The Protective Effects And Underlying Mechanisms Of Roflumilast In Polymicrobial Sepsis

Objective:Sepsis is a life-threatening syndrome accompanied by an overwhelming inflammatory response and organ dysfunction.It is characteristic of high morbidity and mortality rate.Roflumilast is a selective phosphodiesterase 4(PDE4)inhibitor and it has strong anti-inflammation effect.In this study,we evaluated the effects of roflumilast in mice with cecal ligation and puncture(CLP)-induced sepsis,and investigated the underlying mechanism.Methods:1.We used CLP method to establish sepsis model in mice.Mice were randomly divided into five groups:sham-operated group,CLP group,CLP plus a low dose roflumilast group receiving 0.3 mg/kg roflumilast,CLP plus a middle dose roflumilast group treated with 1.0 mg/kg roflumilast,and CLP plus a high dose roflumilast group receiving 3.0 mg/kg roflumilast.Roflumilast was administered orally once daily for seven consecutive days before CLP surgery.Mice were monitored for 7 days after the induction of sepsis to assess the survival rate and the clinical signs were scored according to the pathological symptoms of mice.Statistical method was used in the survival rate and clinical score to analyze whether roflumilast had protective effects in septic mice.2.At 24 h after CLP,the blood,peritoneal lavage fluid,lung,spleen and liver samples were collected,and the bacterial colonies were counted by surface spread plate method.Moreover,the levels of tumor necrosis factor alpha(TNF-a)and interleukin-6(IL-6)in plasma,peritoneal lavage fluid and tissue samples were detected by ELISA assay.Then we measured the plasma concentration of alanine aminotransferase(ALT),aspartate aminotransferase(AST),and lactate dehydrogenase(LDH)in mice,and observe the pathological change in lung,spleen and liver sections by H&E staining.Changes among groups were analyzed to further assess the effects of roflumilast on the pathogenesis of sepsis.3.At 24 h after CLP,liver homogenate of mice was collected.The concentrations of cyclic adenosine monophosphate(cAMP)were measured by ELISA assay,and the expressions of cAMP-response element binding protein(CREB),p-CREB,nuclear factor-kappa B(NF-κB),NF-κB inhibitory protein alpha(IκB-a),p38 mitogen-activated protein kinase(p38 MAPK),p-p38 MAPK,Janus kinase 1(JAK1),p-JAK1,Janus Kinase 2(JAK2),p-JAK2,signal transducer and activator of transcription 3(STAT3)and p-STAT3 were detected by Western blot.Changes in these signaling molecules were analyzed to assess the underlying mechanism of roflumilast in CLP mice.Results:1.Roflumilast had protective effects on CLP mice.First,the sham mice survived during the 7 days of observation,and the survival rate of CLP mice was dropped significantly,while roflumilast pretreatment could increase the survival rate of CLP mice.Secondly,the sham mice had the lowest clinical score,and a rapidly increasing symptom score was obtained in CLP mice,while roflumilast pretreatment could decrease the clinical observation score in CLP mice.2.The protective effects of roflumilast involved in several aspects in sepsis.First,CLP mice displayed substantially higher levels of bacterial load compared with sham mice.Treatment with roflumilast substantially reduced the bacterial load in blood,peritoneal fluid,lung,spleen and liver samples compared with the CLP mice.Secondly,compared with sham mice,the levels of IL-6 and TNF-a in CLP mice was significant increase,while roflumilast could significantly decrease the levels of IL-6,TNF-a in blood,peritoneal lavage fluid,lung,spleen and liver of CLP mice.Thirdly,compared with sham mice,the levels of AST and ALT in plasma of CLP mice were significant increase,while roflumilast could significantly decrease the levels of AST and ALT,suggesting that roflumilast could improve the liver damage of CLP mice,which was further confirm by histopathological observations using H&E staining.In addition,we found that roflumilast could also improve the lung and spleen damages in CLP mice.3.The protective effect of roflumilast involved in several signal pathways.First,compared with sham mice,the accumulation of cAMP and phosphorylation of CREB were significant decrease,while roflumilast could increase the levels of cAMP and phosphorylation of CREB,suggesting that the protective effect of roflumilast might be involved in cAMP/CREB pathway.Secondly,after 24 h of CLP,the degradation of IκB-α,the nuclear translocation of NF-κB p65 and the phosphorylation of p38 MAPK was significantly increased,and roflumilast could inhibit such changes in CLP mice,suggesting that the protective effect of roflumilast might be involved in inhibiting the NF-κB and p38 MAPK pathway.Thirdly,after 24 h of CLP,the phosphorylation of JAK1,JAK2,STAT3 and the nuclear translocation of STAT3 was significantly increased,and roflumilast could inhibit such changes in CLP mice,suggesting that the protective effect of roflumilast might be involved in inhibiting the STAT3 pathway.Conclusion:1.Roflumilast could increase the survival rate and improve the clinical signs in CLP mice.2.The protective effect of roflumilast might be involved in reducing the bacterial load,decreasing the levels of pro-inflammation IL-6 and TNF-a,and reducing the levels of ALT,AST and LDH to improve tissue damage.3.The protective effect of roflumilast might be involved in activating the cAMP/CREB pathway,and inhibiting the NF-κB,p38 MAPK,and STAT3 pathways.