Influence Of MS19 On The Activity Of Macrophages And Inflammation Response Possible By Targeting PIK3R1 3'UTR

Macrophages are one kind of so important immunocytes,equipped with the functions of engulf and kill.This phagocytic function provides a first defense in innate immunity.Macrophages also dispose of pathogens and infected cells targeted by an adptive immune response.The micro-environment and different signaling factors around macrophages can activate different functional macrophages,the procedure of activation was called macrophages polarization.After polarized,macrophages are divided into classically activated macrophages(M1 type)and alternatively activated macrophages(M2 type).LPS and IFN-? can induce macrophages toward M1 polarized,after polarization the cells with high levels of IL-12 and low levels of IL-10,with a function of encourage inflammation.There are different stimuli for M2 type and reports point out that they may closely connected with chronic infectious diseases.For M2 is common increasing IL-10 production and TGF-? production but IL-12 production is turned off,which plays an anti-inflammatory role.Macrophages inflammatory proteins are crucial for immune responses towards infection and inflammation.They activate granulocytes and further enhance immune response and inflammation.Phosphatidylinositide 3-kinases are a family of enzymes involved in cellular functions such as cell growth,proliferation,differentiation,motility,survival and intracellular trafficking.Macrophages polarization is closely related to PI3K/AKT pathway.Meanwhile,PI3 K is crucial for macrophages growth and migration.It's a potential therapeutic target for the treatment of diseases involving macrophages,including inflammation.PIK3R1 codes a regulatory subunit of PI3 K,mainly coded three proteins: p85??p55??p50?.Proteins coded by PIK3R1,except constitute functional PI3 K as a regulatory subunit,also exert self-function respectively because of the redundant expression in cells.A single-stranded DNA,acting as Immunosuppression,is called suppressive ODN.If these sup-ODNs are just right complementary to DNA or m RNA of immunostimulant protein,block and change expression or translation,the sup-ODNs can also be called antisense ODN(ASO).Sup-ODNs are found in eukaryotic chromosome telomeres and bacterias.Our lab designed series of ODNs according to telomeres and human microsatellite DNA,naming them MS ODNs.MS19 is one kind of them.In the model of acute lung injury(ALI)caused by flu virus,MS19 is found that significantly lessen acute lung pathological injury in mice infected,inhibit the weight loss of mice and decreased the mortality rates of the mice.However,the mechanism of rescuing mice remains unclear.In the study,we blast the sequence of MS19 in mouse nucleotides database and find a key gene(PIK3R1,which 3'UTR having a completely complementary with MS19).The proteins coded by PIK3R1 play an important role in cells.It maybe a key pathway refers to the function of MS19.Combining with the time(infected by virus for 2,4 days)of injection and the site(intraperitoneal injection)of injection in the former experiments,we speculate that the target cells are probably peritoneal macrophages.In order to verify if MS19 can complementary paired PIK3R1 to disturb coding proteins in macrophages,we answer it from the following 3 aspects:(Raw264.7 cells derived from mouse ascites can be the desired tool cells to study this issue.)1,The primary investigation about MS19 affect the level of PIK3R1 m RNA in Raw264.7Firstly,we confirmed the target sequence do exist in Raw264.7;Secondly,real-time PCR detected in the condition of ODNs treat cells for 2h,MS19 group can do decline the level of target gene m RNA when compared with control.2,Exploration for the mechanism of the influence of MS19 on PIK3R1.We cloned the PIK3R1 3'UTR from mice genome and constructed vector for transcription in vitro and report plasmid.Taking advantages of transcription in vitro to amplify many m RNA of PIK3R1 3'UTR and mimic the condition of cells inside to observe the interaction of MS19 and m RNA.Results showed MS19 can change the moving ratio of PIK3R1 3'UTR m RNA.In the experiments of transient cotransfection,MS19 had a specific interference on the expression of the report protein(carried the PIK3R1 3'UTR).3,Effect on the function of Raw264.7Through the experiments of Western blot and flow cytometry,we found that MS19 declined the protein,p85?,without a statistic meaning.With the test of q RT-PCR,we observed MS19 early influence on the cytokines in Raw264.7.The found was that MS19 can downregulate PIK3R1 m RNA and IL-10 m RNA,a kind of sup-inflammatory factor,upregulate CXCL10 m RNA and had no effect on Akt m RNA,the down stream factor in PI3 K signal pathway,and i NOS m RNA,macrophage to type M1 polarization.In the experiments of adhesion and wound healing,MS19 was found a negative function in macrophage adhesion and migration.