The Beneficial Effects And Mechanisms Of Some Anti-complement Agentson Lupus Prone Mice

1. Beneficial effect of Bupleurum polysaccharides on autoimmune-prone MRL/1pr miceAim:To determine the beneficial effect of Bupleurum polysaccharides (BPs) on autoimmune-prone MRL/lpr mice and further support the efficacy of BPs on systematic lupus erythematosus (SLE). To undercover the mechanism of BPs effect by detecting its in vivo effects on macrophages and complement.Methods:Mice were randomly grouped into six groups:Normal (Balb/c), Model (MRL/lpr), BPs 15,30,60 mg/kg (MRL/lpr) and Prednisone (MRL/lpr). Mice were orally treated from the age of 12 weeks for three months. For evaluation of BPs efficacy, survival rate and body weight were recorded; the index of spleen, thymus and lymph node were measured; the serum anti-dsDNA, ssDNA, histone and total IgG level were tested by ELISA; the serum creatine level was tested using CreatinineJaffe method kit; Proteinuria was measured by Coomassiebrilliant blue test; Glomerular injury was blindly semiquantified by a renalpathologist; IgG deposits were measured by immunohistochemistry. To examine the effect of BPs on macrophages, RT-PCR and Western were used to test the expression of IFN-γ, IL-6, MCP-1, MHCII, F4/80 and C3 in the kidney.Results:a) Effect of BPs on survival rate and body weight.Compared with model group, BPs 15 mg·kg-1 (87.5%),30 mg·kg-1 (100%) and 60 mg-kg-1 (100%) groups had increased survival rate, while there was no significant difference in body weight.b) Effect of BPs on lymphadenopathyThere was no significantdifference in lymphadenopathy among five groups until 16weeks of age. BPs 60mg-kg-1 significantly delayed thelymphadenopathy after 8 weeks of treatment and prednisoneafter 9 weeks of treatment (P< 0.05).c) BPs Decreased Organ Index of MRL/lpr Mice.The indexof lymph node, thymus, and spleen increased significantlyin model group when compared with controlgroup. BPs inhibited lymph node swelling (P< 0.01),administration of 30 and 60 mg·kg-1BPs inhibitedthymus swelling (P< 0.05), while prednisone treatmentsignificantly decreased the index of lymph node, thymus, andspleen (P< 0.05).d) BPs Reduced Autoantibody and Total IgG Levels in MRL/lprMice.Anti-dsDNA,anti-ssDNA, and anti-histone antibody levels weresignificantly elevated in the model group compared with control group(P< 0.001) and were significantly reducedby BPs 15,30,60, or prednisone 5 mg·kg-1 (P<0.001).Furthermore, total IgG levels were assayed. Similarly,total IgG levels were significantly elevated in model groupcompared with control group (P<0.001) and were reduced by BPs 15,30,60, orprednisone 5 mg·kg-1 significantly (P< 0.001).e) BPs ameliorated renal injury.In comparison with control group, model group exhibitedsignificant increase in the level of urinary protein (P<0.001), indicating a certain degree of kidney dysfunction.However, BPs treatment resulted in a reduction in the levelsof urinary protein.Vehicle-treated MRL/lpr mice developeddiffuse proliferative glomerulonephritis presented as diffusemesangial matrix expansion, profound mesangial cell proliferation,and focal segmental glomerulosclerosis. Cellularcrescents and global glomerulosclerosis were often seen, andthe number of both resident cells and infiltrating leukocyteswas increased in glomeruli. Most animals revealed profoundtubulo interstitial inflammation as characterized byperiglomerular and diffuse interstitial leukocyte infiltrates,tubular atrophy, and intraluminal cast formation. Treatmentwith BPs suppressed lupus nephritis as documented bya significant reduction of the glomerulonephritis scoresthat encompasses glomerular cell proliferation, endocapillaryhypercellularity, crescent formation, sclerosis, tubularatrophy and casts.Vehicle-treatedMRL/lpr mice showed a patchy dense immunoperoxidaseindicative ofmesangial and tubulointerstitial IgG deposition.In contrast, BPs treatment significantly decreased IgG deposition.f) BPs Suppressed Inflammatory Mediators and MarkersExpression in Kidney.The expressionof IFN-y, IL-6, MCP-1 mRNA, and protein was greatlyelevated in vehicle-treated MRL-lpr mice and was reducedin varying degree from BP-treated mice compared with thevehicle-treated mice. BPs treatment reduced the mRNA andprotein expression of the surface marker MHC-II and F4/80. C3 levels were also reduced in the kidney of BPstreatment group, as was shown in Western blotting results.Conclusion:this study demonstrated that BPs improveslupus nephritis mainly by suppressing abnormal autoimmunityof SLE. Our analysis proves the therapeutic efficacy ofBPs in the treatment of SLE in MRL/lpr mice.2. Effects of Arnebia eachromapolysaccharides on immune cells and MRL/lpr mice.Aim:To examine the effects of Arnebia euchromapolysaccharides (CAEP) on macrophage, dendritic cell and splenic cell of Balb/c mice, and to provide support for its in vivo effect. To study the effect of CAEP on MRL/lpr mice and find its mechanism and target.Methods:The effects of CAEP on cell viability and production of cytokines of RAW264.7, peritoneal resident macrophage, peritoneal recruited macrophage and dendritic cell:cells were stimulated with LPS (TLR4), imiquimod (TLR7) or CpG (TLR9), and treated with CAEP (40、80μg/ml). Cell viability was measured by the MTT assay. The production of NO by RAW264.7 was measured by Griess. The expression of IL-6 and TNF-a by macrophage, IL-6, TNF-a and IL12p40 by dendritic cell were measured by ELISA.The effects of CAEP on surface marker expression of dendritic cell:cells were stimulated with LPS, and the surface expression of TLR4, MHCII and CD86 were measured with Flow Cytometry.The effects of CAEP on cell viability and production of cytokines of splenic cell: cells were stimulated with ConA, and treated with CAEP (10/20、40、80μg/ml).Cell viability was measured by the MTT assay. The expression of IFNy was measured by ELISA.MRL/lpr mice were grouped into two groups:Model and CAEP 60mg/kg. Mice were orally administered with normal saline or CAEP from the age of 12 weeks for a month. For evaluation of CAEP efficacy, survival rate and body weight were recorded; the index of spleen, thymus and lymph node were measured; Proteinuria was measured by Coomassiebrilliant blue test.To test CAEP in vivo effect on immune cells, cell surface expression of MHCII and CD86 on splenic dendritic cell and macrophage were measured by flow cytometry. Splenic cells were cultured, and the supernatant expression of IFNy was measured by ELISA.Results:a) Effects of CAEP on RAW264.7.CAEP inhibited the viability of RAW264.7 (P<0.05), but promoted the production of NO (P<0.01). The viability of RAW264.7 decreased after LPS stimulation, but the NO production increased (P<0.001). Compared to LPS group, CAEP inhibited the production of NO (P<0.001). Imiquimod and CpG promoted the production of NO (P<0.001). Compared to imiquimod, CAEP inhibited the cell viability, increased NO production (P<0.01). Compared to CpG, CAEP promoted the NO production (P<0.001) but had no effect on cell viability.b) Effects of CAEP on peritoneal resident macrophage of Balb/c mice.Compared to control group, CAEP significantly promoted the cell viability and expression of IL-6 and TNF-a(P<0.001). The cell viability and expression of IL-6 and TNF-a increased after LPS stimulation (P<0.001). Compared to LPS group, CAEP further increased the viability and IL-6 production (P<0.001). Imiquimodincreased the cell viability and expression of IL-6 (P<0.001) while had no effect on TNF-a production. Compared to imiquimod group, CAEP further increased the viability and production of IL-6 and TNF-a (P<0.001). The cell viability and expression of IL-6 and TNF-a increased after CpG stimulation (P<0.001).Compared to CpG group, CAEP further increased the viability and IL-6 production (P<0.05), but inhibited TNF-α(P<0.01).c) Effects of CAEP on peritoneal recruited macrophageof Balb/c mice.Compared to control group, CAEP 80μg/mlsignificantly promoted the cell viability (P<0.05). CAEP increased the expression of IL-6 and TNF-α(P<0.001). The cell viability and expression of IL-6 and TNF-a increased after LPS stimulation (P<0.001). Compared to LPS group, CAEP increased the viability, decreased IL-6 production (no significance), had no effect on TNF-a. Imiquimoddecreased the cell viability (P<0.01), increased the expression of IL-6 and TNF-α (P<0.001). Compared to imiquimod group, CAEP had no effect on the viability but decreased the production of IL-6 and TNF-α (P<0.05). The cell viability decreased and expression of IL-6 and TNF-a increased after CpG stimulation (P<0.001).Compared to CpG group, CAEP increased the viability inhibited TNF-a(P<0.01), but had no effect on IL-6.d) Effects of CAEP on dendritic cellof Balb/c mice.Compared to control group, CAEP had no effect on the cell viability, the production of IL-6 and TNF-a is relatively low by dendritic cells, and CAEP had no effect. But CAEP decreased the largely produced IL 12p40 (P<0.01). The cell surface marker TLR4, MHCII and CD86 were not affected by CAEP. The expression of IL-6, TNF-a and IL12p40, but not cell viability increased after LPS stimulation (P<0.01), so as to the expression of TLR4, MHCII and CD86. Compared to LPS group, CAEP had no effect on the viability, decreased IL-6, IL 12p40 (P<0.01) and TNF-a(no significance)production, decreased the expression of TLR4,MHCII and CD86. Imiquimodincreased the cell viability and the expression of IL-6, TNF-a and IL 12p40 (P<0.001). Compared to imiquimod group, CAEP had no effect on the viability but decreased IL-6, IL 12p40 (P<0.001) and TNF-a(no significance)production. The cell viability remained the same but the expression of IL-6, TNF-a and IL12p40 increased after CpG stimulation (P<0.001).Compared to CpG group, CAEP had no effect on the viability but inhibited IL-6, TNF-a and IL12p40 production (P<0.01).e) Effects of CAEP on splenic cellof Balb/c mice.CAEP 20,40,80μg/ml promoted splenic cell viability without the stimulation of ConA, while had no effect on splenic cell with the stimulation of ConA.ConA 2.5μg/ml significantly increased the production of IFNy. CAEP inhibited ConA-induced IFNy production in a dose-independent manner. While as to the IFNy increase induced by ConA 5μg/ml, only CAEP 80μg/ml had inhibitory effect.f) In vitro effect of CAEP on immune cells of MRL/lpr mice.CAEP promoted peritoneal resident macrophage viability and IL-6 production without or with stimulation of LPS or CpG. CAEP 80μg/ml activated macrophage with stimulation of imiquimod.CAEP had no effect on dendritic cell viability, but suppressed IL-6 production of dendritic cell without stimulation of CpG. LPS increased expression of MHCII and CD86 expression of dendritic cell, while CAEP had no effect. ConA increased IFNy production of splenic cell, while CAEP had no effect on splenic cell viability or secretion.Conclusion:CAEP promoted the function of resident macrophage, while inhibited the function of induced macrophage, suggesting the immunomodulatory effect of CAEP on macrophage. CAEP suppressed pro-inflammatory function of dendritic cell, and might further suppress the function of splenic cell. CAEP had stronger inhibitory effect against TLR9 ligands.CAEP had no therapeutic efficacy on MRL/lpr mice, and had different effect on immune cells between Balb/c and MRL/lpr mice.