Neuronal degeneration and oxidative stress in the SNc of 6-OHDA intoxicated rats; improving role of silymarin long-term treatment.

Progressive loss in dopaminergic neurons (DA) of substantia nigra pars compacta (SNc) leads to Parkinson's disease with a hypothesis of oxidative stress generation. The present study was conducted to determine the long-term efficacy of silymarin (SM) post-treatment on 6-OHDA-induced oxidative stress in the SNc of male rats. Male Wistar rats were received 6-OHDA (8 µg/rat) into SNc. After 3 weeks, as recovery period, the animals were treated with i.p. injection of SM at different doses of 100, 200, or 300 mg/kg for 15 days. At the end of the treatment, motor function, neuronal cell count, antioxidant enzymes, and lipid peroxidation and tyrosine hydroxylase (TH) activities were evaluated in the ventral midbrain tissue. The 6-OHDA significantly decreased (p ≤ 0.05) motor function, antioxidant enzyme activity, GSH level, and GSH/GSSG ratio and caused an augmentation in GSSG and lipid peroxidation level. The 6-OHDA also reduced the population of neurons and TH expression. The SM repaired the 6-OHDA-induced motor impairment, antioxidant enzyme suppression, and TH down-regulation. All three doses of SM could restore the MDA level to the normal range in the 6-OHDA-lesioned rats and could reversed the effect of 6-OHDA on GSH, GSSG level, and GSH/GSSG ratio. The SM treatment significantly and dose-dependently increased (p ≤ 0.001) the total number of surviving neurons in the SNc. Silymarin chronic treatment restored the brain's antioxidant capacity and salvaged neurons from oxidative stress-induced neurodegeneration. The SM could also improve motor function in parkinsonian animals by increasing TH expression. These results recommend that application of SM over initial clinical stages may depict a hopeful approach versus PD. However, more research is needed to confirm this issue.

Targeted Co-Delivery of Docetaxel and cMET siRNA for Treatment of Mucin1 Overexpressing Breast Cancer Cells.

Purpose: Targeted treatment of breast cancer through combination of chemotherapeutic agents and siRNA had been drawing much attention in recent researches. This study was carried out to evaluate mucin1 aptamer-conjugated chitosan nanoparticles containing docetaxel and cMET siRNA on SKBR3 cells. Methods: Nano-drugs were characterized by transmission electron microscope, Zetasizer and loading efficiency calculation. siRNA entrapment onto nanoparticles, stability of siRNA-loaded nanoparticles and conjugation of mucin1 aptamer to nanoparticles were evaluated via separate electrophoresis. Cellular uptake of the targeted nanoparticles was evaluated through GFP-plasmid expression in mucin1+ SKBR3 vs. mucin1- CHO cells. Protein expression, cell viability and gene expression were assessed by Western Blotting, MTT assay, and Quantitative Real Time-PCR, respectively. Results: Characterization of nano-drugs represented the ideal size (110.5± 3.9 nm), zeta potential (11.6± 0.8 mV), and loading efficiency of 90.7% and 88.3% for siRNA and docetaxel, respectively. Different gel electrophoresis affirmed the conjugation of aptamers to nanoparticles and entrapment of siRNA onto nanoparticles. Increased cellular uptake of aptamer-conjugated nanoparticles was confirmed by GFP expression. cMET gene silencing was confirmed by Western Blotting. The significant (p ≤0.0001) impact of combination targeted therapy vs. control on cell viability was shown. Results of Quantitative Real Time-PCR represented a remarkably decreased (p ≤0.0001) expression of the studied genes involving in tumorigenicity, metastasis, invasion, and angiogenesis (STAT3, IL8, MMP2, MMP9, and VEGF) by targeted combination treatment vs. control. Conclusion: The mucin1 aptamer-conjugated chitosan nanoparticles, containing docetaxel and cMET siRNA, is suggested for treatment of mucin1+ metastatic breast cancer cells. However, further studies should be conducted on animal models.

Silymarin prevents apoptosis through inhibiting the Bax/caspase-3 expression and suppresses toll like receptor-4 pathway in the SNc of 6-OHDA intoxicated rats.

BACKGROUND AND PURPOSE: Several lines of evidence show that apoptosis, oxidative stress and neuroinflammation are associated with the development of Parkinson's disease (PD). In the present study, we investigated the effect of pre-treatment with silymarin (SM) on oxidative stress, apoptosis and toll-like receptor 4 (TLR4) expression in substantia nigra pars copmacta (SNc) of 6-hydroxydopamine (6-OHDA)-lesioned rats. METHODS: Animals were pretreated with 100, 200 or 300 mg/kg of SM daily for 5 days and at 6th day 6-OHDA (8 µg/2 µl) was infused unilaterally into the central region of the SNc. RESULTS: 6-OHDA decreased the total glutathione and antioxidant enzymes activity in the SNc. Interestingly, we found that 6-OHDA caused to TLR4 up regulation. The SNc levels of glutathione, superoxide dismutase, glutathione peroxidase, glutathione reductase and catalase were significantly higher in the SM pretreated rats. SM strongly decreased 6-OHDA-induced elevation of SNc apoptosis, caspase-3 and Bax/Bcl-2 ratio. Furthermore, SM markedly (p < 0.001) prevented from SNc over expression of TLR4 caused by 6-OHDA. A significantly high positive correlation was seen between TLR4 activity with caspase-3 protein levels (r = 0.896, P < 0.01), Bax protein levels (r = 0.96, P < 0.01). CONCLUSION: Pre-treatment of 6-OHDA-lesioned rats with SM reduces SNc neuronal apoptosis possibly through inhibition of TLR4 over expression. Further clinical study should be carried out to prove potential application of SM for protection against PD in susceptible individuals.

In vitro and in vivo evaluation of the mechanisms of citalopram-induced hepatotoxicity.

Even though citalopram is commonly used in psychiatry, there are several reports on its toxic effects. So, the current study was designed to elucidate the mechanisms of cytotoxic effects of in vitro and in vivo citalopram treatment on liver and the following cytolethal events. For in vitro experiments, freshly isolated rat hepatocytes were exposed to citalopram along with/without various agents. To do in vivo studies liver function enzyme assays and histological examination were performed. In the in vitro experiments, citalopram (500 µM) exposure demonstrated cell death, a marked elevation in ROS formation, mitochondrial potential collapse, lysosomal membrane leakiness, glutathione (GSH) depletion and lipid peroxidation. In vivo biochemistry panel assays for liver enzymes function (AST, ALT and GGTP) and histological examination confirmed citalopram (20 mg/kg)-induced damage. citalopram-induced oxidative stress cytotoxicity markers were significantly prevented by antioxidants, ROS scavengers, MPT pore sealing agents, endocytosis inhibitors, ATP generators and CYP inhibitors. Either enzyme induction or GSH depletion were concomitant with augmented citalopram-induced damage both in vivo and in vitro which were considerably ameliorated with antioxidants and CYP inhibitors. In conclusion, it is suggested that citalopram hepatotoxicity might be a result of oxidative hazard leading to mitochondrial/lysosomal toxic connection and disorders in biochemical markers which were supported by histomorphological studies.

Effect of WR-1065 on 6-hydroxydopamine-induced catalepsy and IL-6 level in rats.

OBJECTIVES: Neuroinflammation and oxidative stress play a key role in pathogenesis of Parkinson's disease (PD). In the present study we investigated the effect of reactive oxygen species (ROS) scavenger WR-1065 on catalepsy and cerebrospinal fluid (CSF) level of interleukin 6(IL-6) and striatum superoxide dismutase (SOD) activity in 6-hydroxydopamine (6-OHDA) induced experimental model of PD. MATERIALS AND METHODS: Seventy two male Wistar rats were divided into 9 equal groups and 6-OHDA (8 µg/2 µl/rat) was infused unilaterally into substantia nigra pars copmacta (SNc) to induce PD. Catalepsy was measured by standard bar test, CSF level of IL-6 was assessed by enzyme-linked immunosorbent assay (ELISA) method and SOD activity measured by spectrophotometric method. In pre-treatment groups WR-1065 (20, 40 and 80 µg/2 µl/rat/day, for 3 days) was infused into the SNc before 6-OHDA administration and 21 days later, as a recovery period, behavioral and molecular assay tests were done. RESULTS: Our results showed that pre-treatment with WR-1065 improved (P<0.001) 6-OHDA-induced catalepsy in a dose dependent manner. In 6-OHDA-lesioned animals SOD activity in SNc and CSF level of IL-6 was decreased markedly (P<0.001) when compared with non-lesioned group, while pre-treatment with WR-1065(P<0.001) restored their levels up to the normal range. CONCLUSION: Our study indicated that pre-treatment with WR-1065 could modulate catalepsy and IL-6 level in 6-OHDA-lesioned rats. Also WR1065 could increase SOD activity up to normal range. It can be regarded as an anti-oxidative drug in prevention or adjunctive therapy of PD.

Testosterone ameliorates streptozotocin-induced memory impairment in male rats.

AIM: To study the effects of testosterone on streptozotocin (STZ)-induced memory impairment in male rats. METHODS: Adult male Wistar rats were intracerebroventricularly (icv) infused with STZ (750 µg) on d 1 and d 3, and a passive avoidance task was assessed 2 weeks after the first injection of STZ. Castration surgery was performed in another group of rats, and the passive avoidance task was assessed 4 weeks after the operation. Testosterone (1 mg·kg(-1)·d(-1), sc), the androgen receptor antagonist flutamide (10 mg·kg(-1)·d(-1), ip), the estrogen receptor antagonist tamoxifen (1 mg·kg(-1)·d(-1), ip) or the aromatase inhibitor letrozole (4 mg·kg(-1)·d(-1), ip) were administered for 6 d after the first injection of STZ. RESULTS: STZ administration and castration markedly decreased both STL1 (the short memory) and STL2 (the long memory) in passive avoidance tests. Testosterone replacement almost restored the STL1 and STL2 in castrated rats, and significantly prolonged the STL1 and STL2 in STZ-treated rats. Administration of flutamide, letrozole or tamoxifen significantly impaired the memory in intact rats, and significantly attenuated the testosterone replacement in improving STZ- and castration-induced memory impairment. CONCLUSION: Testosterone administration ameliorates STZ- and castration-induced memory impairment in male Wistar rats.

Silymarin improved 6-OHDA-induced motor impairment in hemi-parkisonian rats: behavioral and molecular study.

BACKGROUND: Neuroinflammation and oxidative stress has been shown to be associated with the development of Parkinson disease (PD). In the present study, we investigated the effect of intraperitoneal (i.p.) administration of silymarin, on 6-OHDA-induced motor-impairment, brain lipid per-oxidation and cerebrospinal fluid (CSF) levels of inflammatory cytokine in the rats. RESULTS: The results showed that silymarin is able to improve motor coordination significantly (p < 0.001) in a dose dependent manner. There was a significant (p < 0.001) increase in MDA levels of 6-OHDA-lesioned rats whereas; in silymarin (100, 200 and 300 mg/kg, i.p. for 5 days) pre-treated hemi-parkinsonian rats MDA levels was decreased markedly (p < 0.001). Furthermore the CSF levels of IL-1ß was decreased (p < 0.001) in silymarin (100, 200 and 300 mg/kg) pre-treated rats up to the range of normal non-parkinsonian animals. CONCLUSION: We found that pre-treatment with silymarin could improve 6-OHDA-induced motor imbalance by attenuating brain lipid per-oxidation as well as CSF level of IL-1ß as a pro-inflammatory cytokine. We suggest a potential prophylactic effect for silymarin in PD. However, further clinical trial studies should be carried out to prove this hypothesis.

Effect of glucosamine HCl on dissolution and solid state behaviours of piroxicam upon milling.

Piroxicam is a non-steroidal anti-inflammatory drug that is characterised by low solubility and high permeability. In order to improve the drug dissolution rate, the co-grinding method was used as an approach to prepare piroxicam co-ground in the carriers such as glucosamine hydrochloride. As, this amino sugar (glucosamine HCl) has been shown to decrease pain and improve mobility in osteoarthritis in joints, therefore, the incorporation of glucosamine in piroxicam formulations would be expected to offer additional benefits to patients. The effect of the order of grinding on the dissolution of piroxicam was also investigated. Co-ground drug and glucosamine were prepared in different ratios using a ball mill. The samples were then subjected to different grinding times. In order to investigate the effect of the grinding process on the dissolution behaviour of piroxicam, the drug was ground separately in the absence of glucosamine. Mixtures of ground piroxicam and unground D-glucosamine HCl were prepared. Physical mixtures of piroxicam and glucosamine were also prepared for comparison. The properties of prepared co-ground systems and physical mixtures were studied using a dissolution tester, FTIR, SEM, XRPD and DSC. These results showed that the presence of glucosamine HCl can increase dissolution rate of piroxicam compared to pure piroxicam. Generally, all dissolution profiles showed the fastest dissolution rate when ground piroxicam was mixed with unground glucosamine. This was closely followed by the co-grinding of piroxicam with glucosamine where lower grinding times showed the fastest dissolution. The solid state studies showed that the grinding of piroxicam for longer times had no effect on polymorphic form of piroxicam, whereas mixtures of piroxicam-glucosamine ground for longer times (60 min) converted piroxicam polymorph II to polymorph I.

Buspirone improves the anti-cataleptic effect of levodopa in 6-hydroxydopamine-lesioned rats.

In Parkinson's disease (PD), prolonged exposure to L-3,4-dihydroxyphenylalanine (L-DOPA) results in motor fluctuations, such as the on-off phenomenon, and L-DOPA-induced dyskinesia. Previously, we found that activation of 5-HT(1A) in the substantia nigra pars compacta (SNc) decreased catalepsy in parkinsonian rats. In the current investigation, we attempted to evaluate the effect of buspirone on the anti-cataleptic effect of L-DOPA in 6-hydroxydopamine (6-OHDA)-lesioned male Wistar rats. Catalepsy was induced by the unilateral infusion of 6-OHDA (8 µg/2 µl/rat) into the central region of the SNc. After a 3-week recovery period, rats received L-DOPA intraperitoneally (ip; 15 mg/kg) twice daily for 20 days, and the anti-cataleptic effect of L-DOPA was assessed by the bar test at days 5, 10, 15 and 20. The results showed that L-DOPA had an anti-cataleptic effect only until day 15, and its effect was abolished on day 20. On day 21, these rats were co-treated with three different doses of buspirone (0.1, 0.5 and 2.5 mg/kg, ip) and L-DOPA (15 mg/kg, ip). At a dose of 0.5 mg/kg, buspirone improved the anti-cataleptic effect of L-DOPA. Furthermore, the effect of buspirone (0.5 mg/kg, ip) on the anti-cataleptic effect of L-DOPA (15 mg/kg, ip) was reversed by 1-(2-methoxyphenyl)-4-(4-phthalimidobutyl)piperazine hydrobromide (NAN-190; 0.5 mg/kg, ip), a 5-HT(1A) receptor antagonist. From these results, it may be concluded that buspirone improves the anti-cataleptic effect of L-DOPA in a 6-OHDA-induced animal model of PD through the activation of 5-HT(1A) receptors. In this regard, further investigations should be undertaken to clarify the exact mechanism of the interaction between 5-HT(1A) and dopaminergic neurons.

Buspirone attenuates tolerance to analgesic effect of morphine in mice with skin cancer.

Adjuvant drugs that can delay tolerance to morphine analgesia may lead to improved management of pain in chronic disease such as cancer. This study was aimed to investigate effect of buspirone, as a partial agonist of 5-HT1A receptor, on tolerance induced to morphine analgesic effect in animals with skin cancer. Study was carried on female Swiss albino mice. For skin tumorigensis, mice were treated with single dose of 7,12-dimethylbenz(a)anthracene (DMBA) and promoted by multiple dose of croton oil. Tolerance to morphine analgesia was induced by daily subcutaneous (sc) injection of morphine (5mg/kg for 30 days) and assayed by using the hot plate method. Results obtained from this study showed that pain threshold in mice with skin cancer were significantly lower. Tolerance to analgesic effect of morphine (5 mg/kg, sc) was appeared at day 15, whereas, in normal and skin tumor bearing mice co-treated daily with morphine (5 mg/kg, sc) and three different intraperitoneal (ip) doses of buspirone (5, 7.5 and 10 mg/kg) tolerance was observed at days 25 and 30. In conclusion our data indicate that concurrent use of morphine with buspirone may produce good cancer pain control and attenuate development of tolerance.

Effect of fluoxetine on tolerance to the analgesic effect of morphine in mice with skin cancer.

Adjuvant drugs that attenuate or inhibit the development of tolerance to morphine may lead to improved management of pain in chronic diseases such as cancer. The aim of this study was to investigate effect of fluoxetine, a specific 5-HT (5-hydroxytryptamine, serotonin) reuptake inhibitor, on tolerance induced to the analgesic effect of morphine in mice with skin cancer. The study was carried out on female Swiss albino mice. For skin tumorigensis, mice were initiated with a single dose of 7,12-dimethylbenz(a)anthracene (DMBA) and promoted by multiple doses of croton oil. Tolerance to morphine analgesia was induced by daily subcutaneous (sc) injections of morphine (5 mg/kg for 30 days) and assayed using the hot plate method. Results obtained from this study showed that pain thresholds in mice with skin cancer were significantly lower. Tolerance to the analgesic effect of morphine (5 mg/kg, sc) appeared at day 15, whereas in normal and skin tumor bearing mice co-treated daily with morphine (5 mg/kg, sc) and three different intraperitoneal (ip) doses of fluoxetine (0.16, 0.32 and 0.64 mg/kg) tolerance was observed at days 20, 25 and 30, respectively. In conclusion, our data indicate that concurrent use of morphine with fluoxetine may produce good cancer pain control and attenuate the development of tolerance.

Evaluation of in vitro-in vivo correlation and anticonvulsive effect of carbamazepine after cogrinding with microcrystalline cellulose.

PURPOSE: Carbamazepine is a poor water soluble drug and its bioavailability is limited by dissolution rate. Dissolution, serum concentration and anticonvulsive effect of the drug have been evaluated after cogrinding with microcrystalline cellulose. A cogrinding technique was used to increase the dissolution, serum concentrations and anticonvulsive effect of the drug. A novel deconvolution technique of in vitro in vivo correlation was evaluated. METHODS: The drug coground with microcrystalline cellulose, the corresponding physical mixture, unground and ground drug powder were subjected to dissolution measurement. Coground and unground drug serum concentrations were investigated in rabbits. Also the anticonvulsive effects of the latter preparations were assessed in mice. For elucidation of observed in vitro and in vivo differences FT-IR spectroscopy, X-ray diffraction patterns and DSC thermograms of the preparations were studied. RESULTS: The dissolution of the coground was the highest (percent dissolved in the first 20 minutes, %D20', was 97.5). The unground drug powder exhibited the lowest dissolution (%D20'=40). The difference was reflected in their corresponding area under the mean serum concentration curves between 0-16 hr (118.96 vs 54.17 microg x hr/ml) as well as protection abilities against strychnine and electrically induced seizures. The onset of tonic seizures induced by strychnine was increased between 40-140% in the case of the coground system depending on dose and time of carbamazepine administration. CONCLUSION: Cogrinding was an effective technique in increasing carbamazepine dissolution due to reduced crystallinity as seen in X-ray pattern, enhanced wettability and decreased particle size, which in turn resulted in increased serum concentrations and its anticonvulsive effect. A novel simple deconvolusion technique not requiring intravenous data denoted as the double reciprocal area method was used to establish correlation between in vitro and in vivo parameters.

Effects of central and peripheral depletion of serotonergic system on carrageenan-induced paw oedema.

The role of serotonergic system was investigated on peripheral inflammation induced by intraplantary injection of carrageenan. Para-chlorophenylalanine (PCPA) was administered intracerebroventriculary (50, 100 microg/rat) or intraperitoneally (150 mg/kg, 3 days) and 2 or 24 h later, respectively, inflammation was induced by injection of carrageenan. Paw oedema was decreased significantly in PCPA-treated (100 microg/rat, i.c.v.) rats compared to control groups. Injection of exogenous serotonin (i.c.v.) by dose of 0.70 nmol/10 microl/rat, but not the dose of 0.35 nmol/10 microl/rat, 15 min after induction of inflammation completely reversed the anti-inflammatory effects of PCPA. Myeloperoxidase activity in inflamed paws was reduced significantly in groups received PCPA (either i.c.v. or i.p.) compared to controls. Exogenous serotonin (0.70 nmol/10 microl/rat) reduced inflammatory response when injected (i.c.v.) 30 min before or 30 min after the induction of inflammation. Injection of serotonin at the time of induction of inflammation had no inflammatory/anti-inflammatory effect. These results suggest that serotonin, as a neurotransmitter in central nervous system, may be involved in modulating peripheral inflammation.