Experimental study on anti-endotoxin activity of a tetrahydropyrimidine derivative, ZL-5015 / 中国病理生理杂志

[ ABSTRACT] 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-1βand 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 40μmol/L) inhibited the expression of IL-1βand TNF-αat 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-1βand TNF-α, and promotion of the expression of anti-inflammatory cytokine IL-10.

Optimization of a lipopolysaccharide-stimulated nitric oxide production model in mouse peritoneal macrophages / 南方医科大学学报

<p><b>OBJECTIVE</b>To optimize the experimental model of nitric oxide (NO) production in mouse peritoneal macrophages in response to lipopolysaccharides (LPS) stimulation.</p><p><b>METHODS</b>Mouse resident peritoneal macrophages were collected by lavaging the peritoneal cavity of mice with Hank's solution and stimulated with Pseudomonas aeruginosa LPS for NO production. NO concentration in the culture supernatants was measured with Griess Reagent. The influences of cell density, LPS concentration, LPS stimulation duration and culture medium volume on NO production were investigated. Finally, the feasibility of the model was confirmed with specific anti-inflammatory drugs.</p><p><b>RESULTS</b>The density of macrophages produced the most significant effect on NO production (P<0.001), and optimal results were obtained at the macrophage density of 6×10(6) cells/ml with a volume of 100 µl in each well in 96-well plate. At a LPS concentration below 1 µg/ml, NO production increased proportionally with the increment of LPS concentration (P<0.001), but the increment of NO production declined obviously at LPS concentrations beyond 1 µg/ml, and the peak NO production occurred at a LPS concentration of 10 µg/ml. NO production also increased significantly with the prolongation of LPS stimulation (P<0.05), and the increments were greater within 24-48 h than those in 48-72 h. NO content in the culture supernatant was associated with the medium volume, and the highest level occurred in a system volume of 100 µl. Aspirin (1 mmol/L), dexamethasone (10 µmol/L), and cyclosporin A (10 µmol/L) all significantly inhibited LPS-stimulated production of NO in mouse resident peritoneal macrophages (P<0.001).</p><p><b>CONCLUSIONS</b>Macrophage density, LPS concentration, and the duration of LPS stimulation are the main factors affecting LPS-stimulated NO production in mouse resident peritoneal macrophages. The optimal results can be obtained with a macrophage density of 5×10(6) cells/ml (100 µl per well), LPS concentration of 10 µg/ml, LPS stimulation duration of 24 h or 48 h, and a culture medium volume of 100 to 200 µl.</p>

Experimental study of the anti-inflammatory and analgesic effects of diethyl 1,3-dicyclohexyl- 1,2,3,6-tetrahydropyrimidine-4,5-dicarboxylate / 南方医科大学学报

<p><b>OBJECTIVE</b>To study the anti-inflammatory and analgesic activities of diethyl 1,3-dicyclohexyl-1,2,3,6-tetrahydropyrimidine-4,5-dicarboxylate (ZL-5010) in vivo and in vitro.</p><p><b>METHODS</b>The analgesic effect of ZL-5010 was evaluated by acetic acid-induced writhing response in mice, and the anti-inflammatory effects was assessed in mice with xylene-induced ear edema and in rats with carrageenan-induced paw edema. Mouse peritoneal exudate cells activated by bacterial lipopolysaccharides (LPS) were used to evaluate the anti-inflammatory effect of ZL-5010 in vitro. The levels of interleukin-1β (IL -1β) and tumor necrosis factor-α (TNF-α) in the cell culture supernatant were measured using enzyme-linked immunosorbent assay (ELISA).</p><p><b>RESULTS</b>At the doses of 0.25 and 0.5 mmol/kg, ZL-5010 administered by gavage once daily for 3 days significantly reduced acetic acid-induced writhing frequency and suppressed xylene-induced ear edema in mice, and alleviated paw edema induced by carrageenan in rats (P<0.05). The agent also inhibited the production of the pro-inflammatory cytokines IL-1β and TNF-α by LPS-induced mouse peritoneal exudate cells in vitro, with the statistically significant minimum effective concentrations of 10 and 20 µmol/L, respectively (P<0.05).</p><p><b>CONCLUSION</b>ZL-5010 administered by gavage has anti-inflammatory and analgesic effects in mice and rats, and in mouse peritoneal exudate cell cultures, the agent also inhibits the production of the pro-inflammatory cytokines IL-1β and TNF-α.</p>

Counteraction of Ganoderma Lucidum Polysaccharide on the Inhibition of Mice Splenocyte Proliferation and IL-1? and IL-2 mRNA Expression Induced by Prostaglandin E2 / 广州中医药大学学报

Objective To observe the counteraction of Ganoderma lucidum polysaccharide(GLP) on the inhibition of mice splenocyte proliferation and IL-1? and IL-2 mRNA expression induced by prostaglandin E2(PGE2).Methods Mixed lymphocyte culture reaction(MLR) method was used for the experiment.The lymphocyte proliferation was determined by MTT method,and the levels of IL-1? and IL-2 mRNA expression were evaluated by semi-quantitative reverse transcriptase polymerase chain reaction(RT-PCR).Results After co-culture with PGE2 for 48 hours,the splenocyte proliferation was inhibited to some extent,and the difference was significant when the concentration of PGE2 was over 10?mol/L(P