Assessing the Safety and Therapeutic Efficacy of Cannabidiol Lipid Nanoparticles in Alleviating Metabolic and Memory Impairments and Hippocampal Histopathological Changes in Diabetic Parkinson's Rats.

Diabetic Parkinson's disease (DP) is a progressive neurodegenerative disease with metabolic syndrome that is increasing worldwide. Emerging research suggests that cannabidiol (CBD) is a neuropharmacological compound that acts against this disease, especially CBD in nano-formulation. The safety of cannabidiol lipid nanoparticles (CBD-LNP) was evaluated by assessing in vitro cytotoxicity in neurons and therapeutic outcomes in a DP animal model, including metabolic parameters and histopathology. CBD-LNPs were fabricated by using a microfluidization technique and showed significantly lower cytotoxicity than the natural form of CBD. The DP rats were induced by streptozotocin followed by a 4-week injection of MPTP with a high-fat diet. Rats were treated orally with a vehicle, CBD, CBD-LNP, or levodopa for 4 weeks daily. As a result, vehicle-treated rats exhibited metabolic abnormalities, decreased striatal dopamine levels, and motor and memory deficits. CBD-LNP demonstrated reduced lipid profiles, enhanced insulin secretion, and restored dopamine levels compared to CBD in the natural form. CBD-LNP also had comparable efficacy to levodopa in ameliorating motor deficits and memory impairment in behavior tests. Interestingly, CBD-LNP presented migration of damaged neuronal cells in the hippocampus more than levodopa. These findings suggest that CBD-LNP holds promise as an intervention addressing both metabolic and neurodegenerative aspects of DP, offering a potential therapeutic strategy.

Stress-induced changes in cognitive function and intestinal barrier integrity can be ameliorated by venlafaxine and synbiotic supplementations.

Stress profoundly impacts various aspects of both physical and psychological well-being. Our previous study demonstrated that venlafaxine (Vlx) and synbiotic (Syn) treatment attenuated learned fear-like behavior and recognition memory impairment in immobilized-stressed rats. In this study, we further investigated the physical, behavior, and cellular mechanisms underlying the effects of Syn and/or Vlx treatment on brain and intestinal functions in stressed rats. Adult male Wistar rats, aged 8 weeks old were subjected to 14 days of immobilization stress showed a decrease in body weight gain and food intake as well as an increase in water consumption, urinary corticosterone levels, and adrenal gland weight. Supplementation of Syn and/or Vlx in stressed rats resulted in mitigation of weight loss, restoration of normal food and fluid intake, and normalization of corticosterone levels. Behavioral analysis showed that treatment with Syn and/or Vlx enhanced depressive-like behaviors and improved spatial learning-memory impairment in stressed rats. Hippocampal dentate gyrus showed stress-induced neuronal cell death, which was attenuated by Syn and/or Vlx treatment. Stress-induced ileum inflammation and increased intestinal permeability were both effectively reduced by the supplementation of Syn. In addition, Syn and Vlx partly contributed to affecting the expression of the glial cell-derived neurotrophic factor in the hippocampus and intestines of stressed rats, suggesting particularly protective effects on both the gut barrier and the brain. This study highlights the intricate interplay between stress physiological responses in the brain and gut. Syn intervention alleviate stress-induced neuronal cell death and modulate depression- and memory impairment-like behaviors, and improve stress-induced gut barrier dysfunction which were similar to those of Vlx. These findings enhance our understanding of stress-related health conditions and suggest the synbiotic intervention may be a promising approach to ameliorate deleterious effects of stress on the gut-brain axis.

Oral calcium and vitamin D supplements differentially alter exploratory, anxiety-like behaviors and memory in male rats.

Oral calcium and calcium plus vitamin D supplements are commonly prescribed to several groups of patients, e.g., osteoporosis, fracture, and calcium deficiency. Adequate and steady extracellular calcium levels are essential for neuronal activity, whereas certain forms of calcium supplement (e.g., CaCO3) probably interfere with memory function. However, it was unclear whether a long-term use of ionized calcium (calcium chloride in drinking water ad libitum), vitamin D supplement (oral gavage) or the combination of both affected anxiety and memory, the latter of which was probably dependent on the hippocampal neurogenesis. Here, we aimed to determine the effects of calcium and/or vitamin D supplement on the anxiety- and memory-related behaviors and the expression of doublecortin (DCX), an indirect proxy indicator of hippocampal neurogenesis. Eight-week-old male Wistar rats were divided into 4 groups, i.e., control, calcium chloride-, 400 UI/kg vitamin D3-, and calcium chloride plus vitamin D-treated groups. After 4 weeks of treatment, anxiety-, exploration- and recognition memory-related behaviors were evaluated by elevated pulse-maze (EPM), open field test (OFT), and novel object recognition (NOR), respectively. The hippocampi were investigated for the expression of DCX protein by Western blot analysis. We found that oral calcium supplement increased exploratory behavior as evaluated by OFT and the recognition index in NOR test without any effect on anxiety behavior in EPM. On the other hand, vitamin D supplement was found to reduce anxiety-like behaviors. Significant upregulation of DCX protein expression was observed in the hippocampus of both calcium- and vitamin D-treated rats, suggesting their positive effects on neurogenesis. In conclusion, oral calcium and vitamin D supplements positively affected exploratory, anxiety-like behaviors and/or memory in male rats. Thus, they potentially benefit on mood and memory in osteoporotic patients beyond bone metabolism.

Atomoxetine and escitalopram migrate the derangement of the temporomandibular joint morphologic and histologic changes in rats exposed to stress-induced depression.

PURPOSE: The purpose of this in vivo study was to determine the effects of stress-induced depression and antidepressants on depressive-like behavior, microstructure, and histomorphology of the temporomandibular joint (TMJ) using rats. METHODS: Experimentally induced depression in rats was created before being treated with two antidepressants; escitalopram (selective-serotonin-reuptake inhibitors) and atomoxetine (norepinephrine-reuptake inhibitors). Micro-computed tomography (Micro-CT) was performed to measure the change in bone volume and bone porosity of the condyle. Further histological evaluation of the condylar cartilage was performed. RESULTS: Micro-CT scanning revealed a decrease in bone volume in the depression group. The bone porosity percentage significantly increased in both the escitalopram and atomoxetine groups compared with the control group and the depression group. Histopathological analysis showed increased thickness of cartilage layers in the depression group. In the atomoxetine group, there was a significant increase in the pre-hypertrophic and hypertrophic layer thickness and cell count, but a significant decrease in proteoglycans. CONCLUSION: The present study findings indicated the change in TMJ characteristics, especially on the superficial part of the condylar head in the depression group. Concerning the applicability of the different antidepressants, depression with the treatment of atomoxetine has the most disadvantages due to bone porosity and cartilaginous condyle changes.

Enhanced wound healing properties of guar gum/curcumin-stabilized silver nanoparticle hydrogels.

Biocompatible materials that act as scaffolds for regenerative medicine are of enormous interest. Hydrogel-nanoparticle composites have great potential in this regard, however evaluations of their wound healing and safety in vivo in animal studies are scarce. Here we demonstrate that a guar gum/curcumin-stabilized silver nanoparticle hydrogel composite is an injectable material with exceptional wound healing and antibacterial properties. We show that the curcumin-bound silver nanoparticles themselves exhibit low cytotoxicity and enhance proliferation, migration, and collagen production in in vitro studies of human dermal fibroblasts. We then show that the hydrogel-nanoparticle composite promotes wound healing in in vivo studies on rats, accelerating wound closure by > 40% and reducing bacterial counts by 60% compared to commercial antibacterial gels. Histopathology indicates that the hydrogel composite enhances transition from the inflammation to proliferation stage of healing, promoting the formation of fibroblasts and new blood vessels, while target gene expression studies confirm that the accelerated tissue remodeling occurs along the normal pathways. As such these hydrogel composites show great promise as wound dressing materials with high antibacterial capacity.

Lethality of MalE-LacZ hybrid protein shares mechanistic attributes with oxidative component of antibiotic lethality.

Downstream metabolic events can contribute to the lethality of drugs or agents that interact with a primary cellular target. In bacteria, the production of reactive oxygen species (ROS) has been associated with the lethal effects of a variety of stresses including bactericidal antibiotics, but the relative contribution of this oxidative component to cell death depends on a variety of factors. Experimental evidence has suggested that unresolvable DNA problems caused by incorporation of oxidized nucleotides into nascent DNA followed by incomplete base excision repair contribute to the ROS-dependent component of antibiotic lethality. Expression of the chimeric periplasmic-cytoplasmic MalE-LacZ72-47 protein is an historically important lethal stress originally identified during seminal genetic experiments that defined the SecY-dependent protein translocation system. Multiple, independent lines of evidence presented here indicate that the predominant mechanism for MalE-LacZ lethality shares attributes with the ROS-dependent component of antibiotic lethality. MalE-LacZ lethality requires molecular oxygen, and its expression induces ROS production. The increased susceptibility of mutants sensitive to oxidative stress to MalE-LacZ lethality indicates that ROS contribute causally to cell death rather than simply being produced by dying cells. Observations that support the proposed mechanism of cell death include MalE-LacZ expression being bacteriostatic rather than bactericidal in cells that overexpress MutT, a nucleotide sanitizer that hydrolyzes 8-oxo-dGTP to the monophosphate, or that lack MutM and MutY, DNA glycosylases that process base pairs involving 8-oxo-dGTP. Our studies suggest stress-induced physiological changes that favor this mode of ROS-dependent death.

Control of zinc homeostasis in Agrobacterium tumefaciens via zur and the zinc uptake genes znuABC and zinT.

The Agrobacterium tumefaciens zinc uptake regulator (Zur) was shown to negatively regulate the zinc uptake genes znuABC, encoding a zinc transport system belonging to the ATP-binding cassette (ABC) transporter family, and zinT, which encodes a periplasmic zinc-binding protein. The expression of znuABC and zinT was inducible when cells were grown in medium containing a metal chelator (EDTA), and this induction was shown to be specific for zinc depletion. The expression of znuABC was reduced in response to increased zinc in a dose-dependent manner, and zinT had a less pronounced but similar pattern of zinc-regulated expression. The inactivation of zur led to constitutively high expression of znuABC and zinT. In addition, a zur mutant had an increased total zinc content compared to the WT NTL4 strain, whereas the inactivation of zinT caused a reduction in the total zinc content. The zinT gene is shown to play a dominant role and to be more important than znuA and znuB for A. tumefaciens survival under zinc deprivation. ZinT can function even when ZnuABC is inactivated. However, mutations in zur, znuA, znuB or zinT did not affect the virulence of A. tumefaciens.

Roles of Agrobacterium tumefaciens membrane-bound ferritin (MbfA) in iron transport and resistance to iron under acidic conditions.

Agrobacterium tumefaciens membrane-bound ferritin (MbfA) is a member of the erythrin (Er)-vacuolar iron transport family. The MbfA protein has an Er or ferritin-like domain at its N terminus and has been predicted to have five transmembrane segments in its C-terminal region. Analysis of protein localization using PhoA and LacZ reporter proteins supported the view that the N-terminal di-iron site is located in the cytoplasm whilst the C-terminal end faces the periplasm. An A. tumefaciens mbfA mutant strain had 1.5-fold higher total iron content than the WT strain. Furthermore, multi-copy expression of mbfA reduced total iron content two- and threefold in WT and mbfA mutant backgrounds, respectively. These results suggest that MbfA may function as an iron exporter rather than an iron storage protein. The mbfA mutant showed 10-fold increased sensitivity to the iron-activated antibiotic streptonigrin, implying that the mutant had increased accumulation of intracellular free iron. Growth of the mbfA mutant was reduced in the presence of high iron under acidic conditions. The expression of mbfA was induced highly in cells grown in iron-replete medium at pH 5.5, further supporting the view that mbfA is involved in the response to iron under acidic conditions. A. tumefaciens MbfA may play a protective role against increased free iron in the cytoplasm through iron binding and export, thus preventing iron-induced toxicity via the Fenton reaction.

Cysteine desulphurase-encoding gene sufS2 is required for the repressor function of RirA and oxidative resistance in Agrobacterium tumefaciens.

The Agrobacterium tumefaciens genome contains a cluster of genes that are predicted to encode Fe-S cluster assembly proteins, and this cluster is known as the sufS2BCDS1XA operon. sufS2 is the first gene in the operon, and it was inactivated to determine its physiological function. The sufS2 mutant exhibited a small colony phenotype, grew slower than the wild-type strain and was more sensitive to various oxidants including peroxide, organic hydroperoxide and superoxide. The sufS2 gene was negatively regulated by iron response regulator (Irr) and rhizobial iron regulator (RirA) under low and high iron conditions, respectively, and was inducible in response to oxidative stress. The oxidant-induced expression of sufS2 was controlled by Irr, RirA and an additional but not yet identified mechanism. sufS2 was required for RirA activity in the repression of a sufS2 promoter-lacZ fusion. RirA may use Fe-S as its cofactor. sufS2 disruption may cause a defect in the Fe-S supply and could thereby affect the RirA activity. The three conserved cysteine residues (C91, C99 and C105) in RirA were predicted to coordinate with the Fe-S cluster and were shown to be essential for RirA repression of the sufS2-lacZ fusion. These results suggested that sufS2 is important for the survival of A. tumefaciens.

Identification of amino acid residues important for the function of Agrobacterium tumefaciens Irr protein.

The key amino acid residues that influence the function of the Agrobacterium tumefaciens iron response regulator protein (Irr(At) ) were investigated. Several Irr(At) mutant proteins containing substitutions in amino acids corresponding to candidate metal- and haem-binding sites were constructed. The ability of the mutant proteins to repress the promoter of the membrane bound ferritin (mbfA) gene was investigated using a promoter-lacZ fusion assay. A single mutation at residue H94 significantly decreased the repressive activity of Irr(At) . Multiple mutation analysis revealed the importance of H45, H65, the HHH motif (H92, H93 and H94) and H127 for the repressor function of Irr(At) . H94 is essential for the iron responsiveness of Irr(At) . Furthermore, the Irr(At) mutant proteins showed differential abilities to complement the H(2) O(2) -hyper-resistant phenotype of an irr mutant.

Agrobacterium tumefaciens membrane-bound ferritin plays a role in protection against hydrogen peroxide toxicity and is negatively regulated by the iron response regulator.

An Agrobacterium tumefaciens membrane-bound ferritin (mbfA) mutant was generated to assess the physiological functions of mbfA in response to iron and hydrogen peroxide (H(2) O(2) ) stresses. Wild-type and the mbfA mutant strains showed similar growth under high- and low-iron conditions. The mbfA mutant was more sensitive to H(2) O(2) than wild-type strain. Expression of a functional mbfA gene could complement the H(2) O(2) -hypersensitive phenotype of the mbfA mutant and a rhizobial iron regulator (rirA) mutant, suggesting that MbfA protects cells from H(2) O(2) toxicity by sequestering intracellular free iron, thus preventing the Fenton reaction. The expression of mbfA could be induced in response to iron and to H(2) O(2) treatment. The iron response regulator (irr) also acted as a repressor of mbfA expression. An irr mutant had high constitutive expression of mbfA, which partly contributed to the H(2) O(2) -hyperresistant phenotype of the irr mutant. The data reported here demonstrate an important role of A. tumefaciens MbfA in the cellular defence against iron and H(2) O(2) stresses.