雷公藤多甙对急性移植物抗宿主病小鼠的作用

作者：于艳秋,张义侠,金玉楠,卢晓梅,王巍　　作者单位：中国医科大学基础医学院病理生理教研室，沈阳 110001; 1中国医科大学附属医院消化内科，沈阳 110001

　　【摘要】本研究应用雷公藤多甙防治小鼠急性移植物抗宿主病。对受致死量照射的C57BL/6受鼠，注入供鼠BABC/c骨髓与脾淋巴细胞的混合液，并给予雷公藤多甙、环孢素A、甲氨蝶呤处理。用免疫组织化学法、流式细胞术和ELISA法检测各组小鼠T淋巴细胞、黏附分子、细胞因子。结果表明： 所有异基因对照组小鼠，在30天内死于aGVHD,而雷公藤多甙组(19/21)、环孢素A+甲氨蝶呤组(13/21)和雷公藤多甙+环孢素A组(17/21)的大部分小鼠存活时间超过了30天，并没有明显的aGVHD症状表现。雷公藤多甙、环孢素A、甲氨蝶呤可明显降低皮肤、肺组织CD3+、CD4+、CD8+、CD11a+、CD18+细胞(P<0.05)和脾组织CD3+, CD4+, CD8+, CD4+CD11a+, CD4+CD18+, CD8+CD11a+ , CD8+CD18+细胞(P<0.05)。而小肠组织CD3+、CD4+、CD8+细胞变化不明显(P>005)。同时，雷公藤多甙可降低受鼠血清中IL-2、TNFα浓度和脾组织IL-2、TNFα的mRNA的表达(P<0.05);上调血清中IL-10的水平(P<0.05)，但对IL-4的作用不显著(P>0.05)。结论：雷公藤多甙具有明显抗aGVHD作用并同时保留抗白血病效应，其作用机制与调节促炎/抑炎细胞因子的分泌和黏附分子的表达，以及抑制T淋巴细胞作用有关。
　　【关键词】 雷公藤多甙;移植物抗宿主病; T淋巴细胞; 黏附分子;细胞因子

　　This project was supported by Young Fundation of State Administration of Traditional Chinese Medicine Fund (国家中医药局青年科学基金)(No.2000-J-Q-06)　Glucosidorum Tripterygii Tororum (GTT) is a kind of Chinese traditional medicine (CTM), for the first time used in China as an immunoregulatory agent which is often used in the treatment of autoimmunological diseases such as rheumatoid arthritis (RA) and nephrotic syndrome. Our previous experiment showed that GTT can decrease the population of lymphocytes, inhibit the secretion of IL-2 and ex[x]pression of IL-2R, down-regulate the sensitivity of T cells to IL-2, and inhibit the ex[x]pression of adhesion molecules (CD11a, CD18) on the surface of T cells. And it can prolong the survival time of allogeneic transplanted skin. Its effect on anti-rejection is weaker than CsA and stronger than methylprednisolone. And, the combination of GTT with them shows a much more stronger effect, indicating that GTT can moderately or obviously inhibit the function of T cells and the secretion of cytokines[1]. Furthermore, GTT may have anti-inflammatory and anti-tumor effects. Graft-Versus-Host disease (GVHD), especially acute Graft-Versus-Host disease (aGVHD) is a major lethal complication after bone marrow transplantation (BMT). Therefore, this study was aimed to adopt GTT or combine GTT with CsA in order to explore the role of GTT on aGVHD and its mechanism.

　　Materials and Methods

　　Mice

　　Donor mice BALB/c(H-2d), female; syngeneic transplantation recipient mice BALB/c(H-2d), male; allogeneic transplantation recipient mice C57BL/6(H-2b) male. All of them were 8-12 weeks old and weight of about 20 g (provided by Institute of Zoology, Chinese Academy of Sciences).

　　Induction of aGVHD

　　The recipients received whole body irradiation by a li- near accelerator (total dose is 1800 cGy, rate of dose is 200 Gy/min, SIMENZ, Primus, Germany) for 6-8 hours before transplantation. Bone marrow and spleen cells of female BALB/c(H-2d) were prepared in RPMI 1640 medium. Both BALB/c and C57BL/6 recipients were injected with 1×106 bone marrow cells and 5×106 lymphocytes via caudal vein.

　　Assignment and process

　　After transplantation, the recipients were randomly divided into following 5 groups: syn-BMT group, allo-BMT group, CsA+MTX group, GTT group and GTT+CsA group. All recipients were bred in aseptic room. Their food and water were sterilized by autoclaving, and gentamicin (320 mg/L) and erythromycin (250 mg/L) were added into the drinking water.The recipients in syn-BMT group and allo-BMT group were intraperitoneally administrated with 0.9% NS 0.2 ml; in CsA+MTX group with CsA 2.5 mg/kg everyday and MTX 7 mg/m2 on the day 1,3, 5 and 9; in GTT group with GTT 5 mg/kg; in GTT+CsA group with GTT 2.5 mg/kg and CsA 2.5 mg/kg. GTT was dissolved into 1 mg/ml by Tween 80 before injection.

　　Evaluation of aGVHD and Histopathology

　　The recipient mice were weighed three times weekly and observed for clinical symptoms of a GVHD (weight loss, lassitude, inappetence, depilation and hunch back). The recipient mice of allo-BMT group had the significant clinical symptoms of aGVHD, such as weight loss, lassitude, inappetence, depilation and hunched posture, whereas recipient mice of syn-BMT group had not. The recipient mice of GTT, GTT+CsA and CsA+MTX groups showed only weight loss, furthermore, inappetence could be observed in GTT and GTT+CsA group.On day 14 after BMT, the tissue samples of skin, lung and intestine were taken from all recipient mice and immediately fixed in 10% formaldehyde, then em[x]bedded in paraffin and cut into slices, stained with hematoxylin and eosin, and examined microscopically.

　　Immunohistochemistry

　　The tissue samples were fixed in 10% formaldehyde, em[x]bedded in paraffin, and cut into slices, and then dewaxed, soaked in 10% H2O2 for 10 minutes and dripped with 10% serum of calf. The slices were stained with FITC conjugated monoclonal anti-mouse CD3, CD4, CD8, and PE conjugated monoclonal anti-mouse CD11a,CD18,each 8μl (Sigma). All the slices were incubated in dark for 1 hour, washed with PBS for three times, sealed by glycerd. The infiltration of lymphocytes was observed with fluorescent microscopy and the photos were taken. And then, the pictures were processed with Real Time Image Processing System (LUZEX F NIRECO/NIKON, Japan) and the fluorescent area of every field of vision was calculated (×105/μm2 ).

　　Flow cytometry analysis

　　The spleens were removed and single-cell suspensions were prepared to determine the number of T lymphocytes in spleen. The spleen cell suspensions were depleted of erythrocytes by lysis with 0.83% Tris-buffered ammonium choride, then washed twice in NaN3-Hanks' solution. Spleen cells (1×107/ml) were incubated with FITC-conjugated monoclonal anti-mouse CD3, CD4, CD8 antibodies and PE conjugated monoclonal anti-mouse CD11α, CD18 antibodies. Surface ex[x]pression was analyzed on a FACStar flow cytometer (Becton Dickinson) using the LYSIS program (Becton Dickinson). Values shown are the percentage of positive cells in the lymphocyte-gated cell population from the spleen of recipients to determine the percentage of CD3+, CD4+, CD8+, CD4+ CD11α+, CD4+ CD18+, CD8+ CD11α+, CD8+ CD18+ cells by FACS.

　　ELISA

　　Blood of recipient mice was collected using heparinocoated syringes, anticoagulated with EDTA and diluted once. The concentration of IL-2, TNF-α, IL-4 and IL-10 were determined by ELISA kits.(DIACLONE).

　　RT-PCR

　　According to the TRIZOL's instruction, total mRNA of spleen was respectively isolated from mice of each groups. The primers of IL-2, IL-4, TNF-α and IL-10 were designed according to the sequence reported by GenBank (producted by TaKaRa company, Japan), shown in Table 1. One microgram of RNA from each group was reverse-transcribed into cDNA using a reverse transc[x]ription system (Promega. Access RT-PCR system). PCR reactions were then performed using primers for β-actin, IL-2, TNFα, IL-4 and IL-10 (TaKaRa Japan). Table 1. Sequences of RT-PCR primers(略)

　　The conditions for RT-PCR reaction of IL-2 were as follows: 45 minutes at 48℃, 3 minutes at 94℃, then repeated the cycle of 1 minutes at 94℃ , 5 minutes at 62℃ ,1.5 minutes at 72℃ for 35 cycles, final extension for 7 minutes at 72℃.The conditions for IL-4 and IL-10 were similar except the annealing step, at 64℃, the annealing step for TNF-α was set at 48℃.In order to determine the product of RT-PCR reaction, 10 μl product was taken for polyacrylamide electrophoresis and stained with ethidium bromide. The gel was scanned and analyzed by UVP gel-image analytic system. The calculating formula for the level of relative mRNA ex[x]pression of IL-2, IL-4, TNF-α and IL-10 was as follows: The ex[x]pression a certain mRNA equals the ratio of scanned value of the gene to that of β-actin.

　　Statistical analysis

　　All determineted values were presented in the form of means ±standard deviation and the significance of the differences between every group was determined by t-test.

　　Results

　　Survival rates of the recipients

　　The survival rates of the recipients were counted on 30 days after transplantation were shown in Figure 1.

　　Pathology of target organs

　　Epithelial cells of skin and mucous membranes, lung and intestinal crypts are primary tissues damaged during the pathobiological course of aGVHD. Tissue samples of the skin, lung, intestine of the recipient mice were also analyzed for pathology associated with murine aGVHD (Figure 2).

　　C57BL/6 mice were induced for aGVHD and then treated. A-F, sections of the skin, lung and intestine were removed from allo-BMT and GTT-treated mice on day-14 post-transplant, and stained with HE.

　　In the tissue slices from the mice of allo-BMT group, massive infiltration of inflammatory cells can be seen in skin, pulmonary interstitium and intestinal wall (A, B, C). In slices of lung tissue (B),we can also observe erythrocytes diffused in alveoli and formation of bullae. Destruction of villi and dilation of gut can be seen in intestinal slices. In contrast, GTT-treated mice (D, E, F) had a decrease of inflammatory cells both in skin and lungs, and the formation of pulmonary bullae was not observed. But the lesions in intestine were not improved, compared with allo-BMT mice.

　　T lympocyte infiltration and CD11a+, CD18+ cells in target organ

　　The results obtained by image analyzer showed that GTT and GTT+CsA can obviously decrease the number of infiltrated CD3+, CD4+, CD8+ cells and the ex[x]pression of adhesion molecules CD11α and CD18 (Figure 3A and 3B) in skin and lung. While in intestine, the declination of CD3+, CD4+, CD8+ cells and adhesion molecules had no statistical significance, which may be mainly attributable to the strong toxicity of GTT on gastrointestinal tract. Interestingly, CsA+MTX had an influence on T cells and their subgroups in intestine, that was obviously better than GTT and GTT+CsA, but no obviously differential effect on skin and lung, compared with those two groups. This study also observed some differences between GTT and GTT+CsA , but without statistical significance.

　　FACS was used to determine the amount of T cells, their subgroups and the adhesion molecules (Figure 4). The results showed that GTT and GTT+CsA groups could decrease splenic T cells and their subgroups, and the ex[x]pression of adhesion molecules.

　　Serum concentration of IL-2 ,IL-4,TNF-α and IL-10

　　The results of serum level of IL-2, IL-4, TNF-α and IL-10 detected by ELISA were shown in Figure 5. The concentrations of IL-2 and TNF-α in serum obviously decreased (P<0.05), but the concentration of IL-10 dramatically increased (P<0.05) by GTT and GTT+CsA treatment.

　　ex[x]pression of IL-2, IL-4, TNF-α and IL-10 mRNA in splenic cells

　　RT-PCR revealed that GTT and GTT+CsA treatment lowered the transc[x]ription level of IL-2 and TNFα which can facilitate aGVHD in spleens (P<0.05);on the other hand, the level of IL-10 mRNA inhibiting aGVHD showed a significant increase (P<0.05). The change of IL-4 was not obvious (P>0.05) (Figure 6).

　　Discussions

　　The pathological mechanism of aGVHD is very complicated. The fundamental reason for aGVHD is the difference in MHCs between donor and recepient, in which T cells play a key role. CD8+ T cells (mainly CTL cells) can attack target cells directly, and CD4+ T cells (Th1, Th2) can promote aGVHD by secreting cytokines[2,3]. The recent researches indicated that the basis of aGVHD is the imbalanced cytokine network, being summarized by "cytokine storm". The term was used and appropriately defined in aGVHD[4-6]. The imbalance between aGVHD promoting cytokines such as IL-1, IL-2, TNF-α , and the inhibiting cytokines such as IL-4, IL-6 and IL-10 may be the main mechanism of aGVHD[7,8,9]. The amount of cytokines and their secreting cells (mainly T cells and their subgroups) plays an important role in aGVHD and also is an important index for evaluating the curative effect. In this study, it was observed that the mice of allo-BMT group showed typical aGVHD pathological features and clinical symptoms. The number of T cells and their subgroup and the ex[x]pression of adhesion mo-lecules (in skin, lung and intestine) surpass that of syn-BMT group remarkably (P<0.05). On day 30 after transplantation, the survival rate was improved by GTT treatment (P<0.05). And GTT could alleviate the pathological changes of the inflammatory cell infiltration in skin, lung and intestine. Meanwhile, the generalized immune function of mice was suppressed. In our experiment, GTT could act against aGVHD through modulating mRNA transc[x]ription and protein synthesis of cytokines.Besides, the survival rates of mice in CsA+MTX group (13/21), and in GTT+CsA group (17/21) were obviously lower than that in GTT (19/21) (P<0.05). It could be related with the stronger side effect of combination usage of CsA+MTX or GTT+CsA. And intestinal tissue showed no significant alleviation in GTT group, although GTT may decrease the amount of CD3+, CD4+, CD8+ cells and adhesion molecules CD11α, CD18 in intestinal tissue ,but no statistical significance, It may be caused by the toxicity of GTT on gastrointestinal tract. And how to overcome such toxicity needs a further study.In conclusion, GTT could increase the survival rate of aGVHD mice and alleviate the injury of organs and tissue so that it has a significant effect on anti-aGVHD. The effect of GTT on anti-aGVHD may be related to the following factors: (1) GTT can decrease the infiltration of CD3+, CD4+, CD8+ T cells and the ex[x]pression of adhesion molecules CD11α, CD18 in local target organs of aGVHD mice; (2) GTT can inhibit the generalized immune function. It decreases the number of T lymphocytes, their subgroups and the ex[x]pression of adhesion molecules on their surface in spleen; (3) GTT can up-regulate the ex[x]pression of aGVHD-inhibiting cytokines IL-4, IL-10 and their mRNA; down-regulate that of aGVHD-activating cytokines IL-2, TNF-α and their mRNA.

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