Triptolide( TP) is isolated from the traditional Chinese medicine Tripterygium wilfordii,which exhibits notable immuneregulative effect. Th17 cells involve in inflammatory response and Treg cells contribute to immune tolerance. They both play an important role in immune response. Previous studies have investigated that TP induced hepatic Th17/Treg imbalance. However,the effect of TP on spleen Th17/Treg cells remains unclear. Therefore,the aim of present study was to investigate the effect of TP on Th17/Treg cells in spleen. In this study,the effect of TP on the proliferation of splenic lymphocyte was detected by cytotoxicity test in vitro. After different concentrations of TP( 2. 5,5,20,40 nmol·L~(-1)) were given to splenic lymphocyte,cytokines secreted from the supernatant of splenic lymphocyte were detected by cytometric bead array,and the expression of suppressor of cytokine signaling( SOCS) mRNA was detected by qRT-PCR. Female C57 BL/6 mice were continuously observed for 24 h after treatment of 500 μg·kg-1 TP. The effects of TP on the splenic tissue structure and the percentage of Th17/Treg cells were examined. The results showed that the IC50 of TP was19. 6 nmol·L~(-1) in spleen lymphocytes. TP inhibited the secretion of IL-2 and IL-10 and induced the expression of SOCS-1/3 mRNA in spleen lymphocytes at the dosage of 2. 5 and 5 nmol·L~(-1) after 24 h in vitro. Administration of TP at dosage of 500 μg·kg-1 had no significant spleen toxicity in vivo. TP treatment increased the percentage of Th17 cells after 12 h and inhibited the proportion of Treg cells after 12 and 24 h. In conclusion,TP reduced the secretion of IL-2 and IL-10 through SOCS-1/3 signaling pathway,thereby induced the percentage of Th17 cells and inhibited the percentage of Treg cells.
Animals ; Cytokines ; metabolism ; Diterpenes ; pharmacology ; Epoxy Compounds ; pharmacology ; Female ; Mice ; Mice, Inbred C57BL ; Phenanthrenes ; pharmacology ; Signal Transduction ; Spleen ; cytology ; drug effects ; Suppressor of Cytokine Signaling 1 Protein ; metabolism ; Suppressor of Cytokine Signaling 3 Protein ; metabolism ; T-Lymphocytes, Regulatory ; cytology ; Th17 Cells ; cytology

To explore the role of methotrexate (MTX) in regulating the number of regulatory T cells (Treg) and the mRNA expression of transcription factor Foxp3.
 Methods: 1) We analyzed the number of Treg and the mRNA expression of Foxp3 by flow cytometry (FCM) and quantitative real-time PCR (qRT-PCR) respectively in patients with psoriasis vulgaris, patients with psoriasis vulgaris after the 8-week treatment of MTX, and healthy people. 2) BALB/c female mice were smeared with imiquimod (IMQ) cream for 6 days. We recorded the change of the lesion in mice every day. The morphological changes of lesion in mice were evaluated by the psoriasis area and severity index (PASI) and HE staining. 3) The mouse model was randomly divided into a control group and an MTX group. The MTX group was treated with different doses of MTX (38.5 and 77.0 nmol/L) on the third day of this experiment. The morphological changes of lesion in mice were evaluated by PASI and HE staining. We tested the number of Treg and the expression level of Foxp3 mRNA in splenic lymphocytes.
 Results: 1) The number of Treg and the expression level of Foxp3 mRNA were lower in psoriasis vulgaris patients than those in the healthy control group (P<0.05). After 8-week treatment of MTX, the number of Treg was increased (P<0.05) and Foxp3 mRNA level was up-regulated (P<0.01). 2) Typical psoriasis-like skin lesions, such as red scaly skin plaque were found after topical application of IMQ. Both the number of Treg in the splenic lymphocytes of mice and the Foxp3 mRNA level of Treg were reduced by IMQ (P<0.01 and P<0.05). 3) Different doses of MTX for mice showed the ability to improve skin lesion, increase the number of Treg in the spleen of mice and Foxp3 mRNA level in psoriatic dermatitis of mice (P<0.05).
 Conclusion: MTX is able to regulate the number of Treg and Foxp3 mRNA expression in psoriasis.
Adjuvants, Immunologic ; pharmacology ; Aminoquinolines ; pharmacology ; Animals ; Case-Control Studies ; Female ; Forkhead Transcription Factors ; metabolism ; Humans ; Imiquimod ; Immunosuppressive Agents ; administration & dosage ; pharmacology ; Lymphocyte Count ; Methotrexate ; administration & dosage ; pharmacology ; Mice ; Mice, Inbred BALB C ; Psoriasis ; drug therapy ; immunology ; metabolism ; pathology ; RNA, Messenger ; metabolism ; Random Allocation ; Spleen ; cytology ; T-Lymphocytes, Regulatory ; cytology ; drug effects ; metabolism

OBJECTIVETo observe the effects of ulinastatin on immune function of splenic CD4(+) T lymphocytes and CD4(+) CD25(+) regulatory T lymphocytes (Tregs) and content of high mobility group box 1 (HMGB1) in peripheral blood of severely burned rats, and to analyze the possible mechanisms.

METHODSNinety-six male SD rats were divided into sham injury group, burn group, and ulinastatin group according to the random number table, with 32 rats in each group. Rats in sham injury group were sham injured on the back by immersing in 37 ℃ warm water for 12 s. Rats in burn group and ulinastatin group were inflicted with 30% total body surface area full-thickness scald (hereinafter referred to as burn) on the back by immersing in 94 ℃ hot water for 12 s. Immediately after injury, rats in each group were intraperitoneally injected with saline (40 mL/kg), meanwhile rats in ulinastatin group were intraperitoneally injected with ulinastatin (4×10(4) U/kg), once per 12 h, till post injury hour 72. Eight rats of each group were respectively selected on post injury day (PID) 1, 3, 5, and 7 to collect abdominal aortic blood samples. Serum content of HMGB1 was detected by enzyme-linked immunosorbent assay (ELISA). And then, rats of the 3 groups were sacrificed immediately to collect spleens and separate CD4(+) CD25(+) Tregs and CD4(+) T lymphocytes. Flow cytometer was used to detect positive expression rates of cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) and forkhead-winged helix transcription factor p3 (Foxp3) in CD4(+) CD25(+) Tregs. Content of IL-10 in culture supernatant of CD4(+) CD25(+) Tregs, and content of interleukin 2 (IL-2), IL-4, and γ interferon (IFN-γ) in culture supernatant of CD4(+) T lymphocytes was detected by ELISA. The proliferative activity of CD4(+) T lymphocytes was determined by microplate reader. The sample number of above-mentioned experiments was 8 at each time point in each group. Data were processed with analysis of variance of factorial design and LSD test.

RESULTS(1) Compared with that in sham injury group, serum content of HMGB1 of rats in burn group was significantly increased from PID 1 to 7 (with P values below 0.01). Compared with that in burn group, serum content of HMGB1 of rats in ulinastatin group was significantly decreased from PID 1 to 7 (with P values below 0.01). (2) Compared with those in sham injury group, the positive expression rates of CTLA-4 and Foxp3 in CD4(+) CD25(+) Tregs and content of IL-10 in culture supernatant of CD4(+) CD25(+) Tregs of rats in burn group were significantly increased from PID 1 to 7 (with P values below 0.01), peaking on PID 3 [(65±10)%, (76±10)%, and (28.2±4.4) pg/mL respectively]. These 3 indexes of rats in sham injury group on PID 3 were (45±7)%, (46±7)%, and (11.2±2.3) pg/mL respectively. Compared with those in burn group, the positive expression rates of CTLA-4 and Foxp3 in CD4(+) CD25(+) Tregs and content of IL-10 in culture supernatant of CD4(+) CD25(+) Tregs of rats in ulinastatin group were significantly decreased from PID 1 to 7 (P<0.05 or P<0.01), reaching the nadir on PID 7 [(43±6)%], PID 1 [(50±8)%], and PID 7 [(12.4±3.4) pg/mL] respectively. These 3 indexes of rats in burn group on PID 7, 1, and 7 were (58±8)%, (71±9)%, and (19.7±2.8) pg/mL respectively. (3) Compared with those in sham injury group, the content of IL-2 and IFN-γ in culture supernatant of CD4(+) T lymphocytes of rats was significantly decreased, while the content of IL-4 in culture supernatant of CD4(+) T lymphocytes of rats was significantly increased in burn group from PID 1 to 7, with P values below 0.01. Compared with that in burn group, the content of IL-2 and IFN-γ in culture supernatant of CD4(+) T lymphocytes of rats was significantly increased, while the content of IL-4 in culture supernatant of CD4(+) T lymphocytes of rats was significantly decreased in ulinastatin group from PID 1 to 7, P<0.05 or P<0.01. (4) Compared with that in sham injury group, the proliferative activity of CD4(+) T lymphocytes of rats in burn group was significantly decreased from PID 1 to 7 (with P values below 0.01). Compared with that in burn group, the proliferative activity of CD4(+) T lymphocytes of rats in ulinastatin group was significantly increased from PID 1 to 7 (with P values below 0.01).

CONCLUSIONSUlinastatin can weaken the immunosuppressive function mediated by splenic CD4(+) CD25(+) Tregs in severely burned rats, and improve proliferative function and secretory function of splenic CD4(+) T lymphocytes, which may be attributed to the inhibiting effect of ulinastatin on the release of HMGB1 in large amount.

Animals ; Burns ; drug therapy ; CTLA-4 Antigen ; metabolism ; Enzyme-Linked Immunosorbent Assay ; Flow Cytometry ; Forkhead Transcription Factors ; metabolism ; Glycoproteins ; pharmacology ; HMGB1 Protein ; blood ; Interferon-gamma ; metabolism ; Interleukin-10 ; metabolism ; Interleukin-2 ; metabolism ; Interleukin-4 ; metabolism ; Male ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Spleen ; drug effects ; T-Lymphocytes, Regulatory ; cytology ; drug effects

OBJECTIVETo study the effect of glycolytic inhibitor 3-Bromopyruvate (3-BrPA) on the proliferation and apoptosis of mouse spleen lymphocytes and explore its mechanism.

METHODSAn one-way mixed lymphocyte culture (MLC) system was established, including BALB/c mouse spleen cells (H-2d) as stimulator and C57BL/6 mouse spleen cells (H-2b) as responder. With treatment of 3-BrPA at different concentrations (0-200 μmol/L), lymphocyte proliferation capacity was detected by the CCK-8 method, the expression of CD3, CD4, and CD8 by flow cytometry, and the concentrations of cytokine interleukin (IL)-4 and interferon (IFN)-γ in the supernatant by ELISA.

RESULTSAt a middle or high dose (over 20 μmol/L), 3-BrPA displayed a dose-dependent inhibitory effect on lymphocyte proliferation in the MLC system. The 50% inhibitory concentration (IC50) were 48.6, 41.2, and 41.9 μmol/L after 24, 36, and 48 h culture, respectively. With treatment of 50 μmol/L 3-BrPA, the IFN-γ level [(164.25 ± 20.14) ng/L] was significantly lower, compared with control [(277.61 ± 18.46) ng/L]. The IL-4 level [(31.06 ± 6.06) ng/L] was significantly higher, compared with control [(28.64 ± 3.97) ng/L]. Consequently, the IFN-γ/IL-4 ratio decreased significantly.

CONCLUSIONThese results indicate that 3-BrPA had a significant inhibitory effect on the proliferation of mouse spleen lymphocytes cultured in MLC system, accompanied with the Th2-biased secretion of cytokines.

Animals ; Apoptosis ; drug effects ; Cell Proliferation ; drug effects ; Cells, Cultured ; Interferon-gamma ; metabolism ; Interleukin-4 ; metabolism ; Lymphocyte Culture Test, Mixed ; Male ; Mice ; Mice, Inbred BALB C ; Mice, Inbred C57BL ; Pyruvates ; pharmacology ; Spleen ; cytology ; metabolism

A series of phthalazine ketone compounds were synthesized and the structures were confirmed by H NMR and HR-MS spectrum. All target compounds were obtained through 7 steps, including selective reduction, nitration, bromination, ring enlargement, reduction, Knoevenagel and acylated reaction. The compounds were evaluated for their immunosuppressive effects of T-cell proliferation and inhibitory activity of IMPDH type II in vitro, as well as their structure-activity relationship were assessed. Several compounds exhibited strong immunosuppressive properties, especially compounds 7f and 7h, with IC50 values of 0.093 micromol x L(-1) and 0.14 micromol x L(-1) respectively, which were superior to mycophenolic acid. The information obtained from the studies may be useful for further research on the immunosuppressive agents.
Animals ; Cell Proliferation ; drug effects ; Female ; IMP Dehydrogenase ; metabolism ; Immunosuppressive Agents ; chemical synthesis ; chemistry ; pharmacology ; Inhibitory Concentration 50 ; Mice ; Mice, Inbred BALB C ; Phthalazines ; chemical synthesis ; chemistry ; pharmacology ; Spleen ; cytology ; Structure-Activity Relationship ; T-Lymphocytes ; drug effects

OBJECTIVETo investigate the effects and mechanisms of Gong-tone music on the immunological function in rats with the Chinese medicine syndrome of Liver (Gan)-qi stagnation and Spleen (Pi)-qi deficiency (LSSD).

METHODSTwenty five male Wistar rats of SPF grade were randomly divided into 5 groups: normal group, model group, Xiaoyao Powder () group, Gong-tone group and combined group (the combination of Gong-tone and Xiaoyao Powder), with 5 rats in each group. The rat model for the Chinese medicine syndrome of LSSD was induced by chronic bandage and irregular diet. The course of treatment was 21 days. After the treatment, the levels of serum gastrin and IgG were detected by enzyme-linked immunoabsorbent assay (ELISA). Phagocytosis of macrophages was detected by the neutral red uptake assay and T cell proliferation was investigated by 3-(4,5-dimethylthiazolyl)-2,5-diphenyltetrazolium bromide (MTT) assay.

RESULTSThe serum gastrin, macrophage phagocytosis, IgG level and proliferation ability of T cells in the model group were significantly decreased compared with those in the normal group (P <0.05). Compared with those in the model group, the serum levels of gastrin, macrophage phagocytosis, IgG level and proliferation ability of T cells in Gong-tone, Xiaoyao Powder, and combined groups were significantly increased (P <0.05). The combined group was superior to either Gong-tone group or Xiaoyao Powder group.

CONCLUSIONGong-tone music may upregulate the immunological function and play a role in adjuvant therapy in the Chinese syndrome of LSSD.

Animals ; Auditory Perception ; Behavior, Animal ; Body Weight ; Cell Proliferation ; Depression ; blood ; immunology ; Gastrins ; blood ; Immunoglobulin G ; blood ; Liver ; immunology ; Macrophages ; cytology ; Male ; Music ; Phagocytosis ; Qi ; Rats ; Rats, Wistar ; Spleen ; immunology ; Syndrome ; T-Lymphocytes ; cytology ; drug effects ; metabolism

This study was aimed to explore the effect of cordyceps sinensis enhancing lymphocyte proliferation and surface CD marker expression in simulated microgravity environment. The splenic lymphocytes were separated from mice and cultured in the rotary cell culture system simulated microgravity environment. The cells were treated with different concentration of cordyceps sinensis solution (0, 6.25, 12.5, 25 and 50 µg/ml) for 24, 48 and 72 h respectively, then the cells were harvested, and analyzed for cell proliferation and the expression of cell surface markers (CD4 and CD8). The results showed that under simulated microgravity environment, the lymphocyte proliferation was inhibited. When the concentration of cordyceps sinensis was 25 or 50 µg/ml, the lymphocyte proliferation, CD4 and CD8 expressions all increased, but 50 µg/ml cordyceps sinensis could inhibit the proliferation ability with the time prolonging. It is concluded that the suitable concentration of cordyceps sinensis displayed the ability to enhance the lymphocyte proliferation and CD marker expression in simulated microgravity environment. These results may be valuable for screening drugs which can be potentially against immunosuppression under simulated microgravity.
Animals ; CD4 Antigens ; metabolism ; CD8 Antigens ; metabolism ; Cell Proliferation ; drug effects ; Cells, Cultured ; Cordyceps ; Immune Tolerance ; Lymphocyte Activation ; drug effects ; Lymphocytes ; drug effects ; metabolism ; Mice ; Mice, Inbred C57BL ; Polysaccharides ; pharmacology ; Spleen ; cytology ; Weightlessness Simulation

BACKGROUNDThe common γ chain (γc) plays a critical role in regulating proliferation, differentiation, and apoptosis of peripheral T-cells. It was previously confirmed that blocking the γc signal can successfully induce transplant tolerance in a murine model. Here we investigated the potential mechanism.

METHODSSplenocytes from C57BL/6 mice were transfused into T-cell deficient Balb/c nude mice that were reconstituted with syngeneic wild-type T-cells labeled with 5-carboxyfluorescein diacetate succinimidyl ester (CFSE). After 24 hours, recipients received i.p. injection of mixture of anti-γc mAbs, or with isotype control IgG2a. The labeled T-cells were harvested from recipient spleens after 12 and 48 hours. T-cell proliferation and apoptosis were detected by flow cytometry.

RESULTST-cell proliferation was markedly inhibited and apoptotic T cells could be detected at 12 hours after the mAbs injection. Proliferation was inhibited at 48 hours, but the proportion of apoptotic T-cells was not more than at 12 hours. In the control group, however, T-cells actively proliferated and no significant apoptosis was detected at either time point.

CONCLUSIONSThe results suggested that blockade of γc signals can synergize with donor splenocyte transfusion and lead to inhibition of antigen-specific T-cell proliferation and induction of apoptotic T-cell death. This protocol may develop a novel approach to induce donor-specific tolerance.

Animals ; Antibodies, Monoclonal ; pharmacology ; Apoptosis ; drug effects ; Cells, Cultured ; Flow Cytometry ; Fluoresceins ; Immune Tolerance ; drug effects ; Interleukin Receptor Common gamma Subunit ; antagonists & inhibitors ; metabolism ; Lymphocyte Activation ; drug effects ; Male ; Mice ; Mice, Inbred BALB C ; Mice, Inbred C57BL ; Mice, Nude ; Signal Transduction ; drug effects ; Spleen ; cytology ; Succinimides ; T-Lymphocytes ; cytology ; drug effects

OBJECTIVETo study the effect of Chinese drugs for invigorating qi and tonifying Shen (IQTS) on expression of CD4+CD25+ regulatory T cells in spleen and maternal-fetal interface of abortion-prone mice during pregnancy.

METHODSCBA female mice were mated with DBA/2 male mice to establish abortion-prone models, which were randomly divided into 4 groups, the negative control group (fed with normal saline), the positive control group (treated with CsA), the Chinese medicine group (treated with IQTS), and the Chinese and Western medicine group (treated with IQTS+CsA). Mice were sacrificed in batches on the 9th and the 14th day of gestation, their splenic and decidual tissues were taken out to analyse CD4+CD25+ regulatory T-cell expression by flow cytometry.

RESULTSCompared with the negative control group, the expression of splenic CD4+CD25+ regulatory T all significantly increased on the 9th day of gestation (P < 0.01 or P < 0.05). There was no statistical difference in intergroup comparison of the three treatment groups (P > 0.05). Compared with the negative control group, the expression of splenic CD4+CD25+ regulatory T all significantly increased on the 14th day of gestation (P < 0.01 or P < 0.05). Of them, its expression was the highest in the Chinese and Western medicine group, showing significant difference from that in the Chinese medicine group and the positive group (P < 0.01). The difference between the Chinese medicine group and the positive group was insignificant (P > 0.05). On day 9 of gestation, compared with the negative control group, the expressions of CD4+CD25+ regulatory T in maternal-fetal interface increased in the three treated groups, showing no statistical significance (P > 0.05). Its expression was ordered from high to low in sequence as the Chinese and Western medicine group, the positive control group, the Chinese medicine group, and the negative control group. On day 14 its expression was obviously enhanced in the Chinese and Western medicine group, showing statistical difference from that in the negative control group (P < 0.05). But its expression was obviously enhanced in the Chinese medicine group and the positive group, showing insignificant difference from that in the negative group. The same sequence was found in the percentage of CD4+CD25+ T cells in CD4+ T cells.

CONCLUSIONSChinese drugs for IQTS could up-regulate the expression of CD4+CD25+ regulatory T in spleen of abortion-prone mice in the early and late pregnancy stages. When combined with CsA, it also could up-regulate its expression in maternal-fetal interface in the mid and late pregnancy stages, suggesting that Chinese drugs for IQTS are facilitate to maintain the immune tolerance state in mice during pregnancy.

Abortion, Spontaneous ; drug therapy ; immunology ; metabolism ; Animals ; Drugs, Chinese Herbal ; pharmacology ; therapeutic use ; Female ; Male ; Mice ; Mice, Inbred CBA ; Phytotherapy ; Pregnancy ; Spleen ; cytology ; drug effects ; T-Lymphocytes, Regulatory ; cytology ; drug effects ; metabolism

OBJECTIVETo study the ex vivo effect of sirolimus on capacity of splenic dendritic cells (DC) from traumatized mice in inducing T cell responses.

METHODSTwenty-four BALB/c mice were divided into control group and trauma group according to the random number table, with 12 mice in each group. Mice in trauma group were bled followed by closed femur fracture after anaesthesia, while mice in control group were only anaesthetized without injury. Twenty-four hours later DC were isolated from spleens and divided into 4 subgroups: sirolimus devoid control (trauma) groups [consisted of cells from control (trauma) groups, without sirolimus treatment] and sirolimus treated control (trauma) groups [consisted of cells from control (trauma) groups, treated with 10 microg/L sirolimus for 6 hours]. Then their autophagic activity, DC-induced mixed lymphocyte reaction (MLR) were measured and recorded as fluorescence intensity (FI) value and absorbance value respectively. The expression of major histocompatibility complex class (MHC) II and costimulatory molecules CD40, CD80, and CD86 on DC surface were measured with flow cytometry. IL-12p40, IL-12p70 and IL-10 levels in lipopolysaccharide-stimulated DC supernatants were determined by ELISA. Data were processed with one-way analysis of variance.

RESULTS(1) Compared with those of sirolimus devoid control group (FI value = 22 +/- 6), DC autophagic activity (FI value = 13 +/- 2) and DC-induced MLR in mice from sirolimus devoid trauma group were significantly weakened (F = 212.836, P < 0.05). Compared with those of sirolimus devoid control (trauma) groups, DC autophagic activity in mice from sirolimus treated control (trauma) groups (FI = 45 +/- 8, 44 +/- 8 respectively) were significantly strengthened (F = 212.836, P < 0.05 or P < 0.01). MLR in mice from sirolimus treated trauma group was stronger than that from sirolimus devoid trauma group (with F value respectively 101.426, 86.533, P values all below 0.05). (2) Compared with those of sirolimus devoid control group [MHC II (85 +/- 6)%, CD40 (8 +/- 1)%], the expressions of MHCII [(60 +/- 9)%] and CD40 [(4 +/- 1)%] on DC surface from sirolimus devoid trauma group were significantly reduced (with F value respectively 37.918, 40.426, P values all below 0.05). The expression of MHCII from sirolimus treated trauma group [(78 +/- 7)%] was higher than that from sirolimus devoid trauma group (F = 37.918, P < 0.05). (3) IL-12p40, IL-12p70 secretion by DC from sirolimus devoid trauma group [(120 +/- 13), (10 +/- 3) pg/mL] were significantly reduced as compared with those from sirolimus devoid control group [(200 +/- 25), (20 +/- 6) pg/mL, with F value respectively 218.646, 310.253, P values all below 0.05]. Compared with those from sirolimus devoid control (trauma) groups, IL-12p40 [(560 +/- 34), (540 +/- 29) pg/mL], IL-12p70 [(55 +/- 8), (60 +/- 11) pg/mL] secretion by DC from sirolimus treated control (trauma) groups were obviously enhanced (with F value respectively 218.646, 310.253, P values all below 0.01), while IL-10 secretion levels were significantly decreased (F = 246.108, P < 0.01).

CONCLUSIONSSirolimus can partially ameliorate DC functions ex vivo in traumatized mice, and further enhance the capacity of DC in inducing T cell responses.

Animals ; B7-1 Antigen ; metabolism ; B7-2 Antigen ; metabolism ; CD40 Antigens ; metabolism ; Dendritic Cells ; drug effects ; immunology ; metabolism ; Histocompatibility Antigens Class II ; immunology ; Interleukin-12 Subunit p40 ; metabolism ; Lymphocyte Culture Test, Mixed ; Male ; Mice ; Mice, Inbred BALB C ; Sirolimus ; pharmacology ; Spleen ; cytology ; immunology ; T-Lymphocytes ; immunology ; Wounds and Injuries ; immunology