Combination Methotrexate And Cyclophosphamide Therapy According To Cell Cycle On Mice With Induced Sj(?)gren's Syndrome

BackgroundsSj(?)gren's syndrome(SS) is an autoimmune disorder that affects multiple exocrine glands, particular those that produce moisture to coat exposed epithelia such as the oral and ocular surfaces.It is associated with lymphocytic infiltration of the salivary and lacrimal glands and eventual atrophy of these tissues,leading to a loss of fluid production.It may affect multiple organs and tissues such as joints,skin,kidneys,heart,lungs,blood vessels,and the brain.It can cause extreme exhaustion,fevers,skin rashes,and can lead to scars,renal failure,central nervous system lesions and death.The diversity and complexity of symptoms in this multi-tissue disorder increase the difficulty of accurate diagnosis and effective treatment.SS results in a significant decrease in the quality of life for patients.Importantly,SS is considered under-diagnosed,under-treated and underresearched.Therefore,SS has considerable significance from a public health perspective.The role of apoptosis in loss of glandular tissue in SS is less clear.Environmental and genetic factors appear to contribute to the etiology of SS,although the evidence is relatively premature. T-cell-mediated cytotoxicity and autoantibodies are important in loss of gland function.There is also a failure to remove autoimmune T-cells at the level of thymic selection,resistance of T-cells within the gland to undergo apoptosis,aberrant expression of increased levels of cell adhesion molecules on glandular epithelial cells(facilitating infiltration of autoimmune lymphocytes to glands),up regulation of human leukocyte antigen(HLA)-DR,and polyclonal activation of B-lymphocytes.Glandular epithelial cells contribute to the autoimmune process by secreting pro-inflammatory cytokines.At present,there is no known cure for SS,nor is there a specific treatment to restore gland function.Treatment is generally symptomatic and supportive.For SS,xerostomia and xerophthalmia,artificial lubricants are commonly used as saliva or tear substitutes.It was suggested that gene therapy,haemopoietic stem cell transplant and biological agents might be some of the future treatments for SS by inducing the growth and differentiation of glands. However,long-term adverse effects have not been evaluated for these therapies.Autoimmune disorders such as SS and lupus are associated with three major destructive cell-based mechanisms:apoptosis of target cells,autoantigen expression/autoantibody production,and production of pro-inflammatory cytokines.If developed,strategies simultaneously targeting these three systems could provide novel preventive and therapeutic measures to combat autoimmune disorders.To expore new herapy has very important significance.Combination therapy is being increasingly accepted in the treatment of SS.However, the choice of combining immunodepressants can not be expected effective in daily practice. Followed successfully combination therapy in leukaemia,according to the theory of cell cycle, we combined low-dose methotrexate(MTX) with cyclophosphamide(CTX) intermittently to treat SS patients.The effectivity and security of new combination therapy has been proved in our clinicl practice.MTX is a cell cycle special stage agent(CCSA),while CTX is a cell cycle nonspecial stage agent(CCNSA).MTX prevent G₀/G₁ stage cell changing to S stage cell.CTX is a difunctionality aldyl agent,killing every stage cell of cell cycle.We presume that MTX make most of cell be blocked in G₀/G₁ stage,and CTX will kill more G₀/G₁ stage cell by given every three week.Side effect will reduce.ObjectiveFor the purpose of from clinical practice to theory,we design an empirical study on SS model mice for 24 weeks to explore effects and possible mechanisms of methatrxate combined with low dose cyclophosphamide intermittently.The objectives of our study are the following.Firstly,to investigate the effect of combination project through observing eccrine glands function and related organ pathology after given MTX and CTX.Secondly,to further explore the synergistic effect of combination project through detecting the proinflammaton cytokines TNF-αand IL-1β, and immunological regulatory factor BAFF and BR3 expression in induced mice model of SS.Methods1.A total of 75 C57BL/6j mice and 40 Lewis rats.P3+S4 ingredients of Lewis rat's eccrine glands homogenate were injected in female C57BL/6 mice.The mice model of SS were set up through immunity induce 4 times.2.After established the SS mice model successfully,they were randomly divided into six SS model group,including MTXLow-dose treatment group(0.02mg/w),MTXhigh-dose treatment group(0.06mg/w),CTXpause treatment group(1.2mg/3w),CTXqod treatment group(0.6mg/2d), MTXlow-dose+CTXpause treatment group(MTX 0.02mg/w,CTX 1.2mg/21d).All of the mice were killed on 24^th weeks after the first immunization.Mice of MTX and CTX_qod group were given by intragastrically.CTX_pause group was given by intraperitoneal injection.Selecting the four groups(CONTROL_SS,MTX_Low-dose,CTX_pause,MTX_low-dose+CTX_pause) to explored the synergistic effect of MTX and CTX.3.Treatment effects were assessed clinically,histologically,serumal and urinous inflammation. The serum and urine TNF-α,IL-1β,BAFF level respectively after the first immunization 6week, 24week,were tested by ELISA.At the end of the experiment,the salivary and lacrimal gland specimens were exposed,fixed,wrapped and cut into slices.All slices were detectd pathology by HE staining.The changes of cytokines(TNF-α,IL-1β,BAFF,BR3) within cell expression were examined by immunohistochemistry method and RT-PCR.All data were analyzed statistically by SPSS 13.0 for windows.Results1.SS mice successfully established.It was clear in decreaseing saliva production and presenting other clinical histological characters.SS model were established in 100%of mice 6 weeks after the first immunization.2.18 weeks after the first immunization,the improvement of the salivary secretion of CTX_qod and MTX_low-dose+CTX_pause treatment group was higher than other groups,which showed statistically significant difference versus SS model group,MTX_low-dose group,CTX_pause group(P＜0.01).CTX_pause treatment group versus MTX_high-dose group,showed no significant difference statistically.3.Compared with the SS model control group,HE staining showed that the lymphocytic infiltration of exocrine glands was decreased in the treatment group.In the single treatment group,MTX_low-dose group and CTX_pause group,heavy inflammatory of cell infiltration were found,and the arrangement of acinar and vessel was disordered.In the CTX_qod and MTX_low-dose+ CTX_pause group,light amount of inflammatory of cell infiltration were found.The pathology degree is CONTROL_SS≥MTX_low-dose≈MTX_high-dose≥CTX_pause≈MTX_low-dose+CTX_pause≈CTX_qod＞CONTROL_normal in turn.The focus score(FS) is CONTROL_SS＞MTX_low-dose≥MTX_high-dose≥CTX_pause≥MTX_low-dose+CTX_pause≈CTX_qod≥CONTROL_normal in turn.4.By ELISA assay,6 weeks after the first immunization,mice serum and urine TNF-α,IL-1β, BAFF level was higher in SS model group than that in the normal control group(P＜0.01).Mice serum and urine TNF-α,IL-1βlevel of 24^th weeks were decreased in combination treatment group and CTX_qod than the other treatment group,which showed statistically significant(P＜0.05).There was no statistic difference between the combination treatment group and CTX_qod group.5.It was found through RT-PCR and immunohistochemistry assey that the mRNA and protein level of TNF-α,IL-1β,BAFF in SS mice was significantly higher than that of control,which suggests that in situ cytokine overexpression contribute to the lymphocytic infiltration of the glands.The expression intensity of cytokines in the treatment groups were decreased markedly(P＜0.01).In the CTX_qod and MTX_low-dose+CTX_pause group,levels were lower than other groups, which showed statistically significant(P＜0.01).There was no statistically difference on the level of BR3 among different treatment groups.Conclusion1.The study using induced-type SS mice demonstrated that MTX and CTX can inhibit lymphocytic infiltration of the salivary and lacrimal glands,leading to an increase of fluid production.2.It showed that both cytokines associated with pro-inflammation(TNF-α,IL-1β) and immunological regulation(BAFF) were inhibited at the transcriptional level by MTX and CTX, and potently inhibited production of protein,which suggests that the two drugs be useful to prevent or ameliorate diseases associated with cytokine overexpression.It suggests that modulation of signaling via BAFF pathways may be a mechanism of action.However,the contributions of BAFF/BR3 signal modifying properties have not been fully elucidated.3.Collectively,our studie is to examine the effects of MTX together with CTX intermitently according cell cycle in the treatment of SS.The results demonstrate that MTX and CTX have a range of beneficial activities.Combination therapy was more effective than single-agent therapy. It showed inhibitory effects of MTX and CTX on cytokine-mediated inflammatory pathways were interaction,synergistic effect mainly.