Abstract
Sepsis refers to a highly lethal organ dysfunction stemming from the host immune system failing to control invasion of pathogenic organisms. Dysregulation in host acute inflammatory response is the primary contributor to the pathophysiology of sepsis and associated organ dysfunction. Substance P (SP) is a tachykinin neuropeptide encoded by the Tac1 gene in mammals. SP has been recognized as a significant mediator in many inflammatory disorders by activating its receptor named neurokinin 1 receptor (NK1R). Recently, the increased SP–NK1R signalling has been linked to the pathogenesis of sepsis and associated organ impairment by promoting the acute pro–inflammatory response in the host. The principal aim of this thesis was to bridge the knowledge gaps on how the increased SP–NK1R signalling affects the acute inflammatory response and contributes to the pathogenesis of acute liver and lung injury in sepsis in the host.
Results presented in this thesis showed that the biosynthesis of SP and the expression of NK1R significantly increased in the liver and lungs in mice with caecal ligation and puncture (CLP) surgery–induced sepsis. The increased SP–NK1R signalling was related to increased inflammatory injury in the liver and lungs and worse physical condition of mice following CLP surgery–induced sepsis. Following these initial observations, the mechanism by which the SP–NK1R axis contributes to acute liver and lung injury in sepsis was investigated. The extracellular signal–regulated kinase 1/2 (ERK1/2)–nuclear factor kappa B (NF–κB) cascade has been shown to amplify the acute inflammatory response in many pathological conditions, including sepsis. The concentration of phosphorylated ERK1/2 in the liver and lungs in mice significantly was increased following CLP surgery–induced sepsis. In addition, the nuclear translocation of phosphorylated NF–κB was upregulated in the liver and lungs in mice with CLP surgery–induced sepsis. Inhibiting the SP–NK1R axis either by the genetic deletion of the Tac1 gene, the pharmacological blockade of NK1R using L703606, or the combination of these two methods suppressed the activation of the ERK1/2–NF–κB cascade in the liver and lungs in mice with CLP surgery–induced sepsis.
Increased infiltration of neutrophils in the liver and lungs is commonly observed in mice with CLP surgery–induced sepsis. Thus, this thesis further investigates how the SP–NK1R axis promotes neutrophil infiltration in these tissue in mice with CLP surgery–induced sepsis. Considering the recruitment process of neutrophils is predominantly orchestrated by multiple pro–inflammatory mediators, the expression of several cytokines, chemokines, and adhesion molecules were measured in the liver and lungs in mice with CLP surgery–induced sepsis. The production of TNF–α, IL–1β, IL–6, MCP–1, MIP–2, ICAM1, and VCAM1 was upregulated in the liver and lungs in mice with CLP surgery–induced sepsis. The activity of myeloperoxidase was also increased in the liver and lungs in mice in mice with CLP surgery–induced sepsis. On the other hand, these CLP surgery–induced alterations in the liver and lungs in mice were mitigated by blocking the SP–NK1R axis.
In an inflammatory state, activated vascular endothelial cells express more adhesion molecules, promoting the rolling neutrophils to attach to these activated vascular endothelial cells, leading to an increased inflammatory response. The expression of ICAM1 and VCAM1 on the liver sinusoidal endothelial cells and the pulmonary vascular endothelial cells was upregulated in mice with CLP surgery–induced sepsis. In contrast, blocking the SP–NK1R axis attenuated CLP surgery–induced increase in the expression of ICAM1 and VCAM1 on the vascular endothelial cells in the liver and lungs in mice
Dysregulated activation and polarization in tissue–resident macrophages can result in aberrant inflammatory response. The expression of SP and NK1R was significantly upregulated in lipopolysaccharide (LPS)–treated RAW 264.7 cells. Blocking the interaction between SP and NK1R in the RAW 264.7 cells using L703606 attenuated LPS stimulation–induced increase in the expressions of TNF–α, IL–1β, IL–6, MCP–1, and MIP–2 in these macrophages. Moreover, L703606 treatment inhibited LPS stimulation–induced activation of the ERK1/2–NF–κB cascade in the RAW 264.7 cells. Furthermore, blocking the SP–NK1R axis further increased the phagocytotic activity in LPS–treated RAW 264.7 cells.
Aberrant ferroptosis is also a major contributor to the uncontrolled inflammatory response in sepsis, as it can impair the function of the immune system. In vivo, CLP surgery increased the accumulation of iron and the production of MDA in the liver and lungs in mice. However, CLP surgery suppressed the expression of Nrf2 and Gpx4 and reduced the concentration of GSH in these tissues in mice. In contrast, blocking the SP–NK1R signalling reversed CLP surgery–induced increase in ferroptosis in the liver and lungs in mice. In vitro, LPS stimulation led to a significant increase in iron accumulation and MDA production in the RAW 264.7 cells. The concentrations of Nrf2, Gpx4, and GSH also decreased in LPS–treated RAW 264.7 cells. In contrast, blocking the SP–NK1R axis reversed LPS stimulation–induced increase in ferroptosis in the RAW 264.7 cells and protected the RAW 264.7 cells against LPS stimulation–induced cytotoxicity.
In conclusion, this thesis demonstrated the pro–inflammatory impacts of the SP–NK1R signalling on the pathogenesis of sepsis and associated acute liver and lung injury via several mechanisms. Firstly, the increased SP–NK1R signalling activates the ERK1/2–NF–κB cascade and upregulates the expressions of multiple pro–inflammatory mediators, thereby promoting the infiltration of neutrophils in the liver and lungs in mice. Secondly, the increased SP–NK1R signalling activates the vascular endothelial cells in the liver and lungs to express more adhesion molecules, which also contributes to the infiltration of neutrophils in these tissues in mice. Thirdly, the dysregulation of the activation and polarization of the tissue–resident macrophages also underlie the pro–inflammatory effects of the increased SP–NK1R signalling on acute liver and lung injury in mice with CLP surgery–induced sepsis. Finally, dysregulated ferroptosis is also involved in the pro–inflammatory impacts of the increased SP–NK1R signalling on acute liver and lung injury in mice with CLP surgery–induced sepsis. Collectively, this thesis highlights a potential role of blocking the SP–NK1R axis as a therapeutic approach for sepsis and associated organ injury.