Biomarkers of Cellular Senescence and Aging: Current State-of-the-Art, Challenges and Future Perspectives.

Population aging has increased the global prevalence of aging-related diseases, including cancer, sarcopenia, neurological disease, arthritis, and heart disease. Understanding aging, a fundamental biological process, has led to breakthroughs in several fields. Cellular senescence, evinced by flattened cell bodies, vacuole formation, and cytoplasmic granules, ubiquitously plays crucial roles in tissue remodeling, embryogenesis, and wound repair as well as in cancer therapy and aging. The lack of universal biomarkers for detecting and quantifying senescent cells, in vitro and in vivo, constitutes a major limitation. The applications and limitations of major senescence biomarkers, including senescence-associated ß-galactosidase staining, telomere shortening, cell-cycle arrest, DNA methylation, and senescence-associated secreted phenotypes are discussed. Furthermore, explore senotherapeutic approaches for aging-associated diseases and cancer. In addition to the conventional biomarkers, this review highlighted the in vitro, in vivo, and disease models used for aging studies. Further, technologies from the current decade including multi-omics and computational methods used in the fields of senescence and aging are also discussed in this review. Understanding aging-associated biological processes by using cellular senescence biomarkers can enable therapeutic innovation and interventions to improve the quality of life of older adults.

Korean Chestnut Honey Suppresses HSV-1 Infection by Regulating the ROS-NLRP3 Inflammasome Pathway.

Herpes simplex virus 1 (HSV-1) is double-stranded DNA virus that belongs to the Orthoherpesviridae family. It causes serious neurological diseases of the central nervous system, such as encephalitis. The current U.S. Food and Drug Administration (FDA)-approved drugs for preventing HSV-1 infection include acyclovir (ACV) and valacyclovir; however, their long-term use causes severe side effects and often results in the emergence of drug-resistant strains. Therefore, it is important to discover new antiviral agents that are safe and effective against HSV-1 infection. Korean chestnut honey (KCH) has various pharmacological activities, such as antioxidant, antibacterial, and anti-inflammation effects; however, antiviral effects against HSV-1 have not yet been reported. Therefore, we determined the antiviral activity and mechanism of action of KCH after HSV-1 infection on the cellular level. KCH inhibited the HSV-1 infection of host cells through binding and virucidal steps. KCH decreased the production of reactive oxygen species (ROS) and calcium (Ca2+) following HSV-1 infection and suppressed the production of inflammatory cytokines by inhibiting nuclear factor kappa-light-chain-enhancer of activated B cells (NF-кB) activity. Furthermore, we found that KCH inhibited the expression of the nod-like receptor protein 3 (NLRP3) inflammasome during HSV-1 infection. Taken together, the antiviral effects of KCH occur through multiple targets, including the inhibition of viral replication and the ROS-mediated NLRP3 inflammasome pathway. Our findings suggest that KCH has potential for the treatment of HSV-1 infection and related diseases.

ApoE4-dependent lysosomal cholesterol accumulation impairs mitochondrial homeostasis and oxidative phosphorylation in human astrocytes.

Recent developments in genome sequencing have expanded the knowledge of genetic factors associated with late-onset Alzheimer's disease (AD). Among them, genetic variant ε4 of the APOE gene (APOE4) confers the greatest disease risk. Dysregulated glucose metabolism is an early pathological feature of AD. Using isogenic ApoE3 and ApoE4 astrocytes derived from human induced pluripotent stem cells, we find that ApoE4 increases glycolytic activity but impairs mitochondrial respiration in astrocytes. Ultrastructural and autophagy flux analyses show that ApoE4-induced cholesterol accumulation impairs lysosome-dependent removal of damaged mitochondria. Acute treatment with cholesterol-depleting agents restores autophagic activity, mitochondrial dynamics, and associated proteomes, and extended treatment rescues mitochondrial respiration in ApoE4 astrocytes. Taken together, our study provides a direct link between ApoE4-induced lysosomal cholesterol accumulation and abnormal oxidative phosphorylation.

Understanding the relationship between cancer associated cachexia and hypoxia-inducible factor-1.

Cancer-associated cachexia (CAC) is a multifactorial disorder characterized by an unrestricted loss of body weight as a result of muscle and adipose tissue atrophy. Cachexia is influenced by several factors, including decreased metabolic activity and food intake, an imbalance between energy uptake and expenditure, excessive catabolism, and inflammation. Cachexia is highly associated with all types of cancers responsible for more than half of cancer-related mortalities worldwide. In healthy individuals, adipose tissue significantly regulates energy balance and glucose homeostasis. However, in metastatic cancer patients, CAC occurs mainly because of an imbalance between muscle protein synthesis and degradation which are organized by certain extracellular ligands and associated signaling pathways. Under hypoxic conditions, hypoxia-inducible factor-1 (HIF-1α) accumulated and translocated to the nucleus and activate numerous genes involved in cell survival, invasion, angiogenesis, metastasis, metabolic reprogramming, and cancer stemness. On the other hand, the ubiquitination proteasome pathway is inhibited during low O2 levels which promote muscle wasting in cancer patients. Therefore, understanding the mechanism of the HIF-1 pathway and its metabolic adaptation to biomolecules is important for developing a novel therapeutic method for cancer and cachexia therapy. Even though many HIF inhibitors are already in a clinical trial, their mechanism of action remains unknown. With this background, this review summarizes the basic concepts of cachexia, the role of inflammatory cytokines, pathways connected with cachexia with special reference to the HIF-1 pathway and its regulation, metabolic changes, and inhibitors of HIFs.

Vitisin B inhibits influenza A virus replication by multi-targeting neuraminidase and virus-induced oxidative stress.

The development of drug-resistant influenza and new pathogenic virus strains underscores the need for antiviral therapeutics. Currently, neuraminidase (NA) inhibitors are commonly used antiviral drugs approved by the US Food and Drug Administration (FDA) for the prevention and treatment of influenza. Here, we show that vitisin B (VB) inhibits NA activity and suppresses H1N1 viral replication in MDCK and A549 cells. Reactive oxygen species (ROS), which frequently occur during viral infection, increase virus replication by activating the NF-κB signaling pathway, downmodulating glucose-6-phosphate dehydrogenase (G6PD) expression, and decreasing the expression of nuclear factor erythroid 2-related factor 2 (Nrf2) antioxidant response activity. VB decreased virus-induced ROS generation by increasing G6PD expression and Nrf2 activity, and inhibiting NF-κB translocation to the nucleus through IKK dephosphorylation. In addition, VB reduced body weight loss, increased survival, decreased viral replication and the inflammatory response in the lungs of influenza A virus (IAV)-infected mice. Taken together, our results indicate that VB is a promising therapeutic candidate against IAV infection, complements existing drug limitations targeting viral NA. It modulated the intracellular ROS by G6PD, Nrf2 antioxidant response pathway, and NF-κB signaling pathway. These results demonstrate the feasibility of a multi-targeting drug strategy, providing new approaches for drug discovery against IAV infection.

Unlocking Prognostic Genes and Multi-Targeted Therapeutic Bioactives from Herbal Medicines to Combat Cancer-Associated Cachexia: A Transcriptomics and Network Pharmacology Approach.

Cachexia is a devastating fat tissue and muscle wasting syndrome associated with every major chronic illness, including cancer, chronic obstructive pulmonary disease, kidney disease, AIDS, and heart failure. Despite two decades of intense research, cachexia remains under-recognized by oncologists. While numerous drug candidates have been proposed for cachexia treatment, none have achieved clinical success. Only a few drugs are approved by the FDA for cachexia therapy, but a very low success rate is observed among patients. Currently, the identification of drugs from herbal medicines is a frontier research area for many diseases. In this milieu, network pharmacology, transcriptomics, cheminformatics, and molecular docking approaches were used to identify potential bioactive compounds from herbal medicines for the treatment of cancer-related cachexia. The network pharmacology approach is used to select the 32 unique genes from 238 genes involved in cachexia-related pathways, which are targeted by 34 phytocompounds identified from 12 different herbal medicines used for the treatment of muscle wasting in many countries. Gene expression profiling and functional enrichment analysis are applied to decipher the role of unique genes in cancer-associated cachexia pathways. In addition, the pharmacological properties and molecular interactions of the phytocompounds were analyzed to find the target compounds for cachexia therapy. Altogether, combined omics and network pharmacology approaches were used in the current study to untangle the complex prognostic genes involved in cachexia and phytocompounds with anti-cachectic efficacy. However, further functional and experimental validations are required to confirm the efficacy of these phytocompounds as commercial drug candidates for cancer-associated cachexia.

SMP30-mediated synthesis of vitamin C activates the liver PPARα/FGF21 axis to regulate thermogenesis in mice.

The vitamin-C-synthesizing enzyme senescent marker protein 30 (SMP30) is a cold resistance gene in Drosophila, and vitamin C concentration increases in brown adipose tissue post-cold exposure. However, the roles of SMP30 in thermogenesis are unknown. Here, we tested the molecular mechanism of thermogenesis using wild-type (WT) and vitamin C-deficient SMP30-knockout (KO) mice. SMP30-KO mice gained more weight than WT mice without a change in food intake in response to short-term high-fat diet feeding. Indirect calorimetry and cold-challenge experiments indicated that energy expenditure is lower in SMP30-KO mice, which is associated with decreased thermogenesis in adipose tissues. Therefore, SMP30-KO mice do not lose weight during cold exposure, whereas WT mice lose weight markedly. Mechanistically, the levels of serum FGF21 were notably lower in SMP30-KO mice, and vitamin C supplementation in SMP30-KO mice recovered FGF21 expression and thermogenesis, with a marked reduction in body weight during cold exposure. Further experiments revealed that vitamin C activates PPARα to upregulate FGF21. Our findings demonstrate that SMP30-mediated synthesis of vitamin C activates the PPARα/FGF21 axis, contributing to the maintenance of thermogenesis in mice.

Yuk-Gunja-Tang attenuates neuronal death and memory impairment via ERK/CREB/BDNF signaling in the hippocampi of experimental Alzheimer's disease model.

Yuk-Gunja-Tang (YG) is the Korean traditional medicine in East Asia for gastrointestinal disorders. In the present study, we determined the protective effects of YG on glutamate-induced cytotoxicity in HT22 hippocampal neuronal cells and mice with scopolamine-induced memory impairment. In vitro assessments were performed using a cell viability assay, flow cytometry, and Western blotting, while in vivo assessments were performed in C57BL/6 mice administered with YG for 7 days and injected with scopolamine (1 mg/kg) for 7 days. We assessed the memory function using the Y-maze, novel object recognition, and passive avoidance tests. Protein expression analyses and histological analyses were performed using hippocampal tissues. YG treatment significantly restored cell viability against glutamate-induced apoptosis. It significantly suppressed glutamate-induced reactive oxygen species accumulation and mitochondrial dysfunction. It also increased Bcl-2 protein expression and decreased HO-1 protein expression. It activated the extracellular signal-regulated kinase/cAMP response element binding protein (ERK/CREB) signaling pathway and increased the expression of brain-derived neurotrophic factor (BDNF) under excitotoxic conditions. In the scopolamine-injected mice, YG ameliorated memory impairment in the Y-maze, novel object recognition, and passive avoidance tests; restored dysfunction in the acetylcholine, acetylcholinesterase expression levels; reduced neuronal damage in Nissl staining; and increased BDNF and phosphorylated ERK and CREB levels in Western blotting and immunofluorescence staining. Thus, YG exerted neuroprotective effects by activating ERK/CREB/BDNF signaling in the hippocampus, indicating its potential cognition-enhancing effects, especially in Alzheimer's disease.

Quercus acuta Thunb. Suppresses LPS-Induced Neuroinflammation in BV2 Microglial Cells via Regulating MAPK/NF-κB and Nrf2/HO-1 Pathway.

Microglial activation-mediated neuroinflammation is associated with the pathogenesis of neurodegenerative disorders. Therefore, the management of microglial cell activation and their inflammatory response is an important therapeutic approach for preventing neurodegenerative diseases. Quercus acuta Thunb. (QA) (Fagaceae) is a tree found in Korea, China, and Japan. The current study investigated the anti-neuroinflammatory effects of QA and its mechanism of action in lipopolysaccharide (LPS)-stimulated BV2 microglial cells. Pretreatment with a methanol extract of dried QA stems (QAE) inhibited the production of nitric oxide and proinflammatory cytokines and decreased the expression of inducible nitric oxide synthase, cyclooxygenase-2 in LPS-stimulated BV2 microglial cells. Furthermore, it inhibited the phosphorylation and degradation of inhibitory κBα and decreased the nuclear translocation and phosphorylation of nuclear factor-κB (NF-κB). Moreover, QAE inhibited the phosphorylation of extracellular signal-regulated kinase, p38 and c-Jun N-terminal kinase, which is known as mitogen-activated protein kinase (MAPK). Additionally, QAE treatment increased heme oxygenase-1 (HO-1) expression by activating the nuclear factor erythroid 2-related factor 2 (Nrf2) signaling, thereby ameliorating LPS-induced intracellular hydrogen peroxide production. Finally, it was found that catechin and taxifolin, two phytochemicals of QAE, also reduced the expression of inflammatory mediators. These findings suggest that QA is beneficial for preventing microglia-mediated neuroinflammatory response through the inhibition of NF-κB, MAPK and the activation of Nrf2/HO-1 signaling pathways.

Vaccinium bracteatum Thunb Extract Inhibits HSV-1 Infection by Regulating ER Stress and Apoptosis.

Herpes simplex Type 1 (HSV-1) is a neurotropic virus that infects the peripheral and central nervous system. Usually, after primary infection in epithelial cells, HSV-1 migrates retrograde to the peripheral nervous system (PNS), where it establishes a latent infection. HSV-1 can remain latent in the nervous system, and its reactivation in the brain can rarely cause acute HSV-1 encephalitis, often a life-threatening condition, or asymptomatic reactivations that could lead to neuronal damage and ultimately neurodegenerative disorders. Acyclovir and related nucleoside analogs have been used as therapeutic agents for HSV-1 infection, but resistance to the drug can arise, and the protective effect of HSV-1 on brain cells is limited. Therefore, there is an urgent need for research into safe and effective new antiviral agents that can protect brain cells from the damage that is caused by HSV-1 infection. Vaccinium bracteatum Thunb. (VBT) is widely distributed in Korea and China, and has pharmacological actions such as anti-inflammatory, antioxidant, and antidiabetic activity. Studies on the antiviral effect of VBT on HSV-1 infection have not been reported so far. Therefore, we sought to determine the HSV-1 antiviral effect and molecular mechanism of VBT at the cellular level. We confirmed that VBT repressed the VP16 and IE genes in both Vero and SK-N-SH cells. We also found that the generation of HSV-1 virions was inhibited by VBT treatment. VBT inhibited the activities of the HSV-1-induced endoplasmic reticulum (ER) stressors PERK, ATF4, and CHOP. We confirmed that VBT inhibited the activity of apoptosis factors by regulating the expression of death receptor (DR) after HSV-1 infection. As HSV-1 is closely associated with brain diseases, the study of the antiviral drug effects and mechanism of VBT is meaningful. Further studies using animal models of infection will also be performed to determine the potential of VBT as an antiviral agent.

Menthae Herba Attenuates Neuroinflammation by Regulating CREB/Nrf2/HO-1 Pathway in BV2 Microglial Cells.

Chronic inflammation and oxidative stress cause microglia to be abnormally activated in the brain, resulting in neurodegenerative diseases such as Alzheimer's disease (AD). Menthae Herba (MH) has been widely used as a medicinal plant with antimicrobial, anti-inflammatory, and antioxidant properties. In this study, we sought to evaluate the effects of MH on the inflammatory response and possible molecular mechanisms in microglia stimulated with lipopolysaccharide (LPS). Transcriptional and translational expression levels of the proinflammatory factors were measured using ELISA, RT-qPCR, and Western blot analysis. MH extract inhibited the production of proinflammatory enzymes and mediators nitric oxide (NO), NO synthase, cyclooxygenase-2, tumor necrosis factor-α, and interleukin-6 in LPS-stimulated cells. Our molecular mechanism study showed that MH inhibited the production of reactive oxygen species (ROS) and the phosphorylation of mitogen-activated protein kinase and nuclear factor (NF)-κB. In contrast, MH activated HO-1 and its transcriptional factors, cAMP response element-binding protein (CREB), and the nuclear factor erythroid 2-related factor 2 (Nrf2) signaling pathways. Thus, MH reduces ROS and NF-κB-mediated inflammatory signaling and induces CREB/Nrf2/HO-1-related antioxidant signaling in microglia. Together, these results may provide specific prospects for the therapeutic use of MH in the context of neuroinflammatory diseases, including AD.

Samhwangsasim-tang attenuates neuronal apoptosis and cognitive decline through BDNF-mediated activation of tyrosin kinase B and p75-neurotrophin receptors.

BACKGROUND: Samhwangsasim-tang (SST) is a traditional medicine used to treat hypertension and arteriosclerosis. Additionally, due to the effects of its constituent herbs, SST is considered effective for memory-related disorders. PURPOSE: We investigated the effects of SST on neuronal survival and memory in glutamate-induced hippocampal cells and in a mouse model of scopolamine-induced memory impairment. METHODS: SST components were identified using 3D-ultra performance liquid chromatography (3D-UPLC). In vitro, we induced glutamate-induced excitotoxicity in HT22 cells after SST pretreatment. We used a cell counting kit-8 and cell cytotoxicity assay, flow cytometry, and western blotting to test the protective effects of SST on neuronal death. In vivo, C57BL/6J mice were administered with 150 and 300 mg/kg SST once daily for 7 days and then intraperitoneally injected with 1 mg/kg scopolamine for 7 days to induce cognitive impairment. We then measured cognitive behavior using a novel object recognition test (NORT) and passive avoidance test (PAT) and analyzed the histological and protein changes. RESULTS: Our results showed that treatment with 50 and 100 µg/ml SST provided significant protection against glutamate-induced cell death. Flow cytometry and western blotting results suggested that 100 µg/ml SST treatment reduced oxidative stress and mitochondrial dysfunction. SST treatment also increased brain-derived neurotrophic factor (BDNF), its receptor, TrkB receptor, and cAMP-response element binding protein (CREB) activation while reducing the P75NTR and JNK signaling activation. Our in vivo results showed that SST administration improved cognitive impairment, similar to donepezil treatment (as a positive control), in NORT and PAT. SST and donepezil decreased neuronal cell death and apoptosis, and acetylcholine levels were increased in the scopolamine-treated hippocampus. Additionally, SST promoted CREB phosphorylation and BDNF maturation while reducing JNK and P75NTR activation; in contrast, donepezil did not alter levels of these proteins in the scopolamine-treated mouse hippocampus. CONCLUSION: Our results suggest that SST has neuroprotective effects to attenuate neuronal cell death and oxidative stress through CREB/JNK signaling via BDNF activation. SST may regulate endogenous survival factors in the hippocampus, which may be a safe and potential clinical treatment for cognitive impairment in AD.

Banhasasim-Tang Ameliorates Spatial Memory by Suppressing Oxidative Stress through Regulation of ERK/p38 Signaling in Hippocampus of Mice.

Since ancient times, Banhasasim-tang (BHS) has been used to treat functional dyspepsia in East Asia. Here, we aimed to determine the protective action of BHS on hippocampal neurons against oxidative stress. We investigated the functional effect of BHS on a scopolamine-induced mouse model, and molecular analysis was performed in glutamate-induced HT22 cells. We observed that BHS administration ameliorated memory dysfunction in scopolamine-treated mice. BHS administration also increased neuronal survival and acetylcholine activity and phosphorylation of extracellular signal-regulated kinase (ERK) and cAMP response element-binding protein (CREB) in the hippocampus of mice. In hippocampal cells, BHS treatment rescued glutamate-induced cytotoxicity, apoptosis, and oxidative stress. We observed an increase of HO-1 and a decrease of Nrf2 protein expression in glutamate-induced oxidative stress; however, the expression level of these proteins was significantly rescued by BHS treatment. BHS treatment also regulated phosphorylation of p38, p53, ERK, and CREB. Therefore, our data indicated that BHS may reduce oxidative stress through regulation of ERK-CREB and p38-p53 signaling in the hippocampus, resulting in decreased neuronal damage and improved memory in rodent models of neurodegenerative disease.

Quercus acuta Thunb. (Fagaceae) and Its Component, Isoquercitrin, Inhibit HSV-1 Replication by Suppressing Virus-Induced ROS Production and NF-κB Activation.

HSV-1 is a neurotropic virus that replicates lytically during acute infection and establishes latency in peripheral neurons. Currently, the clinically approved compounds for the prevention of HSV-1 infection include acyclovir and penciclovir; however, long-term use of the drug is associated with serious side effects, and drug-resistant strains often appear. Therefore, it is important to find a safe and novel antiviral agent for HSV-1 infection. Quercus acuta Thunb. (Fagaceae) (QA) is widely distributed as an ornamental and dietary plant in Korea, Taiwan, China, and Japan. Thus far, the effects of QA extract and its active ingredients are known to have antioxidant, antibacterial, and anti-inflammatory activity, but studies of possible antiviral effects have not been reported. We studied the antiviral effects and molecular mechanism of QA after HSV-1 infection at the cellular level. We confirmed that QA suppresses ROS expression after HSV-1 infection and also suppresses inflammatory cytokine expression through inhibition of NF-кB activity. In addition, we found that QA increases the phosphorylation activity of IRF3 through induction of TBK1 activity during HSV-1 infection. QA exhibits an antiviral effect, and we confirmed through UPLC-DAD-mass spectrometer (MS)/MS analysis that it contains five main components: catechin, chlorogenic acid, fraxin, isoquercitrin, and taxifolin. Of these, isoquercitrin was confirmed to exhibit an antiviral effect on SK-N-SH cells through ICP27 inhibition. Overall, our results suggest that QA is a novel inhibitor with antiviral effects against HSV-1 infection and may be used specifically to prevent and treat of herpes simplex virus encephalitis infection.

Herbal Medicine for Patients with Cognitive Impairment: An Observational Study.

PURPOSE: The potential effects of herbal medicine for patients with cognitive disorders have been reported in various human and animal studies. This study aimed to explore the effect of herbal medicine treatment according to the Korean Medicine (KM) pattern identification for patients with mild cognitive impairment and early dementia. PATIENTS AND METHODS: Twenty patients with mild cognitive impairment or mild dementia who planned to receive herbal medicine treatment were enrolled. Herbal formulae were prescribed based on the KM pattern for 12-24 weeks. Seoul Neuropsychological Screening Battery II (SNSB-II) and Montreal Cognitive Assessment (MoCA) were assessed at the baseline, after 12 weeks, and after 24 weeks (Trial registration: cris.nih.go.kr, KCT0004799). RESULTS: Herbal medicine products, including Yukmijihwang-tang, Samhwangsasim-tang, Palmul-tang, Banhasasim-tang, and Yukgunja-tang, were prescribed to the patients. Among the SNSB-II five cognitive function domains, the T scores for language, visuospatial function, memory, and frontal/executive function increased over time. The MoCA score also improved following the treatment (mean difference 4.23 [95% CI: 2.60, 5.86], p < 0.0001 at 12-week follow-up compared to the baseline). Considering the KM pattern scores, phlegm-dampness and fire-heat scores tended to improve after the treatment. No serious adverse events related to the intervention were reported. CONCLUSION: The potential effect of herbal medicine formulae products on improving cognitive functions in patients with cognitive impairment was observed. Further research is needed to objectify the KM pattern identification process and evaluate the KM pattern-related signs and symptoms.

Unusual Bicyclo[3.2.1]Octanoid Neolignans from Leaves of Piper crocatum and Their Effect on Pyruvate Dehydrogenase Activity.

Three undescribed bicyclo[3.2.1]octanoid neolignan glucosides, along with a known neolignan, were isolated from the leaves of Piper crocatum Ruiz & Pav. Their chemical structures were elucidated using extensive spectroscopic analyses including 1D and 2D NMR experiments and HR-ESI-MS analysis, as well as through comparison with previously reported data. Two compounds were assessed for their inhibitory effect against pyruvate dehydrogenase E1α S300 phosphorylation. The fluorescent image suggested that both compounds (60 µM) revealed a stronger inhibition effect than the positive control (dichloroacetate, DCA 5 mM), with IC50 values of 99.82 µM and 80.25 µM, respectively.

Pyruvate dehydrogenase kinase 1 and 2 deficiency reduces high-fat diet-induced hypertrophic obesity and inhibits the differentiation of preadipocytes into mature adipocytes.

Obesity is now recognized as a disease. This study revealed a novel role for pyruvate dehydrogenase kinase (PDK) in diet-induced hypertrophic obesity. Mice with global or adipose tissue-specific PDK2 deficiency were protected against diet-induced obesity. The weight of adipose tissues and the size of adipocytes were reduced. Adipocyte-specific PDK2 deficiency slightly increased insulin sensitivity in HFD-fed mice. In studies with 3T3-L1 preadipocytes, PDK2 and PDK1 expression was strongly increased during adipogenesis. Evidence was found for epigenetic induction of both PDK1 and PDK2. Gain- and loss-of-function studies with 3T3-L1 cells revealed a critical role for PDK1/2 in adipocyte differentiation and lipid accumulation. PDK1/2 induction during differentiation was also accompanied by increased expression of hypoxia-inducible factor-1α (HIF1α) and enhanced lactate production, both of which were absent in the context of PDK1/2 deficiency. Exogenous lactate supplementation increased the stability of HIF1α and promoted adipogenesis. PDK1/2 overexpression-mediated adipogenesis was abolished by HIF1α inhibition, suggesting a role for the PDK-lactate-HIF1α axis during adipogenesis. In human adipose tissue, the expression of PDK1/2 was positively correlated with that of the adipogenic marker PPARγ and inversely correlated with obesity. Similarly, PDK1/2 expression in mouse adipose tissue was decreased by chronic high-fat diet feeding. We conclude that PDK1 and 2 are novel regulators of adipogenesis that play critical roles in obesity.

PAR2 Deficiency Induces Mitochondrial ROS Generation and Dysfunctions, Leading to the Inhibition of Adipocyte Differentiation.

Protease-activated receptor 2 (PAR2) is a member of G-protein-coupled receptors and affects ligand-modulated calcium signaling. Although PAR2 signaling promotes obesity and adipose tissue inflammation in high fat- (HF-) fed conditions, its role in adipocyte differentiation under nonobesogenic conditions needs to be elucidated. Here, we used several tissues and primary-cultured adipocytes of mice lacking PAR2 to study its role in the development of adipose tissues. C57BL/6J mice with PAR2 deficiency exhibited a mild lipodystrophy-like phenotype in a chow diet-fed condition. When adipocyte differentiation was examined using primary-cultured preadipocytes, PAR2 deficiency led to a notable decrease in adipocyte differentiation and related protein expression, and PAR2 agonist treatment elevated adipocyte differentiation. Regarding the mechanism, PAR2-deficient preadipocytes exhibited impaired mitochondrial energy consumption. Further studies indicated that calcium-related signaling pathways for mitochondrial biogenesis are disrupted in the adipose tissues of PAR2-deficient mice and PAR2-deficient preadipocytes. Also, a PAR2 antagonist elevated mitochondrial reactive oxygen species and reduced the MitoTracker fluorescent signal in preadipocytes. Our studies revealed that PAR2 is important for the development of adipose tissue under basal conditions through the regulation of mitochondrial biogenesis and adipocyte differentiation.

Mori ramulus and its Major Component Morusin Inhibit Herpes Simplex Virus Type 1 Replication and the Virus-Induced Reactive Oxygen Species.

Herpes simplex virus type 1 (HSV-1) is ubiquitous in many populations despite the use of acyclovir or related nucleoside analogs for treating infection. Drug resistance impairs the treatment of HSV-infected individuals who have immune deficits, underscoring the need for new safe and effective antiviral agents. Mori ramulus (the young twig of Morus alba L.) has long been used to treat diseases in Korea, Japan, and China. Recent studies have reported multiple pharmacological activities of Mori ramulus and its constituent morusin, but their effects on HSV-1 remain unknown. Here, we found that treatment with Mori ramulus ethanol extract (MRE) significantly reduced the replication of fluorescently labeled HSV-1 in Vero cells and inhibited the expression of HSV-1 envelope glycoprotein D (gD) and tegument protein VP16. MRE, furthermore, blocked HSV-1-induced production of reactive oxygen species (ROS), and this mediated the inhibition of viral replication. We identified morusin as the active antiviral component of MRE and found that morusin post-treatment was sufficient to inhibit viral gD and VP16 in addition to HSV-1-induced ROS production. Therefore, the inhibition of HSV-1-induced ROS may explain the antiviral activity of MRE against HSV-1. MRE or its component morusin may be potentially developed for anti-HSV-1 agents.

Astrocytic pyruvate dehydrogenase kinase-2 is involved in hypothalamic inflammation in mouse models of diabetes.

Hypothalamic inflammation plays an important role in disrupting feeding behavior and energy homeostasis as well as in the pathogenesis of obesity and diabetes. Here, we show that pyruvate dehydrogenase kinase (PDK)-2 plays a role in hypothalamic inflammation and its sequelae in mouse models of diabetes. Cell type-specific genetic ablation and pharmacological inhibition of PDK2 in hypothalamic astrocytes suggest that hypothalamic astrocytes are involved in the diabetic phenotype. We also show that the PDK2-lactic acid axis plays a regulatory role in the observed metabolic imbalance and hypothalamic inflammation in mouse primary astrocyte and organotypic cultures, through the AMPK signaling pathway and neuropeptidergic circuitry governing feeding behavior. Our findings reveal that PDK2 ablation or inhibition in mouse astrocytes attenuates diabetes-induced hypothalamic inflammation and subsequent alterations in feeding behavior.