| Objective:1. To investigate the effects of STCB against endotoxin-induced toxicity and inflammation.2. To investigate the effect of STCB on immune function.3. To explore the potential of the saponin in treating endotoxemia and SIRS (systemic inflammatory response syndrome).Methods:1. Water saturated n-butyl alcohol was used to isolate the crude saponin from Tupistra chinensis Baker (STCB).2.64normal KM mice were randomly divided into4groups:model saline group, positive control group (Dex,5mg/kg) and STCB (200,400mg/kg) groups, and the mouse model of endotoxin-induced death was established by intraperitoneal (ip) administration of KM mice with lipopolysaccharides.(LPS) from Pseudomonas aeruginosa in dose of60mg/kg. The mouse survival rate and survival time were recorded.3.40normal KM mice were randomly divided into5groups:model saline group, positive control group (Dex,5mg/kg) and STCB (200,400mg/kg) groups. The mouse model of endotoxemia was established by intraperitoneal (ip) administration with lipopolysaccharides (LPS) from Pseudomonas aeruginosa in dose of10mg/kg, and the treatment was given once daily for7consecutive days. The serum levels of interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) in endotoxemia mice were measured by enzyme-linked immunosorbent assay (ELISA).4. Mouse peritoneal exudate cells activated with LPS were used as an in vitro inflammatory model, which was intervened with STCB at the concentrations of10,20and40μg/ml. The cells were incubated at37℃for16h, and then the levels of IL-1β and TNF-a in the culture supernatants were measured by ELISA.5.40normal KM mice were randomly divided into5groups:DTH model group, positive control group (Dex,10mg/kg) and STCB (200,400and800mg/kg) groups. STCB was given intragastrically (ig) for7consecutive days. Mouse model of DTH was established by priming the mice with1%dinitro-fluorobenzene (DNFB) applied to the shaved abdomen skin and attacking to the right ear7days later.24hours after attack, the animals were sacrificed and the pieces of the double ears, thymus and spleen were weighed to evaluate the influence of STCB on cellular immune function.6.40normal mice were randomly divided into5groups, including model group, positive control group and STCB (200,400and800mg/kg) groups. Animals were gastrically administered with vehicle or investigated drugs once daily for7consecutive days. On the next day after treatment, every mouse was then administered intraperitoneally with20%RRBCs (rabbit red blood cells) in a total volume of0.2ml. On the sixth day after priming, the serum samples of the animals were collected for haemolysin assay.7. Mouse spleen cells were isolated and cultured in the presence of STCB and Con A or LPS, which are T or B lymphocyte stimulant respectively. After cultured for48h at37℃, the cell metabolic or proliferative activity was assayed with MTT colorimetry.8. Mouse spleen cells were isolated and cultured in the presence of Con A, and then intervened with STCB at the concentraation of20,40and80μg/ml, incubated at37℃for48h, and then the levels of IL-2in the culture supernatants were measured by ELISA.9. SPSS13.0statistical software was used for statistical analysis. For more than one sample mean comparison, ANOVA (One-Way ANOVA) was applied after testing of homogeneity of variance. If variance was of homogeneity, LSD method for pairwise comparison between groups was adopted. When variance was of heterogeneity, Welch test was applied at first and then DunnettT3method for pairwise comparisons between groups was employed. Significant level was set at α=0.05and P<0.05was considered statistically significant.Results:1.30minutes after injected by endotoxin in the dose of60mg/kg, the mice appeared to be short of breath and acted slowly with fur losing its luster. With the passage of time, the animal started to huddle together and refused feeding. Some of the animals were dying and wet at perineum.20hours later, the animals became to die, and the surviving animals gradually returned to normal after48hours. In the model of endotoxin-induced lethal shock, the survival rates of mice prophylactically treated with STCB (200and400mg/kg, in5consecutive days) were higher compared with that in model group, but no statistical difference was observed (χ2=1.412, P=0.494). The survival time, however, was much longer in the treated group (P=0.051,P=0.028).2. The serum levels of IL-1β and TNF-α in model group were47and67times higher than those in saline control (P<0.001,P<0.001), and the levels in STCB-treated mice (200and400mg/kg, in5consecutive days) were significantly lower, with IL-1β decreased by24%and42%(P=0.247, P=0.006), and TNF-α by31%and41%(P=0.047, P=0.004), respectively compared with those in model group. STCB (10,20and40μg/ml) remarkably inhibited lipopolysaccharidesinduced TNF-α production by peritoneal exudate cells in vitro (P=0.023, P<0.001, P<0.001). High dose of STCB (40μg/ml) inhibited IL-1β production (P=0.013).3. The swelling degree and the thymus index in DTH mice only decreased obviously in the group of high dose of STCB (P=0.018, P=0.020). The spleen index were not affected influenced (P=0.062).4. STCB at high concentrations (80μg/ml) could significantly inhibit the Con A or LPS-induced proliferative response of mouse spleen cells (metabolic activity) with statistical significance (P<0.001, P<0.001), and inhibit IL-2production induced by Con A (P=0.011).The median concentration inhibited the proliferative response induced by Con A or LPS with statistically significance (P=0.001, P<0.001), but could not obviously affect the IL-2production (P=0.061). The low level of STCB could not either affect the spleen cells activity or IL-2production (P=0.136, P=0.061,P=0.330).Conclusion:1. The mouse models of endotoxin-induced death and endotoxemia were established by intraperitoneal (ip) administration with lipopolysaccharides (LPS) from Pseudomonas aeruginosa (60and10mg/kg) successfully.2. STCB could prolong the survival time of the mice treated with lethal dose of LPS remarkably.3. STCB could significantly reduce the serum levels of IL-1β and TNF-α in mice with endotoxemia, and inhibited the function of activated macrophages.4. The mouse model of delayed type hypersensitivity was established successfully. 5. STCB at high concentration showed immunodepressant effect, but the median and low concentration showed no statistical significance.6. STCB at high concentration could inhibit the production of IL-2by spleen cells, indicating inhibition of IL-2production being involved in the STCB immunodepressant mechanism. |
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