Nuciferine Protects against Obesity-Induced Nephrotoxicity through Its Hypolipidemic, Anti-Inflammatory, and Antioxidant Effects.

High-fat diets (HFD) could cause obesity, trigger lipid accumulation, and induce oxidative stress and inflammation, leading to kidney damage. This study aimed to elucidate the protective effects of nuciferine on HFD-caused nephrotoxicity and explore the underlying mechanisms in Kunming mice and palmitic acid-exposed HK-2 cells. In obese mice, nuciferine notably alleviated HFD-induced chronic renal dysfunction and delayed renal fibrosis progression and podocyte apoptosis, as evidenced by the increased expressions of renal function factors BUN, CRE, and UA and the decreased expressions of key protein factors TGF-ß1, p-Samd3, Wnt-1, and ß-catenin. Nuciferine also effectively attenuated HFD-induced renal lipid accumulation via the AMPK-mediated regulation of FAS and HSL expressions and suppressed inflammation and oxidative stress via the AMPK-mediated Nrf-2/HO-1 and TLR4/MyD88/NF-κB pathways. In addition, consistent with the results of animal experiments, nuciferine remarkably reversed cell damage and attenuated lipid accumulation, inflammation, and oxidative stress in palmitic acid-exposed HK-2 cells through the AMPK-mediated signaling pathway. Therefore, nuciferine could be a new food-derived protective agent to offset obesity and correlative kidney damage.

Saturated fatty acid promotes calcification via suppressing SIRT6 expression in vascular smooth muscle cells.

BACKGROUND: This study aims to investigate the effects of saturated free fatty acid on calcification and SIRT6 expression in vascular smooth muscle cells (VSMCs) and the role of SIRT6 in regulating VSMC calcification. METHODS: Sprague-Dawley rats were randomly allocated to two groups: rats with normal diet (ND) and high-fat diet (HFD) from 4 to 12 weeks. At 12 weeks, part rats randomly selected from ND and HFD were administrated with vitamin D3 and nicotine to establish a model of vascular calcification. Thoracic aortas were collected from treatment rats at 16 weeks for assaying vascular calcification and related protein expression. Primary VSMCs isolated from Sprague-Dawley rats were used for investigating the effects of palmitic acid on VSMCs' calcification, apoptosis and target protein expression. RESULTS: HFD-facilitated calcification in medial aorta, with decreased SIRT6 expression in VSMCs of aortas. Palmitic acid decreased SIRT6 expression while increased calcification, apoptosis and protein expression of BMP2 and RUNX2 in primary VSMCs. Overexpression of SIRT6 could, partially or completely, rescue the palmitic acid-induced elevation of calcification, apoptosis and expression of BMP2 and RUNX2. CONCLUSION: This study demonstrated that vascular calcification induced by HFD was linked to the palmitic acid-induced downregulation of SIRT6. Overexpression of SIRT6 could decrease palmitic acid-induced calcification and apoptosis in VSMCs.

A small-molecule JNK inhibitor JM-2 attenuates high-fat diet-induced non-alcoholic fatty liver disease in mice.

BACKGROUND: The prevalence of non-alcoholic fatty liver disease (NAFLD) has been deemed a leading cause of end-stage liver disease. As a member of the mitogen-activated protein kinase family, c-Jun N-terminal kinase (JNK) has been shown to play an important role in the pathogenesis of NAFLD. Here, we identified a novel JNK inhibitor, JM-2, and evaluated its therapeutic effects against NAFLD both in vitro and in vivo. METHODS: In vitro, JNK was blocked by JM-2 in PA-challenged hepatocytes. C57BL/6 mice were fed a high-fat diet for 6 months to develop NAFLD. Mice were treated with JM-2 by intragastric administration. RESULTS: In primary hepatocytes and AML-12 cells, JM-2 treatment significantly suppressed palmitic acid (PA)-induced JNK activation and PA-induced inflammation and cell apoptosis. In addition, JM-2 restricted the production of fibrosis- and lipid metabolism-related genes in PA-challenged hepatocytes. We evaluated the curative effect of JM-2 against NAFLD using a high-fat diet (HFD)-fed mouse model. Based on our findings, JM-2 administration significantly protected the mouse liver from HFD-induced inflammation, lipid accumulation, fibrosis, and apoptosis, accompanied with reduced JNK phosphorylation in the liver tissue. CONCLUSION: JM-2 affords a significant protective effect against HFD-induced NAFLD by inhibiting JNK activation and is potential to be developed as a candidate drug for NAFLD treatment.

Palmitic acid induces intestinal lipid metabolism disorder, endoplasmic reticulum stress and inflammation by affecting phosphatidylethanolamine content in large yellow croaker Larimichthys crocea.

In the 21st century, intestinal homeostatic imbalance has emerged as a growing health challenge worldwide. Accumulating evidence reveals that excessive intake of saturated fatty acid (SFA) induces intestinal homeostatic imbalance. However, the potential molecular mechanism is still unclear. In the present study, we found that palm oil or palmitic acid (PA) treatment disturbed lipid metabolism homeostasis and triggered endoplasmic reticulum (ER) stress and inflammation in the intestine or intestinal cells of large yellow croaker (Larimichthys crocea). Interestingly, PA treatment significantly decreased phosphatidylethanolamine (PE) content in the intestinal cells. PE supplementation decreased triglyceride content in the intestinal cells induced by PA treatment by inhibiting fatty acid uptake and lipogenesis. PE supplementation suppressed ER stress. Meanwhile, PE supplementation alleviated inflammatory response through p38 MAPK-p65 pathway, reducing the damage of intestinal cells caused by PA treatment to some extent. Our work revealed that intestinal homeostatic imbalance caused by PA treatment was partly due to the decrease of PE content. PE consumption might be a nutritional strategy to regulate intestinal homeostasis in fish and even human beings.

Inhibition of the cleaved half of tRNAGly enhances palmitic acid-induced apoptosis in human trophoblasts.

Palmitic acid (PA) induces apoptosis in the human trophoblast cell line HTR8/SVneo. However, the molecular mechanism underlying this effect remains unclear. Although small noncoding RNAs are involved in trophoblast growth and invasion during early pregnancy, the functional roles of tRNA-derived species are currently unknown. Therefore, the purpose of this study was to examine the involvement of tRNA-derived species in PA-induced apoptosis in human trophoblasts. In this study, we investigate the expression and function of tRNA-derived stress-induced RNAs (tiRNAs) in HTR8/SVneo. We determined the expression of tiRNAs in HTR8/SVneo cells in response to PA. Then, we transfected inhibitor of target tiRNA in HTR8/SVneo with or without PA to examine the tRNA-derived species-regulated intracellular signal transduction by detecting calcium homeostasis, mitochondrial membrane potential, and signaling proteins. We found that the expression of tRNAGly-derived tiRNAs decreased in PA-treated human trophoblasts. Moreover, inhibition of tiRNAGlyCCC/GCC enhanced the PA-induced apoptosis along with the induction of DNA fragmentation and mitochondrial depolarization. Inhibition of tiRNAGlyCCC/GCC enhanced the expression of endoplasmic reticulum stress-related proteins and increased Ca2+ levels in the cytoplasm and mitochondria. Moreover, the levels of cytochrome c released from the mitochondria were synergistically affected by tiRNAGlyCCC/GCC inhibitor and PA. Furthermore, artificial regulation of ANG inhibited the expression of tiRNAGlyCCC/GCC and similar effects were observed upon the inhibition of tiRNAGlyCCC/GCC in human trophoblasts. These results suggest that tiRNAGlyCCC/GCC might be the molecule via which PA induces its effects in human trophoblasts.

Raphani Semen (Raphanus sativus L.) Ameliorates Alcoholic Fatty Liver Disease by Regulating De Novo Lipogenesis.

In this study, we investigated the pharmacological effect of a water extract of Raphani Semen (RSWE) on alcoholic fatty liver disease (AFLD) using ethanol-induced AFLD mice (the NIAAA model) and palmitic acid (PA)-induced steatosis HepG2 cells. An RSWE supplement improved serum and hepatic triglyceride (TG) levels of AFLD mice, as well as their liver histological structure. To explore the molecular action of RSWE in the improvement of AFLD, we investigated the effect of RSWE on four major pathways for lipid homeostasis in the liver: free fatty acid transport, lipogenesis, lipolysis, and ß-oxidation. Importantly, RSWE decreased the mRNA expression of de novo lipogenesis-related genes, such as Srebf1, Cebpa, Pparg, and Lpin1, as well as the protein levels of these factors, in the liver of AFLD mice. That these actions of RSWE affect lipogenesis was confirmed using PA-induced steatosis HepG2 cells. Overall, our findings suggest that RSWE has the potential for improvement of AFLD by inhibiting de novo lipogenesis.

Inhibition of lncRNA TCONS_00077866 Ameliorates the High Stearic Acid Diet-Induced Mouse Pancreatic ß-Cell Inflammatory Response by Increasing miR-297b-5p to Downregulate SAA3 Expression.

Long-term consumption of a high-fat diet increases the circulating concentration of stearic acid (SA), which has a potent toxic effect on ß-cells, but the underlying molecular mechanisms of this action have not been fully elucidated. Here, we evaluated the role of long noncoding (lnc)RNA TCONS_00077866 (lnc866) in SA-induced ß-cell inflammation. lnc866 was selected for study because lncRNA high-throughput sequencing analysis demonstrated it to have the largest fold-difference in expression of five lncRNAs that were affected by SA treatment. Knockdown of lnc866 by virus-mediated shRNA expression in mice or by Smart Silencer in mouse pancreatic ß-TC6 cells significantly inhibited the SA-induced reduction in insulin secretion and ß-cell inflammation. According to lncRNA-miRNAs-mRNA coexpression network analysis and luciferase reporter assays, lnc866 directly bound to miR-297b-5p, thereby preventing it from reducing the expression of its target serum amyloid A3 (SAA3). Furthermore, overexpression of miR-297b-5p or inhibition of SAA3 also had marked protective effects against the deleterious effects of SA in ß-TC6 cells and mouse islets. In conclusion, lnc866 silencing ameliorates SA-induced ß-cell inflammation by targeting the miR-297b-5p/SAA3 axis. lnc866 inhibition may represent a new strategy to protect ß-cells against the effects of SA during the development of type 2 diabetes.

Vitamin K2 as a New Modulator of the Ceramide De Novo Synthesis Pathway.

The aim of the study was to evaluate the influence of vitamin K2 (VK2) supplementation on the sphingolipid metabolism pathway in palmitate-induced insulin resistant hepatocytes. The study was carried out on human hepatocellular carcinoma cells (HepG2) incubated with VK2 and/or palmitic acid (PA). The concentrations of sphingolipids were measured by high-performance liquid chromatography. The expression of enzymes from the sphingolipid pathway was assessed by Western blotting. The same technique was used in order to determine changes in the expression of the proteins from the insulin signaling pathway in the cells. Simultaneous incubation of HepG2 cells with palmitate and VK2 elevated accumulation of sphinganine and ceramide with increased expression of enzymes from the ceramide de novo synthesis pathway. HepG2 treatment with palmitate and VK2 significantly decreased the insulin-stimulated expression ratio of insulin signaling proteins. Moreover, we observed that the presence of PA w VK2 increased fatty acid transport protein 2 expression. Our study showed that VK2 activated the ceramide de novo synthesis pathway, which was confirmed by the increase in enzymes expression. VK2 also intensified fatty acid uptake, ensuring substrates for sphingolipid synthesis through the de novo pathway. Furthermore, increased concentration of sphingolipids, mainly sphinganine, inhibited insulin pathway proteins phosphorylation, increasing insulin resistance development.

Involvement of miR-3180-3p and miR-4632-5p in palmitic acid-induced insulin resistance.

Insulin resistance is defined as a failure to trigger the activation of the PI3K-AKT pathway by normal levels of insulin; therefore, it is well linked to metabolic disorders. Although multiple mechanisms contribute to insulin resistance, one major cause is elevated concentrations of plasma free fatty acids, which are known to suppress insulin signaling. However, the underlying mechanism is still elusive. Here, we found that palmitic acid increased the expression of two miRNAs, miR-3180-3p and miR-4632-5p, in HepG2 cells. Transfection of HepG2 cells with miR-3180-3p or miR-4632-5p reduced insulin-induced activation of the PI3K-AKT pathway. Moreover, palmitic acid or two miRNAs inhibited insulin-induced phosphorylation of Tyr612 on IRS-1 without affecting insulin receptor activation. Therefore, two miRNAs are suggested to be involved in palmitic acid-induced insulin resistance through suppression of insulin-induced IRS-1 phosphorylation. Identification of miR-3180-3p and miR-4632-5p targets could provide valuable information for the development of therapeutic drugs for type 2 diabetes.

Epidermal Growth Factor Modulates Palmitic Acid-Induced Inflammatory and Lipid Signaling Pathways in SZ95 Sebocytes.

Epidermal growth factor (EGF) acts as a paracrine and autocrine mediator of cell proliferation and differentiation in various types of epithelial cells, such as sebocytes, which produce the lipid-rich sebum to moisturize the skin. However, sebum lipids via direct contact and by penetrating through the epidermis may have regulatory roles on epidermal and dermal cells as well. As EGF receptor (EGFR) is expressed throughout the proliferating and the lipid-producing layers of sebaceous glands (SGs) in healthy and acne-involved skin, we investigated the effect of EGF on SZ95 sebocytes and how it may alter the changes induced by palmitic acid (PA), a major sebum component with bioactive roles. We found that EGF is not only a potent stimulator of sebocyte proliferation, but also induces the secretion of interleukin (IL)6 and down-regulates the expression of genes involved in steroid and retinoid metabolism. Importantly, when applied in combination with PA, the PA-induced lipid accumulation was decreased and the cells secreted increased IL6 levels. Functional clustering of the differentially regulated genes in SZ95 sebocytes treated with EGF, PA or co-treated with EGF+PA further confirmed that EGF may be a potent inducer of hyperproliferative/inflammatory pathways (IL1 signaling), an effect being more pronounced in the presence of PA. However, while a group of inflammatory genes was up-regulated significantly in EGF+PA co-treated sebocytes, PA treatment in the absence of EGF, regulated genes only related to cell homeostasis. Meta-analysis of the gene expression profiles of whole acne tissue samples and EGF- and EGF+PA -treated SZ95 sebocytes showed that the EGF+PA co-activation of sebocytes may also have implications in disease. Altogether, our results reveal that PA-induced lipid accumulation and inflammation can be modulated by EGF in sebocytes, which also highlights the need for system biological approaches to better understand sebaceous (immuno)biology.

Role of HO-1 against Saturated Fatty Acid-Induced Oxidative Stress in Hepatocytes.

Increased circulating levels of free fatty acids, especially saturated ones, are involved in disease progression in the non-alcoholic fatty liver. Although the mechanism of saturated fatty acid-induced toxicity in the liver is not fully understood, oxidative stress may be deeply involved. We examined the effect of increased palmitic acid, the most common saturated fatty acid in the blood, on the liver of BALB/c mice via tail vein injection with palmitate. After 24 h, among several anti-oxidative stress response genes, only heme oxygenase-1 (HO-1) was significantly upregulated in palmitate-injected mice compared with that in vehicle-injected mice. Elevation of HO-1 mRNA was also observed in the fatty liver of high-fat-diet-fed mice. To further investigate the role of HO-1 on palmitic acid-induced oxidative stress, in vitro experiments were performed to expose palmitate to HepG2 cells. SiRNA-mediated knockdown of HO-1 significantly increased the oxidative stress induced by palmitate, whereas pre-treatment with SnCl2, a well-known HO-1 inducer, significantly decreased it. Moreover, SB203580, a selective p38 inhibitor, reduced HO-1 mRNA expression and increased palmitate-induced oxidative stress in HepG2 cells. These results suggest that the HO-1-mediated anti-oxidative stress compensatory reaction plays an essential role against saturated fatty acid-induced lipotoxicity in the liver.

Palmitoleic acid ameliorates palmitic acid-induced proinflammation in J774A.1 macrophages via TLR4-dependent and TNF-α-independent signallings.

Adipose tissue resident macrophages play an important role in the regulation of the inflammatory response. Monounsaturated fatty acids assist in the prevention of cardiovascular diseases via an anti-inflammatory effect. However, the mechanisms by which monounsaturated fatty acids, such as palmitoleic acid, regulate the inflammatory response has not been well investigated. In this study, we found that a high concentration of palmitic acid induced J774A.1 murine macrophages toward a pro-inflammatory state, possibly through the activation of the TLR2 or TLR4 genes, and their downstream signaling pathways. In contrast, palmitoleic acid induced a protective effect against inflammation in macrophage of non-obese rodents by inducing an alternative activation pathway via reducing TLR2 or TLR4 signaling. This study indicates that the balance of palmitic acid (saturated fatty acid) and palmitoleic acid (monounsaturated fatty acid) effects macrophage activation. The potential therapeutic impact of palmitoleic acid to ameliorate non-obese-mediated inflammation warrants further investigation.

Palmitic acid-rich oils with and without interesterification lower postprandial lipemia and increase atherogenic lipoproteins compared with a MUFA-rich oil: A randomized controlled trial.

BACKGROUND: Interesterified (IE) fats are widely used in place of trans fats; however, little is known about their metabolism. OBJECTIVES: To test the impact of a commonly consumed IE compared with a non-IE equivalent fat on in vivo postprandial and in vitro lipid metabolism, compared with a reference oil [rapeseed oil (RO)]. METHODS: A double-blinded, 3-phase crossover, randomized controlled trial was performed in healthy adults (n = 20) aged 45-75 y. Postprandial plasma triacylglycerol and lipoprotein responses (including stable isotope tracing) to a test meal (50 g fat) were evaluated over 8 h. The test fats were IE 80:20 palm stearin/palm kernel fat, an identical non-IE fat, and RO (control). In vitro, mechanisms of digestion were explored using a dynamic gastric model (DGM). RESULTS: Plasma triacylglycerol 8-h incremental area under the curves were lower following non-IE compared with RO [-1.7 mmol/L⋅h (95% CI: -3.3, -0.0)], but there were no differences between IE and RO or IE and non-IE. LDL particles were smaller following IE and non-IE compared with RO (P = 0.005). Extra extra large, extra large, and large VLDL particle concentrations were higher following IE and non-IE compared with RO at 6-8 h (P < 0.05). No differences in the appearance of [13C]palmitic acid in plasma triacylglycerol were observed between IE and non-IE fats. DGM revealed differences in phase separation of the IE and non-IE meals and delayed release of SFAs compared with RO. CONCLUSIONS: Interesterification did not modify fat digestion, postprandial lipemia, or lipid metabolism measured by stable isotope and DGM analysis. Despite the lower lipemia following the SFA-rich fats, increased proatherogenic large triacylglycerol-rich lipoprotein remnant and small LDL particles following the SFA-rich fats relative to RO adds a new postprandial dimension to the mechanistic evidence linking SFAs to cardiovascular disease risk.

Lipotoxic stress alters the membrane lipid profile of extracellular vesicles released by Huh-7 hepatocarcinoma cells.

Extracellular vesicles (EVs) are well-known mediators in intercellular communication playing pivotal roles in promoting liver inflammation and fibrosis, events associated to hepatic lipotoxicity caused by saturated free fatty acid overloading. However, despite the importance of lipids in EV membrane architecture which, in turn, affects EV biophysical and biological properties, little is known about the lipid asset of EVs released under these conditions. Here, we analyzed phospholipid profile alterations of EVs released by hepatocarcinoma Huh-7 cells under increased membrane lipid saturation induced by supplementation with saturated fatty acid palmitate or Δ9 desaturase inhibition, using oleate, a nontoxic monounsaturated fatty acid, as control. As an increase of membrane lipid saturation induces endoplasmic reticulum (ER) stress, we also analyzed phospholipid rearrangements in EVs released by Huh-7 cells treated with thapsigargin, a conventional ER stress inducer. Results demonstrate that lipotoxic and/or ER stress conditions induced rearrangements not only into cell membrane phospholipids but also into the released EVs. Thus, cell membrane saturation level and/or ER stress are crucial to determine which lipids are discarded via EVs and EV lipid cargos might be useful to discriminate hepatic lipid overloading and ER stress.

Sulforaphane Attenuates Nonalcoholic Fatty Liver Disease by Inhibiting Hepatic Steatosis and Apoptosis.

Nonalcoholic fatty liver disease (NAFLD) is characterized by lipotoxicity and ectopic lipid deposition within hepatocytes. Sulforaphane (SFA), an active compound used for inhibiting tumors, was found to have the potency to improve lipid metabolism. However, its molecular mechanisms on ameliorating NAFLD are still incompletely understood. This research evaluated if SFA could inhibit hepatic steatosis and apoptosis. The effects of SFA on cell viability, lipid accumulation, triglyceride (TG) contents, apoptosis, ceramide contents, and reactive oxygen species (ROS) levels were analyzed in palmitic acid (PA)-treated HepG2 cells and high-fat diet (HFD)-fed mice. The related molecular mechanisms were further explored in hepatocytes. The results showed SFA alleviated lipid accumulation and regulated AMPK/SREBP1c/FAS signaling pathway in PA-stressed HepG2 cells. In addition, SFA alleviated PA-mediated apoptosis, downregulated the expressions of cleaved caspase 3, as well as reduced ceramide contents and ROS levels. Moreover, SFA treatment reduced HFD-induced body weight gain, alleviated insulin resistance, decreased serum TG, total cholesterol (TC), and alanine aminotransferase (ALT) levels, and prevented lipid deposition and apoptosis in the liver. This study showed SFA suppressed lipid deposition and apoptosis both in vitro and in vivo, indicating that SFA may be a potential candidate for preventing and treating NAFLD.

Parathyroid hormone ameliorates osteogenesis of human bone marrow mesenchymal stem cells against glucolipotoxicity through p38 MAPK signaling.

Diabetes mellitus (DM)-induced glucolipotoxicity is a factor strongly contributing to alveolar bone deficiency. Parathyroid hormone (PTH) has been identified as a main systemic mediator to balance physiological calcium in bone. This study aimed to uncover PTH's potential role in ameliorating the osteogenic capacity of human bone marrow mesenchymal stem cells (HBMSCs) against glucolipotoxicity. Optimal PTH concentrations and high glucose and palmitic acid (GP) were administered to cells, followed by alkaline phosphatase (ALP) staining and ALP activity assay. Quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR) and Immunoblot were carried out for assessing mRNA and protein amounts, respectively. Cell counting kit-8 (CCK-8) and flow cytometry were performed for quantitating cell proliferation. Osteogenesis and oxidative stress were determined, and the involvement of mitogen-activated protein kinase (MAPK) signaling was further verified. About 1-50 mmol/ml GP significantly inhibited the osteogenic differentiation of HBMSCs. 10-9 mol/L PTH was found to be the optimal concentration for HBMSC induction. PTH had no effects on HBMSC proliferation, with or without GP treatment. PTH reversed inadequate osteogenesis and excessive oxidative stress in GP-treated HBMSCs. Mechanistically, PTH activated p38 MAPK signaling, while inhibiting p38 MAPK-suppressed PTH's beneficial impacts on HBMSCs. Collectively, PTH promotes osteogenic differentiation in HBMSCs against glucolipotoxicity via p38 MAPK signaling.

Dickkopf­1/cysteine­rich angiogenic inducer 61 axis mediates palmitic acid­induced inflammation and apoptosis of vascular endothelial cells.

Cardiovascular diseases (CVDs) are a major cause of mortality around the world, and the presence of atherosclerosis is the most common characteristic in patients with CVDs. Cysteine­rich angiogenic inducer 61 (CCN1) has been reported to serve an important role in the pathogenesis of atherosclerotic lesions. The aim of the present study was to investigate whether CCN1 could regulate the inflammation and apoptosis of endothelial cells induced by palmitic acid (PA). Dickkopf­1 (DKK1) is an important antagonist of the Wnt signaling pathway, which can specifically inhibit the classic Wnt signaling pathway. Firstly, the mRNA and protein expression levels of CCN1 were detected. Additionally, endothelial nitric oxide (NO) synthase (eNOS), DKK1, ß­catenin, and inflammation­ and apoptosis­associated proteins were measured. Detection of NO was performed using a commercial kit. The expression levels of inflammatory cytokines were assessed to explore the effect of CCN1 on PA­induced inflammation. TUNEL assay was used to detect the apoptosis of endothelial cells. The results revealed that PA upregulated the expression levels of CCN1, inflammatory cytokines and pro­apoptotic proteins in endothelial cells. PA decreased the production of NO, and the levels of phosphorylated­eNOS, whereas knockdown of CCN1 partially abrogated these effects triggered by PA. Furthermore, the Wnt/ß­catenin signaling pathway was activated in PA­induced endothelial cells; however, the levels of DKK1 were downregulated. Overexpression of DKK1 could reduce CCN1 expression via inactivation of the Wnt/ß­catenin signaling pathway. In conclusion, knockdown of CCN1 attenuated PA­induced inflammation and apoptosis of endothelial cells via inactivating the Wnt/ß­catenin signaling pathway.

Berberine alleviates palmitic acid­induced podocyte apoptosis by reducing reactive oxygen species­mediated endoplasmic reticulum stress.

Lipid accumulation in podocytes can lead to the destruction of cellular morphology, in addition to cell dysfunction and apoptosis, which is a key factor in the progression of chronic kidney disease (CKD). Berberine (BBR) is an isoquinoline alkaloid extracted from medicinal plants such as Coptis chinensis, which has been reported to have a lipid­lowering effect and prevent CKD progression. Therefore, the present study aimed to investigate the effect of BBR on palmitic acid (PA)­induced podocyte apoptosis and its specific mechanism using an in vitro model. Cell death was measured using the Cell Counting Kit­8 colorimetric assay. Cell apoptotic rate was assessed by flow cytometry. The expression of endoplasmic reticulum (ER) stress­ and apoptosis­related proteins was detected by western blotting or immunofluorescence. Reactive oxygen species (ROS) were evaluated by 2',7'­dichlorofluorescein diacetate fluorescence staining. The results of the present study revealed that BBR treatment decreased PA­induced podocyte apoptosis. In addition, 4­phenylbutyric acid significantly reduced PA­induced cell apoptosis and the expression of ER stress­related proteins, which indicated that ER stress was involved in PA­induced podocyte apoptosis. In addition, N­acetylcysteine inhibited PA­induced excessive ROS production, ER stress and cell apoptosis of podocytes. BBR also significantly reduced PA­induced ROS production and ER stress in podocytes. These results suggested that PA mediated podocyte apoptosis through enhancing ER stress and the production of ROS. In conclusion, BBR may protect against PA­induced podocyte apoptosis, and suppression of ROS­dependent ER stress may be the key mechanism underlying the protective effects of BBR.

Efeito da adição do ácido palmítico e da vitamina E ao diluidor Tris-gema na criopreservação de sêmen canino / Effect of addition of palmitic acid and Vitamin E to the Tris-egg yolk diluter in canine semen cryopreservation

Canine sperm is a very delicate cell that is quite susceptible to oxidative stress since the cytoplasm is restricted and features little antioxidant reserves. Furthermore, the sperm membrane has some polyunsaturated fatty acids sensitive to lipid peroxidation, which makes it important to addition antioxidant substances to the diluter aiming at decreasing such stress to the sperm cell, particularly during seminal cryopreservation. Several antioxidants have been used in this process in some domestic animal's species, however, the use of palmitic acid has been little reported in works on cryopreservation of semen of the canine species. Hence, this study aimed to assess the effect of addition antioxidants palmitic acid and vitamin E to the Tris-egg yolk diluter on the semen quality of dogs after thawing. Samples were collected from the ejaculates of 4 adult dogs, apparently healthy, of the American Pit Bull Terrier breed of kennels in the city of Teresina, PI, places where the pre-freezing procedures of the dog's semen were performed. The samples were diluted in Tris citric acid fructose (3.28 g Tris-hydroxymethyl-aminomethane, 1.78 g citric acid monohydrate and 1.25 g D-fructose), dissolved in 100 mL distilled water, and added 20% egg yolk and 6% glycerol, at the concentration of 100x106 sptz/mL. The semen samples were divided into 3 mL aliquots to form 3 experimental groups: G1 - Only Tris-egg yolk (Control group); G2 - Tris-egg yolk + 100 µM palmitic acid; and G3 - Tris-egg yolk + 116 µM vitamin E. Semen was collected weekly over a period of little over 2 months. After thawing, thermorresistance test (TTR) was carried out at 0, 30, 60, and 90 min to assess spermatics motility and vigor, in addition to analysis of integrity of plasma membrane, acrosomal membrane and mitochondrial activity of the sperm, using fluorescent probes. These assessments were performed out at the Animal Reproduction Biotechnology Laboratory (LBRA/UFPI). In the TTR, G2 and G3 didn't exhibit significant results for spermatics motility or vigor when compared with the control group. The palmitic acid and vitamin E also had no significant effects on the parameters of acrosomal membrane integrity or mitochondrial activity. However, sperm cryopreserved with the addition of palmitic acid exhibited significant differences for plasma membrane integrity, providing greater protection to the sperm cells in G2. The palmitic acid is one of the most saturated fatty acids in human semen, with reports of great proportions also in the seminal plasma of dogs. Its main role is to protect the plasma membrane from external damage, improving viability and fertility of the sperm after cryopreservation. Data is scarce in the literature on the composition of fatty acids in canine semen and regarding the use of palmitic acid as a seminal antioxidant in that species, which grants further studies aiming to investigate such valuable information for canine reproduction. It is concluded that addition palmitic acid at 100µM concentration to the Tris-egg yolk diluter was able to preserve the integrity of the plasma membrane during the process of cryopreservation of canine semen.(AU)

Coniferaldehyde ameliorates the lipid and glucose metabolism in palmitic acid-induced HepG2 cells via the LKB1/AMPK signaling pathway.

Impaired lipid and glucose metabolism in the liver is a crucial characteristic of nonalcoholic fatty liver disease (NAFLD). Coniferaldehyde (CA), a kind of phenolic compound found in many edible plants, has multiple biological and pharmacological functions. However, since the effect and molecular mechanism of CA on hepatic lipid and glucose metabolism disorders in NAFLD remain unknown, this study investigated its impact on the lipid and glucose metabolism of palmitic acid (PA)-induced HepG2 cells. Compared with the HepG2 cells treated only with PA, supplementation with 25, 50, and 100 µM CA reduced the levels of intracellular triglyceride (by 7.11%, 19.62%, and 31.57%) and total cholesterol (by 8.46%, 23.32%, and 27.17%), and enhanced glucose uptake (by 40.91%, 57.49%, and 61.32%) and intracellular glycogen content (by 12.75%, 41.27%, and 53.77%). Moreover, CA supplementation downregulated the expression of sterol regulatory element-binding protein-1, fatty acid synthase, and stearoyl-CoA desaturase 1 related to lipogenesis while upregulating the expression of carnitine palmitoyltransferase 1α related to fatty acid oxidation. CA supplementation also upregulated the glucose transporter 2 protein expression and phosphorylation of glycogen synthase kinase 3ß while downregulating the phosphorylation of glycogen synthase. Most importantly, most of these effects of CA were reversed by pretreatment with AMP-activated protein kinase (AMPK) inhibitor and small interfering RNA-liver kinase B1 (LKB1). In conclusion, CA ameliorated the lipid and glucose metabolism in PA-induced HepG2 cells via the LKB1/AMPK signaling pathway. PRACTICAL APPLICATION: In this study, coniferaldehyde appeared to be effective in ameliorating hepatic lipid and glucose metabolism disorders in nonalcoholic fatty liver disease by reducing the levels of intracellular triglyceride and total cholesterol and enhancing glucose uptake and intracellular glycogen content via the LKB1/AMPK signaling pathway in vitro. Therefore, our findings provide new evidence in support of that supplementation with coniferaldehyde or food rich in coniferaldehyde might be considered as a viable dietary intervention strategy for preventing and treating nonalcoholic fatty liver disease.