Resveratrol Pristane Induced Lupus Mice Role Research

Background Systemic lupus erythematosus (SLE) is a multisystemic autoimmune disease characterized by autoantibodies to components of the cell nucleus. Renal damage occurs in most patients with SLE. The mechanism of SLE is thought to be the auto-antibodies produced by B cell which is stimulated by abnormal activated CD4+T cell.Current therapeutics of SLE are directed at suppressing humoral immunity and production of auto-antibodies and Th cell and B lymphycte such as glucocortoids, CTX, azathioprine, cyclosprin and mycophenolate mofetil. They suppress immunological system non-selectively and non-specifically and would result in severe side effect. The new generation of biological agents targeting a variety of immune modulatory molecules are under development, However the long-term beneficial and detrimental effects remain unknown. A substantial proportion of SLE patients reponds poorly to these drugs, SLE still severely threat the health of mankind in the world. More effective and safe drugs are needed to control SLE.Resveratrol,3,5,4-trihydroxystilbene, is a natural antimicrobial compound found in various plants and fruits. Resveratrol attracted great attention following the discovery of its cardioprotective properties. It possesses anti-inflammation, antioxidation and blood fat-regulatory properties and can results in gene silence. It has been reported that resveratrol can inhibit several experimental autoimmune diseases including collagen-induced arthritis, encephalomyelitis, colitis. It can inhibit the activation and proliferation of T cell and B cell in vitro, induce their apoptosis, Inhibit antibody secretion by B cell in vitro. It can inhibit the expression of CD40, CD80,CD86, MHC I and II molecules. It can also inhibit COX-2. those imply resveratrol may have potential beneficial effect on SLE.However, until now the effect of resveratrol on SLE and pristant-induced lupus is still unknown. Here we conducted the study To evaluvate whether resveratrol can prevent the development of pristant-induced lupus.Methods1. Female BALB/c mice at age9-10weeks were randomly classified into2groups:(1) Pristane-induced lupus:BALB/c mice received a single intraperitoneal injection of0.5ml of pristane.(2) Controls:BALB/c mice received a single intraperitoneal injection of0.5ml of0.9%sodium chloride. The development was monitored until7months. Proteinuria, autobodies (ANA, anti-ds-DNA antibody, anti-nRNP/Sm antibody), kidney pathology and immunoglobuin deposition were examined.2. Female BALB/c mice at age9-10weeks were randomly classified into3groups:(1) Resveratrol A:Ten mice were feed with50mg/kg/d resverarol from the second day after pristane injection.(2) Resveratrol B:Ten mice were feed with75mg/kg/d resverarol from the second day after pristane injection.(3) Model Controls:Ten mice were injected with pristane. The development was monitored until7months. Proteinuria, autobodies (ANA, anti-ds-DNA antibody, anti-nRNP/Sm antibody), kidney pathology and immunoglobuin deposition were examined.3.(1) Splenic mononuclear cell (SMC) from pristane-induced lupus mice were activated with ConA, anti-CD3or anti-CD3/CD28with or without resveratrol for6-12hour (CD69) or36-48hour (CD71). It was stained with anti-CD4-PerCP and anti-CD69-FITC or anti-CD71-FITC to investigate the activation of CD4+T cell by flow cytometry.(2) Freshly isolated CD4+T cell from SMC were cultured with or without resveratrol for36hours. It was stained with annexin V/PI to evaluate the apoptosis of CD4+T cell.(3) Splenic mononuclear cell or freshly isolated CD4+T lymphocytes were stained with CFSE and cultured with ConA (for splenic mononuclear cell) or IL-2/anti-CD3/CD28(for CD4+T cell) with or without resverarol in vitro for4days to investigate the proliferation by flow cytometry.(4) Freshly isolated SMC were activated with PMA and ionomycine after having been cultured with or without resveratrol for2hours. It was stained with anti-CD4-PerCP, anti-IL-4-PE, anti-IFN-y-APC and anti-IL-17-Alexa Fluor488to investigate Th1, Th2and Th17by flow cytometry.Results1. Pristane-induced lupus mice had proteinuria(8/10), rash(5/10), and high level of ANA, anti-ds-DNA and anti-RNP/Sm antibody in serum, which were significally higher than that in controls. Histological analysis demenstrated glomerulonephritis and IgG/M deposition in kidney from Pristane-induced lupus mice which were not detected in Controls.2. Mice in Model Control had proteinuria(8/10), rash(5/10), glomerulonephritis and IgG/M deposition in kidney. Mice in Resveratrol A had no rash, decreased proteinuria (4/10). Histological analysis demenstrated marked reduced pathological lesion in glomerulus and marked reduced IgG/M deposition in kidney compared with Model Control. A similar effect was also observed in Resveratrol B. No statistical differences were detected between the two resveratrol prevention groups. ANA, anti-ds-DNA, anti-RNP/Sm antibody level in resveratrol prevention groups were lower than that in the control, but there were no significantly statistical difference among them. 3. The levels of CD69of CD4+T lymphocyte were significantly increased after being activated with ConA, anti-CD3and anti-CD3/CD28respectively. Resveratrol (10μM,20μM,40μM,80μM) significantly decreased the levels of CD69of CD4+T lymphocyte activated with anti-CD3in dose-dependent manner, and resveratrol (20μM,40μM,80μM) significantly decreased the levels of CD69of CD4+T lymphocyte activated with ConA, anti-CD3/CD28in dose-dependent manner.The levels of CD71of CD4+T lymphocyte were significantly increased after being activated with ConA, anti-CD3and anti-CD3/CD28respectively. Resveratrol (10μM,20μM,40μM,80μM) significantly decreased the levels of CD69of CD4+T lymphocyte activated with ConA, and resveratrol (20μM,40μM,80μM) significantly decreased the levels of CD69of CD4+T lymphocyte activated with anti-CD3, anti-CD3/CD28. The hibition capacity depended on the dosage.4. Resveratrol (10μM,20μM,40μM,80μM) significantly induced the apoptosis of freshly isolated CD4+T cell in dose-dependent manner.5. Resveratrol (10μM,20μM,40μM,80μM) significantly suppressed the proliferation of splenic mononuclear cell in dose-dependent manner (P<0.05), and resveratrol (20μM,40μM,80μM) significantly suppressed the proliferation of CD4+T cell in dose-dependent manner (P<0.05).6. Resveratrol (20μM,40μM,80μM) inhibited Thl and decreased ratio of Thl/Th2in dose-dependent manner (P<0.05). Resveratrol had no effects on Th2and Thl7.ConclusionPristane can induce BALB/c mice to acquire lupus. Resveratrol possesses lupus-preventive effect. It can inhibit activation and proliferation of CD4+T cell, induce their apoptosis, decrease Th1and the ratio of Th1/Th2. Resveratrol may represent a novel approach to the management of SLE.