The Endogenous Negative Regulator MKP-1:A Potential Drug Target For The Treatment Of Sepsis

Sepsis is typically triggered by an overwhelming systemic inflammatory response to pathogenic microorganisms and may lead to multiple organ dysfunction syndrome(MODS)and death.Sepsis is associated with high morbidity and mortality even under optimal critical care.It is the leading cause of death in non-coronary intensive care units.Because sepsis can be induced by different microorganisms,it restricts the corresponding vaccine development.Moreover,the abuse of antibiotics induces drug resistant strains,resulting in the invalidation of antibiotics.Therefore,not even a single drug which is effective for sepsis is developed.It is urgent to develop new methods for the treatment of sepsis.Part 1 Establishment and Test of stable DUSP1 promoter-driven luciferase reporter RAW264.7 cell lineMitogen-activated protein kinase phosphatase 1(MKP-1)is an inducible nuclear phosphatase that dephosphorylates MAPKs,and thus,it is a negative feedback regulator of MAPK activity.MKP-1 has been found as a key endogenous suppressor of innate immune responses,as well as a regulator of the onset and course of adaptive immune responses.Studies on MKP-1 knockout mice have suggested that MKP-1 has a protective role on septic mice.Moreover,MKP-1 expression and protein function have been found to be up-regulated by certain anti-inflammatory drugs,namely by glucocorticoids,and MKP-1 has been shown to mediate many of their antiinflammatory effects.Therefore,MKP-1 is a potential anti-inflammatory drug target.In this study,we wanted to find out some anti-inflammatory drugs which can up-regulate MKP-1 expression and establish an efficient screening system to screen new anti-inflammatory drugs by monitor MKP-1 expression.Therefore,RAW264.7 cells were used to create a stable cell line expressing the promoter of DUSP1 which encode MKP-1linked to a luciferase reporter gene.Thirteen clones were obtained through seeding serial dilutions of cells into 96 well plates.The clones were screened for reporter activity by incubation with LPS for 24h.From the 13 clones obtained,2 showed high induction in the luciferase transcription that reached 7.6-fold and 8.8-fold the control values.Both dexmethasone and Rolipram enhanced LPS-induced luciferase activity.A time course study of luciferase production in the stable cells after LPS stimulation was performed between 6 and 36h.The results showed a time-dependent increase in the luciferase activity and the response peaked at 24h.These results demonstrate that our established cell line can be used as a tool for screening anti-infammatory drugs.Moreover,Rolipram increased MKP-1 promoter activity in this study and may have protective effect in inflammatory diseases such as sepsis and septic shock.Part 2 The Therapeutic Effect of Rolipram on Septic MiceIt is reported that MKP-1 protect mouse from inflammatory disease such as sepsis and septic shock.In part 1 study,Rolipram increased MKP-1 promoter activity after LPS challenge in stable cell line.And the study concerning the therapy of sepsis with Rolipram has not been reported.Therefore,we hypothesized that Rolipram may have protective effect in sepsis and septic shock.In this study,a series of cellular level experiments and animal experiments were conducted to verify this hypothesis.The results showed that Rolipram inhibited inflammatory cytokines resease in LPS challenged RAW264.7 cells.In LPS-induced sepstic mice,pretreatment with Rolipram significantly improved survial from 29.2%to 100%.Treatment with Rolipram improved survival from 36.4%to 100%.Luminex technology were employed to measure cytokine levels and the results showed that Rolipram inhibited LPS-induced proinflammatory cytokines containing TNF-?,IL-1?IL-6,IL-5,IL-12(p40),chemokines MCP-1,IP-10,MIP-1?3,MIP-2,Eotaxin,KC,MIG,LIF,and growth factor VEGF production,and enhanced anti-inflammatory cytokine IL-10 release in mice serum.Histopathological alterations were observed in lung,liver and kidney.Bronchoalveolar lavage fluid(BALF)was analysed for inflammatory cells and total protein indicated that Rolipram significantly prevented LPS-induced lung injury and inflammatory response.Biochemical analysis of hepatic and renal function markers manifested that Rolipram reduced the concentration of AST,ALT,Cr and BUN in serum.LPS-induced nuclear translocation of the NF-kappaB p65 subunit and phosphorylation of ERK,JNK,p38 MAPK was also blocked by Rolipram.Rolipram treatment also improved the survival rate of E.coli-challenged mice and CLP-challenged mice.These data suggest up-regulating the expression of MKP-1 can be used as a strategy for the treatment of inflammatory disease such as sepsis and sepstic shock.The application of Rolipram treatment is a beneficial strategy in sepsis control.Since Rolipram is an approved drug for COPD in the United States,the Drug re-position strategy of Rolipram save a lot of money and time compared to the development of new inflammatory drugs.