S1PR2 inhibition mitigates cognitive deficit in diabetic mice by modulating microglial activation via Akt-p53-TIGAR pathway.

Cognitive deficit is one of the challenging complications of type 2 diabetes. Sphingosine 1- phosphate receptors (S1PRs) have been implicated in various neurodegenerative and metabolic disorders. The association of S1PRs and cognition in type 2 diabetes remains elusive. Microglia-mediated neuronal damage could be the thread propagating cognitive deficit. The effects of S1PR2 inhibition on cognition in high-fat diet and streptozotocin-induced diabetic mice were examined in this work. We further assessed microglial activation and putative microglial polarisation routes. Cognitive function loss was observed after four months of diabetes induction in Type 2 diabetes animal model. JTE013, an S1PR2 inhibitor, was used to assess neuroprotection against cognitive decline and neuroinflammation in vitro and in vivo diabetes model. JTE013 (10 mg/kg) improved synaptic plasticity by upregulating psd95 and synaptophysin while reducing cognitive decline and neuroinflammation. It further enhanced anti-inflammatory microglia in the hippocampus and prefrontal cortex (PFC), as evidenced by increased Arg-1, CD206, and YM-1 levels and decreased iNOS, CD16, and MHCII levels. TIGAR, TP53-induced glycolysis and apoptosis regulator, might facilitate the anti-inflammatory microglial phenotype by promoting oxidative phosphorylation and decreasing apoptosis. However, since p53 is a TIGAR suppressor, inhibiting p53 could be beneficial. S1PR2 inhibition increased p-Akt and TIGAR levels and reduced the levels of p53 in the PFC and hippocampus of type 2 diabetic mice, thereby decreasing apoptosis. In vitro, palmitate was used to imitate sphingolipid dysregulation in BV2 cells, followed by conditioned media exposure to Neuro2A cells. JTE013 rescued the palmitate-induced neuronal apoptosis by promoting the anti-inflammatory microglia. In the present study, we demonstrate that the inhibition of S1PR2 improves cognitive function and skews microglia toward anti-inflammatory phenotype in type 2 diabetic mice, thereby promising to be a potential therapy for neuroinflammation.

Quality by design endorsed atorvastatin-loaded nanostructured lipid carriers embedded in pH-responsive gel for melanoma.

AIM: Our aim was to repurpose atorvastatin for melanoma by encapsulating in a nanostructured lipid carrier matrix to promote tumour cell internalisation and skin permeation. pH-responsive chitosan gel was employed to restrict At-NLCs in upper dermal layers. METHODS: We utilised a quality by design approach for encapsulating At within the NLC matrix. Further, cellular uptake and cytotoxicity was evaluated along with pH-responsive release and ex vivo skin permeation. RESULTS: Cytotoxicity assay showed 3.13-fold enhanced cytotoxicity on melanoma cells compared to plain drug with nuclear staining showing apoptotic markers. In vitro, release studies showed 5.9-fold rapid release in chitosan gel matrix at pH 5.5 compared to neutral pH. CONCLUSIONS: At-NLCs prevented precipitation, promoted skin permeation, and SK-MEL 28 cell internalisation. The localisation of NLCs on the upper dermal layer due to electrostatic interactions of skin with chitosan gel diminished the incidence of untoward systemic effects.

Sphingosine 1 phosphate lyase inhibition rescues cognition in diabetic mice by promoting anti-inflammatory microglia.

Sphingosine-1-phosphate (S1P) is emerging as a crucial sphingolipid modulating neuroinflammation and cognition. S1P levels in the brain have been found to be decreased in cognitive impairment. S1P lyase (S1PL) is the key enzyme in metabolizing S1P and has been implicated in neuroinflammation. This study evaluated the effect of S1PL inhibition on cognition in type 2 diabetic mice. Fingolimod (0.5 mg/kg and 1 mg/kg) rescued cognition in high-fat diet and streptozotocin-induced diabetic mice, as evident in the Y maze and passive avoidance test. We further evaluated the effect of fingolimod on the activation of microglia in the pre-frontal cortex (PFC) and hippocampus of diabetic mice. Our study revealed that fingolimod inhibited S1PL and promoted anti-inflammatory microglia in both PFC and hippocampus of diabetic mice as it increased Ym-1 and arginase-1. The levels of p53 and apoptotic proteins (Bax and caspase-3) were elevated in the PFC and hippocampus of type 2 diabetic mice which fingolimod reversed. The underlying mechanism promoting anti-inflammatory microglial phenotype was also explored in this study. TIGAR, TP53-associated glycolysis and apoptosis regulator, is known to foster anti-inflammatory microglia and was found to be downregulated in the brain of type 2 diabetic mice. S1PL inhibition decreased the levels of p53 and promoted TIGAR, thereby increasing anti-inflammatory microglial phenotype and inhibiting apoptosis in the brain of diabetic mice. Our study reveals that S1PL inhibition could be beneficial in mitigating cognitive deficits in diabetic mice.

Quality by design (QbD) assisted Fabrication & evaluation of Simvastatin loaded Nano-Enabled thermogel for melanoma therapy.

Melanoma is a form of skin cancer that starts in melanocytes. Rampant chemo-resistance, metastasis, and inability to cross the skin barriers and accumulate within the tumor microenvironment render the conventional chemotherapeutic approaches ineffective. Simvastatin (SIM), a cholesterol synthesis inhibitor, has shown tremendous anticancer potential. Due to the lack of therapeutic alternatives, repositioning SIM in melanoma could be beneficial. Incorporating SIM within the nanoparticles promoted increased melanoma cell internalization, apoptosis, and sustained release profile. Further, the incorporation of nanoparticles into the thermogel facilitated depot formation over the upper dermal layers. Sol-to-gel transition at 34 °C was observed with a 14.03-fold increase in viscosity. This could be fruitful in limiting systemic exposure and preventing adverse effects. Entrapment of SIM in the PLGA NPs enhanced the cytotoxicity by 9.38-fold (p less than 0.05). Nuclear staining with DAPI showed blebbing, membrane shrinkage, and apoptosis confirmed by DCFDA and acridine orange/ethidium bromide staining. Ex vivo diffusion studies revealed the accumulation of C-6 loaded nanoparticles incorporated within the thermogel onto the upper dermal layer and depot formation up to 6 h. Thus, we conclude that SIM-loaded nanoparticulate thermogel could be an efficacious therapeutic alternative for melanoma.

Quality by design steered development of Niclosamide loaded liposomal thermogel for Melanoma: In vitro and Ex vivo evaluation.

Melanoma is the most malignant form of skin cancer across the globe. Conventional therapies are currently ineffective which could be attributed to the rampant chemo-resistance, metastasis, inability to cross the skin barriers and accumulate within the tumor microenvironment. This advent brings in the principles of drug repurposing by repositioning Niclosamide (NIC), an anthelmintic drug for skin cancer. Incorporation into the liposomes facilitated enhanced melanoma cell uptake and apoptosis. Cytotoxicity studies revealed 1.756-fold enhancement in SK-MEL-28 cytotoxicity by NIC-loaded liposomes compared to free drug. Qualitative and quantitative cell internalization indicated greater drug uptake within the melanoma cells illustrating the efficacy of liposomes as efficient carrier systems. Nuclear staining showed blebbing and membrane shrinkage. Elevated ROS levels and apoptosis shown by DCFDA and acridine orange-ethidium bromide staining revealed greater melanoma cell death by liposomes compared to free drug. Incorporating NIC liposomes into the thermogel system restricted the liposomes as a depot onto the upper skin layers. Sustained zero order release up to 48 h with liposomes and 23.58-fold increase in viscosity led to the sol-to-gel transition at 33℃ was observed with liposomal thermogel. Ex vivo gel permeation studies revealed that C-6 loaded liposomes incorporated within the thermogel successfully formed a depot over the upper skin layer for 6 h to prevent transdermal delivery and systemic adverse effects. Thus, it could be concluded that NIC loaded liposomal thermogel system could be an efficacious therapeutic alternative for the management of melanoma.

Proteotoxicity and mitochondrial dynamics in aging diabetic brain.

Impaired neuronal proteostasis is a salient feature of both aging and protein misfolding disorders. Amyloidosis, a consequence of this phenomena is observed in the brains of diabetic patients over the chronic time period. These toxic aggregates not only cause age-related decline in proteostasis, but also dwindle its ability to increase or restore the chaperones in response to any stressful condition. Mitochondria acts as the main source of energy regulation and many metabolic disorders such as diabetes have been associated with altered oxidative phosphorylation (OxPhos) and redox imbalance in the mitochondria. The mitochondrial unfolded protein response (UPRmt) acts as a mediator for maintaining the mitochondrial protein homeostasis and quality control during such conditions. Over a long time period, these responses start shutting off leading to proteotoxic stress in the neurons. This reduces the buffering capacity of protein network signalling during aging, thereby increasing the risk of neurodegeneration in the brain. In this review, we focus on the proteotoxic stress that occurs as an amalgamation of diabetes and aging, as well as the impact of mitochondrial dysfunction on the neuronal survival affecting the diabetic brain and its long term consequences on the memory changes.

Neuroprotective effects of silibinin: an in silico and in vitro study.

AIM OF THE STUDY: Astrogliosis is a key contributor for many neurological disorders involving apoptosis, neuroinflammation and subsequent neuronal death. Silibinin, a polyphenol isolated from milk thistle (Silybum marianum), has been shown to suppress the astrocyte activation in various neurodegenerative disorders and also exhibit a neuroprotective role in neuroinflammation-driven oxidative damage. The present study was designed with an aim to investigate the neuroprotective effects of Silibinin against LPS induced oxido-inflammatory cascade and astrocyte activation. MATERIALS AND METHODS: We have used in-silico molecular modelling techniques to study the interaction and binding affinity of silibinin with chemokine receptors associated with neuroinflammation. We have also tested silibinin against LPS induced oxido-inflammatory cascade and astrocyte activation in C6 glia cell lines. RESULTS: In the present study, we found that treatment with silibinin significantly attenuates LPS-oxidative-nitrosative stress in C6 astrocytoma cells. We also observed the significant inhibition of induced astrocyte activity after treatment with silibinin. Moreover, molecular modelling studies have proposed a binding pose of silibinin with binding sites of p38 MAPK, CX3CR1 and P2X4 which is an important downstream cascade involved in glia cell activation and neuroinflammation. CONCLUSIONS: Overall, the findings from the current study suggests that silibinin exhibits neuroprotective activity by attenuating oxidative damage and astrocytes activation.