The Effect Of Simvastatin On Gas Signal Molecules And Apoptosis In Induced Murine Macrophage By Lipopolysaccharide

Objectives: Sepsis is a serious systemic inflammatory responsesyndrome which is caused by infection. It has high incidence, acuteconditions, limited treatment, and with the mortality rate as high as50%.Since the discovery of penicillin, people have concerned from the infectionitself to focusing on the pathophysiological procedure of which leading to thecondition aggravation by systemic inflammatory response. It was found thatcontrolling the excessive inflammatory reaction and disordered immunefunction both caused by sepsis are the key to the treatment.Monocyte/macrophage are important cell effectors which can generate avariety of inflammatory cytokines, and have a very important influence onsepsis immune reaction and inflammatory response. It may be an importantway that effect the inflammatory reaction through interventing and alteringcell cytokine expression. It has been confirmed that the anti-inflammatoryeffect of simvastatin, but the mechanism is still unclear. It has not been clearlyconfirmed whether simvastatin affect inflammatory reaction through themonocyte/macrophage system. The further understanding of themonocyte/macrophage apoptosis state of simvastatin treatment process hasimportant significance for studying on the treatment of sepsis.The three kinds of gas (NO, CO and H2S) generated during inflammatoryresponse is an important signal transduction molecules. The three kinds of gassignal molecules are also considered to be the three important inflammatorymedium, respectively with the main synthase-iNOS(inducible nitric oxidesynthase)、HO-1(heme oxygenase-1)、CSE (cystathionine-gamma-lyase)，forming an independent and interconnected system(NO/iNOS system,CO/HO-1system, H2S/CSE system). They were proved to have close relationswith inflammatory reaction of sepsis in many researches and play key roles of maintaining the balance of pro-inflammation factors and theanti-inflammatory factors. The intervention and regulation for three kinds ofgas signal molecules in the expression of inflammation can significantlyreduce damage of inflammatory reaction and maintain hemodynamic stabilityin sepsis.In recent years, more and more literatures showed that statins can inhibitthe synthesis of iNOS, reduce the production of NO and improve the symptomof poisoning caused by sepsis and septic shock. And it is unknown whetherstatins improves sepsis or septic shock symptoms by acting on else gas signalmolecules but NO, The effect of statins on monocyte/macrophage apoptosisstill lacks of relevant studies.This research is imitating the endotoxin inflammation model in vitrothrough LPS stimulated on murine macrophage RAW264.7cells, makingfurther research the expressive influence of the iNOS/NO, HO-1/CO andCSE/H2S on account of simvastatin treatment，thus confirm the influence ofsimvastatin on gas signal transduction pathway,and observe the effect ofsimvastatin on cell apoptosis, explore the anti-inflammatory mechanism ofsimvastatin, in order to further clarify the role of simvastain in the treatment ofsepsis and septic shock.Method: This study used murine macrophage line called RAW264.7cells as the research object.through LPS stimulated on RAW264.7cells, andestablished endotoxin inflammation model in vitro. Before making theexperimental groups, take normal logarithm growth cell, to choose theappropriate simvastatin concentration effect by MTT methods. Then theexperiment groups are divided: groupⅠ(control group):completely serum-freemedia incubation; group Ⅱ (model group): the concentration of1g/ml LPSin serum-free medium for incubation; group Ⅲ (simvastatin group):completely serum-free media culture medium with simvastatin20μ mol/l,and1g/ml LPS incubation after2h, cell culture for18hours:(1) observeiNOS mRNA, HO-1mRNA, CSE mRNA in each group of expression byRT-PCR method; half quantitative analysis with an average light density ratio (purpose of genes and-actin); statistical analysis of RT-PCR results withcomplete single factor analysis of variance (2) detect early cell apoptosischanges using Annexin V-FITC&PI double staining.Results：1RT-PCR results:1.1iNOS expression comparison: the expression of iNOS in control group、LPS group and simvastatin intervention group, through the analysis ofvariance，exist statistical differences in the three groups (P<0.05). Pairwisecomparison: the expression of iNOS in LPS group is significantly higherthan control group, exist significant difference (P<0.05); the expression ofiNOS in simvastatin intervention group is lower than LPS group, existsignificant difference (P<0.05); the expression of iNOS in simvastatinintervention group is higher than control group, exist significant difference(P <0.05)1.2HO-1expression comparison: the expression of HO-1in control group、LPS group and simvastatin intervention group, through the analysis ofvariance，exist statistical differences in the three groups (P<0.05). Pairwisecomparison: the expression of HO-1in LPS group is higher than controlgroup, exist significant difference (P<0.05); the expression of HO-1insimvastatin intervention group is higher than LPS group, exist significantdifference (P<0.05); the expression of HO-1in simvastatin interventiongroup is higher than control group, exist significant difference (P<0.05)1.3CSE expression comparison: the expression of CSE in control group、LPSgroup and simvastatin intervention group, through the analysis ofvariance，exist statistical differences in the three groups (P<0.05). Pairwisecomparison: the expression of CSE in LPS group is significantly higherthan control group, exist significant difference (P <0.05); the expressionof CSE in simvastatin intervention group is lower than LPS group, existsignificant difference (P<0.05); the expression of CSE in simvastatinintervention group is higher than control group, exist significant difference(P <0.05) 2Detect apoptosis by Annexin V-FITC&PI double staining: control group,LPS group, simvastatin intervention group by using the one-way analysisof variance, early apoptosis rate exist statistical differences in the threegroups (P<0.05). Pairwise comparison: the early apoptosis rate in LPSgroup is higher than control group, exist significant difference (P<0.05);the early apoptosis rate in simvastatin intervention group is higher thanLPS group, exist significant difference (P<0.05); the early apoptosis ratein simvastatin intervention group is higher than control group, existsignificant difference (P<0.05).Conclusions：1After LPS stimulated murine macrophage, the expression of iNOS, HO-1and CSE has significantly increased, which is confirmed that the gas signalmolecules play an important role in the inflammatory response.2Simvastatin can reduce the expression of iNOS and CSE, and increase thexpression of HO-1in activated murine macrophage. we prove thatsimvastatin improves the inflammatory response symptoms by act on thethree kinds of gas signal molecules but NO.3Simvastain can trigger early apoptosis of LPS-treated murine macrophage,probably through the potentiation of apoptosis in activated macrophage,th-us reduce the release of inflammatory mediators.