Anthocyanidin reductase (ANR) is one of the key enzyme in the flavonoid biosynthetic pathway, and its catalytic activity is important for the synthesis of plant anthocyanin. In this study, specific primers were designed according to the transcriptome data of Lonicera japonica Thunb., and the CDS, gDNA and promoter sequences of ANR genes from Lonicera japonica Thunb. and Lonicera japonica Thunb. var. chinensis (Wats.) Bak. were cloned. The results showed that the CDS sequences of LjANR and rLjANR were 1 002 bp, the gDNA sequences were 2 017 and 2 026 bp respectively, and the promoter sequences were 1 170 and 1 164 bp respectively. LjANR and rLjANR both contain 6 exons and 5 introns, which have the same length of exons and large differences in introns. The promoter sequences both contain a large number of light response, hormone response and abiotic stress response elements. Bioinformatics analysis showed that both LjANR and rLjANR encoded 333 amino acids and were predicted to be stable hydrophobic proteins without transmembrane segments and signal peptides. The secondary structures of LjANR and rLjANR were predicted to be mainly consisted of α-helix and random coil. Sequence alignment and phylogenetic analysis showed that LjANR and rLjANR had high homology with Actinidia chinensis var. chinensis, Camellia sinensis and Camellia oleifera, and were closely related to them. The expression levels of LjANR and rLjANR were the highest in flower buds and the lowest in roots. The expression patterns at different flowering stages were similar, with higher expression levels in S1 and S2 stages and then gradually decreased until reaching the lowest level in S4 stage, after a slow increase in S5 stage, the expression levels decreased again. The expression levels of ANR genes in the two varieties showed significant differences in roots, S2 and S5 stages, while the differences in stems, flower buds, S1, S3 and S6 stages were extremely significant. The prokaryotic expression vector pET-32a-LjANR was constructed for protein expression. The target protein was successfully expressed of about 59 kD. This study lays a foundation for further study on the function of ANR gene and provides theoretical guidance for breeding new varieties of Lonicera japonica Thunb.

This study was to investigate the hypoglycemic effect of wogonoside to improve hepatic insulin resistance( IR) and its relative anti-inflammatory mechanism. The stable IR-Hep G2 cell model was established by the combination of 1×10-9 mol·L^-1 insulin and 3. 75×10-6 mol·L^-1 dexamethasone for 48 hours. The changes of glucose consumption in IR-Hep G2 cells with different concentrations of wogonoside( 1,5,10,20,50 μmol·L^-1) at different time points( 30,36,48,54 h) were detected by glucose oxidase assay to determine the optimal onset time. Glycogen content and cell viability were respectively detected by ketone method and CCK-8 method. Cryptothermal protein 3( NLRP3),suppressor of cytokine signaling 3( SOCS3),Toll-like receptor 4( TLR4),nuclear factor( NF-κB),interleukin( IL^-1β),IL-6,tumor necrosis factor( TNF-α) involving in the inflammatory signaling pathway,as well as leptin,Ob-R,p-IRS2/IRS2,p-PI3 K/PI3 K( p85),p-Akt/Akt and glucose transporter( GLUT1/2/4) involving in the insulin signaling pathway were detected in IR-HepG2 cells by Western blot. RESULTS: showed that 20 and 50 μmol·L^-1 wogonoside significantly up-regulated the glucose consumption of IR-HepG2 cells( P<0. 001) as compared with IR model group,and the optimal onset time was 48 h.Wogonoside had no obvious effect on the cell viability of Hep G2 cells. Further studies showed that 20,50 μmol·L^-1 wogonoside respectively increased the glycogen content of IR-HepG2 cells after 48 h treatment,especially in 50 μmol·L^-1 group( P<0. 001). Compared with IR model group,wogonoside not only inhibited the protein expression of inflammatory nuclear transcriptional factors NLRP3,SOCS3,TLR4,NF-κB,but also decreased the expression of downstream inflammatory effect factors IL^-1β,IL-6 and TNF-α． In addition,wogonoside elevated Ob-R,p-IRS2/IRS2,p-PI3 K/PI3 K( p85),p-Akt/Akt and GLUT1/2/4 protein expression,whereas it suppressed leptin expression that was regulated by SOCS3. Wogonoside could promote glucose uptake and increase glycogen content to enhance insulin sensitivity in IR-Hep G2 cells. The hypoglycemic effect may be related to the intervention of NLRP3/SOCS3-TLR4-NF-κB inflammatory pathway and decrease of inflammatory factor expression.
Flavanones ; Glucosides ; Humans ; Insulin Resistance ; NF-kappa B ; NLR Family, Pyrin Domain-Containing 3 Protein ; Suppressor of Cytokine Signaling 3 Protein ; Toll-Like Receptor 4 ; Tumor Necrosis Factor-alpha

To investigate the hypoglycemic effects of baicalin, berberine, puerarin and liquiritin on the insulin resistance (IR) cells. The IR model of HepG2 cells was established by treatment with insulin and dexamethasone for 48 h. Glucose uptake, glycogen content and cell viability were detected with different concentrations of baicalin, berberine, puerarin, liquiritin in IR-HepG2 cells. Compared with IR model group, all of intervened groups significantly increased the glucose consumption, except for liquiritin groups and 1 μmol·L⁻¹ baicalin group. Moreover, 10, 20, 50 μmol·L⁻¹ baicalin, 5, 10, 20, 50 μmol·L⁻¹ berberine and 40, 80, 160 μmol·L⁻¹ puerarin significantly elevated glycogen content in IR-HepG2 cells. Liquiritin did not show obvious hypoglycemic effect. Compared with normal group, the mRNA expression levels of GLUT1 and GLUT4 were decreased in IR-HepG2 cells according to qPCR results. 5, 20 μmol·L⁻¹ berberine decreased the mRNA expression level of GLUT1 in IR-HepG2 cells, whereas 20, 40, 80 μmol·L⁻¹ puerarin significantly elevated the mRNA expression level of GLUT1. Moreover, 10, 20, 50 μmol·L⁻¹ baicalin and 20 μmol·L⁻¹ berberine increased the mRNA expression level of GLUT4. Whereas, 40, 80 μmol·L⁻¹ puerarin decreased the mRNA expression level of GLUT4. Western blot results suggested that 10, 20, 50 μmol·L⁻¹ baicalin significantly increased the protein expressions of GLUT2 and GLUT4, whereas 20, 40, 80 μmol·L⁻¹ puerarin significantly up-regulated GLUT1 and GLUT2 proteins. In addition, 20 μmol·L⁻¹ berberine increased the protein expressions of GLUT2 and GLUT4, whereas 10 μmol·L⁻¹ berberine up-regulated GLUT4 expression. The results preliminarily suggested that baicalin, berberine and puerarin have differentiated hypoglycemic effects, which accelerate glucose transport, increase glycogen synthesis, regulate glucose metabolism and improve hepatic IR.
Berberine ; pharmacology ; Flavonoids ; pharmacology ; Glucose ; Hep G2 Cells ; Humans ; Hypoglycemic Agents ; pharmacology ; Insulin ; Insulin Resistance ; Isoflavones ; pharmacology

This paper aimed to investigate the hypoglycemic effect and relative mechanism of jatrorrhizine in insulin-resistance (IR)-3T3-L1 adipocytes. The 3T3-L1 preadipocytes were used to induce mature adipocytes, then the stable IR model was established with 1 μmol·L⁻¹ dexamethasone. The adipocytes were divided into normal group, IR model group, rosiglitazone positive group and jatrorrhizine group (0.5, 1, 5, 10, 20 μmol·L⁻¹). After different time points (12, 24, 30, 36, 48 h) treatment, glucose content of 3T3-L1 adipocytes was detected by the glucose oxidase peroxidase method and TG content was measured by glycerol phosphate oxidase method, whereas cell viability was detected by CCK-8 assay. Furthermore, the protein expression levels of insulin receptor substrate 2 (IRS2), phosphinositide-3-kinase regulatory subunit 1(PI3KR1), phosphorylated protein B [p-AKT (Ser473)], phosph-AMP-activated protein [p-AMPK (Thr172)], and glucose transporter type 4/1/2 (GLUT4/1/2) were detected by Western blot assay. The results showed that as compared with the normal group, the glucose consumptionwas significantly decreased in IR model group(<0.01); whereas 0.5, 1, 5, 10, 20 μmol·L⁻¹ jatrorrhizine and rosiglitazone group elevated IR-3T3-L1 cells glucose consumption (<0.01) at 36 h and 48 h administration as compared with IR group. The optimal administration time was 48 h for jatrorrhizine. 1, 5, 10, 20 μmol·L⁻¹ of jatrorrhizine decreased the TG content in 3T3-L1 adipocytes for 48 h administration (<0.05). The protein expression levels of IRS2, PI3KR1, p-AKT (Ser473), p-AMPK (Thr172), GLUT4/1/2 were significantly up-regulated by different concentrations of jatrorrhizine and rosiglitazone (<0.01). The results showed that jatrorrhizine increased glucose uptake with elevated glucose consumption, whereas reduced intracellular TG content in IR-3T3-L1 adipocytes. Moreover, it intervened classic insulin signal pathway IRS2/PI3KR1/p-AKT/GLUT4 and increase AMPK protein phosphorylation level for the activation of GLUT1/4 for insulin sensibility. Thus, jatrorrhizine could effectively regulate the GLUTs with multiple manners for hypoglycemic effect.

To investigate the effects of Gegen Qinlian decoction(GQD) in improving adipocytic insulin resistance(IR) and explore its related molecular mechanism. Diabetic rats models were induced by high glucose and high-fat diet with a small dose of streptozotocin, and after GQD treatment for 3 months, blood biochemical indexes such as fasting blood-glucose(FBG), insulin, glycosylated serum protein(GSP) and HOMA-IRI were detected and assessed. After the total RNA was extracted from the adipose tissue of diabetic SD rats, PPARγ, ADPN, GLUT4, GLUT2, ACACA and ACACB mRNA expression levels were separately detected by qPCR. Then, stable IR-3T3-L1 adipocyte model was built with 1 μmol•L⁻¹ dexamethasone. After the cell viability was detected by CCK-8 assay, 5%, 10% and 15% GQD-containing serum(GQD-CS) were respectively used to treat IR-3T-L1 adipocytes for 24 h. The contents of glucose, nonesterified fatty acid(NEFA) and adiponectin in cell culture supernatants were separately detected whereas the intracellular triglyceride(TG) contents of IR-3T3-L1 adipocytes were also measured. The ADPN, PPARγ and GLUT4 mRNA and protein expression levels were respectively detected by qPCR and Western blot in IR-3T3-L1 adipocytes. Results showed that GQD significantly decreased fasting blood glucose, insulin and GSP(P<0.01), and down-regulated HOMA-IRI(P<0.05) after the high-fat diet/streptozotocin-induced diabetic SD rats were treated for three months, with a good hypoglycemic effect. Moreover, PPARγ, ADPN, GLUT4, GLUT2, ACACA and ACACB mRNA expression levels were significantly elevated in the adipose tissue of GQD-treated diabetic SD rats. The 5%, 10% and 15% GQD-CS significantly increased glucose consumption of IR-3T3-L1 adipocytes at 24 h treatment(P<0.01), significantly decreased the intracellular TG content (P<0.01), and down-regulated NEFA to a certain extent but not significantly. Moreover, GQD-CS significantly up-regulated GLUT4 and ADPN expression. The results indicated that GQD could activate PPARγ to ameliorate adipocytic insulin resistance in the diabetic SD rats and IR-3T3-L1 adipocytes.

Objective To investigate the anti-hyperglycemic effect of the Chinese medicine ingredients such as berberine,baicalin,puerarin and liquiritin on the optimal insulin resistance (IR)-HepG2 cell model by oleic acid.It would provide the theoretical basis for the optimization of Chinese medicine prescription or anti-hyperglycemic components combination.Methods Different concentrations (0.1,0.2,0.5,and 1.0 mmol/L) of oleic acid were used to induce HepG2 cells for different time (24,36 and 48 h),the glucose consumption was measured byglucose oxidase assay,and cell viability was detected by CCK-8 assay to define the optimal inducing concentration and time for IR-HepG2 cell model.Then the cell morphological changes were detected by oil red O staining.Finally,the stability of IR-HepG2 cell model was tested.After the IR-HepG2 model was optimally established,the glucose consumption,glycogen content and cell viability were detected after 24 h administration with different concentrations of berberine,baicalin,puerarin and liquiritin by anthrone method,glycerol phosphate oxidase assay and CCK-8 assay respectively.Results The optimal oleic acid-induced concentration was 1 mmol/L and the optimal induced time was 24 h for the IR-HepG2 cell model that could keep stable more than 36 h.Comparing with IR model group,berberine,puerarin and baicalin significantly increased the glucose consumption,whereas liquiritin did not show significant change in the glucose consumption except for 1 μmol/L.Only 160 μmol/L puerarin and 1 μmol/L baicalin significantly inhibited IR-HepG2 cell viability.Moreover,berberine,puerarin,and baicalin significantly elevated the glycogen content;Liquiritin did not change glycogen content significantly.Conclusion The IR-HepG2 cell model could be stably established with 24 h treatment of 1 mmol/L oleic acid.Berberine,puerarin,and baicalin significantly increased the glucose consumption and glycogen content in the IR-HepG2 cells.The results suggest that berberine,baicalin and puerarin maybe perform different pathways of anti-hyperglycemic effects due to different incentives ofIR.

OBJECTIVETo investigate the risk factors for the development of congenital anal atresia in neonates.

METHODSA total of 70 neonates who were admitted to 17 hospitals in Foshan, China from January 2011 to December 2014 were enrolled as case group, and another 70 neonates who were hospitalized during the same period and had no anal atresia or other severe deformities were enrolled as control group. Univariate and multivariate logistic regression analyses were used to investigate the risk factors for the development of congenital anal atresia.

RESULTSThe univariate analysis revealed that the age of mothers, presence of oral administration of folic acid, infection during early pregnancy, and polyhydramnios, and sex of neonates showed significant differences between the case and control groups (P<0.05). The multivariate logistic regression analysis revealed that infection during early pregnancy (OR=18.776) and male neonates (OR=9.304) were risk factors for congenital anal atresia, and oral administration of folic acid during early pregnancy was the protective factor (OR=0.086).

CONCLUSIONSInfection during early pregnancy is the risk factor for congenital anal atresia, and male neonates are more likely to develop congenital anal atresia than female neonates. Supplementation of folic acid during early pregnancy can reduce the risk of congenital anal atresia.

Anus, Imperforate ; etiology ; Female ; Humans ; Infant, Newborn ; Logistic Models ; Male ; Pregnancy ; Risk Factors

The macroscopic characteristics, tissue, caterpillar body wall and powder of Ophiocordyceps xuefengensis in different batch numbers were observed and researched by the macroscopic and microscopic identification methods. The result shows that the morphology, size, abdominal annulations of caterpillar, etc. of 0. xuefengensis are the macroscopic identification characteristics, the caterpillar body surface mycelium, body wall sculpture and crochets on abdominal legs are the microscopic identification characteristics. These characters are stable and regular discriminant features, which are proved to be the identification basis of O. xuefengensis. In addition, The characters such as crochets on abdominal legs arrange in two parallel ellipse rings, the inner crochets are long strip, and the external toes are unciform, are specific.
Animals ; Hypocreales ; cytology ; Moths ; anatomy & histology ; cytology ; microbiology

OBJECTIVETo conduct preliminary investigation to the species and reserves of medicinal plants in Huangfu Mountain, and to provide references to the general survey of those plants for medicine.

METHODCombined with global positioning system (GPS), the program of investigation with grid sampling was used in this resource survey of medicinal plants.

RESULTAfter the preliminary investigation of the plants for medical use of Huangfu Mountain, it is found that there are 103 families with 313 kinds of plants. There are many medicinal plants and large distribution, such as Pseudostellaria heterophylla, Semiaguilegia adoxoides and Pinellia ternate.

CONCLUSIONHuangfu Mount, with so many different kinds of medicinal plants and comfortable environment for part of the medicinal plants to grow, could be developed as a base for planting Chinese herbal medicines.

China ; Conservation of Natural Resources ; methods ; Drugs, Chinese Herbal ; analysis ; pharmacology ; Ecology ; Geographic Information Systems ; instrumentation ; Plants, Medicinal ; chemistry ; classification ; growth & development

CA-125 Antigen ; metabolism ; Cystadenocarcinoma, Serous ; diagnosis ; metabolism ; Female ; Humans ; Membrane Proteins ; metabolism ; Ovarian Neoplasms ; diagnosis ; metabolism ; Proteins ; metabolism