Effect Of Berberine On Vascular Smooth Muscle Cell Calcification

Objective: Vascular calcification is a common pathological manifestation of vascular remodeling disease.VSMC phenotypic switching plays a key role in vascular calcification.VSMCs transdifferentiate to osteoblast-like cells that generated matrix vesicles for calcium-phosphate deposition in the vessel wall.Berberine(BBR)inhibits the Ras/Rac1/Cyclin D/Cdks pathway by activating the AMPK/p53/p21/Cip1 pathway,which in turn inhibits PDGF-BB-induced VSMC proliferation.In this study,we detected the effect of BBR on PDGF-BB-induced calcification of VSMC.Methods:1 VSMC cultureVSMCs were obtained from thoracic aorta of male Sprague-Dawley rats weighing between 60 and 80 g.All of the experiments were performed using3-5 passages of VSMCs.2 Cell viability assayVSMCs were seeded into 96-well plates and pretreated with various concentrations or various time of BBR.Cell viability was measured using CCK8 assay kit.3 Determination of intracellular calcium contentIntracellular calcium content in VSMCs was analyzed according to the manufacturer's instructions with the intracellular calcium content assay kit.The cells were resuspended in PBS containing 2% Triton-X100 and vortexed.The supernatant was collected after centrifugation and each sample was measured by chromatography at 610 nm wavelength.4 ALP activity assayALP activity was analyzed according to the manufacturer's instructions with the ALP activity assay kit.The cells were resuspended in PBS containing 2% Triton-X100,and the supernatant was collected aftercentrifugation.After the protein was quantified,each sample was measured by chromatography at 405 nm wavelength.5 Western blot analysisEqual amounts of protein were separated by 10% SDS-PAGE and electrotransferred to a PVDF membrane.Membranes were blocked,and then incubated with anti-AMPK and p-AMPK.Results:1.Effect of BBR on the viability of VSMCsBBR inhibited cell viability in a concentration-dependent manner.Higher concentrations of BBR(? 100 ?M)significantly inhibited cell viability.Otherwise,BBR inhibited cell viability in atime-dependent manner.After 48 hours of stimulation,the viability of the cells was significantly reduced.Therefore,we chose 50 ?M BBR to stimulate the cells for 24 h for the follow-up experiments.2.Effect of BBR on calcium content of VSMCsAfter PDGF-BB stimulation for 48 h,the increase of intracellular calcium content was obviously promoted.However,compared with PDGF-BB alone,BBR pretreatment inhibited PDGF-BB-induced calcium content.The results showed that BBR can inhibit PDGF-BB-induced VSMC calcification.3.Effect of BBR on ALP activityPDGF-BB significantly stimulated the ALP activity after 48 h stimulation.However,after BBR pretreatment,PDGF-BB-induced ALP activity was inhibited.The results showed that BBR inhibited cell calcification by inhibiting PDGF-BB-induced ALP activity.4.Effect of BBR on AMPK expressionCompared with the control group,AMPK expression gradually increased with the prolongation of stimulation time,peaked at 24 h,and maintained at a high level at 48 h,indicating that PDGF-BB can induce AMPK expression in VSMC.However,after pretreatment with 50 ?M BBR for 24 h,BBR had no effect on AMPK expression with or without PDGF-BB stimulation.5.Effect of BBR on phosphorylation of AMPKThere are no significantly change in AMPK phosphorylation after PDGF-BB stimulation.However,BBR pretreatment promotes AMPK phosphorylation with or without PDGF-BB stimulation.The results showed that BBR may inhibit PDGF-BB-induced VSMC calcification by inducing AMPK phosphorylation.Conclusion:1.BBR inhibits PDGF-BB-induced VSMC calcification.2.BBR inhibits PDGF-BB-induced VSMC calcification by inducing AMPK phosphorylation.