In vitro profiling and molecular dynamics simulation studies of berberine loaded MCM-41 mesoporous silica nanoparticles to prevent neuronal apoptosis.

Neuronal loss in Alzheimer's disease has been reported to display features of apoptosis, pyroptosis (programmed necrosis), or necroptosis. This study thoroughly examines the production and characterization of MCM-41 based berberine (BBR)-loaded porous silica nanoparticles (MSNs) by a modified Stöber method, focusing on their possible role in inhibiting the apoptotic process. Particle size, polydispersity index, morphology, drug loading, zeta potential, entrapment efficiency, and drug release were examined. The formulation was analyzed using various spectroscopic techniques. The surface area was computed by the Brunauer-Emmett-Teller plot. Computational models were developed for molecular dynamics simulation studies. A small PDI value indicated an even distribution of particles at nanoscale sizes (80-100 nm). Results from XRD and SEAD experiments confirmed the amorphous nature of BBR in nanoparticles. Nanoparticles had high entrapment (75.21 ± 1.55%) and drug loading (28.16 ± 2.5%) efficiencies. A negative zeta potential value (-36.861.1 mV) indicates the presence of silanol groups on the surface of silica. AFM findings reveal bumps due to the surface drug that contributed to the improved roughness of the MSNs-BBR surface. Thermal gravimetric analysis confirmed the presence of BBR in MSNs. Drug release was controlled by simple diffusion or quasi-diffusion. Molecular dynamics simulations confirmed the existence of diffused drug molecules. Cellular studies using SH-SY-5Y cells revealed dose-dependent growth inhibition. Fragmented cell nuclei and nuclear apoptotic bodies in DAPI-stained cells exposed to nanoparticles showed an increase in apoptotic cells. Flow cytometry analysis demonstrated a lower red-to-green ratio in SH-SY-5Y cells treated with nanoparticles. This suggests improved mitochondrial health, cellular viability restoration, and prevention of the apoptotic process. This study provides essential data on the synthesis and potential of MSNs loaded with BBR, which may serve as a viable therapeutic intervention for conditions associated with apoptosis.

Molecular and functional mapping of Plekhm1-Rab7 interaction in osteoclasts.

Mutations in PLEKHM1 cause osteopetrosis in humans and rats. The germline and osteoclast conditional deletions of Plekhm1 gene in mice lead to defective osteoclast bone resorption and increased trabecular bone mass without overt abnormalities in other organs. As an adaptor protein, pleckstrin homology and RUN domain containing M1 (PLEKHM1) interacts with the key lysosome regulator small GTPase RAB7 via its C-terminal RUBICON homologous (RH) domain. In this study, we have conducted a structural-functional study of the PLEKHM1 RH domain and RAB7 interaction in osteoclasts in vitro. The single mutations of the key residues in the Plekhm1 RH predicted from the crystal structure of the RUBICON RH domain and RAB7 interface failed to disrupt the Plekhm1-Rab7 binding, lysosome trafficking, and bone resorption. The compound alanine mutations at Y949-R954 and L1011-I1018 regions decreased Plekhm1 protein stability and Rab7-binding, respectively, thereby attenuated lysosome trafficking and bone resorption in osteoclasts. In contrast, the compound alanine mutations at R1060-Q1068 region were dispensable for Rab7-binding and Plekhm1 function in osteoclasts. These results indicate that the regions spanning Y949-R954 and L1011-I1018 of Plekhm1 RH domain are functionally important for Plekhm1 in osteoclasts and offer the therapeutic targets for blocking bone resorption in treatment of osteoporosis and other metabolic bone diseases.

Amyloidogenic Propensity of Metabolites in the Uric Acid Pathway and Urea Cycle Critically Impacts the Etiology of Metabolic Disorders.

Novel insights into the etiology of metabolic disorders have recently been uncovered through the study of metabolite amyloids. In particular, inborn errors of metabolism (IEMs), including gout, Lesch-Nyhan syndrome (LNS), xanthinuria, citrullinemia, and hyperornithinemia-hyperammonemia-homocitrullinuria (HHH) syndrome, are attributed to the dysfunction of the urea cycle and uric acid pathway. In this study, we endeavored to understand and mechanistically characterize the aggregative property exhibited by the principal metabolites of the urea cycle and uric acid pathway, specifically hypoxanthine, xanthine, citrulline, and ornithine. Employing scanning electron microscopy (SEM), transmission electron microscopy (TEM), and atomic force microscopy (AFM), we studied the aggregation profiles of the metabolites. Insights obtained through molecular dynamics (MD) simulation underscore the vital roles of π-π stacking and hydrogen bonding interactions in the self-assembly process, and thioflavin T (ThT) assays further corroborate the amyloid nature of these metabolites. The in vitro MTT assay revealed the cytotoxic trait of these assemblies, a finding that was substantiated by in vivo assays employing the Caenorhabditis elegans (C. elegans) model, which revealed that the toxic effects were more pronounced and dose-specific in the case of metabolites that had aged via longer preincubation. We hence report a compelling phenomenon wherein these metabolites not only aggregate but transform into a soft, ordered assembly over time, eventually crystallizing upon extended incubation, leading to pathological implications. Our study suggests that the amyloidogenic nature of the involved metabolites could be a common etiological link in IEMs, potentially providing a unified perspective to study their pathophysiology, thus offering exciting insights into the development of targeted interventions for these metabolic disorders.

Appraisal of the Antioxidant Activity, Polyphenolic Content, and Characterization of Selected Himalayan Herbs: Anti-Proliferative Potential in HepG2 Cells.

Natural antioxidants derived from plants have played a vital role in preventing a wide range of human chronic conditions and provide novel bioactive leads for investigators in pharmacotherapy discovery. This work was designed to examine the ethnopharmacological role of Urtica dioica (UD), Capsella bursa-pastoris (CBP), and Inula racemosa (IR). The total phenolic and flavonoid contents (TPC and TFC) were illustrated through colorimetric assays, while the antioxidant activity was investigated through DPPH and ABTS assays. The evaluation of phytochemicals by FT-IR of UD and CBP revealed high contents of aliphatic amines, while IR showed a major peak for ketones. The antioxidant activity, TPC and TFC were highest in the ethanol extract of UD, followed by CBP, and IR showed the lowest activity. All of the extracts revealed significant antioxidant capacities along a dosage gradient. Through a HPLC analysis at a wavelength of 280 nm, UD leaves demonstrated an intense peak of quercetin, and the peak for rutin was less intense. CBP (whole plant), instead, demonstrated a major yield of rutin, and a peak for quercetin was not observed in CBP. IR (rhizomes) showed both quercetin and rutin. All of the extracts were significantly cytotoxic to HepG2 cells after 48 h with the trend IR > UD > CBP. The outcomes of this study may be effective in the selection of specific plants as realistic sources of the bioactive components that might be useful in the nutraceutical progression and other biomedical efficacies.

Flavonoids as Promising Neuroprotectants and Their Therapeutic Potential against Alzheimer's Disease.

Alzheimer's disease (AD) is one of the serious and progressive neurodegenerative disorders in the elderly worldwide. Various genetic, environmental, and lifestyle factors are associated with its pathogenesis that affect neuronal cells to degenerate over the period of time. AD is characterized by cognitive dysfunctions, behavioural disability, and psychological impairments due to the accumulation of amyloid beta (Aß) peptides and neurofibrillary tangles (NFT). Several research reports have shown that flavonoids are the polyphenolic compounds that significantly improve cognitive functions and inhibit or delay the amyloid beta aggregation or NFT formation in AD. Current research has uncovered that dietary use of flavonoid-rich food sources essentially increases intellectual abilities and postpones or hinders the senescence cycle and related neurodegenerative problems including AD. During AD pathogenesis, multiple signalling pathways are involved and to target a single pathway may relieve the symptoms but not provides the permanent cure. Flavonoids communicate with different signalling pathways and adjust their activities, accordingly prompting valuable neuroprotective impacts. Flavonoids likewise hamper the movement of obsessive indications of neurodegenerative disorders by hindering neuronal apoptosis incited by neurotoxic substances. In this short review, we briefly discussed about the classification of flavonoids and their neuroprotective properties that could be used as a potential source for the treatment of AD. In this review, we also highlight the structural features of flavonoids, their beneficial roles in human health, and significance in plants as well as their microbial production.

Circadian desynchronization in pregnancy of Golden hamster following long time light exposure: Involvement of Akt/FoxO1 pathway.

Coordination between central and peripheral reproductive clocks in females is poorly understood. Long light is having a hazardous effect on reproductive health. Hence, explored the effect of long-time light exposure (LLD; 16L:8D) on the central and peripheral reproductive (ovary and uterus) clock genes (Bmal1, Clock, Per1, Per2, Cry1 and Cry2) and its downstream regulators (Aanat, Egf, Cx26, Cx43, ERα, pAktS-473, pAktT-308, pFoxO1T-24, 14-3-3, HoxA10, HoxA11 and Pibf) expression in non-pregnant and pregnant Golden hamster. Young adult Golden hamsters were exposed to LLD for 30 days and then were mated. We observed that LLD exposure increased the thickness of the endometrium and reduced myometrium thickness, resembling uterine adenomyosis. In non-pregnant females LLD altered the expressions of clock genes in suprachiasmatic nuclei (SCN), ovary and the uterus along with serum estradiol rhythm. LLD upregulated Egf and downregulated Aanat, Cx26, and Cx43 mRNA levels in uterus. LLD upregulated Akt/FoxO1 phosphorylation and 14-3-3 expressions in the uterus of nonpregnant females. LLD exposure to pregnant females lowered serum progesterone, Aanat, Pibf, Hoxa10, and Hoxa11 mRNA expressions on D4 (peri-implantation) and D8 (post-implantation) resulting in a low implantation rate on D8 (post-implantation). Hence it is evident that the frequent pregnancy anomalies noted under a long light schedule might be due to desynchronization in Aanat, Pibf, Hoxa10, and Hoxa11 as well as the central and peripheral clock genes (Bmal1, Clock, Per1, Per2, Cry1 and Cry2). LLD exposure desynchronized the central and peripheral reproductive clock affecting uterine physiology via Akt/FoxO1 pathway in Golden hamsters. Thus, LLD is a risk factor for female reproductive health and fertility.

Melatonin induces apoptosis and cell cycle arrest in cervical cancer cells via inhibition of NF-κB pathway.

Cervical cancer is the most prevalent cancer in females. Melatonin, a neurohormone has been documented as a promising therapeutic molecule for cervical cancer. However, the underlying molecular mechanism is not known. We explored the dose-dependent anti-tumor response of melatonin against cervical cancer cell lines, HeLa (HPV-18 positive) and SiHa (HPV-16 positive). The anti-cancer effect of melatonin was evaluated by MTT assay, cell imaging, colony formation, DAPI, AO/PI, LDH, Flow cytometry, scratch assay, western blot analysis and real-time PCR. Results of DAPI, AO/PI, LDH, and Annexin/PI staining revealed that melatonin induces apoptosis. The results of cell cycle analysis revealed that melatonin arrests the HeLa and SiHa cells in sub-G1 and G1 phases, respectively. Western blot analysis revealed that melatonin downregulated the expression of pro-inflammatory transcription factor, NF-κB and the expression of COX-2 protein, a key mediator in cell proliferation. In addition, melatonin downregulated the expression of an invasive marker, MMP-9, an antiapoptotic protein, Bcl-2, and upregulated the expression of pro-apoptotic protein, Bax at both transcriptional and translational levels. Overall, the results suggest that melatonin exhibited strong anti-cancer therapeutic potential against human cervical cancer cell line progression possibly through inhibition of NF-κB signalling pathway.

Continuous artificial light potentially disrupts central and peripheral reproductive clocks leading to altered uterine physiology and reduced pregnancy success in albino mice.

AIMS: The mechanism behind clock coordination in female reproductive disorders is poorly understood despite the known importance of coordinated and synchronized timing of central and clocks in reproductive organs. We investigated the effect of continuous artificial light (LL) on the central and peripheral reproductive clock gene (Bmal1, Clock, Per1, Per2 and Cry1) and its downstream regulators (Hgf, PR-A and HOXA10) during non-pregnancy and pregnancy phases of female mice. MAIN METHODS: Mice (n = 60) in two sets, were maintained under continuous light (LL) and natural day cycle (LD;12L: 12D) for both non-pregnant and pregnant study. Tissues from hypothalamus-containing SCN, ovary, uterus and serum were collected at different zeitgeber time points (ZT; at 4-h intervals across 24-h periods). KEY FINDINGS: LL exposure desynchronized the expressions of the clock mRNAs (Bmal1, Clock, Per1, Per2 and Cry1) in SCN, ovary, and uterus along with Hgf mRNA rhythm. LL significantly increased the thickness of endometrial tissues. Furthermore, the pregnant study revealed lower serum progesterone level during peri- and post-implantation under LL along with downregulated expression of progesterone receptor (PR) as well as progesterone dependent uterine Homeobox A-10 (Hoxa10) proteins with lowered pregnancy outcomes. SIGNIFICANCE: Our result suggests that LL disrupted the circadian coordination between central and clock genes in reproductive tissue leading to interrupted uterine physiology and altered pregnancy in mice. This led us to propose that duration of light exposure at work-places or home for females is very important in prevention of pregnancy anomalies.

Role of Oxidative Stress and Metal Toxicity in the Progression of Alzheimer's Disease.

Alzheimer's disease (AD) is one of the life-threatening neurodegenerative disorders in the elderly (>60 years) and incurable across the globe to date. AD is caused by the involvement of various genetic, environmental and lifestyle factors that affect neuronal cells to degenerate over the period of time. The oxidative stress is engaged in the pathogenesis of various disorders and its key role is also linked to the etiology of AD. AD is attributed by neuronal loss, abnormal accumulation of Amyloid-ß (Aß) and neurofibrillary tangles (NFTs) with severe memory impairments and other cognitive dysfunctions which lead to the loss of synapses and neuronal death and eventual demise of the individual. Increased production of reactive oxygen species (ROS), loss of mitochondrial function, altered metal homeostasis, aberrant accumulation of senile plaque and mitigated antioxidant defense mechanism all are indulged in the progression of AD. In spite of recent advances in biomedical research, the underlying mechanism of disruption of redox balance and the actual source of oxidative stress is still obscure. This review highlights the generation of ROS through different mechanisms, the role of some important metals in the progression of AD and free radical scavenging by endogenous molecule and supplementation of nutrients in AD.