| ObjectiveTo investigate the depressant mechanism of calculus bovis cultivated byglucuronidase on atherosclerosis of apolipoprotein E knockout mice, especially the effectand mechanism on the inflammation and oxidative stress in plasma and atheroscleroticlesions.MethodForty male7-8-week-old apolipoprotein E knockout mice fed a high fat diet (15.8%fat and1.25%cholesterol) were randomly divided into four groups: the control group(vehicle treated group, n=10); the low-dose(0.25g/kg·d, CBCG-L), the medium-dose(0.75g/kg·d, CBCG-M), the high-dose(2.25g/kg·d, CBCG-H) CBCG treated group. Beside oneweek adaptive feeding, Vehicle (1-percent-starch) or CBCG1-percent-starch were perfusedonce daily for8weeks. After8weeks of treatment, blood was collected from theretro-orbital sinus of the apolipoprotein E knockout mice without dietary exposure for12h.The whole aorta was dissected and the proximal aorta attached to the heart was used toprepare cross-sections. Serial aortic root cryosections and en face of the whole aorta werestained with oil red O for determination of atherosclerotic plaque area; Plasmaconcentrations of plasma IL-6and TNF-α were determined by ELISA kit according to themanufacturer’s instructions. Plasma levels of malondialdehyde (MDA) and the activity ofparaoxonase-1(PON1) were determined by spectrophotometric method. We also tried todetermine the bioactive compounds, namely Bilirubin and Taurine by ELISA kit accordingto the instructions. Serial aortic root cryosections were stained with specific antibodiesagainst macrophages (MOMA-2), vascular cell adhesion molecule (VCAM), intercellular adhesion molecule (ICAM), tumor necrosis factor-α(TNF-α),4Hydroxynonena(HNE),P-Janus kinase-signal transducers2(P-JAK2), and P-activators of transcription1(P-STAT1).In the vitro experiment, ox-LDL(100μg/ml)was added to the cultured RAW264.7macrophages. After incubation for24h, quantitation of the secreted proinflammatorycytokines, including IL-6and IFN-γ, were performed from aliquots of conditioned mediumby ELISA kit according to the instructions. Protein extracts were subjected to Western blotanalyses using anti-P-JAK2, anti-P-STAT1and SOCS1. In the end, Concentrations ofplasma total cholesterol (TC) and HDL-C were determined by enzymatic methods.Non-HDL-C was calculated as TC minus HDL-C. Livers and kidneys were used to makeformalin-fixed paraffin imbedding and to prepare sections. Then to observe the historicaland pharmacological changes after normal HE for insight into the safety of CBCG．41male C57BL/6J mice at same week of age were used for HDL functionality assay.the C57BL/6J mice fed a high fat diet were randomly divided into3groups, the controlgroup (n=14, vehicle treated group); the medium-dose (n=13,0.75g/kg/d, CBCG-M group)and high-dose (n=14,2.25g/kg/d, CBCG-H) CBCG1-percent-starch treated groups. Vehicle(1-percent-starch) or CBCG1-percent-starch were perfused once daily for12weeks. After12weeks of treatment, blood was collected from the retroorbital sinus of the C57BL/6Jmice without dietary exposure for12h. LDL and HDL3were separated by sequentialultracentrifugation. HDL functionality assay, Cu2+-induced LDL oxidation assay,endothelial cell-monocyte adhesion assay,3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay were determined to analysis the functional properties ofthe HDL particle.ResultsEight weeks Intragastric admini-stration of CBCG decreases atherosclerotic lesionformation both in Serial aortic root cryosections and en face of the whole aorta inapolipoprotein E knockout mice. ELISA assay revealed that the production of tumornecrosis factor-α and interleukin-6were significantly suppressed. Spectrophoto-metricmeasurement showed that plasma levels of MDA were significantly decreased and theactivity of PON-1was significantly improved. The intragastric administration ofCBCG1-percent-starch significantly increased the plasma level of Bilirubin while thechange of Taurine level was not obvious. Quantitative immunohistochemistry imagingrevealed that the positive area of VCAM-1, ICAM-1, TNF-α, MOMA-2and HNE withinthe lesion area was significantly decreased. The result suggested that the intragastricadministration of CBCG suppressed the JAK2/STAT1pathway. Plasma analysis by enzymatic method showed that eight weeks intragastric administration ofCBCG1-percent-starch remarkably increased plasma high-density lipoprotein (HDL)cholesterol in apolipoprotein E knockout mice. HE staining found no significantdifferences between control group and CBCG treated groups. In the vitro experiment, thesecretion of IFN-γ and IL-6by macrophages was significantly reduced by CBCG-treatment. The JAK2/STAT1pathway was suppressed like the vivo experiment. AndSOCS1, the negative regulator of JAK2/STAT1, was suppressed. In addition, CBCGimproved the functional quality of HDL particle in C57BL/6J mice, including reversecholesterol transport(RCT)-promoting, anti-oxidative(prevention of LDL oxidation),endothelial protective as well as anti-inflammatory (induce adhesion of monocyte toHUVECs) properties.Conclusion1. CBCG could reduce atherosclerotic plaque and inhibit the development of arteryatherosclerosis in apolipoprotein E knockout mice.2. The antiatherosclerotic actions of CBCG were linked with a down-regulation offactors important for plasma lipid oxidation and peroxidation and factors important forplasma inflammation.3. The antiatherosclerotic actions of CBCG were linked with a strong reduction ofaortic inflammation (ICAM-1, VCAM-1, TNF-α and MOMA-2) and the suppression ofaortic lipid oxidation(HNE).4. The mechanism of anti-inflammation in the antiatherosclerotic actions of CBCGwas linked with the suppression of JAK2/STAT1/SOCS1.5. The antiatherosclerotic actions of CBCG were linked with improving the functionalquality of HDL particle in C57BL/6J mice, including reverse cholesterol transport(RCT)-promoting, anti-oxidative(prevention of LDL oxidation), endothelial protective aswell as anti-inflammatory (induce adhesion of monocyte to HUVECs) properties. |
PDF Full Text Request