OBJECTIVE: To investigate the effect of biflavonoid 4'-O-methylochnaflavone (MF) on palmitic acid-induced endothelial dysfunction in rat cavernous endothelial cells (RCECs). METHODS: The isolated RCECs were commercially available and randomly divided into four groups: normal+BSA group (NC group), palmitic acid (PA) group, MF group, and icariside Ⅱ (ICA Ⅱ) group. The protein expression levels of protein kinase B (PKB/AKT) and endothelial nitric oxide synthase (eNOS) in each group were evaluated via Western blotting. The differences in the intracellular nitric oxide of RCECs treated by MF or ICA Ⅱ were detected by DAF-FM DA that served as a nitric oxide fluorescent probe. Effects of MF and ICA Ⅱ on cell proliferation of PA-stimulated RCECs were determined via CCK-8 assay. RESULTS: The content of nitric oxide in RCECs was significantly increased after the treatment of MF and ICA Ⅱ in comparison with the NC group (P < 0.05). Moreover, compared with ICA Ⅱ group, MF demonstrated a more obvious effect in promoting nitric oxide production (P < 0.05). Compared with the NC group, the expression levels of eNOS and AKT in the PA group were significantly decreased, indicating that a model for simulating the high-fat environment in vitro was successfully constructed (P < 0.05). Meanwhile, the intervention of MF and ICA Ⅱ could effectively increase the expression of eNOS and AKT, suggesting that MF and ICA Ⅱ could promote the recovery of endothelial dysfunction caused by high levels of free fatty acids (P < 0.05). The results of CCK-8 assays showed that PA could significantly reduce the proli-feration ability of RCECs (P < 0.05). Furthermore, the decreased cell viability induced by PA was significantly elevated by treatment with ICA Ⅱ and MF (P < 0.05). CONCLUSION In RCECs, MF and ICA Ⅱ could effectively increase the content of nitric oxide. The down-regulation of the expression of proteins associated with the AKT/eNOS pathway after PA treatment revealed that this pathway was involved in the development of endothelial dysfunction, which could be effectively reversed by MF and ICA Ⅱ. In addition, the cell proliferation ability was significantly decreased following PA treatment, but MF and ICA Ⅱ could restore the above changes. Overall, biflavonoid MF has an obvious repairing effect on PA-stimulated endothelial dysfunction.
Animals ; Biflavonoids/pharmacology* ; Cells, Cultured ; Endothelial Cells/metabolism* ; Nitric Oxide/pharmacology* ; Nitric Oxide Synthase Type III/pharmacology* ; Palmitic Acid/pharmacology* ; Phosphorylation ; Proto-Oncogene Proteins c-akt/metabolism* ; Rats ; Signal Transduction ; Sincalide/pharmacology*

The present study observed the effect of Guanxin Zhitong Capsules(GXZT) on the lipotoxicity of vascular endothelial cells and investigated the mechanism of GXZT in atherosclerosis treatment. The lipotoxicity model in human umbilical vein endothelial cells(HUVECs) was induced by palmitic acid(PA) stimulation. These cells were divided into a normal control group(NC, 15% normal serum), a model group(PA, 0.6 mmol·L~(-1) PA+15% normal serum), a high-dose GXZT group(GXZT-H, 0.6 mmol·L~(-1) PA+15% GXZT-medicated serum), a medium-dose GXZT group(GXZT-M, 0.6 mmol·L~(-1) PA+10% GXZT-medicated serum+5% normal serum) and a low-dose GXZT group(GXZT-L, 0.6 mmol·L~(-1) PA+5% GXZT-medicated serum+10% normal serum). HUVECs were detected for cell viability by cell counting kit-8(CCK-8) assay, apoptosis by flow cytometry, mitochondrial membrane potential(MMP) by JC-1 labeled laser scanning confocal microscopy, and total and phosphorylated proteins of p38, ERK1/2, and JNK1/2 in the mitogen-activated protein kinases(MAPK) signaling pathway by Western blot. The phosphorylated level was calcula-ted. Compared with the NC group, the PA group showed decreased cell viability and MMP(P<0.01, P<0.01), elevated apoptosis(P<0.01), and up-regulated phosphorylated levels of p38, ERK1/2, and JNK1/2(P<0.01, P<0.01, P<0.01). Compared with the PA group, the GXZT-H, GXZT-M, and GXZT-L groups showed increased cell viability and MMP(P<0.01, P<0.01, P<0.01), reduced apoptosis(P<0.01), and down-regulated protein expression and phosphorylated levels of p38, ERK1/2 and JNK1/2 in the MAPK signaling pathway(P<0.01, P<0.01, P<0.01). In conclusion, the results suggest that GXZT functions via blocking MAPK signaling pathway to relieve the damage of HUVECs induced by PA.
Apoptosis ; Capsules ; Human Umbilical Vein Endothelial Cells/metabolism* ; Humans ; MAP Kinase Signaling System ; Palmitic Acid/toxicity* ; Signal Transduction ; p38 Mitogen-Activated Protein Kinases/metabolism*

OBJECTIVES: High fat-induced podocyte injury is one of the important factors leading to obesity related nephropathy (ORG), but the mechanism is not clear. This study aims to explore the mechanism of period circadian clock 3 (PER3) in the oxidative stress and inflammation induced by palmitic acid (PA) in podocytes. METHODS: The C57BL/6J mice were fed with chow and high-fat diet for 16 weeks. The PER3 expression in kidney tissues were detected in the normal body weight group and the obesity group. The PER3 mRNA and protein expression were detected after the podocytes were induced with different concentrations (0, 50, 150 and 300 μmol/L) of PA for 48 h. The PER3 mRNA and protein expression were detected after the podocytes were induced with 150 μmol/L PA for 0, 24, 36, and 48 h. Triglyceride (TG) levels were examined in the PA group, the adenovirus (ad)-PER3+PA group, and the siRNA-PER+PA group after the podocytes were transfected by Ad-PER3 or small interfering RNA (siRNA)-PER3 for 48 h and subsequently were induced with 150 μmol/L PA for 48 h. The differential gene expression was detected using RNA sequencing (RNA-seq) after podocytes were transfected by siRNA-PER3 (siRNA-PER3 group) and siRNA-control (siRNA-control group), respectively. The mRNA levels of nephrin, podocin, podocalyxin, podoplanin, superoxide dismutase 1 (SOD1), glutathione peroxidase 1 (GPX1), catalase (CAT), and the levels of malondialdehyde (MDA), glutathione (GSH), tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), interleukin-1β (IL-1β) and interleukin-2 (IL-2) were detected after podocytes were transfected with Ad-PER3 or Ad-control for 48 h and then they were induced by 150 μmol/L PA for 48 h. RESULTS: The PER3 was down-regulated in the obesity group compared with the normal body weight group ( CONCLUSIONS PER3 can decrease the PA-induced oxidative stress and inflammatory factor secretion via inhibiting the lipogenesis in podocytes.
Animals ; Circadian Clocks ; Mice ; Mice, Inbred C57BL ; Oxidative Stress ; Palmitic Acid/toxicity* ; Podocytes/metabolism*

Protein palmitoylation, one of post-translation modifications, refers to the addition of saturated 16-carbon palmitic acid to cysteine residues via the thioester bond. It plays key roles in various functional activities, such as the interaction, stability and location of proteins. Heat shock protein 90 (Hsp90), an important molecular chaperone, has been reported to be involved in sperm capacitation. However, it remains unclear whether protein palmitoylation exists in sperm and whether Hsp90 in sperm is palmitoylated under different physiological conditions. In this study, we examined whether the protein palmitoylation is present in mouse cauda epididymis sperm using acyl-biotin exchange method, predicted the potential palmitoylated sites of Hsp90 by the software CSS-Palm 4.0 and detected the palmitoylated Hsp90 in the mouse sperm from caput epididymis and cauda epididymis by immunoprecipitation. We found that some proteins, approximately 50, 65, 72, 85 and 130 kDa, were palmitoylated in mouse cauda epididymis sperm. Five sites in two Hsp90 isoforms were predicted to be palmitoylated. The results also showed that Hsp90 in mouse sperm was palmitoylated and its palmitoylation level was involved in different physiological conditions: the palmitoylation level of cauda epididymis sperm was higher than that of caput epididymis sperm; and the palmitoylation level after capacitation was much higher than that before capacitation. In conclusion, this study reveals that protein palmitoylation is present in mouse sperm and the palmitoylated Hsp90 is associated with different physiological conditions in sperm.
Animals ; Epididymis ; HSP90 Heat-Shock Proteins ; metabolism ; Lipoylation ; Male ; Mice ; Palmitic Acid ; chemistry ; Sperm Capacitation ; Spermatozoa ; metabolism

OBJECTIVETo investigate the effects of berberine (BBR) and cinnamic acid (CA), the main active components in Jiaotai Pill (, JTP), on palmitic acid (PA)-induced intracellular triglyceride (TG) accumulation in NIT-1 pancreatic β cells.

METHODSCells were incubated in culture medium containing PA (0.25 mmol/L) for 24 h. Then treatments with BBR (10 μmol/L), CA (100 μmol/L) and the combination of BBR and CA (BBR+CA) were performed respectively. Intracellular lipid accumulation was assessed by Oil Red O staining and TG content was measured by colorimetric assay. The expression of adenosine monophosphate-activated protein kinase (AMPK) protein and its downstream lipogenic and fatty acid oxidation genes, including fatty acid synthase (FAS), acetyl-coA carboxylase (ACC), phosphorylation acetyl-coA carboxylase (pACC), carnitine acyl transferase 1 (CPT-1) and sterol regulating element binding protein 1c (SREBP-1c) were determined by Western blot or real time polymerase chain reaction.

RESULTSPA induced an obvious lipid accumulation and a significant increase in intracellular TG content in NIT-1 cells. PA also induced a remarkable decrease in AMPK protein expression and its downstream targets such as pACC and CPT-1. Meanwhile, AMPK downstream lipogenic genes including SREBP-1c mRNA, FAS and ACC protein expressions were increased. Treatments with BBR and BBR+CA, superior to CA, significantly reversed the above genes changes in NIT-1 pancreatic β cells. However, the synergistic effect of BBR and CA on intracellular TG content was not observed in the present study.

CONCLUSIONIt can be concluded that in vitro, BBR and BBR+CA could inhibit PA-induced lipid accumulation by decreasing lipogenesis and increasing lipid oxidation in NIT-1 pancreatic β cells.

AMP-Activated Protein Kinases ; metabolism ; Animals ; Berberine ; chemistry ; pharmacology ; Cell Line ; Cinnamates ; chemistry ; pharmacology ; Fatty Acids ; metabolism ; Gene Expression Regulation ; drug effects ; Insulin-Secreting Cells ; drug effects ; metabolism ; Intracellular Space ; metabolism ; Lipogenesis ; drug effects ; genetics ; Mice ; Oxidation-Reduction ; drug effects ; Palmitic Acid ; toxicity ; Triglycerides ; metabolism

Non-alcoholic steatohepatitis (NASH), a metabolic disorder consisting of steatosis and inflammation, is considered the hepatic equivalent of metabolic syndrome and can result in irreversible liver damage. Macrophage-stimulating protein (MSP) is a hepatokine that potentially has a beneficial role in hepatic lipid and glucose metabolism via the activation of AMP-activated protein kinase (AMPK). In the current study, we investigated the regulatory role of MSP in the development of inflammation and lipid metabolism in various NASH models, both in vitro and ex vivo. We observed that MSP treatment activated the AMPK signaling pathway and inhibited lipopolysaccharide (LPS)- and palmitic acid (PA)-induced gene expression of pro-inflammatory cytokines in primary mouse hepatocytes. In addition, MSP treatment resulted in a significant reduction in PA-induced lipid accumulation and inhibited the gene expression of key lipogenic enzymes in HepG2 cells. Upon short hairpin RNA-induced knockdown of RON (the membrane-bound receptor for MSP), the anti-inflammatory and anti-lipogenic effects of MSP were markedly ablated. Finally, to mimic NASH ex vivo, we challenged bone marrow-derived macrophages with oxidized low-density lipoprotein (oxLDL) in combination with LPS. OxLDL+LPS exposure led to a marked inhibition of AMPK activity and a robust increase in inflammation. MSP treatment significantly reversed these effects by restoring AMPK activity and by suppressing pro-inflammatory cytokine gene expression and secretion under this condition. Taken together, these data suggest that MSP is an effective inhibitor of inflammation and lipid accumulation in the stressed liver, thereby indicating that MSP has a key regulatory role in NASH.
AMP-Activated Protein Kinases ; Animals ; Cytokines ; Fatty Liver* ; Gene Expression ; Glucose ; Hep G2 Cells ; Hepatocytes ; In Vitro Techniques ; Inflammation* ; Lipid Metabolism ; Lipogenesis* ; Lipoproteins ; Liver ; Macrophages ; Metabolism ; Mice ; Palmitic Acid

OBJECTIVETo evaluate the effect of palmitic acid (PA) on oxidative stress and activation of inflammasomes in hepatocytes.

METHODSTo test the dose-dependent effect of PA on normal murine hepatocytes AML12, the cells were treated with 0, 0.15, 0.25 and 0.4 mmol/L of palmitic acid (PA). The cells were also divided into blank control group, 0.25 mmol/L PA group and 0.25 mmol/L PA+N-acetylcysteine (NAC) group to examine the effect of reactive oxygen species (ROS) on the activation of inflammasomes. After 24 h of treatment, lipid accumulation, total ROS, mitochondrial ROS, expression and localization of NOX4, and expressions of inflammasomes and IL-1β were detected in the hepatocytes.

RESULTSCompared with the control cells, PA treatment of the cells significantly increased cytoplasmic lipid accumulation, concentrations of total ROS (12 463.09±2.72 vs 6691.23±2.45, P=0.00) and mitochondrial ROS (64.98±0.94 vs 45.04±0.92, P=0.00), and the expressions of NOX4, NLRP3, ASC, caspase-1, and IL-1β (1603.52±1.32 vs 2629.33±2.57, P=0.00). The mitochondria and NOX4 were found to be co-localized in the cytoplasm. NAC obviously reduced cellular ROS level stimulated by PA (7782.15±2.87 vs 5445.6±1.17, P=0.00) and suppressed the expressions of NLRP3, ASC and caspase-1.

CONCLUSIONPA treatment can stimulate lipid accumulation in hepatocytes and induce oxidative stress through NOX4 and mitochondria pathway to activate inflammasomes and stimulate the secretion of IL-1β.

Acetylcysteine ; pharmacology ; Animals ; Carrier Proteins ; metabolism ; Caspase 1 ; metabolism ; Cells, Cultured ; Hepatocytes ; drug effects ; metabolism ; Inflammasomes ; drug effects ; metabolism ; Interleukin-1beta ; metabolism ; Mice ; Mitochondria ; drug effects ; NADPH Oxidase 4 ; NADPH Oxidases ; metabolism ; NLR Family, Pyrin Domain-Containing 3 Protein ; Oxidative Stress ; Palmitic Acid ; pharmacology ; Reactive Oxygen Species ; metabolism

Inhibition of CD36, a fatty acid transporter, has been reported to prevent glucotoxicity and ameliorate high glucose induced beta cell dysfunction. Ezetimibe is a selective cholesterol absorption inhibitor that blocks Niemann Pick C1-like 1 protein, but may exert its effect through suppression of CD36. We attempted to clarify the beneficial effect of ezetimibe on insulin secreting cells and to determine whether this effect is related to change of CD36 expression. mRNA expression of insulin and CD36, intracellular peroxide level and glucose stimulated insulin secretion (GSIS) under normal (5.6 mM) or high glucose (30 mM) condition in INS-1 cells and primary rat islet cells were compared. Changes of the aforementioned factors with treatment with ezetimibe (20 μM) under normal or high glucose condition were also assessed. mRNA expression of insulin was decreased with high glucose, which was reversed by ezetimibe in both INS-1 cells and primary rat islets. CD36 mRNA expression was increased with high glucose, but decreased by ezetimibe in INS-1 cells and primary rat islets. Three-day treatment with high glucose resulted in an increase in intracellular peroxide level; however, it was decreased by treatment with ezetimibe. Decrease in GSIS by three-day treatment with high glucose was reversed by ezetimibe. Palmitate uptake following exposure to high glucose conditions for three days was significantly elevated, which was reversed by ezetimibe in INS-1 cells. Ezetimibe may prevent glucotoxicity in pancreatic β-cells through a decrease in fatty acid influx via inhibition of CD36.
Animals ; Anticholesteremic Agents/*pharmacology ; Antigens, CD36/antagonists & inhibitors/genetics/*metabolism ; Cells, Cultured ; Ezetimibe/*pharmacology ; Flow Cytometry ; Glucose/toxicity ; Insulin/genetics/metabolism/secretion ; Insulin-Secreting Cells/cytology/*drug effects/metabolism ; Male ; Palmitic Acid/metabolism ; RNA, Messenger/metabolism ; Rats ; Rats, Sprague-Dawley ; Reactive Oxygen Species/metabolism ; Real-Time Polymerase Chain Reaction

Mammalian pancreatic β-cells play a pivotal role in development and glucose homeostasis through the production and secretion of insulin. Functional failure or decrease in β-cell number leads to type 2 diabetes (T2D). Despite the physiological importance of β-cells, the viability of β-cells is often challenged mainly due to its poor ability to adapt to their changing microenvironment. One of the factors that negatively affect β-cell viability is high concentration of free fatty acids (FFAs) such as palmitate. In this work, we demonstrated that Yes-associated protein (Yap1) is activated when β-cells are treated with palmitate. Our loss- and gain-of-function analyses using rodent insulinoma cell lines revealed that Yap1 suppresses palmitate-induced apoptosis in β-cells without regulating their proliferation. We also found that upon palmitate treatment, re-arrangement of F-actin mediates Yap1 activation. Palmitate treatment increases expression of one of the Yap1 target genes, connective tissue growth factor (CTGF). Our gain-of-function analysis with CTGF suggests CTGF may be the downstream factor of Yap1 in the protective mechanism against FFA-induced apoptosis.
Actins ; metabolism ; Adaptor Proteins, Signal Transducing ; antagonists & inhibitors ; genetics ; metabolism ; Animals ; Apoptosis ; drug effects ; physiology ; Bridged Bicyclo Compounds, Heterocyclic ; pharmacology ; Cell Line, Tumor ; Connective Tissue Growth Factor ; genetics ; metabolism ; pharmacology ; Cytochalasin D ; pharmacology ; Fatty Acids, Nonesterified ; pharmacology ; HEK293 Cells ; Humans ; Immunohistochemistry ; Insulin-Secreting Cells ; cytology ; drug effects ; metabolism ; Mice ; Microscopy, Fluorescence ; Palmitic Acid ; pharmacology ; Phosphoproteins ; antagonists & inhibitors ; genetics ; metabolism ; RNA Interference ; RNA, Small Interfering ; metabolism ; Rats ; Recombinant Proteins ; genetics ; metabolism ; pharmacology ; Thiazolidines ; pharmacology

OBJECTIVETo investigate the effects of JAZF1 overexpression on the pro-inflammatory cytokines in hepatic steatosis.

METHODSThe model of hepatic steatosis was established by incubating hepatocytes with palmitic acid (PA) at 0, 0.125, 0.25, 0.5 and 1 mM dose and for 0, 6, 12, 24 and 48 hours, after which recombinant adenovirus expressing JAZF1 (Ad-JAZF1) was introduced to up-regulate expression. Triglyceride level was measured by GOD. Cell viability was detected by CCK-8. The mRNA and protein expression of TNF-alpha, MCP-1, IL-8 and JAZF1 was examined by RT-PCR, ELISA, and western blotting.

RESULTSThe PA-treated hepatocytes showed dose-dependent significant increases in TNF-alpha, MCP-1 and IL-8 mRNA expression for doses up to 0.25 mM; there were no significant increases for the highest doses of 0.5 and 1 mM. The 0.25 mM PA-treated hepatocytes showed time-dependent significant increases in TNF-alpha, MCP-1 and IL-8 mRNA expressions (FTNF-alpha = 26.51, FMCP-1 = 57.20, FIL-8 = 353.85, P less than 0.01), with the maximum level reached at 12 h and followed by a gradual decrease with longer treatment times. JAZF1 mRNA and protein expression was markedly increased in hepatocytes infected with Ad-JAZF1 (P less than 0.01). However, the AP-treated hepatocytes with JAZF1 overexpression showed down-regulation of TNF-alpha, MCP-1 and IL-8 mRNA expression (decreased by 89.69%, 77.68%, and 83.21%, respectively) and secretion (37%, 37% and 41%, respectively, P less than 0.01).

CONCLUSIONStimulation of hepatocytes by the PA fatty acid in vitro promotes mRNA expression of TNF-alpha, MCP-1 and IL-8, but overexpression of JAZF1 inhibits the PA-induced expression and secretion of these factors.

Cell Survival ; Chemokine CCL2 ; metabolism ; Cytokines ; metabolism ; Fatty Liver ; Hepatocytes ; drug effects ; metabolism ; Humans ; Interleukin-8 ; metabolism ; Neoplasm Proteins ; metabolism ; Palmitic Acid ; pharmacology ; RNA, Messenger ; metabolism ; Tumor Necrosis Factor-alpha ; metabolism ; Up-Regulation