Chronic Inflammation as an Underlying Mechanism of Ageing and Ageing-Related Diseases.

Age-related chronic inflammation is characterized as the unresolved low-grade inflammatory process underlying the ageing process and various age-related diseases. In this chapter, we review the age-related changes in the oxidative stress-sensitive pro-inflammatory NF-κB signaling pathways causally linked with chronic inflammation during ageing based on senoinflammation schema. We describe various age-related dysregulated pro- and anti-inflammatory cytokines, chemokines, and senescence-associated secretory phenotype (SASP), and alterations of inflammasome, specialized pro-resolving lipid mediators (SPM), and autophagy as major players in the chronic inflammatory intracellular signaling network. A better understanding of the molecular, cellular, and systemic mechanisms involved in chronic inflammation in the ageing process would provide further insights into the potential anti-inflammatory strategies.

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.

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.

PPAR Pan Agonist MHY2013 Alleviates Renal Fibrosis in a Mouse Model by Reducing Fibroblast Activation and Epithelial Inflammation.

The peroxisome proliferator-activated receptor (PPAR) nuclear receptor has been an interesting target for the treatment of chronic diseases. Although the efficacy of PPAR pan agonists in several metabolic diseases has been well studied, the effect of PPAR pan agonists on kidney fibrosis development has not been demonstrated. To evaluate the effect of the PPAR pan agonist MHY2013, a folic acid (FA)-induced in vivo kidney fibrosis model was used. MHY2013 treatment significantly controlled decline in kidney function, tubule dilation, and FA-induced kidney damage. The extent of fibrosis determined using biochemical and histological methods showed that MHY2013 effectively blocked the development of fibrosis. Pro-inflammatory responses, including cytokine and chemokine expression, inflammatory cell infiltration, and NF-κB activation, were all reduced with MHY2013 treatment. To demonstrate the anti-fibrotic and anti-inflammatory mechanisms of MHY2013, in vitro studies were conducted using NRK49F kidney fibroblasts and NRK52E kidney epithelial cells. In the NRK49F kidney fibroblasts, MHY2013 treatment significantly reduced TGF-ß-induced fibroblast activation. The gene and protein expressions of collagen I and α-smooth muscle actin were significantly reduced with MHY2013 treatment. Using PPAR transfection, we found that PPARγ played a major role in blocking fibroblast activation. In addition, MHY2013 significantly reduced LPS-induced NF-κB activation and chemokine expression mainly through PPARß activation. Taken together, our results suggest that administration of the PPAR pan agonist effectively prevented renal fibrosis in both in vitro and in vivo models of kidney fibrosis, implicating the therapeutic potential of PPAR agonists against chronic kidney diseases.

Jagged1/Notch2 controls kidney fibrosis via Tfam-mediated metabolic reprogramming.

While Notch signaling has been proposed to play a key role in fibrosis, the direct molecular pathways targeted by Notch signaling and the precise ligand and receptor pair that are responsible for kidney disease remain poorly defined. In this study, we found that JAG1 and NOTCH2 showed the strongest correlation with the degree of interstitial fibrosis in a genome-wide expression analysis of a large cohort of human kidney samples. Transcript analysis of mouse kidney disease models, including folic-acid (FA)-induced nephropathy, unilateral ureteral obstruction (UUO), or apolipoprotein L1 (APOL1)-associated kidney disease, indicated that Jag1 and Notch2 levels were higher in all analyzed kidney fibrosis models. Mice with tubule-specific deletion of Jag1 or Notch2 (Kspcre/Jag1flox/flox and Kspcre/Notch2flox/flox) had no kidney-specific alterations at baseline but showed protection from FA-induced kidney fibrosis. Tubule-specific genetic deletion of Notch1 and global knockout of Notch3 had no effect on fibrosis. In vitro chromatin immunoprecipitation experiments and genome-wide expression studies identified the mitochondrial transcription factor A (Tfam) as a direct Notch target. Re-expression of Tfam in tubule cells prevented Notch-induced metabolic and profibrotic reprogramming. Tubule-specific deletion of Tfam resulted in fibrosis. In summary, Jag1 and Notch2 play a key role in kidney fibrosis development by regulating Tfam expression and metabolic reprogramming.

Synergistic effect of copper and amino acid mixtures on the production of extracellular matrix proteins in skin fibroblasts.

The stimulation of extracellular matrix (ECM) protein production is an interesting target to maintain normal skin structure and delay skin aging. Copper has been shown to stimulate ECM protein synthesis by activating lysyl oxidase. Although copper increases elastin and collagen synthesis, the effect of copper and amino acid mixtures on gene expression and protein synthesis changes relating to the ECM have not been fully investigated. In this study, we showed that copper ions (Cu2+) and amino acid mixtures significantly increased the expression of genes and proteins related to the ECM in human dermal fibroblasts. The expression of genes involved in ECM production was evaluated through quantitative polymerase chain reaction in the presence of amino acid mixtures containing different Cu2+ concentrations. Cu2+ dose-dependently increased the gene expression of elastin and collagen I. In addition, a mixture of amino acids and Cu2+ increased the protein expression of elastin and collagen I. We further evaluated the effect of Cu2+ with or without amino acids. Although Cu2+ treatment increased the expression of genes encoding ECM proteins, the Cu2+ treatment without amino acids did not increase protein expression in the ECM. Our results demonstrated the synergistic effects of amino acids and a Cu2+ mixture on ECM protein synthesis in dermal fibroblasts.

Interaction between CHOP and FoxO6 promotes hepatic lipid accumulation.

BACKGROUND & AIMS: Endoplasmic reticulum (ER) stress is one of the major causes of hepatic insulin resistance through increasing de novo lipogenesis. Forkhead box O6 (FoxO6) is a transcription factor mediating insulin signalling to glucose and lipid metabolism, therefore, dysregulated FoxO6 is involved in hepatic insulin resistance. In this study, we elucidated the role of FoxO6 in ER stress-induced hepatic lipogenesis. METHODS: Hepatic ER stress responses and lipogenesis were monitored in mice overexpressed with constitutively active FoxO6 allele and FoxO6-null mice. In the in vitro study, HepG2 cells overexpressing constitutively active FoxO6 were treated with palmitate, and then alterations in ER stress and lipid metabolism were measured. RESULTS: FoxO6 activation induced hepatic lipogenesis and the expression of ER stress-inducible genes. The expression and transcriptional activity of peroxisome proliferator-activated receptor γ (PPARγ) were significantly increased in constitutively active FoxO6 allele. Interestingly, we found that the active FoxO6 physically interacted with C/EBP homologous protein (CHOP), an ER stress-inducible transcription factor, which was responsible for PPARγ expression. Palmitate treatment caused the expression of ER stress-inducible genes, which was deteriorated by FoxO6 activation in HepG2 cells. Palmitate-induced ER stress led to PPARγ expression through interactions between CHOP and FoxO6 corresponding to findings in the in vivo study. On the other hand, the expression of PPARα and ß-oxidation were decreased in constitutively active FoxO6 allele which implied that lipid catabolism is also regulated by FoxO6. CONCLUSION: Our data present significant evidence demonstrating that CHOP and FoxO6 interact to induce hepatic lipid accumulation through PPARγ expression during ER stress.

Impairment of PPARα and the Fatty Acid Oxidation Pathway Aggravates Renal Fibrosis during Aging.

Defects in the renal fatty acid oxidation (FAO) pathway have been implicated in the development of renal fibrosis. Although, compared with young kidneys, aged kidneys show significantly increased fibrosis with impaired kidney function, the mechanisms underlying the effects of aging on renal fibrosis have not been investigated. In this study, we investigated peroxisome proliferator-activated receptor α (PPARα) and the FAO pathway as regulators of age-associated renal fibrosis. The expression of PPARα and the FAO pathway-associated proteins significantly decreased with the accumulation of lipids in the renal tubular epithelial region during aging in rats. In particular, decreased PPARα protein expression associated with increased expression of PPARα-targeting microRNAs. Among the microRNAs with increased expression during aging, miR-21 efficiently decreased PPARα expression and impaired FAO when ectopically expressed in renal epithelial cells. In cells pretreated with oleic acid to induce lipid stress, miR-21 treatment further enhanced lipid accumulation. Furthermore, treatment with miR-21 significantly exacerbated the TGF-ß-induced fibroblast phenotype of epithelial cells. We verified the physiologic importance of our findings in a calorie restriction model. Calorie restriction rescued the impaired FAO pathway during aging and slowed fibrosis development. Finally, compared with kidneys of aged littermate controls, kidneys of aged PPARα-/- mice showed exaggerated lipid accumulation, with decreased activity of the FAO pathway and a severe fibrosis phenotype. Our results suggest that impaired renal PPARα signaling during aging aggravates renal fibrosis development, and targeting PPARα is useful for preventing age-associated CKD.

Quantitative Proteomic Analysis of Changes Related to Age and Calorie Restriction in Rat Liver Tissue.

Calorie restriction (CR) is the most frequently studied mechanism for increasing longevity, protecting against stress, and delaying age-associated diseases. Most studies have initiated CR in young animals to determine the protective effects against aging. Although aging phenomena are well-documented, the molecular mechanisms of aging and CR remain unclear. In this study, we observe changes in hepatic proteins upon age-related and diet-restricted changes in the rat liver using quantitative proteomics. Quantitative proteomes are measured using tandem mass tag labeling followed by LC-MS/MS. We compare protein levels in livers from young (6 months old) and old (25 months old) rats with 40% calorie-restricted (YCR and OCR, respectively) or ad libitum diets. In total, 44 279 peptides and 3134 proteins are identified and 260 differentially expressed proteins are found. Functional enrichment analysis show that these proteins are mainly involved in glucose and fatty acid metabolism-related processes, consistent with the theory that energy metabolism regulation is dependent on age-related and calorie-restricted changes in liver tissue. In addition, proteins mediating inflammation and gluconeogenesis are increased in OCR livers, but not YCR livers. These results show that CR in old rats might not have antiaging benefits because liver inflammation is increased.

Evaluation of Antimelanogenic Activity and Mechanism of Galangin in Silico and in Vivo.

Abnormal pigmentation owing to excessive melanin synthesis can result in serious problems such as freckles, age-spots, and melanoma. Tyrosinase inhibitors have been an interesting target for the treatment of hyperpigmentation because tyrosinase is the rate-limiting enzyme in melanin synthesis. The screening for strong tyrosinase inhibitors led to the finding of the flavonoid galangin, which showed notable inhibitory effects on mushroom tyrosinase. The IC50 value of galangin (3.55±0.39 µM) was lower than that of kojic acid (48.55±1.79 µM), which was used as a positive control. In silico docking simulation and mechanistic studies demonstrated that galangin interacted with the catalytic sites of tyrosinase and competed with tyrosine. In B16F10 melanoma cells stimulated with α-melanocyte stimulating hormone, galangin inhibited tyrosinase activity as well as melanin production. Although high doses of galangin were cytotoxic, no cytotoxic effects were observed at low doses. In addition, the in vivo efficacy of galangin was evaluated in HRM2 melanin-possessing hairless mice. As measured by the skin-whitening index and melanin staining, repeated UVB exposure increased skin melanin synthesis. Galangin application significantly reduced melanogenesis induced by UVB exposure. Collectively, our data indicates that galangin shows strong tyrosinase inhibition activity, which suggests that it may be an effective skin-whitening agent.

A PPAR Pan Agonist, MHY2013 Alleviates Age-Related Hepatic Lipid Accumulation by Promoting Fatty Acid Oxidation and Suppressing Inflammation.

Nonalcoholic fatty liver disease (NAFLD) is frequently observed in obese and aged individuals. Peroxisome proliferator-activated receptors (PPARs) play a role in regulating hepatic lipid accumulation, a hallmark of NAFLD development. A PPAR pan agonist, 2-(4-(5,6-methylenedioxybenzo[d]thiazol-2-yl)-2-methylphenoxy)-2-methylpropanoic acid (MHY2013) has been shown to prevent fatty liver formation and insulin resistance in obese mice (db/db) model. However, the beneficial effects of MHY2013 in aged model remain unknown. In this study, we investigated whether MHY2013 alleviates hepatic lipid accumulation in aged Sprague-Dawley (SD) rats. We confirmed that MHY2013 increased the activities of three PPAR subtypes in HepG2 cells using luciferase assay. When administered orally in aged SD rats, MHY2013 markedly decreased the hepatic triglyceride levels without changes in body weight. Regarding underlying mechanisms, MHY2013 increased the mRNA levels of lipid oxidation-related genes, including carnitine palmitoyltransferase 1 (CPT1) and peroxisomal acyl-CoA oxidase 1 (ACOX1), without apparent change in the mRNA expression of lipogenesis-related genes. Furthermore, MHY2013 significantly increased systemic fibroblast growth factor 21 (FGF21) and adiponectin levels and suppressed inflammatory mRNA expression in the liver. In conclusion, MHY2013 alleviated age-related hepatic lipid accumulation, in part by upregulating ß-oxidation signaling and suppressing inflammation in the liver. Therefore, MHY2013 is a potential pharmaceutical agent for treating age-related hepatic lipid accumulation.

Isolation of tyrosinase and melanogenesis inhibitory flavonoids from Juniperus chinensis fruits.

A new biflavonoid, amentoflavone-7-O-ß-D-glucoside, and thirteen known flavonoids were isolated from the fruits of Juniperus chinensis using a bioactivity-guided method and their tyrosinase inhibitory effects were tested using a mushroom tyrosinase bioassay. Two isolates, hypolaetin-7-O-ß-D-glucoside and quercetin-7-O-α-L-rhamnoside, were found to reduce tyrosinase activity at a concentration of 50 µM. Quercetin-7-O-α-L-rhamnoside attenuated cellular tyrosinase activity and melanogenesis in α-MSH plus IBMX-stimulated B16F10 melanoma cells. Molecular docking simulation revealed that quercetin-7-O-α-L-rhamnoside inhibits tyrosinase activity by hydrogen bonding with residues His85, His244, Thr261, and Gly281 of tyrosinase. Abbreviations: EtOH, ethanol; CH2Cl2, dichloromethane; EtOAc, ethylacetate; n-BuOH, n-butanol; MeOH, metanol; CHCl3,chloroform; DMSO, dimethylsulfoxide; DMEM, Dulbecco's modified Eagle's medium; FBS, fetal bovine serum; α-MSH, α-melanocyte stimulating hormone; L-DOPA, L-3, 4-dihydroxyphenylalanine.

The Standardized Extract of Juniperus communis Alleviates Hyperpigmentation in Vivo HRM-2 Hairless Mice and in Vitro Murine B16 Melanoma Cells.

In European folk medicine, the fruits of Juniperus communis are used in the treatment of skin-related disorders such as skin infection, itching, and psoriasis. Previously, we reported that the EtOAc fraction of J. communis (EAJC) contained tyrosinase inhibition properties in vitro non-cellular experiment. The aim of this study was to evaluate anti-melanogenic effect of standardized EAJC on a hyperpigmentation animal model. Therapeutic effects of EAJC toward skin hyperpigmentation were confirmed by both in vivo experiment and in vitro cell-based assay. Skin depigmenting effect was detected by topical treatment of EAJC for 11 d to HRM-2 melanin-possessing hairless mice. Histologic findings including significantly decreased melanin depositions could be observed in dorsal skin samples of EAJC-treated group. In addition, the EAJC (50 µg/mL) attenuated melanin production through down-regulation of tyrosinase activity and protein expression in B16 murine melanoma cells. According to the phytochemical analysis, EAJC was found to contain hypolaetin-7-O-ß-D-xylopyranoside and isoscutellarein-7-O-ß-D-xylopyranoside as main components. Hypolaetin-7-O-ß-D-xylopyranoside was responsible for the skin-lightening effect of EAJC by reducing the number of melanocytes in dorsal skins of HRM-2 mice. The present study provided direct experimental evidence for skin-lightening effect of EAJC in UV-irradiated hairless mouse model. Therapeutic attempts with the J. communis might be useful in the management of skin pigmentation-related diseases.

The roles of FoxOs in modulation of aging by calorie restriction.

FoxO activity and modifications, such as its phosphorylation, acetylation, and methylation, may help drive the expression of genes involved in combating oxidative stress by causing the epigenetic modifications, and thus, preserve cellular function during aging and age-related diseases, such as diabetes, cancer, and Alzheimer disease. Insulin signaling has been postulated to influence the aging process by increasing resistance to oxidative stress, and slowing the accumulation of oxidative damage. Some antioxidative effects are mediated by a conserved family of forkhead box transcription factors (FoxOs), which in the absence of insulin signaling freely bind to promoters of antioxidant enzymes, superoxide dismutase, and catalase. On the other hand, calorie restriction (CR) extends the lifespans of several species via the insulin pathway, and extends longevity and healthspan in diverse species via a conserved mechanism. CR enhances adaptive stress responses at the cellular and organism levels and extends lifespan in a FoxO-independent manner. Thus, increased modification of FoxO is modulated via the hyperinsulinemia-induced PI3K/Akt pathway during aging, and CR reverses this process. Accordingly, FoxO plays an important role in maintenance of metabolic homeostasis and removal of oxidative stress in the aging process and in the effect of CR on lifespan.

Characterization of a small molecule inhibitor of melanogenesis that inhibits tyrosinase activity and scavenges nitric oxide (NO).

BACKGROUND: Excessive melanin production and accumulation are characteristics of a large number of skin diseases, including melasma, and post-inflammatory hyperpigmentation. During our on-going search for new agents with an inhibitory effect on tyrosinase, we synthesized a new type of tyrosinase inhibitor, 4-(thiazolidin-2-yl)benzene-1,2-diol (MHY-794), which directly inhibits mushroom tyrosinase. METHODS: The inhibitory effect of MHY-794 on tyrosinase activity and nitric oxide (NO) scavenging activity was evaluated in cell free system. Additional experiments were performed using B16F10 melanoma cells to demonstrate the effects of MHY-794 in vitro. HRM2 hairless mice were used to evaluate anti-melanogenic effects of MHY-794 in vivo. RESULTS: MHY-794 effectively inhibited mushroom tyrosinase activity in cell free system. In silico docking simulation also supported the inhibitory effects of MHY-794 on mushroom tyrosinase. MHY-794 also proved to be effective at scavenging nitric oxide (NO), which serves as an important modulator in the melanogenesis signaling pathway. In addition, MHY-794 effectively inhibited SNP (NO donor)-induced melanogenesis by directly inhibiting tyrosinase and diminishing NO-mediated melanogenesis signaling in B16 melanoma cells. The anti-melanogenic effects of MHY-794 were further confirmed in HRM2 hairless mice. Ultraviolet light (UV) significantly up-regulated NO-mediated melanogenesis signaling in HRM2 hairless mice, but MHY-794 effectively inhibited both melanogenesis and diminished UV-induced NO-signaling. CONCLUSIONS: Our results indicate that MHY-794 is highly effective at inhibiting NO-mediated melanogenesis in vitro and in vivo by direct NO scavenging and directly inhibiting tyrosinase activity, and suggest that MHY-794 be considered a new developmental candidate for the treatment of hyper-pigmentation disorders. GENERAL SIGNIFICANCE: MHY-794, which showed great efficacy on NO-mediated melanogenesis by direct NO scavenging as well as direct inhibition of tyrosinase catalytic activity, might be utilized for the development of a new candidate for treatment of the hyper-pigmentation disorders.

Fasting potentiates diclofenac-induced liver injury via inductions of oxidative/endoplasmic reticulum stresses and apoptosis, and inhibition of autophagy by depleting hepatic glutathione in mice.

Diclofenac, a widely used non-steroidal anti-inflammatory drug, can cause liver damage via its metabolic activation by hepatic CYP450s and UGT2B7. Fasting can affect drug-induced liver injury by modulating the hepatic metabolism, but its influence on diclofenac hepatotoxicity is unknown. Thus, we investigated diclofenac-induced liver damage after fasting in mice, and the cellular events were examined. Male ICR mice fasted for 16 h showed the elevation of CYP3A11, but the decreases of UGT2B7, glutathione (GSH), and GSH S-transferase-µ/-π levels in the livers. Diclofenac (200 mg/kg) injection into the mice after 16-h fasting caused more significant liver damage compared to that in the diclofenac-treated fed mice, as shown by the higher serum ALT and AST activities. Diclofenac-promoted hepatic oxidative stress (oxidized proteins, 4-hydroxynonenal, and malondialdehyde), endoplasmic reticulum (ER) stress (BiP, ATF6, and CHOP), and apoptosis (cleaved caspase-3 and cleaved PARP) were enhanced by fasting. Autophagic degradation was inhibited in the diclofenac-treated fasting mice compared to that of the corresponding fed mice. The results suggest that fasting can make the liver more susceptible to diclofenac toxicity by lowering GSH-mediated detoxification; increased oxidative/ER stresses and apoptosis and suppressed autophagic degradation may be the cellular mechanisms of the aggravated diclofenac hepatotoxicity under fasting conditions.

Doxorubicin Attenuates Free Fatty Acid-Induced Lipid Accumulation via Stimulation of p53 in HepG2 Cells.

Non-alcoholic fatty liver disease (NAFLD) is characterized by excessive accumulation of fat in the liver, and there is a global increase in its incidence owing to changes in lifestyle and diet. Recent findings suggest that p53 is involved in the development of non-alcoholic fatty liver disease; however, the association between p53 expression and the disease remains unclear. Doxorubicin, an anticancer agent, increases the expression of p53. Therefore, this study aimed to investigate the role of doxorubicin-induced p53 upregulation in free fatty acid (FFA)-induced intracellular lipid accumulation. HepG2 cells were pretreated with 0.5 µg/mL of doxorubicin for 12 h, followed by treatment with FFA (0.5 mM) for 24 h to induce steatosis. Doxorubicin pretreatment upregulated p53 expression and downregulated the expression of endoplasmic reticulum stress- and lipid synthesis-associated genes in the FFA -treated HepG2 cells. Additionally, doxorubicin treatment upregulated the expression of AMP-activated protein kinase, a key modulator of lipid metabolism. Notably, siRNA-targeted p53 knockdown reversed the effects of doxorubicin in HepG2 cells. Moreover, doxorubicin treatment suppressed FFA -induced lipid accumulation in HepG2 spheroids. Conclusively, these results suggest that doxorubicin possesses potential application for the regulation of lipid metabolism by enhance the expression of p53 an in vitro NAFLD model.

PAR2-mediated cellular senescence promotes inflammation and fibrosis in aging and chronic kidney disease.

Cellular senescence contributes to inflammatory kidney disease via the secretion of inflammatory and profibrotic factors. Protease-activating receptor 2 (PAR2) is a key regulator of inflammation in kidney diseases. However, the relationship between PAR2 and cellular senescence in kidney disease has not yet been described. In this study, we found that PAR2-mediated metabolic changes in renal tubular epithelial cells induced cellular senescence and increased inflammatory responses. Using an aging and renal injury model, PAR2 expression was shown to be associated with cellular senescence. Under in vitro conditions in NRK52E cells, PAR2 activation induces tubular epithelial cell senescence and senescent cells showed defective fatty acid oxidation (FAO). Cpt1α inhibition showed similar senescent phenotype in the cells, implicating the important role of defective FAO in senescence. Finally, we subjected mice lacking PAR2 to aging and renal injury. PAR2-deficient kidneys are protected from adenine- and cisplatin-induced renal fibrosis and injury, respectively, by reducing senescence and inflammation. Moreover, kidneys lacking PAR2 exhibited reduced numbers of senescent cells and inflammation during aging. These findings offer fresh insights into the mechanisms underlying renal senescence and indicate that targeting PAR2 or FAO may be a promising therapeutic approach for managing kidney injury.

Amelioration of Astrocyte-Mediated Neuroinflammation by EI-16004 Confers Neuroprotection in an MPTP-induced Parkinson's Disease Model.

Parkinson's disease (PD) is a neurodegenerative disorder that results in motor impairment due to dopaminergic neuronal loss. The pathology of PD is closely associated with neuroinflammation, which can be characterized by astrocyte activation. Thus, targeting the inflammatory response in astrocytes might provide a novel therapeutic approach. We conducted a luciferase assay on an in-house chemical library to identify compounds with anti-inflammatory effects capable of reducing MPP+-induced NF-κB activity in astrocytes. Among the compounds identified, EI-16004, a novel 3-benzyl-N-phenyl-1H-pyrazole-5-carboxamides, exhibited a significant anti-inflammatory effect by significantly reducing MPP+-induced astrocyte activation. Biochemical analysis and docking simulation indicated that EI-16004 inhibited the MPP+-induced phosphorylation of p65 by attenuating ERK phosphorylation, and EI-16004 reduced pro-inflammatory cytokine and chemokine levels in astrocytes. In vivo studies on the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced PD model in male C57BL/6 mice showed that EI-16004 ameliorated motor impairment and protected against dopaminergic neuronal loss, and EI-16004 effectively mitigated the MPTP-induced astrocyte activation in striatum (STR) and substantia nigra (SN). These results indicate EI-16004 is a potential neuroprotective agent for the prevention and treatment of astrocyte-mediated neuroinflammatory conditions in PD.

Evaluation of in vitro and in vivo anti-melanogenic activity of a newly synthesized strong tyrosinase inhibitor (E)-3-(2,4 dihydroxybenzylidene)pyrrolidine-2,5-dione (3-DBP).

BACKGROUND: Tyrosinase inhibitors have become increasingly important because of their ability to inhibit the synthesis of the pigment melanin. A search for new agents with strong tyrosinase activity led to the synthesis of the tyrosinase inhibitor (E)-3-(2,4-dihydroxybenzylidene)pyrrolidine-2,5-dione (3-DBP). METHODS: The inhibitory effect of 3-DBP on tyrosinase activity and melanin production was examined in murine melanoma B16F10 cells. Additional experiments were performed using HRM2 hairless mice to demonstrate the effects of 3-DBP in vivo. RESULTS: The novel compound, 3-DBP, showed an inhibitory effect against mushroom tyrosinase (IC50=0.53 µM), which indicated that it was more potent than the well-known tyrosinase inhibitor kojic acid (IC50=8.2 µM). When tested in B16F10 melanoma cells treated with α-melanocyte stimulating hormone (α-MSH), 3-DBP also inhibited murine tyrosinase activity, which in turn induced a decrease in melanin production in these cells. The anti-melanogenic effect of 3-DBP was further verified in HRM2 hairless mice. The skin-whitening index (L value) of HRM2 hairless mice treated with 3-DBP before irradiation with UVB was greater than that of UVB-irradiated mice that were not treated with 3-DBP. GENERAL SIGNIFICANCE: The newly synthesized 3-DBP has a potent inhibitory effect on tyrosinase. In addition to an in vitro investigation of the effects of 3-DBP on tyrosinase, in vivo studies using an HRM2 hairless mouse model demonstrated the anti-melanogenic potency of 3-DBP. Our newly synthesized 3-DBP showed efficient tyrosinase inhibitory effect in vivo and in vitro. Our finding suggests that 3-DBP can be an effective skin-whitening agent.