Cassiae Semen improves non-alcoholic fatty liver disease through autophagy-related pathway / 中草药·英文版

OBJECTIVE@#Cassiae Semen (CS, Juemingzi in Chinese) has been used for thousands of years in ancient Chinese history for relieving constipation, improving liver function as well as preventing myopia. Here we aimed to elucidate the anti-steatosis effect and underlying mechanism of CS against non-alcoholic fatty liver disease (NAFLD).@*METHODS@#High-performance liquid chromatography (HPLC) was used to identify the major components of CS water extract. Mice were fed with a high-fat and sugar-water (HFSW) diet to induce hepatic steatosis and then treated with CS. The anti-NAFLD effect was determined by measuring serum biomarkers and histopathology staining. Additionally, the effects of CS on cell viability and lipid metabolism in oleic acid and palmitic acid (OAPA)-treated HepG2 cells were measured. The expression of essential genes and proteins involved in lipid metabolism and autophagy signalings were measured to uncover the underlying mechanism.@*RESULTS@#Five compounds, including aurantio-obtusin, rubrofusarin gentiobioside, cassiaside C, emodin and rhein were simultaneously identified in CS extract. CS not only improved the diet-induced hepatic steatosis in vivo, as indicated by decreased number and size of lipid droplets, hepatic and serum triglycerides (TG) levels, but also markedly attenuated the OAPA-induced lipid accumulation in hepatocytes. These lipid-lowering effects induced by CS were largely dependent on the inhibition of fatty acid synthase (FASN) and the activation of autophagy-related signaling, including AMP-activated protein kinase (AMPK), light chain 3-II (LC3-II)/ LC3-1 and autophagy-related gene5 (ATG5).@*CONCLUSION@#Our study suggested that CS effectively protected liver steatosis via decreasing FASN-related fatty acid synthesis and activating AMPK-mediated autophagy, which might become a promising therapeutic strategy for relieving NAFLD.

Construction and optimization of engineered Bacillus subtilis for surfactin production / 生物工程学报

Surfactin has great potential applications in enhancing oil recovery, agriculture, pharmaceuticals, foods and beverages, and cosmetics due to its extraordinary surface activity, biodegradability, anti-bacterial activity and biocompatibility. Enhancing surfactin production by engineering surfactin-producer and optimizing culture conditions is the key of its industrial production and subsequent applications. In this study, the effect of fatty acid synthesis pathway on surfactin synthesis was investigated, and Bacillus subtilis THBS-2 and THBS-8 with high surfactin titer were constructed by overexpressing key genes involved in the fatty acid synthesis pathway. To optimize culture condition, the amount and adding time of isopropyl-beta-D-thiogalactopyranoside (IPTG) and amino acids were studied, and a two-stage culture method was obtained: IPTG (final concentration: 1.25 mmol/L) and leucine (final concentration: 5 g/L) were added at 3 h, leucine (final concentration 5 g/L) and condensed culture medium (5 mL) were added at 24 h. Applying this strategy, the surfactin titer of B. subtilis THBS-2 reached to 24 g/L in shake flask at 48 h and up to 34 g/L after 68 h fermentation in a 30-L fermentor. The results provide basis for large-scale production and broad application of surfactin.

Action Mechanism of Main Components from Glycyrrhizae Radix et Rhizoma in Improving Insulin Resistance in L6 Rat Myoblasts / 中国实验方剂学杂志

Objective::To determine whether the main components of Glycyrrhizae Radix et Rhizoma can improve insulin resistance by regulating glycogen synthesis, glycolysis pathway and fatty acid synthesis in myoblasts of L6 rat myoblasts. Method::Insulin resistance (IR) model of L6 rat myoblasts was established through incubation with 0.05 mmol·L-1 palmitic acid (PA) for 9 hours. Normal group, model group, glycyrrhizic acid (GA, 25 μmol·L-1) group, 18β-glycyrrhetinic acid (18β-GA, 25 μmol·L-1) group, isoliquiritigenin (ILG, 25 μmol·L-1) group and isoliquiritin (ILQ, 25 μmol·L-1) group were set up, glucose content in supernatant of cell culture medium was detected by glucose kit, myoblasts glycogen content was determined by glycogen detection kit, protein expression levels of Sterol regulatory element binding protein-1c(SREBP-1c), fatty acid synthetase (FAS) and glycogen synthase kinase3β(GSK3β) were detected by Western blot, and the mRNA expressions of key enzymes in glycolysis were detected by quantitative real-time fluorescence polymerase chain reaction(Real-time PCR). Result::Compared with those in the normal group, the glucose consumption rate was significantly down-regulated in model group (P<0.01), the glycogen content was decreased (P<0.05), the protein expressions of Sterol regulatory element binding protein-1c (SREBP-1c) and fatty acid synthase (FAS) were decreased (P<0.05, P<0.01), the mRNA expressions of fructose phosphate kinase 1 (PFK1), pyruvate kinase (PK) and hexokinase (HK) were down-regulated (P<0.05), and the protein expression of glycogen synthase kinase 3 (GSK3β) protein was increased (P<0.05). Compared with model group, GA, 18β-GA and ILG could significantly increase glycogen content in myoblasts of IR-L6 rats (P<0.05, P<0.01). GA, 18β-GA and ILQ could significantly increase the expression of SREBP-1c (P<0.05, P<0.01), and GA, 18β-GA, ILG and ILQ could significantly increase the expression of FAS (P<0.05, P<0.01), the mRNA expressions of PFK1, PK and HK (P<0.05, P<0.01), and down-regulate the protein expression of GSK3β (P<0.05). Conclusion::The main components of licorice improve the insulin resistance by promoting glycolysis and glycogen synthesis and regulating fatty acid synthesis.

RASAL1 induces to downregulate the SCD1, leading to suppression of cell proliferation in colon cancer via LXRα/SREBP1c pathway

BACKGROUND: Recent studies have confirmed that RASAL1 has an antitumor effect in many cancers, but its functional role and the molecular mechanism underlying in colon cancer has not been investigated. RESULTS: We collected human colon cancer tissues and adjacent non-tumor tissues, human colon cancer cell lines LoVo, CaCo2, SW1116, SW480 and HCT-116, and normal colonic mucosa cell line NCM460. RT-qPCR was used to detect the RASAL1 level in the clinical tissues and cell lines. In LoVo and HCT-116, RASAL1 was artificially overexpressed. Cell viability and proliferation were measured using CCK-8 assays, and cell cycle was detected via PI staining and flow cytometry analysis. RASAL1 significantly inhibited the cell proliferation via inducing cell cycle arrest, suppressed cell cycle associated protein expression, and decreased the lipid content and inhibited the SCD1 expression. Moreover, SCD1 overexpression induced and downregulation repressed cell proliferation by causing cell cycle arrest. Additionally, luciferase reporter assays were performed to confirm the direct binding between SREBP1c, LXRα; and SCD1 promoter, we also demonstrated that RASAL1 inhibit SCD1 3'-UTR activity. RASAL1 inhibited tumor growth in xenograft nude mice models and shows inhibitory effect of SCD1 expression in vivo. CONCLUSION: Taken together, we concluded that RASAL1 inhibited colon cancer cell proliferation via modulating SCD1 activity through LXRα/SREBP1c pathway.

Increasing pinosylvin production in Escherichia coli by reducing the expression level of the gene fabI-encoded enoyl-acyl carrier protein reductase

Background: The plant secondary metabolite pinosylvin is a polyphenol from the stilbene family, which have positive effects on human health. Biotechnological production is an attractive alternative for obtaining this stilbene. In Escherichia coli, malonyl-CoA is the precursor for both stilbene and fatty acid syntheses. In this study, with the aim of increasing pinosylvin production, we evaluated a novel approach that is based on reducing the expression of the gene fabI, which encodes the enzyme enoyl-acyl carrier protein reductase that is involved in fatty acid synthesis. Results: A recombineering method was employed to eliminate the chromosomal -35 promoter sequence and the upstream region of the gene fabI in E. coli strain W3110. Analysis, employing RT-qPCR, showed that such modification caused a 60% reduction in the fabI transcript level in the mutant strain W3110Δ-35fabI::Cm compared to the wild type W3110. Synthetic genes encoding a mutant version of 4-coumaroyl-CoA ligase from Streptomyces coelicolor A3 with improved catalytic activity employing cinnamic acid as substrate and a stilbene synthase from Vitis vinifera were cloned to generate the plasmid pTrc-Sc4CL(M)-VvSTS. The production performance of strains W3110Δ-35fabI::Cm/pTrc-Sc4CL(M)-VvSTS and W3110/pTrc-Sc4CL(M)- VvSTS was determined in shake flask cultures with Luria-Bertani medium supplemented with 10 g/L glycerol and 3 mM cinnamic acid. Under these conditions, the strain W3110Δ-35fabI::Cm/pTrc-Sc4CL(M)-VvSTS produced 52.67 mg/L pinosylvin, a level 1.5-fold higher than that observed with W3110/pTrc-Sc4CL(M)-VvSTS. Conclusion: A reduction in the transcript level of fabI caused by the elimination of the -35 and upstream promoter sequences is a successful strategy to improve pinosylvin production in E. coli.

Research progress of sterol regulatory element binding transcription factor 1(SREBP1) in cancer / 中国肿瘤临床

The increase of fatty acid synthesis is the third biggest metabolic phenotype of tumor cells,and sterol regulatory element binding transcription factor 1(SREBP1)is the major nuclear transcription factor involved in lipid metabolism,especially in the synthesis of fatty acid.The expression of SREBP1 is elevated in multiple tumors,which plays an important role in tumor proliferation,apoptosis, invision,drug resistance,energy metabolism etc.In this paper,we will review the research progress on SREBP1 in tumor.

Fatty acid metabolism and tumor occurrence, development and metastasis / 肿瘤研究与临床

In recent years,the relationship between fatty acid metabolism and tumor has become a hot topic and widely recognized.In tumor cell,fatty acid metabolism is important to its growth and metastasis.About synthesis,fatty acid can participate in the synthesis of phospholipids on the membrane of cancer cells and take part in important signal transduction pathways,such as PI3K-AKT-mTOR;in fatty acid catabolism,tumor cells can produce ATP to maintain its growth by β-oxidation;what's more,it could be kept redox homeostasis by producing nicotinamide adenine dinucleotide phosphate hydrogen (NADPH).So,anabolism and catabolism of fatty acid may promote occurrence and development of tumors,but the mechanisms are unclear.This review highlights the effects of fatty acid metabolism on the occurrence,development and metastasis of tumors.

Exploring novel drug targets in fatty acid pathway of Plasmodium falciparum.

The malarial parasite Plasmodium falciparum infects humans and proliferates rapidly inside the host before its detection. The proliferation step requires a large amount of lipids for membrane synthesis. Thus fatty acid biosynthesis occurring in the apicoplast plays an important role in causing cerebral malaria. In this study, we explored and analyzed these pathways using stoichiometric matrix, elementary flux modes and robustness analysis. Based on the above analysis, the robustness of this pathway diminished as the result of virtual enzyme knock out indicating four key enzymes, 3-oxoacyl-ACP synthase, 3-oxoacyl-ACP synthase, 3-oxoacyl-ACP synthase and Glycerol-3-phosphate o-acyl transferase. Among the four, the first three are existing drug targets. Subsequently, we also found that a combinatorial double knock out of these enzymes predicts further reduction in overall pathway enzyme activity. Thus, we propose multi drug targeting as a better way to treat brain malaria.

The effects of Alpha-linolenic acid on the related key gene expressions of fatty acid synthesis in HepG2 cells / 中国比较医学杂志

Objective To investigate the effects of different doses Alpha-Linolenic acid ( ALA ) on the expressions of fatty acid synthesis-related genes in HepG2 cells.Methods HepG2 cells were divided into two groups, normal control group (NC) and high fat group (HF) in which cells were firstly cultured for 36h in the medium contained 0.5mmol/L stearic acid.Real-time quantitative PCR was taken to detect mRNA expression of genes SREBP1C, FAS and ACC which are related to fatty synthesis.While there are significant differences in fatty synthesis, 10%, 20%, 50%, 70% and 100%ALA took the place of stearic acid, 36h later, real-time quantitative PCR and Western blotting were taken to detect mRNA and protein expression of genes related to fatty synthesis.Results SREBP1C mRNA expression of ALA substitution groups were significantly lower than the high-fat group ( P <0.001) .The FAS of 0.5 mmol/L ALA group and 0.35 mmol/L ALA group were significantly lower than the high-fat group (P <0.001).ACC genes mRNA level was not significantly different from high-fat group.SREBP1C and FAS protein expression were significantly lower than the high-fat group, but ACC showed no significant difference.Conclusions Saturated fatty acids promote hepatocyte fatty acid synthesis, ALA abates fatty acid synthesis by inhibiting FAS and SREBP1C gene expression.

Anti-obesity effects of Lysimachia foenum-graecum characterized by decreased adipogenesis and regulated lipid metabolism

Lysimachia foenum-graecum has been used as an oriental medicine with anti-inflammatory effect. The anti-obesity effect of L. foenum-graecum extract (LFE) was first discovered in our screening of natural product extract library against adipogenesis. To characterize its anti-obesity effects and to evaluate its potential as an anti-obesity drug, we performed various obesity-related experiments in vitro and in vivo. In adipogenesis assay, LFE blocked the differentiation of 3T3-L1 preadipocyte in a dose-dependent manner with an IC50 of 2.5 microg/ml. In addition, LFE suppressed the expression of lipogenic genes, while increasing the expression of lipolytic genes in vitro at 10 microg/ml and in vivo at 100 mg/kg/day. The anti-adipogenic and anti-lipogenic effect of LFE seems to be mediated by the inhibition of PPARgamma and C/EBPalpha expression as shown in in vitro and in vivo, and the suppression of PPARgamma activity in vitro. Moreover, LFE stimulated fatty acid oxidation in an AMPK-dependent manner. In high-fat diet (HFD)-induced obese mice (n = 8/group), oral administration of LFE at 30, 100, and 300 mg/kg/day decreased total body weight gain significantly in all doses tested. No difference in food intake was observed between vehicle- and LFE-treated HFD mice. The weight of white adipose tissues including abdominal subcutaneous, epididymal, and perirenal adipose tissue was reduced markedly in LFE-treated HFD mice in a dose-dependent manner. Treatment of LFE also greatly improved serum levels of obesity-related biomarkers such as glucose, triglycerides, and adipocytokines leptin, adiponectin, and resistin. All together, these results showed anti-obesity effects of LFE on adipogenesis and lipid metabolism in vitro and in vivo and raised a possibility of developing LFE as anti-obesity therapeutics.

Reconstitution of Escherichia coli Fatty Acid Biosynthesis Reaction In vitro / 生物化学与生物物理进展

Seven genes (fabD, fabG, fabH, fabA, fabZ, fabB and fabI) of E.coli fatty acid biosynthetic enzymes were cloned by PCR amplifying and appropriate expression vectors were constructed. Under induction assay expression of plasmid encoded proteins was carried out in strain BL21(DE3) and seven enzymes were purified using Ni-NTA agarose resin. In the absence of [2-14C] malonyl-CoA fatty acid synthetic reaction was reconstituted in vitro by adding seven enzymes and co-factors. And several model reactions were established for identification of special fatty acid biosynthetic enzymes. Meanwhile Clostridium acetobutylicium FabZ function was characterized by this method.

Effects of Acupuncture Stimulation on Hyperlipidemia Induced by a Cholesterol-Free, High-Fructose Diet in Rats / 全日本鍼灸学会雑誌

Objective : We examined the effects of acupuncture stimulation on hyper lipidemia induced by a cholesterol-free, high-fructose diet (HFD) in rats.<BR>Methods : Acupoints on the rats' bodies were selected at the positions relative to the human acupoints, such as BL 18, LR 14, CV 12, ST 36 and T13-L1, which starts of the origin of splanchnic nerve and runs at intervals of 1 cm on both sides of the nerve between the spinous process of the thirteenth thoracic vertebra and the first lumber vertebra (T13-L1). Nonacupoints were selected on bi lateral buttocks for rats fed with a normal diet and HFD control. Acupuncture stimulations were administered by the subcutaneous insertion of acus. The stimulation was started with HFD feeding and continued for two weeks.<BR>Results : Feeding with HFD for 2 weeks incresed the levels of total cholesterol (TC), especially in very low density lipoprotein (VLDL) and low density lipoprotein (LDL), free cholesterol (FC), triglyceride (TG) and phospholipiid (PL) in serum. Acupuncture stimulations of BL18, LR14, CV12 and S36 inhibited the increase of TC, while the increase of VLDL·LDL-C was inhibited by the acupuncture stimulation of all acupoints. The stimulation of BL18, LR14 and ST36 inhibited the increase of FC. The stumulation of T13-L1 inhibited the increases of TG, TG in high density lipoprotein and PL. The increase of TG in liver by HFD feeding was inhibited by the stimulations on LR14, T13-L1 and CV 12. The reductions of glucose-6-phosphate dehydrogenase and malic enzyme activities in the liver of rats fed by HFD feeding were enhanced by the stimulation of T13-L1 and S36. The activity of β-oxdation in the liver was slightly increased by the stimulations on LR14 and ST36.<BR>Conclusions : These results suggest that the acupuncture stimulation on BL18, LR14, CV12 and ST36 inhibited the increase of intrinsic cholesterol and enhanced the metabolism of VLDL·LDL-C. In addition, it appears that the mechanism of TG decrease by the stimulation on LR14, T13-L1 and ST36 was related to the inhibition of fatty acid synthesis and the enhancement of fatty acid metabolism in the liver.

Cerulenin induces apoptosis of the cells in oral squamous cell carcinoma / 实用口腔医学杂志

Subjective: To observe apoptosis of the cells in oral sq uamous cell carcinoma(SCC) induced by cerulenin. Methods: SCC TCA-83 cells and fresh tissue of SCC of tongue from 5 patients were expos ed to cerulenin (10 mg/ml) for 24 hours, then the genosome DNA of the cells was extracted and electrophoresed; the fresh tissue of SCC was assessed by TUNEL labeling. Results: DNA gel electrophoresis showed typical apoptic DNA ladders from TCA-83 cells. The rate (%) of TUNEL-positive cells in cerulenin treated tissue of SCC was 29.0?2.6～40.6?16.2,that in the control 2 . 0?1.7～14.7?0.6 (P

Relationship between fatty acid binding proteins, acetyl-CoA formation and fatty acid synthesis in developing human placenta.

The relationship between fatty acid binding proteins, ATP citrate lyase activity and fatty acid synthesis in developing human placenta has been studied. Fatty acid binding proteins reverse the inhibitory efect of palmitoyl-CoA and oleate on ATP citrate lyase and fatty acid synthesis. In the absence of these inhibitors fatty acid binding proteins activate ATP citrate lyase and stimulate [ 1–14 C] acetate incorporation into placental fatty acids indicating binding of endogenous inhibitors by these proteins. Thus these proteins regulate the supply of acetyl-CoA as well as the synthesis of fatty acids from that substrates. As gestation proceeds and more lipids are required by the developing placenta fatty acid binding protein content, activity of ATP citrate lyase and rate of fatty acid synthesis increase indicating a cause and efect relationship between the demand of lipids and supply of precursor fatty acids during human placental development.

Studies on the inhibition a fatty acid synthesis in the chicken liver by adenine compounds in vitro.

The biosynthesis of fatty acids from [1-14C]-acetate in the chicken liver slices in vitro was inhibited by cAMP, adenosine, 5'-AMP, 3'-AMP, ATP, NAD and FAD but not by adenine, guanine or inosine. The minimum structural requirement for inhibition appears to be adenosine. The inhibitory action of adenosine, 5'-AMP and NAD on fatty acid synthesis is likely to be mediated by adenosine or its metabolites since adenosine deaminase reverses the inhibition while it has no effect on the inhibition by cAMP; thus, the inhibitory effect of cAMP is probably not mediated through its hydrolysis products, 5'-AMP, or adenosine.