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Objective@#Leukocyte mediated IL-6 (IL-6) plays an important role in chronic viral hepatitis pathogenesis and related liver diseases, We did a large sample-size case-control study and clinical data analysis to find association between IL-6 single nucleotide polymorphism and HBV susceptibility, and to achieve the detection of the body on the expression of IL-6 for the prevention and treatment of HBV infection.@*Methods@#Totally 848 HBV patients and 894 healthy controls in Shenzhen were selected and rs1800796 genotypes were determined by TaqMan assays.@*Results@#The result showed that rs1800796 polymorphism was associated with susceptibility to HBV infection (P=0.0003, odds ratio (OR)=1.43, the difference was statistically significant.@*Conclusions@#The single nucleotide polymorphism of IL-6rs1800796 locus was associated with the susceptibility of HBV infection in Chinese population, and the rs1800796 G allele is a protective gene for HBV infection.
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Objective To investigate the effects of allele-31 C>T on the binding activity to IL-1βpromoter of the nuclear transcription factor C/EBPβand PU.1 induced by Mycobacterium tuberculosis infection.Methods The electrophoretic mobility shift assay ( EMSA) was performed to explore whether the nuclear transcription factor C/EBPβand PU.1 could bind to -31 region in IL-1βpromoter.The C/EBPβ-and PU.1-expressing vectors were constructed and co-transfected into HeLa cells with IL-1βpromoter luciferase vector.The expression of C/EBPβand PU.1 was confirmed using Western blotting assay, and the promoter activity was determined using Dual-Glo Luciferase system under various transfection conditions. Lentivirus-mediated RNA interference was used to explore the effects of C/EBPβand PU.1 on IL-1βexpression.GraphPad Prism 5.0 was used for data analysis.Results EMSA results showed that both C/EBPβand PU.1 could bind to -31 region in IL-1βpromoter.Both C/EBPβand PU.1 induced by Mycobacterium tuberculosis infection could increase IL-1βpromoter activity, especially for the -31 T allele (t=22.33 and 7.98,PT can induce IL-1βpromoter activity and gene transcription through regulation of binding activity to C/EBPβand PU.1 induced by Mycobacterium tuberculosis infection.
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This study is to evaluate the therapeutic effect of fibroblast growth factor 21 (FGF21) on type 2 diabetic mice model and to provide mechanistic insights into its therapeutic effect. Type 2 diabetic animal model was established with high calorie fat diet and low dose streptozotocin (STZ) injection. Mice were then randomized into 5 groups: model control, FGF21 0.25 and 0.05 μmol x kg(-1) x d(-1) groups, insulin treatment group. Ten age-matched normal KM mouse administered with saline were used as normal controls. Serum glucose, insulin, lipid products and the change of serum and liver tissue inflammation factor levels between five groups of mouse were determined. The results showed that blood glucose, insulin, free fatty acids (FFAs), triglycerides, and inflammatory factor average FGF-21 of type 2 diabetes model group and normal control group were significantly higher (P < 0.01), while compared with insulin group, no difference was significant. Average blood glucose, insulin, blood lipid and inflammatory factor of FGF-21 treatment group compared with type 2 diabetes group was significantly lower (P < 0.01) and insulin group has no difference with the model control group. The results of OGTT and HOMA-IR showed that insulin resistance state was significantly relieved in a dose-dependent manner. Thus, this study demonstrates that FGF-21 significantly remits type 2 diabetic mice model's insulin resistance state and participates in the regulation of inflammatory factor levels and type 2 diabetes metabolic disorders.
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In order to obtain the lead compound for treatment of rheumatoid arthritis (RA), in this study, therapeutic efficacy of three bispecific antibodies (BsAB-1, BsAB-2 and BsAB-3) against both hIL-1beta and hIL-17 were compared on CIA model mice. First, by ELISA method we compared the binding capacity of the three bispecific antibodies to the two antigens. The results showed that all three antibodies could simultaneously bind both antigens, among these antibodies, BsAB-1 was superior over BsAB-2 and BsAB-3. CIA model was established with chicken type II collagen (CII) and developed RA-like symptoms such as ankle swelling, skin tight, hind foot skin hyperemia. The CIA mice were treated with three antibodies once every two days for total of 29 days. Compared with the CIA model mice, the RA-like symptoms of the antibody treated-mice significantly relieved, while the BsAB-1 treated-mice were almost recovered. CII antibody level in the serum and cytokines (IL-2, IL-1beta, IL-17A and TNF-alpha) expression in the spleen were examined. Compared with the CIA model mice, all three antibodies could significantly reduce CII antibody and cytokine expression levels. BsAB-1 antibody was more potent than BsAB-2 and BsAB-3. In summary, BsAB-1 is superior over BsAB-2 and BsAB-3 in amelioration of RA symptoms and regulation of CII antibody production and pro-inflammatory cytokine expression, therefore, BsAB-1 can be chosen as a lead compound for further development of drug candidate for treatment of RA.
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This study aims to investigate the effects of fibroblast growth factor 21 (FGF-21) on learning and memory abilities and antioxidant capacity of D-galactose-induced aging mice. Kunming mice (37.1 +/- 0.62) g were randomly divided into normal control group, model group and FGF-21 high, medium and low dose groups (n = 8). Each group was injected in cervical part subcutaneously with D-galactose 180 mg x kg(-1) x d(-1) once a day for 8 weeks. At the same time, FGF-21-treated mice were administered with FGF-21 by giving subcutaneous injection in cervical part at the daily doses of 5, 2 and 1 mg x kg(-1) x d(-1). The normal control group was given with normal saline by subcutaneous injection in cervical part. At seventh week of the experiment, the learning and memory abilities of mice were determined by water maze and jumping stand tests. At the end of the experiment, the mice were sacrificed and the cells damage of hippocampus was observed by HE staining in each group. Reactive oxygen species (ROS), malondialdehyde (MDA), superoxide dismutase (SOD), glutathione peroxidase (GPx), catalase (CAT) and total antioxidant capacity (T-AOC) in the brain of mice were determined. The results showed that different doses of FGF-21 could reduce the time reaching the end (P < 0.01 or P < 0.05) and the number of touching blind side (P < 0.01 or P < 0.05) in the water maze comparing with the model group. It could also prolong the latency time (P < 0.05) and decrease the number of errors (P < 0.01 or P < 0.05) in the step down test. The result of HE staining showed that FGF-21 could significantly reduce brain cell damage in the hippocampus. The ROS and MDA levels of three different doses FGF-21 treatment group reduced significantly than that of the model group [(5.58 +/- 1.07), (7.78 +/- 1.92), (9.03 +/- 1.77) vs (12.75 +/- 2.02) pmol (DCF) x min(-1) x mg(-1), P < 0.01 or P < 0.05], [(2.92 +/- 0.71), (4.21 +/- 0.81), (4.41 +/- 0.97) vs (5.62 +/- 0.63) nmol x mg(-1) (protein), P < 0.01]. Comparing with the model group, the activities of SOD, GPx, CAT and T-AOC of the three different doses FGF-21 treatment groups were also improved in a dose-dependent manner. This study demonstrates that FGF-21 can ameliorate learning and memory abilities of D-galactose induced aging mice, improve the antioxidant abilities in brain tissue and delay brain aging. This finding provides a theoretical support for clinical application of FGF-21 as a novel therapeutics for preventing aging.
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Previous studies proposed that the synergistic effect of fibroblast growth factor-21 (FGF-21) and insulin may be due to the improvement of insulin sensitivity by FGF-21. However, there is no experimental evidence to support this. This study was designed to elucidate the mechanism of synergistic effect of FGF-21 and insulin in the regulation of glucose metabolism. The synergistic effect of FGF-21 and insulin on regulating glucose metabolism was demonstrated by investigating the glucose absorption rate by insulin resistance HepG2 cell model and the blood glucose chances in type 2 diabetic db/db mice after treatments with different concentrations of FGF-21 or/and insulin; The synergistic metabolism was revealed through detecting GLUT1 and GLUT4 transcription levels in the liver by real-time PCR method. The experimental results showed that FGF-21 and insulin have a synergistic effect on the regulation of glucose metabolism. The results of real-time PCR showed that the effective dose of FGF-21 could up-regulate the transcription level of GLUT1 in a dose-dependent manner, but had no effect on the transcription level of GLUT4. Insulin (4 u) alone could up-regulate the transcription level of GLUT4, yet had no effect on that of GLUT1. Ineffective dose 0.1 mg kg(-1) FGF-21 alone could not change the transcription level of GLUT1 or GLUT4. However, when the ineffective dose 0.1 mg x kg(-1) FGF-21 was used in combination with insulin (4 u) significantly increased the transcription levels of both GLUT1 and GLUT4, the transcription level of GLUT1 was similar to that treated with 5 time concentration of FGF-21 alone; the transcription level of GLUT4 is higher than that treated with insulin (4 u) alone. In summary, in the presence of FGF-21, insulin increases the sensitivity of FGF-21 through enhancing GLUT1 transcription. Vice versa, FGF-21 increases the sensitivity of insulin by stimulating GLUT4 transcription in the presence of insulin. FGF-21 and insulin exert a synergistic effect on glucose metabolism through mutual sensitization.
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Fibroblast growth factor-21 (FGF-21) is an important metabolism regulator, however, whether FGF-21 has effects on cardiovascular remains unclear. In this study, H2O2-induced injury in H9c2 cells was used as a cell model, the anti-apoptosis potential and mechanism of FGF-21 against oxidative injury were evaluated by MTT assay, flow cytometry assay and real-time PCR. The results showed that FGF-21 could increase the cell survival of H2O2-induced injury in H9c2 cells and prevent H9c2 cells from oxidative stress-induced apoptosis. Furthermore, FGF-21 can elevate SOD activity and regulate Bcl-2/Bax expression in H9c2 cells. The results suggest that FGF-21 have protective effect against the H2O2-induced apoptosis in H9c2 cells.
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The aim of the study is to establish a platform to deliver therapeutic proteins into target cells through a polyarginine-based cell penetrating peptide. To facilitate the expression of therapeutic proteins, a pSUMO (Small Ubiquitin-like Modifier)-R9-EGFP (enhanced green fluorescence protein) prokaryotic expression vector was constructed. After induction, the fusion protein SUMO-R9-EGFP was efficiently expressed. To validate the cell penetrating ability of the fusion protein, HepG2 cells were incubated with the purified R9-EGFP or EGFP protein as control, internalization of the fluorescent proteins was examined by either flow cytometry or confocal microscopy. The result obtained by flow cytometry showed that the R9-EGFP fusion protein could efficiently penetrate into the HepG2 cells in a dose and time-dependent manner. In contrast, the fluorescence was barely detected in the HepG2 cells incubated with EGFP control. The fluorescence intensity of the R9-EGFP treated cells reached plateau phase after 1.5 h. The result obtained by confocal microscopy shows that R9-EGFP efficiently entered into the HepG2 cells and was exclusively located in the cytoplasm, whereas, no fluorescence was detected in the cells incubated with the EGFP control. The heparin inhibition experiment showed that heparin could inhibit penetrating effect of the R9-EGFP protein by about 50%, suggesting that the penetrating ability of the fusion protein is heparin-dependent. In summary, the study has established a platform to deliver therapeutic proteins into target cells through a polyarginine-based penetrating peptide.
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Humans , Cell-Penetrating Peptides , Genetics , Pharmacology , Genetic Vectors , Genetics , Green Fluorescent Proteins , Genetics , Hep G2 Cells , Peptides , Genetics , Metabolism , Protein Transport , Recombinant Fusion Proteins , Genetics , PharmacologyABSTRACT
Insulin is the most common medicine used for diabetic patients, unfortunately, its effective time is short, even the long-acting insulin cannot obtain a satisfactory effect. Fibroblast growth factor (FGF)-21 is a recently discovered glucose mediator and expected to be a potential anti-diabetic drug that does not rely on insulin. In this study, db/db mice were used as the type 2 diabetic model to examine whether mFGF-21 has the long-term blood lowering effect on the animal model. The results showed that mFGF-21 could stably maintain the blood glucose at normal level for a long-term in a dose-dependent manner. Administration of mFGF-21 once a day with three doses (0.125, 0.25 and 0.5 mg x kg(-1)) could maintain blood glucose of the model animals at normal level for at least 24 h. Administration of mFGF-21 every two days with the same doses could maintain blood glucose of the model animals at normal level for at least 48 h, although it took longer time for blood glucose to reach to normal level depending on doses used (twenty injections for 0.125 mg x kg(-1) and 0.25 mg x kg(-1) doses, ten injections for 0.5 mg x kg(-1) dose). Surprisingly, the blood glucose of the treated model animals still maintained at normal level for 24 h after the experiment terminated. Glycosylated hemoglobin level of the animals treated with mFGF-21, which represented long-term glucose status, decreased significantly compared to the control group and the insulin group. The results suggest that FGF-21 has potential to become a long-acting and potent anti-diabetic drug.
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This study is to evaluate the therapeutic effect of fibroblast growth factor 21 (FGF21) on hypertension induced by insulin resistance in rats and to provide mechanistic insights into its therapeutic effect. Male Sprague-Dawley (SD) rats were fed with high-fructose (10%) water to develop mild hypertensive models within 4 weeks, then randomized into 4 groups: model control, FGF21 0.25, 0.1 and 0.05 micromol x kg(-1) x d(-1) groups. Five age-matched normal SD rats administrated with saline were used as normal controls. The rats in each group were treated once a day for 4 weeks. Body weight was measured weekly, systolic blood pressure (SBP) was measured noninvasively using a tail-cuff method, insulin sensitivity was assessed using oral glucose tolerance test (OGTT) and HOMA-IR assay. At the end of the treatment, blood samples were collected, and blood glucose, serum cholesterol, serum triglyceride and serum insulin were measured. The results showed that blood pressure of the rats treated with different doses of FGF21 returned to normal levels [(122.2 +/- 3.5) mmHg, P < 0.01] after 4-week treatment, whereas, SBP of untreated (model control) rats maintained a high level [(142.5 +/- 4.5) mmHg] throughout the treatment. The observation of blood pressure in 24 h revealed that SBP of FGF21 treated-rats maintained at (130 +/- 4.5) mmHg vs. (143 +/- 5.5) mmHg for model control (P < 0.01). FGF21 treatment groups improved serum lipids obviously, total cholesterol (TC) and triglyceride (TG) levels decreased significantly to normal levels. The serum NO levels of three different doses FGF21 treatment group were significantly higher than that of the model control group [(7.32 +/- 0.11), (7.24 +/- 0.13), (6.94 +/- 0.08) vs. (6.56 +/- 0.19) micromol x L(-1), P < 0.01], and the degree of improvement showed obvious dose-dependent manner, indicating that FGF21 can significant increase serum NO in fructose-induced hypertension rat model and improve endothelial NO release function. The results of OGTT and HOMA-IR showed that insulin resistance state was significantly relieved in a dose-dependent manner. Thus, this study demonstrates that FGF21 significantly ameliorates blood pressure in fructose-induced hypertension model by relieving insulin resistance. This finding provides a theoretical support for clinical application of FGF21 as a novel therapeutics for treatment of essential hypertension.
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Fibroblast growth factor 21 (FGF21) is a member of FGF family. It has been demonstrated that FGF21 is an independent, safe and effective regulator of blood glucose levels in vivo. In order to improve the activity of FGF21, we exchanged the beta10-beta12 domain of the human FGF21 with that of the mouse FGF21 to construct a novel FGF21 gene (named hmFGF21), and then subcloned hmFGF21 gene into the SUMO expression vector to create pSUMO-hmFGF21 and transformed it into E. coli Rosetta for expression of the fusion protein SUMO-hmFGF21. Both in vitro and in vivo glucose regulation activity of hmFGF21 was evaluated. The SDS-PAGE result showed that compared with wild-type hFGF21, the soluble expression of hmFGF21 increased about 2-fold. HmFGF21 was more potent in stimulation of glucose uptake in HepG2 cells in vitro. The results of anti-diabetic effect on db/db mice demonstrated that hmFGF21 had better efficacy on controlling the blood glucose of the db/db diabetic animals than wild-type hFGF21. These results suggest that the biological properties of FGF21 are significantly improved by optimization.
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Objective To observe the effect of Sopfrology-Lamaze childbirth breathing training on the delivery quality and negative mood of pregnant women.Methods 218 pregnant women were sampled and randomly divided into study group(A group,n =122) and control group(B group,n =96).A group learned the method and technique of Sopfrology-Lamaze childbirth breathing.From 28th week pregnancy to delivery,the times of training was more than 3 per week,and lasted 20 minutes.The rate of cesarean delivery,rate of severe pain,the time of labor,the amount of bleeding after 2h labor,and the dosage of patient controlled analgesia were observed.The selfrating anxiety scale (SAS) and self-rating depression scale(SDS) were used to assess the mood of prengnat women in 28th week pregnancy,before and after labor.Results There were significant difference in rate of cesarean delivery(A group 7.4％,B group 17.7％,x2 =5.46,P ＜ 0.01),rate of severe pain (A group18.8％,B group 59.4％,x2 =37.9,P ＜ 0.05),amount of bleeding after 2h labor (A group (219.43-± 31.47) ml,B group (287.5 ± 37.83)ml,t =14.50,P ＜ 0.05),and the dosage of patient controlled analgesia (A group(13.25 ± 1.89)ml,B group (19.87 ±2.52)ml,t=21.43,P＜0.05) between A group and B group.The SAS and SDS scores were lower in A group (SAS:before labor(44.3 ±7.6),after labor(42.2 ±4.7) ;SDS:before labor(45.2 ±4.7),after labor(42.2 ± 5.1),P ＜ 0.05) than those in B group (SAS:before labor(48.5 ± 6.6),after labor(47.35 ±5.1) ; SDS:before labor(47.7 ± 5.3),after labor (46.6 ± 6.3),P ＜ 0.05).Conclusion Sopfrology-Lamaze childbirth breathing training can improve the delivery quality and negative moods of pregnant women
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The aim of this project is to establish a fibroblast growth factor-21 (FGF-21) signaling pathway targeted cell model, for screening a class of FGF-21 receptor agonists as anti-diabetic candidates. FGF-21 requires beta klotho transmembrane proteins as co-receptor for the activation of tyrosine kinase FGF receptor (FGFR) signaling, thereby activating a series of intracellular signaling pathways and regulating gene transcription for glucose metabolism. Firstly a recombinant plasmid expressing co-receptor beta klotho and EGFP reporter genes was constructed. After introducing the recombinant plasmid into package cells, the cell culture supernatant was used to infect 3T3-L1 cells, which were then screened for stably expressing beta klotho gene. Administration of FGF-21 increased the expression of GLUT1 and stimulated GLUT1-mediated glucose uptake. This novel cell model can be conveniently used in high-throughput drug screening of FGF-21 or FGF-21 analogues.
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Objective To clone the full length staphylococcal protein A(SPA) gene from Staphylococcus aureus (ATCC6538), and subsequently study the gene structure and antibody binding ability.Methods The full length and the functional region of the SPA gene were cloned into pHisSUMO vector respectively, and expressed in E. coli. The full length and the functional fragment of the SPA protein were detected for antibody binding ability and stability. The functional fragment of the SPA protein fused with SUMO was coupled to the CNBr-activated agarose for antibody purification from rabbit serum. Results A variant of the full length SPA gene was cloned, which has been submitted to GenBank (the accession number is EU695225). Two fusion proteins had the same antibody binding ability as the untagged SPA protein. However, the formers was more stable than the latter at the tested conditions. SUMO-SPA conjugated-agarose kept high efficiency for antibody binding. Conclusion To our knowledge, the full length SPA gene of S.aureus(ATCC6538) is a novel variant. The SUMO tag can improve the stability of the functional region of the SPA protein without damaging the antibody binding ability. This fusion protein has been used for antibody purification successfully.
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Objective To evaluate the usefulness of DiversiLab system for genotyping of Acinetobacter baumannii.Methods Fifty-eight non-duplicated clinical Acinetobacter baumannii isolated from 15 cities in China in 2005 were typed by rep-PCR-based DiversiLab system.The results were compared with those of PFGE and multilocus sequence typing. Simpson's index of diversity was used to compare the discriminatory power among DiversiLab system, PFGE and MLST.Results Fifty-eight Acinetobacter baumannii isolates were differentiated into 5 clusters and 25 unique types by DiversiLab system. MLST identified 35 distinct sequence types, which fell into one clone complex of CC22 and 35 singletons, while PFGE resolved 5 pulsotypes and 34 unique types.Simpson's diversity indices for DiversiLab system, MLST and PFGE were O.876, O.944 and 0.961, respectively.Conclusions The discriminatory power of DiversiLab system is lower than that of PFGE and MLST.But as a simple, fast and reproducible typing method, it could be used as a first-line typing tool for the analysis of a large number of isolates.
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Insulin resistance in insulin sensitive organ results in metabolic disorder such as hyperglycemia, hyperinsulinemia and hyper triglyceridemia which are common features of type 2 diabetes.Insulin resistance in liver cells mainly causes impaired glycogen synthesis, failed to suppress glucose production which is the major contribution to hyperglycemia.FGF-21 as a new metabolic regulator can control fasting blood glucose.The mechanism of FGF-21 effects on regulating plasma glucose has little to known.In order to establish an in vitro insulin resistant model of liver cells and evaluate the effects and mechanism of FGF-21 on glucose metabolism in the cell model, HepG2 cells were incubated with 10-7 mol/L insulin for 24 h to build insulin-resistant cell model.To evaluate the cells for insulin resistance, the cells were stimulated with fresh insulin for 24 h and the glucose uptake by these cells was carried out.The insulin-resistant cells were treated with different concentrations of FGF-21 for 24 h and insulin-treated cells were used as a control.The glucose uptake by the cells was detected by the method of glucose oxidizes/peroxides(GOD-POD);the synergy between insulin and FGF-21 was evaluated.The mRNA expression of GLUT1 in the insulin-resistant cells was detected by the real-time PCR.Glycogen synthesis of the cells was examined by the anthrone method.The results showed that HepG2 cells treated with 10-7 mol/L insulin for 24 h became resistant to insulin and the insulin resistance status was maintained for 48 h without change of cell morphology.FGF-21 could stimulate glucose consumption of the insulin-resistant model in a dose-dependent manner.The glucose consumption and glycogen synthesis of the insulin-resistant model were significantly improved by FGF-21 treatment.FGF-21 showed strong synergy with insulin in glucose uptake and glycogen synthesis of the model cells.While the cells became resistant to insulin, FGF-21 could increase the mRNA expression of GLUT1.Thus, It is concluded that FGF-21 stimulates glucose uptake in insulin resistant HepG2 cells through GLUT1 expression, stimulates glycogen synthesis and improves the glucose metabolism in the insulin resistant liver cell model.
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Fibroblast growth factor(FGF)-21 is a novel insulin-independent glucose regulator,and can be a potential therapeutics for treatment of type Ⅱ diabetes.Although FGF-21 was discovered recently,the insight into its biology and therapeutic utility is rapidly evolving.A number of key metabolically-linked molecules and pathways have been suggested to be involved in the mechanism of action of FGF-21,which enables us to renew the understanding of the FGF-21.The aim of this review is to report the update research outcomes.