Wistar Rats BMSCs Induce Macrophage M2 Polarization Through STAT3 And NF-?B Pathways In Inflammation After Hepatic RFA

Background Radio frequency thermal ablation(RFA)is the most commonly used treatment for situ or secondary hepatocellular carcinoma,which accepted in standardized clinical practice [1].For small hepatocellular carcinoma,RFA can be completely cured have reached a consensus.With its basic clinical research progress,gradually expand the indications,but medium and advanced hepatocellular carcinoma(especially diameter ? 3cm)immune response bio-markers in post-RFA inflammatory responses promote local residual tumor growth [2] and remote single or multiple organ metastases [3-6].Bone marrow mesenchymal stem cells(BMSCs)can promote Macrophage M2 polarization [7-9],which can regulate the release of immune response bio-markers,thereby reducing the inflammatory response in the tissue [10],moreover,reduce risk of the recurrence and metastasis after liver radio frequency ablation.Objective By extracting Wistar rat BMSCs and for extraction liver macrophages after radio frequency ablation,BMSCs induce macrophage M2 polarization through STAT3 and NF-?B pathway to regulate the release of inflammatory cytokines and alleviate the inflammatory response.Method 1.Normal rat BMSCs were extracted by density gradient centrifugation combined with adherent method.The antigen phenotypes were detected by flow cytometry,and osteogenic induction and identification were performed.2.36 Wistar rats were randomly divided into 6 groups((1)RFA control group,(2)Post-RFA12 h,(3)Post-RFA24 h,(4)Post-RFA72 h,(5)Post-RFA120 h,(6)Post-RFA168h)to establish liver inflammation model of RFA injury,using tissue Immunofluorescence was used to evaluate the time point at which M2 aggregated expression was highest.3.M? and Post-RFA-M? in the normal group were extracted by density gradient centrifugation combined with adherent method.Cell migration ability was detected by Transwell chamber and scratch test,and the expression of M2-M? was marked by CD163 immunofluorescence staining.4.BMSCs and macrophage Transwell co-culture in vitro divided into 6 groups(Control group A: PBS and D: BMSCs;Macrophage control group B: RFA control M? and C: post-RFA M?;experimental group E: BMSCs + RFA control M? and F: BMSCs + post-RFA M?)Supernatants and cells were harvested after 24 hours.The supernatants were assayed for IL-10 and TNF-? by ELISA;The levels of IL-10 and TNF-? m RNA in macrophages were determined by quantitative RT-PCR.The macrophages were fixed after 48 h,the immunofluorescence assay and flow cytometry detection of CD163 were performed.The proliferation of macrophages was detected by MTT assay.Macrophage migration capacity was detected through Transwell chamber culture macrophages;experimental group were added in STAT3 inhibitor S3I-201,the upper chamber was discarded after co-cultured 48 h,the protein product was detected by Western blot NF-?B p65 / NF-?B p-p65,JAK1 / p-JAK1 and STAT3 / p-STAT3.Result 1.BMSCs with strong growth and continuous subculture were successfully isolated from the tibial bone marrow of healthy Wistar rats.2.Thirty Wistar rats were successfully constructed into a model of inflammation after liver radio frequency ablation.HE staining showed that intrahepatic fibroblasts proliferated significantly at 72 h,nevertheless,immunofluorescence displayed macrophages began to accumulate.There is a significant difference between the two groups after 168 h Statistical significance.3.The macrophages were successfully extracted from the RFA control group and the post-RFA 168 h group respectively,and the strongest migration ability was found at 48 h by scratch and Transwell chamber assay.The immunofluorescence staining of CD163 showed that the positive rate of M2 in 168 h group was 43%.4.After co-cultured with BMSCs and macrophages,the level of IL-10 in group F was significantly increased,however,TNF-? was significantly decreased after ELISA detection and quantitative RT-PCR detection.Immunofluorescence and flow cytometry showed that the positive rate of CD163 in group F.While,the MTT assay showed that BMSCs significantly promoted the proliferation of macrophages;Transwell chamber results showed that the migratory ability in E group and F group was enhanced;Western blot results showed that BMSCs inhibited the expression of NF-?B p65 on macrophages surface and promoted p-JAK1 and p-STAT3 expression,STAT3 inhibitor,BMSCs induced p-STAT3 expression was significantly reduced.Conclusion BMSCs can promote the proliferation and migration of macrophages after RFA and regulate the secretion of immune response bio-markers.BMSCs can induce macrophage polarization to anti-inflammatory repair type M2 through NF-?B and STAT3 signaling pathways.