ABSTRACT
@#Objective To develop and verify a method for detecting the activity of neutralizing antibodies in ELISA antibody positive serum of rats immunized with recombinant human interleukin-1 receptor antagonist(rhIL-1Ra). Methods The SD rats were subcutaneously immunized with 3,20 and 100 mg/kg rhIL-1Ra injection respectively,10 rats in each group,half male and half female,twice a day at an interval of at least 4 h between each dose for 13 consecutive weeks. The blood samples were collected from the jugular vein of rats during the administration period and the recovery period. The serum samples were isolated and detected for the antibody titers by ELISA,and the samples positive for rhIL-1Ra antibody were purified by Protein A chromatographic column. Based on,D10G4·1 cells biological activity assay,a method for the detection of neutralizing antibody activity was developed and verified for the specificity,sensitivity and precision. The neutralizing antibody activity of rhIL-1Ra antibody positive serum determined by ELISA was detected by using the developed method.Results With the increase of doses,the serum antibody titers of rats in various dose groups gradually increased,and there were still antibodies in the recovery period,and the titer was still high. Rabbit anti-rhIL-1Ra monoclonal antibody showed obvious neutralizing effect on rIL-1Ra,while rabbit anti-rIFN-2b monoclonal antibody had no dose-effect relationship with rIL-1Ra. The sensitivity of the method was 171. 93 μg/mL;The CVs of precision verification were not more than 20%. The positive antibody sera detected by ELISA all had neutralizing effect on rhIL-1Ra injection,which was consistent with the results detected by ELISA. Conclusion The method developed in this study has good specificity and high sensitivity in the detection of serum neutralizing antibody activity in rats immunized with rhIL-1Ra,which can be used to detect the serum neutralizing antibody activity of animals with rhIL-1Ra repeated administration.
ABSTRACT
Objective: To evaluate protective effects of recombinant human IL-1Ra (rhIL-1Ra) on acute liver injury in vitro by using D-galactosamine (D-GalN) and HepG2 cells to establish the D-galactosamine (D-GalN)-induced HepG2 cells injury models. Methods:Models of HepG2 cells injury induced by D-GalN was established. HepG2 cells were maintained in mediums which contained different concentration of D-GalN (0.02, 0.2, 2 or 4 mg/mL) for different time (1, 2 or 3 d). Optimized concentration and time of D-GalN were used to analyze cell viability and morphology. A serial dose of rhIL-1Ra (10, 20 or 50 μg/mL) was used to treat HepG2 cells which were challenged with D-GalN. Cell apoptosis and the levels of intracellular reactive oxygen species (ROS) were analyzed in different treatment groups. Real-time PCR was employed to analyze the mRNA levels of IL-1β, IL-6 and TNF-α in cells. ERK1/2 inhibitor (SCH772984) was used to confirm whether ERK1/2 phosphorylation played a critical role in IL-1Ra protecting hepatocytes or not. Results: Cell viability was significantly decreased by D-GalN whose concentration was 4 mg/mL in HepG2 cells after 2 d. Compared with the control group, rhIL-1Ra could significantly improve cell survival and down-regulate the level of ROS in the cells. RhIL-Ra also could suppress expression of pro-apoptotic cytokines factors (IL-1β, IL-6 and TNF-α) induced by D-GalN in HepG2 cells. The results also showed that erk1/2 signaling pathways have certain effect on mediating the injury of rhIL-1Ra to protect hepatocytes. Conclusion: RhIL-1Ra can protect hepatocytes from toxins by directly targeting hepatocytes and inhibit cells apoptosis by activating ERK1/2 pathway in HepG2 cells.
ABSTRACT
Aim: To study the therapeutic effects of recombinant human interleukin 1 receptor antagonist (rhIL-1ra) on type II collagen-induced arthritis (CIA) rats. Methods: SD rats were divided randomly into six groups including normal, model, rhIL-1ra (7.5,30,120 mg·kg-1) and anakinra(120 mg· kg-1) groups. Collagen II emulsion was used to induce CIA model in rats. The body weight was observed once a week. Paw swelling of CIA rats was measured with volume meter. C II induced delayed-type hypersensitivity (DTH) was measured. Meanwhile, the level of anti-C II IgG antibody in serum was assayed by ELISA. Results: The onset of paw swelling was on dlO after injection of emulsion. The level of serum anti-C II IgG antibody was increased significantly in CIA. Pathological changes in joints of CIA rats showed hyperplastic synovium of CIA, inflammatory cells infiltration, pannus, destruction of cartilage and bone. rhIL-1ra(7.5,30,120 mg·kg-1·d-1 x 7 d) and anakinra (120 mg·kg-1·d-1 x 7 d) subcutaneous injection (sc) inhibited inflammatory swelling. rhIL-1ra(30, 120 mg·kg-1·d-1 x 7 d) significantly suppressed the DTH reaction induced with C II in CIA rats. Moreover, rhIL-1ra reduced the level of anti-C II IgG antibody in serum. Pathological examination showed rhIL-1ra(120 mg·kg-1·d-1 x 7 d) significantly improved subchondral inflammation, synovium hyperplasia, pannus and cartilage damage. Conclusion: rhIL-1ra has therapeutic effects on CIA rats.