8-Prenylgenistein Isoflavone in Cheonggukjang Acts as a Novel AMPK Activator Attenuating Hepatic Steatosis by Enhancing the SIRT1-Mediated Pathway.

8-Prenylgenistein (8PG), a genistein derivative, is present in fermented soybeans (Glycine max), including cheonggukjang (CGJ), and exhibits osteoprotective, osteogenic, and antiadipogenic properties. However, the hepatoprotective effects of 8PG and its underlying molecular mechanisms remain largely unexplored. Here, we identified the high binding affinity of 8PG with AMP-activated protein kinase (AMPK) and sirtuin 1 (SIRT1), which acts as a potent AMPK activator that counteracts hepatic steatosis. Notably, 8PG exhibited better pharmacokinetics with greater absorption and higher plasma binding than the positive controls for the target proteins. Moreover, 8PG exerted non-carcinogenic activity in rats and significantly increased AMPK phosphorylation. Compound C, an AMPK inhibitor, did not antagonize 8PG-activated AMPK in HepG2 cells. 8PG significantly attenuated palmitate-induced lipid accumulation and enhanced phosphorylated AMPK and its downstream target, acetyl-CoA carboxylase. Further, 8PG activated nuclear SIRT1 at the protein level, which promoted fatty acid oxidation in palmitate-treated HepG2 cells. Overall, 8PG acts as a potent AMPK activator, further attenuating hepatic steatosis via the SIRT1-mediated pathway and providing new avenues for dietary interventions to treat metabolic dysfunction-associated steatotic liver disease (MASLD).

Exploring 2-mercapto-N-arylacetamide analogs as promising anti-melanogenic agents: in vitro and in vivo evaluation.

Based on the hypothesis that the 2-mercaptoacetamide moiety chelates the copper ions of tyrosinase, 2-mercapto-N-arylacetamide (2-MAA) analogs were designed and synthesized as potential tyrosinase inhibitors. Four 2-MAA analogs showed low IC50 values ranging from 0.95 to 2.0 µM against mushroom tyrosinase, which was 12-26 times lower than that of kojic acid (IC50 value = 24.3 µM). However, according to a copper ion chelation experiment performed, the 2-MAA analogs did not participate in chelation with copper ions. To identify the mode of inhibition of the 2-MAA analogs, kinetic studies were performed, and the results were supported by docking results. In addition, docking simulation results suggested that the 2-MAA analogs strongly inhibited tyrosinase activity because of the hydrogen bonding of the amide NH group and the hydrophobic interaction of the aryl ring instead of chelation with copper ions. In experiments using B16F10 cells, 2-MAA analogs were shown to inhibit melanin production by inhibiting cellular tyrosinase activity. Western blotting showed that in addition to directly inhibiting tyrosinase activity, analog 7 also has an anti-melanogenic effect by inhibiting the expression of microphthalmia-associated transcription factor (MITF) and tyrosinase. The 2-MAA analogs showed no appreciable cytotoxicity against HaCaT and B16F10 cells, making them suitable for dermal applications. In a depigmentation experiment using zebrafish embryos, analogs 1 and 2 showed more potent depigmentation effects than kojic acid even at 1000 times lower concentration than that of kojic acid. These results suggest that the 2-MAA analogs are promising anti-melanogenic agents that can inhibit most tyrosinases in various species.

Design, synthesis, and anti-melanogenic efficacy of 2-mercaptobenzoxazoles with nanomolar tyrosinase activity inhibition.

Tyrosinase is a metalloenzyme that contains copper(II) ions. We designed and synthesized eight known low-molecular-weight 2-mercaptobenzoxazole (2-MBO) analogs as tyrosinase inhibitors. Our focus was on the mercapto functional group, which interacts with copper ions. Analogs 1-3 exhibited mushroom tyrosinase inhibitory activity at the nanomolar level and demonstrated strong potency with extremely low half-maximal inhibitory concentration (IC50) values of 80-90 nM for l-dopa and 100-240 nM for l-tyrosine. Analogs 2, 4, and 5 showed the most potent anti-melanogenic effects in B16F10 cells, and their mode of action was demonstrated by kinetic analysis. Their anti-melanogenic effects were similar to the tyrosinase inhibition results, suggesting that their anti-melanogenic effects could be attributed to their tyrosinase inhibitory ability. Experiments using copper-chelating activity assays and changes in tyrosinase inhibitory activity with and without CuSO4 demonstrated that 2-MBO analogs inhibit tyrosinase activity by chelating the copper ions of tyrosinase. In conclusion, the 2-MBO analogs show potential as anti-melanogenic agents with potent tyrosinase inhibitory activity.

Investigation of the Efficacy of Benzylidene-3-methyl-2-thioxothiazolidin-4-one Analogs with Antioxidant Activities on the Inhibition of Mushroom and Mammal Tyrosinases.

Based on the fact that substances with a ß-phenyl-α,ß-unsaturated carbonyl (PUSC) motif confer strong tyrosinase inhibitory activity, benzylidene-3-methyl-2-thioxothiazolidin-4-one (BMTTZD) analogs 1-8 were prepared as potential tyrosinase inhibitors. Four analogs (1-3 and 5) inhibited mushroom tyrosinase strongly. Especially, analog 3 showed an inhibitory effect that was 220 and 22 times more powerful than kojic acid in the presence of l-tyrosine and l-dopa, respectively. A kinetic study utilizing mushroom tyrosinase showed that analogs 1 and 3 competitively inhibited tyrosinase, whereas analogs 2 and 5 inhibited tyrosinase in a mixed manner. A docking simulation study indicated that analogs 2 and 5 could bind to both the tyrosinase active and allosteric sites with high binding affinities. In cell-based experiments using B16F10 cells, analogs 1, 3, and 5 effectively inhibited melanin production; their anti-melanogenic effects were attributed to their ability to inhibit intracellular tyrosinase activity. Moreover, analogs 1, 3, and 5 inhibited in situ B16F10 cellular tyrosinase activity. In three antioxidant experiments, analogs 2 and 3 exhibited strong antioxidant efficacy, similar to that of the positive controls. These results suggest that the BMTTZD analogs are promising tyrosinase inhibitors for the treatment of hyperpigmentation-related disorders.

MHY446 induces apoptosis via reactive oxygen species-mediated endoplasmic reticulum stress in HCT116 human colorectal cancer cells.

This study investigated the potential of a newly synthesized histone deacetylase (HDAC) inhibitor, MHY446, in inducing cell death in HCT116 colorectal cancer cells and compared its activity with that of suberoylanilide hydroxamic acid (SAHA), a well-known HDAC inhibitor. The results showed that MHY446 increased the acetylation of histones H3 and H4 and decreased the expression and activity of HDAC proteins in HCT116 cells. Additionally, MHY446 was confirmed to bind more strongly to HDAC1 than HDAC2 and inhibit its activity. In vivo experiments using nude mice revealed that MHY446 was as effective as SAHA in inhibiting HCT116 cell-grafted tumor growth. This study also evaluated the biological effects of MHY446 on cell survival and death pathways. The reactive oxygen species (ROS) scavenger N-acetyl-L-cysteine (NAC) confirmed that ROS play a role in MHY446-induced cell death by reducing poly(ADP-ribose) polymerase cleavage. MHY446 also induced cell death via endoplasmic reticulum (ER) stress by increasing the expression of ER stress-related proteins. NAC treatment decreased the expression of ER stress-related proteins, indicating that ROS mediate ER stress as an upstream signaling pathway and induce cell death. While MHY446 did not exhibit superior HDAC inhibition efficacy compared to SAHA, it is anticipated to provide innovative insights into the future development of therapeutic agents for human CRC by offering novel chemical structure-activity relationship-related information.

TLR7 activation by miR-21 promotes renal fibrosis by activating the pro-inflammatory signaling pathway in tubule epithelial cells.

BACKGROUND: Toll-like receptor 7 (TLR7) is an endosomal TLR activated by single-stranded RNA, including endogenous microRNAs. Although TLR7 is known to promote inflammatory responses in pathophysiological conditions, its role in renal fibrosis has not been investigated. Here, we aim to investigate the inflammatory roles of TLR7 in kidney inflammation and fibrosis. METHODS: TLR7 knockout mice (Tlr7 -/-) subjected to AD-induced kidney injury were utilized to examine the role of TLR7 in kidney fibrosis. To elucidate the role of TLR7 in renal epithelial cells, NRK52E rat renal tubule epithelial cells were employed. RESULTS: Under fibrotic conditions induced by an adenine diet (AD), TLR7 was significantly increased in damaged tubule epithelial cells, where macrophages were highly infiltrated. TLR7 deficiency protected against AD-induced tubular damage, inflammation, and renal fibrosis. Under in vitro conditions, TLR7 activation increased NF-κB activity and induced chemokine expression, whereas TLR7 inhibition effectively blocked NF-κB activation. Furthermore, among the known TLR7 endogenous ligands, miR-21 was significantly upregulated in the tubular epithelial regions. In NRK52E cells, miR-21 treatment induced pro-inflammatory responses, which could be blocked by a TLR7 inhibitor. When the TLR7 inhibitor, M5049, was administered to the AD-induced renal fibrosis model, TLR7 inhibition significantly attenuated AD-induced renal inflammation and fibrosis. CONCLUSIONS: Overall, activation of TLR7 by endogenous miR-21 in renal epithelial cells contributes to inflammatory responses in a renal fibrosis model, suggesting a possible therapeutic target for the treatment of renal fibrosis. Video Abstract.

Integrative Transcriptomic Analysis Reveals Upregulated Apoptotic Signaling in Wound-Healing Pathway in Rat Liver Fibrosis Models.

Liver fibrosis, defined by the aberrant accumulation of extracellular matrix proteins in liver tissue due to chronic inflammation, represents a pressing global health issue. In this study, we investigated the transcriptomic signatures of three independent liver fibrosis models induced by bile duct ligation, carbon tetrachloride, and dimethylnitrosamine (DMN) to unravel the pathological mechanisms underlying hepatic fibrosis. We observed significant changes in gene expression linked to key characteristics of liver fibrosis, with a distinctive correlation to the burn-wound-healing pathway. Building on these transcriptomic insights, we further probed the p53 signaling pathways within the DMN-induced rat liver fibrosis model, utilizing western blot analysis. We observed a pronounced elevation in p53 protein levels and heightened ratios of BAX/BCL2, cleaved/pro-CASPASE-3, and cleaved/full length-PARP in the livers of DMN-exposed rats. Furthermore, we discovered that orally administering oligonol-a polyphenol, derived from lychee, with anti-oxidative properties-effectively countered the overexpressions of pivotal apoptotic genes within these fibrotic models. In conclusion, our findings offer an in-depth understanding of the molecular alterations contributing to liver fibrosis, spotlighting the essential role of the apoptosis pathway tied to the burn-wound-healing process. Most importantly, our research proposes that regulating this pathway, specifically the balance of apoptosis, could serve as a potential therapeutic approach for treating liver fibrosis.

Sex-Mediated Differences in TNF Signaling- and ECM-Related Gene Expression in Aged Rat Kidney.

Aging leads to the functional decline of an organism, which is associated with age and sex. To understand the functional change of kidneys depending on age and sex, we carried out a transcriptome analysis using RNA sequencing (RNA-Seq) data from rat kidneys. Four differentially expressed gene (DEG) sets were generated according to age and sex, and Gene Ontology analysis and overlapping analysis of Kyoto Encyclopedia of Genes and Genomes pathways were performed for the DEG sets. Through the analysis, we revealed that inflammation- and extracellular matrix (ECM)-related genes and pathways were upregulated in both males and females during aging, which was more prominent in old males than in old females. Furthermore, quantitative real-time PCR analysis confirmed that the expression of tumor necrosis factor (TNF) signaling-related genes, Birc3, Socs3, and Tnfrsf1b, and ECM-related genes, Cd44, Col3a1, and Col5a2, which showed that the genes were markedly upregulated in males and not females during aging. Also, hematoxylin-eosin (H&E) staining for histological analysis showed that renal damage was highly shown in old males rather than old females. In conclusion, in the rat kidney, the genes involved in TNF signaling and ECM accumulation are upregulated in males more than in females during aging. These results suggest that the upregulation of the genes may have a higher contribution to age-related kidney inflammation and fibrosis in males than in females.

MHY2251, a New SIRT1 Inhibitor, Induces Apoptosis via JNK/p53 Pathway in HCT116 Human Colorectal Cancer Cells.

Sirtuins (SIRTs) belong to the nicotinamide adenine dinucleotide (NAD+)-dependent class III histone deacetylase family. They are key regulators of cellular and physiological processes, such as cell survival, senescence, differentiation, DNA damage and stress response, cellular metabolism, and aging. SIRTs also influence carcinogenesis, making them potential targets for anticancer therapeutic strategies. In this study, we investigated the anticancer properties and underlying molecular mechanisms of a novel SIRT1 inhibitor, MHY2251, in human colorectal cancer (CRC) cells. MHY2251 reduced the viability of various human CRC cell lines, especially those with wild-type TP53. MHY2251 inhibited SIRT1 activity and SIRT1/2 protein expression, while promoting p53 acetylation, which is a target of SIRT1 in HCT116 cells. MHY2251 treatment triggered apoptosis in HCT116 cells. It increased the percentage of late apoptotic cells and the sub-G1 fraction (as detected by flow cytometric analysis) and induced DNA fragmentation. In addition, MHY2251 upregulated the expression of FasL and Fas, altered the ratio of Bax/Bcl-2, downregulated the levels of pro-caspase-8, -9, and -3 proteins, and induced subsequent poly(ADP-ribose) polymerase cleavage. The induction of apoptosis by MHY2251 was related to the activation of the caspase cascade, which was significantly attenuated by pre-treatment with Z-VAD-FMK, a pan-caspase inhibitor. Furthermore, MHY2251 stimulated the phosphorylation of c-Jun N-terminal kinase (JNK), and MHY2251-triggered apoptosis was blocked by pre-treatment with SP600125, a JNK inhibitor. This finding indicated the specific involvement of JNK in MHY2251-induced apoptosis. MHY2251 shows considerable potential as a therapeutic agent for targeting human CRC via the inhibition of SIRT1 and activation of JNK/p53 pathway.

MHY4571, a novel diarylcyclohexanone derivative, exerts anti-cancer activity by regulating the PKA-cAMP-response element-binding protein pathway in squamous cell lung cancer.

BACKGROUND: The protein kinase A (PKA)/cAMP response element-binding protein (CREB) has been suggested to be related to the inhibition of the proliferation of non-small cell lung cancer (NSCLC) cells. This study aimed to investigate the efficacy of a novel diarylcyclohexanone derivative, MHY4571, in regulating the PKA-CREB pathway and to study its anti-tumor role in squamous NSCLC. METHODS: We designed MHY4571 as a novel PKA inhibitor with acceptable in silico ADME properties and tested it in vitro in lung cancer cell lines and in vivo in xenograft and orthotopic mouse models of squamous cell lung carcinoma. RESULTS: MHY4571 inhibited PKA activity (> 70% inhibition) and suppressed the expression of p-PKA and p-CREB dose-dependently. MHY4571 treatment reduced lung cancer cell viability and promoted caspase 3-dependent apoptotic cell death. Orally administered MHY4571 significantly suppressed lung tumor growth in xenograft and orthotopic mouse models. PKA catalytic subunit alpha-silencing by siRNA (siPKA) strongly attenuated CREB phosphorylation; siCREB did not alter PKA protein levels or its phosphorylation, suggesting that PKA is an upstream regulator of CREB activity. MHY4571 acted synergistically with cisplatin (on co-treatment) to induce apoptotic cell death in lung cancer cells. CONCLUSIONS: Our results imply that MHY4571 may be a potential drug candidate for squamous cell lung cancer treatment.

A Novel Class of Potent Anti-Tyrosinase Compounds with Antioxidant Activity, 2-(Substituted phenyl)-5-(trifluoromethyl)benzo[d]thiazoles: In Vitro and In Silico Insights.

Sixteen compounds bearing a benzothiazole moiety were synthesized as potential tyrosinase inhibitors and evaluated for mushroom tyrosinase inhibitory activity. The compound 4-(5-(trifluoromethyl)benzo[d]thiazol-2-yl)benzene-1,3-diol (compound 1b) exhibited the highest tyrosinase activity inhibition, with an IC50 value of 0.2 ± 0.01 µM (a potency 55-fold greater than kojic acid). In silico results using mushroom tyrosinase and human tyrosinase showed that the 2,4-hydroxyl substituents on the phenyl ring of 1b played an important role in the inhibition of both tyrosinases. Kinetic studies on mushroom tyrosinase indicated that 1b is a competitive inhibitor of monophenolase and diphenolase, and this was supported by docking results. In B16F10 murine melanoma cells, 1a and 1b dose-dependently and significantly inhibited melanin production intracellularly, and melanin release into medium more strongly than kojic acid, and these effects were attributed to the inhibition of cellular tyrosinase. Furthermore, the inhibition of melanin production by 1b was found to be partially due to the inhibition of tyrosinase glycosylation and the suppression of melanogenesis-associated genes. Compound 1c, which has a catechol group, exhibited potent antioxidant activities against ROS, DPPH, and ABTS, and 1b also had strong ROS and ABTS radical scavenging activities. These results suggest that 5-(trifluoromethyl)benzothiazole derivatives are promising anti-tyrosinase lead compounds with potent antioxidant effects.

Identification of a Novel Class of Anti-Melanogenic Compounds, (Z)-5-(Substituted benzylidene)-3-phenyl-2-thioxothiazolidin-4-one Derivatives, and Their Reactive Oxygen Species Scavenging Activities.

The rate-determining role of tyrosinase makes it a critical component in the mechanism that is responsible for melanogenesis. Thirteen (Z)-5-(substituted benzylidene)-3-phenyl-2-thioxothiazolidin-4-one ((Z)-BPTT) analogs were designed based on the structural features of two potent tyrosinase inhibitors, viz. (Z)-5-(3-hydroxy-4-methoxybenzylidene)-2-thioxothiazolidin-4-one (5-HMT) and (Z)-2-(2,4-dihydroxybenzylidene)benzo[4,5]imidazo[2,1-b]thiazol-3(2H)-one (compound I). The trisubstituted double bond geometry of the (Z)-BPTT analogs that were generated by Knoevenagel condensation was determined using vicinal 1H and 13C coupling constants in 13C NMR spectra. Four analogs, numbers 1-3 and 6, inhibited mushroom tyrosinase 9 to 29 times more potently than kojic acid did. Kinetic study results indicated that these four analogs inhibited mushroom tyrosinase competitively and this was supported by docking simulation. Also, docking results using human tyrosinase suggested that analogs 2 and 3 might be potent human tyrosinase inhibitors. In vitro studies using B16F10 cells (a melanoma cell line) showed that analogs 1, 2, 3, and 6 inhibited cellular tyrosinase and melanin production more than kojic acid did, without perceptible cytotoxicity. In particular, analog 2, which possesses a catechol group, exerted an extremely potent anti-melanogenic effect. In addition, analog 2 showed strong scavenging activity against DPPH and ABTS radicals. Furthermore, analog 2 not only reduced ROS levels, which induce melanogenesis, but it also suppressed tyrosinase and MITF (microphthalamia-associated transcription factor) protein levels and the expressions of melanogenesis-related genes. These results suggest that analog 2 is an efficient tyrosinase inhibitor that alleviates melanogenesis by dual mechanisms of (i) the inhibition of melanogenesis-related proteins and genes and (ii) the direct inhibition of tyrosinase activity.

MHY2245, a Sirtuin Inhibitor, Induces Cell Cycle Arrest and Apoptosis in HCT116 Human Colorectal Cancer Cells.

Sirtuins (SIRTs), which are nicotinamide adenine dinucleotide-dependent class III histone deacetylases, regulate cell division, survival, and senescence. Although sirtinol, a synthetic SIRT inhibitor, is known to exhibit antitumor effects, its mechanism of action is not well understood. Therefore, we aimed to assess the anticancer effects and underlying mechanism of MHY2245, a derivative of sirtinol, in HCT116 human colorectal cancer cells in vitro. Treatment with MHY2245 decreased SIRT1 activity and caused DNA damage, leading to the upregulation of p53 acetylation, and increased levels of p53, phosphorylation of H2A histone family member X, ataxia telangiectasia and Rad3-related kinase, checkpoint kinase 1 (Chk1), and Chk2. The level of the breast cancer type 1 susceptibility protein was also found to decrease. MHY2245 induced G2/M phase cell cycle arrest via the downregulation of cyclin B1, cell division cycle protein 2 (Cdc2), and Cdc25c. Further, MHY2245 induced HCT116 cell death via apoptosis, which was accompanied by internucleosomal DNA fragmentation, decreased B-cell lymphoma 2 (Bcl-2) levels, increased Bcl-2-asscociated X protein levels, cleavage of poly(ADP-ribose) polymerase, and activation of caspases -3, -8, and -9. Overall, MHY2245 induces cell cycle arrest, triggers apoptosis through caspase activation, and exhibits DNA damage response-associated anticancer effects.

Anti-Inflammatory Effect of IKK-Activated GSK-3ß Inhibitory Peptide Prevented Nigrostriatal Neurodegeneration in the Rodent Model of Parkinson's Disease.

Parkinson's disease (PD) is a progressive movement disorder caused by nigrostriatal neurodegeneration. Since chronically activated neuroinflammation accelerates neurodegeneration in PD, we considered that modulating chronic neuroinflammatory response might provide a novel therapeutic approach. Glycogen synthase kinase 3 (GSK-3) is a multifunctional serine/threonine protein kinase with two isoforms, GSK-3α and GSK-3ß, and GSK-3ß plays crucial roles in inflammatory response, which include microglial migration and peripheral immune cell activation. GSK-3ß inhibitory peptide (IAGIP) is specifically activated by activated inhibitory kappa B kinase (IKK), and its therapeutic effects have been demonstrated in a mouse model of colitis. Here, we investigated whether the anti-inflammatory effects of IAGIP prevent neurodegeneration in the rodent model of PD. IAGIP significantly reduced MPP+-induced astrocyte activation and inflammatory response in primary astrocytes without affecting the phosphorylations of ERK or JNK. In addition, IAGIP inhibited LPS-induced cell migration and p65 activation in BV-2 microglial cells. In vivo study using an MPTP-induced mouse model of PD revealed that intravenous IAGIP effectively prevented motor dysfunction and nigrostriatal neurodegeneration. Our findings suggest that IAGIP has a curative potential in PD models and could offer new therapeutic possibilities for targeting PD.

Organ-differential Roles of Akt/FoxOs Axis as a Key Metabolic Modulator during Aging.

FoxOs and their post-translational modification by phosphorylation, acetylation, and methylation can affect epigenetic modifications and promote the expression of downstream target genes. Therefore, they ultimately affect cellular and biological functions during aging or occurrence of age-related diseases including cancer, diabetes, and kidney diseases. As known for its key role in aging, FoxOs play various biological roles in the aging process by regulating reactive oxygen species, lipid accumulation, and inflammation. FoxOs regulated by PI3K/Akt pathway modulate the expression of various target genes encoding MnSOD, catalases, PPARγ, and IL-1ß during aging, which are associated with age-related diseases. This review highlights the age-dependent differential regulatory mechanism of Akt/FoxOs axis in metabolic and non-metabolic organs. We demonstrated that age-dependent suppression of Akt increases the activity of FoxOs (Akt/FoxOs axis upregulation) in metabolic organs such as liver and muscle. This Akt/FoxOs axis could be modulated and reversed by antiaging paradigm calorie restriction (CR). In contrast, hyperinsulinemia-mediated PI3K/Akt activation inhibited FoxOs activity (Akt/FoxOs axis downregulation) leading to decrease of antioxidant genes expression in non-metabolic organs such as kidneys and lungs during aging. These phenomena are reversed by CR. The results of studies on the process of aging and CR indicate that the Akt/FoxOs axis plays a critical role in regulating metabolic homeostasis, redox stress, and inflammation in various organs during aging process. The benefical actions of CR on the Akt/FoxOs axis in metabolic and non-metabolic organs provide further insights into the molecular mechanisms of organ-differential roles of Akt/FoxOs axis during aging.

Human cardiac stem cells rejuvenated by modulating autophagy with MHY-1685 enhance the therapeutic potential for cardiac repair.

Stem cell-based therapies with clinical applications require millions of cells. Therefore, repeated subculture is essential for cellular expansion, which is often complicated by replicative senescence. Cellular senescence contributes to reduced stem cell regenerative potential as it inhibits stem cell proliferation and differentiation as well as the activation of the senescence-associated secretory phenotype (SASP). In this study, we employed MHY-1685, a novel mammalian target of rapamycin (mTOR) inhibitor, and examined its long-term priming effect on the activities of senile human cardiac stem cells (hCSCs) and the functional benefits of primed hCSCs after transplantation. In vitro experiments showed that the MHY-1685‒primed hCSCs exhibited higher viability in response to oxidative stress and an enhanced proliferation potential compared to that of the unprimed senile hCSCs. Interestingly, priming MHY-1685 enhanced the expression of stemness-related markers in senile hCSCs and provided the differentiation potential of hCSCs into vascular lineages. In vivo experiment with echocardiography showed that transplantation of MHY-1685‒primed hCSCs improved cardiac function than that of the unprimed senile hCSCs at 4 weeks post-MI. In addition, hearts transplanted with MHY-1685-primed hCSCs exhibited significantly lower cardiac fibrosis and higher capillary density than that of the unprimed senile hCSCs. In confocal fluorescence imaging, MHY-1685‒primed hCSCs survived for longer durations than that of the unprimed senile hCSCs and had a higher potential to differentiate into endothelial cells (ECs) within the infarcted hearts. These findings suggest that MHY-1685 can rejuvenate senile hCSCs by modulating autophagy and that as a senescence inhibitor, MHY-1685 can provide opportunities to improve hCSC-based myocardial regeneration.

Protease-activated receptor 2 induces ROS-mediated inflammation through Akt-mediated NF-κB and FoxO6 modulation during skin photoaging.

Long-term exposure to ultraviolet irradiation to skin leads to deleterious intracellular effects, including reactive oxygen species (ROS) production and inflammatory responses, causing accelerated skin aging. Previous studies have demonstrated that increased expression and activation of protease-activated receptor 2 (PAR2) and Akt is observed in keratinocyte proliferation, suggesting their potential regulatory role in skin photoaging. However, the specific underlying molecular mechanism of PAR2 and the Akt/NF-κB/FoxO6-mediated signaling pathway is not clearly defined. In this study, we first used the UVB-irradiated photoaged skin of hairless mice and observed an increase in PAR2 and Gαq expression and PI3-kinase/Akt, NF-κB, and suppressed FoxO6. Consequently, increased levels of proinflammatory cytokines and decreased levels of antioxidant MnSOD was observed. Next, to investigate PAR2-specific roles in inflammation and oxidative stress, we used photoaged hairless mice topically applied with PAR2 antagonist GB83 and photoaged PAR2 knockout mice. PAR2 inhibition and deletion significantly suppressed inflammatory and oxidative stress levels, which were associated with decreased IL-6 and IL-1ß levels and increased MnSOD levels, respectively. Furthermore, NF-κB phosphorylation and decreased FoxO6 was reduced by PAR2 inhibition and deletion in vivo. To confirm the in vivo results, we conducted PAR2 knockdown and overexpression in UVB-irradiated HaCaT cells. In PAR2 knockdown cells by si-PAR2 treatment, it suppressed Akt/NF-κB and increased FoxO6, whereas PAR2 overexpression reversed these effects and subsequently modulated proinflammatory target genes. Collectively, our data define that PAR2 induces oxidative stress and inflammation through Akt-mediated phosphorylation of NF-κB (Ser536) and FoxO6 (Ser184), which could be a critical upstream regulatory mechanism in ROS-mediated inflammatory response.

PPARα Agonist, MHY3200, Alleviates Renal Inflammation during Aging via Regulating ROS/Akt/FoxO1 Signaling.

PPARα is a ligand-dependent transcription factor and its activation is known to play an important role in cell defense through anti-inflammatory and antioxidant effects. MHY3200 (2-[4-(5-chlorobenzo[d]thiazol-2-yl)phenoxy]-2,2-difluoroacetic acid), a novel benzothiazole-derived peroxisome proliferator-activated receptor α (PPARα) agonist, is a synthesized PPARα activator. This study examined the beneficial effects of MHY3200 on age-associated alterations in reactive oxygen species (ROS)/Akt/forkhead box (FoxO) 1 signaling in rat kidneys. Young (7-month-old) and old (22-month-old) rats were treated with MHY3200 (1 mg/kg body weight/day or 3 mg/kg body weight/day) for two weeks. MHY3200 treatment led to a notable decrease in triglyceride and insulin levels in serum from old rats. The elevated kidney ROS level, serum insulin level, and Akt phosphorylation in old rats were reduced following MHY3200 treatment; moreover, FoxO1 phosphorylation increased. MHY3200 treatment led to the increased level of FoxO1 and its target gene, MnSOD. MHY3200 suppressed cyclooxygenase-2 expression by activating PPARα and inhibiting the activation of nuclear factor-κB (NF-κB) in the kidneys of old rats. Our results suggest that MHY3200 ameliorates age-associated renal inflammation by regulating NF-κB and FoxO1 via ROS/Akt signaling.

PAR2 promotes high-fat diet-induced hepatic steatosis by inhibiting AMPK-mediated autophagy.

Protease-activated receptor 2 (PAR2) is a member of G protein-coupled receptors. There are two types of PAR2 signaling pathways: Canonical G-protein signaling and ß-arrestin signaling. Although PAR2 signaling has been reported to aggravate hepatic steatosis, the exact mechanism is still unclear, and the role of PAR2 in autophagy remains unknown. In this study, we investigated the regulatory role of PAR2 in autophagy during high-fat diet (HFD)-induced hepatic steatosis in mice. Increased protein levels of PAR2 and ß-arrestin-2 and their interactions were detected after four months of HFD. To further investigate the role of PAR2, male and female wild-type (WT) and PAR2-knockout (PAR2 KO) mice were fed HFD. PAR2 deficiency protected HFD-induced hepatic steatosis in male mice, but not in female mice. Interestingly, PAR2-deficient liver showed increased AMP-activated protein kinase (AMPK) activation with decreased interaction between Ca2+/calmodulin-dependent protein kinase kinase ß (CAMKKß) and ß-arrestin-2. In addition, PAR2 deficiency up-regulated autophagy in the liver. To elucidate whether PAR2 plays a role in the regulation of autophagy and lipid accumulation in vitro, PAR2 was overexpressed in HepG2 cells. Overexpression of PAR2 decreased AMPK activation with increased interaction of CAMKKß with ß-arrestin-2 and significantly inhibited autophagic responses in HepG2 cells. Inhibition of autophagy by PAR2 overexpression further exacerbated palmitate-induced lipid accumulation in HepG2 cells. Collectively, these findings suggest that the increase in the PAR2-ß-arrestin-2-CAMKKß complex by HFD inhibits AMPK-mediated autophagy, leading to the alleviation of hepatic steatosis.

(E)-1-(Furan-2-yl)-(substituted phenyl)prop-2-en-1-one Derivatives as Tyrosinase Inhibitors and Melanogenesis Inhibition: An In Vitro and In Silico Study.

A series of (E)-1-(furan-2-yl)prop-2-en-1-one derivatives (compounds 1-8) were synthesized and evaluated for their mushroom tyrosinase inhibitory activity. Among these series, compound 8 (2,4-dihydroxy group bearing benzylidene) showed potent tyrosinase inhibitory activity, with respective IC50 values of 0.0433 µM and 0.28 µM for the monophenolase and diphenolase as substrates in comparison to kojic acid as standard compound 19.97 µM and 33.47 µM. Moreover, the enzyme kinetics of compound 8 were determined to be of the mixed inhibition type and inhibition constant (Ki) values of 0.012 µM and 0.165 µM using the Lineweaver-Burk plot. Molecular docking results indicated that compound 8 can bind to the catalytic and allosteric sites 1 and 2 of tyrosinase to inhibit enzyme activity. The computational molecular dynamics analysis further revealed that compound 8 interacted with two residues in the tyrosinase active site pocket, such as ASN260 and MET280. In addition, compound 8 attenuated melanin synthesis and cellular tyrosinase activity, simulated by α-melanocyte-stimulating hormone and 1-methyl-3-isobutylxanthine. Compound 8 also decreased tyrosinase expressions in B16F10 cells. Based on in vitro and computational studies, we propose that compound 8 might be a worthy candidate for the development of an antipigmentation agent.