Studying The Role Of MIP-2 In Inflammatory Responses Of Raw264.7 Cells Exposed To LPS

The mechanism of liver failure is still not very clear.The clarification of liver failure mechanism and the early intervention measures are very important for further treating liver failure and reducing the mortality.When liver failure occurs,the pro-inflammatory cytokines and chemokines released by macrophages play an important role in severe inflammation.At the early stage of inflammation,the released MIP-2 can chemotaxis neutrophils to inflammation sites,and the activated neutrophils release lot of proteolytic enzymes to cause the serious necrosis of liver cells.At the late stage of inflammation,the macrophages release actively the important pro-inflammatory factor,i.e.high mobility group protein 1(HMGB1),resulting inflammation cytokine storm.The aim of present study is to explore the effects of MIP-2,which is an early inflammatory chemokine released by macrophages,on the late fatal inflammation,and to further study the mechanism of liver failure.The present study includes the following three parts:Part I:Screening the most effective mip-2 siRNA sequence.Objective:To screen the best mip-2 siRNA sequence in vitro and to study its function.Methods:Raw264.7 cells were treated with different doses of LPS,the cellular total RNA was extracted at different time points during 24h,mip-2 mRNA relative expression level of Raw264.7 cells was detected by qRT-PCR to determine the optimal LPS dose.After Raw264.7 cells were treated by mip-2 siRNA with different sequences for 24 h,then the optimal dose LPS was used to treat Raw264.7 cells for 2 h,and then cellular total RNA were extracted and mip-2 and HMGB1 mRNA relative expression was detected by qRT-PCR to determine the optimal sequence of siRNA.RAW264.7 cells were interfered by the optimal sequence of siRNA,then were treated with LPS for 2h.The cellular total RNA was extracted.IL-6,TNF-a,IL-1β,iNOS,CCL2,TLR4,HMGB1 mRNA relative expression levels were detected by qRT-PCR.TNF-a,IL-6,IL-1β protein expression levels in cellular culture supernatants were measured by ELISA.RAW264.7 cells were interfered by the optimal sequence of siRNA,then were treated with LPS for 24h.The cellular total RNA and total protein was extracted.The HMGB1 protein expression level of RAW264.7 cells was detected by Western Blot assay and HMGB1 mRNA relative expression levels were detected by qRT-PCR.Result:(1)The mip-2 mRNA expression levels of Raw264.7 cells exposed to LPS at the different doses for 2h were correlated positively to LPS doses.(2)Raw264.7 cells exposed to mip-2 siRNA with different sequences for 24 h were treated by 0.25 μg/mL LPS for 2 h or 24h.Then the mip-2 and HMGB1 mRNA relative expression levels were detected by qRT-PCR to determine si5 as the optimal sequence.(3)mip-2 siRNA can reduce significantly IL-1β,TNF-a and IL-6 protein expression levels in the culture supernatant of RAW264.7 cells exposed to LPS in a dose-dependent manner.(4)mip-2 siRNA can reduce significantly IL-6,TNF-a,iNOS,IL-1β,CCL2 mRNA expression levels.(5)mip-2 siRNA can reduce significantly HMGB1 mRNA or protein levels and the TLR4 mRNA expression level.Conclusion:(1)0.25μg/mL dose LPS is the optimal dose;(2)Interference si-mip-2 sequences labeled as si5 is the optimal sequences;(3)mip-2 siRNA can block RAW264.7 cell inflammation induced by LPS;(4)Down-regulation of HMGB1 expression is one of mechanisms of mip-2 siRNA;(5)mip-2 siRNA can also influence the HMGB1-TLR4 signaling pathways.Part Ⅱ:MIP-2 antibody reduces the pro-inflammatory effects and inhibit the release of HMGB1 in RAW264.7 cells induced by LPS by PI3K/AKTs,JAK/STAT and p38-MAPK signaling pathways.Objective:To study the anti-inflammatory effects of MIP-2 antibody and its mechanismMethods:Raw264.7 cells treated with the MIP-2 antibody or without MIP-2 antibody were exposed to 0.25μg/mL LPS for 24 hours.Then TNF-a,IL-6 and IL-1β protein expression levels in the cell culture supernatant were determined by ELISA.HMGB1 protein expression and the phosphorylation levels PI3K/AKTs,JAK/STAT3 and MAPKs signaling pathway proteins were determined by Western blot assay.RAW264.7 cells treated by PI3K/AKTs,JAK/STAT3 and MAPKs signaling pathway inhibitors for 2 h were exposed to 0.25μg/mL LPS for 24 hours,TNF-α,IL-6 and IL-1β protein expression levels in the cell culture supernatant were measured by ELISA,HMGB1 protein level in RAW264.7 cells was detected by Western blot assay;RAW264.7 cells exposed to 0.25μg/mL LPS were treated with HMGB1 protein inhibitor(EP)at different time point within 0-36 h,respectively,after 36h,TNF-a,IL-6 and IL-1β3 protein expression levels in the cell culture supernatant were measured by ELISA,then RAW264.7 cells exposed to 0.25μg/mL LPS were treated by MIP-2 antibody at different time point within 0-12h or 0-24h,respectively.TNF-a,IL-6,IL-iβ and HMGB1 protein expression levels in the cell culture supernatant were detected by ELISA.Results:(1)MIP-2 antibody could significantly reduce the TNF-α,IL-6 and IL-1βprotein expression in the cell culture supernatant;(2)HMGB1 protein expression level and the phosphorylation levels PI3K/AKTs,JAK/STAT3 and p38-MAPK signaling pathway proteins could be reduced by MIP-2 antibody in a dose-dependent manner.(3)PI3K/AKTs,JAK/STAT3 and p38-MAPK signaling pathway inhibitors could significantly reduce the TNF-α,IL-6 and IL-1β protein expression levels in the cell culture supernatant and HMGB1 protein expression level;(4)RAW264.7 cells were treated by MIP-2 antibody for 6 h,MIP-2 antibody could decrease significantly the protein expression levels of TNF-α,IL-6,IL-1β and HMGB1 in cell culture supernatant.Conclusion:(1)MIP-2 antibody can decrease RAW264.7 cell inflammatory response induced by LPS;(2)The down-regulation of HMGB1 expression is one of anti-inflammation mechanisms of MIP-2 antibody,which is related to PI3K/AKTs,JAK/STAT3 and MAPKs signaling pathways;(3)RAW264.7 cells exposed to LPS for 6h were added by MIP-2 antibody,the inflammation response of RAW264.7 cells could be inhibited.Part Ⅲ:The study on the pro-inflammatory role of recombinant MIP-2 protein in vitro.Objective:To study the pro-inflammation effects of recombinant MIP-2 protein.Methods:RAW264.7 cells exposed to 30 ng/mL mouse recombinant MIP-2 protein for 2 h were treated by LPS for 24 h.TNF-a,IL-6 and IL-1β protein expression levels of cell culture supernatant were determined by ELISA;RAW264.7 cells exposed to 30 ng/mL mouse recombinant MIP-2 protein for 2 h were treated with recombinant MIP-2 protein or without MIP-2 antibody for 24 h.TNF-a,IL-6 and IL-1β protein expression levels of cell culture supernatant were measured by ELISA,HMGB1 protein expression level was detected by Western blot assay.Results:(1)MIP-2 recombinant protein could significantly increase TNF-α,IL-6 and IL1β protein expression levels in cell culture supernatant of cells exposed to LPS;(2)MIP-2 recombinant protein could significantly enhance TNF-α,IL-6 and IL-1βprotein expression levels in cell culture supernatant;(3)MIP-2 recombinant protein could significantly increase HMGB1 protein expression level.Conclusion:(1)MIP-2 recombinant protein can increase RAW264.7 cell inflammatory response caused by LPS;(2)MIP-2 recombinant protein can induce RAW264.7 cell inflammatory response;(3)The enhanced pro-inflammatory mechanism of MIP-2 recombinant protein is related to the increased HMGB1 protein expression.