Induction Of Systemic Lupus Erythematosus Syndrome In Mice And Anti-inflammatory And Immunosuppressive Action Of Tripterine

AIM:To establish an animal model for systemic lupus erythematosus (SLE)-like syndrome in BALB/c mice and to investigate its characteristics. To observe whether tripterine, isolated from Tripterygium Wilfordii Hoog f. in China, had beneficial effects on this experimental systemic lupus erythematosus induced by syngeneic active chromatin in mice. To determine whether tripterine also had beneficial effects on rat adjuvant arthritis(AA), another kind of animal model of autoimmune disease and its effect mechanism.METHODS:BALB/c mice were immunized with active chromatin isolated from ConA-actived syngeneic spleno-lymphocytes, the dose was100μg/mice on d0, d14 and d28 to establish SLE-like model. Plasma samples of mice were tested by enzyme-linked immunosorbent assays (ELISA) for the presence of IgG anti-dsDNA, -ssDNA, anti-histone antibodies and total IgG Hep-2 cells were used to detect the patterns of antinuclear antibodies. Tumor necrosis factor-α (TNF-α ) in serum was measured by ELISA. Nitric oxide (NO) and complement level in serum were measured. Spleno-lymphocyte proliferation assays and the levels of interferon-γ (IFN-γ) in supernatants were tested respectively. Peritoneal cells were cultured and TNF-α in supernatants was tested. Urine protein was measured and kidneys were examined bydirect immunohistochemical method and light microscopy. Red cells, white cells, platelets were counted.BALB/c mice were immunized with active chromatin isolated from concanavalin A-activated syngenetic spleno-lymphocytes on day 0. Tripterine 6 or 12 mg kg^-1 day^-1, or prednisone 5 mg kg^-1 day^-1 was given to BALB/c mice intragastrically from day 35 to day 50. Serum samples of mice were tested by enzyme-linked immunosorbent assays (ELISA) for the presence of IgG anti-dsDNA, -ssDNA, anti-histone antibodies and total IgG. Nitric oxide (NO) level in serum was measured. Spleno-lymphocyte proliferation assays and the levels of interferon-γ (IFN-γ) in supernatants were tested respectively. Peritoneal cells were cultured and IL-10 in supernatants was tested. Urine protein was measured and kidneys were examined by light microscopy.AA was induced in rats by intradermal injection into the right hind footpad with Freund's complete adjuvant. The injection day was regarded as day 0. Sixteen days after inoculation, the animals were selected and distributed in groups(n=10) according to the severity of arthritis, Tripterine5, 10, 20 mg kg^-1 day^-1, or prednisone 5 mg kg^-1 day^-1 was given to rats intragastrically from day 19 to day 24. Paw volume was measured to observe the effect of tripterine on induced paw and non-induced paws, polyarthritis was reflected by arthritis score. Joint were examined by light microscopy. Peritoneal macrophage was collected and was treated with different concentration of tripterine. Nitrite accumulation, as an indicator of NO production was measured in supernatant of peritoneal macrophage using the Griees reagent. The mRNAs of tumor necrosis factor( TNF-α)and interleukin-1(IL-1β) were detected with reverse transcription-polymerase chain reaction (RT-PCR) and protein level of NF-κB was measured by ELISA.RESULTS:Induction and Characteristics of animal model for SLE-like syndrome in BALB/cmiceAnti-nuclear antibodies are associated with lupus nephritis in humans. Activechromatin-treated mice had markedly elevated levels of IgG anti-ds -DNA, anti-ss-DNA, anti-histone and total IgG when tested at d45 and d60(P<0.05), the pattern of antinuclear antibodies detected by Hep-2 cells is homogeneous and cytoplasmic. In nil and resting chromatin groups no significant anti-nuclear antibody response was found in BALB/c mice.In the absence of mitogen, the spleno-lymphocyte proliferation was stronger in the active chromatin -immunized group than those in nil and resting chromatin-immunized groups on d 45 and d 60 (P<0.01). But after LPS or Con A stimulation, the spleno-lymphocyte proliferation was decreased in the active chromatin-immunized group compared with nil and resting chromatin-immunized. Nitric oxide (NO) level in serum was increased in active chromatin -immunized group on d 45 (P<0.05)and d 60 (P<0.01). Levels of interferon-γ (IFN-γ) in culture supernatants of spleno-lymphocyte were lowered and levels of TNF-α in serum and in supernatants of peritoneal cells were decreased in resting and active chromatin-immunized group, especially in the latter.The active chromatin inoculated mice had elevated levels of protein in their urine compared with other two groups. Active chromatin immunized mice all had moderate to severe renal Ig deposition, mostly localized to glomeruli, but no glomerular Ig deposition was present in mice in control group. Under light microscopy kidney sections from active chromatin immunized mice showed segmental or diffuse proliferative glomerular lesion. Proliferation was mainly related to increase in mesangial cells. In addition, mild influx of monocytes was seen in the glomerular capillaries. These changes were not apparent in the glomeruli of control mice. Thrombocytopenia appeared in active chromatin inoculated mice.Therapeutic effect of tripterine on systemic lupus erythematosus syndrome in miceEffect of tripterine on autoantibodies and total IgG productionVehicle-treated group showed progressive increase in autoantibodies and IgG production compared with nil and resting chromatin-immunized group (P<0.001, P<0.05). Treatment with tripterine 6 and 12 mg·kg^-1·d^-1 or prednisone 5mg·kg^-1·d^-1 for15 days caused reduction of the three autoantibodies and IgG levels compared with vehicle-treated group (P<0.01, P<0.001). Tripterine 12 mg·kg^-1·d^-1 had better effect in reducing anti-ssDNA (P=0.027) and anti-histone (P=0.033) antibodies compared with prednisone 5 mg·kg^-1·d^-1. Effect of tripterine on spleno-lymphocyte proliferationSpleno-lymphocyte proliferation in the absence of mitogen was higher in vehicle-treated group compared with that in nil and rest chromatin-treated group (P<0.001). But spleno-lymphocyte proliferation induced by concanavalin A or lipopolysaccharide was markedly lower in vehicle-treated group compared with nil and rest chromatin-treated group (P<0.001).Treatment with tripterine 12 mg·kg^-1·d^-1 for 15 days partly improved spleno-lymphocyte proliferation stimulated by concanavalin A and lipopolysaccharid compared with vehicle group (P<0.05), but had little effect on spleno-lymphocyte proliferation in the absence of mitogen. Treatment with tripterine 6 mg·kg^-1·d^-1 for 15 days had no influence on spleno-lymphocyte proliferation in the presence or absence of mitogen compared with vehicle-treated group . Effect of tripterine on interleukin-10 and interferon- y productionProduction of interleukin-10 from macrophages was elevated but production of IFN-γ from splenocytes was reduced in vehicle-treated group compared with nil-and rest chromatin-treated group (P<0.001, P<0.05).Treatment with tripterine 6 and 12 mg·kg^-1·d^-1 for 15 d inhibited IL-10 production (P<0.001) but had no influence on IFN-γ production compared with vehicle-treated group. Effect of tripterine on urine protein and serum NO levelVehicle-treated group showed progressive increase in albuminuria compared with nil- and rest chromatin-treated group (P<0.001). Treatment with tripterine 12 mg·kg^-1·d^-1 for 15 d caused a marked reduction of urinary albumin compared with vehicle-treated group (P<0.05). Treatment with tripterine 6 mg·kg^-1·d^-1 or prednisone 5 mg·kg^-1·d^-1 for 15 d caused a slight reduction of urinary albumin but the difference was not significant compared with vehicle-treated group.Serum NO level was elevated in vehicle-treated group compared with nil- and rest chromatin-treated group (P<0.001). Treatment with tripterine 12 mg·kg^-1·d^-1 for 15 days caused a marked reduction of NO level (P<0.001) but tripterine 6 mg·kg^-1·d^-1 had no effect compared with vehicle-treated group. Effect of tripterine on glomerular injuryVehicle-treated group showed segmental or diffuse glomerular lesions (n=6). Glomerular lesions were characterized by apparent proliferation of mesangial cells in mesangium and mild influx of monocytes in the glomerular capillaries. These lesions were not apparent in nil- and rest chromatin-treated group.Treatment with tripterine 12 mg·kg^-1·d^-1 for 15 days markedly ameliorated the glomerular injury but tripterine 6 mg·kg^-1·d^-1 had no significant effects.Therapeutic effect of tripterine on adjuvant arthritis in rats and its effect mechanismTherapeutic effect of tripterine on adjuvant arthritisTreatment with tripterine at dosage of 5 , 10, 20 mg kg^-1 day^-1 for 6 days clearly inhibit plantar edema on injected (primary lesion) and contralateral (non-injected) hind paw (secondary lesion), reduced the arthritis score which reflect polyarthritis. Pathological (P<0.05 ) and radiographic examination reveal that tripterine 20 mg kg^-1 day^-1 inhibited the synovial hyperplasia and the pannus, alleviated the destruction of articular cartilages. Effect of tripterine on peritoneal macrophage of adjuvant arthritisIn vitro, TNF-α mRNA (P<0.05 ) and IL-1β mRNA (P<0.01) and NO(P<0.01) expression of peritoneal cells from arthritis (AA) rats induced by LPS weremarkedly inhibited by tripterine (0.1μg/ml～1μg/ml), the NF-kB activity is alsoreduced by tripterine (0.01μg/ml～1μg/ml, P<0.01) and alterations in nuclearmorphology of macrophage detected with Hoechst 33258 were observed (0.1μg/ml).CONCLUSION:The active chromatin-induced SLE-like mouse model was similar to idiopathic SLE inhuman. Tripterine had a beneficial effect on systemic lupus erythematosus induced by active chromatin in BALB/c mice. Tripterine had a beneficial effect on adjuvant arthritis. The therapeutically effect of tripterine on adjuvant arthritis is probably bydecreasing TNF-α mRNA and IL-1β mRNA expression and NF-kB activity.