To explore the effect of ophiopogonin D on main fatty acid metabolic enzymes in human cardiomyocyte AC-16,so as to provide reference for cardiovascular protection mechanism and safe clinical application of Ophiopogon japonicus.CCK-8 (cell counting kit-8) was used to detect the effect of different concentrations of ophiopogonin D on the viability of cardiomyocytes.Meanwhile,the effect of different concentrations of ophiopogonin D on the morphology and quantity of cardiomyocytes was observed under microscope.The effect of ophiopogonin D on the mRNA expression of CYP2J2,CYP4F3,CYP4A11,CYP4A22 and CYP4F2 in cardiomyocytes was detected by RT-PCR.Western blot was used to detect the protein expression of CYP4F3 in different concentrations of ophiopogonin D.Compared with the control group,low-concentration ophiopogonin D had no effect on the viability of cardiomyocytes.However,ophiopogonin D with a concentration of higher than 20μmol·L~(-1)could promote the viability.Under the microscope,ophiopogonin D with a concentration of below 100μmol·L~(-1)had no significant effect on the morphology and number of cardiomyocytes.RT-PCR results showed that compared with the control group,5μmol·L~(-1)ophiopogonin D could slightly up-regulate mRNA expressions of CYP2J2 and CYP4F3,while high-concentration ophiopogonin D (10 and 20μmol·L~(-1)) could significantly induce mRNA expressions of CYP2J2and CYP4F3 in a dose-dependent manner (P<0.05).The same concentration of ophiopogonin D had a little effect on the mRNA expressions of CYP4A11,CYP4A22 and CYP4F2.Western blot results showed that 20μmol·L~(-1)ophiopogonin D could significantly induce the protein expression of CYP4F3 in a dose-dependent manner (P<0.05).Based on the above results,ophiopogonin D (less than100μmol·L~(-1)) has no effect on the viability of AC-16 cardiomyocytes.Ophiopogonin D (less than 100μmol·L~(-1)) can selectively induce the expressions of CYP2J2 and CYP4F3,regulate the metabolic pathway of fatty acid signaling molecules,and thus protecting the cardiovascular system.
Fatty Acids ; Humans ; Myocytes, Cardiac ; Saponins/pharmacology* ; Spirostans/pharmacology*

OBJECTIVETo study the effects of different dietary fatty acid on the expression of nuclear receptor genes in the breast cancer of rats.

METHODSFifty-day-old female Sprague-Dawley rats were fed on eight different diets containing following fatty acids: saturated fatty acid (SFA); monounsaturated fatty acid (MUFA); n-6 polyunsaturated fatty acid (PUFA); n-3 PUFA; 1:1 n-6/n-3; 5:1 n-6/n-3; 10:1 n-6/n-3; 1:2:1 S/M/P (n-6/n-3 at 1:1). The rats were given a single intraperitoneal injection of methyl-nitrosourea (MNU) at 50 mg/kg body weight to establish the rat model of mammary carcinogenesis, the ultrastructure changes of mammary gland cells in rats were observed by transmission electron microscope, the cell proliferation activity was detected by BrdU-labeled immunocytochemistry, and the expression of PPARbeta and PPARgamma mRNA were assayed by RT-PCR.

RESULTSThere was no breast cancer occurring in control groups and the MNU-treated n-3 PUFA group, and the ultrastructure and proliferation activity of mammary gland cells in these groups were normal. In contrast, there appeared obvious marker of adenocarcinomas in mammary gland cells of MNU-induced breast cancer, and a high cell proliferation activity was found in tumor growth-enhancing groups (SFA, MUFA, n-6 PUFA, 5:1 n-6/n-3, 10:1 n-6/n-3 and S/M/P, 21% - 22% of BrdU-labeled cells), while a low cell proliferation activity was detected in rats fed with 1:1 n-6/n-3 diet (13% of BrdU-labeled cells, P < 0.05). Moreover, peroxisome proliferator-activated receptors (PPARs), as important nuclear receptor genes of relating lipid metabolism, the expressions of PPARbeta and PPARgamma mRNA were significantly up-regulated in mammary adipose tissues of MNU-induced breast cancer as compared with the control groups, but the expression levels of peroxisome proliferator-activated receptors (PPARs) in rats fed with 1:1 n-6/n-3 group were lowest (P < 0.05).

CONCLUSIONThe different dietary fatty acid compositions should diversely adjust the expression of PPARs gene in rats, which maybe have an important role in affecting incidence of breast cancer.

Animals ; Fatty Acids ; pharmacology ; Fatty Acids, Omega-3 ; pharmacology ; Fatty Acids, Omega-6 ; pharmacology ; Fatty Acids, Unsaturated ; pharmacology ; Female ; Gene Expression ; Gene Expression Regulation, Neoplastic ; Mammary Neoplasms, Experimental ; genetics ; PPAR gamma ; genetics ; RNA, Messenger ; genetics ; Rats ; Rats, Sprague-Dawley

Mycolic acids (MAs), i.e. 2-alkyl, 3-hydroxy long-chain fatty acids, are the hallmark of the cell envelope of Mycobacterium tuberculosis and are related with antibiotic resistance and host immune escape. Nowadays, they've become hot target of new anti-tuberculosis drugs. There are two main methods to detect MAs, ¹⁴C metabolic labeling thin-layer chromatography (TLC) and liquid chromatograph mass spectrometer (LC-MS). However, the user qualification of ¹⁴C or the lack of standards for LC-MS hampered the easy use of this method. TLC is a common way to analyze chemical substance and can be used to analyze MAs. In this study, we used tetrabutylammonium hydroxide and methyl iodide to hydrolyze and formylate MAs from mycobacterium cell wall. Subsequently, we used diethyl ether to extract methyl mycolate. By this method, we can easily extract and analyze MA in regular biological labs. The results demonstrated that this method could be used to compare MAs of different mycobacterium in different growth phases, MAs of mycobacteria treated by anti-tuberculosis drugs or MAs of mycobacterium mutants. Therefore, we can use this method as an initial validation for the changes of MAs in researches such as new drug screening without using radioisotope or when the standards are not available.
Mycolic Acids/metabolism* ; Chromatography, Thin Layer ; Mycobacterium tuberculosis ; Fatty Acids ; Antitubercular Agents/pharmacology*

OBJECTIVETo investigate the effect of long-term administration of fluvastatin on improvement of ventricular remodeling of rats after myocardial infarction and its mechanism.

METHODSSprague-Dawley rats were subjected to ligation in anterior descending branch of coronary artery and treated with fluvastatin (20 mg.kg(-1) d(-1)) or distilled water for 8 weeks. Doppler echocardiography, hemodynamic study and cardiac histomorphometry were used to estimate the ventricular remodeling and cardiac function. Laser scanning confocal microscope was used to definite the distribution of superoxide anion (O(2)(*-)) and nitrogen monoxide. RT-PCR and immunohistochemistry were used to detect the expression of NOS2 and p22phox in mRNA and protein level. The level of lipid peroxidation, glutathione peroxidase, nitrogen monoxide and total cholesterol were detected too.

RESULTSAdministration of fluvastatin ameliorated left ventricular remodeling without affecting the infarct size [(40 +/- 6 vs 42 +/-5)%, P>0.05]. The level of left ventricular end-diastolic pressure [(18.24 +/-6.58 vs 10.74 +/-4.71) mmHg, P<0.05], right ventricular ameliorated relative weight [(0.92 +/-0.19 vs 0.71 +/-0.13) g/kg, P<0.05], the thickness of left ventricular posterior wall [(3.04 +/-0.28 vs 2.60 +/-0.36) mm, P<0.05] decreased after fluvastatin treatment. The left ventricular ejection fraction was not influenced, the relative lung weight and the left atrium diameter reduced [(5.79 +/-2.92 vs 3.69 +/-0.68) g/kg, (0.55 +/-0.12 vs 0.45 +/-0.04) mm, P<0.05]; the expressions of LPO in the plasma and myocardium [(8.64 +/-0.59 vs 7.71 +/-0.66) U/dl, P<0.05; (3.12 +/-0.38 vs 1.93 +/-0.40) ng/microg.pro, P<0.01] were reduced, and the overexpressed NO was inhibited [(436.87 +/-47.22 vs 313.78 +/-34.35) mg/dl, P<0.01], but the expression of GPx increased [(66.13 +/-8.31 vs 79.78 +/-2.38) mg/dl, P<0.01]. The expression of O(2)(*-) and the activity of NADPH oxidase subunit p22phox increased; NOS2 and its products NO were over-expressed too.

CONCLUSIONVentricular remodeling and hemodynamics are improved profoundly in MI rats treated with fluvastatin. The effect of antioxidative stress of fluvastatin might be involved in the mechanism.

Animals ; Antioxidants ; pharmacology ; Fatty Acids ; pharmacology ; Fatty Acids, Monounsaturated ; pharmacology ; Indoles ; pharmacology ; Male ; Myocardial Infarction ; metabolism ; pathology ; physiopathology ; Rats ; Ventricular Remodeling ; drug effects

Genkwa Fols, Kansui Radix, and Euphorbiae Pekinensis Radix in Shizao Decoction(SZD) are toxic to intestinal tract. Jujubae Fructus in this prescription can alleviate the toxicity, but the mechanism is still unclear. Therefore, this study aims to explore the mechanism. To be specific, 40 normal Sprague-Dawley(SD) rats were classified into the normal group, high-dose and low-dose SZD groups, and high-dose and low-dose SZD without Jujubae Fructus(SZD-JF) groups. The SZD groups were given(ig) SZD, while SZD-JF groups received the decoction without Jujubae Fructus. The variation of body weight and spleen index were recorded. The patho-logical changes of intestinal tissue were observed based on hematoxylin and eosin(HE) staining. The content of malondialdehyde(MDA) and glutathione(GSH) and activity of superoxide dismutase(SOD) in intestinal tissue were measured to evaluate the intestinal injury. Fresh feces of rats were collected to detect intestinal flora structure by 16S ribosomal RNA gene(16S rDNA) sequencing technology. The content of fecal short chain fatty acids and fecal metabolites was determined by gas chromatography-mass spectrometer(GC-MS) and liquid chromatography-mass spectrometer ultra-fast liquid chromatography-quadrupole-time-of-flight mass spectrometer(UFLC-Q-TOF-MS), separately. Spearman's correlation analysis was employed to analyze the differential bacteria genera and differential metabolites. RESULTS:: showed that high-dose and low-dose SZD-JF groups had high content of MDA in intestinal tissue, low GSH content and SOD activity, short intestinal villi(P<0.05), low diversity and abundance of intestinal flora, variation in the intestinal flora structure, and low content of short chain fatty acids(P<0.05) compared with the normal group. Compared with high-dose and low-dose SZD-JF groups, high-dose and low-dose SZD groups displayed low content of MDA in intestinal tissue, high GSH content and SOD activity, recovery of the length of intestinal villi, increased abundance and diversity of intestinal flora, alleviation of dysbacteria, and recovery of the content of short chain fatty acids(P<0.05). According to the variation of intestinal flora and fecal metabolites after the addition of Jujubae Fructus, 6 differential bacterial genera(Lactobacillus, Butyricimonas, Clostridia＿UCG-014, Prevotella, Escherichia-Shigella, Alistipes),4 differential short chain fatty acids(such as acetic acid, propionic acid, butyric acid, valeric acid) and 18 differential metabolites(such as urolithin A, lithocholic acid, and creatinine) were screened out. Beneficial bacteria such as Lactobacillus were in positive correlation with butyric acid and urolithin A(P<0.05). The pathogenic bacteria such as Escherichia-Shigella were in negative correlation with propionic acid and urolithin A(P<0.05). In summary, SZD-JF caused obvious intestinal injury to normal rats, which could lead to intestinal flora disorder. The addition of Jujubae Fructus can alleviate the disorder and relieve the injury by regulating intestinal flora and the metabolites. This study discusses the effect of Jujubae Fructus in relieving the intestinal injury caused by SZD and the mechanism from the perspective of intestinal flora-host metabolism, which is expected to serve as a reference for clinical application of this prescription.
Rats ; Animals ; Rats, Sprague-Dawley ; Propionates/pharmacology* ; Gastrointestinal Microbiome ; Fatty Acids, Volatile/pharmacology* ; Butyrates/pharmacology*

OBJECTIVETo establish an in vitro cell model to investigate hepatic steatosis of non-alcoholic fatty liver disease.

METHODSHepG2 cells cultured in MEM containing 10 % fetal bovine serum were divided into control group and model group. At 7 0%-80 % confluency, HepG2 cells in model group were exposed to a long-chain mixture of free fatty acids (oleate and palmitate) for 24 h, cells in control group were subject to fresh medium. Lipid droplets were observed with oil red O stain and electron microscope, triglyceride and malonaldehyde were detected by respective assay kits.

RESULTA large number of lipid droplet were detected in model HepG2 cells; the level of triglyceride increased. However,malonaldehyde did not increase significantly compared with control group.

CONCLUSIONA large number of lipid droplet were detected in model HepG2 cells; the level of triglyceride increased. However, malonaldehyde did not increase significantly compared with control group.

Culture Media ; pharmacology ; Fatty Acids, Nonesterified ; pharmacology ; Fatty Liver ; Hep G2 Cells ; Humans ; Models, Biological ; Non-alcoholic Fatty Liver Disease ; Oleic Acid ; pharmacology ; Palmitates ; pharmacology

AIMTo examine the liver mechanism with which clenbuterol (CL) is explained how to affect growth metabolism.

METHODSThe technique of chronic poly catheter was used to study the effects of CL (0.8 mg/kg b w) on the hepatic flux of nitrogen, VFA and glucose in 4 sheep.

RESULTSThe urea-nitrogen flux in CL-treated period always was lower than that in control during 24 h. The average flux of urea-nitrogen in hepatic and portal vein were decreased by 16.86% (P < 0.01) and 15.51% (P < 0.05), respectively, compared with that of control. The peptide level in hepatic vein was decreased with the treatment of CL, average flux of peptide was decreased by 38.71% (P < 0.01). But the peptide level of portal vein in CL treatment period was similar to control. Moreover, VFA level in the portal vein was enhanced by CL, the average flux of acetate in portal vein was increased by 19.49% (P < 0.01). No difference of VFA level in hepatic vein was noted between CL-treated period and control. In addition, the glucose flux in hepatic vein was obviously increased with CL treatment, the average flux of glucose was increased by 25.96% (P < 0.01). And glucose flux in portal vein was also elevated during CL-treated period.

CONCLUSIONCL can affect growth metabolism of animal with increasing nitrogen deposition, improving absorption and utilization of VFA and enhancing glucose synthesis in sheep liver.

Animals ; Clenbuterol ; pharmacology ; Fatty Acids, Volatile ; metabolism ; Glucose ; metabolism ; Liver ; drug effects ; metabolism ; Sheep

OBJECTIVETo establish an in vitro cell model for investigating hepatic steatosis in non-alcoholic fatty liver disease.

METHODSL-02 cells cultured in 1640 containing 10% fetal bovine serum were divided into control group and model group. At 70%-80% confluency, L-02 cells in the model group were exposed to a long-chain mixture of free fatty acids (FFA, oleate and palmitate ) for 24 h, and cells in control group were treated with fresh medium. Lipid droplets in the cells were observed and total lipid content was determined with Oil Red O staining. The morphology of lipid droplets, trilyceride level, malonaldehyde content and cell apoptosis rate were evaluated to verify the cell model, and the effect of Huganqingzhi tablet on the lipid droplets was observed.

RESULTSA large number of lipid droplets were found in the cell model, which showed markedly increased level of triglyceride without significant changes of malonadehyde content or cell apoptosis rate. Intervention with two doses of Huganqingzhi tablet significantly decreased the number of lipid droplets and trilyceride content in the cell model.

CONCLUSIONhepatic steatosis L-02 cell model can be established by long-chain mixture of free fatty acids (oleate:spalmitate=2:1) for therapeutic drug studies.

Apoptosis ; Cell Line ; Drugs, Chinese Herbal ; pharmacology ; Fatty Acids, Nonesterified ; chemistry ; Fatty Liver ; Humans ; Malondialdehyde ; analysis ; Non-alcoholic Fatty Liver Disease ; Triglycerides ; analysis

Investigation in our laboratory has been undertaken to study the effect of ethanol on the triglyceride (TG) content in the liver, the free fatty acid (FFA) content in the serum and the glycogen in the liver of rats which were fed on various diets. Four hours after administration of a sing1e dose of glucose (5g/kg BW.) and ethanol (6g/kg BW.) by gavage tube to rats fed a norma1 diet for 20 days then fasted for 18 hours, TG content in the liver increased by 80%, 10% compared to the control. When a sing1e dose of equal amounts of both glucose and ethanol were administered to another group, TG content in the liver was 42% higher than the control. There was no great change in serum FFA content in the glucose treated group as compared with the control, however, there was an increment of serum FFA content in the ethanol treated group and in the group treated with both ethanol and glucose by 81% and 71% of the control, respectively. The results indicate that ethanol administration had an inhibitory effect on the TG accumulation in the liver of rats fed by glucose. There is a correlation between TG accumulation in the liver and FFA content in the serum, and it appears that the ethanol administration did not induce the TG accumulation in the liver but the increment of serum FFA content in rats is probably due to the increased fatty acid mobilization from adipose tissue. However, countercurrent results were observed in the glucose treated group as compared with the ethanol treated group suggesting that glucose administration does induce TG accumulation in the liver but does not increase the serum FFA content in rats. The increment of serum FFA content in rats. The increment of serum FFA content by ethanol treatment was not ameliorated by glucose administration. In the liver perfusion experiment with rats fed both ethanol and various other diets, the results of incorporation of ethanol-1-14C into the total lipid in the high carbohydrate, high fat, low carbohydrate and control diet group were 1925 +/- 257 (cpm/g liver), 1237 +/- 76, 1269 +/- 105, 2041 +/- 74, respective1y. The results indicate that amount of dietary carbohydrate and high fat had an effect on the total lipid accumulation derived from ethanol-1-14C molecule in the liver. Liver glycogen content in the control on rats, high fat, 1ow carbohydrate and high carbohydrate diets were 91.5 +/- 7.9(mg%), 93.0 +/- 1.8, 99.1 +/- 4.4, and 153.7 +/- 26.0, respectively. There were no great differences between each dietary group and the rest control group except in the case of the high carbohydrate group which was over 1.5 times greater than that of the control. The incorporation of labelled ethanol into liver glycogen in the control rats and those on high fatdiet, low carbohydrate diet and high carbohydrate diet were 525, 401, 351 and 806 cpm/g liver, respectively. The increased incorporation of ethanol-1-14C into liver glycogen in the high carbohydrate diet group is thought to be due to the increased gluconeogenesis from acetyl CoA derived from 14C from ethanol because rats were fasted for 18 hours before perfusion. It might be the result of increased gluconegenesis of acetyl CoA derived from ethanol-1-14C by spare action of high carbohydrate on acetyl CoA. During the liver perfusion, 14CO2 production from ethanol-1-14C was higher in the high fat diet and low carbohydrate diet groups than in the control group, however, no great difference was observed between the high carbohydrate and control groups. The higher production of 14CO2 from the single ethanol-1-14C dose in rats on the high fat diet and low carbohydrate diet groups than in the control group is probably due to the increased metabolism of ethanol through Kreb's cycle rather than the incorporation of it into the liver fat.
Animal ; Diet ; Ethanol/metabolism ; Ethanol/pharmacology* ; Fatty Acids, Nonesterified/blood ; Glucose/pharmacology ; In Vitro ; Liver/metabolism* ; Male ; Rats ; Triglycerides/metabolism*

OBJECTIVESTo investigate the effects of glucose and free fatty acids (FFAs) on the proliferation and cell cycle of human vascular endothelial cells in vitro, and to examine whether the combined presence of elevated FFAs and glucose may cross-amplify their individual injurious effects.

METHODSCultured human vascular endothelial cells (ECV304) were incubated with various concentrations of glucose and/or FFAs (palmitate and/or oleate) for 24 - 96 h. Morphologic alterations were observed using a phase contrast microscope and an electron microscope. Inhibition of proliferation was measured by a colorimetric 3-[4, 5-dimethyl thiazol-2-yl]-2, 5-diphenyltetrazolium bromide (MTT) assay. Cell viability was determined using trypan blue exclusion. Distribution of cells along phases of the cell cycle was analyzed by flow cytometry.

RESULTSGlucose 15 or 30 mmol/L, palmitate (PA) 0.25 or 0.5 mmol/L, and oleate (OA) 0.5 mmol/L inhibited proliferation and accelerated death of endothelial cells in a dose-and-time-dependent manner. After treatment with elevated glucose and/or FFAs, the G(0)/G(1) phase cells increased, whereas S phase cells decreased, suggesting that high glucose and/or FFAs mainly arrested endothelial cells at G(0)/G(1) phase. The inhibitive rates of proliferation and population of dead cells in endothelial cells incubated with glucose plus FFAs (glucose 30 mmol/L + PA 0.25 mmol/L, glucose 30 mmol/L + OA 0.5 mmol/L, glucose 30 mmol/L + PA 0.25 mmol/L + OA 0.5 mmol/L) increased more markedly than those treated with high glucose or FFAs (PA and/or OA) alone.

CONCLUSIONBoth high ambient glucose and FFAs can inhibit proliferation and accelerate death of endothelial cells in vitro. These changes were cross-amplified in the combined presence of high levels of glucose and FFAs.

Cell Division ; drug effects ; Cell Survival ; drug effects ; Cells, Cultured ; Endothelium, Vascular ; cytology ; Fatty Acids, Nonesterified ; pharmacology ; Glucose ; pharmacology ; Humans