Bioactive Fractions Isolated from Harungana madagascariensis Lam. and Psorospermum aurantiacum Engl. Regulate Collagen and Melanin Biosynthesis Gene Expression in UVB-Irradiated Cells with Additional Anti-Inflammatory Potential.

BACKGROUND: Harungana madagascariensis (HM) and Psorospermum aurantiacum (PA), used traditionally for skin care, have been reported to upregulate the expression of intracellular antioxidant genes, thereby preventing melanoma and protecting fibroblast cell lines from Ultraviolet B (UVB)-induced intracellular oxidative stress. AIMS: This investigation aimed to identify major compounds in bioactive fractions using bioassay- guided fractionation. METHODS: The anti-inflammatory effect of fractions was determined by measuring their inhibitory activity on 15-lipoxygenase and nitric oxide (NO) in lipopolysaccharide-stimulated RAW 264.7 macrophage cells. Additionally, the anti-aging efficacy of the fractions was determined by assessing the expression of markers for the aging process, i.e., expression of tyrosinase (TYR), tyrosinase-related protein-1 (TRP-1), procollagen type-1 (COL1A1), and matrix metalloproteinase- 1 (MMP-1) in UVB-induced photoaging in skin cell-lines. Furthermore, UHPLCMS- based identification of the bioactive compounds from the most prominent fraction was also carried out. RESULTS: Hexane fraction of HM significantly inhibited (p <0.05) the 15-lipoxygenase (IC50 = 46.80 µg/mL) and NO production (IC50 = 66.55 µg/mL), whereas hexane fraction of PA was effective (p <0.05) in inhibiting 15-lipoxygenase activity (IC50 = 27.55 µg/mL). Furthermore, the hexane fraction of HM and methanol fraction of PA were significantly effective (p <0.05) in reverting the UVB-mediated altered expressions of MMP-1, TYR, TRP-1, and COL1A1. Furthermore, hexane fraction of HM revealed the presence of harunganin and betulinic acid, whereas vismion D, vismin, kenganthranol B, and bianthrone 1a were identified from the methanol fraction of PA. CONCLUSION: Overall, the hexane fraction of HM and methanol fraction of PA displayed effective anti-aging activities, with additional anti-inflammatory effects.

Vitamin-H Channeled Self-Therapeutic P-gp Inhibitor Curcumin-Derived Nanomicelles for Targeting the Tumor Milieu by pH- and Enzyme-Triggered Hierarchical Disassembly.

An effective nanocarrier-mediated drug delivery to cancer cells primarily faces limitations like the presence of successive drug delivery barriers, insufficient circulation time, drug leakage, and decreased tumor penetration capacity. With the aim of addressing this paradox, a self-therapeutic, curcumin-derived copolymer was synthesized by conjugation with PEGylated biotin via enzyme- and acid-labile ester and acetal linkages. This copolymer is a prodrug of curcumin and self-assembles into ∼150-200 nm-sized nanomicelles; it is capable of encapsulating doxorubicin (DOX) and hence can be designated as self-therapeutic. pH- and enzyme-responsive linkages in the polymer skeleton assist in its hierarchical disassembly only in the tumor microenvironment. Further, the conjugation of biotin and poly(ethylene glycol) (PEG) imparts features of tumor specificity and improved circulation times to the nanocarrier. The dynamic light scattering (DLS) analysis supports this claim and demonstrates rapid swelling and disruption of micelles under acidic pH. UV-vis spectroscopy provided evidence of an accelerated acetal degradation at pH 4.0 and 5.0. The in vitro release studies revealed a controlled release of DOX under acidic conditions and curcumin release in response to the enzyme. The value of the combination index calculated on HepG2 cells was found to be <1, and hence, the drug pair curcumin and DOX acts synergistically for tumor regression. To prove the efficiency of acid-labile linkages and the prodrug strategy for effective cancer therapy, curcumin-derived polymers devoid of sensitive linkages were also prepared. The prodrug stimuli-responsive nanomicelles showed enhanced cell cytotoxicity and tumor penetration capability on HepG2 cells as well as drug-resistant MCF-7 cell lines and no effect on normal NIH/3T3 fibroblasts as compared to the nonresponsive micelles. The results were also supported by in vivo evidence on a hepatocellular carcinoma (HCC)-induced nude mice model. An evident decrease in MMP-2, MMP-9, and α-fetoprotein (AFP), the biomarkers specific to tumor progression, was observed along with metastasis upon treatment with the drug-loaded dual-responsive nanomicelles. These observations corroborated with the SGOT and SGPT data as well as the histoarchitecture of the liver tissue in mice.

Melatonin induces Nrf2-HO-1 reprogramming and corrections in hepatic core clock oscillations in Non-alcoholic fatty liver disease.

Melatonin pleiotropically regulates physiological events and has a putative regulatory role in the circadian clock desynchrony-mediated Non-alcoholic fatty liver disease (NAFLD). In this study, we investigated perturbations in the hepatic circadian clock gene, and Nrf2-HO-1 oscillations in conditions of high-fat high fructose (HFHF) diet and/or jet lag (JL)-mediated NAFLD. Melatonin treatment (100 µM) to HepG2 cells led to an improvement in oscillatory pattern of clock genes (Clock, Bmal1, and Per) in oleic acid (OA)-induced circadian desynchrony, while Cry, Nrf2, and HO-1 remain oblivious of melatonin treatment that was also validated by circwave analysis. C57BL/6J mice subjected to HFHF and/or JL, and treated with melatonin showed an improvement in the profile of lipid regulatory genes (CPT-1, PPARa, and SREBP-1c), liver function (AST and ALT) and histomorphology of fatty liver. A detailed scrutiny revealed that hepatic mRNA and protein profiles of Bmal1 (at ZT6) and Clock (at ZT12) underwent corrective changes in oscillations, but moderate corrections were recorded in other components of clock genes (Per1, Per2, and Cry2). Melatonin induced changes in oscillations of anti-oxidant genes (Nrf2, HO-1, and Keap1) subtly contributed in the overall improvement in NAFLD recorded herein. Taken together, melatonin induced reprograming of hepatic core clock and Nrf2-HO-1 genes leads to an improvement in HFHF/JL-induced NAFLD.

HSP60 knockdown exerts differential response in endothelial cells and monocyte derived macrophages during atherogenic transformation.

Ectopic expression of HSP60 in vascular cells is known to activate auto-immune response that is critical to atherogenic initiation. However, the pathogenic relevance of the aberrant HSP60 upregulation in intracellular signaling pathways associated with atherogenic consequences in vascular cells remains unclear. The aim of the present study was to determine the role of endogenous HSP60 in atherogenic transformation of endothelial cells and macrophages. After generating primary evidence of oxidized low density lipoprotein (OxLDL) induced HSP60 upregulation in human umbilical vein endothelial cells (HUVEC), its physiological relevance in high fat high fructose (HFHF) induced early atherogenic remodelling was investigated in C57BL/6J mice. Prominent HSP60 expression was recorded in tunica intima and media of thoracic aorta that showed hypertrophy, lumen dilation, elastin fragmentation and collagen deposition. Further, HSP60 overexpression was found to be prerequisite for its surface localization and secretion in HUVEC. eNOS downregulation and MCP-1, VCAM-1 and ICAM-1 upregulation with subsequent macrophage accumulation provided compelling evidences on HFHF induced endothelial dysfunction and activation that were also observed in OxLDL treated- and HSP60 overexpressing-HUVEC. OxLDL induced concomitant reduction in NO production and monocyte adhesion were prevented by HSP60 knockdown, implying towards HSP60 mediated possible regulation of the said genes. OxLDL induced HSP60 upregulation and secretion was also recorded in THP-1 derived macrophages (TDMs). HSP60 knockdown in TDMs accounted for higher OxLDL accumulation that correlated with altered scavenger receptors (SR-A1, CD36 and SR-B1) expression further culminating in M1 polarization. Collectively, the results highlight HSP60 upregulation as a critical vascular alteration that exerts differential regulatory role in atherogenic transformation of endothelial cells and macrophages.

Carbon nanotube embedded cyclodextrin polymer derived injectable nanocarrier: A multiple faceted platform for stimulation of multi-drug resistance reversal.

A combination of cocktail chemotherapy (CCT), photothermal therapy (PTT) and inhibition of angiogenesis was investigated as an effective approach to combat major challenges of multidrug resistance and non-targeted drug delivery encountered in conventional cancer therapy. An injectable nanocarrier was developed through functionalization of carbon nanotubes (CNTs) with rationally modified carbohydrate (ß-Cyclodextrin-CD) derived pH and thermo responsive polymer. Embedding CNT to CD polymer offers a nanocarrier which effectively demonstrated CCT, high NIR triggered photothermal efficiency, anti-angiogenic potential for selective tumor homing as well as enhanced multi-drug resistance (MDR) reversal with minimal toxic effects on normal cells. The simultaneously loading with curcumin and doxorubicin hydrochloride exhibited synergistic effect for triggering antitumor effect in vitro and demonstrated down regulation of growth factors associated with angiogenesis ex-ovo. In-vivo studies ascertained that the nanocarrier synthesized with the rational for MDR reversal can lead to enhanced cancer cell death via multiple approaches.

Carbon monoxide releasing molecule-A1 improves nonalcoholic steatohepatitis via Nrf2 activation mediated improvement in oxidative stress and mitochondrial function.

Nuclear factor-erythroid 2 related factor 2 (Nrf2)-mediated signaling plays a central role in maintaining cellular redox homeostasis of hepatic cells. Carbon monoxide releasing molecule-A1 (CORM-A1) has been reported to stimulate up-regulation and nuclear translocation of Nrf2 in hepatocytes. However, the role of CORM-A1 in improving lipid metabolism, antioxidant signaling and mitochondrial functions in nonalcoholic steatohepatitis (NASH) is unknown. In this study, we report that CORM-A1 prevents hepatic steatosis in high fat high fructose (HFHF) diet fed C57BL/6J mice, used as model of NASH. The beneficial effects of CORM-A1 in HFHF fed mice was associated with improved lipid homeostasis, Nrf2 activation, upregulation of antioxidant responsive (ARE) genes and increased ATP production. As, mitochondria are intracellular source of reactive oxygen species (ROS) and important sites of lipid metabolism, we further investigated the mechanisms of action of CORM-A1-mediated improvement in mitochondrial function in palmitic acid (PA) treated HepG2 cells. Cellular oxidative stress and cell viability were found to be improved in PA + CORM-A1 treated cells via Nrf2 translocation and activation of cytoprotective genes. Furthermore, in PA treated cells, CORM-A1 improved mitochondrial oxidative stress, membrane potential and rescued mitochondrial biogenesis thru upregulation of Drp1, TFAM, PGC-1α and NRF-1 genes. CORM-A1 treatment improved cellular status by lowering glycolytic respiration and maximizing OCR. Improvement in mitochondrial respiration and increment in ATP production in PA + CORM-A1 treated cells further corroborate our findings. In summary, our data demonstrate for the first time that CORM-A1 ameliorates tissue damage in steatotic liver via Nrf2 activation and improved mitochondrial function, thus, suggesting the anti-NASH potential of CORM-A1.

ß-cyclodextrin based dual-responsive multifunctional nanotheranostics for cancer cell targeting and dual drug delivery.

Multifunctional nanoconjugates possessing an assortment of key functionalities such as magnetism, florescence, cell-targeting, pH and thermo-responsive features were developed for dual drug delivery. The novelty lies in careful conjugation of each of the functionality with magnetic Fe3O4 nanoparticles by virtue of urethane linkages instead of silica in a simple one pot synthesis. Further ß-cyclodextrin (CD) was utilized to carry hydrophobic as well as hydrophilic drug. Superlative release of DOX could be obtained under acidic pH conditions and elevated temperature, which coincides with the tumor microenvironment. Mathematical modelling studies revealed that the drug release kinetics followed diffusion mechanism for both hydrophobic drug and hydrophilic drug. A number of fluorophores onto a single nanoparticle produced a strong fluorescence signal to optically track the nanoconjugates. Enhanced internalization due to folate specificity could be observed by fluorescence imaging. Further their accumulation driven by magnet near tumor site led to magnetic hyperthermia. in vitro studies confirmed the nontoxicity and hemocompatibility of the nanoconjugates. Remarkable cell death was observed with drug-loaded nanoconjugates at very low concentrations in cancer cells. The internalization and cellular uptake of poor bioavailable anticancer agent curcumin were found to be remarkably enhanced on dosing the drug loaded nanoconjugates as compared to free curcumin. Site specific drug delivery due to folate conjugation and subsequent significant suppression in tumor growth was demonstrated by in vivo studies.

Carbon monoxide releasing molecule A-1 attenuates acetaminophen-mediated hepatotoxicity and improves survival of mice by induction of Nrf2 and related genes.

Acute liver injury is frequently associated with oxidative stress. Here, we investigated the therapeutic potential of carbon monoxide releasing molecule A-1 (CORM A-1) in oxidative stress-mediated liver injury. Overnight-fasted mice were injected with acetaminophen (APAP; 300 mg/kg; intraperitoneally) and were sacrificed at 4 and 12 h. They showed elevated levels of serum transaminases, depleted hepatic glutathione (GSH) and hepatocyte necrosis. Mice injected with CORM A-1 (20 mg/kg) 1 h after APAP administration, had reduced serum transaminases, preserved hepatic GSH and reduced hepatocyte necrosis. Mice that received a lethal dose of APAP (600 mg/kg), died by 10 h; but those co-treated with CORM A-1 showed a 50% survival. Compared to APAP-treated mice, livers from those co-treated with CORM A-1, had upregulation of Nrf2 and ARE genes (HO-1, GCLM and NQO-1). APAP-treated mice had elevated hepatic mRNA levels of inflammatory genes (Nf-κB, TNF-α, IL1-ß and IL-6), an effect blunted in those co-treated with CORM A-1. In tert-butyl hydroperoxide (t-BHP)-treated HepG2 cells, CORM A-1 augmented cell viability, reduced oxidative stress, activated the nuclear factor erythroid 2-related factor 2 (Nrf2) and anti-oxidant response element (ARE) genes. The molecular docking profile of CO in the kelch domain of Keap1 protein suggested that CO released from CORM A-1 mediated Nrf2 activation. Collectively, these data indicate that CORM A-1 reduces oxidative stress by upregulating Nrf2 and related genes, and restoring hepatic GSH, to reduce hepatocyte necrosis and thus minimize liver injury that contributes to an overall improved survival rate.