Ubiquitin C-terminal Hydrolase L1 And Galangin Regulate The LPS-stimulated Inflammation

BackgroundSepsis is highly lethal in clinically,which caused by bacterial infection.When lipopolysaccharide is released into blood,the inflammatory reaction will be activated and may be expanded via NF-κB and MAPKs pathway.Finally,a waterfall effect produce and cause many organ dysfunction.However,there is still no effective treatment for sepsis untill now.Based on the understanding of the pathogenesis of sepsis,we sought to find ways to interfere sepsis.Ubiquitin proteasome system regulates protein degradation,which participates in many physiological aspects in cells.Ubiquitin C-terminal hydrolase-L1(UCH-L1),a member of the ubiquitin proteasome family,is found in many human tissues,including nerve cells,endothelial cells,smooth muscle cells and so on.At present,many studies have confirmed that abnormal expression of UCH-L1 is associated with some diseases,such as neurological disorders,ischemia,traumatic brain injury,cancer and Parkinson,etc.Study also found that UCH-L1 may play an important role in nephritis and vascular endothelial cell inflammation.Thus,we need to do further research to recognise the relationship between UCH-L1 and sepsis.ObjectivesIn this study,we constructed an inflammatory cell model of sepsis by LPSinduced human acute monocytic leukemia cell line(THP-1).We use the model to investigate the relationship between UCH-L1 and LPS-induced inflammatory response and its possible mechanism.Methods1.Using enzyme-linked immunosorbent assay(ELISA)to test the level of TNF-α,IL-1β and IL-6 in LPS-stimulated(100ng/m L)THP-1 with different concentrations of LDN57444(10,20,30 μM)at different time points(6,12,24 hours).The effect of LDN57444(10,20,30 μM)on cell viability was measured by MTS assay.Using Western blot to detect the expression of UCH-L1 in THP-1 induced by LPS at different concentrations(0-200ng/m L).2.Applying Western blot to study the level of p-ERK,p-P38,p-JNK,ERK,P38,JNK in LPS-induced(100ng/m L)THP-1 cells which is inhibited by LDN57444(20 μM).Samely,we detecte the nuclear translocation of p65,IκBα via Western blot.The nuclear translocation of P65 in THP-1 stimulated by LPS was analyzed by immunofluorescence confocal microscopy.3.After transfecting UCH-L1 si RNA(40n M)for 48 h,using LPS induced THP-1 for 12 hours.ELISA was applied to test the release of inflammatory factor TNF-α,IL-1β and IL-6.After knockouting of UCH-L1,MTS assay was used to detecte THP-1 cell viability.4.Same as 3.,using Western blot to detect the expression of p-ERK,p-P38,pJNK,ERK,P38,JNK,IκBα and p65 translocated from cytoplasm to nucleus.Results 1.Comparing with LPS group,LDN57444 inhibited the release of inflammatory cytokines TNF-α,IL-1β and IL-6(p<0.05).LDN57444 had no significant effect on viability in LPS-induced THP-1.As the concentrations of LPS(0-200ng/m L)growing,the expression of UCH-L1 increased in THP-1 induced by LPS(p<0.05).2.LDN57444 down-regulated the expression of p-ERK,p-P38,p-JNK in THP-1 cells which is stimulated by LPS,P38 increased(p<0.05),the level of ERK,JNK have no outstanding change(p>0.05).Western blot showed LDN57444 reduced the level of nuclear translocation of P65(p<0.05),The expression of IκBα had increased(p<0.05).Immunofluorescence confocal microscopy also certified that LDN57444 inhibit the nuclear translocation of p65 in the same model.3.Comparing with negative si RNA+LPS group,the level of TNF-α、IL-1β and IL-6 in UCH-L si RNA +LPS group had decreased(p<0.05).UCH-L1 si RNA had no significant effect on cell viability(p>0.05).4.Different from negative si RNA group,the level of UCH-L1 in the group which transfected with UCH-L1 si RNA had downregulated(p<0.05).UCH-L1 si RNA inhibited the expression of p-ERK,p-P38,p-JNK and the nuclear translocation of p65 in LPS-induced THP-1 cells(p<0.05),IκBα had upregulated,but the level of ERK,P38,JNK had no obvious change(p>0.05).Conclusions UCH-L1 increases the inflammatory response of LPS induced THP-1,and its mechanism is related to increase the P38,JNK,IκBα phosphorylation and the nuclear translocation of P65.Background Sepsis caused by uncontroled inflammation may lead to multiple organ dysfunction The incidence,morbidity and mortality rate of the disease are increasing year by year,which is one of the most lethal diseases in ICU.The mechanism of sepsis has not been fully elucidated,but it is believed that the basic mechanism is the uncontrolled inflammatory response.Galangin is a kind of flavonols compounds,and have many researchs in ischemic injury,antiviral,anti-tumor,analgesic,antioxidant,anti-inflammatory and other aspects.Our previous studies have indicated that galangin can inhibit the inflammatory reflect on the LPS-induced THP-1,but rarely research to explore galangin to LPSstimulated mancrophage inflammatory response.Inflammation is a process of inflammatory pathways through expanding step by step,finally prompted macrophages release a matrix metalloproteinases(MMPs),ect.These inflammatory factors increase vascular endothelial cell permeability,futher increase the release of inflammatory factors,and eventually make the endothelial cell damage,lack of oxygen,tissue necrosis.Via activating the NF-κB and MAPK pathway,LPS stimulate the inflammatory cells to prompte many inflammatory mediators gene synthesis and increase the release of inflammatory medium.We detect the relationship between galangin and inflammation in sepsis to offer more evidence about galagin to intervene sepsis.Objectives In this research,we use lipopolysaccharide(LPS)to stimulate macrophages to explore the effect of galangin on the inflammation.Methods1.The macrophages were generated by PMA-induced THP-1 cells,then were pretreated with galangin at different concentrations.The level of TNF-α,IL-1β,IL-6 released by LPS-induced macrophages were tested by ELISA.2.LPS-induced macrophages proliferation was detected by MTS assay.Cell apoptosis was determined with Annexin V-FITC /PI double staining by flow cytometry.3.Appling by ELISA,we mesured the level of TIMP-1 expressed by LPSstimulated macrophages.Western blot was used to analysis the protein levels of MMP-2 and MMP-9.Results 1.Compared with the group treated with LPS(500ng/m L)alone,galangin within 2.5-20μmol/L could inhibit the expression of TNF-α,IL-1β,IL-6(P<0.05).2.Galagin within 0-20μmol/L was not toxic to macrophages(P>0.05),and don’t cause apoptosis and necrosis of the macrophages(P>0.05).3.Galagin had no effect on the expression of TIMP-1 released by macrophages(P>0.05).But Galangin within 10-20μmol/L could suppress the levels of MMP-2 and MMP-9(P<0.05).Conclusions Galangin decreased the levels of inflammatory factor including TNF-α,IL-1β,IL-6,MMP-2,MMP-9 in LPS-induced macrophages.