Obesity Exacerbates Coxsackievirus Infection via Lipid-Induced Mitochondrial Reactive Oxygen Species Generation.

Coxsackievirus B3 (CVB3) infection causes acute pancreatitis and myocarditis. However, its pathophysiological mechanism is unclear. Here, we investigated how lipid metabolism is associated with exacerbation of CVB3 pathology using high-fat diet (HFD)-induced obese mice. Mice were intraperitoneally inoculated with 1×106 pfu/mouse of CVB3 after being fed a control or HFD to induce obesity. Mice were treated with mitoquinone (MitoQ) to reduce the level of mitochondrial ROS (mtROS). In obese mice, lipotoxicity of white adipose tissue-induced inflammation caused increased replication of CVB3 and mortality. The coxsackievirus adenovirus receptor increased under obese conditions, facilitating CVB3 replication in vitro. However, lipid-treated cells with receptor-specific inhibitors did not reduce CVB3 replication. In addition, lipid treatment increased mitochondria-derived vesicle formation and the number of multivesicular bodies. Alternatively, we found that inhibition of lipid-induced mtROS decreased viral replication. Notably, HFD-fed mice were more susceptible to CVB3-induced mortality in association with increased levels of CVB3 replication in adipose tissue, which was ameliorated by administration of the mtROS inhibitor, MitoQ. These results suggest that mtROS inhibitors can be used as potential treatments for CVB3 infection.

Ultrasound-activated particles as CRISPR/Cas9 delivery system for androgenic alopecia therapy.

Compared to a plasmid, viral, and other delivery systems, direct Cas9/sgRNA protein delivery has several advantages such as low off-targeting effects and non-integration, but it still has limitations due to low transfer efficiency. As such, the CRISPR/Cas9 system is being developed in combination with nano-carrier technology to enhance delivery efficiency and biocompatibility. We designed a microbubble-nanoliposomal particle as a Cas9/sgRNA riboprotein complex carrier, which effectively facilitates local delivery to a specific site when agitated by ultrasound activation. In practice, we successfully transferred the protein constructs into dermal papilla cells in the hair follicle of androgenic alopecia animals by microbubble cavitation induced sonoporation of our particle. The delivered Cas9/sgRNA recognized and edited specifically the target gene with high efficiency in vitro and in vivo, thus recovering hair growth. We demonstrated the topical application of ultrasound-activated nanoparticles for androgenic alopecia therapy through the suppression of SRD5A2 protein production by CRISPR-based genomic editing.

Differential Mechanism of ATP Production Occurs in Response to Succinylacetone in Colon Cancer Cells.

Our aim was to verify the potential ability of succinylacetone (SA) to inhibit mitochondrial function, thereby suppressing cancer cell proliferation. SA treatment caused apoptosis in HCT116 and HT29 cells, but not in SW480 cells, with mitochondria playing a key role. We checked for dysfunctional mitochondria after SA treatment. Mitochondria of HT29 cells were swollen, indicating damage, whereas in HCT116 cells, several mitochondria had a diminished size. Damaged mitochondria decreased ATP production and induced reactive oxygen species (ROS) in the cells. To understand SA-induced reduction in ATP production, we investigated the electron transfer chains (ETC) and pyruvate dehydrogenase kinase (PDK) activity, which prevents the transfer of acetyl-CoA to the TCA (tricarboxylic acid) cycle by inhibiting PDH (pyruvate dehydrogenase) activity. In each cell line, the inhibitory mechanism of ATP by SA was different. The activity of complex III consisting of the mitochondrial ETCs in HT29 cells was decreased. In contrast, PDH activity in HCT116 cells was reduced. Nicotinamide nucleotide transhydrogenase (NNT)-removing reactive oxygen species (ROS) was upregulated in HT29 cells, but not in HCT116 cells, indicating that in HT29 cells, a defense mechanism was activated against ROS. Collectively, our study showed a differential mechanism occurs in response to SA in colon cancer cells.

2-Phenyl-8-(1-phenylallyl)-chromenone compounds have a pan-PPAR modulator pharmacophore.

Adiponectin is an adipocytokine with insulin-sensitizing, anti-atherogenic, and anti-inflammatory properties. Adiponectin secretion-inducing compounds have therapeutic potential in a variety of metabolic diseases. Phenotypic screening led to the discovery that 5,7-dihydroxy-8-(1-(4-hydroxy-3-methoxyphenyl)allyl)-2-phenyl-4H-chromen-4-one (compound 1) had adiponectin secretion-inducing activity during adipogenesis in human bone marrow mesenchymal stem cells (hBM-MSCs). Compound 1 was originally reported to be an anti-cancer chemical isolated from natural honeybee propolis, and its adiponectin secretion-inducing activity was found in non-cytotoxic concentrations. In a target identification study, compound 1 and its potent synthetic derivative compound 5 were shown to be novel pan-peroxisome proliferator-activator receptor (PPAR) modulators. Molecular docking models with PPARs have indicated that the binding modes of chromenone compounds preferentially interacted with the hydrophobic ligand binding pocket of PPARs. In addition, chromenone compounds have been shown to result in different phenotypic outcomes in the transcriptional regulation of lipid metabolic enzymes than those of selective PPAR mono-agonists for PPARα, PPARγ, and PPARδ. In line with the pharmacology of adiponectin and PPAR pan-modulators, compounds 1 and 5 may have diverse therapeutic potentials to treat cancer and metabolic diseases.

Structure analysis and antioxidant activities of an amylopectin-type polysaccharide isolated from dried fruits of Terminalia chebula.

Dried fruits of Terminalia chebula (TF) are used as herbal medicine for diverse symptoms, and their bioactivities are known to involve antioxidant activities. The aim of this study was to elucidate the structure and antioxidant effects of an active TF polysaccharide (TFP). The neutral polysaccharide (named as TFP-a) isolated by ion-exchange chromatography was a homogenous α-Glc-rich polysaccharide (over 70% α-Glc, 534.9 kDa, PDI 1.36) with a porous and flake-like morphology. Linkage and NMR data comprehensively showed that TFP-a was an amylopectin-type polysaccharide with (1→4)-α-Glc(p) backbone branched at C6/C2. Side chains were composed of (1→4)-ß-Gal(p) substituted with α-Ara(f), ß-GalUA(p), ß-GalUA(p)-Me, and α-Rham(p). In antioxidant activity assays, TFP-a exhibited potent and concentration-dependent antioxidant effects, including DPPH and superoxide radical scavenging and reducing power. We concluded that TFP-a is an amylopectin-type polysaccharide that may be used as a potential natural antioxidant.

Lecithin nano-liposomal particle as a CRISPR/Cas9 complex delivery system for treating type 2 diabetes.

BACKGROUND: Protein-based Cas9 in vivo gene editing therapeutics have practical limitations owing to their instability and low efficacy. To overcome these obstacles and improve stability, we designed a nanocarrier primarily consisting of lecithin that can efficiently target liver disease and encapsulate complexes of Cas9 with a single-stranded guide RNA (sgRNA) ribonucleoprotein (Cas9-RNP) through polymer fusion self-assembly. RESULTS: In this study, we optimized an sgRNA sequence specifically for dipeptidyl peptidase-4 gene (DPP-4) to modulate the function of glucagon-like peptide 1. We then injected our nanocarrier Cas9-RNP complexes directly into type 2 diabetes mellitus (T2DM) db/db mice, which disrupted the expression of DPP-4 gene in T2DM mice with remarkable efficacy. The decline in DPP-4 enzyme activity was also accompanied by normalized blood glucose levels, insulin response, and reduced liver and kidney damage. These outcomes were found to be similar to those of sitagliptin, the current chemical DPP-4 inhibition therapy drug which requires recurrent doses. CONCLUSIONS: Our results demonstrate that a nano-liposomal carrier system with therapeutic Cas9-RNP has great potential as a platform to improve genomic editing therapies for human liver diseases.

Active Turnover of Heme in Hibernation Period in Mammals.

Heme oxygenase (HO)-1 plays an important role during hibernation by catalyzing the degradation of heme to biliverdin/bilirubin, ferrous iron, and carbon monoxide, which activates the protective mechanisms against stress. In this context, it was important to analyze the metabolic processes of heme. Nevertheless, to date, no study has approached on biosynthesis of heme. Therefore, our study aims to understand the process of heme biosynthesis, which regulates cell survival in conditions of hypothermia and calorie restriction (CR). During hibernation, the mRNA levels of enzymes responsible for de novo heme biosynthesis were increased in the liver tissue of a Syrian hamster model of hibernation. Moreover, heme trafficking and iron metabolism were found to be more active, as assessed based on the changes in the levels of heme transporter and ferroportin mRNA. The levels of HO-1, a powerful antioxidant, were also upregulated during hibernation. Additionally, increased levels of Sirt-1 mRNA were also observed. These enzymes are known to act as cellular metabolic sensors that activate the cytoprotective mechanisms. These results indicate that HO-1 induction, brought about by the upregulation of heme during the pre-hibernation period, may protect against external stress. Here, we describe heme catabolism during hibernation by analyzing the regulation of the key molecular players involved in heme metabolism. Therefore, this study offers a new strategy for the better regulation of intracellular heme concentrations during hypothermia and other stresses.

Aquaporin 11-Dependent Inhibition of Proliferation by Deuterium Oxide in Activated Hepatic Stellate Cells.

Deuterium oxide (D2O) has been reported to be active toward various in vitro cell lines in combination with phytochemicals. Our objective was to describe, for the first time, the effect of D2O on the proliferation of hepatic stellate cells (HSCs). After D2O treatment, the p53-cyclin-dependent kinase (CDK) pathway was stimulated, leading to inhibition of the proliferation of HSCs and an increase in the [ATP]/[ADP] ratio. We also evaluated the role of aquaporin (AQP) 11 in activated HSCs. We found that D2O treatment decreased AQP11 expression levels. Of note, AQP11 levels elevated by a genetic approach counteracted the D2O-mediated inhibition of proliferation. In addition, the expression levels of AQP11 negatively correlated with those of p53. On the other hand, cells transfected with an AQP11-targeted small interfering RNA (siRNA) showed enhanced inhibition of proliferation. These findings suggest that the inhibition of cell proliferation by D2O in activated HSCs could be AQP11 dependent. Our previous studies have documented that bisdemethoxycurcumin (BDMC) induces apoptosis by regulating heme oxygenase (HO)-1 protein expression in activated HSCs. In the current study, we tested whether cotreatment with BDMC and D2O can modulate the AQP11-dependent inhibition of cell proliferation effectively. We observed that D2O cotreatment with BDMC significantly decreased cell proliferation compared to treatment with D2O alone, and this effect was accompanied by downregulation of HO-1 and an increase in p53 levels.

DDQ-Promoted Mild and Efficient Metal-Free Oxidative α-Cyanation of N-Acyl/Sulfonyl 1,2,3,4-Tetrahydroisoquinolines.

A mild and highly efficient metal-free oxidative α-cyanation of N-acyl/sulfonyl 1,2,3,4-tetrahydroisoquinolines (THIQs) has been accomplished at an ambient temperature via DDQ oxidation and subsequent trapping of N-acyl/sulfonyl iminium ions with (n-Bu)3SnCN. Employing readily removable N-acyl/sulfonyl groups as protecting groups rather than N-aryl ones enables a wide range of applications in natural product synthesis. The synthetic utility of the method was illustrated using a short and efficient formal total synthesis of (±)-calycotomine in three steps.

3,5-Diethoxy-3'-Hydroxyresveratrol (DEHR) Ameliorates Liver Fibrosis via Caveolin-1 Activation in Hepatic Stellate Cells and in a Mouse Model of Bile Duct Ligation Injury.

Hepatic stellate cells (HSCs) are involved in the pathogenesis of liver fibrosis. Resveratrol, 3,5,4'-trihydroxystilbene, is a dietary polyphenol found in natural food products. Here, we evaluated the anti-proliferative effects of a synthetic resveratrol derivative, 3,5-diethoxy-3'-hydroxyresveratrol (DEHR), on HSCs. Flow cytometry and Western blot analyses showed that DEHR induces apoptosis through the upregulation of cleaved caspase-3 and poly (ADP-ribose) polymerase expression and reduction in the level of an anti-apoptotic protein B-cell lymphoma 2 (Bcl2). As caveolin-1 (CAV1), a competitive inhibitor of heme oxygenase 1 (HO-1), is related to apoptotic proteins in hepatic cells, we focused on the role of CAV1 in DEHR-induced apoptosis in HSCs through Western blot analyses. Our results showed that the inhibitory effect of DEHR on cell viability was stronger in HO-1 siRNA-transfected cells but weakened in CAV1 siRNA-transfected cells. Collagen concentration was significantly reduced, whereas CAV1 expression increased after treatment of a bile duct ligation injury-induced liver fibrosis model with DEHR for four weeks. We confirmed that DEHR treatment significantly reduced fibrous hyperplasia around the central veins, using hematoxylin and eosin and Sirius red staining. DEHR ameliorates liver fibrosis in vitro and in vivo, possibly through a mechanism involving CAV1.

Long-term safety outcome of systemic immunosuppression in pig-to-nonhuman primate corneal xenotransplantation.

BACKGROUND: Safety concerns exist for corneal recipients under immunosuppression. We report long-term safety results of porcine corneal xenotransplantation under immunosuppression in nonhuman primates. METHODS: Systemic monitoring data from 49 Chinese rhesus macaques that received pig corneal transplant between 2009 and 2018 were retrospectively reviewed. The recipients were divided into 4 groups depending on the systemic immunosuppressants used: (a) conventional steroid group; costimulation blockade groups ([b] anti-CD154 antibody, [c] anti-CD40 antibody); and (d) commercially available immunosuppressants (anti-CD20 antibody, tacrolimus, basiliximab) group. We compared results of general condition monitoring; hematologic, biochemical, and electrolyte tests; and Rhesus Cytomegalovirus infection monitoring. RESULTS: All recipients recovered from early weight loss. White blood cell counts significantly decreased at 6 months in the steroid and anti-CD154 groups. Abnormal liver and kidney function and electrolyte imbalance were not observed in all groups. The mean value of Rhesus Cytomegalovirus DNA copies was consistently lower than 200 copies/mL, and antibody titers did not change over time in all groups. Tacrolimus-associated thrombotic microangiopathy was developed in one case, which resolved after discontinuation of tacrolimus. In 2017, a simian varicella virus outbreak led to clinical signs in 5 that received immunosuppressive therapies, of which 3 died. CONCLUSION: Costimulatory blockade-based and anti-CD20 antibody/tacrolimus-based immunosuppressive therapies seem to be comparably safe with steroid therapy in nonhuman primates receiving corneal xenotransplantation, as they did not reactivate Rhesus Cytomegalovirus and maintained manageable systemic status. Although reactivation is rare, antiviral prophylaxis for simian varicella virus should be considered in immunocompromised hosts.

Comparative efficacy of anti-CD40 antibody-mediated costimulation blockade on long-term survival of full-thickness porcine corneal grafts in nonhuman primates.

Porcine corneas may be good substitutes for human corneas in donor shortage. Therefore, we evaluated the efficacy and safety of an anti-CD40 antibody-based regimen compared with an anti-CD20 antibody-based regimen on the survival of full-thickness corneas in pig-to-rhesus xenotransplant. Thirteen Chinese rhesuses underwent full-thickness corneal xenotransplant. Six were administered anti-CD40 antibody, and the others were administered anti-CD20 antibody, basiliximab, and tacrolimus. Graft survival and changes in lymphocyte, donor-specific and anti-Galα1,3Galß1,4GlcNAc-R (αGal) antibody, and aqueous complement levels were evaluated. Treatment with the anti-CD40 antibody (>511, >422, >273, >203, >196, 41 days) and anti-CD20 antibody (>470, 297, >260, >210, >184, 134, >97 days) resulted in long-term survival of grafts. In the anti-CD20 group, the number of activated B cells was significantly lower than that in the anti-CD40 group, and the level of aqueous complements at 6 months was significantly higher than the preoperative level. There were no differences in the levels of T cells or donor-specific and anti-αGal antibodies between the 2 groups. In the anti-CD20 group, 3 primates had adverse reactions. In conclusion, both the anti-CD40 antibody- and the anti-CD20 antibody-based protocols were effective for the long-term survival of full-thickness corneal xenografts, but the anti-CD40 antibody-based treatment had fewer adverse effects.

The novel resveratrol derivative 3,5-diethoxy-3',4'-dihydroxy-trans-stilbene induces mitochondrial ROS-mediated ER stress and cell death in human hepatoma cells in vitro.

Resveratrol (3,5,4'-trihydroxy-trans-stilbene) is a well-known polyphenol that is present in grapes, peanuts, pine seeds, and several other plants. Resveratrol exerts deleterious effects on various types of human cancer cells. Here, we analyzed the cell death-inducing mechanisms of resveratrol-006 (Res-006), a novel resveratrol derivative in human liver cancer cells in vitro. Res-006 was more effectively suppressed the viability of HepG2 human hepatoma cells than resveratrol (the IC50 values were 67.2 and 354.8 µmol/L, respectively). Co-treatment with the ER stress regulator 4-phenylbutyrate (0.5 mmol/L) or the ROS inhibitor N-acetyl-L-cysteine (NAC, 1 mmol/L) significantly attenuated Res-006-induced HepG2 cell death, suggesting that pro-apoptotic ER stress and/or ROS may govern the Res-006-induced HepG2 cell death. We further revealed that treatment of HepG2 cells with Res-006 (65 µmol/L) immediately elicited the dysregulation of mitochondrial dynamics and the accumulation of mitochondrial ROS. It also collapsed the mitochondrial membrane potential and further induced ER stress and cell death. These events, except for the change in mitochondrial morphology, were prevented by the exposure of the HepG2 cells to the mitochondrial ROS scavenger, Mito-TEMPO (300-1000 µmol/L). The results suggest that Res-006 may kill HepG2 cells through cell death pathways, including the ER stress initiated by mitochondrial ROS accumulation. The cell death induced by this novel resveratrol derivative involves crosstalk between the mitochondria and ER stress mechanisms.

Hepatoprotective Flavonoids in Opuntia ficus-indica Fruits by Reducing Oxidative Stress in Primary Rat Hepatocytes.

BACKGROUND: Liver disorder was associated with alcohol consumption caused by hepatic cellular damages. Opuntia ficus-indica fruit extracts (OFIEs), which contain betalain pigments and polyphenols including flavonoids, have been introduced as reducing hangover symptoms and liver protective activity. OBJECTIVE: To evaluate hepatoprotective activity of OFIEs and isolated compounds by high-speed countercurrent chromatography (HSCCC). MATERIALS AND METHODS: The extract of O. ficus-indica fruits was fractionated into methylene chloride and n-butanol. The n-butanol fraction was isolated by HSCCC separation (methylene chloride-methanol-n-butanol-water, 5:4:3:5, v/v/v/v). The hepatoprotective activity of OFIEs and isolated compounds was evaluated on rat primary hepatocytes against ethanol-induced toxicity. Antioxidative parameters such as glutathione reductase and glutathione peroxidase (GSH-Px) enzymes and the GSH content were measured. RESULTS: Two flavonoids, quercetin 3-O-methyl ester (1) and (+)-taxifolin, and two flavonoid glycosides, isorhamnetin 3-O-ß-d-glucoside (3) and narcissin (4), were isolated from the n-butanol fraction by HSCCC separation. Among them, compound 2 significantly protected rat primary hepatocytes against ethanol exposure by preserving antioxidative properties of GR and GSH-Px. CONCLUSIONS: OFIEs and (+)-taxifolin were suggested to reduce hepatic damage by alcoholic oxidative stress. SUMMARY: Hepatoprotective Flavonoids were isolated from Opuntia ficus-indica by high -speed countercurrent chromatography (HSCCC).

Cytotoxic pterosins from Pteris multifida roots against HCT116 human colon cancer cells.

Two new pterosin glycosides, (2S,3S)-pterosin C 3-O-ß-d-(4'-(E)-caffeoyl)-glucopyranoside (1) and (2S,3S)-pterosin C 3-O-ß-d-(6'-(E)-p-coumaroyl)-glucopyranoside (2), were isolated from Pteris multifida (Pteridaceae) roots along with ten known pterosin compounds (3-12). The chemical structures of the isolated compounds were elucidated by extensive analysis of the 1D, 2D NMR, HRESIMS, and CD spectroscopic data. The cytotoxicities of 1-12 against HCT116 human colorectal cancer cell line were evaluated. Among the isolates, compound 1 showed moderate antiproliferative activity in HCT116 cells with an IC50 value of 8.0±1.7µM. Additionally, 1 induced the upregulation of the caspase-9 and procaspase-9 levels in Western blots and increased the annexin V/propidium iodide (PI)-positive cell population in flow cytometry.

Lignans from Opuntia ficus-indica seeds protect rat primary hepatocytes and HepG2 cells against ethanol-induced oxidative stress.

Bioactivity-guided isolation of Opuntia ficus-indica (Cactaceae) seeds against ethanol-treated primary rat hepatocytes yielded six lignan compounds. Among the isolates, furofuran lignans 4-6, significantly protected rat hepatocytes against ethanol-induced oxidative stress by reducing intracellular reactive oxygen species levels, preserving antioxidative defense enzyme activities, and maintaining the glutathione content. Moreover, 4 dose-dependently induced the heme oxygenase-1 expression in HepG2 cells.

Stereoisomer-specific anticancer activities of ginsenoside Rg3 and Rh2 in HepG2 cells: disparity in cytotoxicity and autophagy-inducing effects due to 20(S)-epimers.

Autophagy has been an emerging field in the treatment of hepatic carcinoma since anticancer therapies were shown to ignite autophagy in vitro and in vivo. Here we report that ginsenoside Rg3 and Rh2, major components of red ginseng, induce apoptotic cell death in a stereoisomer-specific fashion. The 20(S)-forms of Rg3 and Rh2, but not their respective 20(R)-forms, promoted cell death in a dose-dependent manner accompanied by downregulation of Bcl2 and upregulation of Fas, resulting in apoptosis of HepG2 cells with poly ADP ribose polymerase cleavage. The LD50 value [45 µM for Rg3(S), less than 10 µM for Rh2(S)] and gross morphological electron microscopic observation revealed more severe cellular damage in cells treated with Rh2(S) than in those treated with Rg3(S). Both Rg3(S) and Rh2(S) also induced autophagy when undergoing induced apoptosis. Inhibition of autophagy with lysosomotrophic agents significantly potentiated the cellular damage, implying a favorable switch of the cell fate to tumor cell death. Blocking intracellular calcium with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetrakis(acetoxymethyl ester) (BAPTA-AM) restored the cell death induced by both Rg3(S) and Rh2(S). Our results suggest that the 20(S)-forms of Rg3 and Rh2 in red ginseng possess more potent antitumor activity with autophagy than their 20(R)-forms via calcium-dependent apoptosis.

Bisdemethoxycurcumin Induces apoptosis in activated hepatic stellate cells via cannabinoid receptor 2.

Activated Hepatic Stellate Cells (HSCs), major fibrogenic cells in the liver, undergo apoptosis when liver injuries cease, which may contribute to the resolution of fibrosis. Bisdemethoxycurcumin (BDMC) is a natural derivative of curcumin with anti-inflammatory and anti-cancer activities. The therapeutic potential of BDMC in hepatic fibrosis has not been studied thus far in the context of the apoptosis in activated HSCs. In the current study, we compared the activities of BDMC and curcumin in the HSC-T6 cell line and demonstrated that BDMC relatively induced a potent apoptosis. BDMC-induced apoptosis was mediated by a combinatory inhibition of cytoprotective proteins, such as Bcl2 and heme oxygenase-1 and increased generation of reactive oxygen species. Intriguingly, BDMC-induced apoptosis was reversed with co-treatment of sr144528, a cannabinoid receptor (CBR) 2 antagonist, which was confirmed with genetic downregulation of the receptor using siCBR2. Additionally, incubation with BDMC increased the formation of death-induced signaling complex in HSC-T6 cells. Treatment with BDMC significantly diminished total intracellular ATP levels and upregulated ATP inhibitory factor-1. Collectively, the results demonstrate that BDMC induces apoptosis in activated HSCs, but not in hepatocytes, by impairing cellular energetics and causing a downregulation of cytoprotective proteins, likely through a mechanism that involves CBR2.