In order to understand the structural characteristics of squalene synthase genes in the triterpenoids biosynthesis pathway of Crataegus pinnatifida, the squalene synthase genes of C. pinnatifida was cloned and analyzed by bioinformatics and prokaryotic expression. Two squalene synthase genes CpSQS1 and CpSQS2 were cloned from C. pinnatifida fruit by RT-PCR. The ORF length of CpSQS1 and CpSQS2 were 1 239 bp and 1 233 bp respectively, encoding 412 aa and 410 aa respectively. CpSQS1 and CpSQS2 were predicted to be stable acidic proteins by online tools. The secondary structure was mainly composed of α-helix structure, and the tertiary structure was predicted by homology modeling. Structural functional domain analysis showed that 35-367 aa of CpSQS1 and CpSQS2 cDNA containing conserved trans-isoprenyl pyrophosphate synthase domains. Transmembrane domain analysis predicted that two transmembrane domains were founded in CpSQS1 and CpSQS2. The squalene synthase amino sequence of C. pinnatifida had higher homology with the known SQS of Salvia miltiorrhiza and Glycyrrhiza glabra. Phylogenetic tree analysis showed that CpSQS1 and CpSQS2 were clustered into one branch of MdSQS1 and MdSQS2, which were consistent with the phylogenetic rule. Prokaryotic expression vector pGEX-4 T-1-CpSQS1 and pGEX-4 T-1-CpSQS2 were transformed into Escherichia coli Transetta(DE3) for induction, and the target protein was successfully expressed at 65 kDa. The expression levels of CpSQS2 were significantly higher than that of CpSQS1 in three different developmental stages of C. pinnatifida. In this study, the full-length cDNA sequences of C. pinnatifida SQS1 and SQS2 were cloned and analyzed for the first time, which provided the foundation for further study on the metabolic pathway of C. pinnatifida triterpenoids.
Amino Acid Sequence ; Cloning, Molecular ; Crataegus/genetics* ; Farnesyl-Diphosphate Farnesyltransferase/genetics* ; Fruit/enzymology* ; Phylogeny ; Plant Proteins/genetics*

OBJECTIVEIn order to investigate the possible mechanism of its function to degrade lipid, we detect the effects of hawthorn flavanone to the influence on blood-fat levels and adipogenesis genes transcription expression in fat and muscle tissue of hyperlipoidemia mouse.

METHODIn this experiment, a total of 48 mouse were randomised to four groups and irrigated with two different concentrations (1.5 g kg(-1) body weight and 3.0 g kg(-1) body weight) of hawthorn flavanone, and killed in 0 h, 1 h, 2 h and 4 h. To estimate the content of TC, TG and HCL-C in blood: Total RNA was isolated from adipose and muscle, Real-time RT-PCR was used to analyze expression changes of adipogenesis genes (SREBP-1c, FAS, HSL and TGH) with time series; to analyze the correlation between TG in blood and some kinds of adipogenesis genes and the ratio of FAS/HARMEAN (HSL, TGH) mRNA in adipose.

RESULTHawthorn flavanone was able to cut down the level ofTC, TG and HDL significantly in blood and achieved the lowest level at 1 h. In adipose tissue, hawthorn flavanone up-regulated FAS, HSL and TGH, and achieved the level of significance (P<0.05), the expression level of FAS and TGH was ascend after 1 h, but HSL descend. The expression level of SREBP-1c was descend rapidly and achieved the level of significance after treating with hawthorn flavanone at 1 h (P<0.05), after that it rise again to even higher than the level of before treatment. After treating with hawthorn flavanone, the ratio of FAS/HARMEAN (HSL, TGH) in adipose was significantly descend and achieved the lowest level at 1 h (P<0.01), but it was descendsubsequently. In muscle tissue, hawthorn flavanone was able to significantly up-regulated the expression of FAS and HSL and lower dose group showed greater increasing, the change of SREBP-1c was similar in adipose tissue except the more heavily upgrade.

CONCLUSIONHawthorn flavanone had the function of depressing the concentration of blood-fat, it co-adjusted lipid metabolism of animal by regulating the transcription expression of FAS, HSL, TGH and SREBP-1c especially HSL and SREBP-1c transcription level.

Adipose Tissue ; drug effects ; metabolism ; Animals ; Crataegus ; chemistry ; Flavanones ; pharmacology ; Gene Expression Regulation ; drug effects ; Hyperlipidemias ; blood ; genetics ; Lipids ; blood ; Lipogenesis ; drug effects ; Lipolysis ; drug effects ; genetics ; Male ; Mice ; RNA, Messenger ; genetics ; metabolism ; Sterol Regulatory Element Binding Protein 1 ; genetics ; Triglycerides ; blood ; Up-Regulation ; drug effects ; fas Receptor ; genetics

OBJECTIVETo observe the influence of vitexia-rhamnoside (V-R) on vasomotor factor expression of endothelial cell (EC) damaged by hypoxia and reoxygenation.

METHODThe cultured human umbilical vein endothelial cells (HUV ECs) were subject to ischemia and reperfusion following hypoxia and reoxygenation. The levels of ET-1, NO and NOS intracellular in culture supertanants were measured by radioimmunity, Griess and immunohistochemistry, respectively. And the gene expressions of ET-1 and NOS intracellular were measured by reverse transcriptase-polymerase chain reaction.

RESULTV-R at different doses markedly increased the gene expression and activity of NOS, enhanced the level of vaso-dilating factor NO, and significantly decreased the gene expression and production of vaso-constricting factor ET-1 of EC.

CONCLUSIONWe have demonstrated that V-R had the regulatory effect on the expression of vaso-active substances of EC damaged by hypoxia and reoxygenation in the levels of protein and gene transcription of cytokines.

Apigenin ; isolation & purification ; pharmacology ; Cell Hypoxia ; Cells, Cultured ; Crataegus ; chemistry ; Endothelial Cells ; metabolism ; Endothelin-1 ; biosynthesis ; genetics ; Gene Expression ; Humans ; Nitric Oxide ; biosynthesis ; genetics ; Nitric Oxide Synthase ; biosynthesis ; genetics ; Plant Leaves ; chemistry ; Plants, Medicinal ; chemistry ; RNA, Messenger ; biosynthesis ; genetics ; Umbilical Veins ; cytology

OBJECTIVETo investigate the effect of total flavone of haw leaves (TFHL) on the expression of nuclear factor erythroid-2 related factor (Nrf2) and other related factors in nonalcoholic steatohepatitis (NASH) rats induced by high-fat diet and then to further discuss the mechanism of TFHL's prevention against NASH.

METHODSHigh-fat diet was fed to 40 rats to establish the NASH model. Then model rats were intragastrically administrated with 40, 80, 160 mg/(kg•day) TFHL, respectively. The pathological changes of liver tissues in NASH rats were detected by oil red O and hematoxylin-eosin (HE) stainings. The expression of Nrf2 in rat liver was examined through immunohistochemistry. The level of 8-iso-prostaglandin F2α in serum was detected through enzyme linked immunosorbent assay (ELISA). The mRNA and protein levels of Nrf2 and other related factors in liver tissue were measured by real-time reverse transcriptionpolymerase chain reaction and western blot.

RESULTSLipid deposition, hepatic steatosis, focal necrosis in lobular inflammation and ballooning degeneration were emerged in livers of NASH rats. The 8-iso-prostaglandin F2α in the serum of NASH rats increased significantly compared with the control group (P<0.05). The mRNA of Nrf2, hemeoxyenase1 (HO-1) and the mRNA and protein levels of quinine oxidoreductase (NQO1) in NASH rats liver tissue showed a striking increase, while the mRNA levels of Keap1, r-glutamylcysteine synthethase (rGCS) and glutathione S-transferase (GST) were significantly decreased compared with the control group (P<0.05). After TFHL treatment, 8-iso-prostaglandin F2α level in serum significantly decreased, and Nrf2 mRNA and protein levels in hepatocytes nucleus enhanced compared with the model group (P<0.05 or 0.01). Meanwhile the Keap1 mRNA, the mRNA and protein levels of HO-1, NQO1 antibody, rGCS antibody, GST increased after TFHL treatment (P<0.05 or 0.01).

CONCLUSIONSNrf2 and other related factors were involved in development of NASH, and they also served as an important part in its occurrence. By regulating expression of Nrf2 and other related factors, TFHL may play a role in antioxidative stress and prevention of NASH.

Animals ; Cell Nucleus ; drug effects ; metabolism ; Crataegus ; chemistry ; Dinoprost ; metabolism ; Flavones ; pharmacology ; therapeutic use ; Lipids ; chemistry ; Liver ; drug effects ; metabolism ; pathology ; NF-E2-Related Factor 2 ; genetics ; metabolism ; Non-alcoholic Fatty Liver Disease ; drug therapy ; genetics ; pathology ; Phytotherapy ; Plant Leaves ; chemistry ; RNA, Messenger ; genetics ; metabolism ; Rats, Sprague-Dawley

OBJECTIVETo test whether 7 herbs stimulate human pregnane X receptor (PXR)-mediated CYP3A4 transcription.

METHODTransient cotransfection reporter gene assays were performed with human PXR expression plasmids and a reporter plasmid containing the XRES in the CYP3A4 gene promoter in HepG2 cells.

RESULTThe aqueous extracts of Chrysanthemi Flos, Lycii Fructus, and Salviae Miltiorrhizae Radix et Rhizoma, and the methanol extracts of Chrysanthemi Flos, Crataegi Fructus, Lycii Fructus, Lonicerae Japonicae Flos, Dioscoreae Rhizoma,and Salviae Miltiorrhizae Radix et Rhizoma, activated human PXR-mediated transcription.

CONCLUSIONThe aqueous extracts of Chrysanthemi Flos, Lycii Fructus, and Salviae Miltiorrhizae Radix et Rhizoma, and the methanol extracts of Chrysanthemi Flos, Crataegi Fructus, Lycii Fructus, Lonicerae Japonicae Flos, Dioscoreae Rhizoma, and Salviae Miltiorrhizae Radix et Rhizoma are inducers of CYP3A4 by activating PXR, and thus may influence the metabolism of other substrates on CYP3A4.

Cell Line ; Chrysanthemum ; Crataegus ; Cytochrome P-450 CYP3A ; drug effects ; genetics ; metabolism ; Dioscorea ; Drugs, Chinese Herbal ; pharmacology ; Gene Transfer Techniques ; Genes, Reporter ; Hep G2 Cells ; Humans ; Lonicera ; Lycium ; Medicine, Chinese Traditional ; Plant Extracts ; pharmacology ; Receptors, Steroid ; drug effects ; genetics ; metabolism ; Salvia miltiorrhiza

OBJECTIVETo study the expression of uncoupling protein 2 (UCP2) in liver of rats with nonalcoholic steatohepatitis (NASH) induced by fat-rich diet, and the effect of total flavones of hawthorn leafon (TFHL) on UCP2.

METHODThe NASH model of rat was induced by 12 weeks of fat-rich diet. Subsequently the rats were administrated with TFHL in accordance with 250, 125 mg x kg(-1) x d(-1) and the Essentiale N with 195.4 mg x kg(-1) x d(-1). The change of liver pathological. The levels of serum ALT and AST, the content of TG, CHOL, MDA and T-AOC activity of liver and were evaluated. The UCP2mRNA expression in liver was detected with RT-PCR, and the contents of UCP2 were examined with ELISA.

RESULTThere are severe steatosis, inflammatory cellular infiltration in the liver of the NASH models. The levels of serum ALT, AST and the contents of TG, CHOL, MDA and UCP2 in the model group were higher than those of in the normal groop. The expression of UCP2mRNA was obviously enhanced and the activity of T-AOC decreased. The expression of UCP2 mRNA of rats was positively correlation with the contents of MDA, TNF-alpha. The inflammation activity in rat liver, the contents of MDA and UCP2, the expression of UCP2 mRNA in the administrated groups were obviously lower than those in the model group, while the activity of T-AOC was higher than that of model.

CONCLUSIONTFHL may alleviate liver injury by means of the suppression of Oxidative stress/lipid peroxidation reaction and the overexpression of UCP2 in liver, which could prevent the further development of NASH.

Animals ; Crataegus ; chemistry ; Enzyme-Linked Immunosorbent Assay ; Fatty Liver ; drug therapy ; metabolism ; Flavones ; chemistry ; therapeutic use ; Gene Expression ; drug effects ; Ion Channels ; genetics ; Liver ; drug effects ; metabolism ; Male ; Mitochondrial Proteins ; genetics ; Plant Leaves ; chemistry ; Polymerase Chain Reaction ; Rats ; Rats, Sprague-Dawley ; Uncoupling Protein 2

OBJECTIVE: To determine the effects of hawthorn extract on serum lipid levels, pathological changes in aortic atherosclerosis plaque, inflammatory factors, and apoptosis-related protein and mRNA expression in apolipoprotein E gene knockout (ApoE) mice. METHODS: Thirty-six ApoE mice were fed with a high-fat diet starting at the age of 8 weeks. Mice were randomly divided into 3 groups by a random number table including model group, hawthorn extract group, and simvastatin group, 12 mice in each group. Twelve 8-week-old C57BL/6 mice were fed a basic diet and served as control. The mice in the control and model groups were administered 0.2 mL saline daily, the mice in the hawthorn extract and simvastatin groups were administered with 50 mg/kg hawthorn extract or 5 mg/kg simvastatin daily for 16 weeks. After 16 weeks, plasma lipids including total cholesterol (TC), triglyceride (TG), low-density lipoprotein cholesterol (LDL-C) and high-density lipoprotein cholesterol (HDL-C) were determined by an enzymatic assay. Aortic atherosclerotic lesions were observed by light microscopy, scanning and transmission electron microscopy, respectively. Plasma levels of monocyte chemoattractant protein-1 (MCP-1), interleukin-1β (IL-1β), adiponectin (APN), and hypersensitive C-reactive protein (hs-CRP) were measured by enzyme-linked immunosorbent assay (ELISA). Protein and mRNA expressions of Bax and Bcl-2 in the aorta were assessed by Western blotting and quantitative real-time polymerase chain reaction (qRT-PCR), respectively. RESULTS: Compared to the control group, the plasma levels of TC, TG and LDL-C were significantly increased and HDL-C were significantly decreased in the model group (P<0.01). Compared to the model group, treatment with hawthorn extract significantly decreased the plasma levels of TC, TG, and LDL-C and increased the plasma level of HDL-C in ApoE mice (P<0.01). The levels of MCP-1, IL-1ß, and hs-CRP in the model group were significantly increased and APN was significantly decreased compared with the control group (P<0.01). Compared to the model group, treatment with hawthorn extract decreased the levels of MCP-1, IL-1ß, and hs-CRP and increased the APN level (P<0.01). Compared to the control group, the protein and mRNA expression of Bax in the model group were significantly increased and the expression of Bcl-2 was significantly decreased (P<0.01). Hawthorn extract also reduced the protein and mRNA expression of Bax and increased the Bcl-2 expression in the aorta (P<0.01). CONCLUSION Hawthorn extract has anti-atherosclerosis and stabilizing unstable plaque effects. The mechanism may be related to the inflflammation and apoptosis signaling pathways.
Animals ; Aorta ; pathology ; ultrastructure ; Apoptosis ; drug effects ; Atherosclerosis ; blood ; complications ; drug therapy ; Crataegus ; chemistry ; Inflammation ; blood ; complications ; drug therapy ; Inflammation Mediators ; metabolism ; Lipids ; blood ; Male ; Mice, Inbred C57BL ; Plant Extracts ; pharmacology ; therapeutic use ; RNA, Messenger ; genetics ; metabolism ; bcl-2-Associated X Protein ; metabolism