Mechanisms of the role of fibroblast growth factor 21 in attenuating insulin resistance / 药学学报

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.

Therapeutic efficacy of three bispecific antibodies on rheumatoid arthritis mice models / 药学学报

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.

FGF-21 protects H9c2 cardiomyoblasts against hydrogen peroxide-induced oxidative stress injury / 药学学报

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.

The synergistic effect of FGF-21 and insulin on regulating glucose metabolism and its mechanism / 药学学报

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.

Effect of FGF-21 on learning and memory ability and antioxidant capacity in brain tissue of D-galactose-induced aging mice / 药学学报

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.

The long lasting effect of the murine fibroblast growth factor-21 on blood glucose control of diabetic animals / 药学学报

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.

Therapeutic effect of fibroblast growth factor 21 on hypertension induced by insulin resistance / 药学学报

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.

A study on paternity testing with 96 autosomal SNPs / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To explore the feasibility of applying autosomal single nucleotide polymorphisms (SNPs) on parentage testing.</p><p><b>METHODS</b>All SNP genotyping results of HapMap (r27) were downloaded from the website. With self-made computer programs, SNPs were extracted when their minor allele frequency (MAF) were ≥ 0.30 among all of the 11 HapMap populations. Ninety-six SNPs were chosen and integrated into the Illumina Goldengate bead arrays on the condition that no linkage disequilibrium was found between them. Three father-child-mother trios (9 samples in total) were tested with the arrays. Cumulative paternity index (CPI) was then calculated and compared with genotyping results using 15 short tandem repeats (STRs)(Identifiler(TM)).</p><p><b>RESULTS</b>Family 1 was found to have nine SNPs or seven STRs that did not conform to the Mendelian laws, Family 2 had 13 such SNPs or seven STRs, and Family 3 only had one such SNP but no STR. For Family 3, when all of the 96 SNPs were used in combine, the CPI was 1207, which had contrasted with the CPI by the 15 STRs, i.e., 355 869.</p><p><b>CONCLUSION</b>When applied to paternity testing, the paternity exclusion (PE) value for a SNP is usually less than 1/3 of that of a STR. The proportion of SNPs not comforming to the Mendelian laws for the tested SNPs may not be as high as that of inconsistent STRs over all tested STRs. Because of the low mutation rate of a SNP, the CPI will be greatly reduced even if one SNP did not conform to the Mendelian laws. Therefore, highly accurate testing methods are required to reduce artificial errors when applying SNPs for paternity testing.</p>

Optimization and characterization of a novel FGF21 mutant / 药学学报

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.

Establishment of a novel cell model targeted on FGF-21 receptor for screening anti-diabetic drug candidates / 药学学报

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.