| Background: Atorvastatin(ATO)is safe and effective in the treatment of chronic subdural haematoma(CSDH),and the combination of ATO with low-dose dexamethasone(DEX)was more effective in the treatment of CSDH than ATO monotherapy.In addition,studies have revealed the high expression of angiogenesis-related proteins and miR-144-5p in the haematoma of CSDH patients.However,little is known about the underlying molecular mechanisms.Thus,this study was designed to investigate the underlying mechanisms of the improved efficacy of this combination therapy and the possibility of this mechanism being utilized to optimize clinical treatment.The role of macrophages,cytokines,angiogenesis-related proteins and miR-144-5p in haematoma were analyzed.Methods: In the first part,mass spectrometry was performed to assess the drug concentration in the haematoma and serum of CSDH patients after treatment with ATO alone or combined with low-dose DEX,and the concentrations of vascular endothelial growth factor(VEGFA)were quantified by sandwich ELISA kits.Flow cytometry was conducted to explore the differentiation of macrophages in the haematoma.In the second part,mass spectrometry was performed to assess the drug distribution and concentration in cultured macrophages after treatment with ATO or DEX in vitro.Flow cytometry was conducted to explore the effects of ATO and DEX on the differentiation and apoptosis of macrophages.The expression of cytokines,chemokines and angiogenesis-related proteins was specifically evaluated using a proteome profiler array,western blot or ELISA.In the last part,the expression of miR-144-5p in THP-1 macrophages was measured by q RT-PCR after incubated with exosomes from haematoma isolated by hypercentrifugation.After knockdown or overexpression of miR-144-5p in THP-1macrophages in vitro,the expression of VEGFA was detected by ELISA.After knockdown or overexpression of miR-144-5p in the perihaematoma tissues of SDH rats,the absorption of haematoma was evaluated by MRI,and the expressions of ANG-1,ANG-2 and VEGFA were detected by western blot.Results: In the first part,the presence of DEX,ATO,o-ATO and p-ATO were detected in the haematoma of CSDH patients.For those patients who switched to surgery after unsuccessful conservative treatment,the concentrations ATO and p-ATO in the haematoma were significantly higher than that in the serum.Additionally,the concentrations of ATO,o-ATO and p-ATO in the haematoma demonstrated notably greater levels in the combination therapy group than in the ATO monotherapy group.A much larger number of M1-polarized macrophages than those of the M2 phenotype observed in the haematoma of CSDH patients,and the concentration of VEGFA in haematoma was significantly higher than those in peripheral blood.These evidences suggest the presence of a strong pro-inflammatory environment in the haematoma.In the conservatively treated patients who had a good prognosis,the VEGFA concentration in serum was markedly reduced after administration of the drug.Interestingly,no changes in patients who failed to conservative treatment and switched to surgery were observed after medication,suggesting that VEGFA may be a biomarker related to the prognosis of CSDH.For conservatively treated patients who had good efficacy,the reduction in serum VEGFA levels was generally greater for patients who took the combination regimen than for those who received ATO monotherapy.Moreover,the concentration of VEGFA in serum notably increased after surgery,suggesting that adjuvant drug therapies may also be needed for patients undergoing surgery.In the second part,ATO was detected to bind to the membrane of THP-1macrophages and enter into the cytoplasm in vitro.And the concentration of ATO in macrophages showed significantly greater levels in the combination group than in the ATO group both bound to the membrane of THP-1 macrophages and entered the cells.Decreased numbers of M1-polarized macrophages and increased numbers of M2-polarized macrophages were observed when ATO plus DEX was applied while apoptosis induced by high-dose DEX was avoided.The combined treatment decreased both the basal and LPS-induced VEGFA release in macrophages.The LPS-induced IL-1β,IL-6,IL-8 and and other angiogenesis-related factors were markedly decreased after treatment with the combination regimen.Levels of the angiogenesis inhibitor TSP-1 and the vascular maturation promoting factors ET-1 and PTX3 were significantly elevated by combined DEX therapy.In the last part,the CSDH haematoma contained a large number of exosomes,and the expression of miR-144-5p in THP-1 macrophages was significantly increased after incubated with these exosomes.Neither knockdown nor overexpression of miR-144-5p affected the expression of VEGFA in THP-1 macrophages.Overexpression of miR-144-5p inhibited the absorption of haematoma in SDH rats,and it also increased the expression of ANG-2,decreased the expression of ANG-1,but did not affect the expression of VEGFA.Conclusions: 1.It is the first time to confirm that ATO entered the subdural haematoma and macrophages,and the concentrations of ATO inside both the haematoma and macrophages were increased by DEX.The effect of ATO on anti-inflammation-induced dysfunctional angiogenesis was significantly enhanced by DEX,with a rapid decrease in levels of VEGFA and other inflammatory angiogenic factors by regulating the differentiation of macrophages,while the side effects caused by high-dose DEX was avoided.2.The reason for unsuccessful conservative treatment in some patients may be that their VEGFA level does not decreased after medication.Prognosis and a decrease in the serum VEGFA level after medication were closely correlated,which may be valuable in predicting the efficacy of conservative management.3.Exosomes derived from CSDH haematoma were rich in miR-144-5p,which inhibited the absorption of haematoma in SDH rats by affecting the expression of ANG-1 and ANG-2 and by regulating the permeability and maturation of the neovascular.But the expression of VEGFA was not regulated by miR-144-5p. |
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