| Part I: MSCs-secreted TGF-β1 regulates lipopolysaccharidestimulated macrophage M2-like polarization via the Akt/FoxO1 pathwayObjective: To clarify the role and related mechanisms of TGF-β1 secreted by MSCs in the induction of LPS-stimulated macrophages to M2 phenotype.Methods:(1)Different concentrations of LPS were used to stimulate macrophages,and the effect of LPS on macrophage viability was detected by the CCK-8.After clarifying the optimal stimulation concentration of LPS to promote macrophage polarization,the appropriate concentration LPS stimulated macrophages at different times,then the macrophage phenotype and expression of inflammatory factors were detected.(2)Macrophages stimulated with LPS and MSCs were indirectly co-cultured in the Transwell system,and then treated with TGF-β1 receptor inhibitors,and then macrophage phenotype and inflammatory factors were detected.(3)Co-culture of recombinant TGF-β1 with LPS-stimulated macrophages to detect macrophage phenotype and expression of inflammatory factors.(4)The protein chip was used to screen the potential mechanism of MSCs paracrine TGF-β1 induced LPS-stimulated macrophages to M2 type polarization,combined with Akt inhibitor and FoxO1 inhibitor pretreatment to detect changes in pathway proteins and macrophage phenotype.(5)Changes in macrophage phagocytic capacity during MSCs-induced M2 polarization in macrophages were detected by flow cytometry.Results:(1)LPS 500 ng/ml significantly increased the viability of macrophages,and when the LPS concentration reached 1000 ng/ml,the viability of macrophages was significantly reduced;In comparison with the control group,LPS 500 ng/ml significantly induced macrophages to M1 type polarization for 24 h,accompanied by increased expression of proinflammatory cytokines(IL-6 and IL-1β)in a timedependent manner p<0.05).(2)MSCs induced LPS-stimulated macrophages to M2-like phenotype through paracrine,while inhibiting the expression of IL-6 and IL-1β,increasing the expression of anti-inflammatory cytokine IL-10,and TGF-β1 receptor inhibitors reversed the above changes(p<0.05).(3)Recombinant TGF-β1 induced LPSstimulated macrophages to M2-like phenotype,inhibited the expression of IL-6 and IL-1β,while increased the expression of IL-10(p<0.05).(4)Protein chip and Western blot confirmed that TGF-β1 secreted by MSCs activated the Akt/FoxO1 pathway,and TGF-β1 secreted by MSCs promoted phosphorylation of the downstream Akt pathway,which in turn induced FoxO1 nuclear translocation and increased expression in the cytoplasm.Pretreatment with Akt inhibitors and FoxO1 inhibitors significantly inhibited the phenotype regulation of TGF-β1 secreted by MSCs on LPS-stimulated macrophages.(5)TGF-β1 secreted by MSCs affects the phagocytic capacity of LPSstimulated macrophages through the Akt/FoxO1 signaling pathway.Conclusions: MSCs paracrine TGF-β1 induced LPS-stimulated macrophages to M2 type polarization by activating the Akt/FoxO1 signaling pathway,reducing inflammation.Part II: Overexpressing TGF-β1 in mesenchymal stem cells attenuated inflammation and organ dysfunction in CLPinduced septic miceObjective: To determine the effect of TGF-β1 overexpressing MSCs on organ injury and systemic inflammatory response in cecal ligation and puncture(CLP)-induced septic mice.Methods: Mouse MSCs stably transfected with TGF-β1 was constructed.(1)In order to verify the transfection efficiency,we used fluorescence microscope to detect the expression of GFP,RT-PCR to detect the expression of TGF-β1 gene and ELSIA to detect the concentration of TGF-β1 in the cell culture supernatant.(2)Septic mice model was replicated by CLP method,and different treatments were given by group after the model was replicated for 6h.After 24 hours,the mice were sacrificed,then the lung,liver and spleen tissue samples were taken,H&E staining for histopathological examination to assess the level of tissue damage;Calculated the ratio of lung wet weight to body weight(LWW/BW)to evaluate pulmonary edema.(3)ELISA was used to detect the levels of inflammatory factors IL-6,IL-1β and IL-10 in peripheral blood to evaluate the degree of systemic inflammation.(4)Immunohistochemistry was used to evaluate the infiltration of macrophages in the tissue.(5)Immunohistochemistry and flow cytometry were used to detect the effect of MSC-TGF-β1 transplantation on the phenotype of macrophages in the lung tissue of septic mice.(6)In vitro detection of the effect of MSC-TGF-β1 on the regulation of macrophage phenotype and the expression of inflammatory factors.(7)The CLP method was used to replicate the septic mice model,and MSC-TGF-β1 pretreated macrophages were infused through the tail vein.H&E staining was performed for histopathological examination to assess the degree of tissue damage,and LWW/BW was calculated to evaluate pulmonary edema.(8)Clodronate disodium liposomes were used to deplete the macrophages in the lung tissues of mice,and CLP was used to replicate the sepsis mouse model.MSC-TGF-β1 was infused via the tail vein.The mice were sacrificed 24 hours later.The lung tissue of mice were stained with H&E to evaluate the degree of damage of each tissue by histopathological examination.Results:(1)Lentivirus-mediated overexpression of TGF-β1 in MSCs,the fluorescence microscope observation showed that the transfection efficiency was up to 90%(p<0.05).In copmarison with the MSC-GFP group,the TGF-β1 m RNA expression in MSC-TGF-β1 group was significantly increased(p<0.05);Meanwhile,the ELISA results showed that the TGF-β1 concentration in MSC-TGF-β1 group was increased than that in MSCGFP group(p<0.05).(2)The effect of MSC-TGF-β1 transplantation on the pathology of lung,liver and spleen in septic mice: compared with the MSC-GFP group,the infusion of MSC-TGF-β1 significantly aggravateed the histopathological impairment of the lung、liver and spleen tissue(p<0.05);the effect of MSC-TGF-β1 transplantation on pulmonary vascular permeability in septic mice: Compared with MSC-GFP group,the LWW/BW of septic mice was significantly reduced in MSC-TGF-β1 transplantation group(p<0.05).(3)The effect of MSC-TGF-β1 transplantation on systemic inflammation in septic mice: Compared with the MSC-GFP group,the plasma levels of IL-1β and IL-6 in the MSC-TGF-β1 treatment group were significantly reduced(p<0.05),while the level of IL-10 were significantly increased(p<0.05).(4)The effect of MSC-TGF-β1 transplantation on macrophage infiltration in lung,liver and spleen tissues of septic mice: Compared with the MSC-GFP group,the expression of macrophages in lung,liver and spleen tissue was significantly reduced after MSC-TGF-β1 transplantation(p<0.05).(5)The effects of MSC-TGF-β1 transplantation on the phenotype of macrophage in lung tissue of septic mice: Compared with the MSC-GFP group,the expression of M1 phenotypic markers(CD86 and i NOS)in lung tissue of septic mice was significantly reduced,while the expression of M2 phenotypic markers(CD206 and Arg-1)was obviously increased in MSC-TGF-β1 transplantation group(p<0.05).(6)In vitro experiments,compared with the MSC-GFP group,the MSC-TGF-β1 treatment group significantly inhibited the expression of M1 phenotype macrophages in LPS-stimulated peritoneal macrophages and promoted the expression of M2 phenotype macrophages(p<0.05).In addition,in comparision with the MSCGFP group,the expression levels of IL-1β and IL-6 in the MSC-TGF-β1 group were significantly reduced(p<0.05),while the level of IL-10 was significantly increased(p<0.05).(7)The effect of infusion of MSC-TGF-β1 pretreated macrophages on the pathology of the lung,liver and spleen of septic mice: Compared with the MSC-GFPpretreated macrophage group,infusion of MSC-TGF-β1 pretreated macrophages improved lung,liver and spleen injury and reduced tissue injury scores in septic mice,and LWW/BW was significantly reduced(p<0.05).(8)Flow cytometry results showed that clodronate disodium liposomes can significantly reduce the expression of macrophages in mouse lung tissue.MSC-TGF-β1 can significantly improve lung tissue damage in septic mice,while the therapeutic effect of MSC-TGF-β1 was significantly weakened in the macrophage depletion group.Conclusions: Under sepsis conditions,TGF-β1 overexpressing MSCs can enhance the therapeutic effects of MSCs on organ injury and excessive inflammation in CLPinduced septic mice;the benifical effects of MSC-TGF-β1 may be achieved by inhibiting macrophages infiltration in the tissue and inducing macropahges polarization to M2 phenotype. |
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