Effects Of Berberine On Macrophage Foam Cell Formation May Be Related To ACE2-Ang-(1-7)-MAS Receptor Axis

Objective：To observe the effects of berberine on the macrophage foam cellinduced by ox-LDL, including morphology, lipid content, oxidative stressand inflammation, and to explore whether its mechanism may be related toACE2-Ang-(1-7)-MAS axis.Methods：1. Establishment of foam cells model：20、40、60、80μg·ml-1ox-LDLwere used to induce RAW264.7into foam cells, the intracellular lipidaccumulation was observed by oil red O staining, quantitative analysis bythe values of OD of intracellular lipid droplets amounts, determined theoptimal concentration of ox-LDL for establishment of foam cells model.2. RAW264.7cells were cultured in different concentrations of1，2.5，5，10，20，40，80μM berberine respectively with24h,48h and72h, cellproliferation was determined by MTT assays.3. Experimental groups： control group：RAW264.7without any administration；model group：RAW264.7cultured with60μg·ml-1ox-LDL for24h；berberine groups：RAW264.7were co-treated with60μg·ml-1ox-LDLand different concentrations berberine (2.5,5,10μM) for24h.4. Effects of berbebrine on the intracellular lipid accumulation in foamcells was observed by oil red O staining, quantitative analysis by the valuesof OD of intracellular lipid droplets amounts.5. Measured the content of total cholesterol (TC) and cholesterol ester(CE) content, the ratio of CE/TC to decide the effects of berberine on thecholesterol content in foam cells.6. The contents of malondialdehyde (MDA), superoxide dismutase(SOD), glutathione (GSH) were determined by Chemocolorimetry.7. The content of tumor necrotic factor-α (TNF-α) was measured byELISA and the content of NO was determined by Chemocolorimetry.8. Real-Time PCR was used to detect ACE2and MAS mRNAexpressions.9. The content of Ang-(1-7)) was measured by ELISA.Results：1. The macrophages became larger and rounder after treated withox-LDL (20、40、60、80μg·ml-1) for24h by oil red O staining, the amountof lipid droplets in the cytoplasm increased in dose-dependent. 2. MTT assay results showed that berberine inhibit the proliferation ofRAW264.7in time-does-dependent manner.3. Oil red O staining showed the positive cells were decreased, andintracellular lipid droplets and cholesterol in dose-dependant decreased aftertreat with different concentrations of berberine.4. Compared with the control group, the content of MDA wassignificantly increased (P<0.01), and the contents of SOD and GSH weredecreased (P<0.05, P<0.01) in model group; while berberine groups showedthat MDA contents were increased(P<0.01), SOD and GSH contents weredecreased (P<0.05, P<0.01) compared with model group.5. Compared with the control group, the contents of TNF-α and NOwere significantly increased (P<0.01) in model group; berberine groupsshowed the contents of TNF-α and NO were increased (P<0.01) comparedwith model group.6. Real-time PCR showed the expression of ACE2and MAS in modelgroup were significantly decreased (P<0.01) compared with control group;ACE and MAS mRNA expression were increased after berberine treatmentcompared with model group (P<0.05, P<0.01).7. Ang-(1-7) content detected by ELISA was significantly decreased inmodel group compared with control group(P<0.01); while the levels ofAng-(1-7) were increased in dose-dependent after berberine treatment (P<0.01).Conclusion：Berberine may inhibit the macrophage foam cell formation induced byox-LDL, reduce intracellular lipid droplets and cholesterol content, andhave anti-oxidation and anti-inflammatory effects, which may be related toincrease the function of ACE2-Ang-(1-7)-MAS receptor axis.