The Study On The Effect And Mechanism Of Acetylcholinesterase Inhibitor Galanthamine On Sepsis Inflammation

Sepsis is the most common complications of trauma,burns,shock,infection,and major surgery.The severe sepsis can develop into septic shock and multiple organ dysfunction syndrome(MODS).Sepsis has become one of the important causes of death in critical ill patients,and it is a difficult problem for critical care medicine. Because the nature of sepsis the out-of-control inflammatory response,the task of the research on drug is the development of specific inhibitors,blocking agents,or neutralizers of inflammatory mediators.The researchers try to stop on each specific aspect of the occurrence and development of sepsis.Although a lot of experiments in animals achieved satisfactory results,most of the clinical applications are unsuccessful.Vagus nerve is an important parasympathetic,and its fiber widely distributes in reticuloendothelial system organs,like the lung,the liver,the spleen,the kidney,and the intestines.Besides regulating the activities of these organs,a recent study showed that electrical stimulation of the branch of vagus nerve could inhibit the synthesis of tumor necrosis factor-α(TNF-α) of the liver,spleen,heart tissue in endotoxemic rats and reduce the TNF-αlevel in plasma,and thus reduce the incidence of septic shock. Vagal neurotransmitter acetylcholine can inhibit endotoxin-stimulated macrophages in releasing TNF-α,IL-interleukin(IL)-1β,IL-6 and IL-18,but does't affect the synthesis and release of anti-inflammatory cytokines IL-10.After the resection of vagus nerve,the synthesis and release of TNF-αis significantly increased,the endotoxin lethality in experimental animals is also enhanced.These studies indicate that the vagus nerve excited can reduce the systemic inflammatory response.Because its transmitters is acetylcholine,is is called cholinergic anti-inflammatory pathway.Theα7 nicotinic acetylcholine receptor(α7nAChR) expresses in the surface of macrophages, monocytes,endothelial cells,which plays an important role in the connection between the cholinergic system and the immune system.Cholinergic anti-inflammatory pathway provides us with a new the idea for the sepsis treatment,that's activating cholinergic anti-inflammatory pathway.There are two ways for activating cholinergic anti-inflammatory pathway:one is the direct electrical stimulation of vagus nerve,and the other one is drug.Central cholinergic system can excite central muscarinic(M) acetylcholine receptors(especially the M1 subtype) or increase the central release of acetylcholine (emotional self-regulation of presynaptic M2 receptors),activate the cholinergic anti-inflammatory pathway,increase in vagus nerve activity,and reduce the release of TNF,but the directly excited peripheral M receptor can't produce anti-inflammatory effects.There are a variety of ways for exciting the central cholinergic system,including using acetylcholine precursors,acetylcholinesterase inhibitors(AchEI),selective cholinergic agonist,or regulators of acetylcholine release.As we all know,that AchEI is effective in treating Alzheimer disease(AD),and so far is the only one drug for the treatment of AD,which has approved by the United States Food and Drug Administration.Galantamine is the only drug with such mechanism among this kind of drugs.Besides inhibiting the degradation of acetylcholine,galantamine can stimulate brain nerve cells to release more acetylcholine.The active ingredient of galantamine was first extracted from a plant,and at present it can be synthesized by chemical methods.In 2001,The United States Food and Drug Administration approved low or medium dose of galanthamine in the treatment of Alzheimers Disease,and now galanthamine has entered into the clinical in China.By using rats with abdominal infection induced by endotoxin and in vitro cell culture,the study is to explore the effect and mechanism of galanthamine on inflammatory response,and to explore new ways for the treatment of sepsis.The study was divided into two parts:the first part is about the effect and mechanism of galanthamine(cholinesterase inhibitors)on inflammatory response,and the second is about the effect of galanthamine on the RAW264.7 activation after stimulated by endotoxin.Objective:The study is to explore galanthamine(cholinesterase inhibitors) on the circulating TNF in rats with abdominal infection induced by endotoxin,the effecft of vagus nerve on inhibitory effect of galanthamine,and the effect of galanthamine on macrophage activation after stimulated by endotoxin,and to explain the mechanism of galanthamine on the regulating inflammation.Methods:Chapter one The chapter is about the effect of galanthamine on the circulating TNF in rats with abdominal infection induced by endotoxin and effects of vagus nerve on galanthamine inhibiting inflammatory response.The models of rats with abdominal infection induced by endotoxin and the animal models of bilateral cervical vagotomy were produced.In the first part,the rats were randomly divided into control group (n=8 ),endotoxin + galanthamine 1(LPS+Gal 1,n=8 ),endotoxin + galanthamine 2(LPS+Gal 2,n=8 ),endotoxin + galanthamine 3(LPS+Ga3 1,n=8 ),and in the second part,the rats were randomly divided into Sham group(n=8),vagotomy group (Vg group,n=8),vagotomy + endotoxin group(Vg+LPS,n=8),vagotomy + endotoxin + galanthamine group(Vg+LPS+Gal,n=12).The plasma TNF was determined in every group.Sigmastat software was used for statistical analysis. ANOVA and Newman-keuls test was used.P<0.05 was considered as significantly different.Chapter two The second chapter is about the effect of galanthamine on the macrophage activation after stimulated by endotoxin.The in vitro culture of mouse macrophage cell line RAW264.7 was used.The cell line was preincubated with different concentrations of GLA and stimulated with endotoxin.The TNF,HMGB-1 by Western Blot,HMGB-1mRNA,p-NF-κB,and p-I-κB were determined.1.RAW264.7 was preincubated with different concentrations of GLA(Final Concentration10,30,100,300,1000,10000 nM) for 24 hours,and then added with 100 ng/ml endotoxin for another 4 hours.The supernatant was collected for the measurement of TNF.2.RAW264.7 was preincubated with different concentrations of GLA(Final Concentration 10,30,100,300,1000,10000 nM) for 24 hours,and then added with 100 ng/ml endotoxin for another 24 hours.The supernatant was collected for the measurement of HMGB-1 by Western Blot.3.RAW264.7 was preincubated with different concentrations of GLA(Final Concentration 10,30,100,300,1000,10000 nM) for 24 hours,and then added with 100 ng/ml endotoxin for another 24 hours.The cells were collected and RNA of cells was extracted.Th real-time fluorescent PCR was applicated for quantitative assessment of PCR amplification,and the espression of HMGB-1 mRNA was quantitatively analyzed.4.RAW264.7 was preincubated with different concentrations of GLA(Final Concentration 10,30,100,300,1000,10000 nM) for 24 hours,and then added with 100 ng/ml endotoxin for another 2 hours.The cells were collected for the measurement of P-NF-κB and p-I-κB by Western Blot.Sigmastat software was used for statistical analysis.ANOVA and Newman-keuls test was used.P<0.05 was considered as significantly different.Results:Chapter one The level of circulating TNF was obviously increased in rats with abdominal infection induced by endotoxin(p<0.001);after added with galanthamine, the level of circulating TNF was significantly decreased,there was significant difference compared with rats with abdominal infection induced by endotoxin(p<0.05 ). The 3mg/kg galanthamine had the most significant inhibition on the circulating TNF. Compared with sham group,the circulating TNF in rats with vagotomy was increased. Galanthamine obviously decreased the circulating TNF in rats with abdominal infection induced by endotoxin,vagotomy in rats weakened the galanthamine inhibitory effet on TNF.Chapter two The TNF secreted by normal cultured RAW264.7 was very low, and the endotoxin stimulation increased the TNF secretion of RAW264.7(p<0.001). within the concentration of 10,30,100,300nM,galanthamine dose-dependently inhibited TNF release.The 300nM galanthamine inhibited LPS-induced TNF release up to 30%.Chapter three Compared to controls,LPS-challenged rats had significantly increased the levels of phosphorylated NF-kB in lung tissues.High- and moderate-dose of edaravone pretreatment resulted in a drastic down-regulation of phospho-NF-kappaB. Edaravone significantly attenuated LPS-induced phospho-NF-kappaB expression in dose-dependent.HMGB-1 existed in the normal cultured RAW264.7,and the level of HMGB-1 in supernatant was significantly increased after stimulated with endotoxin.Galanthamine inhibited the extracellular secretion of HMGB-1 in RAW264.7.The real-time quantitative PCR was used for quantitative determination of HMGB-1mRNA in RAW264.7.At 24 hours after stimulated by endotoxin,the level of HMGB-1mRNA in RAW264.7 had no significant change,and the preincubation of different dose of galanthamine also had no significant effect on the level of HMGB-1mRNA in RAW264.7.The levels of p-NF-KB and p-I-κB in RAW264.7 were significantly increased after stimulated by endotoxin,and this showed the activation of NF-κB pathway.The preincubation of different dose of galanthamine can obviously down-regulate p-NF-κB and p-I-κB,and showed a dose-dependent,and had the consistent dose-dependent with galanthamine inhibiting TNFαsecretion.The 300nM galanthamine had the maximum inhibitory effect.Conclusion:Galanthamine(cholinesterase inhibitor) can decrease the circulating TNF in rats Conclusion:Galanthamine(cholinesterase inhibitor) can decrease the circulating TNF in rats with abdominal infection induced by endotoxin,and vagotomy in rats can weaken the galanthamine inhibitory effet on TNF.Stimulated by endotoxin,RAW264.7 cells produces substantial TNF and HMGB-1,and galanthamine inhibites endotoxin -induced TNF release and reduces the secretion of HMGB-1 in RAW264.7.The inhibitory effect of galanthamine on TNF shows a dose-dependent,and the best dose is ~300nM, which is consistent with study of the galanthamine on the neuronal apoptosis. Galanthamine can reduce endotoxin-induced the pathway activation of macrophage NF-kappaB signaling.Galanthamine has role of inflammation control,so galanthamine may have regulatory effect or even treatment effect on the inflammatory diseases(such as sepsis).