Crocin attenuates the lipopolysaccharide-induced neuroinflammation via expression of AIM2 and NLRP1 inflammasome in an experimental model of Parkinson's disease.

The underlying mechanisms of inflammasome activation and the following dopaminergic neuron loss caused by chronic neuroinflammation remain entirely unclear. Therefore, this study aimed to investigate the impact of crocin on the inflammasome complex within an experimental model of Parkinson's disease (PD) using male Wistar rats. PD was induced by the stereotaxic injection of lipopolysaccharide (LPS), and crocin was intraperitoneally administrated one week before the lesion, and then treatment continued for 21 days. Open field (OF) and elevated plus maze tests were applied for behavioral assays. Furthermore, hematoxylin and eosin (H&E) and immunostaining were performed on whole brain tissue, while dissected substantia nigra (SN) was used for immunoblotting and real-time PCR to evaluate compartments involved in PD. The time spent in the center of test was diminished in the LPS group, while treatment with 30 mg/kg of crocin significantly increased it. H&E staining showed a significant increase in cell infiltration at the site of LPS injection, which was ameliorated upon crocin treatment. Notably, crocin-treated animals showed a reduced number of caspase-1 and IL-1ß positive cells, whereas the number of positive cells was increased in the LPS group (P < 0.05). A significant decrease in tyrosine hydroxylase (TH) expression was also found in the LPS group, while crocin treatment significantly elevated its expression. IL-1ß, IL-18, NLRP1, and AIM2 genes expression significantly increased in the LPS group. On the other hand, treatment with 30 mg/kg of crocin significantly downregulated the expression levels of these genes along with NLRP1 (P < 0.05). In summary, our findings suggest that crocin reduces neuroinflammation in PD by diminishing IL-1ß and caspase-1 levels, potentially by inhibiting the expression of AIM2 and NLRP1 genes.

Long-term administration of metformin ameliorates age-dependent oxidative stress and cognitive function in rats.

BACKGROUND: Aging is an inevitable physiological process, associated with a decline in cognitive function. Recently, metformin, as the first-line treatment for type II diabetes, has been shown to increase the life expectancy of diabetic patients. Therefore, researchers are paying increasing attention to its anti-aging properties. Oxygen free radicals are responsible for oxidative stress, which is a prominent factor in age-associated diseases. This study aimed to evaluate the effects of long-term administration of metformin on age-dependent oxidative stress and cognitive function. METHODS: In this experimental study, 32 normal (nondiabetic) male Wistar rats were randomly assigned into control and metformin groups (n = 16 per group). The metformin group received 100 mg/kg of metformin in drinking water daily for six months. The shuttle box test was used for the passive avoidance task in 24-month-old rats. For the biochemical assay, the total antioxidant capacity (TAC) and malondialdehyde (MDA) level were measured. Nissl and TUNEL staining were also used for histopathological assessments. Data were analyzed using independent t-test. RESULTS: The present findings revealed that metformin significantly reduced the MDA level and increased the TAC in the hippocampus of the metformin group (p < 0.05). The survival of hippocampal CA1 neurons was significantly higher in the metformin group as compared to the control group, while the number of TUNEL-positive neurons decreased significantly (p < 0.05). On the other hand, metformin markedly improved the passive avoidance memory in the metformin group as compared to the control group (p < 0.05). CONCLUSION: It can be concluded that long-term metformin intake, by modulating the oxidant/antioxidant mechanisms, prevents the loss of hippocampal neurons caused by age-dependent oxidative stress and improves memory.

Withania coagulans extract attenuates oxidative stress-mediated apoptosis of cerebellar purkinje neurons after ischemia/reperfusion injury.

Cerebral ischemia/reperfusion (I/R) is known to increase reactive oxygen species (ROS) generation, consequences of oxidative stress (OS), and neuronal death in the susceptible brain areas including the cerebellum. Newly, remarkable attention has been paid to a natural diet with the capability to scavenge ROS. Withania coagulans root extract (WCE) is rich in components with antioxidants properties. Therefore, this study aimed to evaluate the effect of WCE on cerebellar Purkinje cells (PCs) against OS-mediated apoptosis after I/R injury. In this experimental study 64 male adult Wistar rats were randomly divided into 4 groups (n = 16) as follows: control, sham, I/R, and WCE 1000 + I/R. I/R animals were pretreated with daily administration of hydro-alcoholic WCE (1000 mg/kg) or distilled water as a vehicle for 30 days before I/R injury. After 72 h, the animals were sacrificed, the cerebellum tissue was removed and used for biochemical (CAT, SOD, GPx, and MDA levels) and histopathological (Nissl and TUNEL staining) assays. Findings showed that the MDA level and the number of apoptotic neurons significantly increased and viable Purkinje neurons decreased in I/R injury (p < 0.05). Administration of 1000 mg/kg WCE reduced MDA level and enhanced antioxidants activity including CAT, SOD, and GPx significantly. In addition, intact surviving PCs increased. At the same time, TUNEL-positive neurons decreased significantly in the WCE pre-treated group (p < 0.05). These findings suggest that WCE can counteract cerebral I/R-induced OS and associated neuronal death by enhancement of ROS scavenging and antioxidant capacity. It appears that pre-treatment with 1000 mg/kg WCE for thirty days can protect PCs against OS-mediated apoptosis after I/R injury.

Evaluation of the neuroprotective effects of electromagnetic fields and coenzyme Q10 on hippocampal injury in mouse.

Electromagnetic fields (EMFs) are reported to interfere with chemical reactions involving free radical production. Coenzyme Q10 (CoQ10) is a strong antioxidant with some neuroprotective activities. The purpose of this study was to examine and compare the neuroprotective effects of EMF and CoQ10 in a mouse model of hippocampal injury. Hippocampal injury was induced in mature female mice (25-30 g), using an intraperitoneal injection of trimethyltin hydroxide (TMT; 2.5 mg/kg). The experimental groups were exposed to EMF at a frequency of 50 Hz and intensity of 5.9 mT for 7 hr daily over 1 week or treated with CoQ10 (10 mg/kg) for 2 weeks following TMT injection. A Morris water maze apparatus was used to assess learning and spatial memory. Nissl staining and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) tests were also performed for the histopathological analysis of the hippocampus. Antiapoptotic genes were studied, using the Western blot technique. The water maze test showed memory improvement following treatment with CoQ10 and coadministration of CoQ10 + EMF. The Nissl staining and TUNEL tests indicated a decline in necrotic and apoptotic cell count following treatment with CoQ10 and coadministration of CoQ10 + EMF. The Western blot study indicated the upregulation of antiapoptotic genes in treatment with CoQ10, as well as coadministration. Also, treatment with EMF had no significant effects on reducing damage induced by TMT in the hippocampus. According to the results, EMF had no significant neuroprotective effects in comparison with CoQ10 on hippocampal injury in mice. Nevertheless, coadministration of EMF and CoQ10 could improve the neuroprotective effects of CoQ10.

The neuroprotective effects of hydro-alcoholic extract of olive (Olea europaea L.) leaf on rotenone-induced Parkinson's disease in rat.

Parkinson's disease (PD) is an age-related disease in which dopaminergic neurons in the nigrostriatal pathway are destroyed, resulting in movement and behavioral problems. Oxidative stress and the generation of reactive oxygen species play key roles in neurodegenerative diseases, such as PD. Rotenone (ROT) is a common pesticide that induces oxidative stress. Olive leaves extract (OLE) has antioxidant and neuroprotective effects. Thus, the aim of this study was to investigate the neuroprotective effects of OLE on ROT-induced oxidative stress in the midbrain of a rat model of PD. Ninety-six Wistar rats were randomly divided into the following 6 groups (n = 16 rats/group): Control, Sham, ROT, and 3 ROT + OLE (75, 150, and 300 mg/kg/daily) groups. ROT (2.5 mg/kg/48 h) was injected subcutaneously, and vehicle or OLE was orally administered for 30 days. The animals were then sacrificed, and their brains were removed. Biochemical measures, including the levels of catalase (CAT), glutathione peroxidase (GPx), superoxide dismutase (SOD), and malondialdehyde (MDA), and the number of tyrosine hydroxylase (TH)-positive neurons were determined, and behavioral (rotarod and hanging) tests were conducted. The balance and muscle strength of the OLE (150 and 300 mg/kg)-treated groups were significantly improved. Treatment with OLE prevented the increases in the levels of MDA, significantly improved the SOD, CAT, and GPx levels in the midbrain, and prevented the depletion of the TH-positive neurons. These findings suggested that OLE has neuroprotective properties and that it might be useful for preventing the death of dopaminergic neurons in patients with PD.

In vitro differentiation of neural stem cells derived from human olfactory bulb into dopaminergic-like neurons.

This study describes a new accessible source of neuronal stem cells that can be used in Parkinson's disease cell transplant. The human olfactory bulb contains neural stem cells (NSCs) that are responsible for neurogenesis in the brain and the replacement of damaged cellular components throughout life. NSCs are capable of differentiating into neuronal and glial cells. We isolated NSCs from the olfactory bulb of brain-death donors and differentiated them into dopaminergic neurons. The olfactory bulb tissues obtained were cultured in Dulbecco's modified Eagle's medium/nutrient mixture F12, B27 supplemented with basic fibroblast growth factor, epidermal growth factor and leukemia inhibitory factor. The NSCs and proliferation markers were assessed. The multipotentiality of olfactory bulb NSCs was demonstrated by their capacity to differentiate into neurons, oligodendrocytes and astrocytes. To generate dopaminergic neurons, olfactory bulb NSCs were differentiated in neurobasal medium, supplemented with B27, and treated with sonic hedgehog, fibroblast growth factor 8 and glial cell-derived neurotrophic factor from the 7th to the 21st day, followed by detection of dopaminergic neuronal markers including tyrosine hydroxylase and aromatic l-amino acid decarboxylase. The cells were expanded, established in continuous cell lines and differentiated into the two classical neuronal phenotypes. The percentage of co-positive cells (microtubule-associated protein 2 and tyrosine hydroxylase; aromatic l-amino acid decarboxylase and tyrosine hydroxylase) in the treated cells was significantly higher than in the untreated cells. These results illustrate the existence of multipotent NSCs in the adult human olfactory bulb that are capable of differentiating toward putative dopaminergic neurons in the presence of trophic factors. Taken together, our data encourage further investigations of the possible use of olfactory bulb NSCs as a promising cell-based therapeutic strategy for Parkinson's disease.

Withania coagulans Extract Induces Cell Apoptosis and Inhibits COX-2 Expression in a Rat Model of Benign Prostatic Hyperplasia.

BACKGROUND: Phytotherapy is a popular treatment option in cases of benign prostatic hyperplasia (BPH), with many different herbal products being used for the treatment of this condition. Withania coagulans (WC) is an herbal medicine that has shown anti-tumoral, anti-inflammatory, and antioxidant effects. OBJECTIVES: This study examined the effect of Withania coagulans extract (WCE) on prostatic cell apoptosis and cyclooxygenase-2 (COX-2) expression in cases of benign prostatic hyperplasia (BPH) in rats. METHODS: Forty Wistar rats were equally divided into five groups: control, sham, BPH, BPH + WCE, and BPH + CLX (celecoxib) as a positive control group. The induction of BPH was achieved via the subcutaneous injection of 3 mg/kg of testosterone propionate (TP) daily for 28 days. The animals received WCE, celecoxib, or distilled water by oral gavage accompanied by the TP injection. After four weeks, the prostate glands of the rats were weighed to measure the prostatic index (PI). The ventral lobes of the prostates were dissected and processed with paraffin blocks in order to study the number of mast cells. A TUNEL analysis was performed to evaluate the cell apoptosis, while the expression of COX-2 was examined using immunohistochemistry. RESULTS: BPH was obvious in the ventral lobe of the prostate, and the administration of WCE markedly decreased the PI and the number of mast cells (P < 0.001) in the BPH rats. Additionally, the WCE treatment induced prostatic cell apoptosis when compared to the BPH group. Furthermore, following the WCE treatment, the expression of COX-2 in the prostatic tissues was significantly decreased when compared to the BPH groups. CONCLUSIONS: According to the results of this study, WCE was effective in the treatment of BPH in rats. It may therefore have beneficial effects in the treatment of patients with BPH.

Neuroprotective effects of Withania coagulans root extract on CA1 hippocampus following cerebral ischemia in rats.

OBJECTIVE: Oxygen free radicals may be implicated in the pathogenesis of ischemia reperfusion damage. The beneficial effects of antioxidant nutrients, as well as complex plant extracts, on cerebral ischemia-reperfusion injuries are well known. This study was conducted to determine the effects of the hydro-alcoholic root extract of Withania coagulans on CA1 hippocampus oxidative damages following global cerebral ischemia/reperfusion in rat. MATERIALS AND METHODS: Male Wistar rats were randomly divided in five groups: control, sham operated, Ischemia/ Reperfiusion (IR), and Withania Coagulans Extract (WCE) 500 and 1000mg/kg + I/R groups. Ischemia was induced by ligation of bilateral common carotid arteries for 30 min after 30 days of WCE administration. Three days after, the animals were sacrificed, their brains were fixed for histological analysis (NISSL and TUNEL staining) and some samples were prepared for measurement of malondialdehyde (MDA) level and superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx) activity in hippocampus. RESULTS: WCE showed neuroprotective activity by significant decrease in MDA level and increase in the SOD, CAT and GPx activity in pretreated groups as compared to I/R groups (p<0.001). The number of intact neurons was increased while the number of TUNEL positive neurons in CA1 hippocampal region in pretreated groups were decreased as compared to I/R group (p<0.001). CONCLUSION: WCE showed potent neuroprotective activity against oxidative stress-induced injuries caused by global cerebral ischemia/ reperfusion in rats probably by radical scavenging and antioxidant activities.

Antiproliferative and Antioxidant Effects of Withania coagulans Extract on Benign Prostatic Hyperplasia in Rats.

BACKGROUND: Benign prostate hyperplasia (BPH) is a common urological disorder in elderly men. Phytotherapy is frequently used to alleviate the symptoms of this condition. OBJECTIVES: The present study investigated the effect of Withania coagulans extract (WCE), which is known to have antioxidant, anti-inflammatory, antihyperglycemic, and anti-cancer properties, on testosterone-induced BPH in rats. MATERIALS AND METHODS: Forty Wistar rats were divided into five groups (each n = 8): the control group, the untreated BPH group, and three WCE-treated groups (WCE250, 500, and 1000). BPH was induced with 3 mg/kg subcutaneous injections of testosterone propionate for four weeks. WCE was concomitantly administrated by oral gavage. At the end of the induction schedule, the animals were sacrificed and their prostate glands were dissected, weighed, and fixed for histological examination (H&E and proliferating cell nuclear antigen [PCNA] staining). Half of each sample was prepared for measurement of malondialdehyde (MDA) and total antioxidant capacity (TAC) levels in the prostate. RESULTS: The present study revealed that BPH caused elevation of MDA levels, suppression of TAC levels, and increased PCNA expression in the prostate gland. Interestingly, in a dose-dependent manner, WCE caused decreased MDA levels and increased TAC levels in the prostate gland, compared to the untreated BPH group. Histopathological examinations showed a reduction in PCNA expression in the prostate epithelium of the WCE animals. CONCLUSIONS: W. coagulans inhibits the development of BPH can be useful for the treatment of this condition.

Different effects of olive leaf extract on antioxidant enzyme activities in midbrain and dopaminergic neurons of Substantia Nigra in young and old rats.

OBJECTIVES: Study of the effects of olive leaf extract on antioxidant enzyme activities in midbrain and dopaminergic neurons of Substantia Nigra in young and old rats. METHODS: Male wistar rats age 4 and 18 months were randomized into control and experimental groups. A single daily dose of 50 mg/kg of olive leaf extract was administered orally by gavage to each rat for 6 months. The control group received only distilled water. All rats were sacrificed 2 hours after the last gavage and their midbrains were separated for Malondialdehyde (MDA) and antioxidant enzyme activitiy analysis. TUNEL assay and immunohistochemical (IHC) staining were used for evaluation of the number of neurons in the Substantia Nigra. RESULTS: The level of Catalase, Glutathione Peroxidase and Superoxide Dismutase enzyme activity were significantly increased in experimental young and old groups compared to their control groups. However the level of Superoxide Dismutase enzyme activity was significantly increased in experimental old group when compared to control group (P< 0.05), the level of Superoxide Dismutase enzyme activity was not significantly changed in young groups. MDA level was decreased significantly in experimental young and old rats compared to their control groups. Histological analysis demonstrated that the number of neurons in Substantia Nigra of experimental old group was more than the control group (P<0.05). The number of apoptotic cells was significantly decreased in experimental old group compared to the corresponding control group (P<0.05). In IHC and TUNEL assay, no change was observed in the number of neurons between experimental and control young groups. CONCLUSION: Long term treatment with olive leaf extract increases antioxidant enzyme activity and protects the neurons in Substantia Nigra against oxidative stress.

Evaluation of effect of oleuropein on skin wound healing in aged male BALB/c mice.

OBJECTIVE: Olive oil and olive leaf extract are used for treatment of skin diseases and wounds in Iran. The main component of olive leaf extract is Oleuropein. This research is focused on the effects of Oleuropein on skin wound healing in aged male Balb/c mice. MATERIALS AND METHODS: In this experimental study, Oleuropein was provided by Razi Herbal Medicine Institute, Lorestan, Iran.Twenty four male Balb/c mice, 16 months of age, were divided equally into control and experimental groups.Under ether anesthesia, the hairs on the back of neck of all groups were shaved and a 1 cm long full-thickness incision was made.The incision was then left un-sutured. The experimental group received intradermal injections with a daily single dose of 50 mg/kg Oleuropein for a total period of 7 days.The control group received only distilled water. On days 3 and 7 after making the incision and injections, mice were sacrificed, and the skin around incision area was dissected and stained by hematoxylin and eosin (H&E) and Van Gieson's methods for tissue analysis.In addition, western blot analysis was carried out to evaluate the level of vascular endothelial growth factor (VEGF) protein expression. The statistical analysis was performed using SPSS (SPSS Inc., Chicago, USA). The t test was applied to assess the significance of changes between control and experimental groups. RESULTS: Oleuropein not only reduced cell infiltration in the wound site on days 3 and 7 post incision, but also a significant increase in collagen fiber deposition and more advanced re- epithelialization were observed (p<0.05) in the experimental group as compared to the control group. The difference of hair follicles was not significant between the two groups at the same period of time. Furthermore, western blot analysis showed an increased in VEGF protein level from samples collected on days 3 and 7 post-incision of experimental group as compared to the control group (p<0.05). CONCLUSION: These results suggest that Oleuropein accelerates skin wound healing in aged male Balb/c mice. These findings can be useful for clinical application of Oleuropein in expediting wound healing after surgery.

Antioxidant role of oleuropein on midbrain and dopaminergic neurons of substantia nigra in aged rats.

BACKGROUND: Oleuropein is a phenolic compound which is present in the olive leaf extract. The purpose of the present study was to investigate the neuroprotective effect of oleuropein as an antioxidant agent on the substantia nigra in aged rats. METHODS: Twenty 18-month-old Wistar rats (450-550 g) were randomly divided into control and experimental groups. The experimental group received a daily single dose of 50 mg/kg of oleuropein by oral gavage for 6 months. The control group received only distilled water. All rats were sacrificed two hours after the last gavage and the brains were removed and midbrains were cut. One part of the midbrains were homogenized and centrifuged. The tissue supernatant was assayed for lipid peroxidation (LPO) and antioxidant enzyme activities. The other part of midbrains fixed and embedded in paraffin, then processed for Nissl and immunohistochemistry (IHC) staining. Data was analyzed using SPSS by t-test. Differences were considered significant for P<0.05. RESULTS: The level of LPO in midbrain of the rats was decreased significantly in the experimental group, but superoxide dismutase, catalase and glutathione peroxidase activities were increased in experimental group compared to control group (P<0.05). Morphometric analyses showed significantly that the experimental group had more neurons in substantia nigra pars compacta (SNc) either in Nissl or IHC staining when compared to control (P<0.05). CONCLUSION: The results of the present study indicate that treatment of the old rats with oleuropein reduces the oxidative damage in SNc by increasing the antioxidant enzyme activities.

Therapeutic effects of oleuropein on wounded skin in young male BALB/c mice.

INTRODUCTION: Oleuropein is generally the most abundant phenolic compound in olive leaves. In this study, therapeutic effects of oleuropein were studied on wounded skin in young male Balb/c mice. METHODS: Four-month-old male Balb/c mice were randomized into 2 groups, a control and an experimental group. Under ether anesthesia, hair on the neck of mice in both groups was shaved and 1-cm long full-thickness incisions were made and left unsutured. The experimental group was injected intradermally with a daily single dose of oleuropein (50 mg/kg) for a total of 7 days. The control group received only distilled water. On days 3 and 7 post-incision, mice were sacrificed and skin around the area of the incisions were dissected and processed for hematoxylin and eosin and Van Gieson's staining. Portions of dissected tissues were also lysed and used for western blot analysis to evaluate the level of vascular endothelial growth factor (VEGF) protein expression. RESULTS: The analyses showed oleuropein reduced cell infiltration into the wound sites on day 3 and 7 postincision; however, it significantly increased collagen fiber deposition and caused faster reepithelialization when compared to the control group (P < 0.05). Furthermore, western blot analysis showed a significant increase in VEGF protein level compared to the control group (P < 0.05). CONCLUSION: In summary, oleuropein showed healing effects on wounded skin by accelerating the reepithelialization process, enhancing collagen fiber generation, and increasing the blood supply to the wounded area by upregulation of VEGF protein expression.