Lysophosphatidic Acid Induces Podocyte Pyroptosis in Diabetic Nephropathy by an Increase of Egr1 Expression via Downregulation of EzH2.

Podocyte damage and renal inflammation are the main features and pathogenesis of diabetic nephropathy (DN). Inhibition of lysophosphatidic acid (LPA) receptor 1 (LPAR1) suppresses glomerular inflammation and improves DN. Herein, we investigated LPA-induced podocyte damage and its underlying mechanisms in DN. We investigated the effects of AM095, a specific LPAR1 inhibitor, on podocytes from streptozotocin (STZ)-induced diabetic mice. E11 cells were treated with LPA in the presence or absence of AM095, and the expression of NLRP3 inflammasome factors and pyroptosis were measured. A chromatin immunoprecipitation assay and Western blotting were performed to elucidate underlying molecular mechanisms. Gene knockdown by transfecting small interfering RNA was used to determine the role of the transcription factor Egr1 (early growth response protein 1) and histone methyltransferase EzH2 (Enhancer of Zeste Homolog 2) in LPA-induced podocyte injury. AM095 administration inhibited podocyte loss, NLRP3 inflammasome factor expression, and cell death in STZ-induced diabetic mice. In E11 cells, LPA increased NLRP3 inflammasome activation and pyroptosis via LPAR1. Egr1 mediated NLRP3 inflammasome activation and pyroptosis in LPA-treated E11 cells. LPA decreased H3K27me3 enrichment at the Egr1 promoter in E11 cells by downregulating EzH2 expression. EzH2 knockdown further increased LPA-induced Egr1 expression. In podocytes from STZ-induced diabetic mice, AM095 suppressed Egr1 expression increase and EzH2/H3K27me3 expression reduction. Collectively, these results demonstrate that LPA induces NLRP3 inflammasome activation by downregulating EzH2/H3K27me3 and upregulating Egr1 expression, resulting in podocyte damage and pyroptosis, which may be a potential mechanism of DN progression.

Bavachin alleviates diabetic nephropathy in db/db mice by inhibition of oxidative stress and improvement of mitochondria function.

Diabetic nephropathy (DN) is a major complication of diabetes mellitus. Psoralea corylifolia L. seed (PCS) is a traditional medicine effective against various diseases. In this study, we aimed to investigate the effect of bavachin, the major active component of PCS, on DN in db/db mice. Bavachin (10 mg/kg/day) was administered orally to 12-week-old male db/db mice for 6 wk. For in vitro experiments, SV40 MES13 cells were treated with bavachin in the presence of 25 mM glucose. Food and water intake and urine mass were significantly increased in db/db mice compared to wild-type CON mice, but bavachin administration significantly reduced these increases. Urinary microalbumin, blood urea nitrogen, and creatinine clearance which were significantly increased in db/db mice, were also decreased by bavachin administration. Glomerular area and collagen deposition in the kidney were significantly decreased in db/db mice following bavachin administration. Increased renal levels of fibrotic factors, fibronectin, COL1A1, and α-SMA, were reduced following bavachin administration. Protein expressions of antioxidant enzymes, namely SOD2, catalase, and HO-1, and mitochondrial function-related factors, namely SIRT1, PGC1α, Nrf1, and mtTFA, were reduced in the kidney tissues of db/db mice compared to wild-type CON mice, and bavachin administration upregulated these protein expressions. In vitro studies also showed that bavachin decreases mitochondria ROS production, increases the expression of PGC-1α and SIRT1, and decreases the expression of α-SMA in high glucose-treated SV40 MES13 cells. Based on these results, bavachin improved DN by inhibiting oxidative stress and enhancing mitochondrial function.

The Inhibition of Autophagy and Pyroptosis by an Ethanol Extract of Nelumbo nucifera Leaf Contributes to the Amelioration of Dexamethasone-Induced Muscle Atrophy.

Muscle atrophy is characterized by a decline in muscle mass and function. Excessive glucocorticoids in the body due to aging or drug treatment can promote muscle wasting. In this study, we investigated the preventive effect of Nelumbo nucifera leaf (NNL) ethanolic extract on muscle atrophy induced by dexamethasone (DEX), a synthetic glucocorticoid, in mice and its underlying mechanisms. The administration of NNL extract increased weight, cross-sectional area, and grip strength of quadriceps (QD) and gastrocnemius (GA) muscles in DEX-induced muscle atrophy in mice. The NNL extract administration decreased the expression of muscle atrophic factors, such as muscle RING-finger protein-1 and atrogin-1, and autophagy factors, such as Beclin-1, microtubule-associated protein 1A/1B-light chain 3 (LC3-I/II), and sequestosome 1 (p62/SQSTM1) in DEX-injected mice. DEX injection increased the protein expression levels of NOD-like receptor pyrin domain-containing protein 3 (NLRP3), cleaved-caspase-1, interleukin-1beta (IL-1ß), and cleaved-gasdermin D (GSDMD), which were significantly reduced by NNL extract administration (500 mg/kg/day). In vitro studies using C2C12 myotubes also revealed that NNL extract treatment inhibited the DEX-induced increase in autophagy factors, pyroptosis-related factors, and NF-κB. Overall, the NNL extract prevented DEX-induced muscle atrophy by downregulating the ubiquitin-proteasome system, autophagy pathway, and GSDMD-mediated pyroptosis pathway, which are involved in muscle degradation.

Bavachin and Corylifol A Improve Muscle Atrophy by Enhancing Mitochondria Quality Control in Type 2 Diabetic Mice.

Type 2 diabetes reduces muscle mass and function. Chronic inflammation and mitochondrial dysfunction play critical roles in muscle atrophy pathogenesis. Here, we investigated the effects of bavachin and corylifol A from Psoralea corylifolia L. seeds on muscle atrophy in dexamethasone-treated mice and in db/db mice. Bavachin and corylifol A enhanced muscle strength and muscle mass in dexamethasone-treated mice. In diabetic mice, they enhanced muscle strength and cross-sectional areas. Bavachin and corylifol A suppressed inflammatory cytokine (interleukin-6 and tumor necrosis factor-α) expression levels by downregulating nuclear factor-κB phosphorylation. They decreased the muscle atrophic factor (myostatin, atrogin-1, and muscle RING finger-1) expression levels. They activated the AKT synthetic signaling pathway and induced a switch from fast-type glycolytic fibers (type 2B) to slow-type oxidative fibers (types I and 2A). They increased mitochondrial biogenesis and dynamic factor (optic atrophy-1, mitofusin-1/2, fission, mitochondrial 1, and dynamin 1-like) expression levels via the AMP-activated protein kinase-peroxisome proliferator-activated receptor gamma coactivator 1-alpha signaling pathway. They also improved mitochondrial quality by upregulating the mitophagy factor (p62, parkin, PTEN-induced kinase-1, and BCL2-interacting protein-3) expression levels. Therefore, bavachin and corylifol A exert potential therapeutic effects on muscle atrophy by suppressing inflammation and improving mitochondrial function.

LPA/LPAR1 signaling induces PGAM1 expression via AKT/mTOR/HIF-1α pathway and increases aerobic glycolysis, contributing to keratinocyte proliferation.

AIMS: Psoriasis is a chronic inflammatory skin disease and lysophosphatidic acid (LPA) has recently been reported to contribute to its pathogenesis through keratinocyte proliferation. In this study, we investigated the metabolic changes in the LPA-induced keratinocyte hyperproliferation and underlying mechanisms. MAIN METHODS: HaCaT or HEKa cells were treated with LPA in the presence or absence of various chemical inhibitors. The glycolysis stress was measured using the Seahorse extracellular flux analyzer. Gene knockdown by siRNA transfection was used to investigate the role of LPAR1, PGAM1, and HIF-1α in LPA-induced metabolic changes. We confirmed the expression of PGAM1 and HIF-1α in imiquimod (IMQ)-induced psoriasis-like BALB/c mice. KEY FINDINGS: LPA increased aerobic glycolysis; however, treatment with ki16425, or LPAR1 knockdown inhibited LPA-induced glycolysis in HaCaT cells. LPA increased the expression of glycolytic enzyme PGAM1 through LPAR1. PGAM1 knockdown in HaCaT cells suppressed LPA-induced cell proliferation, changes in cell cycle factor expression, and inhibited LPA-induced aerobic glycolysis. LPA sequentially activated AKT, mTOR, its downstream target, p70 S6K, and increased HIF-1α expression through LPAR1. HIF-1α knockdown inhibited LPA-induced PGAM1 expression and aerobic glycolysis. A6730 also decreased LPA-induced activation of mTOR and p70 S6K and LPA-induced increases in HIF-1α and PGAM1 expression. Ki16425 suppressed the increased expression of PGAM1 and HIF-1α in the lesions of IMQ-induced psoriasis-like mice. In primary keratinocytes, LPA/LPAR1 signaling also induced AKT-mediated aerobic glycolysis. SIGNIFICANCE: Collectively, the results demonstrated that LPA induces aerobic glycolysis via AKT/mTOR/HIF-1α-dependent PGAM1 expression during keratinocytes proliferation and this might be one of the mechanisms of psoriasis development.

Prevention of LPS-Induced Acute Kidney Injury in Mice by Bavachin and Its Potential Mechanisms.

Acute kidney injury (AKI) is a serious complication of sepsis with a rapid onset and high mortality rate. Bavachin, an active component of Psoralea corylifolia L., reportedly has antioxidant, anti-apoptotic, and anti-inflammatory effects; however, its beneficial effects on AKI remain undetermined. We investigated the protective effect of bavachin on lipopolysaccharide (LPS)-induced AKI in mice and elucidated the underlying mechanism in human renal tubular epithelial HK-2 cells. Increased serum creatinine and blood urea nitrogen levels were observed in LPS-injected mice; however, bavachin pretreatment significantly inhibited this increase. Bavachin improved the kidney injury score and decreased the expression level of tubular injury markers, such as neutrophil gelatinase-associated lipocalin (NGAL) and kidney injury molecule-1 (KIM-1), in both LPS-injected mice and LPS-treated HK-2 cells. LPS-induced oxidative stress via phosphorylated protein kinase C (PKC) ß and upregulation of the NADPH oxidase (NOX) 4 pathway was also significantly decreased by treatment with bavachin. Moreover, bavachin treatment inhibited the phosphorylation of MAPKs (P38, ERK, and JNK) and nuclear factor (NF)-κB, as well as the increase in inflammatory cytokine levels in LPS-injected mice. Krüppel-like factor 5 (KLF5) expression was upregulated in the LPS-treated HK-2 cells and kidneys of LPS-injected mice. However, RNAi-mediated silencing of KLF5 inhibited the phosphorylation of NF-kB, consequently reversing LPS-induced KIM-1 and NGAL expression in HK-2 cells. Therefore, bavachin may ameliorate LPS-induced AKI by inhibiting oxidative stress and inflammation via the downregulation of the PKCß/MAPK/KLF5 axis.

Reactive oxygen species induce HNF-4α expression via the ASK1-CREB pathway, promoting ChREBP expression and lipogenesis in hepatocytes.

AIMS: The production of reactive oxygen species (ROS) increases with aging and is associated with liver diseases. ChREBP is involved in lipogenic gene expression in hepatocytes, and we hypothesized that increases in ROS production would induce liver metabolic diseases via regulation of ChREBP expression. MAIN METHODS: Mechanism studies were conducted using H2O2 induced HepG2 cells and primary hepatocytes from old mouse. KEY FINDINGS: We detected increases in ChREBP expression, lipogenic gene (FAS and SCD1) expression, and intracellular triglyceride (TG) levels in H2O2-treated HepG2 cells, which were inhibited by ChREBP knockdown. ChREBP expression and intracellular TG levels were higher in primary hepatocytes from old mice than in those of young mice. Compared with old wild-type mice, intracellular TG levels were significantly reduced in primary hepatocytes from old ChREBP knockout mice. Furthermore, H2O2 stimulation was found to induce HNF-4α expression in HepG2 cells and this expression was higher in primary hepatocytes from old mice. ChIP-qPCR assays indicated that binding of HNF-4α to ChREBP promoter was significantly increased by H2O2 treatment in HepG2 cells. Further study revealed that H2O2-promoted increases in ChREBP expression and intracellular TG levels were significantly inhibited by HNF-4α knockdown. H2O2 treatment also significantly increased ASK1 expression, whereas ASK1 knockdown inhibited H2O2-induced HNF-4α expression. SIGNIFICANCE: Collectively, our data indicate that an increase in ROS production can induce HNF-4α expression via the ASK1 pathway and subsequently enhances the expression of ChREBP, thereby contributing to lipogenesis. This pathway may be involved in the elevated liver lipid levels associated with aging and oxidative stress.

Gomisin G improves muscle strength by enhancing mitochondrial biogenesis and function in disuse muscle atrophic mice.

Disuse muscle atrophy is characterized by a decrease in muscle mass and strength and an increase in glycolytic muscle fiber type. Although Schisandra chinensis extract has beneficial effects on muscle atrophy induced by various conditions (e.g., dexamethasone and aging), the effect of gomisin G, a lignan component of S. chinensis, on disuse muscle atrophy is unclear. Here, we induced disuse muscle atrophy through wire immobilization of the hind legs in mice followed by the oral administration of gomisin G. The cross-sectional area and muscle strength in disuse muscle atrophic mice were increased by gomisin G; however, the total muscle mass did not increase. Gomisin G decreased the expression of muscle atrophic factors (myostatin, atrogin-1, and MuRF1) but increased the expression of protein synthesis factors (mTOR and 4E-BP1). In H2O2-treated C2C12 myotubes, the level of puromycin incorporation (as a marker of protein synthesis) gradually increased in a dose-dependent manner by gomisin G. Furthermore, gomisin G induced a muscle fiber switch from fast-type glycolytic fibers (type 2B) to slow-type oxidative fibers (type I, 2A) in the gastrocnemius (GA) muscle as proved a decrease in the expression of TnI-FS and an increase in the expression of TnI-SS. Gomisin G increased mitochondrial DNA content and ATP levels in the GA muscle and COX activity in H2O2-treated C2C12 myotubes, improving mitochondrial function. Mechanistically, mitochondrial biogenesis is regulated by gomisin G via the Sirt 1/PGC-1α signaling pathway, targeting NRF1 and TFAM. These data suggest that gomisin G has a potential therapeutic effect on disuse muscle atrophy.

Lysophosphatidic Acid Promotes Epithelial-Mesenchymal Transition in Kidney Epithelial Cells via the LPAR1/MAPK-AKT/KLF5 Signaling Pathway in Diabetic Nephropathy.

The epithelial-mesenchymal transition (EMT) is a differentiation process associated with fibrogenesis in diabetic nephropathy (DN). Lysophosphatidic acid (LPA) is a small, naturally occurring glycerophospholipid implicated in the pathogenesis of DN. In this study, we investigated the role of LPA/LPAR1 signaling in the EMT of tubular cells as well as the underlying mechanisms. We observed a decrease in E-cadherin and an increase in vimentin expression levels in the kidney tubules of diabetic db/db mice, and treatment with ki16425 (LPAR1/3 inhibitor) inhibited the expression of these EMT markers. Ki16425 treatment also decreased the expression levels of the fibrotic factors fibronectin and alpha-smooth muscle actin (α-SMA) in db/db mice. Similarly, we found that LPA decreased E-cadherin expression and increased vimentin expression in HK-2 cells, which was reversed by treatment with ki16425 or AM095 (LPAR1 inhibitor). In addition, the expression levels of fibronectin and α-SMA were increased by LPA, and this effect was reversed by treatment with ki16425 and AM095 or by LPAR1 knockdown. Moreover, LPA induced the expression of the transcription factor, Krüppel-like factor 5 (KLF5), which was decreased by AM095 treatment or LPAR1 knockdown. The expression levels of EMT markers and fibrotic factors induced by LPA were decreased upon KLF5 knockdown in HK-2 cells. Inhibition of the extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and serine-threonine kinase (AKT) pathways decreased LPA-induced expression of KLF5 and EMT markers. In conclusion, these data suggest that LPA contributes to the pathogenesis of diabetic nephropathy by inducing EMT and renal tubular fibrosis via regulation of KLF5 through the LPAR1.

Effect of BBT-877, a novel inhibitor of ATX, on a mouse model of type 1 diabetic nephropathy.

Diabetic nephropathy (DN) is one of the common microvascular complications of diabetes. Autotaxin (ATX) is an enzyme with lysophospholipase D activity, producing lysophosphatidic acid (LPA). LPA signaling has been implicated in renal fibrosis, thereby inducing renal dysfunction. BBT-877 is an orally administered small molecule inhibitor of ATX. However, its effect on DN has not been studied so far. In this study, we investigated the effect of BBT-877, a novel inhibitor of ATX, on the pathogenesis of DN in a mouse model of streptozotocin (STZ)-induced diabetes. BBT-877 treatment significantly reduced albuminuria, albumin-to-creatinine ratio (ACR), neutrophil gelatinase-associated lipocalin (NGAL), and glomerular volume compared to the STZ-vehicle group. Interestingly, BBT-877 treatment attenuated hyperglycemia and dyslipidemia in STZ-induced diabetes mice. In the liver, the expression levels of ß-oxidation-related genes such as PPAR α and CPT1 were significantly decreased in STZ-induced diabetic mice. However, this effect was reversed by BBT-877 treatment. BBT-877 treatment also suppressed mRNA levels of pro-inflammatory cytokines IL-6, MCP-1, and TNF-α and protein levels of fibrotic factors (TGF-ß, fibronectin, CTGF, and collagen type Ι alpha Ι (COL1A1)) in the kidneys of STZ-induced diabetic mice. In conclusion, our results indicate that BBT-877 is effective in preventing the pathogenesis of DN by reducing systemic blood glucose levels and inhibiting inflammation and fibrosis in the renal tissue of diabetes mice. These novel findings suggest that inhibition of ATX may be a potential therapeutic target for DN.

Psoralea corylifolia L. seed extract attenuates dexamethasone-induced muscle atrophy in mice by inhibition of oxidative stress and inflammation.

ETHNOPHARMACOLOGICAL RELEVANCE: The seeds of Psoralea corylifolia (PCS), also called "Boh-Gol-Zhee" in Korean, have been used in traditional medicine. PCS is effective for the treatment of vitiligo, cancer, inflammatory diseases, neurodegenerative diseases, kidney diseases, and musculoskeletal diseases. AIM OF THE STUDY: In this study, we validated the beneficial effects of PCS extract on dexamethasone (DEX)-induced muscle atrophy in mice. MATERIALS AND METHODS: DEX (20 mg/kg/day, 10 days) was intraperitoneally injected into C57BL/6 male mice to induce muscular atrophy. Oral administration of PCS extract (200 or 500 mg/kg/day) was started 2 days before DEX injection and continued for 12 days. RESULTS: PCS extract inhibited DEX-induced decrease in body and muscle weight, grip strength, and cross-sectional area of the tibialis anterior. PCS extract significantly increased the mRNA and protein expression levels of myosin heavy chain 1, 2A, and 2X in DEX-administered mice. DEX administration significantly increased the levels of muscle atrophy factors atrogin-1, muscle RING-finger protein-1, and myostatin, which were inhibited by the PCS extract. Additionally, PCS extract increased the expression of muscle regeneration factors, such as myoblast determination protein 1, myogenin, and embryonic myosin heavy chain, and muscle synthesis markers, such as protein kinase B and mammalian target of rapamycin signaling molecules. PCS extract also significantly decreased the DEX-induced production of 4-hydroxynonenal, an oxidative stress marker. Furthermore, PCS extract recovered superoxide dismutase 2, glutathione peroxidase, and catalase activities, which were significantly reduced by DEX administration. Moreover, DEX-induced activation of nuclear factor kappa-light-chain-enhancer of activated B cells and expression of cytokines, such as tumor necrosis factor α and monocyte chemoattractant protein-1, significantly decreased after PCS extract administration. CONCLUSIONS: Here, we demonstrated that PCS extract administration protected against DEX-induced muscle atrophy. This beneficial effect was mediated by suppressing the expression of muscle degradation factors and increasing the expression of muscle regeneration and synthesis factors. This effect was probably due to the inhibition of oxidative stress and inflammation. These results highlight the potential of PCS extract as a protective and therapeutic agent against muscle dysfunction and atrophy.

Inhibition of ChREBP ubiquitination via the ROS/Akt-dependent downregulation of Smurf2 contributes to lysophosphatidic acid-induced fibrosis in renal mesangial cells.

BACKGROUND: Mesangial cell fibrosis, a typical symptom of diabetic nephropathy (DN), is a major contributor to glomerulosclerosis. We previously reported that the pharmacological blockade of lysophosphatidic acid (LPA) signaling improves DN. Although LPA signaling is implicated in diabetic renal fibrosis, the underlying molecular mechanisms remain unclear. Here, the role of carbohydrate-responsive element-binding protein (ChREBP) in LPA-induced renal fibrosis and the underlying mechanisms were investigated. METHODS: Eight-week-old wild-type and db/db mice were intraperitoneally injected with the vehicle or an LPAR1/3 antagonist, ki16425 (10 mg/kg), for 8 weeks on a daily basis, following which the mice were sacrificed and renal protein expression was analyzed. SV40 MES13 cells were treated with LPA in the presence or absence of ki16425, and the expression of ChREBP and fibrotic factors, including fibronectin, TGF-ß, and IL-1ß, was examined. The role of ChREBP in the LPA-induced fibrotic response was investigated by ChREBP overexpression or knockdown. The involvement of Smad ubiquitination regulatory factor-2 (Smurf2), an E3 ligase, in LPA-induced expression of ChREBP and fibrotic factors was investigated by Smurf2 overexpression or knockdown. To identify signaling molecules regulating Smurf2 expression by LPA, pharmacological inhibitors such as A6370 (Akt1/2 kinase inhibitor) and Ly 294002 (PI3K inhibitor) were used. RESULTS: The renal expression of ChREBP increased in diabetic db/db mice, and was reduced following treatment with the ki16425. Treatment with LPA induced the expression of ChREBP and fibrotic factors, including fibronectin, TGF-ß, and IL-1ß, in SV40 MES13 cells, which were positively correlated. The LPA-induced expression of fibrotic factors increased or decreased following ChREBP overexpression and knockdown, respectively. The production of reactive oxygen species (ROS) mediated the LPA-induced expression of ChREBP and fibrotic factors, and LPA decreased Smurf2 expression via Traf4-mediated ubiquitination. The LPA-induced expression of ubiquitinated-ChREBP increased or decreased following Smurf2 overexpression and knockdown, respectively. Additionally, Smurf2 knockdown significantly increased the expression of ChREBP and fibrotic factors. The pharmacological inhibition of Akt signaling suppressed the LPA-induced alterations in the expression of ChREBP and Smurf2. CONCLUSION: Collectively, the results demonstrated that the ROS/Akt-dependent downregulation of Smurf2 and the subsequent increase in ChREBP expression might be one of the mechanisms by which LPA induces mesangial cell fibrosis in DN.

TGF-ß activates NLRP3 inflammasome by an autocrine production of TGF-ß in LX-2 human hepatic stellate cells.

Inflammation contributes to the pathogenesis of liver disease, and inflammasome activation has been identified as a major contributor to the amplification of liver inflammation. Transforming growth factor-beta (TGF-ß) is a key regulator of liver physiology, contributing to all stages of liver disease. We investigated whether TGF-ß is involved in inflammasome-mediated fibrosis in hepatic stellate cells. Treatment with TGF-ß increased priming of NLRP3 inflammasome signaling by increasing NLRP3 levels and activating TAK1-NF-kB signaling. Moreover, TGF-ß increased the expression of p-Smad2/3-NOX4 in LX-2 cells and consequently increased ROS content, which is a trigger for NLRP3 inflammasome activation. Elevated expression of NEK7 and active caspase-1 was also shown in TGF-ß-induced LX-2 cells, and this level was reduced by (5Z)-oxozeaenol, a TAK inhibitor. Finally, TGF-ß-treated cells significantly increased TGF-ß secretion levels, and their production was inhibited by IL-1ß receptor antagonist treatment. In conclusion, TGF-ß may represent an endogenous danger signal to the active NLRP3 inflammasome, by which IL-1ß mediates TGF-ß expression in an autocrine manner. Therefore, targeting the NLRP3 inflammasome may be a promising approach for the development of therapies for TGF-ß-induced liver fibrosis.

Ethanol Extract of Liriope platyphylla Root Attenuates Non-Alcoholic Fatty Liver Disease in High-Fat Diet-Induced Obese Mice via Regulation of Lipogenesis and Lipid Uptake.

Non-alcoholic fatty liver disease (NAFLD) is a common metabolic disorder that causes excess lipid accumulation in the liver and is the leading cause of end-stage liver disease. Liriope platyphylla is a medicinal herb that has long been used to treat cough, obesity, and diabetes. However, the effect of Liriope platyphylla on NAFLD has not been studied. The aim of this study was to investigate the effect of Liriope platyphylla root ethanolic extract (LPE) on hepatic lipid accumulation in high-fat diet (HFD)-induced obese mice. Six-week-old C57BL/6 male mice were fed a HFD for 8 weeks and then treated with LPE (100 or 250 mg/kg/day) by oral gavage for another 8 weeks. Body weight gain and liver weight were significantly lower in the 250 mg/kg LPE-treated HFD group than in the vehicle-treated HFD group. Histological analysis of liver sections demonstrated that LPE treatment reduced lipid accumulation compared to the vehicle treatment. The serum total cholesterol, AST, and ALT levels significantly decreased in the LPE-treated HFD group compared to those in the vehicle-treated HFD group. The LPE significantly decreases the protein expression levels of SREBP1, ACC, p-ACC, FAS, and SCD1, which are involved in lipogenesis, and PPARγ, CD36/FAT, and FATP5, which are involved in fatty acid uptake, both in vivo and in vitro. Thus, LPE may attenuate HFD-induced NAFLD by decreasing lipid accumulation by inhibiting lipogenesis and fatty acid uptake.

Effects of Psoralea corylifolia L. seed extract on AGEs-induced cell proliferation and fibrotic factor expression in mesangial cells.

Diabetic nephropathy is a microvascular complication of diabetes that is characterized by mesangial expansion and thickening of the glomerular basement membrane. The production of advanced glycation end products (AGEs) increases in diabetic patients. Activation of the receptor of AGE (RAGE) signaling pathway induces mesangial expansion via the reactive oxygen species (ROS)-mediated production of pro-inflammatory and extracellular matrix molecules. The Psoralea corylifolia L. seed (PCS) is a widely used herbal medicine with various biological activities. The current study investigated the effect of PCS extract on mesangial cell proliferation and the RAGE signaling pathway in SV40 MES 13 cells. SV40 MES 13 cells were harvested after treatment with various concentrations of PCS extract at 10 µg/ml AGEs for 24 h. The results revealed that the PCS extract inhibited AGEs-induced mesangial cell proliferation and cyclin protein expression in a concentration-dependent manner. In addition, the AGEs-induced expression of fibrotic factors, such as transforming growth factor ß, fibronectin and collagen, was reduced in mesangial cells after exposure to the PCS extract. The PCS extract also reduced RAGE expression and inhibited the expression of its downstream signaling pathways, such as NADPH oxidase, intracellular ROS and phospho-NF-κB. In conclusion, the data suggested that the PCS extract attenuated AGEs-induced renal mesangial cell proliferation and fibrosis via the suppression of oxidative stress and the downregulation of inflammatory and fibrotic factor expression.

Combination of PD98059 and TGF-ß1 Efficiently Differentiates Human Urine-Derived Stem Cells into Smooth Muscle Cells.

Pluripotent adult stem cells have potential applications in cell therapy and tissue engineering. Urine-derived stem cells (UDSCs) differentiate into various cell types. Here, we attempted to differentiate human UDSCs (hUDSCs) into smooth muscle cells (SMCs) using transforming growth factor-beta 1 (TGF-ß1) and/or PD98059, an extracellular signal-regulated kinase (ERK) inhibitor. Both quantitative polymerase chain reaction (qPCR) and Western blot analysis showed that the expression of messenger ribonucleic acid (mRNA) and proteins for alpha-smooth muscle actin (α-SMA), calponin (CNN1), and smooth muscle myosin heavy chain (SM-MHC), which are specific markers for SMCs, increased on day 9 after differentiation and again on day 14. The differentiated cells from human UDSCs (hUDSCs) with a combination of TGF-ß1 and PD98059 showed the highest expression of SMC marker proteins. Immunocytochemical staining performed to assess the molecular expression revealed CNN and α-SMA colocalizing in the cytoplasm. The cells that differentiated from hUDSCs with a combination of TGF-ß1 and PD98059 showed the strongest expression for CNN1, α-SMA, and SM-MHC. Functional testing of the differentiated cells revealed a stronger contractile capacity for the cells differentiated with a combination of PD98059 and TGF-ß1 than those differentiated with a single factor. These results suggest the combination of PD98059 and TGF-ß1 to be a more effective differentiation method and that differentiated SMCs could be used for restoring the functions of the sphincter muscle or bladder.

Lysophosphatidic Acid Mediates Imiquimod-Induced Psoriasis-like Symptoms by Promoting Keratinocyte Proliferation through LPAR1/ROCK2/PI3K/AKT Signaling Pathway.

Psoriasis is a chronic inflammatory skin disease. Recently, lysophosphatidic acid (LPA)/LPAR5 signaling has been reported to be involved in both NLRP3 inflammasome activation in macrophages and keratinocyte activation to produce inflammatory cytokines, contributing to psoriasis pathogenesis. However, the effect and molecular mechanisms of LPA/LPAR signaling in keratinocyte proliferation in psoriasis remain unclear. In this study, we investigated the effects of LPAR1/3 inhibition on imiquimod (IMQ)-induced psoriasis-like mice. Treatment with the LPAR1/3 antagonist, ki16425, alleviated skin symptoms in IMQ-induced psoriasis-like mouse models and decreased keratinocyte proliferation in the lesion. It also decreased LPA-induced cell proliferation and cell cycle progression via increased cyclin A2, cyclin D1, cyclin-dependent kinase (CDK)2, and CDK4 expression and decreased p27Kip1 expression in HaCaT cells. LPAR1 knockdown in HaCaT cells reduced LPA-induced proliferation, suppressed cyclin A2 and CDK2 expression, and restored p27Kip1 expression. LPA increased Rho-associated protein kinase 2 (ROCK2) expression and PI3K/AKT activation; moreover, the pharmacological inhibition of ROCK2 and PI3K/AKT signaling suppressed LPA-induced cell cycle progression. In conclusion, we demonstrated that LPAR1/3 antagonist alleviates IMQ-induced psoriasis-like symptoms in mice, and in particular, LPAR1 signaling is involved in cell cycle progression via ROCK2/PI3K/AKT pathways in keratinocytes.

Schisandrae chinensis Fructus Extract Ameliorates Muscle Atrophy in Streptozotocin-Induced Diabetic Mice by Downregulation of the CREB-KLF15 and Autophagy-Lysosomal Pathways.

Type 1 diabetes mellitus is an autoimmune disease caused by the destruction of pancreatic beta cells. Many patients with type 1 diabetes experience skeletal muscle wasting. Although the link between type 1 diabetes and muscle wasting is not clearly known, insulin insufficiency and hyperglycemia may contribute to decreased muscle mass. In this study, we investigated the therapeutic effect of the ethanolic extract of Schisandrae chinensis Fructus (SFe) on muscle wasting in streptozotocin (STZ)-induced diabetic mice. STZ-diabetic C57BL/6 mice (blood glucose level ≥300 mg/dL) were orally administered SFe (250 or 500 mg/kg/day) for 6 weeks. We observed that SFe administration did not change blood glucose levels but increased gastrocnemius muscle weight, cross-sectional area, and grip strength in STZ-induced diabetic mice. Administration of SFe (500 mg/kg) decreased the expression of atrophic factors, such as MuRF1 and atrogin-1, but did not alter the expression of muscle synthetic factors. Further studies showed that SFe administration decreased the expression of KLF15 and p-CREB, which are upstream molecules of atrophic factors. Examination of the expression of molecules involved in autophagy-lysosomal pathways (e.g., p62/SQSTM1, Atg7, Beclin-1, ULK-1, LC3-I, and LC3-II) revealed that SFe administration significantly decreased the expression of p62/SQSTM1, LC3-I, and LC3-II; however, no changes were observed in the expression of Atg7, Beclin-1, or ULK-1. Our results suggest that SFe ameliorated muscle wasting in STZ-induced diabetic mice by decreasing protein degradation via downregulation of the CREB-KLF15-mediated UPS system and the p62/SQSTM1-mediated autophagy-lysosomal pathway.

Dulaglutide improves muscle function by attenuating inflammation through OPA-1-TLR-9 signaling in aged mice.

Dulaglutide, a glucagon-like peptide-1 receptor (GLP-1R) agonist, is widely used to treat diabetes. However, its effects on muscle wasting due to aging are poorly understood. In the current study, we investigated the therapeutic potential and underlying mechanism of dulaglutide in muscle wasting in aged mice. Dulaglutide improved muscle mass and strength in aged mice. Histological analysis revealed that the cross-sectional area of the tibialis anterior (TA) in the dulaglutide-treated group was thicker than that in the vehicle group. Moreover, dulaglutide increased the shift toward middle and large-sized fibers in both young and aged mice compared to the vehicle. Dulaglutide increased myofiber type I and type IIa in young (18.5% and 8.2%) and aged (1.8% and 19.7%) mice, respectively, compared to the vehicle group. Peroxisome proliferator-activated receptor-gamma coactivator-1α (PGC-1α), a master regulator of mitochondrial biogenesis, decreased but increased by dulaglutide in aged mice. The expression of atrophic factors such as myostatin, atrogin-1, and muscle RING-finger protein-1 was decreased in aged mice, whereas that of the myogenic factor, MyoD, was increased in both young and aged mice following dulaglutide treatment. In aged mice, optic atrophy-1 (OPA-1) protein was decreased, whereas Toll-like receptor-9 (TLR-9) and its targeting inflammatory cytokines (interleukin-6 [IL-6] and tumor necrosis factor-α [TNF-α]) were elevated in the TA and quadriceps (QD) muscles. In contrast, dulaglutide administration reversed this expression pattern, thereby significantly attenuating the expression of inflammatory cytokines in aged mice. These data suggest that dulaglutide may exert beneficial effects in the treatment of muscle wasting due to aging.

Synthesis and anti-diabetic activity of novel biphenylsulfonamides as glucagon receptor antagonists.

Type 2 diabetes is characterized by chronic hyperglycemia. Insulin, a hormone secreted from pancreatic ß-cells, decreases blood glucose levels, and glucagon, a hormone secreted from pancreatic α-cells, increases blood glucose levels by counterregulation of insulin through stimulation of hepatic glucose production. In diabetic patients, dysregulation of glucagon secretion contributes to hyperglycemia. Thus, inhibition of the glucagon receptor is one strategy for the treatment of hyperglycemia in type 2 diabetes. In this paper, we report a series of biphenylsulfonamide derivatives that were designed, synthesized, and then evaluated by cAMP and hepatic glucose production assays as glucagon receptor antagonists. Of these, compound 7aB-3 decreased glucagon-induced cAMP production and glucagon-induced glucose production in the in vitro assays. Glucagon challenge tests and glucose tolerance tests showed that compound 7aB-3 significantly inhibited glucagon-induced glucose increases and improved glucose tolerance. These results suggest that compound 7aB-3 has therapeutic potential for the treatment of type 2 diabetes.