugs that are often co‐abused with it, leading to heightened neurotoxicity.     

Mephedrone, an abused psychoactive component of 'bath salts' and methamphetamine congener, does not cause neurotoxicity to dopamine nerve endings of the striatum

Mephedrone (4-methylmethcathinone) is a β-ketoamphetamine with close structural analogy to substituted amphetamines and cathinone derivatives. Abuse of mephedrone has increased dramatically in recent years and has become a significant public health problem in the United States and Europe. Unfortunately, very little information is available on the pharmacological and neurochemical actions of mephedrone. In light of the proven abuse potential of mephedrone and considering its similarity to methamphetamine and methcathinone, it is particularly important to know if mephedrone shares with these agents an ability to cause damage to dopamine nerve endings of the striatum. Accordingly, we treated mice with a binge-like regimen of mephedrone (4 × 20 or 40 mg/kg) and examined the striatum for evidence of neurotoxicity 2 or 7 days after treatment. While mephedrone caused hyperthermia and locomotor stimulation, it did not lower striatal levels of dopamine, tyrosine hydroxylase or the dopamine transporter under any of the treatment conditions used presently. Furthermore, mephedrone did not cause microglial activation in striatum nor did it increase glial fibrillary acidic protein levels. Taken together, these surprising results suggest that mephedrone, despite its numerous mechanistic overlaps with methamphetamine and the cathinone derivatives, does not cause neurotoxicity to dopamine nerve endings of the striatum.     

Mechanisms involved in the neurotoxic and cognitive effects of developmental methamphetamine exposure

Methamphetamine exposure in utero leads to a variety of higher‐order cognitive deficits, such as decreased attention and working, and spatial memory impairments in exposed children (Piper et al., 2011; Roussotte et al., 2011; Kiblawi et al., 2011). As with other teratogens, the timing of methamphetamine exposure greatly determines its effects on both neuroanatomical and behavioral outcomes. Methamphetamine exposure in rodents during the third trimester human equivalent period of brain development results in distinct and long‐lasting route‐based and spatial navigation deficits (Williams et al., 2003; Vorhees et al., 2005, 2008, 2009;). Here, we examine the impact of neonatal methamphetamine‐induced neurotoxicity on behavioral outcomes, neurotransmission, receptor changes, plasticity proteins, and DNA damage. Birth Defects Research (Part C) 108:131–141, 2016. © 2016 Wiley Periodicals, Inc.     

The nNOS inhibitor,AR-R17477AR,prevents the loss of NF68 immunoreactivity induced by methamphetamine in the mouse striatum

The present study examined the time-course and regionally-selective changes in the levels of the neurofilament protein NF68 in the mouse brain induced by methamphetamine (METH). The ability of low ambient temperature, or of the specific neuronal nitric oxide synthase (nNOS) inhibitor AR-R17477AR, to protect against both long-term striatal NF68 and dopamine loss induced by METH (3 mg/kg, i.p.) was also studied. Seven days after METH administration (3, 6 and 9 mg/kg, i.p., three times at 3 h intervals), mice showed a reduction of about 40% in immunoreactivity for NF68 in the striatum. This effect was not produced in cortex after METH administration at the dose of 3 mg/kg. No difference from controls was observed when measurements were carried out 1 h and 24 h after the last METH injection at the dose of 3 mg/kg. The loss of NF68 immunoreactivity seems to be associated with the long-term dopamine depletion induced by METH, since no change in serotonin concentration is observed in either the striatum or cortex 7 days after dosing. Animals kept at a room temperature of 4 degrees C showed a loss of NF68 similar to those treated at 22 degrees C but an attenuation of dopamine depletion in the striatum. Pre-treatment with AR-R17477AR (5 mg/kg, s.c.) 30 min before each of the three METH (3 mg/kg, i.p.) injections provided complete protection against METH-induced loss of NF68 immunoreactivity and attenuated the decrease in striatal dopamine and HVA concentrations by about 50%. These data indicate that both the reduction of NF68 immunoreactivity and the loss of dopamine concentration are due to an oxidative stress process mediated by reactive nitrogen species, and are not due to changes in body temperature.     

Synthesis of a molecularly imprinted polymer for the solid‐phase extraction of betulin and betulinic acid from plane bark

Introduction – Plant extracts are usually complex mixtures of various polarity compounds and their study often includes a purification step, such as solid‐phase extraction (SPE), to isolate interest compounds prior analytical investigations. Molecularly imprinted polymers (MIPs) are a new promising type of SPE material which offer tailor‐made selectivity for the extraction of trace active components in complex matrices. Numerous specific cavities that are sterically and chemically complementary of the target molecules, are formed in imprinted polymers. A molecularly imprinted polymer (MIP) was synthesised in order to trap a specific class of triterpene, including betulin and betulinic acid from a methanolic extract of plane bark. Methodology – Imprinted polymers were synthesised by thermal polymerisation of betulin as template, methacrylic acid (MAA) or acrylamide (AA) as functional monomer, ethylene glycol dimethacrylate as crosslinking agent and chloroform as porogen. Afterwards, MAA‐ and AA‐MIPs were compared with their non‐imprinted polymers (NIPs) in order to assess the selectivity vs betulin and its derivatives. Recovered triterpenes were analysed by HPLC during MIP‐SPE protocol. Results – After SPE optimisation, the MAA‐imprinted polymer exhibited highest selectivity and recovery (better than 70%) for betulin and best affinity for its structural analogues. Thus, a selective washing step (chloroform, acetonitrile) removed unwanted matrix compounds (fatty acids) from the SPE cartridge. The elution solvent was methanol. Finally, the MAA‐MIP was applied to fractionate a plane bark methanolic extract containing betulin and betulinic acid. Conclusion – This study demonstrated the possibility of direct extraction of betulin and its structural analogues from plant extracts by MIP technology. Copyright © 2009 John Wiley & Sons, Ltd.     

Biotransformation optimization of betulin into betulinic acid production catalysed by cultured Armillaria luteo‐virens Sacc ZJUQH100‐6 cells

Aims: Betulinic acid has attracted attention in terms of its important biological and pharmacological characteristics. The main objective of this work was to optimize the variables of biotransformation process in order to enhance betulinic acid production from betulin catalysed by fungus Armillaria luteo‐virens Sacc ZJUQH100‐6. Methods and Results: Fractional factorial design and response surface methodology were applied to optimize the main parameters that affect betulinic acid production in the growing‐cells system. Results indicated that the addition of Tween 80 and substrate concentration were identified as the significant factors on betulinic acid formation, and the central composite experimental design was then adopted to derive a statistical model for optimizing biotransformation conditions. The optimum conditions were observed at pH 6·0, 0·57% Tween 80, 15 mg l^?1 betulin and at 3 days of stage of inoculation. Conclusions: Under the optimized conditions, the highest productivity of betulinic acid predicted was 9·32%, which increased by 74·53% in comparison with that of the nonoptimized. The verified experiment revealed that the model can well simulate betulin biotransformation. Moreover, the bioconversion of betulin and betulin‐28‐monooxygenase activities was compared between the optimized and the nonoptimized conditions. Significance and Impact of the Study: Current data imply that betulinic acid production from betulin can be effectively enhanced through biotransformation optimization strategy.     

Effect of ibogaine on serotonergic and dopaminergic interactions in striatum from mice and rats

The effect of ibogaine (Endabuse, NIH 10567) on serotonin uptake and release, and on serotonergic modulation of dopamine release, was measured in striatal tissue from rats and mice. Two hours after treatment in vivo with ibogaine (40 mg/kg i.p.), the uptake of labeled [³H]serotonin and [³H]dopamine uptake in striatal tissue was similar in the ibogaine-treated animal to that in the control. The 5HT_1B agonist CGS-12066A (10^–5 M) had no effect on stimulation-evoked tritium release from mouse or rat striatal tissue preloaded with [³H]serotonin; however, it elevated tritium efflux from striatal tissue preloaded with [³H]dopamine. This increase was not seen in mice treated with ibogaine 2 or 18 hours previously, or in rats treated 2 hours before. Dopamine autoreceptor responses were not affected by ibogaine pretreatment in either mouse or rat striatal tissue; sulpiride increased stimulation-evoked release of tritium from tissue preloaded with [³H]dopamine. The long-lasting effect of ibogaine on serotonergic functioning, in particular, its blocking of the 5HT_1B agonist-mediated increase in dopamine efflux, may have significance in the mediation of its anti-addictive properties.Special issue dedicated to Dr. Sidney Ochs.     

Increases in cytoplasmic dopamine compromise the normal resistance of the nucleus accumbens to methamphetamine neurotoxicity

Methamphetamine (METH) is a neurotoxic drug of abuse that damages the dopamine (DA) neuronal system in a highly delimited manner. The brain structure most affected by METH is the caudate–putamen (CPu) where long-term DA depletion and microglial activation are most evident. Even damage within the CPu is remarkably heterogenous with lateral and ventral aspects showing the greatest deficits. The nucleus accumbens (NAc) is largely spared of the damage that accompanies binge METH intoxication. Increases in cytoplasmic DA produced by reserpine, l -DOPA or clorgyline prior to METH uncover damage in the NAc as evidenced by microglial activation and depletion of DA, tyrosine hydroxylase (TH), and the DA transporter. These effects do not occur in the NAc after treatment with METH alone. In contrast to the CPu where DA, TH, and DA transporter levels remain depleted chronically, DA nerve ending alterations in the NAc show a partial recovery over time. None of the treatments that enhance METH toxicity in the NAc and CPu lead to losses of TH protein or DA cell bodies in the substantia nigra or the ventral tegmentum. These data show that increases in cytoplasmic DA dramatically broaden the neurotoxic profile of METH to include brain structures not normally targeted for damage by METH alone. The resistance of the NAc to METH-induced neurotoxicity and its ability to recover reveal a fundamentally different neuroplasticity by comparison to the CPu. Recruitment of the NAc as a target of METH neurotoxicity by alterations in DA homeostasis is significant in light of the important roles played by this brain structure.     

Treatment with 7-nitroindazole enhances kainic acid induced cholinergic neurotoxicity in the rat striatum: a neuroprotective role for neuronal nitric oxide

1. In this study we investigated the effect of 7-nitroindazole (7-NI), a preferential inhibitor of neuronal nitric oxide synthase (nNOS), on kainic acid (KA) induced neurotoxicity in rats. Choline acetyltransferase activity (CAT), a cholinergic marker, and histological changes were employed to assess neurotoxicity.2. In control rats, the local intrastriatal injection of 0.5 g of KA reduced CAT from 22.9 ± 2.2 to 14.7 ± 2.0 nmol/h/mg tissue ((38 ± 6)% reduction) (P < 0.001). Greater reductions in CAT were observed with 1 and 2 g of KA ((70 ± 6)% and (80 ± 3)%, respectively). 7-NI aggravated KA-induced cholinergic and histological damage. KA reduced CAT by (68.2 ± 4)% in 7-NI-treated rats, by (38 ± 6)% in saline-treated controls, and by (41 ± 4)% in peanut-oil- (7-NI-vehicle-) treated rats (P = 0.0047).3. After KA, CAT activity averaged 14.3 ± 2.0 in peanut-oil-treated rats and 7.9 ± 1.0 nmol/h/mg tissue in 7-NI- (peanut-oil-) treated rats (P = 0.015). Similarly to changes in CAT, 7-NI treatment aggravated KA-induced histological changes indicative of neuronal damage (acute ischemic neuronal changes, disorganization of myelinated fibers bundle, and vacuolation changes of the neuropil). Treatment with 7-NI was not associated with increased mortality.4. Our findings suggest that neuronal NO plays a neuroprotective action on excitotoxicity.     

Impairment of lysosomal integrity by B10, a glycosylated derivative of betulinic acid, leads to lysosomal cell death and converts autophagy into a detrimental process

In this study, we report a novel mechanism of action for a cytotoxic derivative of betulinic acid (BA). B10 is a semi-synthetic glycosylated derivative of BA selected for its enhanced cytotoxic activity. Interestingly, although B10 induces apoptosis, caspase-3 downregulation incompletely prevents B10-induced cell death, Bcl-2 overexpression fails to protect cells and DNA fragmentation rates do not reflect cell death rates in contrast to cytoplasmic membrane permeabilization. These results implicate that apoptotic and non-apoptotic cell death coexist upon B10 treatment. Unexpectedly, we found that B10 induces autophagy and also abrogates the autophagic flux. B10 destabilizes lysosomes as shown by Lysotracker Red staining and by cathepsin Z and B release from lysosomes into the cytoplasm. Consistently, the cathepsin inhibitor Ca074Me significantly decreases B10-induced cell death, further supporting the fact that the release of lysosomal enzymes contributes to B10-triggered cell death. Downregulation of ATG7, ATG5 or BECN1 by RNAi significantly decreases caspase-3 activation, lysosomal permeabilization and cell death. Thus, by concomitant induction of autophagy and inhibition of the autophagic flux, B10 turns autophagy into a cell death mechanism. These findings have important implications for the therapeutic exploitation of BA derivatives, particularly in apoptosis-resistant cancers.     

Modifications in muscarinic, dopaminergic and serotonergic receptors concentrations in the hippocampus and striatum of epileptic rats

The present study was undertaken in order to investigate the muscarinic (M(1)), dopaminergic (D(1) and D(2)) and serotonergic (5-HT(2)) receptors densities in hippocampus and striatum of Wistar rats after status epilepticus (SE) induced by pilocarpine. The control group was treated with 0.9% saline. An other group of rats received pilocarpine (400 mg/kg, s.c.) and both groups were sacrificed 1 h after treatment. The results have shown that pilocarpine administration and resulting SE produced a downregulation of M(1) receptor in hippocampus (41%) and striatum (51%) and an increase in the dissociation constant (K(d)) values in striatum (42%) alone. In both areas the 5-HT(2) receptor density remained unaltered, but a reduction (50%) and an increase (15%) in the K(d) values were detected in striatum and hippocampus, respectively. D(1) and D(2) receptor densities in hippocampus and striatum remained unaltered meanwhile K(d) values for D(1) receptor declined significantly, 33% in hippocampus and 26% in striatum. Similarly, K(d) values for D(2) decreased 55% in hippocampus and 52% in striatum. From the preceding results, it is clear that there is a possible relation between alterations in muscarinic receptor density and others systems studied as well as they suggest that changes in dissociation constant can be responsible for the establishment of pilocarpine-induced SE by altering the affinity of neurotransmitters such as acetylcholine, dopamine and serotonine.     

Contrasting responses by basal ganglia met-enkephalin systems to low and high doses of methamphetamine in a rat model

The influence of methamphetamine (METH) on basal ganglia met-enkephalin (Menk) was studied by determining levels of this peptide in striatal, pallidal and nigral regions after administering a single low (0.5 mg/kg) or high (10 mg/kg) dose of this stimulant. The Menk levels in the striatal and pallidal areas were reduced and increased after the low- and high-dose METH treatments, respectively, 12 h after drug administration in all striatal and pallidal regions examined. The low-dose effect appeared to be principally influenced by increased activation of the dopamine D2-like receptor, while the high-dose effect seemed to result from dominance of D1-like receptor activation. However, both effects required coactivation of D1- and D2-like receptors. For the most part, both low- and high-dose METH-induced changes in Menk tissue content were fully recovered by 24 h. The Menk levels were not significantly altered in the substantia nigra 3-24 h after either METH treatment. Results reported herein indicated that striatal and pallidal Menk pathways respond differently after acute treatment with low or high doses of METH.     

Effect of biotic and abiotic elicitors on production of betulin and betulinic acid in the hairy root culture of Betula pendula Roth

Abstract

Betulin (B) and betulinic acid (BA) are two triterpenoids with a wide range of biological and medicinal activities in different organs of Betula pendula. This research aimed to increase the accumulation of B and BA in the hairy root culture of B. pendula by seven biotic and abiotic elicitors. Hairy root was induced in the stem’s inner bark of B. pendula using the C58C1 strain in the WPM (Woody Plant Medium). The effects of different concentrations of elicitors and different time of root harvest in hairy root culture of B. pendula showed that highest level of growth index (GI), B, and BA was acquired in treated hairy roots with chitosan (CTS), chlorocholine chloride (CCC) and chitosan nano-fiber (CTS NF). Highest GI of B. pendula hairy roots was 13 that was obtained in the roots treated with CTS 150?mg l^?1 on the 8th day. The highest content of BA was 1.3?mg g^?1 DW after treatment with 1?mg l^?1CCC on the 4th and 6th days and 200?mg l^?1CTS NF on the 10th day. The highest B content (0.94?mg g^?1DW) was obtained in the treated hairy root by 2?mg l^?1 CCC after 4 and 6?days.     

The putative multidrug resistance protein MRP‐7 inhibits methylmercury‐associated animal toxicity and dopaminergic neurodegeneration in Caenorhabditis elegans

Parkinson's disease (PD) is the most prevalent neurodegenerative motor disorder worldwide, and results in the progressive loss of dopamine (DA) neurons in the substantia nigra pars compacta. Gene–environment interactions are believed to play a significant role in the vast majority of PD cases, yet the toxicants and the associated genes involved in the neuropathology are largely ill‐defined. Recent epidemiological and biochemical evidence suggests that methylmercury (MeHg) may be an environmental toxicant that contributes to the development of PD. Here, we report that a gene coding for the putative multidrug resistance protein MRP‐7 in Caenorhabditis elegans modulates whole animal and DA neuron sensitivity to MeHg. In this study, we demonstrate that genetic knockdown of MRP‐7 results in a twofold increase in Hg levels and a dramatic increase in stress response proteins associated with the endoplasmic reticulum, golgi apparatus, and mitochondria, as well as an increase in MeHg‐associated animal death. Chronic exposure to low concentrations of MeHg induces MRP‐7 gene expression, while exposures in MRP‐7 genetic knockdown animals results in a loss of DA neuron integrity without affecting whole animal viability. Furthermore, transgenic animals expressing a fluorescent reporter behind the endogenous MRP‐7 promoter indicate that the transporter is expressed in DA neurons. These studies show for the first time that a multidrug resistance protein is expressed in DA neurons, and its expression inhibits MeHg‐associated DA neuron pathology.