Bakuchiol initiates autophagy to reduce β-phosphoglycerol-induced calcification of mouse vascular smooth muscle cells

Abstract:

Objective To screen drugs that can initiate cellular autophagy among 36 selected monomers of traditional Chinese medicine， and to explore the effects of Bakuchiol on mouse vascular smooth muscle cell autophagy and calcification.Methods The human brain glioma cells（U87） were treated at different concentrations by flavonoids extracted from Chinese herbal medicine for 72 hours， the semi-inhibition concentration（IC₅₀） of U87 cells were treated by MTT method，and U87 cells were treated at different concentration gradients under conditions less than IC₅₀ concentration. The activation of cellular autophagy activity was observed by observing the fluorescent signals of U87 cell GFP-LC3［microtube-related protein 1 light chain 3（LC3）］ and the method of Western blots（WB） to detect autophagy activity and to screen for drugs that can initiate autophagy. Further experiments were conducted to explore the effects of Bakuchiol initiation on vascular smooth muscle cell calcification after autophagy. The calcification model of vascular smooth muscle cells（VSMCs） in mice was constructed using β-GP，involving control group（CTR） and calcification group（CAL），and the expression of Runx2，BMP2 and LC3 was detected by WB. After intervention with Bakuchiol， they were divided into 4 groups：CTR，CAL，calcification with Bakuchiol group（CAL + Administration），and control with Bakuchiol group （CTR + Administration），respectively，with WB detection for LC3，Runx2 and BMP2，and with Alizarin red to observe the formation of cell calcification nodules. In animal experiments，a high-fat diet was used to build a calcification model of the aorta in mice using vitamin D through intraperitoneal injection，and interventions were made in mice using intraperitoneal injection of Bakuchiol. The mice were divided into four groups：CTR，CAL，CTR + Administration and CTR + Administration. The deposition calcium in the artery medium membrane was observed via Von Kossa staining，and the thickness of the membrane in the aorta in mice was observed using the hematoxylin-eosin（HE） staining method.Results The observed fluorescence intensity and WB cues of LC3 showed that：Asarinim，Bavachin，Juncusol，Mullberrin，Ziyuglycoside 2，Bakuchiol，Corylifolinin，Schisandrin A，and Schisandrin B could significantly increase the expression of autophagy（P<0.05）. WB results suggested a significant increase in the expression of BMP2，Runx2，and LC3 in calcified vascular smooth muscle cells（P<0.05）. WB results after intervention with Bakuchiol suggested a decrease in BMP2 and Runx2 expression（P<0.05），and an increase in expression of LC3-Ⅱ（P<0.05）. The results showed a significant decrease in calcium nodule deposition（P<0.05）. In animal experiments，the results of HE staining after the intervention of Bakuchiol showed a significant increase in the thickness of the medium membrane than the CAL group，and the Von Kossa results hinted that the Bakuchiol could significantly reduce calcium deposition.Conclusion Bakuchiol can activate the autophagy of VSMCs and affect the process of aortic calcification in mice.