IL-32 Level In Sepsis Patients And The Inhibitory Effect Of PB1 To LPS Leaded THP-1 Sourced Macrophages

Objective : Sepsis is a severe problem in Emergency Medicine,which is characterised by fast progressing,high fatality rate and a rising morbidity year on year.The pathogenisis of sepsis is largely contributed to LPS produced from gram negative bacilli erruption and through stimulating the toll-like receptor 4(TLR4)signaling,LPS induce monocytes and macrophages to produce significant amount of cytokines,leading to overreaction of inflammation which in turn causes sepsis.Recently,IL-32--a new cytokine was found in research,which signal transduction is closely linked to TLR4 pathway.IL-32 and TNF-α induce the secretion of each other,thus formed a production circle,causing elevated and long lasting inflammation response.To further understand the role of IL-32 and TNF-α in sepsis development we studied the relation of IL-32 and TNF-α level in real patient,and experimented the effect of natural product Procyanidin B1(PB1)and novel TLR4 inhibitor CLI-095 on IL-32 and TNF-α production through the setting up of a sepsis cell model.Methods:Clinical study: Based on the diagnosis of sepsis,among all the patients admitted to our emergency intensive care unit(EICU)during 2015.05-2016.04,32 cases of sepsis patients were selected and divided into death group and survival group depends on the outcome.Collect their blood sample in the morning at day 1,3,5,7,14,IL-32 and TNF-α concentration were detected through ELISA method;Cell experiment: THP-1 cells were transformed into macrophages through incubation with phorbol ester(PMA)for 48 h and submitted to all the following cell experiments.After use CCK-8 to test the safe working range of PB1,we select 4 different concentration of PB1: 2.5,5,10,20μg/ml,and 2 different concentration of CLI-095: 5,10μg/ml to incubate with THP-1 sourced macrophages for 4h before treating each group with LPS 100ng/ml for another 18 h.Then cell culture supernatants were collected and tested for IL-32 and TNF-α through ELISA.All data were analysed through SPSS 22.0.Take P<0.05 as significant.Results:In clinical study,IL-32 and TNF-α serum concentration of the death group is significant higher than survival group(P<0.05).In cell experiments,we completed the cell transformation from THP-1 cells into macrophages and the set up of a sepsis cell model to imitate the sepsis progress in human.The blocking of TLR4 by CLI-095 inhibited LPS leaded both IL-32 and TNF-α production in THP-1 sourced macrophages and PB1 exerted the similar effect.The best working concentration of PB1 in LPS stimulated THP-1 sourced macrophages is 10μg/ml.Conclusion:The concentration of IL-32 and TNF-αin patients serum indicates the outcome to some extent.The blocking of TLR4 signaling by CLI-095 inhibited both IL-32 and TNF-α production in THP-1 sourced macrophages stimulated by LPS.PB1 has a clear inflammation inhibitory effect by reducing both IL-32 and TNF-α production in THP-1 sourced macrophages stimulated by LPS.