Sirtuin 1 is involved in oleic acid-induced calf hepatocyte steatosis via alterations in lipid metabolism-related proteins.

Sirtuin 1 (SIRT1), an NAD-dependent protein deacetylase, plays a central role in the control of lipid metabolism in nonruminants. However, the role of SIRT1 in hepatic lipid metabolism in dairy cows with fatty liver is not well known. Thus, we used isolated primary bovine hepatocytes to determine the role of SIRT1 in protecting cells against oleic acid (OA)-induced steatosis. Recombinant adenoviruses to overexpress (AD-GFP-SIRT1-E) or knockdown (AD-GFP-SIRT1-N) SIRT1 were used for transduction of hepatocytes. Calf hepatocytes isolated from five female calves (1 d old, 30 to 40 kg) were used to determine both time required and the lowest dose of OA that could induce triacylglycerol (TAG) accumulation. Analyses indicated that 0.25 mM OA for 24 h was suitable to induce TAG accumulation. In addition, OA not only led to an increase in TAG, but also upregulated mRNA and protein abundance of sterol regulatory element-binding transcription factor 1 (SREBF1) and downregulated SIRT1 and peroxisome proliferator-activated receptor-gamma coactivator 1 alpha (PPARGC1A). Thus, these in vitro conditions were deemed optimal for subsequent experiments. Calf hepatocytes were cultured and incubated with OA (0.25 mM) for 24 h, followed by adenoviral AD-GFP-SIRT1-E or AD-GFP-SIRT1-N transduction for 48 h. Overexpression of SIRT1 led to greater protein and mRNA abundance of SIRT1 along with fatty acid oxidation-related genes including PPARGC1A, peroxisome proliferator-activated receptor alpha (PPARA), retinoid X receptor α (RXRA), and ratio of phospho-acetyl-CoA carboxylase alpha (p-ACACA)/total acetyl-CoA carboxylase alpha (ACACA). In contrast, it resulted in lower protein and mRNA abundance of genes related to lipid synthesis including SREBF1, fatty acid synthase (FASN), apolipoprotein E (APOE), and low-density lipoprotein receptor (LDLR). The concentration of TAG decreased due to SIRT1 overexpression. In contrast, silencing SIRT1 led to lower protein and mRNA abundance of SIRT1, PPARGC1A, PPARA, RXRA, and greater protein and mRNA abundance of SREBF1, FASN, APOE, and LDLR. Further, those responses were accompanied by greater content of cellular TAG and total cholesterol (TC). Overall, data from these in vitro studies indicated that SIRT1 is involved in the regulation of lipid metabolism in calf hepatocytes subjected to an increase in the supply of OA. Thus, it is possible that alterations in SIRT1 abundance and activity in vivo contribute to development of fatty liver in dairy cows.

Low Expression of Sirtuin 1 in the Dairy Cows with Mild Fatty Liver Alters Hepatic Lipid Metabolism.

Dairy cows usually experience negative energy balance coupled with an increased incidence of fatty liver during the periparturient period. The purpose of this study was to investigate the effect of hepatic steatosis on the expression of the sirtuin 1 (SIRT1), along with the target mRNA and protein expressions and activities related to lipid metabolism in liver tissue. Control cows (n = 6, parity 3.0 ± 2.0, milk production 28 ± 7 kg/d) and mild fatty liver cows (n = 6, parity 2.3 ± 1.5, milk production 20 ± 6 kg/d) were retrospectively selected based on liver triglycerides (TG) content (% wet liver). Compared with the control group, fatty liver cows had greater concentrations of cholesterol and TG along with the typically vacuolated appearance and greater lipid droplets in the liver. Furthermore, fatty liver cows had greater mRNA and protein abundance related to hepatic lipid synthesis proteins sterol regulatory element binding proteins (SREBP-1c), long-chain acyl-CoA synthetase (ACSL), acyl-CoA carbrolase (ACC) and fatty acid synthase (FAS) and lipid transport proteins Liver fatty acid binding protein (L-FABP), apolipoprotein E (ApoE), low density lipoprotein receptor (LDLR) and microsomal TG transfer protein (MTTP) (p < 0.05). However, they had lower mRNA and protein abundance associated with fatty acid ß-oxidation proteins SIRT1, peroxisome proliferator-activated receptor co-activator-1 (PGC-1α), peroxisome proliferator-activated receptor-α (PPARα), retinoid X receptor (RXRα), acyl-CoA 1 (ACO), carnitine palmitoyltransferase 1 (CPT1), carnitine palmitoyltransferase 2 (CPT2) and long- and medium-chain 3-hydroxyacyl-CoA dehydrogenases (LCAD) (p < 0.05). Additionally, mRNA abundance and enzyme activity of enzymes copper/zinc superoxide dismutase (Cu/Zn SOD), catalase (CAT), glutathione peroxidase (GSH-Px) and manganese superoxide dismutase (Mn SOD) decreased and mRNA and protein abundance of p45 nuclear factor-erythroid 2 (p45 NF-E2)-related factor 1 (Nrf1), mitochondrial transcription factor A (TFAM) decreased (p < 0.05). Lower enzyme activities of SIRT1, PGC-1α, Cu/Zn SOD, CAT, GSH-Px, SREBP-1c and Mn SOD (p < 0.05) and concentration of reactive oxygen species (ROS) were observed in dairy cows with fatty liver. These results demonstrate that decreased SIRT1 associated with hepatic steatosis promotes hepatic fatty acid synthesis and inhibits fatty acid ß-oxidation. Hence, SIRT1 may represent a novel therapeutic target for the treatment of the fatty liver disease in dairy cows.

Immune Regulation of RAW264.7 Cells In Vitro by Flavonoids from Astragalus complanatus via Activating the NF-κB Signalling Pathway.

The current study aimed at investigating the effects of flavonoids from Astragalus complanatus (FAC) on the proliferation, the contents, and gene expression levels of cytokines, secretion of surface stimulating factors, cell cycle, and the expression level of the NF-κB signalling pathway in RAW264.7 cells. Our results revealed that compared with control group, the contents of IL-6, IL-1ß, TNF-α, and NO and the mRNA expression levels of IL-6, IL-1ß, TNF-α, and iNOS in FAC-treated groups significantly increased (p < 0.01). Moreover, FAC induced macrophage activation to release the above-mentioned mediators partly involved in NF-κB/MAPK signalling pathways. Therefore, FAC regulates immune function in RAW264.7 cells via activating the NF-κB signalling pathway. FAC could be applicable for agriculture, drug research, and food industry as a potent immune-modulatory agent.

Immune regulation mechanism of Astragaloside IV on RAW264.7 cells through activating the NF-κB/MAPK signaling pathway.

The present study was designed to investigate the effects of Astragaloside IV (ASIV) on the immune functions of RAW264.7 cells. Compared with control group, the concentrations of interleukin 1ß (IL-1ß), tumor necrosis factor α (TNF-α) and nitric oxide (NO) were higher in the 100µg/mL ASIV-treatment group. The interleukin 6 (IL-6) concentration was significantly higher in the 50 and 100µg/mL ASIV-treatment groups. The relative mRNA expression levels of IL-1ß, TNF-α and inducible nitric oxide synthase (iNOS) were significantly higher in the 50 and 100µg/mL ASIV-treatment groups. The relative mRNA expression levels of IL-6 in the 100µg/mL ASIV-treatment group were significantly higher. In contrast, the relative mRNA expression levels of interleukin 4 (IL-4) and IL-6 markedly reduced in ASIV-treatment groups. Furthermore, ASIV promoted the secretion of CD40 and CD86 and increased the number of cells in G2/M phase. The apoptosis of RAW264.7 cells was decreased in ASIV-treatment groups. The protein levels of cyclin D1, CDK4 and CDK6, p50 and p-p65 increased in a dose-dependent manner. The ratio of p50/ß-actin was significantly higher in the 50 and 100µg/mL ASIV-treatment groups, and p-p65/p65 was significantly higher in the 25, 50 and 100µg/mL ASIV-treatment groups. The phosphorylation levels of p38, ERK and JNK increased, and the protein expression of total p38, ERK and JNK decreased in a dose-dependent manner. These effects of ASIV were alleviated by PDTC. ASIV enhances the immune function of RAW264.7 cells by activating the NF-κB/MAPK signaling pathway.

[The evaluation of four-year highly active antiretroviral therapy in HIV-1 infected patients].

OBJECTIVE: To observe that antiretroviral efficacy, immune reconstitution of four-year highly active antiretroviral therapy (HAART), and evaluate its side effect in Chinese HIV-1-infected patients. METHODS: A total of 258 HIV-1 infected patients, given HAART regimens composed of two nucleoside reverse transcriptase inhibitor (NRTI) and one non-nucleoside reverse transcriptase inhibitor (NNRTI) for mean 51.5 months, measured HIV RNA viral load (VL) and the counts of CD(4)(+) T cell, CD(8)(+) T cell at the baseline and 6, 12, 24, 36 and 48 months after HAART initiation, respectively, monitoring side effect, blood routine, main biochemical parameters, and other disadvantageous accidents during the 51.5-month treatment. RESULTS: Plasma HIV-1 RNA level was determined by fluorescent quantitative polymerase chain reactions (FQ-PCR) at the baseline and 6, 12, 24, 36 and 48 months after starting HAART, and showed 5.27, 2.97, 2.74, 2.62, 2.67 and 2.75 lg (copies/ml), respectively. The counts of CD(4)(+) T cell from (127 ± 63) cells/µl at the baseline increased to (190 ± 115), (248 ± 93), (269 ± 127), (296 ± 156) and (317 ± 195) cells/µl at 6, 12, 24, 36 and 48 months after starting HAART. A total of 149 treated patients (57.8%)had gastrointestinal side effects, peripheral polyneuropathy, various rashes, central nervous system disorders, fever or baldness. Twenty-two patients changed one of three medicines to another because toxicity. Sixteen changed the regimen to the second line HAART for lactic acidosis or other serious toxicities. CONCLUSIONS: A total of 258 HIV-1 infected Chinese patients treated with two NRTI and one NNRTI as first line HAART regimen during mean 51.5 months, showed a good antiretroviral efficacy and immune reconstitution, but a few side-effects at the parts of patients. It is necessary to treat adverse effect and change HAART regimen for severe toxicity in time.