The Effects Of Estradiol In The Development Of Systemic Erythematosus Lupus

The Effects of Estradiol in the Development of Systemic Erythematosus LupusObjectiveSystemic erythematosus lupus (SLE) is a chronic autoimmue disease. There are about one million patients in China, and multisystem and many organs of the patients are involved. The desease has severe impact on the body and psyche. SLE is characterized with autoantibodies such as antinuclear antibody (ANA), antihistone antibody (AHA) and others in serum and followed with kidney involved. If detected with immunofluorescence or electron microscope one hundred percent of the patients' kidneys were involved and as is called lupus nephritis (LN) and it has unfavourable prognosis. Up to now the pathogenesy of SLE is not clear, but it is thought that this disease has relations with genetics, female sex hormones, environmental agents, and so on.Animal models are necessary to explore the pathogenesy, diagnosis and treatment of SLE. SLE model mice have two categories. The first kind of animal models develop SLE spontaneously, and the latter need to be induced artificially. In addition the former falls ill slowly and has different pathological lesions, and the latter fall ill quickly and is easily controlled and have high consistency in clinical manifestation. So we induced mouse SLE model by injecting activated homologous splenic cells.More and more data showed there are apoptosis disturbances in SLE patients, in this situation, overacting apoptosis leads to a large quantity nucleosome released, the increased nucleosome sensitize autoreactive T lymphocytes and so is B lymphoctes activated. These were convinced in Clq, SAP and Dnasel gene knockout mice. But other researchers showed apoptosis repression could also make autoreactive lymphocytes escaping apoptosis and result into the development of SLE. So any formal apoptosis disturbance could be the beginning factor of SLE. Bcl-2 and NFkappaB are two main factors to controlling cell apoptosis. So in our experiment we detected splenic cells apoptosis and the expression of Bcl-2 and NFkappaB to reveal the mechanism of SLE.SLE has dominance in females and especially in reproductive females. All these phenomena show that female hormones have relations with SLE. There are some reports about the corelations of female hormones and autoimmune diseases. Now estrogen receptors in immune cells have been found and this show that estrogen has direct effect on immune cell through estrogen receptor, and so estrogen can influence SLE by this way. But among these studies about relations of estrogen and SLE, there have not coincident conclusions. This perhaps caused by the objects studied insufficient and the different physiological phase of them. So we use ovariectomized inbred strain Balb/c mice as our research objects and so can eliminate the interference of estrogen secreted from ovaries. We observe the influence of estradiol on the development of SLE through controlling the level of estradiol in vivo by injecting different doses of estradiol benzoate.In the development of SLE, the change of cytokine network has important impact on the course of SLE. Many factors have relations with SLE including the disproportion of Th1 and Th2 cytokines, increasing or decreasing of cytokines and augment or weakening of the rivalry or the synergistic action of cytokines. Studies about the changes of cytokines in SLE have been reported already and most of these reports considered that SLE is predominant in Th2 cytokines, but there are different opinions. However researches on proinflammatary cytokine interleukin-1(IL-1), interleukin-2 (IL-2) and turner necrosis factor alpha (TNF-α) were less reported. But these cytokines have been reported changed in SLE and so demonstrated they participate in the development of SLE. In our experiments we want to reveal the pathogenecy of SLE through detecting the changes of proinflammatary cytokines at different level of estradiol in vivo.Moreover, it is generally thought that the metabolism of estrogen is abnormal in SLE patients. Aromatase is the key enzyme transforming androgen to estrogen in different local tissue. Researches showed that aromatase expressing in the pituitary of rat has sex differences and is concerned with estrous cycle and is regulated by estrogen. Another study confirmed that proinflammatary cytokines (TNF-α,IL-6,IL-1) in rheumatoid arthromeningitis can obviously stimulate aromatase activity, and so androgen decrease and estrogen increase in local tissue. In our experiments we want to explore the pathogenecy of SLE through detecting the changes of proinflammatory cytokines and aromatase mRNA expression in renal tissue at different estradiol in vivo when SLE developed.MethodsI Induction of SLE mice model and the change of splenic cells apoptosis and its correlated regulating mechanism1. Preparing of activated homologuous series splenic cellBalb/c mice were sacrificed by cervical vertebra dislocation under anesthesia with aether. Spleens removed under asepsis were minced through a metal mesh to obtain single-cell suspensions. The cells were washed twice in phosphate-buffered saline (PBS) and counted. Cell density were regulated to 2×10⁶/ml with RPMI1640 containing 10% fetal calf serum. 500μg/ml ConA were added to the cells suspension and its final concentration is 5μg/ml. The splenic cell suspension were incubated at 5% CO₂, 37℃. After cultured for 72h activated splenic cells were cllocted and regulated to 2×10⁸/ml with isotonic Na chloride. Unactivated splenic cells suspensions without concanavalin A were prepared in the same condition as control.2. Grouping and treatment of Animals72 Balb/c mice were randomly grouped to 3 groups, 24 mice each group:â‘ Mice of model group (group MI) were injected 5×10⁷ the same inbred strain splenic cells activated by concanavalin A (ConA) subcutaneouly three times, once a week.â‘¡Mice of splenic cell control group (group CC) were injected the same dose unactivated splenic cell at the same position and in the same time.â‘¢Mice of normal control group (group NC) were injected isotonic Na chloride as control.3. Identification of SLE model mice(1) Collection of sampleIn the 4th week, 6th week, 8th week and 10th week after the first immunization, six mice of each group were randomly selected to be detected including antinuclear antibodies, antihistone antibodies in serum and pathological changes of renal tissue. Blood samples were centrifuged at 4500 rpm for 20 min at 4℃after collecting from the eyes under anaesthesia with ether, and serum was stored at -20℃until assays were performed. The samples of kidneys were removed also and divided to three sections. The first section was fixed with 10% buffered Formalin. The second part was snap frozen in liquid nitrogen followed by immunofluoresce assay. The third one was fixed in 3% glutaral buffer solution and prepared for electron microscopy.(2) Antinuclear antibody (ANA) and antihistone antibody (AHA) in peripheral blood were detected by enzyme linked immunosorbent assay (ELISA).(3) Observation of pathological changes in renal tissues of SLE model miceâ‘ Morphological changes of renal tissue were observed by light microscope after paraffin section followed hematoxylin-eosin staining.â‘¡IgG IC deposit in renal tissue were detected by direct immunofluorescence assay.â‘¢The ultrastructure changes of kidney were observed with transmission electron microscope.4. Apoptosis of splenic cells were detected by terminal deoxynucleotidyl transferase mediated nick end labeling (Tunel).5. Expression of Bcl-2 and NFkappaB of splenic cells were detected by immunocytochemistry. â…¡Influence of estradiol on induction of SLE model of Balb/c mice1. Grouping and treatment of Animals168 Balb/c mice were randomly divided to 7 groups, 24 mice each group. Group A is sham operation control, group B is operation group control, group C is sham operation model, group D is operation model and group E, F and G are respectively low, middle and high dose of estradiol (E2). The mice of group A, B, C and D were intramuscularly injected arachis oil as control after sham operation or ovariectomy. And the mice of group E, F and G were intramuscularly injected 0.1μg, 1μg and 10μg each mouse once two days. The mice of group C, D, E, F and G were induced SLE by injected activated the same inbred strain splenic cell after pretreated with arachis oil or E2 one week.2. Collection of samplesIn the 4th week, 6th week, 8th week and 10th week after the first immunization, six mice of each group were randomly selected to be detected including autoantibodies in serum and pathological changes of renal tissue. Blood samples were centrifuged at 4500 rpm for 20 min at 4℃after collecting from the eyes under anaesthesia with ether, and serum was stored at -20℃until assays were performed. The samples of kidneys were removed also and divided to four sections. The first section was snap frozen in liquid nitrogen followed by immunofluoresce scopy. The second part was fixed in 3% glutaral buffer solution and prepared for electron microscopy. The third one was fixed with 10% buffered Formalin for hematoxylin and eosin staining after paraffin section. The last one was levigated and the supernatant of homogenate was stored at -20℃for advanced detection.3. E2 in peripheral blood or kidney tissue and ANA and AHA in peripheral blood was detected by ELISA assay.4. Observation of pathological changes in renal tissues of SLE model mice(1) Morphological changes of renal tissue were observed by light microscope after paraffin section followed hematoxylin-eosin staining.(2) IgG IC deposit in renal tissue were detected by direct immunofluorescence assay.(3) The ultrastructure changes of kidney were observed with transmission electron microscope.â…¢Influence of estradiol on proinflammatory cytokines and the expression of aromatase mRNA in kidney of SLE mice1. Grouping and treatment of Animals were same to二,1,.2. Collection of samplesIn the 4th week, 6th week, 8th week and 10th week after the first immunization, six mice of each group were randomly selected to be detected. Blood samples were centrifuged at 4500 rpm for 20 rain at 4℃after collecting from the eyes under anaesthesia with ether, and serum was stored at -20℃until assays were performed. Kidneys were removed and divided to two sections. The first section was levigated and the supematant of homogenate was stored at -20℃for advanced detection. The scond section was prepared for detecting aromatase mRNA expression.3. Proinflammatory cytokines (IL-1, IL-8 and TNF-alpha) in peripheral blood and renal tissue were detected by ELISA.4. Detection of Aromatase mRNA expression in renal tissueAromatase mRNA in renal tissue was detected by reverse transcriptase polymerase chain reaction (RT-PCR). Total RNA was isolated from the kidney of the experimental mice using acid-guanidinium-phenol-chloroform method, cDNA were synthesized with oligo dT. The resulting cDNA samples were PCR-amplified using the following set of primers: Forward primer sequence 5'- GTATGAACGATCCGTCAAGG-3', reverse primer sequence 5'-AGCCGTCAATTACGTCATCC-3'. The PCR reaction consisted of 10μl cDNA, 0.5μl Taq DNA polymerase in a final volume of 50μl. The PCR were performed 25 cycles under the following conditions: 94^C for 30 second, 55"C for 1 minute, and 72℃for 1 minute. After an extra 10 minutes extension period at 72℃in the final cycle, the amplified DNA fragments were separated by 1.5% agarose gel electrophoresis.ResultsI Induction of SLE mice model and apoptosis of Splenic cells and its correlated regulating mechanism1. Levels of ANA and AHA in peripheral blood of SLE model miceANA and AHA were found in serum of every mice of group MI in the 4th week after firstly vaccinated with activated splenic cells and the level of them increased gradually and began to decrease in the 10th week. No mice of group NC and group CC were found ANA and AHA in serum.2. Pathological changes of renal tissues of SLE model mice(1) Morphological changes of renal tissueUsing light microscope, we found that the mice of group MI develop glomerular nephritis. Compared with that of the 4th week, the pathological integrates of the 10th week increased obviously (P＜0.01). There were not abnormal changes in group NC and group CC.(2) Immune complex deposit in kidneyUsing immunofluorescence assay we found flavo-green plaque deposit in the renal glomeruli of the mice of group MI and they distributed linearly and enhanced more and more. But there are not immunocomplex deposits in the kidney of the mice of group NC and group CC.(3) Ultrastructure changes of kidneyUsing transmission electron microscope we found the glomerulus sieve pore membrane of kidney fused, endothelial cell fall off, intercapillary cell proliferating and building in basal membrane and basal membrane deposited with immunocomplex. The renal ultrastructures of group NC and CC were perfectly.3. Apoptosis of splenic cells of SLE model miceCompared with group NC and group CC, there are lower apoptosis percentage of splenic cells in the mice of group MI.4. Expression of Bcl-2 and NFkappaB of splenic cells of SLE model miceCompared with group NC and group CC, there are higher apoptosis percentage of splenic cells in the mice of group MI.â…¡Influence of estradiol on SLE modeling of Balb/c mice1. Levels of E2 in peripheral blood and renal tissue of SLE model miceAfter ovariectomy the level of E2 of group B decreased obviously. Compared with that of group A and group B respectively, the level of E2 of group C and group D was higher and this demonstrated that inflammation can promote the level of E2. Among the other three groups, the level of E2 of group G is highest, the next is that of group F and the last is that of group E and compared with each other, there is obvious discrepancy. The level of E2 in renal tissue had the same changing tendency of that in serum, but the level has great disparity.2. Levels of autoantibodies in peripheral blood of SLE model miceThere are not ANA and AHA detected in serum of the mice of group A and group B. We found that ANA and AlIA appear in serum of the mice of the other 5 groups. Autoantibodies of group C and group D increase firstly in the 4th and 6th week and then decreased in the 10th week. This phenomenon coincides with typical immune response course. But autoantibodies in group E, group F and group G didn't decrease in the 10th week.3. Pathological changes of renal tissue of SLE model mice(1) Morphological changes of renal tissueThe mice vaccinated with activated splenic cell developed glomerular nephritis, that of group G was most serious, and the follows are group F and group E. pathological changes of group C is similar to that of group D.(2) Immune complex deposit in kidneyUsing direct immunofluorescence assay we found plaque deposits stained with fluorescein isothiocyanate (FITC) in glomerulus of kidney on the frozen section and the fluorescence intensity increased gradually in all the mice immunized with activated splenic cell. Among these groups, that of group G is the most serious.(3) Ultrastructure changes of kidneyWe found ultrastructure pathological changes including glomerular basal membrane, pore membrane, intercapillary cells, endotheliocytes and electron-dense deposits in the mice vaccinated with activated cells. These pathological changes coincide with the level of E2 and become more and more critical.â…¢Influence of estradiol on proinflammatory cytokines and the expression of aromatase mRNA in SLE mice1. Changes of proinflammatory cytokines in serum and renal tissue(1) Changes of IL-1 in serum and renal tissueThe level of IL- 1 in serum of the mice of group B increased compared with that of group A, The level of IL-1 in group C and group D increased in the 4th week and 6th week and then decreased in the 10th week and reached the level of group A. But the tendency did not appear in group E, group F and group G and there is obvious distinctition every each other, the mice of group G have the highest level. In the kidney tissue, the level of I1-1 has the same changing tendency, but it is higher in kidney tissue than that in serum. (2) Changes of IL-8 in serum and renal tissueThere was not obvious change of IL-8 in serum, only that of group C and group D increased in the 4th week and the 6th week. Compared with the level of IL-8 in kidney tissue of the mice of group A, that of the other five groups increased distinctly. The level of group C and D began to increase in the 4th week and then decreased in the 8th week. The level of group G was the highest among the other three groups.(3) Changes of TNF-αin serum and renal tissueCompared with that of group A, the level of TNF-αin serum of the mice of group B increased greatly, but the level of TNF-αof the other groups decreased obviously, and there are not difference among group C, D, E, F and G. Unlike the change tendency of TNF-αin serum, compared with that of group A, the level of TNF-αof all the other six groups increased but there are not obvious distinction among group E, F and G.2. Changes of aromatase mRNA expressionWe have observed GAPDH 530bp and aromatase gene 428bp straps by electrophoresis after PCR, and GAPDH as reference. The results of image analysis manifested that aromatase mRNA of group A expressed lowly, and compared with group A and B, that of group C and D expressed increasely. There are two straps in group E, F and G, and followed with the level of E2 increase in vivo, the expression of aromatase mRNA increased too.Conclutions1. Balb/c mouse were successfully induced to develop SLE when vaccinated with homologous splenic cell activated by Con A.2. Splenic cells apoptosis repression could be the beginning factor of SLE mice induced by activated homologous splenic cells.3. Upregulated Bcl-2 by NFkappaB leads to splenic cells apoptosis repression in SLE model mice.4. Estradiol in vivo could be decreased after ovarectomy, but could not prevent Balb/c mouse to develop SLE.5. Estradiol is the evidently distinctive promoting agent in Balb/c mouse SLE modeling.6. Estradiol influences the development of SLE via regulating proinflammatory cytokines.7. Estradiol influences the development of SLE via augmenting the expression of aromatase mRNA.