Novel Therapeutic Approach in PEGylated Chitosan Nanoparticles of Apigenin for the Treatment of Cancer via Oral Nanomedicine.

The goal of this study was to optimize and formulate apigenin (APG)-loaded pegylated chitosan nanoparticles (PEGylated-CNPs) via ionic gelation techniques using the Box-Behnken design (BBD). Three individual variables, X1(chitosan: TPP concentration), X2 (PEG-400 concentration), and X3 (sonication time), were investigated for their influence on response variables (Y1-particle size (PS); Y2-drug entrapment efficiency (DEE); and Y3-zeta potential (ZP). The optimized formula of APG-PEGylated CNPs was picked from the statistical design and was then examined for physical, morphological, release characterization, anti-oxidant, and anti-tumor potential. The average PS, PDI, %DEE, and ZP were found to be 139.63 ± 5.67 nm, 0.296 ± 0.014, 79.55 ± 3.12%, and 24.68 ± 1.84 mV, respectively. The optimized APG formulation was chosen and reformulated based on the desirability function. Results of the observed and predicted values of responses through the BBD process were found to be nearly identical. The resulting APG-PEGylated CNPs were spherical and smooth, according to surface morphology studies. The release study revealed that PEGylated-CNPs exhibited biphasic release patterns distinguished by an initial burst release of APG only at early phases accompanied by a delayed release near 24 h. Furthermore, APG-PEGylated CNPs demonstrated statistically increased antioxidant activities and cytotoxicity against MCF-7 cells compared to pure APG. Based on the findings, it is possible to conclude that BBD was efficient in optimizing the PEGylated CNPs formulation and recognizing the impacts of formulation variables. In conclusion, the developed formulation has a significant potential for anticancer therapy.

MyD88-mediated signaling intercedes in neurogenic muscle atrophy through multiple mechanisms.

Skeletal muscle atrophy is a debilitating complication of many chronic disease states and disuse conditions including denervation. However, molecular and signaling mechanisms of muscle wasting remain less understood. Here, we demonstrate that the levels of several toll-like receptors (TLRs) and their downstream signaling adaptor, myeloid differentiation primary response 88 (MyD88), are induced in skeletal muscle of mice in response to sciatic nerve denervation. Muscle-specific ablation of MyD88 mitigates denervation-induced skeletal muscle atrophy in mice. Targeted ablation of MyD88 suppresses the components of ubiquitin-proteasome system, autophagy, and FOXO transcription factors in skeletal muscle during denervation. We also found that specific inhibition of MyD88 reduces the activation of canonical nuclear factor-kappa (NF-κB) pathway and expression of receptors for inflammatory cytokines in denervated muscle. In contrast, inhibition of MyD88 stimulates the activation of non-canonical NF-κB signaling in denervated skeletal muscle. Ablation of MyD88 also inhibits the denervation-induced increase in phosphorylation of AMPK without having any effect on the phosphorylation of mTOR. Moreover, targeted ablation of MyD88 inhibits the activation of a few components of the unfolded protein response (UPR) pathways, especially X-box protein 1 (XBP1). Importantly, myofiber-specific ablation of XBP1 mitigates denervation-induced skeletal muscle atrophy in mice. Collectively, our experiments suggest that TLR-MyD88 signaling mediates skeletal muscle wasting during denervation potentially through the activation of canonical NF-κB signaling, AMPK and UPR pathways.

Circulating Placental Alkaline Phosphatase Expressing Exosomes in Maternal Blood Showed Temporal Regulation of Placental Genes.

Background: Analysis of placental genes could unravel maternal-fetal complications. However, inaccessibility to placental tissue during early pregnancy has limited this effort. We tested if exosomes (Exo) released by human placenta in the maternal circulation harbor crucial placental genes. Methods: Placental alkaline phosphate positive exosomes (ExoPLAP) were enriched from maternal blood collected at the following gestational weeks; 6-8th (T1), 12-14th (T2), 20-24th (T3), and 28th-32nd (T4). Nanotracking analysis, electron microscopy, dynamic light scattering, and immunoblotting were used for characterization. We used microarray for transcriptome and quantitative PCR (qPCR) for gene analysis in ExoPLAP. Results: Physical characterization and presence of CD63 and CD9 proteins confirmed the successful ExoPLAP enrichment. Four of the selected 36 placental genes did not amplify in ExoPLAP, while 32 showed regulations (n = 3-8/time point). Most genes in ExoPLAP showed significantly lower expression at T2-T4, relative to T1 (p < 0.05), such as NOS3, TNFSF10, OR5H6, APOL3, and NEDD4L. In contrast, genes, such as ATF6, NEDD1, and IGF2, had significantly higher expression at T2-T4 relative to T1. Unbiased gene profiling by microarray also confirmed expression of above genes in ExoPLAP-transcriptome. In addition, repeated measure ANOVA showed a significant change in the ExoPLAP transcriptome from T2 to T4 (n = 5/time point). Conclusion: Placental alkaline phosphate positive exosomes transcriptome changed with gestational age advancement in healthy women. The transcriptome expressed crucial placental genes involved in early embryonic development, such as actin cytoskeleton organization, appropriate cell positioning, DNA replication, and B-cell regulation for protecting mammalian fetuses from rejection. Thus, ExoPLAP in maternal blood could be a promising source to study the placental genes regulation for non-invasive monitoring of placental health.

Expression of Toll-like Receptor 2 and 4 in Peripheral Blood Neutrophil Cells from Patients with Chronic Obstructive Pulmonary Disease.

OBJECTIVES: Chronic obstructive pulmonary disease (COPD) is one of the leading causes of morbidity and mortality around the world. Preliminary studies have evaluated the association between innate immunity including Toll-like receptors (TLRs) and airway samples of patients with COPD. The role of TLRs in peripheral blood neutrophils is poorly understood. Hence, this study aimed to investigate the role of TLR2 and TLR4 in peripheral blood neutrophils of COPD patients. METHODS: A total of 101 COPD cases and an equal number of healthy controls participated in this case-control study. Peripheral blood neutrophils were isolated from all participants and cultured for 24 hours through lipopolysaccharide (LPS) stimulation. The gene expressions of TLR2 and TLR4 were assessed by real-time polymerase chain reaction. The protein levels of interleukin (IL)-8 and matrix metalloproteinase (MMP)-9 were measured in neutrophils cell culture supernatants using enzyme-linked immunosorbent assay (ELISA). RESULTS: The levels of IL-8 and MMP-9 were significantly higher in patients with COPD compared to healthy controls. Similarly, the gene expression of TLR2 and TLR4 were increased in LPS stimulated peripheral blood neutrophils of patients with COPD. Smoke pack years was positively correlated with IL-8 levels and negatively correlated with forced expiratory volume in the first second % (r = -0.33; p = 0.023) and FEV1/forced vital capacity (FVC) (r = -0.27; p = 0.011). CONCLUSIONS: The increased expression of TLR2 and TLR4 suggests its role in disease pathogenesis of COPD. Smoke pack years was negatively associated with spirometric parameters in COPD patients. This may help to predict the smokers without COPD who risk developing the condition in the future.

Intranasal exposure to silica nanoparticles induces alterations in pro-inflammatory environment of rat brain.

Silica nanoparticles (SiNPs) are being used increasingly in biomedical and industrial fields; however, their adverse effects on human health have not been fully investigated. In this study, we focused on some of the toxicological aspects of SiNPs by studying oxidative stress and pro-inflammatory responses in the frontal cortex, corpus striatum and hippocampus regions of rat brain. Wistar rats were exposed to SiNPs of size 80 nm and 10 nm at a dose of 150 µg/50 µL phosphate-buffered saline/rat for 30 days. The results indicated a significant increase of lipid peroxide levels and hydrogen peroxide content in various regions of the treated rat brain. Moreover, these changes were accompanied with a significant decrease in the activities of manganese superoxide dismutase, glutathione reductase, catalase and reduced glutathione in different brain regions, suggesting impaired antioxidant defence system. Furthermore, SiNPs exposure not only increased messenger RNA (mRNA) and protein expression of nuclear factor-κB (NF-κB) but also significantly increased the mRNA and protein levels of tumour necrosis factor α (TNF-α), interleukin 1ß (IL-1ß) and monocyte chemoattractant protein 1 (MCP-1) in different regions of rat brain. Cumulatively, these data suggest that SiNPs induced the activation of NF-κB and increased the expression of TNF-α, IL-1ß and MCP-1 in rat brain, possibly via redox-sensitive cellular signalling pathways.

Role of Endoplasmic Reticulum Stress and Unfolded Protein Responses in Health and Diseases.

Endoplasmic reticulum (ER) is the site of protein synthesis, protein folding, maintainance of calcium homeostasis, synthesis of lipids and sterols. Genetic or environmental insults can alter its function generating ER stress. ER senses stress mainly by three stress sensor pathways, namely protein kinase R-like endoplasmic reticulum kinase-eukaryotic translation-initiation factor 2α, inositol-requiring enzyme 1α-X-box-binding protein 1 and activating transcription factor 6-CREBH, which induce unfolded protein responses (UPR) after the recognition of stress. Recent studies have demonstrated that ER stress and UPR signaling are involved in cancer, metabolic disorders, inflammatory diseases, osteoporosis and neurodegenerative diseases. However, the precise knowledge regarding involvement of ER stress in different disease processes is still debatable. Here we discuss the possible role of ER stress in various disorders on the basis of existing literature. An attempt has also been made to highlight the present knowledge of this field which may help to elucidate and conjure basic mechanisms and novel insights into disease processes which could assist in devising better future diagnostic and therapeutic strategies.

Aluminum Activates PERK-EIF2α Signaling and Inflammatory Proteins in Human Neuroblastoma SH-SY5Y Cells.

Aluminum is the third most abundant element present in the earth's crust and human exposure to it is possible due to industrialization, utensils, medicines, antiperspirants, etc. Evidences suggest involvement of aluminum in a variety of neurodegenerative disorders including Alzheimer's disease. Endoplasmic reticulum (ER) stress has been implicated in various neurological disorders. ER stress may be a result of impaired calcium homeostasis due to perturbed redox balance and is known to elicit inflammation through the activation of unfolded protein response (UPR). In the present study, we aimed to investigate the role of aluminum in ER stress-mediated activation of inflammatory responses in neuroblastoma cells. Lactate dehydrogenase (LDH) release assay revealed that aluminum compromised the membrane integrity of neuroblastoma cells, probably due to membrane damage, as indicated by enhanced levels of lipid peroxidation (LPO). Besides this, our results clearly demonstrated elevated reactive oxygen species (ROS) levels and a weakened antioxidant defence system manifested by decrease in catalase (CAT) activity and cellular glutathione (GSH). Moreover, we studied the expression of key apoptosis-related proteins, ER stress-mediated activation of UPR, and its downstream inflammatory pathway. It was observed that aluminum potentially enhanced protein levels of PERK, EIF2α, caspase 9, caspase 3, and inflammatory markers like NF-κB, NLRP3, HMGB1, and nitric oxide (NO). Furthermore, aluminum altered TNFα, IL1ß, IL6, and IL10 mRNA levels as well. The overall findings indicated that aluminum mediates UPR activation through ER stress, which results in induction of inflammatory pathway and apoptotic proteins in neuronal cells.

Aluminium induced endoplasmic reticulum stress mediated cell death in SH-SY5Y neuroblastoma cell line is independent of p53.

Aluminium (Al) is the third most abundant element in the earth's crust and its compounds are used in the form of house hold utensils, medicines and in antiperspirant etc. Increasing number of evidences suggest the involvement of Al+3 ions in a variety of neurodegenerative disorders including Alzheimer's disease. Here, we have attempted to investigate the role of Al in endoplasmic reticulum stress and the regulation of p53 during neuronal apoptosis using neuroblastoma cell line. We observed that Al caused oxidative stress by increasing ROS production and intracellular calcium levels together with depletion of intracellular GSH levels. We also studied modulation of key pro- and anti-apoptotic proteins and found significant alterations in the levels of Nrf2, NQO1, pAKT, p21, Bax, Bcl2, Aß1-40 and Cyt c together with increase in endoplasmic reticulum (ER) stress related proteins like CHOP and caspase 12. However, with respect to the role of p53, we observed downregulation of its transcript as well as protein levels while analysis of its ubiquitination status revealed no significant changes. Not only did Al increase the activities of caspase 9, caspase 12 and caspase 3, but, by the use of peptide inhibitors of specific and pan-caspases, we observed significant protection against neuronal cell death upon inhibition of caspase 12, demonstrating the prominent role of endoplasmic reticulum stress generated responses in Al toxicity. Overall our findings suggest that Al induces ER stress and ROS generation which compromises the antioxidant defenses of neuronal cells thereby promoting neuronal apoptosis in p53 independent pathway.