| Objective and significanceSystemic lupus erythematosus (SLE) is one of chronic and systemic autoimmune diseases which can cause multiple system damage, with a large number of different anti-nuclear antibody-based autoantibodies. SLE has variety of clinical manifestations, only involving a single organ in early, such as the skin, kidneys, joints, also can affect multiple organs. The early symptoms of this disease is the most common joint and skin manifestations, The early symptoms of this disease is the most common joint and skin manifestations,followed by fever, light sensitivity, Raynaud’s phenomenon, nephritis and serositis. SLE is widely distributed around the world, The worldwide prevalence of approximately is about17to48/100000, Mainland China, the survey shows that the prevalence rate of SLE is about40to70/10million. SLE occurred in young women, prevalent in the15to45-year-old, the male to female ratio is about1:7to9. The pathogenesis of SLE is not clear, the factors associated with the disease mainly include genetic, viral infection, environment, medicine and so on. The pathogenesis of this disease remain unclear, but a large number of studies show that SLE is an autoimmune disease.Interaction of multiple genetic factors, environment, virus that changes its structure body,or Immunoreactive cell mutation,due to the disorder of immune regulation. Immune responses against its own tissues (including the humoral and cellular immunity), normal tissue is destroyedby by its own immune cells or autoantibodies.In recent years a large number of studies have found that CD4+T cells were divided into four subgroups according to the development, different fuctions of the cytokine secretion. Including Thl cells, Th2cells, Treg cells and Th17cells subgroup. Th17cells named by secreting IL-17. In addition, it is also secretes of IL-17F, IL-21, IL-22and TNF a. These cytokines can induce local cytokine secretes Inflammatory substances and chemotactic factor to attract neutrophils.Playing an important role in process of chronic inflammation, resulting in defense against extracellular bacterial infection and autoimmune disease. Regulatory T cell is one of CD4+T cell subsets found in human and mouse in recent years,which has a significant inhibitory effect. It can be applied to a variety of target cells through different pathways. Treg cells can regulate the immune response negatively, then stabilizing the immune balance and Maintaining the immune tolerance. According to the different cell factor and the surface markers, Treg cells can be divided into CD4+CD25+Treg cells, Trl and Th3and other subtypes. CD4+CD25+Treg cells are thought to the most important T cell subtype in regulatory T cells family.FoxP3is the specific marker for CD4+CD25+Treg, It plays a key role in the process of. development and function in regulatory T cells. Many studies have confirmed that the Increased expression of FoxP3can induce differentiation of CD4+CD25-T cells into CD4+CD25+Treg cells. Th17cells and Treg cells are derived from the initial T cells. The differentiation and function are mutual inhibition, keeping both in balance under the normal circumstances to maintain immune steady state.The Immune balance of Th17/Treg cells is an important complement to Thl/Th2immune balance theory. The imbalance between them may lead to the occurrence of autoimmune disease. We speculate that the incidence of imbalance in the number and function of Th17cells and/Treg cells may be involved in SLE.Most of SLE patients need long-term use of glucocorticoid, immunosuppressive agents, but many of its toxic side effect is easy to cause patient injury again. So the compliance of patients is poor,limiting its clinical application.Total glucosides of paeony(TGP) is the effective component extracted from the root of the Paeonia lactiflora Pall,which is the traditional Chinese Medicine. It mainly contains Paeoniflorin, Albiflorin, hydroxy-paeoniflorin, peony glucoside, benzoylpaeoniflorin composition. The paeoniflorin content accounted for more than90%of total glucosides. It is generally considered to be the main effective component of total glucosides of paeony. Modern pharmacological research show that TGP has many ways of bidirectional to regulate the autoimmune reaction, Such as inhibition of monocyte macrophages to produce, the secretion of TNF-α and IL-1, Scavenging oxygen free radicals, effect of cell proliferation, anti-inflammatory, Relieve pain, Stress resistance and protection of liver. In recent years, It has been widely used in the treatment of rheumatoid arthritis,SLE and other rheumatic diseases.Its adverse reactions were mild and tolerable. Review of the literature, we have not yet found reports about the effect of TGP on the balance of Thl7/Treg.Flow cytometry (FCM) is a new technology since the seventy’s. It combines computer technology, laser technology, fluid mechanics, chemistry, immunology in cells. At the same time it has the function of analysis and sorting of cells. It can not only measure the size of cells,the internal particle character, and it can also be used to detect cell surface and cytoplasmic antigens and the level of intracellular DNA and RNA. It can Analysis the groups of cells at the level of single cell, detect and analysis a large number of cells in a short period of time, collect, store and process data and multi parameter quantitative analysis. It can be classified collection and sorting of a subpopulation of cells. The sorting purity is greater than95%. FCM is widely used in hematology, oncology, immunology, pharmacology and molecular biology.This study used the lupus-like mice which is belong to strain of MRL/lpr to research. Study on the effects of different concentrations of TGP against double-stranded DNA antibody and urine protein in lupus-like mice. Using the technology of flow cytometry to find the effect of TGP on number of Th17cells and Treg cells in spleen lymphocyte. Preliminary study on regulation effect and immune mechanism of TGP on SLE in imbalance of Th17/Treg cells from peripheral lymphoid organs. To further improve the SLE peripheral Th17/Treg cells imbalance mechanism theory. Fill in the blanks on the theory of TGP’s regulation on the balance of peripheral Th17/Treg cell in SLE at home and abroad.Research methods1、Using ELIS A to detect the concentration of ds-DNA antibody which is belong to IgG type.2、Using coomassie brilliant blue method to determinate the protein content of mouse urine.3、Using FCM to detecte the percentage of CD4+CD25+Foxp3+T/CD4+T cells,CD4+IL-17+T/CD4+T cells and CD4+CD25+Foxp3+T/CD4+IL-17+T cells in the three groups. And analyze the correlation between urine protein and Th17cells and Treg cells.Research content and process1、The grouping method:The20MRL/lpr mice were purchased from the Experimental Animal Center of Guangdong Province. female,3months, quarantine is qualified. These mice have been raised in the SPF laboratory animal room of the Experimental Animal Center of Guangdong Province during the whole experiment. When the anti ds-DNA antibody and urine protein was positive, Descripte the incidence of SLE in mice. Using random digits table divide these mice into three groups,including high TGP concentration (7), TGP low concentration group (7mice) and control group (6mice). Dose was calculated according to the<Between the animal and human body surface area reduced equivalent dose ratio table>. High levels of TGP group were given orally121mg/kg TGP suspension, Low concentration of TGP group were given orally81mg/kg TGP suspension, The control group was given the same volume of purified water by gavage. All one times a day, for30consecutive days.2、Research methods:(1) We observed daily the hair of mice, behavior, mental state, feeding, and activities generally in each group and recorded daily. (2) ELISA was used to detect the concentration of anti ds-DNA antibody in peripheral blood of the lupus mice.Using Coomassie brilliant blue method to detect the concentration of urine protein in mice. When two of them were positive descript that the lupus model was successfully established.Collected the24-hour urine after treatment for zeroth days, fourteenth days, thirtieth days and placed in the-80℃. Using the Coomassie brilliant blue method to detect the content of urinary protein.(3)Sacrificed all the mice after treatment30days,dissected out the spleen, collected single cell suspensions from spleen.Using FCM to detecte the percentage of CD4+CD25+Foxp3+T/CD4+T cells,CD4+IL-17+T/CD4+T cells and CD4+CD25+Foxp3+T/CD4+IL-17+T cells in the three groups. And analyze the correlation between urine protein and Th17cells and Treg cells.3、Statistical analysis(1)Analyzed all data by using SPSS13.0statistical software:The measurement data using mean±standard deviation said.(2) Compared the content of urinary protein and anti ds-DNA antibody by using the analysis of variance for repeated measurement design.(3)Compared the percentage of CD4+CD25+Foxp3+T cells/CD4+T cells,CD4+IL-17+T cells/CD4+T cells and CD4+CD25+Foxp3+T cells/CD4+IL-17+T cells in the three groups by using one-way analysis of variance. When the data are consistent with the homogeneity of variance, using LSD method to analyze the multiple comparisons between groups. When the data are not consistent with the homogeneity of variance, using Welch method,and using the correcting method(Dunnett’s T3) to analyze the multiple comparisons between groups.(4) Analyzed the correlation between urine protein and Th17cells and Treg cells by using the analysis of bivariate correlation. When the data obey the normal distribution using the Pearson correlation analysis, otherwise, using Spearman correlation analysis.(5) Took P<0.05as statistically significant difference.results 1、General condition:Feeding, behavior and the hair were normal in three groups before the treatment. With the passage of time the mice of control group gradually appeared mental burnout,settled,the reduce of feeding times and activities, and decreases of hair gloss.Four of these mice’s neck back hair was falling. Low and high concentration of TGP groups had no obvious change,only hair gloss decline compared with the previous. All mice’s eyes, ears, nose and mouth showed no abnormality secretion in the course of the experiment. All the mice’s feces and urine were normal. All the mice survived.2、concentration of anti ds-DNA antibody:There was no significant difference in concentration of anti ds-DNA antibody of the three groups of mice before treatment (P>0.05). After two weeks of treatment, the level of antibody to ds-DNA of low concentration of TGP group was higher than the other two groups, and it was significantly higher than high concentration of TGP group (P=0.048); The level of anti ds-DNA antibody in TGP treated groups were significantly higher than that of the control group four weeks after medication (P=0.000,P=0.000).High TGP concentration of anti ds-DNA antibody concentration increased gradually with the passage of time, the level in fourth weeks was significantly higher than that in0and2weeks (P=0.002, P=0.005); Low concentration of TGP group of anti ds-DNA antibody concentration at the end of the second week,4weeks was significantly higher than that of0weeks (P=0.019, P=0.000), that in second weeks and fourth weeks, there was no significant difference (P=0.933); The anti ds-DNA antibody level of control group was increased first and then decreased, but the difference is not statistically significant (P=0.460, P=0.257, P=0.122).3、Concentration of urinary protein:Before and14days after treatment, there was no significant difference between the three groups with urinary protein concentration (P>0.05).30days after treatment, high concentration of TGP group was significantly lower than that of TGP in low concentration group (P=0.005), high TGP concentration and low concentration group were significantly lower than those of the control group (P=0.014、P=0.04).After30days of high TGP concentration in comparison with the administration after14days and before treatment, the urine protein changes were significant differences (P=0.003, P=0.006). Thirtieth days compared with fourteenth days and thirtieth days compared with zeroth weeks of control mice urinary protein, the urine protein changes were significant differences (P=0.048, P=0.007)4、Percentages of CD4+CD25+Foxp3+Tcells/CD4+T cells in the spleen lymphocytes:The percentages of CD4+CD25+Foxp3+T cell/CD4+T cells in spleen lymphocyte were increased gradually with increasing concentration of TGP. The levels were:0.49±0.13%、0.53±0.04%、0.97±0.20%. Among them, the percentage of TGP in high concentration group was significantly higher than that of TGP in low concentration group (P=0.031).High concentration of TGP group was significantly higher than that of pure water control group (P=0.025). Low concentration of TGP group was higher than control group, but the difference was not statistically significant (P=0.84)5、Percentages of CD4+IL-17+Tcells/CD4+T cells in the spleen lymphocytes:The percentages of CD4+IL-17+T/CD4+T cells in spleen lymphocyte were decreased gradually with the increase of TGP concentration. The levels were:6.02±3.06%、4.63±1.21%、3.20±0.83%. But there were no significant difference among the groups (PH-L=0.598, PH-C=0.321, PL-C=0.607, P>0.05)6、Percentage of CD4+IL-17+T cells/CD4+CD25+Foxp3+T cells in the spleen lymphocytes:The percentages of CD4+IL-17+T cells/CD4+CD25+Foxp3+T cells in spleen lymphocyte were increase gradually with the decreased of TGP concentration. The levels were:4.27±1.28、8.91±2.20、12.38±3.82. Ratio in high concentration of TGP group was significantly higher than that in pure water control group (P=0.049). Ratio in low concentration of TGP group was lower than that of pure water control group,but higher than the high concentration of TGP group (P=0.357、P=0.224), but the difference was not statistically significant.7、Correlation analysis of urine protein respectively with CD4+IL-17+T cells/CD4+Tcells and CD4+CD25+Foxp3+Tcells/CD4+Tcells.The percentage of CD4+CD25+Foxp3+T/CD4+T cells was negatively correlated with the concentration of thirtieth day mouse urinary protein (r=-0.481, P=0.032), There was no significant correlation between the percentage of CD4+IL-17+Tcells/CD4+Tcells and urinary protein (r=0.301, P=0.198).Conclusion1、TGP can improve the general situation of the lupus model mice(MRL/lpr). Such as to maintain a normal state of activities and feeding, keep hair gloss. The results indicated that TGP has some improvement on the disease state of lupus mice and can improve the quality of life of lupus mice.2、TGP can increase the concentration of anti ds-DNA antibody in peripheral blood of lupus mice, but reduce the content of urine protein. This shows that TGP can Improve the lupus renal damage, and protect the kidney function may not be realized by reducing the anti ds-DNA antibody. The antibody of ds-DNA could not the only pathogenic factor of lupus nephritis.3、The immune imbalance in Th17/Treg cell does exist in peripheral lymphocytes of SLE, and which is correlation with disease severity.4、TGP can decrease the levels of CD4+IL-17+T cells in lymphocyte of spleen cells, and improve the level of CD4+CD25+Foxp3+T cells. It indicate that TGP can correct the lupus/Treg mice Th17cell immune imbalance, then play the role of anti-inflammatory and immune regulation. 5、The percentage of CD4+CD25+Foxp3+T/CD4+T cells was negatively correlated with the concentration of thirtieth day mouse urinary protein. It suggests that CD4+CD25+Foxp3+Tcells is involved in the protection of lupus renal damage.6、TGP has the ability to regulate the immune balance of Th17/Treg in SLE. The ability of TGP to up-regulate Treg cells was more obviously than down-regulate Th17cells. It suggests that TGP up-regulates the Treg cells at first and then through regulate the immune system of Treg/Th17cells to reduce the differentiation of Th17cells.7、In the immune balance of Thl/Th2/Th17/Treg cells in early,TGP may promote Th2and Treg first, and then regulate the Thl/Th2/Th17Treg cells negatively. |
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