OBJECTIVE: To investigate the effect of palmitic acid (PA) on autophagy in neonatal rat cardiomyocytes (NRCMs) and explore the underlying mechanism. METHODS: NRCMs were isolated and cultured for 24 h before exposure to 10% BSA and 0.1, 0.3, 0.5, or 0.7 mmol/L PA for 24 h. After the treatments, the expressions of Parkin, PINK1, p62, LC3Ⅱ/ LC3Ⅰ, cGAS, STING and p-IRF3/IRF3 were detected using Western blotting and the cell viability was assessed with CCK8 assay, based on which 0.7 mmol/L was selected as the optimal concentration in subsequent experiments. The effects of cGAS knockdown mediated by cGAS siRNA in the presence of PA on autophagy-related proteins in the NRCMs were determined using Western blotting, and the expressions of P62 and LC3 in the treated cells were examined using immunofluorescence assay. RESULTS: PA at different concentrations significantly lowered the expressions of Parkin, PINK1, LC3 Ⅱ/LC3 Ⅰ and LC3 Ⅱ/LC3 Ⅰ+Ⅱ (P < 0.05), increased the expression of p62 (P < 0.05), and inhibited the viability of NRCMs (P < 0.05). Knockdown of cGAS obviously blocked the autophagy-suppressing effect of PA and improved the viability of NRCMs (P < 0.05). CONCLUSION PA inhibits autophagy by activating the cGAS-STING-IRF3 pathway to reduce the viability of NRCMs.
Animals ; Animals, Newborn ; Autophagy ; Myocytes, Cardiac ; Nucleotidyltransferases/pharmacology* ; Palmitic Acid/pharmacology* ; Rats

OBJECTIVE: To determine the volatile constituents and their contents in the roots of 5 cultivated Angelica dahurica and one wild A. dahurica and analyze the chemical relationship among the plants of A. dahurica. METHODS: The essential oil was extracted from the roots of 5 cultivated plants of Angelica dahurica and one wild A. dahurica by water steam distillation. Gas chromatography-mass spectrometry (GC-MS) was used to separate and identify all the volatile oil components in the extracts, and their relative contents were calculated with area normalization method. We also conducted clustering analysis and principal component analysis of the volatile oil components. RESULTS: We identified a total of 81 compounds from the roots of the 6 plants of Angelica dahurica, including 27 in Chuanbaizhi (Angelica dahurica cv. 'Hangbaizhi'), 34 in Hangbaizhi (Angelica dahurica cv. 'Hangbaizhi'), 24 in Qibaizhi (Angelica dahurica cv. 'Qibaizhi'), 32 in Yubaizhi (Angelica dahurica cv.'Qibaizhi'), 28 in Bobahizhi (Angelica dahurica cv.'Qibaizhi'), and 34 in Xinganbaizhi (Angelica dahuirca). These compounds included, in the order of their relative contents (from high to low), alkanes, olefins, esters, organic acids and alcohols. Among the common components found in the roots of all the plants of A. dahurica, nonylcyclopropane, cyclododecane and hexadecanoic acid were identified as the volatile oil components that showed the highest relative contents. Clustering analysis of the volatile oil components showed that wild Angelica dahurica (Xing'anbaizhi) and the 5 cultivated Angelica dahurica (Chuanbaizhi, Hangbaizhi, Qibaizhi, Yubaizhi, Bobaizhi) could be divided into two groups, and the cultivated Angelica dahurica could be divided into two subgroups: Chuanbaizhi, Yubaizhi and Hangbahizhi were clustered in one subgroup, and Qibaizhi and Bobaizhi in another. The results of principal component analysis was consistent with those of clustering analysis. CONCLUSION The main volatile oil components and their contents vary among the 6 plants of A. dahurica. Nonylcyclopropane, cyclododecane and hexadecanoic acid are the most abundant volatile oil components in all the plants of A. dahurica, which can be divided into two clusters.
Angelica/chemistry* ; Gas Chromatography-Mass Spectrometry ; Oils, Volatile/analysis* ; Palmitic Acid/analysis* ; Plant Roots/chemistry*

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*

OBJECTIVE: To investigate effects of Jiangtang Sanhuang tablet (JTSHT) for regulating blood glucose and alleviating islet cell damage in db/db mice and its protective effects against endoplasmic reticulum stress (ERS) and autophagy induced by glycolipid toxicity. METHODS: Forty db/db mice were randomized into 4 groups for daily intragastric administration of saline, JTSHT of 2.64 and 1.32 g/kg, and metformin at 0.225g/kg for 8 weeks, using 10 C57BL/6J mice as the normal control. After the treatments, the metabolic indexes of the mice were measured, and morphological changes of the islet cells were observed. A mouse islet cell line (MIN6) was exposed to high glucose (22 mmol/L glucose) and 0.1 mmol/L palmitic acid, followed by treatment with the sera from JTSHT- or saline- treated SD rats, alone or in combination with SP600125, and the changes in cell apoptosis, ERS and autophagy were evaluated using flow cytometry, RT-qPCR and Western blotting. RESULTS: In db/db mice, treatment with JTSHT significantly improved glucose and lipid metabolism (P < 0.05) and suppressed progressive weight gain (P < 0.05) without significant effect on drinking water volume (P > 0.05). JTSHT was also found to promote repair of islet cell injuries. In the cell experiments, high glucose exposure significantly increased apoptosis rate of MIN6 cells (P < 0.05), which was obviously lowered by treatment with JTSHT-treated rat serum (P < 0.05). Western blotting showed that JTSHT significantly reduced the level of ERS and autophagy caused by glycolipid toxicity in MIN6 cells (P < 0.05). Interference with ERS using SP600125 significantly attenuated the protective effect of JTSHT against MIN6 cell injury, apoptosis and autophagy induced by glycolipid toxicity (P < 0.05). CONCLUSION JTSHT has protective effects against glycolipid toxicity in MIN6 cells possibly by inhibiting ERS and autophagy.
Animals ; Anthracenes ; Apoptosis ; Autophagy ; Blood Glucose ; Diabetes Mellitus ; Drinking Water ; Drugs, Chinese Herbal ; Endoplasmic Reticulum Stress ; Glucose/pharmacology* ; Glycolipids/pharmacology* ; Islets of Langerhans ; Metformin ; Mice ; Mice, Inbred C57BL ; Palmitic Acid/pharmacology* ; Tablets/pharmacology*

This study aimed to investigate the effect of lipopolysaccharide (LPS) on lipophagy in hepatocytes and the underlying mechanism. Human hepatoma cell line HepG2 was cultured in vitro, treated with 0.1 mmol/L palmitic acid (PA), and then divided into control group (0 μg/mL LPS), LPS group (10 μg/mL LPS), LPS+DMSO group and LPS+RAPA (rapamycin, 10 μmol/L) group. Lipid accumulation in hepatocytes was observed by oil red O staining. The autophagic flux of the cells was assessed using confocal laser scanning microscope after being transfected with autophagy double-labeled adenovirus (mRFP-GFP-LC3). The level of intracellular lipophagy was visualized by the colocalization of lipid droplets (BODIPY 493/503 staining) and lysosomes (lysosome marker, lysosomal associated membrane protein 1, LAMP1). The expression levels of mammalian target of rapamycin (mTOR), phosphorylated mTOR (p-mTOR), ribosome protein subunit 6 kinase 1 (S6K1), p-S6K1, LC3II/I and P62 protein were examined by Western blot. The results showed that the number of red lipid droplets stained with oil red O was significantly increased in LPS group compared with that in control group (P < 0.001). Moreover, in LPS group, the number of autophagosomes was increased, while the number of autophagolysosomes and the colocalization rate of LAMP1 and BODIPY were significantly decreased (P < 0.05). Meanwhile, the ratios of p-mTOR/mTOR and p-S6K1/S6K1, the ratio of LC3II/LC3I and the protein expression of P62 were significantly increased (P < 0.05) in LPS group. Furthermore, compared with LPS+DMSO group, RAPA treatment obviously reduced the number of lipid droplets and autophagosomes, and raised the number of autophagolysosomes and the colocalization rate of LAMP1 and BODIPY (P < 0.05). In conclusion, the results demonstrate that LPS inhibits lipophagy in HepG2 cells via activating mTOR signaling pathway, thereby aggravating intracellular lipid accumulation.
Autophagy ; Hep G2 Cells ; Humans ; Lipopolysaccharides ; Palmitic Acid ; Signal Transduction ; TOR Serine-Threonine Kinases

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*

PURPOSE: Protein overloading in the endoplasmic reticulum (ER) leads to endoplasmic reticulum stress, which exacerbates various disease conditions. Emodin, an anthraquinone compound, is known to have several health benefits. The effect of emodin against palmitic acid (PA) - induced ER stress in HepG2 cells was investigated. METHODS: HepG2 cells were treated with varying concentrations of palmitic acid to determine the working concentration that induced ER stress. ER stress associated genes such as ATF4, XBP1s, CHOP and GRP78 were checked using RT- PCR. In addition, the expression levels of unfolded protein response (UPR) associated proteins such as IRE1α, eIF2α and CHOP were checked using immunoblotting to confirm the induction of ER stress. The effect of emodin on ER stress was analyzed by treating HepG2 cells with 750 µM palmitic acid and varying concentrations of emodin, then analyzing the expression of UPR associated genes. RESULTS: It was evident from the mRNA and protein expression results that palmitic acid significantly increased the expression of UPR associated genes and thereby induced ER stress. Subsequent treatment with emodin reduced the mRNA expression of ATF4, GRP78, and XBP1s. Furthermore, the protein levels of p-IRE1α, p-elF2α and CHOP were also reduced by the treatment of emodin. Analysis of sirtuin mRNA expression showed that emodin increased the levels of SIRT4 and SIRT7, indicating a possible role in decreasing the expression of UPR-related genes. CONCLUSION: Altogether, the results suggest that emodin could exert a protective effect against fatty acid-induced ER stress and could be an agent for the management of various ER stress related diseases.
Emodin ; Endoplasmic Reticulum Stress ; Endoplasmic Reticulum ; Hep G2 Cells ; Immunoblotting ; Insurance Benefits ; Palmitic Acid ; Polymerase Chain Reaction ; RNA, Messenger ; Sirtuins ; Unfolded Protein Response

PURPOSE: The skin pH is maintained by epidermal lactate, free fatty acids (FFAs), and free amino acids (FAAs). As a significant determinant of skin health, the skin pH is increased (less acidic) under abnormal and aged skin conditions. In a search for dietary alternatives that would promote an acidic skin pH, this study investigated the dietary effects of Lactobacillus plantarum CJLP55 isolated from Korean kimchi on the skin pH, and epidermal levels of lactate, FFAs, and FAAs in adult subjects. METHODS: Seventy eight subjects (mean age 24.9 ± 0.5 years, range 19 ~ 37 years) were assigned randomly to ingest CJLP55, Lactobacillus strain from kimchi, (n = 39, CJLP group) or placebo supplements (n = 39, placebo group) for 12 weeks in a double-blind, placebo-controlled trial. Skin pH and epidermal levels of lactate, FFAs and FFAs were assessed at 0, 6 and 12 weeks. RESULTS: Although significant decreases in skin pH were observed in both the CJLP and placebo groups at 6 weeks, the skin pH was decreased significantly only in the CJLP group at 12 weeks. In parallel, the epidermal level of lactate in the CJLP group was also increased by 25.6% at 12 weeks. On the other hand, the epidermal level of FAAs were not altered in the CJLP and placebo groups, but the epidermal level of total FFAs, including palmitic acid and stearic acid, was lower in the CJLP group than in the placebo group over 12 weeks. The changes in the other FFAs, such as palmitoleic acid and oleic acid, were similar in the CJLP and placebo groups over 12 weeks. CONCLUSION: Overall, a dietary supplement of CJLP55 promotes acidic skin pH with a selective increase in epidermal lactate in adult subjects.
Adult ; Amino Acids ; Dietary Supplements ; Fatty Acids, Nonesterified ; Hand ; Humans ; Hydrogen-Ion Concentration ; Lactic Acid ; Lactobacillus plantarum ; Lactobacillus ; Oleic Acid ; Palmitic Acid ; Skin

BACKGROUND/OBJECTIVES: The NAD+ precursor nicotinamide riboside (NR) is a type of vitamin B3 found in cow's milk and yeast-containing food products such as beer. Recent studies suggested that NR prevents hearing loss, high-fat diet-induced obesity, Alzheimer's disease, and mitochondrial myopathy. The objective of this study was to investigate the effects of NR on inflammation and mitochondrial biogenesis in AML12 mouse hepatocytes. MATERIALS/METHODS: A subset of hepatocytes was treated with palmitic acid (PA; 250 µM) for 48 h to induce hepatocyte steatosis. The hepatocytes were treated with NR (10 µM and 10 mM) for 24 h with and without PA. The cell viability and the levels of sirtuins, inflammatory markers, and mitochondrial markers were analyzed. RESULTS: Cytotoxicity of NR was examined by PrestoBlue assay. Exposure to NR had no effect on cell viability or morphology. Gene expression of sirtuin 1 (Sirt1) and Sirt3 was significantly upregulated by NR in PA-treated hepatocytes. However, Sirt1 activities were increased in hepatocytes treated with low-dose NR. Hepatic pro-inflammatory markers including tumor necrosis factor-alpha and interleukin-6 were decreased in NR-treated cells. NR upregulated anti-inflammatory molecule adiponectin, and, tended to down-regulate hepatokine fetuin-A in PA-treated hepatocytes, suggesting its inverse regulation on these cytokines. NR increased levels of mitochondrial markers including peroxisome proliferator-activated receptor γ coactivator-1α, carnitine palmitoyltransferase 1, uncoupling protein 2, transcription factor A, mitochondrial and mitochondrial DNA in PA-treated hepatocytes. CONCLUSIONS: These data demonstrated that NR attenuated hepatic inflammation and increased levels of mitochondrial markers in hepatocytes.
Adiponectin ; alpha-2-HS-Glycoprotein ; Alzheimer Disease ; Animals ; Beer ; Carnitine O-Palmitoyltransferase ; Cell Survival ; Cytokines ; DNA, Mitochondrial ; Fatty Liver ; Gene Expression ; Hearing Loss ; Hepatocytes ; Inflammation ; Interleukin-6 ; Mice ; Milk ; Mitochondria ; Mitochondrial Myopathies ; Niacin ; Niacinamide ; Obesity ; Organelle Biogenesis ; Palmitic Acid ; Peroxisomes ; Sirtuin 1 ; Sirtuins ; Transcription Factors ; Tumor Necrosis Factor-alpha