The Study Of The Inhibitory Effects By CsA On The Immunity And NF-κB Pathway Activation In The Brain Of STZ-induced Diabetic Rats

The study of the inhibitory effects by CsA on the immunity and NF-κB pathway activation in the brain of STZ-induced diabetic ratsBackgrounds In our clinical practice, many biopsies are being carried out. Thepathological evidence showed that there were depositions of immunoglobulins andcomplements which would definitely damage the tissues they deposited on. On theother hand, however, there were many complications in DM, such as diabeticnepropathy, diabetic retinopathy, diabetic neuropathy and so on. The stroke anddiabetic encephalopathy are believed to be the main manifestations of central nervoussystem (CNS) in diabetes mellitus (DM). To make it clear, many organs have beeninvestigated in the project by patho-immunehistochemistry to study if there wasautoimmune injuries. Meanwhile, cyclosporine was also administered to thoseSTZ-diabetic rats to evaluate if those injuries could be prevented or even diminishedby the treatment with immuno-depressive agent, cyclosporine A.It has been believed for a long period of time that oxidative stress is the maincause of chronic diabetic complications including diabetic encephalopathy. From themetabolic point of view, anti-oxidative therapy is effective in decreasing radicalproduction, but not in chronic diabetic complications. We have found that DM isaccompanied by many autoimmune diseases. Our previous work has proved that therewere autoimmune injuries to the multiple organs in STZ-induced diabetes rats, at thesame time, depositon of immunoglobulins was found in kidneys, skins and muscles ofthe diabetics by biopsy and many recent studies also suggested that immunologicinjuries might play a more important role in the pathogenesis of DM. Therefore wepresumed that there might be a close correlation between the occurrence of diabetichyperglycemia, oxidative stress and abnormal immunity, diabetes and chroniccomplications may be the results of autoimmune leisions to multiple organs. It hasbeen confirmed that the brain has its relatively independent of immune system. Thecurrent study is just a part of the whole project. There is a possibility that thefunctions of blood brain barrier (BBB) might be impaired in DM, and oxidative stress would be easily occur and activate NF-κB in the brain. Do abnormal humoral and cellimmunity induce immune and inflammatory activation in the brain and result indiabetic pathologic abnormalities by the activation of NF-κB pathway? Our workhave proved that cyclosporine A (CsA) could decrease depositon of immunoglobulinsin multiple organs and inhibit higher expression of many inflammatory cytokines suchas monocyte chemoattractant protein (MCP)-1 and matrix metalloproteinases (MMPs)in diabetic rats.Does CsA work on the immune activation in CNS? Related studiedhave not been reported.Objectives 1. morphological changes of the brain, activation extent of astrocytesand complement expression as well as the intervening effects of CsA were studied. 2.The relationship between autoimmune injuries and oxidative stress was evaluated andthe effects of the activation of immunity on oxidative stress was also studied,including NF-κB pathway and related cytokines as well as the effects of CsA in thebrain of STZ-induced diabetic rats. The autoimmune injuries are now confirmed to bethe one of the factors involved either in the development or the pathogenesis of DM.The present study may provide experimental evidence to support the immunodepressive treatment for the DM patients with those chronic complications related tothe brain. Our conclusive data may be helpful for supporting the hypothesis thatinfection-autoimmune-inflammation-organ injuries-dysfunctions of organs may be acommon cascade for the pathogenesis of many diseases while DM is just one of them.Materials and methods Sprague-Dawley rats were randomly divided into thefollowing groups: control group(CON group), diabetic group without anytreatment(DM group), insulin-treated group(INS group) and three diabetic groupstreated with low(0.5mg/kg.d), middle(1mg/kg.d), high(2mg/kg.d) dose of CsA(ML,MM and MH group). The DM rat models were made by intravenous administration ofSTZ (50 kg/kg) from caudal vein. After modeling, The three CsA-treated groups weregiven CsA intervention through subcutaneous injection respectively post modeling asthe above corresponding dose respectively while the insulin-treated group treated with2～6 IU/kg.d intermediate-acting insulin so as to keep glycemia at about 10mmol/L.All the rats were monitored simultaneously and sacrificed at the end of 16 weeks postmodeling. Morphological changes of the brain were observed by hematoxylin andeosin staining (HE staining), the deposition of immunoglobulins was detected byimmunohistochemistry and immunofluorescence, the immunohistochemical expression of GFAP and complement 3b in the brain were studied. The content oflipid peroxide product—Malonaldehyde (MDA), the activities of anti-oxidativeenzymes including total superoxide dismutase (T-SOD), CuZn-SOD, glutathioneperoxidase (GSH-Px) and catalase (CAT) were measured in brain homogenaterespectively. At the same time, the expression of NF-κB, tumor necrosis factor-alpha(TNF-α) and MCP-1 on the protein and mRNA level in the brain were detected byimmunohistochelnistry and TNF-αand MCP-1 mRNA expression on the gene levelby RT-PCR.respectively.Results There were not significant changes in the brain in 16-week diabetic ratsmorphologically. Compared with CON group, there were obvious deposition of IgG,IgA on the vessel walls in the cortex and thalamus in DM group, higher GFAP,C3b,NF-κB,TNF-αand MCP-1 expression on the protein level and TNF-αandMCP-1 expression on the mRNA gene level in the brain were also found. At the sametime, the content of MDA was significantly higher while the activities of allanti-oxidative enzymes were obviously decreased in the brain. Compared with DMgroup, the changes of the above parameters in CsA groups were improved in differentdegrees, especially those in middle and high dose of CsA group.Conclusions 1. There is a strong evidence to show that there was deposition ofimmunoglobulins (Igs) in the brain of diabetic rats, probably resulting fromautoimmune injuries. CsA can make the deposition of Igs declined or even vanishedby its depressive effects or by regulating the immunity of the body. 2. There are anextensive activation of astrocytes and higher complement expression in the brain ofdiabetic rats. The data suggest that CsA might have an inhibitory effects on extensiveactivation of astrocytes and complement expression in the brain. 3. There areOxidative stress, activation of NF-κB and related inflammatory cytokines pathway inthe brain of diabetic rats, which might be one of the mechanisms of abnormalsystemic immunity to activate immune reaction in central nervous system andeventually contribute to neuronal damage and diabetic complications in the brain. Asan immune depressive agent, CsA may prevent the autoimmune injuries to organs firstand then the oxidative stress and the activation of NF-κB pathway would be blockedeventually, As the result, the immune and inflammatory damage to the brain ofdiabetic rats may be protected. 4. The autoimmune injuries may be a initiating step to cause the oxidative stress happened to the brain.