Engineering Of Recombinant Human Fibroblast Growth Factor 21 And Its Treatment On Non-alcoholic Fatty Liver Disease Mice

Fibroblast growth factor 21 (FGF21), a new member of FGF family, is a novel regulator of glucose and lipid metabolism, specific acting on liver, pancreas, adipose tissue. It can decrease blood glucose and lipoprotein, improve insulin resistance and isletβcell function, showing therapeutical action on type 2 diabetes mellitus, obesity, atherosclerosis,and fatty liver diseases.Non-alcoholic fatty liver disease (NAFLD) is syndrome with liver histopathologic findings, including hepatic steatosis, inflammatory infiltration, and fibroplasia, which did not followed by excessive alcohol consumption and other virus infection. To date, it is emergency request in developing novel molecular to treat NAFLD. Previous reports demonstrated that FGF21 can effectively decrease the serum triglyceride, cholesterol, low density lipoprotein, with the potential in treatment of NAFLD. In present study, we expressed the soluble rhFGF21 in Escherichia coli, and mouse 3T3-L1 adipocytes were used to determine the glucose uptake activity of rhFGF21. Then, mouse experimental NAFLD models was established to evaluate the therapeutical effect of FGF21 and related mechanisms.So as to provide experimental evidences for its production and application.The cDNA fragment coded the his-tagged fgf21 was inserted into vector pET-3c and recombinant expression vector pET-fgf21 was transformated into Origami B (DE3). The optimal soluble expression conditions were selected as induction with 1 mM IPTG in culture medium at 20℃for 20 h. The soluble recombinant hFGF21 was expressed, and take up approximately 22.1% of the total supernant bacterial proteins.Based on the criteria for manufacturing of genetically engineered product, rhFGF21 would be used for process study on 50-liter scale fermentation and purification.Glucose would be the source of carbon, while yeast extracts and peptone were selected to be the source of nitrogen. Micro-elements, such as 0.3 g/L magnesium sulfate,2.5 g/L dipotassium hydrogen phosphate, 1.0 g/L monobasic potassium phosphate, and 4.0 g/L NaCl were added into fermented culture media.Optimal culture temperature for fermentation of bacterial strains of rhFGF21 would be set at 37℃, and the induction was set at 20℃. Fermentation cultivations using a glucose-pH-stat strategy were carried out in order to achieve high cell densities and high-level expression of rhFGF21. During the process of fermentation, the dissolved oxygen concentration was maintained above 30% of air saturation, and pH was adjusted at 7.0 by controlling the flow acceleration rate of glucose and NaOH. When the wet cell weight was up to 30 g/L and the pH was raised to 7.0, IPTG was added to the fermentation broth to a final concentration of 1 mM. By keeping optimal growth conditions for cell growth in the bioreactor, a high density of biomass of 51.3 g/L wet cell weight was achieved after 20 h induction and the expression level of rhFGF21 was up to 33.4% in soluble form.The target protein was purified by the combination of Ni-NTA affinity chromatography and Sephadex S-100 resin,3%(w/v) trehalose solution was able to prevent rhFGF21 from degradation effectively. By the optimal purification conditions,7,455 mg of rhFGF21 was obtained and the yield was 213 mg/L. The purity of rhFGF21 detected by HPLC was higher than 97%. The preliminary biochemical characterization of rhFGF21 was confirmed by Western blot, MALDI-TOF/MS, Peptide Map Finger (PMF) analysis.Based on the properties and pharmacokinetic characteristics of FGF21, mouse 3T3-L1 adipocytes and HepG2 cells were selected for this study to analyze in vitro biological activities of rhFGF21.The rhFGF21 stimulated glucose uptake in differentiated mouse 3T3-L1 adipocytes in a dose-dependent manner(EC50≈2.21 nM), comparable to that of the commercial hFGF21, demonstrating the biological function of rhFGF21 was well maintained during the purification process. The rhFGF21 strongly promoted GLUT1 mRNA expression for 24 h in differentiated mouse 3T3-L1 adipocytes in dose of 0.5μg/ml. The rhFGF21 showed no proliferation effect in HepG2 cells in range of 0.125～32μg/ml. But in range of 0.25～4μg/ml, rhFGF21 significantly enhanced the viability of HepG2 cells depressed by LY294002, demonstrating that rhFGF21 could activate the AKT pathway. The study, based on pharmacokinetic characteristics of FGF21, selected differentiated mouse 3T3-L1 adipocytes for activity assessment of final purified rhFGF21.A mouse NAFLD model induced by high fat diet was established to evaluate the therapeutical effect of rhFGF21. Three treated groups (0.15mg/kg/d,0.5mg/kg/d, 1mg/kg/d) were showed significant weight reduction after 30 days treatment. The plasma fasting glucose, fasting insulin, HOMA-IR levels were significant decreased after rhFGF treated, compaired to the HFD (high fat diet) model group, demonstrating the rhFGF could improved insulin resisitance level in NAFLD mice. Serum concentration of TG, TC, HDL-C, LDL-C, AST, ALT, ALP were retrieved to normal level after treatment, compaired to HFD group. Liver ROS (reactive oxygen species) production and mitochondria damage were alleviated after treatment, predicting that the rhFGF21 protected hepatic celld from oxidative stress, and maintained physio-structure.After treatment of rhFGF21, liver pathological changes including micro vesicular steatosis, hepatocyte hydropicde generation, infiltration, fibroplasia were alleviated and improved, compaired to the HFD group.In NAFLD mice,treatment of rhFGF21 could significant upregulated the liver SIRT1 and LDLR expression, and down regulated the key moleculars of lipid metabolism including ACC1, ACC2, FAS and SCD1, demonstrating that FGF21 inhibited lipogenesis, promoted fatty acid oxidation, reduced liver TG accumulation by affecting the ACC-MA pathway.