Protective Effects And Mechanism Of Vitamin K3 On Septic Mice Via Inhibition Of Monocyte Pyroptosis

Objective: Sepsis is a systemic inflammatory response syndrome caused by bacterial infection,which includes severe sepsis and septic shock,with high mortality rate.At present,there is a lack of effective therapeutic drugs in clinic.Pyroptosis is a newly discovered,inflammatory programmed cell death mediated by caspase-1 and caspase-11.It is characterized by cell swelling,membrane rupture and up regulation of inflammatory factors.More and more evidence show that pyroptosis play crucial roles in the pathogenesis of sepsis.Therefore,it may provide a new strategy for the treatment of sepsis through targeting cell pyroptosis with small compounds.Vitamin K is a kind of naphthoquinones with isoprene side chain.In addition to its important role in blood coagulation,its anti-inflammatory activity has been widely investigated in recent years.In our previous work,a total of 155 compounds from the microbial natural product compound library was screened for the anti-pyroptotic activity.We found that vitamin K3 could significantly inhibit pyroptosis in THP-1 monocytes.In this study,we will further study the role of vitamin K3 in THP-1 pyroptosis and explore its mechanism of action.In addition,the protective effect of vitamin K3 on septic mouse model was also investigated.Our study may provide novel evidence for the treatment of sepsis and the research and development of anti-pyroptotic drugs.Methods:1.Screening of the anti-pyroptotic activity of vitamin K family in THP-1 cellsTHP-1 cells were cultured and were pre-treated with vitamin K1,K2 and K3 at different concentrations for 24 hours.The toxic effects of different vitamin Ks in THP-1 were detected by CCK8 and relative lactate dehydrogenase(LDH)release.THP-1 pyroptosis was induced by LPS combined with nigericin.The protective effects of vitamin K on THP-1 pyroptosis were evaluated by CCK8,relative LDH release,pyroptosis-associated proteins assay with western blot,and morphological observation.2.The protective effect of vitamin K3 on THP-1 pyroptosis THP-1 cells were pre-treated with different concentrations of vitamin K3.CCK8,relative LDH release and calcein AM/PI staining were used to determine the dose response of THP-1 cells to vitamin K3.The inhibition of vitamin K3 on the activation of NLRP3,capase-1,GSDMD and IL-1β was detected by western blot.The m RNA expression of pro-inflammatory cytokines IL-1β and IL-18 was detected by real-time PCR.3.Mechanism of vitamin K3 in regulation of THP-1 pyroptosisTHP-1 cells were pre-treated with different concentrations of vitamin K3 followed by LPS stimulation.The inhibitory activity of vitamin K3 on NF-κB and MAPK(including ERK,JNK and p38)signal pathways was determined by western blot.4.Protective effect of vitamin K3 on LPS-induced sepsis in miceTo evaluate the protective effect of vitamin K3 in vivo,the LPS-induced sepsis model was established in mice.Vitamin K3 at 25 mg/kg and 50 mg/kg was injected intraperitoneally.HE staining was used to analyze the tissue damage and immune cell infiltration in lungs,kidneys and livers.Serum LDH content was detected and serum IL-1β levels was measured by ELISA.Results:1.At the concentration range of 0-200 μM,vitamin K1 or vitamin K2 had no obvious toxic effects on THP-1 cells.Vitamin K3 had no obvious cytotoxicity below 50 μM.With the treatment of 50-200 μM vitamin K3,the cell viability was significantly decreased,and the relative LDH release was significantly increased,indicating obvious cytotoxicity induced by vitamin K3.At the concentrations of0-200 μM,vitamin K1 or K2 had no obvious inhibitory effect on THP-1pyroptosis,whereas 25-200 μM vitamin K3 could significantly inhibit LPS puls nigericin induced THP-1 pyroptosis.2.Within the concentration range of 5-30 μM,vitamin K3 could increase THP-1cell viability,decrease relative LDH release and increase Calcein AM/PI live/dead cell ratio in a concentration-dependent manner.Vitamin K3concentration-dependently suppressed the expression of NLPR3,inhibited the degradation and activation of GSDMD and Caspase-1,and reduced IL-1β content in the supernatant.In addition,vitamin K3 also down-regulated the expression of IL-1β and IL-18 m RNA levels.3.At the concentration range of 5-30 μM,vitamin K3 concentration-dependently suppressed LPS-induced phosphorylation of p65 and IκBα subunits,inhibited the degradation of IκBα,thus inhibiting the activation of NF-κB signaling pathway.Vitamin K3 also significantly inhibited the phosphorylation of JNK,but not p38 or ERK1/2 pathways.4.Pretreatment with 25 and 50 mg/kg vitamin K3 significantly improved the acute lung,kidney and liver injury,and reduced the tissue infiltration of inflammatory cells in LPS-challenged mice.Pretreatment with vitamin K3 also reduced serum LDH and IL-1β levels in septic mice.Conclusions:1.Vitamin K3 significantly suppresses THP-1 pyroptosis by inhibiting the classical caspase-1 pyroptosis signal pathway.2.Vitamin K3 prevents cell pyroptosis probably through inhibiting the activation of NF-κB and JNK signaling pathways.3.Vitamin K3 may play a protective role in tissue damage and systemic inflammation during sepsis by inhibiting the pyroptosis of monocytes.