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 investigate the effects of intragastric administration of human interferon-α (hIFN-α)-transformed Bifidobacterium on immune functions of mice.

METHODSThe E.coli-Bifidobacterium shuttle expression vector containing hIFN-α gene was constructed and transformed into Bifidobacterium. The hIFN-α-transformed Bifidobacterium suspension (1010 /ml) was prepared after induction with 0.2% L-arabinose for hIFN-α expression and administered intragastrically in male Balb/C mice at the dose of 0.1 ml every other day for 2 weeks, with the mice receiving empty vector-transformed Bifidobacteria as the negative control and those having an equal volume of saline as the blank control. The percentages of mononuclear cell subsets in the thymus, spleen and blood were detected in the mice by flow cytometry, and the serum levels of IL-4, IL-12, IFN-γ and TNF-α were assayed using mouse cytokine FlowCytomix Kit.

RESULTSThe percentages of CD3⁺CD8⁺ and CD4⁺CD8⁺ cells in the thymus, CD3⁺CD4⁺, CD3⁺CD8⁺ and CD4⁺CD8⁺ cells in the spleen, and CD3⁺CD8⁺ cells in the blood all increased significantly in IFN group as compared with those in the negative and blank control groups (P<0.01 or 0.05). The serum level of IFN-γ also increased significantly (P<0.05) while IL-4 level remained unchanged in IFN group compared with those in the two groups.

CONCLUSIONIntragastric administration of hIFN-α-transformed Bifidobacterium promotes lymphocyte proliferation and maturation and increases the serum levels of Th1 cytokines in mice.

Animals ; Bifidobacterium ; Cell Proliferation ; Genetic Vectors ; Humans ; Interferon-alpha ; pharmacology ; Interferon-gamma ; blood ; Interleukin-12 ; blood ; Interleukin-4 ; blood ; Lymphocyte Activation ; drug effects ; Male ; Mice ; Mice, Inbred BALB C ; Recombinant Proteins ; pharmacology ; Spleen ; cytology ; Th1 Cells ; cytology ; Thymus Gland ; cytology ; Tumor Necrosis Factor-alpha ; blood

Thymopentin (TP5) and bursopentin (BP5) are both immunopotentiators. To explore whether the TP5-BP5 fusion peptide (TBP5) has adjuvant activity or not, we cloned the TBP5 gene and confirmed that the TBP5 gene in a recombinant prokaryotic expression plasmid was successfully expressed in Escherichia coli BL21. TBP5 significantly promoted the proliferation of thymic and splenic lymphocytes of mice. The potential adjuvant activity of the TBP5 was examined in mice by coinjecting TBP5 and H9N2 avian influenza virus (AIV) inactivated vaccine. HI antibody titers, HA antibodies and cytokines levels (IL-4 and IFN-γ) were determined. We found that TBP5 markedly elevated serum HI titers and HA antibody levels, induced the secretion of both IL-4 and IFN-γ cytokines. Furthermore, virus challenge experiments confirmed that TBP5 contributed to inhibition replication of the virus [H9N2 AIV (A/chicken/Jiangsu/NJ07/05)] from mouse lungs. Altogether, these findings suggest that TBP5 may be an effective adjuvant for avian vaccine and that this study provides a reference for further research on new vaccine adjuvants.
Adjuvants, Immunologic ; pharmacology ; Animals ; Antibodies, Viral ; blood ; Cell Proliferation ; drug effects ; Influenza A Virus, H9N2 Subtype ; drug effects ; physiology ; Influenza Vaccines ; immunology ; Interferon-gamma ; immunology ; Interleukin-4 ; immunology ; Lymphocytes ; drug effects ; Mice ; Oligopeptides ; immunology ; Orthomyxoviridae Infections ; drug therapy ; Recombinant Fusion Proteins ; immunology ; Spleen ; cytology ; Thymopentin ; immunology ; Thymus Gland ; cytology ; Vaccines, Inactivated ; immunology ; Virus Replication

The present study sought to investigate the anti-inflammation and immunoloregulation effect of 17 Guizhi Fuling capsule ingredients. The anti-inflammatory ingredients on LPS-induced RAW264. 7 cell injury were assessed with ELISA and immunofluorescence. The release of IL-1β, TNF-α, PGE2 were detected with ELISA and the expression of COX-2 was detected with immunofluorescence. The effects of them on promoting splenic lymphocyte proliferation were assessed with MTT and Hoechst 33342 staining method. The results showed that 15 ingredients had obviously anti-inflammatory activity on LPS- induced injury and play the immunoloregulation roles. This study suggested that the 15 ingredients may be the active ingredients on pelvic infection.
Animals ; Anti-Inflammatory Agents ; pharmacology ; Capsules ; pharmacology ; Cyclooxygenase 2 ; immunology ; Drugs, Chinese Herbal ; administration & dosage ; Immunologic Factors ; pharmacology ; Inflammation ; drug therapy ; Interleukin-1beta ; immunology ; Macrophages ; drug effects ; enzymology ; immunology ; Male ; Mice ; Mice, Inbred BALB C ; Spleen ; cytology ; drug effects ; immunology ; Tumor Necrosis Factor-alpha ; immunology

Tripterygium glycosides preparation which extracted from the traditional Chinese herb Tripterygium wilfordii (TWHY), was widely used to treat the autoimmune diseases. Previous works demonstrated that TWHF had potent anti-inflammatory and immunosuppressive properties. But the different quality and high incident rate of side effects of different manufactures inhibited its clinical application. Since TWHF had been generally known to play a therapeutical effect by synergism of multiple constituents, it was necessary to build the relationship between the HPLC fingerprint and bioactivity so as to ensure the quality safety and efficacy. The HPLC fingerprint showed that description and content of peaks from different manufactures were diverse. Only 11 common peaks were found. In this study, mice spleen cells stimulated by Con A were used to test the proliferation inhibition bioactivity of TWHF preparations, which were incubated with 30, 15, 7.5, 3.75, 1.88 and 0.94 mg x L(-1) TWHF preparations for 48 h. The results showed that mice spleen cells proliferation was inhibited by all TWHF preparations significantly compared with the control group, which suggested the TWHF preparations showed immune suppress activity. The TWHF preparations from 7 manufacture showed different IC50 value, which might belong to different contents which showed in the HPLC fingerprint. Moreover, a relationship between the HPLC fingerprint and the bioactivity were established to identify important constituents by grey relational analysis (GRA). The result showed that all the contents were relative with the IC50, especially No. 5 and 10 peaks, but No. 1 peak, which was proved to be triptolide, had few contribute to the inhibition of mice spleen cells proliferation. The study of relationship between the HPLC fingerprint and the IC50 by GRA could help to investigate mechanism of bioactive and provide an evidence for the quantification of multi-constituents.
Animals ; Anti-Inflammatory Agents ; analysis ; pharmacology ; Cell Proliferation ; drug effects ; Chromatography, High Pressure Liquid ; Drugs, Chinese Herbal ; analysis ; pharmacology ; Glycosides ; analysis ; pharmacology ; Male ; Mice ; Mice, Inbred BALB C ; Spleen ; cytology ; drug effects ; Tripterygium ; chemistry

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

The purpose of this paper is to study the effect of kinetin (Kn) on immunity and splenic lymphocyte proliferation in vitro of aging rats induced by D-galactose (D-gal). Fifty SD rats were randomly divided into five groups: control group, aging model group, Kn low dose group, Kn middle dose group and Kn high dose group. The aging model group was proposed by napes subcutaneous injection of D-gal (125 mg/kg) for 45 d, and anti-aging groups were intragastrically administered with 5, 10, 20 mg/kg of Kn respectively from day 11. IgG, IgA, IgM contents of serum, the apoptosis percentage, stimulation index (SI) and proliferation index (PI) of splenic lymphocyte in vitro were evaluated. The results showed that the apoptosis percentage of splenic lymphocyte in aging model rats was higher, the serum IgG, IgA and IgM contents, SI and PI were lower than control group. Kn significantly decreased the apoptosis percentage of splenic lymphocyte, while increased the serum IgG, IgA and IgM contents, SI and PI in aging model group. These results suggest that Kn could inhibit the apoptosis, while promote the proliferation of splenic lymphocyte, and then effectively enhance the immune power of the aging rats and slow down the aging process.
Aging ; drug effects ; immunology ; Animals ; Antibodies ; blood ; Apoptosis ; Cell Proliferation ; drug effects ; Galactose ; adverse effects ; Kinetin ; pharmacology ; Lymphocytes ; cytology ; Rats ; Rats, Sprague-Dawley ; Spleen ; cytology

Immunogenicity for medical devices of animal origin is the key and difficult point during immune safety evaluation for these devices. This paper firstly investigated the effect of collagen sponge of animal origin on mouse splenic lymphocyte transformation and proliferation, and then analyzed the influence factors on the MTT method and CFSE method. The results showed that collagen sponge extract cannot significantly induce transformation and proliferation of mouse splenic lymphocyte in vitro.
Animals ; Cells, Cultured ; Collagen ; pharmacology ; Lymphocyte Activation ; drug effects ; Mice ; Porifera ; chemistry ; Spleen ; cytology