Role Of Proanthocyanidins On THP-1Cells With LPS

Background and purpose:Sepsis is the most common complications, patients who has a special disease,such as serious infections,sepsis shock, burn,and so on, may suffer from multiple organdysfunction syndrome(MODS). Sepsis has a rapid onset and high mortality.At present,we have gradually attained a consistent viewpoint about the mechanism in thepathogenesis of sepsis,the systemic inflammatory response syndrome (SIRS) is the mostimportant reason for the formation of sepsis and multiple organ failure.MAPK signal transduction pathway exists in most of the cells, the p38(38kDaprotein) is a kind of mitogen activated protein kinase (MAPK),and plays an importantrole in the inflammatory response that is related to cell reaction system. numerousinflammatory factors can be producted in SIRS. TNF-is one of the most importantinflammatory factors, some studies reported that procyanidins may be treated as ananti-inflammatory agent during monocyte-macrophage induced by LPS. Itsanti-inflammatory mechanism may be related to the inhibition of nuclear transcriptionfactor transformation.We regarded THP-1cells with LPS as the sepsis cell model in vitro. This studyindicated that procyanidins has significant protective effect on the sepsis cell modelwith non-toxic function. While procyanidins also can restrain the release ofinflammatory factors such as TNF-. With the treatment of procyanidins, we tried toexplain the relationship between TNF-and p38-MAPK pathway.Methods:THP-1cells were cultured in vitro (37℃and5%CO2hatch box). In thelogarithmic phase, we added1ug/ml LPS to set up the cell model of sepsis. Firstly,weuse CKK-8to research whether procyanidins can affect the proliferation of the THP-1cells. We chose three time points,namely6h,12h,24h,according to the different drugconcentrations,we grouped at each time point.We used CCK-8test to comfirm the effect of procyanidins on THP-1with LPS as well as to select different time of adding drugs.According to different time, we explored the different drug concentrations for the wholestudy.Secondly, we did the ELISA test to detect the release quantity of TNF-and chosethe best drug concentration of procyanidins. Finally, we found the expression of P-p38and NF-κB protein via the measurement of westernblotting. And the results revealedprocyanidins suppressed the release of TNF-,trying to explain the relationship betweenTNF-and p38-MAPK pathway.Results:1.To determine the proliferation activity on cells, we did the CCK-8test and measuredabsorbance value at450nm by the enzyme-linked immune detector. We used thevitality value (%) to express the results. Compared with control group, differentconcentrations of procyanidins did not significantly impact on proliferation activity ofTHP-1cells (P>0.05).2.(1) CCK-8test showed that for6h,12h,24h, the cell survival rate (%)in differentconcentration drug+LPS group were lower than the control group, with statisticalsignificance (P <0.05).(2) At different treatment time, with the increasing concentration of procyanidins, thesurvival rate that measured by CCK-8kit in different groups decreased, then graduallypresented a increasing trend. When the concentration of procyanidins drugs was100ug/ml, cell survival rate fell again. To save resources and to simplify the operation, wechose12h group with50ug/ml and100ug/ml procyanidins for the rest part of thestudy.3.(1)We did the Elisa method to detect the release quantity of TNF-in the supernatantof THP-1cells induced by LPS. Compared with control group, TNF-relative proteinconcentration in experimental group was more obvious (P <0.05);(2) The simple LPS group released the highest relative protein concentration of TNF-.While TNF-relative protein of concentration50ug/ml and100ug/ml procyanidinstreatment group was lower than the simple LPS group (P<0.05), indicating both theseprocyanidins concentrations decreased the release of TNF-by LPS-induced THP-1cells;(3) Compared the results from50ug/ml and100ug/ml concentration of procyanidinstreatment group, inhibitive effect of100ug/ml treatment group is more obvious (P <0.05), we selected this concentration for the study of protein level. 4.(1) Westernblotting detection was used to explore P-p38expression in different groups.Compared with other groups, the control group had the lowest P-p38expression (P <0.05). Simple LPS group’s P-p38expression is at the highest level.(2)NF-κB protein expression was detected by westernblotting. The results showedthat different treatment group and the control group were significantly different (P <0.05). THP-1cells in simple LPS group had the highest expression. Procyanidinstreatment group had the lowest expression. The changing of NF-κB protein expressionin different groups was similar with that of P-p38.Conclusion:1.Sepsis is caused by systemic inflammatory response against infection factors. LPS,the outer membrane of gram-negative bacterium, is a major cause of sepsis. This studyset up the sepsis cell model by inducing THP-1with LPS, and showed that procyanidinshad a protective effect on the sepsis cell model without toxic side effects in a larger drugconcentration scope.2.Procyanidins can effectively inhibit the release of TNF-.The relativeanti-imflammation mechanism may be related to the p38-MAPK signal transductionpathway.