Defective mitophagy and induction of apoptosis by the depleted levels of PINK1 and Parkin in Pb and ß-amyloid peptide induced toxicity.

Exposure to lead (Pb), an environmental pollutant, is closely associated with the development of neurodegenerative disorders through oxidative stress induction and alterations in mitochondrial function. Damaged mitochondria could be one of the reasons for the progression of Alzheimer's Disease (AD). Mitophagy is vital in keeping the cell healthy. To know its role in Pb-induced AD, we investigated the PINK1/Parkin dependent pathway by studying specific mitophagy marker proteins such as PINK1 and Parkin in differentiated SH-SY5Y cells. Our data have indicated a significant reduction in the levels of PINK1 and Parkin in cells exposed to Pb and ß-amyloid peptides, both Aß (25-35) and Aß (1-40) individually and in different combinations, resulting in defective mitophagy. Also, the study unravels the status of mitochondrial permeability transition pore (MPTP), mitochondrial mass, mitochondrial membrane potential (MMP) and mitochondrial ROS production in cells treated with individual and different combination of Pb and Aß peptides. An increase in mitochondrial ROS production, enhanced MPTP opening, depolarization of membrane potential and reduced mitochondrial mass in the exposed groups were observed. Also, in the present study, we found that Pb and ß-amyloid peptides could trigger apoptosis by activating the Bak protein, which releases the cytochrome c from mitochondria through MPTP that further activates the AIF (apoptosis inducing factor) and caspase-3 proteins in the cytosol. The above findings reveal the potential role of mechanisms like PINK1/Parkin mediated mitophagy and dysfunctional mitochondria mediated apoptosis in Pb induced neurotoxicity.

Mitochondria-Mediated Moderation of Apoptosis by EGCG in Cytotoxic Neuronal Cells Induced by Lead (Pb) and Amyloid Peptides.

The developmental, epigenetic, and epidemiological studies on lead (Pb) toxicity have reported a strong connection between lead exposure and the progression of Alzheimer's disease (AD). The amyloid peptides were the main triggering elements, in the generation of extracellular plaques through which multiple cellular signaling events such as apoptosis and primarily oxidant-antioxidant balancing system will be affected, which leads to neuronal cell death. Our previous studies indicated that epigallocatechin gallate (EGCG), abundantly present in green tea, was found to be effective in alleviating the metal-induced neurotoxicity at the cellular level in terms of cell viability and apoptosis The aim of this study was to explore the protective mechanism of EGCG on the markers of oxidant-antioxidant system and mitochondria, which are involved in metal-induced neuronal cell death. Initially, the IC50 values for lead(Pb-5 µM), amyloid peptides (AP(1-40)-60 µM; AP(1-40)-8 µM), and EC50 value for EGCG(50 µM) were determined by both time- (12 h, 24 h, 48 h) and concentration-dependent manner and analyzed by MTT assay. The experimental groups were designed initially by treating with Pb and APs individually and in different combinations along with the presence of EGCG and are compared to the Pb and AP treated group without EGCG exposure. The cell lysates were used for analyzing oxidative stress markers by standardized laboratory protocol and the expression of mitochondrial markers such as VDAC and cytochrome C which were analyzed by both western blot and real-time PCR. Our results indicate that the EGCG-treated group has shown a significant increase in antioxidant marker expression levels (GSH, SOD, catalase, vitamin C) and a decrease in oxidative stress marker (NOS, MDA) levels when compared to the group without EGCG treatment (p < 0.05). Similarly, a significant decrease in expression levels of VDAC and cytochrome c were observed in the EGCG-treated group when compared to the group without EGCG treatment (p < 0.05). Our approach revealed that EGCG protects SH-SY5Y cells from Pb- and AP-induced cytotoxicity by regulating voltage-dependent anion channel (VDAC) expression and oxidant-antioxidant system and inhibits neuronal cell death.

Inactivation of GAP-43 due to the depletion of cellular calcium by the Pb and amyloid peptide induced toxicity: An in vitro approach.

Environmental pollutant, Lead (Pb) is known to induce neurotoxicity in human. The central nervous system is the most vulnerable to the minute levels of Pb induced toxicity. Pb has been linked to Alzheimer's disease (AD) as a probable risk factor, as it shows epigenetic and developmental link associated with Alzheimer's disease-like pathology. Beta amyloid peptides were considered as the crucial factors in the beta amyloid plaque formation in Alzheimer's disease brain. In this context, we investigated the molecular mechanism involved in the development of Pb induced Alzheimer's disease in in vitro. Previous data from our studies have reported that Pb in the presence of beta Amyloid peptide (1-40) and (25-35) induces more apoptosis than individual exposures. Here, to further evaluate the molecular mechanism underlying Pb induced Alzheimer's disease; we focussed on the involvement of calcium signalling in inducing cell death. Our experimental observations suggesting that Pb in the presence of beta amyloid peptide alters intracellular calcium levels, which leads to the increased beta-secretase activity, which further promotes the generation of beta amyloid peptides. It also showed depression in the levels of GAP-43 expression, inhibition of PKC activity and altering synaptic activity further leads to cell death.

Enhanced bioavailability of cinnarizine nanosuspensions by particle size engineering: Optimization and physicochemical investigations.

Cinnarizine (CIN), a poorly soluble drug with erratic bioavailability due to pH dependent solubility has limited advantage to formulate oral solid dosage forms in subject having low gastric acidity. In present study precipitation-ultrasonication was used to fabricate nanosuspensions of cinnarizine stabilized by Poly vinyl alcohol (PVA) to enhance the bioavailability. We investigated the effects of PVA concentration (X1) and solvent to antisolvent ratio (X2) on the quality attributes like mean particle size (Y1); % drug content (Y2); and time required to 90% drug release (Y3) via 3(2) factorial design. The morphology of nanosuspensions was found almost spherical by SEM observation. DSC and FT-IR studies revealed lack of significant interactions between CIN and PVA. Nanosuspensions of mean particle size 621.08 nm was achieved. The dissolution rate obtained from all formulations were markedly higher than pure CIN. Response surface methodology and optimized polynomial equations were used to select the optimal formulation i.e. 0.2% W/V of X1 and 1:42 of X2 to get the desired response Y1; 636.78 nm, Y2; 95.24% and Y3; 7.09 min that were in reasonable agreement with the observed value. The in-vivo study in rat demonstrated that Cmax and AUC0→12 values of nanosuspension were approximately 2.8-fold and 2.7-fold greater than that of reference preparation respectively.

Effects of carbohydrate polymers in self-microemulsified tablets on the bioavailability of atorvastatin: In vitro-in vivo study.

AIMS: The concentrations of crospovidone (CP), maltodextrin and microcrystalline cellulose (MCC) have been optimized in the development of self-microemulsified tablets (SMET) to improve the oral bioavailability of an anti-hyperlipidemic drug, atorvastatin, and the in-vivo pharmacokinetic parameters of the optimized SMET were compared with those of a commercial tablet in rabbits. MAIN METHODS: Self microemulsified liquids (SELS) were prepared with oleic acid, Span 40 and Tween 80. SELS were converted into SMET by adsorption, followed by compression using factors such as CP, maltodextrin and MCC, which were optimized through a 2(3)-factorial design considering responses such as the disintegration time and, the times for 50% and 80% of the drug to be released. KEY FINDINGS: The results indicated that CP and MCC were inversely related to the responses, while maltodextrin was directly related to the responses. The droplet size of the disintegrated SMET oil globules was within 2.73 to 4.77 µm. The Cmax and AUC0-∞ of the optimized SMET were found to be 32.5% and 38.8% higher, respectively, than those of the commercial tablet. SIGNIFICANCE: The present results indicate that the bioavailability of the SMET of atorvastatin is better than the commercial formulation.

Formulation and process optimization of naproxen nanosuspensions stabilized by hydroxy propyl methyl cellulose.

In present study precipitation-ultrasonication was used to obtain nanosuspensions of poorly water-soluble drug, naproxen (NPX). We investigated the effects of HPMC concentration (X1) and time of ultrasonication (X2) on imperative attributes like mean particle size (Y1), % drug content (Y2), and time required to 90% drug release (Y3) via 3(2) factorial design. The morphology of nanosuspensions was found almost spherical by SEM observation. DSC and XRD studies suggested slight crystalline change in drug. FT-IR revealed lack of significant interactions between NPX and HPMC. Nanosuspensions of mean particle size 530.55 nm was achieved. Dissolution rate obtained from all nanosuspensions were markedly higher than pure NPX. Response surface methodology and optimized polynomial equations were used to select optimal formulation i.e. 1.36%W/V of X1 and 13.9 min of X2 to get desired response Y1; 727.97 nm, Y2; 95.59% and Y3; 8.67 min that were in reasonable agreement with observed value.