Rosemary essential oil potentiates the antitumour activity of 5-fluorouracil in human colorectal carcinoma cells.

OBJECTIVES: Improving response rates in colorectal cancer (CRC) is an urgent clinical need. This study aimed to explore the synergistic action of Lebanese rosemary essential oil (REO) and 5-fluorouracil (5-FU) in HCT116 CRC cells. METHODS: We tested the cell viability of monotherapy and combination therapy. The combination index was calculated using CompuSyn software to evaluate drug-drug interactions and the level of synergistic cytotoxicity. We also evaluated cell migration and cytopathology. Furthermore, cell apoptosis-related proteins (i.e. Bax and Bcl-2) were measured by Western blot analysis. KEY FINDINGS: The REO/5-FU combination synergistically reduced cell viability, effectively decreased cell migration, and increased the Bax/Bcl-2 ratio in HCT116 cells. This triggered a proapoptotic morphology and initiated an apoptotic cascade in HCT116 cells, as indicated by a higher Bax/Bcl-2 ratio. CONCLUSIONS: Our results provide evidence of the REO/5-FU combination as a better approach to improve 5-FU anticancer efficacy and allow the use of lower 5-FU doses due to the adjuvant effect of REO.

Transcranial Irradiation Mitigates Paradoxical Sleep Deprivation Effect in an Age-Dependent Manner: Role of BDNF and GLP-1.

The growing prevalence of aged sleep-deprived nations is turning into a pandemic state. Acute sleep deprivation (SD) accompanies aging, changing the hippocampal cellular pattern, neurogenesis pathway expression, and aggravating cognitive deterioration. The present study investigated the ability of Near Infra Red (NIR) light laser to ameliorate cognitive impairment induced by SD in young and senile rats. Wistar rats ≤ 2 months (young) and ≥ 14 months (senile) were sleep-deprived for 72 h with or without transcranial administration of NIR laser of 830 nm. Our results showed that NIR photobiomodulation (PBM) attenuated cognitive deterioration made by SD in young, but not senile rats, while both sleep-deprived young and senile rats exhibited decreased anxiety (mania)-like behavior in response to PBM. NIR PBM had an inhibitory effect on AChE, enhanced the production of ACh, attenuated ROS, and regulated cell apoptosis factors such as Bax and Bcl-2. NIR increased mRNA expression of BDNF and GLP-1 in senile rats, thus facilitating neuronal survival and differentiation. The present findings also revealed that age exerts an additive factor to the cellular assaults produced by SD where hippocampal damages made in 2-month rats were less severe than those of the aged one. In conclusion, NIR PBM seems to promote cellular longevity of senile hippocampal cells by combating ROS, elevating neurotrophic factors, thus improving cognitive performance. The present findings provide NIR as a possible candidate for hippocampal neuronal insults accompanying aging and SD.

Photomodulatory effects in the hypothalamus of sleep-deprived young and aged rats.

Insufficient sleep is associated with impaired hypothalamic activity and declined attentional performance. In this study, alterations in the hypothalamus of REM sleep-deprived (SD) young and aged rats, and the modulatory effect of near-infrared (NIR) laser were investigated. Forty-eight male Wistar rats (24 young at 2 months and 24 senile at 14 months) were divided into three groups: the control, the SD group subjected to 72 hr of sleep deprivation, and the transcranial-NIR laser-treated (TLT) group subjected to SD for 72 hr and irradiated with 830 nm laser. The hypothalamic levels of oxidative stress, inflammatory biomarkers, antioxidant enzymes, mitochondrial cytochrome C oxidase (CCO), apoptotic markers (BAX, BCL-2), and neuronal survival-associated genes (BDNF, GLP-1) were evaluated. Furthermore, the hypothalamic tissue alterations were analyzed via histological examination. The results revealed that TLT treatment has enhanced the antioxidant status, prevented oxidative insults, suppressed neuroinflammation, regulated CCO activity, reduced apoptotic markers, and tuned the survival genes (BDNF & GLP-1) in hypothalamic tissue of SD young and aged rats. Microscopically, TLT treatment has ameliorated the SD-induced alterations and restored the normal histological features of hypothalamus tissue. Moreover, the obtained data showed that SD and NIR laser therapy are age-dependent. Altogether, our findings emphasize the age-dependent adverse effects of SD on the hypothalamus and suggest the use of low-laser NIR radiation as a potential non-invasive and therapeutic approach against SD-induced adverse effects in young and aged animals.

Neural stem cells characterization in the vagal complex of adult ovine brain: A combined neurosphere assay/RTqPCR approach.

Neural stem cells are the effectors of adult neurogenesis, which occurs in discrete restricted areas of adult mammalian brain. In ovine species, like in rodents, in vivo incorporation of labeled DNA precursor led to characterize neurogenic proliferation in the subventricular zone and progeny migration and differentiation into the olfactory bulb. The present study addresses directly the existence of neural stem cells in the neurogenic niche of the vagal centre (area postrema) by in vitro neurosphere assay and RT-qPCR of specific markers on ex-vivo adult tissue explants, comparatively with the canonical neurogenic niche: the subventricular zone (SVZ) of the forebrain. Explants defined from the neuroanatomical patterns of in vivo BrdU incorporation yielded expandable and self-renewing spheres from both SVZ and AP. Within SVZ though, the density of sphere-forming cells was higher in ventral SVZ (SVZ-V) than in its latero-dorsal (SVZ-D) and lateral (SVZ-L) regions, which differs from the distributions of neural stem cells in mouse and swine brains. Consistently, RT-qPCR of the biomarker of neural stem cells, Sox2, yields highest expression in SVZ-V ahead of SVZ-D, SVZ-L and AP. These results are discussed with regard to previously published dynamics of adult ovine neurogenesis in vivo, and in light of corresponding features in other mammalian species. This confirms existence of neurogenetic plasticity in the vagal complex of adult mammals.

Garden Cress Seed Oil Abrogates Testicular Oxidative Injury and NF-kB-Mediated Inflammation in Diabetic Mice.

Diabetes mellitus is a metabolic disorder associated with various complications encompassing male reproductive dysfunction. The present study aimed to investigate the therapeutic potential of biologically active Lepidium sativum seed oil (LSO) against the testicular dysfunction associated with streptozotocin (STZ)-induced diabetes. Male adults (n = 24) were divided into four groups: control, LSO-administered, diabetic (D), and LSO-treated diabetic (D+LSO) groups. LSO was extracted from L. sativum seeds, and its chemical composition was determined using GC-MS. Serum testosterone levels, testicular enzymatic antioxidants (catalase (CAT) and superoxide dismutase (SOD)), an oxidative stress (OS) biomarker, malondialdehyde (MDA), pro-inflammatory markers (NF-kB, IL-1, IL-6, and TNF-α), and the expression level of NF-kB were assessed. In addition, histopathological changes were evaluated in testicular tissues. The results obtained showed that the chemical composition of LSO indicated its enrichment mainly with γ-tocopherol (62.1%), followed by 2-methylhexacosane (8.12%), butylated hydroxytoluene (8.04%), 10-Methylnonadecane (4.81%), and δ-tocopherol (3.91%). Moreover, LSO administration in the D+LSO mice significantly increased testosterone levels and ameliorated the observed testicular oxidative damage, inflammatory response, and reduced NF-kB expression compared to the diabetic mice. Biochemical and molecular analyses confirmed the histological results. In conclusion, LSO may prevent the progression of diabetes-induced impairment in the testes through inhibition of the OS- and NF-kB-mediated inflammatory response.

Effect of Epigallocatechin-3-gallate on Stress-Induced Depression in a Mouse Model: Role of Interleukin-1ß and Brain-Derived Neurotrophic Factor.

Epigallocatechin 3-gallate (EGCG) is a natural polyphenolic antioxidant in green tea leaves with well-known health-promoting properties. However, the influence of EGCG on a chronic animal model of depression remains to be fully investigated, and the details of the molecular and cellular changes are still unclear. Therefore, the present study aimed to investigate the antidepressant effect of EGCG in mice subjected to chronic unpredictable mild stress (CUMS). After eight consecutive weeks of CUMS, the mice were treated with EGCG (200 mg/kg b.w.) by oral gavage for two weeks. A forced swimming test (FST) was used to assess depressive symptoms. EGCG administration significantly alleviated CUMS-induced depression-like behavior in mice. EGCG also effectively decreased serum interleukin-1ß (IL-1ß) and increased the mRNA expression levels of brain-derived neurotrophic factor (BDNF) in the hippocampal CA3 region of CUMS mice. Furthermore, electron microscopic examination of CA3 neurons in CUMS mice showed morphological features of apoptosis, loss or disruption of the myelin sheath, and degenerating synapses. These neuronal injuries were diminished with the administration of EGCG. The treatment effect of EGCG in CUMS-induced behavioral alterations was comparable with that of clomipramine hydrochloride (Anafranil), a tricyclic antidepressant drug. In conclusion, our study demonstrates that the antidepressive action of EGCG involves downregulation of serum IL-1ß, upregulation of BDNF mRNA in the hippocampus, and reduction of CA3 neuronal lesions.

Immunomodulatory assessment of Portulaca oleracea L. extract in a mouse model of colitis.

Ulcerative colitis (UC) is a gastrointestinal inflammatory disease with a multifactorial pathophysiology. This study aims to investigate the immunomodulatory effect of Portulaca oleracea leaf ethanolic extract (POE) on acetic acid (AA)-induced UC in mice. Experimental animals received oral doses of POE (200 mg/kg for 7 days) after an induction of colitis by intrarectal AA administration. In mice with AA-induced UC treated with POE, the results revealed a significant modulation in body weight and colon length. Moreover, treatment with POE downregulated the interleukin 1, 6, and 17, tumor necrosis factor-alpha, gamma interferon, and nuclear factor-kappa B levels compared with the colitis group. Furthermore, POE markedly inhibited histological damage, decreased myeloperoxidase activity and reduced fecal calprotectin level compared with the colitis group. These data are consistent with the reduction in total bacterial content in the colon. Taken together, treatment with POE may reduce colonic inflammation by alleviating the immune response and inhibiting the severity of colitis. The HPLC analysis of POE resulted in the identification of seven medicinal compounds comprising two phenolic acids (ferulic and caffeic acids) and five flavonoids (kaempferol, quercetin, rutin, narenginin and hesperidin). Subsequent analysis of POE by GC-MS revealed ten phytocomponents; the major percentages were hexadecenoic acid, methyl ester (29.8119%), α-linolenic acid (25.8431%), 16-octadecenoic acid, methyl ester (15.1578%) and α-tocopherol (10.7848%). Delta-lactams and alkanes were the minor components. Such natural plant-derived substances and their probable synergistic action appear to contribute to a promising therapeutic protocol for colitis.

Thymoquinone ameliorates age-related hearing loss in C57BL/6J mice by modulating Sirt1 activity and Bak1 expression.

Age-related hearing loss (AHL) is the most common sensory disorder of aged population. Currently, one of the most important sources of experimental medicine for AHL is medicinal plants. This study performed the first investigation of the effect of thymoquinone (TQ), a potent antioxidant, on AHL. Here, we used inbred C57BL/6J mice (B6 mice) as a successful experimental model of the early onset of AHL. The behavioral assessment of hearing revealed that the injection of a high dose of TQ (40 mg/kg; TQ40) significantly improved the auditory sensitivity of B6 mice at all tested frequencies (8, 16 and 22 kHz). Histological sections of cochlea from B6 mice injected with a low dose (20 mg/kg; TQ20) and high dose showed relatively less degenerative signs in the modiolus, hair cells and spiral ligaments, the main constituents of the cochlea. In addition, TQ40 completely restored the normal pattern of hair cells in B6 mice, as shown in scanning electron micrographs. Our data indicated that TQ20 and TQ40 reduced levels of Bak1-mediated apoptosis in the cochlea of B6 mice. Interestingly, the level of Sirt1, a positive regulator of autophagy, was significantly increased in B6 mice administered TQ40. In conclusion, TQ relieves the symptoms of AHL by downregulating Bak1 and activating Sirt1 in the cochlea of B6 mice.

Combination of docosahexaenoic acid and Ginko biloba extract improves cognitive function and hippocampal tissue damages in a mouse model of Alzheimer's disease.

Alzheimer's disease (AD) is one of the most common neurodegenerative diseases characterized by a progressive loss of memory and other cognitive functions among elder people. Nowadays, natural antioxidants have been used to recover the quality of life for those with AD. In this study, we investigated, for the first time, the combined effect of docosahexaenoic acid (DHA) and Ginkgo bilobastandardized extract (EGb761) on AD mice. AD was induced in adult male albino mice with AlCl3 (20 mg/kg b.w, i.g.) and D-galactose (D-gal; 120 mg/kg, i.p.) for 90 days. 30 days after induction, mice were treated with DHA (200 mg/kg b.w., i.g.) and EGb761 (200 mg/kg b.w., i.g.) for two months. Our data revealed that the dual treatment of DHA and EGb761 significantly improved cognitive memory and spatial learning abilities in AD-induced mice. The drug treatments preserved the hippocampal CA3 architecture and restored neuronal ultrastructural alterations. Expression of protein phosphatase 2A (PP2A), the most implicated protein phosphatase in AD neurodegeneration, was highly upregulated in the CA3 hippocampus of AD mice treated with DHA and EGb761. Intriguingly, TNF-α expression was significantly reduced in the same group. In conclusion, our findings proved that the combined effect of DHA and EGb761 tended to be potent against the neurodegenerative effect of AlCl3 and D-gal. The applied treatment enhanced neuronal survival and cognitive functions via upregulation of PP2A and restoration of TNF-α expression.

Neuropeptide RNA localization in tissue sections.

In situ hybridization has become a routine technique to provide insights into RNA localization. However, different protocols exist for multiple purposes, and it is, therefore, important to clearly define specific needs to choose the most suitable one(s). For instance, in situ hybridization can target different types of RNA, including mRNA or small noncoding RNA such as micro RNA (miRNA). Detection protocols are developed for light or electron microscopy and can be combined with immunocytochemistry to study RNA coexpression with proteins or peptides. In this chapter, we present some protocols to illustrate the diversity of in situ hybridization methods. We focus on the detection of mRNA or miRNA and show that the protocols are quite similar but use dedicated probe types, namely, oligo- or riboprobes and locked nucleic-acid probes.

Bidirectional integrative regulation of Cav1.2 calcium channel by microRNA miR-103: role in pain.

Chronic pain states are characterized by long-term sensitization of spinal cord neurons that relay nociceptive information to the brain. Among the mechanisms involved, up-regulation of Cav1.2-comprising L-type calcium channel (Cav1.2-LTC) in spinal dorsal horn have a crucial role in chronic neuropathic pain. Here, we address a mechanism of translational regulation of this calcium channel. Translational regulation by microRNAs is a key factor in the expression and function of eukaryotic genomes. Because perfect matching to target sequence is not required for inhibition, theoretically, microRNAs could regulate simultaneously multiple mRNAs. We show here that a single microRNA, miR-103, simultaneously regulates the expression of the three subunits forming Cav1.2-LTC in a novel integrative regulation. This regulation is bidirectional since knocking-down or over-expressing miR-103, respectively, up- or down-regulate the level of Cav1.2-LTC translation. Functionally, we show that miR-103 knockdown in naive rats results in hypersensitivity to pain. Moreover, we demonstrate that miR-103 is down-regulated in neuropathic animals and that miR-103 intrathecal applications successfully relieve pain, identifying miR-103 as a novel possible therapeutic target in neuropathic chronic pain.