地塞米松对早期脓毒症大鼠肝脏Toll样受体4基因表达干预性研究
摘要
目的:
本实验以大鼠盲肠结扎穿孔法(cecal ligation and puncture,CLP)造成腹腔感染复制脓毒症模型,观察脓毒症早期大鼠肝脏TLR4表达的变化与LPS的关系,并以地塞米松(Dexamethasone,DXM)干预,初步探讨大鼠肝脏中TLR4表达与肝脏损伤的关系,及DXM干预的影响。
方法:
1.采用大鼠盲肠结扎穿孔(CLP)模型复制脓毒症。动物分为正常对照组(n=10)、假手术组(n=10)、CLP组(n=30,分为2、6、8、12、24、48h组)、杀菌性/通透性增加蛋白(BPI)治疗组(n=20),CLP组分别于术后相应时间点处死动物,留取肝脏标本,以半定量逆转录多聚酶链反应技术(RT-PCR)及相关软件分析不同组TLR4mRNA、ELISA法检测血清肿瘤坏死因子-α(TNF-α)水平,同时检测血清丙氨酸转移酶(ALT)、天冬氨酸转氨酶(AST),评估肝脏损伤情况。
2.采用大鼠盲肠结扎穿孔(CLP)模型复制脓毒症。将56只大鼠随机分为DXM干预组1(DXM1,n=40,CLP术前半小时腹腔注射DXM 10mg/kg),DXM干预组2(DXM2,n=16,均分为3组,DXM2a、DXM2b分别于CLP术前半小时腹腔注射DXM 0.1mg/kg、1mg/kg),实验1为对照。DXM干预组1于CLP后2、6、8、12、24h处死动物,DXM干预组2于术后
Sepsis results from the inability of the immune system to limit bacterial spread during an ongoing infection. Massive bacterial load overrides the inhibitory mechanisms controlling inflammation. While normally helping to eradicate pathogens from a local infection of peripheral tissues , inflammation during sepsis develops into a systemic syndrome with multiple manifestations such as tissue injury, increased vascular permeability, and, ultimately, multi-organ dysfunction syndrome (MODS) and shock. Mortality rates of septic patients are high (up to 70%), and the costs of treatment have been calculated to amount to more than $15 billion per year in the US alone. Since antibiotics carry the risk of promoting the release of bacterial products and thus to exacerbate the shock syndrome during sepsis, the development of alternative treatments and the improvement of current regimens for treating septic patients are of high priority.Our current understanding of the pathogenesis of sepsis indicates that bacteria and bacterial products activate an uncontrolled network of host-derived mediators, such as pro-inflammatory cytokines, that can lead ultimately to multi-organ failure, cardiovascular collapse and death. The innate immune system has developed a series of conserved receptors, known as pattern-recognition receptors (PRRs), that recognize specific
pathogen-associated molecular patterns (PAMPs), thereby allowing the innate immune system to distinguish self-molecules from pathogen- associated non-self structures and initiate the host defence response 1. The family of Toll-like receptors (TLRs) is receiving considerable attention as potential regulators and controllers of the immune response through their ability to recognize pathogen-associated molecular patterns.On the basis of the key role of TLRs in the recognition of microbial components, it is clear that an inappropriate TLR response to bacterial signals could have important consequences during infection, leading to exaggerated responses, such as sepsis.Antagonists for TLR proteins might, therefore, be useful tools to counteract the harmful pro-inflammatory response that is associated with systemic microbial infections.Of the ten mumbers of TLRS family ,TLR-4 as a receptor for Lipopoly- saccharide (LPS) has been paid great attention to the relations with sepsis since it has been found so far. In addition to the influences to the immune system, blocking TLRs may be a potential therapeutic method in sepsis.In order to understand the role of TLR4 in the septic rats during the early phase ,a two- part study is carried out.Objective: To investigate the expression of the TLR4 mRNA in liver after severe abdominal infection and the intervention effects of Dexamethasone . Methods:
1.70 healthy male SD rats were randomly divided into four groups as follows :normal control group ,sham operation group ,CLP group and BPI treated group. Rats were sacrificed at 2,6,8,12,24and 48h; the TLR4 mRNA expressions in liver were measured at various intervals by reverse-transcription PCR, TNF-amRNA was messured by ELISA. Meanwhile, the serum transaminases (ALT and AST) were detected.2.56 healthy male SD rats were randomly divided into four groups as follows :DXMl(n=40,pretreated with DXM lOmg/kg 30min before CLP);DXM2(DXM2a, DXM2b) which were pretreated with DXM 0.1mg/kg> 1 mg/kg separately) .DXM1 were sacrificed at 2, 6,8,12 and 24h. DXM2 were sacrificed at 12h. TLR4> TNF-amRNA, ALT AST and the liver histopathology were detected. Results:1. The expression of TLR 4 mRNA was up-regulated after CLP,and it got to the peak at 8~12h,then ,it went down to get to the normal level after 48h.The TNF-a mRNA, ALT and AST of the CLP group were significantly high than the normal control group and the sham operation group,as well as the BPI treated group.2. The expression of TLR 4 mRNA was down-regulated in the DXM pretreated group,and the TNF-a mRNA was down-regulated significantly together with the TLR4 in comparison with the rats CLP alone (P < 0.01).The levels of ALT and AST in DXM pretreated group were muc
本实验以大鼠盲肠结扎穿孔法(cecal ligation and puncture,CLP)造成腹腔感染复制脓毒症模型,观察脓毒症早期大鼠肝脏TLR4表达的变化与LPS的关系,并以地塞米松(Dexamethasone,DXM)干预,初步探讨大鼠肝脏中TLR4表达与肝脏损伤的关系,及DXM干预的影响。
方法:
1.采用大鼠盲肠结扎穿孔(CLP)模型复制脓毒症。动物分为正常对照组(n=10)、假手术组(n=10)、CLP组(n=30,分为2、6、8、12、24、48h组)、杀菌性/通透性增加蛋白(BPI)治疗组(n=20),CLP组分别于术后相应时间点处死动物,留取肝脏标本,以半定量逆转录多聚酶链反应技术(RT-PCR)及相关软件分析不同组TLR4mRNA、ELISA法检测血清肿瘤坏死因子-α(TNF-α)水平,同时检测血清丙氨酸转移酶(ALT)、天冬氨酸转氨酶(AST),评估肝脏损伤情况。
2.采用大鼠盲肠结扎穿孔(CLP)模型复制脓毒症。将56只大鼠随机分为DXM干预组1(DXM1,n=40,CLP术前半小时腹腔注射DXM 10mg/kg),DXM干预组2(DXM2,n=16,均分为3组,DXM2a、DXM2b分别于CLP术前半小时腹腔注射DXM 0.1mg/kg、1mg/kg),实验1为对照。DXM干预组1于CLP后2、6、8、12、24h处死动物,DXM干预组2于术后
Sepsis results from the inability of the immune system to limit bacterial spread during an ongoing infection. Massive bacterial load overrides the inhibitory mechanisms controlling inflammation. While normally helping to eradicate pathogens from a local infection of peripheral tissues , inflammation during sepsis develops into a systemic syndrome with multiple manifestations such as tissue injury, increased vascular permeability, and, ultimately, multi-organ dysfunction syndrome (MODS) and shock. Mortality rates of septic patients are high (up to 70%), and the costs of treatment have been calculated to amount to more than $15 billion per year in the US alone. Since antibiotics carry the risk of promoting the release of bacterial products and thus to exacerbate the shock syndrome during sepsis, the development of alternative treatments and the improvement of current regimens for treating septic patients are of high priority.Our current understanding of the pathogenesis of sepsis indicates that bacteria and bacterial products activate an uncontrolled network of host-derived mediators, such as pro-inflammatory cytokines, that can lead ultimately to multi-organ failure, cardiovascular collapse and death. The innate immune system has developed a series of conserved receptors, known as pattern-recognition receptors (PRRs), that recognize specific
pathogen-associated molecular patterns (PAMPs), thereby allowing the innate immune system to distinguish self-molecules from pathogen- associated non-self structures and initiate the host defence response 1. The family of Toll-like receptors (TLRs) is receiving considerable attention as potential regulators and controllers of the immune response through their ability to recognize pathogen-associated molecular patterns.On the basis of the key role of TLRs in the recognition of microbial components, it is clear that an inappropriate TLR response to bacterial signals could have important consequences during infection, leading to exaggerated responses, such as sepsis.Antagonists for TLR proteins might, therefore, be useful tools to counteract the harmful pro-inflammatory response that is associated with systemic microbial infections.Of the ten mumbers of TLRS family ,TLR-4 as a receptor for Lipopoly- saccharide (LPS) has been paid great attention to the relations with sepsis since it has been found so far. In addition to the influences to the immune system, blocking TLRs may be a potential therapeutic method in sepsis.In order to understand the role of TLR4 in the septic rats during the early phase ,a two- part study is carried out.Objective: To investigate the expression of the TLR4 mRNA in liver after severe abdominal infection and the intervention effects of Dexamethasone . Methods:
1.70 healthy male SD rats were randomly divided into four groups as follows :normal control group ,sham operation group ,CLP group and BPI treated group. Rats were sacrificed at 2,6,8,12,24and 48h; the TLR4 mRNA expressions in liver were measured at various intervals by reverse-transcription PCR, TNF-amRNA was messured by ELISA. Meanwhile, the serum transaminases (ALT and AST) were detected.2.56 healthy male SD rats were randomly divided into four groups as follows :DXMl(n=40,pretreated with DXM lOmg/kg 30min before CLP);DXM2(DXM2a, DXM2b) which were pretreated with DXM 0.1mg/kg> 1 mg/kg separately) .DXM1 were sacrificed at 2, 6,8,12 and 24h. DXM2 were sacrificed at 12h. TLR4> TNF-amRNA, ALT AST and the liver histopathology were detected. Results:1. The expression of TLR 4 mRNA was up-regulated after CLP,and it got to the peak at 8~12h,then ,it went down to get to the normal level after 48h.The TNF-a mRNA, ALT and AST of the CLP group were significantly high than the normal control group and the sham operation group,as well as the BPI treated group.2. The expression of TLR 4 mRNA was down-regulated in the DXM pretreated group,and the TNF-a mRNA was down-regulated significantly together with the TLR4 in comparison with the rats CLP alone (P < 0.01).The levels of ALT and AST in DXM pretreated group were muc
引文
1. Williams DL, Ha T, Li C, et al: Inhibiting early activation of tissue nuclear factor-kB and nuclear factor interleukin 6 with (1-3)-β-D-glucan increases long-term survival in polymicrobial sepsis. Surgery 1999; 126: 54-65
2. Manuel PR, Josefa R., Manuel MR, et al. Vascular endothelial growth factor production in peritoneal macrophages of cirrhotic patients: regulation by cytokines and bacterial lipopolysaccharide. Hepatology, 1999, 29: 1057-1063
3. Michael DJ, Rena FB, Kunitaro F, et al. Lipopolysaccharide and D2 galactosamine2induced hepatic injury is mediated by TNF2 α and not by Fas ligand. Am J Physiol Regulatory Integrative Comp Physiol, 2000, 278 :R1196-R1201
4. Takehiko U, Matthias F, Gavin E A, et al. Toll2like receptor 4 is involved in the mechanism of early alcohol2induced liver injury in mice. Hepatology, 2001, 34: 101-108
5. Qureshi ST, Gros P, Malo D: Host resistance to infection: Genetic control of lipopolysaccharide responsiveness by Toll-like receptor genes. Trends Genet, 1999; 15:291-294
6. Medzhitov R, Janeway CA: An ancient system of host defense. Curr Opin Immunol, 2000; 10:12-15
7. Medzhitov R, Janeway CA: Self-defense: The fruit fly style. Proc Natl Acad Sci U S A, 1998; 95:429-430
2. Manuel PR, Josefa R., Manuel MR, et al. Vascular endothelial growth factor production in peritoneal macrophages of cirrhotic patients: regulation by cytokines and bacterial lipopolysaccharide. Hepatology, 1999, 29: 1057-1063
3. Michael DJ, Rena FB, Kunitaro F, et al. Lipopolysaccharide and D2 galactosamine2induced hepatic injury is mediated by TNF2 α and not by Fas ligand. Am J Physiol Regulatory Integrative Comp Physiol, 2000, 278 :R1196-R1201
4. Takehiko U, Matthias F, Gavin E A, et al. Toll2like receptor 4 is involved in the mechanism of early alcohol2induced liver injury in mice. Hepatology, 2001, 34: 101-108
5. Qureshi ST, Gros P, Malo D: Host resistance to infection: Genetic control of lipopolysaccharide responsiveness by Toll-like receptor genes. Trends Genet, 1999; 15:291-294
6. Medzhitov R, Janeway CA: An ancient system of host defense. Curr Opin Immunol, 2000; 10:12-15
7. Medzhitov R, Janeway CA: Self-defense: The fruit fly style. Proc Natl Acad Sci U S A, 1998; 95:429-430