Studio ex-vivo sugli effetti indotti dalla modulazione farmacologica della cascata di segnalazione FAK-Pyk2 nel rene e fegato di animali esposti al danno da setticemia

Sepsis is a life-threatening organ dysfunction caused by a dysregulated host response to infection. It is characterized by dysfunction of one or more organs even distant from the primary site of infection and is a serious global health problem, considerable one of the major public health concerns. It involves both excessive and prolonged inflammation and immune suppression and is notable for failure to restore normal homeostasis, resulting in possible multi-organ dysfunction and death. Recent findings have demonstrated the pathological role of two non-receptor protein tyrosine kinases, focal adhesion kinase (FAK) and proline-rich tyrosine kinase (Pyk2), in experimental models investigating inflammation in atherosclerosis, endometriosis, asthma, and cancer. In this thesis project, we evaluated the involvement of this signaling pathway in the pathogenesis of sepsis and the potential benefits by administering the dual FAK-Pyk2 reversible inhibitor, PF562271 (PF271), in the dysregulated inflammatory and cytokine response. The study was conducted at University of Turin (Italy) and at Federal University of Santa Catarina (Brazil), where an in vivo sepsis model was set up in 53 male five-month-old mice by CLP (caecal ligation and puncture) surgery, split in two different protocols, once for an acute evaluation, and the other for a long-term evaluation. Mice weighing 30-35 g were kept under standard laboratory conditions for four weeks before the start of the experimentation and were randomised in three different groups: Sham + Vehicle (n=10), CLP + Vehicle (n=10), and CLP + PF562271 (n=5). The septic mice group underwent CLP surgery while for the Sham group was performed laparotomic surgery without applying CLP procedure. After surgical procedures, an analgesic agent (Carprofen, 5 mg/kg, s.c.) and resuscitation fluid (NaCl 0.9%) were administered to each mouse to reproduce the hemodynamic phase seen in sepsis and one hour after surgeries, they received once either Vehicle or PF562271 subcutaneously at the dose of 25 mg/kg. Twenty-four hours after surgery, mice were anesthetized and euthanized by cardiac exsanguination, after which ex vivo analysis were performed, and serum biomarkers (ALT, AST, urea, creatinine and lactate) were measured to assess organ function and damage. The thesis mainly focused on the analysis by Western Blot technique, performed on kidney and liver samples, of the overexpression of FAK and Pyk2 in the experimental sepsis model, which was also associated with increased expression of another inflammatory marker, NLRP3. All this led to higher expression of pro-inflammatory (TNF-alpha, IL-1beta, IL-17 and IL-6) and anti-inflammatory (IL-10) cytokines, adhesion molecules (ICAM-1, VCAM-1, E-Selectin), the enzyme NOS-2 and Myeloperoxidase. All parameters were found to be attenuated in mice treated with the dual inhibitor PF271. Treatment with PF271 through inhibiting the activation of FAK-Pyk2 was successful in attenuating sepsis-induced inflammatory abnormalities. In conclusion, the present project showed for the first time, the importance of the role of the FAK-Pyk2 pathway in the context of inflammation and organ dysfunction induced by sepsis, and how, therefore, the administration of selective dual inhibitors of this cascade may represent a potential innovative pharmacological approach for the treatment of sepsis-related multiorgan failure.