Effect And Mechanism Of Intravenous Anesthetics On Endotoxin-stimulated Proinflammatory Cytokine Response Of Murine Celiac Macrophages

Objective To observe the concerntration and time dependent effects of endotoxin on proinflammatory cytokine; To evaluate the effect of intravenous anesthetics on the endotoxin-stimulated proinflammatory cytokine response and investigate themechanism--the effect of intravenous anesthetics on the activation of NF-KB.Methods Murine celiac macrophages which had been isolated were maintained in RPMI1640 medium and then perform the experiments as follows: 1. Concerntration-dependent effects of endotoxin on proinflammatory cytokines: Incubate the macrophages with Ips at different concentrations for 4 hours, then analyse the expression level of TNF-a, II-6 and II-8 mRNA using RT-PCR. 2. Time-dependent effects of endotoxin on proinflammatory cytokines: 10ng/ml Ips incubate the macrophages, 1 to 6 hours later analyse the expression level of TNF-a, II-6 and II-8 mRNA.3. Effect of intravenous anesthetics on the expression of proinflammatory cytokine mRNA and the activation of NF-KB. Macrophages were randomly assigned to one of seven groups(n=5) bellow: (a) negative control; (b) endotoxin group, receiving 10ng/ml Ips ; (c) anesthetics group, only incubate macrophages with one of following anesthetics-propofol, ketamine, thiopentone.midazolam or etomidate; (d) (e) (f) (g)groups: Ips+four concentrations anesthetics above, pretreat macrophages with different concerntrations of anesthetics for 2 hours and then incubate it with 10ng/ml Ips 1 or 4hours. 1 hours later, analyse the expression level of TNF-amRNA using RT-PCR; analyse the level of phosphorylated NF-KB P65 and kB using Western bolting; 4 hours later , analyse the expression level of II-6, II-8 mRNA using RT-PCR Results 1. Lps induced TNF-a, IL-6 and IL-8 mRNA expression in a concentration -dependent manner at concentrations 0.1-10ng/ml and reached a plateau with concentration 10ng/ml. 2. TNF-a, IL-6 and L-8 mRNA expressionreached a plateau 2 hours , 4 hours and 6 hours after the stimulation of 10ng/ml Ips. 3. Expression of TNF-a, IL-6 and IL-8 mRNA increased rapidly after stimulated by Ips, with TNF-a mRNA being attenuated by propofol at concentrations 0.5, 5, 50 and 500p.g/ml; by ketamine at concentrations 20, 200 and 500|ag/ml; by thiopentone at concentrations 500ng/ml and 1mg/ml; with IL-6 mRNA being attenuated by propofol at concentrations 0.5, 5, 50 and 500|j.g/ml; by ketamine at concentrations 200ng/ml and 500ng/ml; with IL-8 mRNA being attenuated by ketamine at concentrations 200ng/ml and 500(ig/ml, but being promoted by midazolam at concentrations 1, 10 and 100|ag/ml g/ml; Etomidate showed no influence on the expression of TNF-a, IL-6, IL-8 mRNA 4. After stimulated by Ips, the level of phosphorylated NF-KB P65 move up sharply, but this effect is significantly being suppressed by ketamine at concentrations 20, 200 and 500|ig/ml; by thiopentone at concentrations 500ng/ml and 1mg/ml. Propofol, midazolam and etomidate did not affect the activation of NF-KB. 5. Lps stimulation led to the degradation of IxB, but its expression increased in macrophages being treated with Ips+ketamine at 20, 200 or 500(ig/ml and lps+ thiopentone at 500ng/ml or 1mg/ml. Conclusion: Propofol at clinical-relevant concentrations can suppress the expression of proinflammatary cytokine of macrophages stimulated by Ips, but this effect is not throuth the inhibition on NF-KB activation; Ketamine and thiopentone can suppress the expression of proinflammatary cytokine throuth the inhibitation of NF-KB activation. Midazolam and etomidate did not affect the activation of NF-KB, as well as showed no effect on the expression of proinflammatary cytokine.