Mechanisms for a decrease in mortality of LPS-challenged mice by rhynchophylline

AIM: To observe effect of rhynchophylline （Rhy） on mortality and organ injury in endotoxemic mice and further investigate the mechanisms of its actions. METHODS: Male mice were randomly assigned into control, LPS, Rhy +LPS and Rhy group, and injected subcutaneously with normal saline (0.05 mL/10 g), or rhynchophylline once a day for 3 d, 1 h after subcutaneously treatment on day 3, LPS (20 mg/kg) or normal saline was injected intraperitoneally. Survival rate was recorded every 12 h for 6 d. In another experiment, 12 h after LPS injection, the left lung and intestine tissue sections were prepared for histological analysis and the right lung were used to determine the ratio of wet to dry lung tissue weight (W/D),the serum was collected to detect the concentrations of alanine aminotransferase（ALT）, aspartate aminotransferase (AST ), bloodureanitrogen (BUN) and creatinine (Cr). In addition, the concentrations of tumor necrosis factor-α (TNF-α), interleukin-1β(IL-1β) and interleukin-10 (IL-10) in serum at 2 h after LPS challenge were detected by enzyme-linked immunosorbent assay. The concentration of NO in serum at 8 h was detected by enzymic method. The effect of Rhy on survival rate of mice subjected to cecal ligation and puncture (CLP) was also observed. RESULTS: Mortality of mice challenged with LPS alone was higher significantly than that in control at 24 h after LPS challenge, pretreated with Rhy at a dose of 8 or 16 mg/kg increased markedly the survival rate of LPS-challenged mice. However, Rhy at a dose of 8 mg/kg significantly increased mortality of mice subjected to CLP. In the histological analysis, severe inflammation was observed both in the lung and intestine tissues in the LPS group. LPS elevated lung W/D, the levels of ALT, AST, BUN, Cr, TNF-α, IL-1β, IL-10 and NO in serum. Pretreatment with Rhy had no obvious improvement in the lung and intestine tissue injury, and no significant depression in the lung W/D and the serum levels of ALT, AST, BUN, Cr, IL-1β, IL-10 and NO, but decreased the level of TNF-α in serum significantly in LPS -treated mice. CONCLUSION: Pretreatment with Rhy reduces the mortality in endotoxemic mice, but not decrease the mortality of mice challenged with CLP, at least in part, through inhibiting the synthesis and secretion of TNF-α.     

Effects of thymopeptide and 4 polysaccharides isolated from Chinese medicine on immune functions of spleen and tumor tissues in U14 cervical cancer-bearing mice

AIM: To identify the enhancement of relatively specific immune responses by thymopeptide and 4 polysaccharides isolated from traditional Chinese medicine in the mice bearing cervical cancer. METHODS: The model of cervical cancer-bearing mice was established by implantation of cervical cancer U14 cells into the armpit of the right forelimb of the mice. Beginning from the 2nd day of tumor implantation, 4 polysaccharides Lycium barbarum polysaccharide (LBP), Angelica sinensis polysaccharide (ASP), Ganodema lucidum polysaccharide (GLP) and Panax ginseng polysaccharide (PGP)], thymopeptide or saline solution were intragastric administered for 21 days according to the experimental design. The animals were then killed, the tumor inhibitory rate and spleen index in treatment groups were compared with those in control group. The splenic T-lymphocytes (TLC) and natural killer (NK) cells, and the content of IFN-γ and IL-10 in the tumor tissues were also determined by flow cytometry and immunohistochemical methods. RESULTS: The order of tumor inhibitory rates was: ASP < PGP < thymopeptide < LBP < GLP. The effects of GLP, LBP and thymopeptide were statistically different from that of control group. No difference of tumor inhibition between PGP/ASP groups and control group was observed. The order of spleen index was: ASP < tumor control group < thymopeptide < GLP < PGP < LBP, that of CD4/CD8 enhancement was: PGP < ASP < GLP < thymopeptide < LBP, and that of CD49b⁺ NK hyperplasia was: ASP < thymopeptide < LBP < GLP < PGP,with statistically significant differences among the groups. The changes of IFN-γ and IL-10 in the tumor tissues showed that Th1/Th2 in thymopeptide group, LBP group and GLP group shifted to Th1. No shift to Th1 in PGP group and ASP group was observed. CONCLUSION: LBP, GLP and thymopeptide show significant inhibitory effects on tumor growth in U14 cervical tumor-bearing mice and can enhance the immune functions in the animals.     

Expression of Th cytokines in peripheral blood before and after splenectomy in immune thrombocytopenia patients

AIM: To investigate the changes of Th cytokines before and after splenectomy in immune thrombocytopenia (ITP) patients. METHODS: The QuantiGene Plex method was used to measure the mRNA expression of Th1, Th2 (IL-4, IL-5, IL-6 and IL-10), Th3 (transforming growth factor β₁,TGF-β₁), and Th17 (IL-17) cytokines in peripheral blood of ITP patients before and after laparoscopic splenectomy and those in peripheral blood of healthy controls. RESULTS: The mRNA level of IL-2 was significantly decreased in ITP patients before operation compared with the healthy controls, whereas IL-17 was obviously over-expressed. No significant difference of the other cytokines between preoperative group and the normal controls was found. After splenectomy, the expression levels of both IL-2 and TGF-β₁ were significantly higher than those in preoperative group and the normal controls. IL-2 was also significantly increased after operation, but was still lower than that in the normal controls. No significant difference of other cytokines between postoperative group and healthy controls was observed. In addition, The Th1 cytokines (IL-2 and IFN-γ) were found to be positively correlated (r=0.647, P<0.01) in preoperative patients, while no correlation was found between the other cytokines. There was a positive correlation between IL-2 and IFN-γ (r=0.787, P<0.01) in postoperative patients. IL-17 also had positive correlations with IL-2 (r=0.554,P<0.01) and IFN-γ (r=0.461,P<0.05) in ITP patients after operation, respectively. CONCLUSION: There is an imbalance of Th cytokines in ITP patients. The mechanism of splenectomy for treating ITP may be associated with the balance regulation of Th cytokines.     

Effects of glucosidorum tripterygii tororum on cytokine productions in graft-versus-host disease mice

AIM:To observe the effect of glucosidorum tripterygii tororum (GTT) on cytokine productions in acute graft-versus-host disease (aGVHD) mice. METHODS: C₅₇BL/6 mice were exposed to radiation delivered by a linear accelerator. To establish a aGVHD model, the cell suspensions, which were obtained from bone marrow and spleen of the BALB/C mice, were transplanted to the radiated C₅₇BL/6 mice. The recipients were treated with GTT, GTT+CsA and CsA+MTX. The serum concentrations of IL-2, TNF-α, IL-4 and IL-10 were determined by ELISA. RESULTS:The survival rate on day 11 in GTT group (9/10) was higher than in allogeneic bone marrow transplatation (allo-BMT) group (8/19). The concentrations of IL-2 and TNF-α in GTT group were significantly lower, but the concentration of IL-10 was remarkably higher than that in allo-BMT group (P＜0.05). However, the concentration of IL-4 showed no changed in all groups (P＞0.05). CONCLUSION:GTT inhibited aGVHD development by regulating the production of cytokines in the host.     

Dynamic expression of CD30 on peripheral blood lymphocytes in mice after a cecal ligation and puncture

AIM: To investigate the details of Th2 cell differentiation in septic mice. METHODS: Experimental septic mice were induced by cecal ligation and puncture (CLP). The exression of CD30 on CD4⁺T cells at different time after CLP were estimated by flow cytometry following three-color immunofluorescent staining.RESULTS: CD30 expression on CD4⁺T cell was different at each time point. The highest expression was showed at 38 h after CLP and declined later, which matched the changes in mortality of the animals. CONCLUSION: During sepsis, differentiation of Th2 cell changed with the development of sepsis and might be associated with the severity of the disease.     

Effects of glucocorticoid on miRNA-155 expression in CD4+T cells of asthmatic mice

AIM:To investigate the effects of glucocorticoid on the regulation of microRNA-155 (miRNA-155) expression in the CD4⁺ T cells of asthmatic mice. METHODS:The ovalbumin (OVA)-induced asthma mouse model was established and the mice were treated with glucocorticoid. The effects of glucocorticoid on the pulmpnary histopathological changes, the expression of miRNA-155 in the lung tissues and CD4⁺T cells, and the levels of cytokines in the bronchoal-veolar lavage fluid (BALF) were evaluated. RESULTS:The results of RT-qPCR showed that the expressions of miRNA-155 in the lung tissues and CD4⁺T cells from the spleen of asthmatic mice were significantly increased, and the level of miRNA-155 in the CD4⁺T cells was significantly increased with the increase in the allergen exposure time (P<0.01). HE and PAS staining showed that OVA significantly increased inflammatory cell infiltration as compared with control group, and the peribronchial and perivascular inflammation and mucus secretion of proliferative goblet cells were significantly reduced after glucocorticoid treatment. Glucocorticoid treatment inhibited the increase in the proportion of CD4⁺ CD8^- cells in the spleen and decreased the accumulation of CD4⁺ T cells in the lung tissues of asthmatic mice (P<0.01). After glucocorticoid treatment, the levels of interleukin-4 (IL-4), IL-5 and IL-13 in BALF were decreased, while the level of interferon-γ was increased significantly (P<0.01). CONCLUSION:Glucocorticoid reduces the accumulation of CD4⁺ T cells and inhibits the expression of miRNA-155 in the lung tissues and spleen CD4⁺ T cells of asthmatic mice.     

Calreticulin expression is necessary for protective immune effect of T-cell vaccine in EAE mice

AIM:To study the role of cell membrane ectopic calreticulin (CALR) expression on the protective immunie effect of T-cell vaccine (TCV) on experimental autoimmune encephalomyelitis (EAE). METHODS:EAE model was established by myelin oligodendrocyte glycoprotein 35-55 (MOG_35-55) immunization in C57BL/6 mice, and the mice were immunized with MOG_35-55-specific CALR⁺ and CALR^- T-lymphocytes. Symptomatic scores were compared at the maximum of the disease. On the 15th day after immunization, the proportion of CD4⁺ CD25⁺ Foxp3⁺ regulatory T cells (Treg) in the spleen, and the expression of interferon-γ (IFN-γ), interleukin-4 (IL-4), IL-10 and IL-17A in the serum were measured. RESULTS:Increased expression of CALR in activated T cells after γ-irradiation was observed. Blockade of CALR on the vaccinating T-cell surface reduced the protective effect of TCV. Furthermore, blockade of CALR reduced the number of Treg in the spleen and up-regulated pro-inflammatory cytokines. CONCLUSION:CALR expression in the T cells is necessary for the protective immunity induced by TCV in EAE mice.     

Therapeutic effect of ulinastatin and berberine on burn sepsis in rats

AIM: To investigate the combination therapeutic effect of ulinastatin and berberine on burn sepsis in the rats.METHODS: The rats with burn sepsis were administrated with ulinastatin or berberine or combination therapy. The survival rate and body weight of the rats were measured. The levels of IL-6, IL-10 and TNF-α were examined by ELISA. The morphological changes of the lung were observed by hematoxylin and eosin staining. The content of bacteria in the liver, lung and spleen tissues was detected.RESULTS: Combination of ulinastatin and berberine significantly increased the survival rate and body weight in the rats with burn sepsis. Moreover, combination therapy inhibited the elevation of IL-6 and TNF-α levels, whereas increased the IL-10 level. Combination therapy protected the structure of hepatic lobule, and further reduced the bacterial content in the liver, lung and spleen. CONCLUSION: Combination of ulinastatin and berberine improves the symptoms of burn sepsis by regulating the levels of IL-6, IL-10 and TNF-α and inhibiting the content of bacteria.     

Effects of pentoxifylline on ventricular remodeling and cardiac function of dilated cardiomyopathy rats

AIM: To explore the effects of pentoxifylline (PTX) on ventricular remodeling and cardiac function in dilated cardiomyopathy (DCM) rats.METHODS: Lewis rats were randomly allocated to a myocin-induced dilated cardiomyopathy (DCM) group receiving saline (n=10), a DCM group receiving PTX (PTX group; 25 mg·kg^-1·d^-1, ip, for 30 days, n=10) or healthy control group (n=10). The levels of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6) and IL-10 in the blood plasma were analyzed by ELISA. The extent of fibrosis was estimated using Massons staining and immunohistochemistry analyses. Cardiac structure and function were measured by echocardiography.RESULTS: PTX decreased plasma levels of TNF-α and IL-6, and increased IL-10 level in DCM animals compared with DCM group ［TNF-α: (7.21±0.24) μg/L vs (19.30±1.31) μg/L, P<0.01; IL-6: (119.60±36.58) ng/L vs (189.50±13.25) ng/L, P<0.05; IL-10: (41.26±3.27) μg/L vs (32.45±4.32) μg/L, P<0.05］. Collagen volume fraction (CVF), perivascular collagen area (PVCA) and collagen Ⅰ/Ⅲ ratio were lower in PTX group than those in DCM group ［CVF: (16.45±3.01)% vs (23.33±4.43)%, P<0.05; PVCA: 4.58±2.10 vs 13.74±4.29, P<0.05; Ⅰ/Ⅲ ratio: 2.84±0.67 vs 4.22±0.54, P<0.01］. Left ventricular end-diastolic dimension reduced ［(6.11±0.51) mm vs (6.46±0.28) mm, P<0.05］ and left ventricular ejection fraction elevated ［(77.29±5.20)% vs (62.73±10.11)%, P<0.01］ by PTX compared with DCM.CONCLUSION: PTX modulates plasma levels of inflammatory cytokines, delays the ventricle remodeling and improves the heart function in DCM rats.     

The cytotoxicity of nitric oxide induced by inflammatory cytokine in combination with LPS in endothelial cells

AIM: To explore the mechanism underlying inducible nitric oxide (NO) caused injury of endothelial cells during inflammation. METHODS:The activity of iso-enzymes of NO synthase (NOS), NO level and iNOS expression were examined using NADPH method, Griess reaction and RT-PCR, respectively. Furthermore, the lactate dehydrogenase (LDH) release rate, malondialdehyde (MDA) content were also measured. RESULTS:Co-administration of cytokines (TNF-α 5×10⁵ U/L, IL-1β 2×10⁵ U/L, INF-γ 2×10⁵ U/L) and LPS (10 mg/L) caused an obvious increase in NOS activity, NO levels (about two-fold) and a significant injury of the cells. At the same time, a significant increase in iNOS mRNA was also detected. Wheareas, treatment of the cells separately with cytokines or LPS for 24 h had no significant effect on NOS activity and NO level in cell lysates, however, it caused a significant increase in LDH release and MDA content. Also, the effect of cytokines and LPS on cell viability was concentration-and time-dependent. L-NMMA, a inhibitor of NOS, can suppress inducible NO production and protect cells against NO induced injury. CONCLUSION:Co-administration of cytokines (TNF-α, IL-1β and INF-γ) and LPS significant activated iNOS and NO production which, in turn, induced oxidative reaction in endothelial cells.     

Experimental study on anti-endotoxin activity of a tetrahydropyrimidine derivative,ZL-5015

AIM: To investigate the protective effect of 1, 3-dicyclopentyl-1, 2, 3, 6-tetrahydropyrimidine-4, 5-dicarboxylic acid diethyl ester (ZL-5015) on lethal endotoxin-challenged mice and to explore the underlying mechanism. METHODS: Mouse model of lethal endotoxin challenge and endotoxemia were established by intraperitoneal administration of lipopolysaccharide (LPS) at a dose of 70 mg/kg to the C57BL/6J mice. Mouse peritoneal macrophages stimulated with LPS (10 mg/L) were used as an in vitro inflammatory model. The levels of interleukin-1β (IL-1β), interleukin-10 (IL-10) and tumor necrosis factor-α (TNF-α) were measured by enzyme-linked immunosorbent assay (ELISA). Real-time PCR was used to evaluate the mRNA expression of the cytokines. RESULTS: Prophylactic treatment of the mice with ZL-5015 (100 and 200 mg/kg, ig) slightly increased the survival rate, extended the survival time, decreased the serum levels of IL-1β and TNF-α, and increased the serum level of IL-10 in the early stage of endotoxemia as compared with model group. The results of in vitro study demonstrated that treatment of the endotoxin-stimulated mouse peritoneal macrophages with ZL-5015 (10, 20 and 40 μmol/L) inhibited the expression of IL-1β and TNF-α at both mRNA and protein levels but promoted the expression of IL-10 at both mRNA and protein levels. CONCLUSION: The tetrahydropyrimidine derivative ZL-5015 shows a moderate anti-endotoxin effect by increasing the survival rate and extending the survival time of the mice challenged by endotoxin, which may result from inhibition of the expression of pro-inflammatory cytokines such as IL-1β and TNF-α, and promotion of the expression of anti-inflammatory cytokine IL-10.     

Role of IL-23/IL-17 inflammatory axis in the pathogenesis of psoriasis-like lesions induced by imiquimod in mice

AIM: To observe the dynamic changes of IL-23/IL-17 inflammatory axis in psoriasis-like lesions of mice induced by imiquimod (IMQ).METHODS: BALB/c female mice were randomly divided into control group and IMQ group. The morphological changes of lesional skin in mice were evaluated according to the psoriasis area and severity index (PASI) and HE staining. cytokine antibody chips were used to determine the cytokine changes in serum and lesions. The mRNA and protein expression of cytokines were analyzed by cytometric bead array, real-time PCR and Western blotting. Moreover, the changes of cellular constituents in the peripheral blood and splenic cells of mice were detected by flow cytometry.RESULTS: Typical psoriasis-like skin lesions, such as red scaly skin plaques, caused by topical IMQ showed a parabolic dynamic change. There was a dynamic increase in proinflammatory cytokines of the IL-23/IL-17 axis in IMQ-treated skin. IMQ application resulted in elevated expression of cytokines related with IL-23/IL-17 inflammatory axis,Th1-type cytokines,Th2-type cytokines and Treg-type cytokines at day 4. IMQ-treated BALB/c mice showed an increased pericentage of dentric cells in peripheral blood and spleen compared with control animals. Percentages of Th17 and Treg in IMQ-treated mice were increased by 3~4 times and twice as compared with control mice, respectively.CONCLUSION: The skin lesions, histopathological features and cytokine changes in mice induced by IMQ are similar to human psoriasis, which are suitable for investigating the pathogenesis of psoriasis as a psoriasis-like model. IL-23/IL-17 axis is involved in the formation of psoriasis-like skin lesions in mice induced by IMQ and presents a dynamic change. Besides, Th1 cell-mediated inflammatory response is also activated in the formation of lesional skin, accompanied by the increase expression of Th2 and Treg cytokines in a feedback mechanism.     

2,3,5,4’-Tetrahydroxystilbene-2-O-β-D-glucoside protects against LPC-induced endothelial cell injury

AIM: To observe the protective effect of 2,3,5,4’-tetrahydroxystilbene-2-O-β-D-glucoside (TSG) on lysophosphatidylcholine (LPC)-induced vascular endothelial cell injury. METHODS: The 3rd and 4th generations of human umbilical vein endothelial cells (HUVECs) were cultured in vitro and propagated. The cells were randomly divided into 3 groups: control group, model group (LPC) and experimental group (TSG+LPC). The cells in control group were not treated with any reagent. To establish endothelial cell injury model, LPC was administered to HUVECs at concentration of 10 mg/L and incubated with the cells for 24 h. In TSG+LPC group, TSG was administered to HUVECs at concentrations of 10.0, 1.0 and 0.1 μmol/L 1 h before administration of LPC, and then the cells were incubated for 24 h. The cell viability, the content of asymmetric dimethyl arginine (ADMA) and NO, and apoptotic rate were detected. RESULTS: Compared with control group, ADMA content in the cell culture supernatants and apoptotic rate of the HUVECs in LPC group were significantly increased, while the NO content and cell viability were notably decreased. Compared with LPC group, ADMA content and apoptotic rate in TSG+LPC group was significantly decreased, while the NO content and cell viability were notably increased. CONCLUSION: TSG may protect LPC-injured vascular endothelial cells by attenuating the expression of ADMA and enhancing the production of NO, thus inhibiting apoptosis and increasing cell survival rate.     

Effects of cholecystokinin octapeptide on expression of IL-1β, IL-6, IL-4 and IL-10 in lipopolysaccharide-attacked mice

AIM:To observe the effects of cholecystokinin octapeptide (CCK-8) on the expression of proinflammatory cytokines IL-1β, IL-6 and anti-inflammatory cytokines IL-10, IL-4 in LPS- attacked mice. METHODS:Kunming mice were randomly assigned and injected intraperitoneally with LPS alone or/and CCK-8 at different time points. The expression of IL-1β, IL-6, IL-10 and IL-4 in the serum and lung tissues were assayed by ELISA and RT-PCR. RESULTS:The expression of IL-1β, IL-6, IL-10 and IL-4 were upregulated in LPS-attacked mice. Pre-treatment of CCK-8 decreased both IL-1β and IL-6 expression and augmented IL-10 and IL-4 expression in LPS-attacked mice. CONCLUSIONS:CCK-8 exerts an anti-inflammatory effect by inhibiting the expression of IL-1β, IL-6 and increasing the expression of IL-10, IL-4 in LPS-attacked mice, which could alleviate the inflammatory response in lung tissue.     

Effect of 1α, 25-dihydroxyvitamin D3 on T helper cell 17 and expression of related cytokines in penetrating keratoplasty in mice

AIM: To evaluate the effects of 1α, 25-dihydroxyvitamin D3 on T helper cell 17 (Th17 cells) and its related cytokines in a mouse model of corneal allograft transplantation. METHODS: C57BL/6 mice were transplanted with corneal grafts from BALB/c mice and treated intraperitoneally with 1.0 μg 1α, 25-dihydroxyvitamin D3 or soybean oil every other day after operation. The transparency of the corneal grafts was evaluated for potential rejection signs by slit lamp biomicroscopy and histopathology. The expression levels of IL-17, RORγt and IFN-γ in the spleen were measured by real-time PCR. Moreover, the protein expression of RORγt and IL-17 in the peripheral blood was analyzed by Western blotting. IL-17 and IFN-γ in peripheral blood were measured by flow cytometry. RESULTS: 1α, 25-dihydroxyvitamin D3 significantly inhibited the rejection of the corneal allograft and reduced the numbers of inflammatory infiltrates in the corneal graft. In the spleen, 1α, 25-dihydroxyvitamin D3 treatment reduced the expression levels of IL-17, RORγt and IFN-γ. In the peripheral blood, 1α, 25-dihydroxyvitamin D3 treatment downregulated the expression levels of RORγt, IL-17 and IFN-γ. CONCLUSION: The effects of 1α, 25-dihydroxyvitamin D3 on suppressing corneal transplantation-induced allograft rejection in mice are closely associated with its modulation on IL-17 and related cytokine RORγt.     

Pilot study on the function of anti-bacterium immunity of the genome DNA of mycobacterium tuberculosis H37Ra strain

AIM: To determine whether the celiac macrophages are activated and the production of nitric oxide, and expression of the anti- tuberculous cytokines after macrophage activation by immunized intracutaneously with the genome DNA of mycobacterium tuberculosis H37Ra strain and mycobacterium bovis-bacille Calmette-Guerin(BCG). METHODS: Male C57BL/6 mice were immunized intracutaneously with the genome DNA of mycobacterium tuberculosis H37Ra strain and BCG. The production of NO and H₂O₂, the expression levels of IL-12 and TNF-α by mouse celiac macrophages with or without IFN-γ stimulation were determined by Griesss method, chemical method and ELISA assay respectively. RESULTS: 30 d and 60 d after intracutaneous vaccination, the genome DNA of mycobacterium tuberculosis H37Ra strain effectively induced the secretion of IL-12 and TNF-α in macrophages, a significant difference was observed compared to that in the un-immunized group, but without any difference compared with BCG-immunized group of mice. In addition, it also induced macrophages to produce NO and H₂O₂ with significant difference to the un-immunized group, but without any difference with BCG -immunized group of mice. IFN-γ demonstrated an intensive effect on the production of nitric oxide and cytokines from macrophages. CONCLUSION: It is concluded that the intracutaneous vaccination with the genome DNA of mycobacterium tuberculosis H37Ra strain induces activation of macrophages and generates strong immune responses, and this effect is not significant difference from immunizing with BCG.     

Establishment of a mouse model of sepsis associated encephalopathy and preliminary research of cognitive dysfunction in this model

AIM: To establish the mouse model of sepsis-associated encephalopathy (SAE) and the preliminary research of cognitive dysfunction in this model. METHODS: SPF male C57BL/6J mice of 8~10 weeks old were selected. The first part of the experiment divided the mice into 4 groups randomly, namely control group, cecal ligation and puncture (CLP)1 group and CLP2 group (CLP was performed with 7 and 12 syringe needle respectively). The mice in sham operation group were only laparotomy. In the second part of the experiment, the mice were randomly divided into control group, sham operation group and CLP group. The Kaplan-Meier method was used to analyze the postoperative survival rate of the mice in the first part experiment. The neurobehavioral scores were used to evaluate the neurobehavioral changes of the mice. The Morris water maze test and the passive avoidance experiment were used to detect the changes of cognitive memory function in the mice. The pole test and the wire suspension test were used to test the motor coordination of the mice. The serum levels of prostaglandin E2 (PGE₂) were measured by ELISA. RESULTS: In the first part of the experiment, the CLP mice showed obvious symptoms such as lethargy, piloerection, chills and anorexia. The 48 h mortality in CLP1 group and CLP2 group were 20% and 30% respectively. In the 2 parts of the experiments, the neurobehavioral scores of the CLP mice were significantly lower than those in control group and the sham operation group (P<0.01). In CLP mice, the escape latency time of the Morris water maze was significantly prolonged (P<0.01), the target quadrant dwell time and the number of crossing platforms were decreased (P<0.01), the scores in the suspension experiment and the pole test were significantly reduced (P<0.01), the activity of the mice was decreased or even did not enter the darkroom in the step-through test (P<0.05). In the second part of the experiment, the serum level of PGE₂ in the mice after CLP was significantly increased (P<0.01). CONCLUSION: A stable mouse model of sepsis-associated encephalopathy is successfully established by cecal ligation and puncture with 12 syringe needle. The SAE mouse model established by this method is useful for investigating the learning and memory cognitive dysfunction.     

Association between development of CD4+CD25+ regulatory T cells and thymus CD4-CD25+ cells

AIM: To explore the correlation between development of CD4⁺CD25⁺ regulatory T cells (CD4⁺CD25⁺ Tr) and thymus CD4^-CD25⁺ cells. METHODS: The ratios of CD4⁺CD25⁺ regulatory T cells to CD4+ T cells in thymus, spleen, lymph node and peripheral blood of mice from birth to mature and also the ratios of CD4^-CD25⁺ cells to CD4^- T cells in thymus were measured by flow cytometry. Purified CD4⁺CD25⁺ T cells and CD4⁺CD25^- T cells were labeled with CFDA-SE, and then stimulated with various kinds of stimulators. RESULTS: The percentages of CD4⁺CD25⁺ Tr in mouse spleen, lymph nodes and peripheral blood increased gradually, but not in thymus, from day one to week 10 of the age with rapid rising from day one to week 1. The percentages of CD4^-CD25⁺ cells in mouse thymus were quite high on day one after birth, and decreased rapidly from day one to week 1. Both CD4⁺CD25⁺ Tr and CD4⁺CD25^- T cells showed no proliferation in response to ConA, while CD4⁺CD25⁺ Tr showed a transient enlargement of cell size. Both CD4⁺CD25⁺ Tr and CD4⁺CD25^- T cells underwent proliferation in response to PDB plus ionomycin. CD4⁺CD25^- T cells, but not CD4⁺CD25⁺ Tr, showed a proliferative response to the stimulation of coated anti-CD3 plus soluble anti-CD28 antibody, however, CD4⁺CD25⁺ Tr showed significant proliferation and CD4⁺CD25^- T cells showed a stronger response in addition of high dose of IL-2. CONCLUSION: The thymus CD4^-CD25⁺ cells are probably the precursor of CD4⁺CD25⁺ Tr during cell development.     

Effect of forsythiaside A on immune function in rats with ulcerative colitis

AIM To investigate the effect of forsythiaside A (FA) on immune function in rats with ulcerative colitis and its related mechanism. METHODS Healthy SD rats were randomly divided into 5 groups: control group (no treatment, normal feeding), model group (establishment of rat ulcerative colitis model), and low, medium and high doses of FA groups (treatment of the model rats with FA at 5 mg/kg, 20 mg/kg and 80 mg/kg, respectively). The malondialdehyde (MDA) content and superoxide dismutase (SOD) activity in rat colon tissues were measured by colorimetry, and the serum levels of tumor necrosis factor-α (TNF-α), interleukin-2 (IL-2) and IL-4 were detected by ELISA. The spleen index and thymus index, the percentages of CD3⁺, CD4⁺ and CD8⁺ T-lymphocytes in peripheral blood mononuclear cells （PBMC）, the serum IgA and IgG levels, and the serum complement C3 and C4 levels were also determined. RESULTS The colon tissues of the rats in model group showed obvious inflammation and ulceration, indicating that the animal model was successfully established. Compared with model group, the colonic inflammation and ulceration were significantly attenuated in FA groups, among which the high dose had the best effect. Compared with control group, the spleen index and thymus index of the rars in model group were decreased (P<0.05), MDA content in colon tissues was increased (P<0.05), and SOD activity in colon tissues was decreased (P<0.05). The levels of CD3⁺, CD4⁺, CD8⁺ and CD4⁺/CD8⁺ T-lymphocytes in PBMC, and the serum levels of C3, C4 and IL-4 were decreased (P<0.05), while the serum levels of IgA, IgG, TNF-α, and IL-2 were increased in model group as compared with control group. Furthermore, the spleen index and thymus index of the rats in FA groups were increased (P<0.05), the MDA content in the colon tissues was decreased (P<0.05), and the SOD activity in the colon tissues was increased (P<0.05). The levels of CD3⁺, CD4⁺, CD8⁺ and CD4⁺/CD8⁺ T-lymphocytes in PBMC, and the serum levels of C3, C4 and IL-4 were increased (P<0.05), while serum IgA, IgG, TNF-α and IL-2 levels were decreased in FA groups as compared with model group (P<0.05). CONCLUSION Forsythiaside A effectively attenuates the colonic lesions in rats with ulcerative colitis, and its mechanism may be related to reinforcement of oxygen free radical scavenging power, alleviation of inflammatory response, and enhancement of immune function.