Association of PCSK-9 with the Biomarkers of Type-2 Diabetes and its Complications in the Indian Population: a Pilot Study.

BACKGROUND: Proprotein convertase subtilisin/kexin type-9 (PCSK-9) is a serine protease with profound effects on plasma LDL-C, the major risk factor for cardiovascular diseases (CVDs). However, plasma PCSK-9 level and its association with the biomarkers of CVDs, diabetes, and associated complications have not yet been reported in the northeastern population of India. METHODS: Of the total cohort (n = 233), we analyzed healthy controls (HC; n = 50), freshly diagnosed type-2-diabetes mellitus (T2DM-FD; n = 46), T2DM treated (T2DM-T; n = 49), diabetic nephropathy (T2DM-N; n = 43), and diabetic dyslipidemia (T2DM-DL; n = 45) subjects. Plasma PCSK-9 and other biological determinants associated with T2DM, CVD, and nephrotic dysfunction were assessed. RESULTS: The level of plasma PCSK-9 in HC, T2DM-FD, T2DM-T, T2DM-N, and T2DM-DL groups was found to be 184.1 ± 13.83, 183.1 ± 24.4.3, 241.8 ± 75.42, 403.7 ± 85.94, and 641.3 ± 135.5 ng/mL, respectively, indicating its role in the severity of the here-mentioned complications. Moreover, plasma PCSK-9 levels further showed a significant correlation with the biomarkers of hyperglycemia, particularly HbA1c, as well as LDL-C in T2DM-FD, T2DM-N, and T2DM-DL subjects of the Indian population, while moderate association in T2DM-T subjects. CONCLUSIONS: Our first-of-its-kind clinical study aiming to quantify the circulatory PCSK-9 level in the Indian population concluded that elevated PCSK-9 was significantly associated with the severity of diabetes and associated complications. Moreover, such elevation in PCSK-9 might be attributed to the lipid- and glucoselowering medication-induced SREBP-2-dependent mechanisms. Since our conclusion is based on a pilot study, further cohort studies in larger populations of India are required to get a generalization regarding the role of PCSK-9 in DM and associated complications.

Naturally occurring organosulfur compounds effectively inhibits PCSK-9 activity and restrict PCSK-9-LDL-receptor interaction via in-silico and in-vitro approach.

The present study intended to divulge the potential role of garlic-derived organosulfur compounds (OSCs) in targeting PCSK-9 and averting its interaction with the EGF-A portion of LDL-R via in-vitro and in-silico analysis. Our in-silico screening data showed that 3-(Propylsulfinyl)-L-alanine (PSA), S-Ethyl-L-cysteine (SEC), alliin, and S-Allyl-L-cysteine (SAC) exhibited higher binding energy (-7.05, -7.00, -6.65, and -6.31 Kcal/mol, respectively) against PCSK-9, among other selected OSCs. Further, the protein-protein interaction study of PCSK-9-OSCs-complex with EGF-A demonstrated a similar binding pattern with E-total values ranging from -430.01 to -405.6 Kcal/mol. These results were further validated via in-vitro analysis which showed that SEC, SAC, and diallyl trisulphide (DAT) exhibited the lowest IC50 values of 4.70, 5.26, and 5.29 µg/mL, respectively. In conclusion, the presented data illustrated that SEC, SAC, and DAT were the best inhibitors of PCSK-9 activity and may have the potential to improve the LDL-R function and lower the circulatory LDL-C level.

Association of circulatory PCSK-9 with biomarkers of redox imbalance and inflammatory cascades in the prognosis of diabetes and associated complications: a pilot study in the Indian population.

AbstractsBesides the profound role of proprotein convertase subtilisin/kexin type-9 (PCSK-9) in LDL-C regulation, its association with other metabolic complications cannot be disregarded. The co-existence of redox imbalance and inflammatory cascades has greatly reflected the etiology of hyperglycemia. Therefore, we studied the association of PCSK-9 with inflammation and oxidative stress biomarkers to predict its role in the prognosis of diabetes and its associated complications in the Indian population. This pilot study examined a total of n = 187 subjects: healthy controls (HC; n = 50), diabetic without complication (T2DM; n = 49), diabetic nephropathy (T2DM-N; n = 43), and diabetic dyslipidemic (T2DM-DL; n = 45) subjects. The relationship between circulatory PCSK-9 levels and inflammation and redox imbalance biomarkers has been explored. The significant positive association of elevated PCSK-9 level with the inflammatory (i.e. IL-1ß, IL-6, TNF-α, and CRP) and oxidative stress marker (i.e. XOD, CD, LOOH, and MDA) was observed in T2DM-N and T2DM-DL subjects. Whereas single regression analysis depicted that PCSK-9 was inversely associated with the FRAP and PON-1 in T2DM-N and T2DM-DL subjects. Furthermore, no significant correlation was detected in both T2DM and HC subjects. We found a significant relationship between these prognostic biomarkers with an elevated level of PCSK-9 in T2DM-N and T2DM-DL subjects. PCSK-9 is a nontraditional biomarker in diabetes that may help identify patients at risk of developing secondary complications of diabetes in the Indian population. However, further large cohort validation studies are needed.

Antiglycation and chemopreventive effects of leaf extracts of Ficus palmata Forssk. found in the Hail region of Saudi Arabia.

The antioxidant and antiglycation activities of the Ficus leaf extracts were evaluated using in vitro assays. The antioxidant activity was determined using the α, α-diphenyl-ß-picrylhydrazyl and 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) radical scavenging assays. In vitro ferric reducing activity was evaluated using the ferric reducing antioxidant power assay. The antiglycation potential of the extract was evaluated using dinitrophenylhydrazine, thiobarbituric acid and protein thiol assays. The inhibition of the formation of advanced glycation end products (AGEs) was detected using AGE-specific fluorescence with a fluorescence spectrophotometer. This study was aimed at investigating the potential of Ficus palmata Forssk. leaf extracts, which have abundant bioactive constituents, including polyphenols and flavonoids, in inhibiting glycation and cancer. The results show that the aqueous and methanolic Ficus leaf extracts are rich in phenolic and flavonoid compounds. Both extracts showed potent antioxidant activities. Furthermore, the methanolic extract showed antiglycation activities, as assessed using an in vitro model of bovine serum albumin glycation with D-ribose. The polyphenol- and flavonoid-rich Ficus leaf extracts exhibit antiglycation, chemopreventive and antioxidant activities and have potential for use in the treatment of diseases, such as cancer, which involve oxidative and glycative impairment of cellular proteins.

Differential impact of glycation on apolipoprotein A-I of high-density lipoprotein: a review.

Hyperglycemia is a poorly controlled diabetic condition, affects about 70% of people all round the world. In the year 2015, about 41.5 crore people were diabetic and is expected to reach around 64.3 crore by the year 2040. Cardiovascular diseases (CVDs) are considered as one of the major risk factors that cause more than half of the death of diabetic patients and promote related comorbidities. Atherosclerosis and amyloidosis are the prime factors linked with CVDs. Apolipoprotein A-I (ApoA-I) of HDL has protective action against CVDs, participates in reverse cholesterol transport mechanism and lipid metabolism, but gets easily glycated under prolonged hyperglycemic aura, i.e. glycation. ApoA-I has a potent role in maintenance of glucose level, providing a compelling link between diabetes and CVDs. Increased protein glycation in people with diabetes promotes atherosclerosis, which might play possible role in promotion of protein aggregation by altering the protein structure and its conformation. Here, we intend to investigate the mechanistic behavior of ApoA-I under the menace of glycation and its impact on ApoA-I structure and function that possibly link with aggregation or amyloidosis.

Ezetimibe attenuates experimental diabetes and renal pathologies via targeting the advanced glycation, oxidative stress and AGE-RAGE signalling in rats.

The current in-vivo study was premeditated to uncover the protective role of ezetimibe (EZ) against advanced glycation endproducts (AGEs)-related pathologies in experimental diabetes. Our results showed that EZ markedly improved the altered biochemical markers of diabetes mellitus (DM) (FBG, HbA1c, insulin, microalbumin, and creatinine) and cardiovascular disease (in-vivo lipid/lipoprotein level and hepatic HMG-CoA reductase activity) along with diminished plasma carboxymethyl-lysine (CML) and renal fluorescent AGEs level. Gene expression study revealed that EZ significantly down-regulated the renal AGEs-receptor (RAGE), nuclear factor-κB (NFκB-2), transforming growth factor-ß (TGF-ß1), and matrix metalloproteinase-2 (MMP-2) mRNA expression, however, the neuropilin-1 (NRP-1) mRNA expression was up-regulated. In addition, EZ also maintained the redox status via decreasing the lipid peroxidation and protein-bound carbonyl content (CC) and increasing the activity of high-density lipoprotein (HDL)-associated-paraoxonase-1 (PON-1) and renal antioxidant enzymes as well as also protected renal histopathological features. We conclude that EZ exhibits antidiabetic and reno-protective properties in diabetic rats.

Carvacrol protects against carbonyl osmolyte-induced structural modifications and aggregation to serum albumin: Insights from physicochemical and molecular interaction studies.

The robust use of osmolytes (i.e., polyols and sugars) in the key therapeutic regimens/formulations has questioned their impact beyond the stability of therapeutic proteins as these osmolytes trigger structural alterations into proteins including misfolding and subsequent aggregation into amyloid fibrils. Therefore, the current study is the first to delineate the inhibitory effect of carvacrol (CRV) on the carbonyl osmolyte-induced aggregation as well as structural alterations to the bovine serum albumin (BSA) via a set of physicochemical as well as artificial intelligence (AI)-based molecular docking studies. Our initial findings from physicochemical investigations revealed that CRV exhibits substantial protection to BSA under carbonyl osmolyte stress as evident by the compromised hyperchromicity, Schiff's bases, carbonyl and hydroxymethyl furfural content, reduced fluorescent signals, low Rayleigh scattering and prevention of covalent modifications at Lys and Arg residues. The protection against aggregate formation by CRV was further confirmed through the reduced amyloid-specific congo red absorbance as well as fluorescent signals recorded after adding the fibril-specific extrinsic fluorophore probes (i.e., ThT and ANS). The AI-based molecular docking analysis further revealed that CRV (ΔG: -4.96 kcal/mol) competes with d-fructose (ΔG: -4.40 kcal/mol) to mask the Lys and Arg residues to restrict the osmolyte-mediated protein modifications. In conclusion, CRV exhibits substantial protective impact against carbonyl osmolyte-induced structural alterations and protein misfolding and aggregation.

Soyasapogenol-B as a Potential Multitarget Therapeutic Agent for Neurodegenerative Disorders: Molecular Docking and Dynamics Study.

Neurodegenerative disorders involve various pathophysiological pathways, and finding a solution for these issues is still an uphill task for the scientific community. In the present study, a combination of molecular docking and dynamics approaches was applied to target different pathways leading to neurodegenerative disorders such as Alzheimer's disease. Initially, abrineurin natural inducers were screened using physicochemical properties and toxicity assessment. Out of five screened compounds, a pentacyclic triterpenoid, i.e., Soyasapogenol B appeared to be the most promising after molecular docking and simulation analysis. Soyasapogenol B showed low TPSA (60.69), high absorption (82.6%), no Lipinski rule violation, and no toxicity. Docking interaction analysis revealed that Soyasapogenol B bound effectively to all of the targeted proteins (AChE, BuChE MAO-A, MAO-B, GSK3ß, and NMDA), in contrast to other screened abrineurin natural inducers and inhibitors. Importantly, Soyasapogenol B bound to active site residues of the targeted proteins in a similar pattern to the native ligand inhibitor. Further, 100 ns molecular dynamics simulations analysis showed that Soyasapogenol B formed stable complexes against all of the targeted proteins. RMSD analysis showed that the Soyasapogenol B-protein complex exhibited average RMSD values of 1.94 Å, 2.11 Å, 5.07 Å, 2.56 Å, 3.83 Å and 4.07 Å. Furthermore, the RMSF analysis and secondary structure analysis also indicated the stability of the Soyasapogenol B-protein complexes.

Potential dual inhibitors of PCSK-9 and HMG-R from natural sources in cardiovascular risk management.

Atherosclerotic cardiovascular disease (ASCVD) stands amongst the leading causes of mortality worldwide and has attracted the attention of world's leading pharmaceutical companies in order to tackle such mortalities. The low-density lipoprotein-cholesterol (LDL-C) is considered the most prominent biomarker for the assessment of ASCVD risk. Distinct inhibitors of 3-hydroxy-3-methyl-glutaryl-CoA reductase (HMG-R), the chief hepatic cholesterogenic enzyme, are being used since last seven decades to manage hypercholesterolemia. On the other hand, discovery and the association of proprotein convertase subtilisin/kexin type-9 (PCSK-9) with increased ASCVD risk have established PCSK-9 as a novel therapeutic target in cardiovascular medicine. PCSK-9 is well reckoned to facilitate the LDL-receptor (LDL-R) degradation and compromised LDL-C clearance leading to the arterial atherosclerotic plaque formation. The currently available HMG-R inhibitors (statins) and PCSK-9 inhibitors (siRNA, anti-sense oligonucleotides, and monoclonal antibodies) have shown great promises in achieving LDL-C lowering goals, however, their life long prescriptions have raised significant concerns. These deficits associated with the synthetic HMG-R and PCSK-9 inhibitors called for the discovery of alternative therapeutic candidates with potential dual HMG-R and PCSK-9 inhibitory activities from natural origins. Therefore, this report firstly describes the mechanistic insights into the cholesterol homeostasis through HMG-R, PCSK-9, and LDL-R functionality and then compiles the pharmacological effects of natural secondary metabolites with special emphasis on their dual HMG-R and PCSK-9 inhibitory action. In conclusion, various natural products exhibit atheroprotective effects via targeting HMG-R and PCSK-9 activities and lipoprotein metabolism, however, further clinical assessments are still warranted prior their approval for ASCVD risk management in hypercholesterolemic patients.

Exploring the Binding Pattern of Geraniol with Acetylcholinesterase through In Silico Docking, Molecular Dynamics Simulation, and In Vitro Enzyme Inhibition Kinetics Studies.

Acetylcholinesterase (AChE) inhibition is a key element in enhancing cholinergic transmission and subsequently relieving major symptoms of several neurological and neuromuscular disorders. Here, the inhibitory potential of geraniol and its mechanism of inhibition against AChE were elucidated in vitro and validated via an in silico study. Our in vitro enzyme inhibition kinetics results show that at increasing concentrations of geraniol and substrate, Vmax did not change significantly, but Km increased, which indicates that geraniol is a competitive inhibitor against AChE with an IC50 value 98.06 ± 3.92 µM. All the parameters of the ADME study revealed that geraniol is an acceptable drug candidate. A docking study showed that the binding energy of geraniol (-5.6 kcal mol-1) was lower than that of acetylcholine (-4.1 kcal mol-1) with AChE, which exhibited around a 12.58-fold higher binding affinity of geraniol. Furthermore, molecular dynamics simulation revealed that the RMSD of AChE alone or in complex with geraniol fluctuated within acceptable limits throughout the simulation. The mean RMSF value of the complex ensures that the overall conformation of the protein remains conserved. The average values of Rg, MolSA, SASA, and PSA of the complex were 3.16 Å, 204.78, 9.13, and 51.58 Å2, respectively. We found that the total SSE of AChE in the complex was 38.84% (α-helix: 26.57% and ß-sheets: 12.27%) and remained consistent throughout the simulation. These findings suggest that geraniol remained inside the binding cavity of AChE in a stable conformation. Further in vivo investigation is required to fully characterize the pharmacokinetic properties, optimization of dose administration, and efficacy of this plant-based natural compound.

Glycyrrhizic Acid Scavenges Reactive Carbonyl Species and Attenuates Glycation-Induced Multiple Protein Modification: An In Vitro and In Silico Study.

The current study is aimed at studying the inhibitory effect of glycyrrhizic acid (GA) on D-ribose-mediated protein glycation via various physicochemical analyses and in silico approaches. Being a potent free radical scavenger and a triterpenoid saponin, GA plays a vital role in diminishing the oxidative stress and thus could be an effective inhibitor of the nonenzymatic glycation process. Our data showed that varying concentrations of GA inhibited the in vitro BSA-AGEs via inhibiting the formation of fructosamines, fluorescent AGEs, scavenging protein carbonyl and hydroxymethyl furfural (HMF) content, and protection against D-ribose-induced modification of BSA as evident by increased free Arg and Lys residues in GA-treated Gly-BSA samples. Moreover, GA also attenuated D-ribose-induced alterations in the secondary structure of BSA by protecting the α-helix and ß-sheet conformers and amide-I band delocalization. In addition, GA attenuated the modification in ß-cross amyloid structures of BSA and in silico molecular interaction study too showed strong binding of GA with higher number of Lys and Arg residues of BSA and binding energy (ΔG) of -8.8 Kcal/mol, when compared either to reference standard aminoguanidine (AG)-BSA complex (ΔG: -4.3 Kcal/mol) or D-ribose-BSA complex (ΔG: -5.2 Kcal/mol). Therefore, GA could be a new and favorable inhibitor of the nonenzymatic glycation process that ameliorates AGEs-related complications via attenuating the AGE formation and glycation-induced multiple protein modifications with a reduced risk of adverse effects on protein structure and functionality; hence, it could be investigated at further preclinical settings for the treatment and management of diabetes and age-associated complications.

High-Throughput Screening and Molecular Dynamics Simulation of Natural Product-like Compounds against Alzheimer's Disease through Multitarget Approach.

Alzheimer's disease (AD) is a progressive neurological disorder that affects 50 million people. Despite this, only two classes of medication have been approved by the FDA. Therefore, we have planned to develop therapeutics by multitarget approach. We have explored the library of 2029 natural product-like compounds for their multi-targeting potential against AD by inhibiting AChE, BChE (cholinergic pathway) MAO-A, and MOA-B (oxidative stress pathway) through in silico high-throughput screening and molecular dynamics simulation. Based on the binding energy of these target enzymes, approximately 189 compounds exhibited a score of less than -10 kcal/mol against all targets. However, none of the control inhibitors exhibited a binding affinity of less than -10 kcal/mol. Among these, the top 10 hits of compounds against all four targets were selected for ADME-T analysis. As a result, only F0850-4777 exhibited an acceptable range of physicochemical properties, drug-likeness, pharmacokinetics, and suitability for BBB permeation with high GI-A and non-toxic effects. The molecular dynamics study confirmed that F0850-4777 remained inside the binding cavity of targets in a stable conformation throughout the simulation and Prime-MM/GBSA study revealed that van der Waals' energy (ΔGvdW) and non-polar solvation or lipophilic energy (ΔGSol_Lipo) contribute favorably towards the formation of a stable protein-ligand complex. Thus, F0850-4777 could be a potential candidate against multiple targets of two pathophysiological pathways of AD and opens the doors for further confirmation through in vitro and in vivo systems.

Insights into pharmacological mechanisms of polydatin in targeting risk factors-mediated atherosclerosis.

Polydatin (PD) is a monocrystalline metabolite from the underground parts of Polygonum cuspidatum Sieb. et Zucc., a member of the Polygonaceae family, which has been traditionally used in Asian countries as both foodstuffs and medicine. PD, also reckoned as pieceid, 3,4',5-trihydroxystilbene-3-ß-D-glucoside, (E)-piceid, (E)-polydatin, and trans-polydatin. It possesses potent biological activities i.e. analgesic, anti-inflammatory, antidiabetic, anticancer, and anti-atherosclerotic properties. The initial part of this report specifically explains distinct sequential mechanisms underlying the initiation and development of atherosclerotic plaques and later part deals with the pharmacological efficacy of PD in the management of major cardiac event i.e. atherosclerotic cardiovascular diseases (ASCVD) via modulation of a set of molecular mechanisms i.e. antioxidant potential, lipid and lipoprotein metabolism including total cholesterol (TC) and low density lipoprotein (LDL) levels, ß-hydroxy-ß-methyl-glutaryl-CoA reductase (HMG-R) expression and functionality, SIRT signalling, LDL-receptor (LDL-R), LDL oxidation status (Ox-LDL), effects on endothelial cells (ECs), smooth muscle cells (SMCs), macrophage, foam cell formation and plaque stabilization, inflammatory signalling pathways and hypertension. In contrast, one of the major insight into the potential cardioprotective molecular mechanism is the PD-mediated targeting of proprotein convertase subtilisin/kexin type-9 (PCSK-9) and LDL-R pathway, both at transcriptional and protein functional level, which makes it a better alternative therapeutic medicinal candidate to treat hypercholesterolemia, especially for the patients facing inadequate lipid lowering with classical HMG-R inhibitors (statins) and statin intolerance. Finally, to sum up the whole, we concluded that PD may be promoted from alternative to mainstream medicine in targeting risk factors mediated ASCVD.

A biochemical & biophysical study on in-vitro anti-glycating potential of iridin against d-Ribose modified BSA.

Non-enzymatic protein glycation results in the formation of advanced glycation end products (AGEs) leads to the pathogenesis of long-term diabetic complications. Iridin (ID), an antioxidant, plays an important role in protecting against oxidative stress and could therefore be an efficacious anti-glycating regimen. Herein, we assessed the anti-glycating potential of ID against d-ribose induced glycation of bovine serum albumin (BSA) by various biophysical and biochemical techniques. Our results from several physicochemical assays advocated that ID was able to evidently prevent the AGEs generation via reducing hyperchromicity, early glycation products (EGPs), carbonyl content (CC), hydroxymethyl furfural (HMF) content, production of fluorescent AGEs, protection against loss of secondary structure (i.e. α-helix and ß-sheets) of proteins, increasing the free lysine and free arginine content, reduced binding of congo red (CR), and reduced thioflavin T (ThT) and 8-aninilo-1-napthalene sulphonate (ANS)-specific fuorescence in glycated-BSA (Gly-BSA). On the basis of these findings, we concluded that ID possesses the significant anti-glycation potential and may be established as a remarkable anti-AGEs therapeutic agent. Further in-vivo and clinical studies are still warranted to uncover the therapeutic effects of ID against age-related as well as metabolic diseases.

Modulatory role of HMG-CoA reductase inhibitors and ezetimibe on LDL-AGEs-induced ROS generation and RAGE-associated signalling in HEK-293 Cells.

AIMS: Advanced glycation end products (AGEs) trigger intracellular reactive oxygen species (ROS) generation, activation of receptor for AGEs (RAGE) expression/functionality and RAGE-associated signalling pathways which influence the diabetic-cum-atherosclerotic complications, whereas, the atherosclerosis progression is greatly influenced by hepatic ß-Hydroxy-ß-methyl-glutaryl-Co-A reductase (HMG-R) activity. The present report was premeditated to uncover the regulatory role of HMG-R inhibitors and ezetimibe (EZ) in attenuating the LDL-AGEs-induced pathogenicity via targeting cellular-ROS and RAGE-associated signalling in HEK-293 cells. MAIN METHODS: The MTT assay was used to assess either the cytotoxic or cytoprotective impact of each HMG-R inhibitors, EZ, and LDL-AGEs, whereas, quantification of ROS was performed by DCFDA method. The qRT-PCR was used to detect the mRNA level of RAGE, neuropilin-1 (NRP-1) and other RAGE-associated genes like MMP-2, NF-κB, and TGFß-1. KEY FINDINGS: The HMG-R inhibitors do not exert any cytotoxicity in HEK-293 cells, whereas, and LDL-AGEs negatively affected the cell viability of HEK-293 cells. However, viability of LDL-AGEs-treated HEK-293was markedly retained after simultaneous treatment with our test inhibitors. Further, DCFDA staining showed that LDL-AGEs-induced ROS was also suppressed upon treatment with our test inhibitors in HEK-293 cells. qRT-PCR analysis reflected that these inhibitors suppress the RAGE, NF-κB, TGFß-1, and MMP-2 expression, whereas, the NRP-1 was up-regulated by these compounds in LDL-AGEs-exposed HEK-293 cells. SIGNIFICANCE: The above pharmacological effects signify that HMG-R inhibitors and EZ (alone or in combination) may implied in the treatment of AGEs-induced oxidative stress and tissue damage in diabetic complications via targeting intracellular-ROS, NRP-1 functionality and RAGE-associated genes i.e. NF-κB, TGFß-1, and MMP-2.

Ginkgolide B Mediated Alleviation of Inflammatory Cascades and Altered Lipid Metabolism in HUVECs via Targeting PCSK-9 Expression and Functionality.

The potential of oxidized-LDL (Ox-LDL) to elicit inflammatory responses in macrophages leading to the atherosclerosis (AS) progression is well known. Since proprotein convertase subtilisin/Kexin-9 (PCSK-9), the posttranslational regulator of LDL-receptor, is associated with elevated LDL in the circulation, the present report was aimed to uncover the ameliorative effects of Ginkgolide B, a terpenic lactone from Ginkgo biloba, against Ox-LDL-induced alterations in cholesterol metabolism in HUVECs. Consequently, our results demonstrated that incubation with Ox-LDL significantly upregulated the PCSK-9 expression in HUVECs, which was significantly downregulated, both at mRNA and protein level, after Ginkgolide B treatment via subsequent suppression of sterol element binding protein (SREBP-2) expression. Moreover, Ginkgolide B-mediated inhibition of PCSK-9 activity was also validated by in silico methods which revealed that it interferes the PSCK-9 interaction with LDL-receptor (LDL-R). Interestingly, Ox-LDL-induced LDL-R expression was further enhanced by Ginkgolide B treatment in HUVECs. Moreover, Ginkgolide B treatment lead to downregulation of lectin-like Ox-LDL receptor (LOX-1) and NADPH oxidase (NOX-4) expression which was upregulated in Ox-LDL-treated HUVECs, along with the attenuation of mitochondrial ROS generation. Furthermore, Ginkgolide B significantly inhibited the augmented expression of intercellular adhesion molecule-1 (ICAM-1) and vascular adhesion molecule-1 (VCAM-1) in Ox-LDL-activated HUVECs. Ginkgolide B also significantly ameliorated the inflammatory response in Ox-LDL-activated HUVECs by suppressing the expression of IL-1α, IL-1ß, IL-6, CXCL-1, CXCL-2, and monocyte chemotactic protein (MCP-1), at mRNA and protein level. Our in vitro and in silico study established that Ginkgolide B alleviated the Ox-LDL-induced inflammatory cascades and altered lipid metabolism in HUVECs by suppressing the PCSK-9 and, thus, could be established as a treasured alternative therapeutic candidate in the atherosclerosis management.

Phyllanthus virgatus forst extract and it's partially purified fraction ameliorates oxidative stress and retino-nephropathic architecture in streptozotocin-induced diabetic rats.

Diabetic retinopathy and nephropathy are questionably the most dreaded complications of diabetes; contribute to serious morbidity and mortality. The current study was undertaken with the aim of exploring the anti-lipoperoxidative and antioxidant status including nephroprotective and retinoprotective potential of Phyllanthus virgatus methanolic extract and its partially purified fraction in streptozotocin (STZ)-induced diabetic stressed rats. Diabetes was induced by intraperitoneal injection of Streptozotocin (60mg/kg B. Wt of rat). Among all the treatment groups, P. virgatus methanolic extract and its partially purified fraction at a dose of 50mg/kg (PET-1) and 0.5mg/kg (CT-1), respectively, showed significant protection against STZ-induced diabetic oxidative stress in rats with marked amelioration in lipid peroxidation byproducts level, antioxidant enzymes, nephroprotective and retinoprotective effects and plasma total antioxidant levels after treatment of 28 days. The combined results demonstrated significant protection against STZ-induced oxidative stress, nephropathy and retinopathy condition by P. virgatus methanolic extract and its bioactive compound.

MHC binding peptides for designing of vaccines against Japanese encephalitis virus: A computational approach.

Japanese encephalitis (JE), a viral disease has seen a drastic and fatal enlargement in the northern states of India in the current decade. The better and exact cure for the disease is still in waiting. For the cause an in silico strategy in the development of the peptide vaccine has been taken here for the study. A computational approach to find out the Major Histocompatibility Complex (MHC) binding peptide has been implemented. The prediction analysis identified MHC class I (using propred I) and MHC class II (using propred) binding peptides at an expectable percent predicted IC (50) threshold values. These predicted Human leukocyte antigen [HLA] allele binding peptides were further analyzed for potential conserved region using an Immune Epitope Database and Analysis Resource (IEDB). This analysis shows that HLA-DRB1*0101, HLA-DRB3*0101, HLA-DRB1*0401, HLA-DRB1*0102 and HLA-DRB1*07:01% of class II (in genotype 2) and HLA-A*0101, HLA-A*02, HLA-A*0301, HLA-A*2402, HLA-B*0702 and HLA-B*4402% of HLA I (in genotype 3) bound peptides are conserved. The predicted peptides MHC class I are ILDSNGDIIGLY, FVMDEAHFTDPA, KTRKILPQIIK, RLMSPNRVPNYNLF, APTRVVAAEMAEAL, YENVFHTLW and MHC class II molecule are TTGVYRIMARGILGT, NYNLFVMDEAHFTDP, AAAIFMTATPPGTTD, GDTTTGVYRIMARGI and FGEVGAVSL found to be top ranking with potential super antigenic property by binding to all HLA. Out of these the predicted peptide FVMDEAHFTDPA for allele HLA-A*02:01 in MHC class I and NYNLFVMDEAHFTDP for allele HLA-DRB3*01:01 in MHC class II was observed to be most potent and can be further proposed as a significant vaccine in the process. The reported results revealed that the immune-informatics techniques implemented in the development of small size peptide is useful in the development of vaccines against the Japanese encephalitis virus (JEV).

Ginkgolide B ameliorates oxidized low-density lipoprotein-induced endothelial dysfunction via modulating Lectin-like ox-LDL-receptor-1 and NADPH oxidase 4 expression and inflammatory cascades.

The current study was undertaken to delineate the protective effect of Ginkgolide B, a phyto-constituent from Ginkgo biloba, on oxidized (ox)-LDL-induced endothelial dysfunction via targeting Lectin-like ox-LDL-receptor-1 (LOX-1), NADPH oxidase 4 (NOX-4), and other inflammatory proteins. Our results have shown that Ginkgolide B downregulated the expression of LOX-1 in ox-LDL-treated human umbilical vein endothelial cells (HUVECs) and RAW246.7 murine macrophages which ultimately resulted in decreased cholesterol deposits in HUVECs and RAW264.7. Moreover, Ginkgolide B suppressed the enhanced NOX4 expression, which was associated with attenuation of ROS generation in ox-LDL-stimulated HUVECs and RAW264.7 cells. Ginkgolide B also ameliorated the endothelial dysfunction by inhibiting the augmented expression of monocyte chemotactic protein-1 (MCP-1), intercellular adhesion molecule-1 (ICAM-1), and vascular cell adhesion molecule-1 (VCAM-1) in ox-LDL-activated HUVECs. Furthermore, the enhanced expression of many inflammatory cytokines in ox-LDL-induced RAW264.7 macrophages, both at transcription and protein level, was significantly down-regulated after Ginkgolide B treatment. Ginkgolide B also illustrated atheroprotective property via suppressing the augmented expression of matrix metalloproteinase-1 and cyclooxygenase-2 in ox-LDL-stimulated RAW264.7 macrophages. In summary, our study has established that Ginkgolide B ameliorates endothelial dysfunction via targeting LOX-1, NOX-4, MCP-1, ICAM-1, and VCAM-1 along with the markers associated with inflammatory cascades and thus could be promoted as a valuable therapeutic agent in prevention and management of atherosclerosis.

Antiglycation study of HMG-R inhibitors and tocotrienol against glycated BSA and LDL: A comparative study.

Non-enzymatic glycation mediated advanced glycation end products (AGEs) generation results in the pathogenesis of diabetic complications and atherosclerotic cardiovascular disease (ASCVD) which is greatly influenced by 3-hydroxy-3-methyl-glutaryl Co-A reductase (HMG-R) activity. HMG-R inhibitors, statins, are well known for reducing mortality and morbidity of ASCVD in patients with diabetes due to their pleiotropic effects independent of cholesterol lowering. Due to distinct chemical structures, various statins may play important role in the inhibition of AGEs mediated pathologies. Herein, we evaluated the anti-glycating potential of atorvastatin (AT), rosuvastatin (RT), pitavastatin (PT), fluvastatin (FT), simvastatin (ST) alone as well in combination with ezetimibe (EZ) and tocotrienol (TT) against d-ribose mediated BSA and LDL glycation by various physicochemical approaches. Our data suggested that AT, TT, RT, EZ, EZ-AT, and EZ-RT were able to substantially inhibit the AGEs formation via modulation of hyperchromicity, fluorogenic AGEs, % contribution of α-helix and ß-sheets to protein secondary structure, amide-I band stretching, carbonyl and HMF content in Gly-BSA as well as Gly-LDL. On the basis of above findings, we concluded that HMG-R inhibitors and TT, alone or in combination with EZ, may be established as terrific therapeutic agents for the patients suffering from AGEs induced diabetic cum ASCVD complications.