补骨脂酚启动自噬减轻β-GP诱导的小鼠主动脉中膜钙化

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

摘要:

目的 在选定的36种中药单体中筛选能够启动细胞自噬的药物；探究补骨脂酚对小鼠血管平滑肌细胞自噬的影响以及对血管平滑肌细胞钙化的影响。方法 从中草药中提取的黄酮类化合物以不同浓度处理人脑胶质瘤细胞（human glioma cells，U87）72 h，用MTT法得到药物对U87细胞的半抑制浓度（half maximal inhibitory concentration，IC₅₀），并以小于IC₅₀浓度的条件下以不同浓度梯度处理U87细胞，观察U87细胞GFP-LC3（微管相关蛋白1轻链3（Microtubule-associated protein light chain 3，LC3）的荧光信号和Western blot检测自噬指标LC3从而观察细胞自噬活动激活情况，以筛选出能够启动自噬的药物。后续选取补骨脂酚进行进一步实验，探究药物启动自噬后对血管平滑肌细胞钙化的影响。使用β-GP构建小鼠血管平滑肌细胞（vascular smooth muscle cells，VSMCs）钙化模型，分为对照组（C-CTR组）和钙化组（C-CAL组），并通过Western blot检测Runx2、BMP2及LC3的表达。使用补骨脂酚干预后，分为对照组（C-CTR组）、钙化组（C-CAL组）、钙化+补骨脂酚组（C-CAL+Administration组）、对照+补骨脂酚组（C-CTR+Administration组）4组，分别用Western blot检测LC3、Runx2和BMP2，用茜素红染色观察细胞钙化结节的形成。在动物实验中使用高脂饮食同时在腹腔注射维生素D的方式构建小鼠主动脉钙化模型，使用腹腔注射补骨脂酚的方式对小鼠进行干预，分为对照组（CTR组）、钙化组（CAL组）、钙化+补骨脂酚组（CAL+Administration组）、对照+补骨脂酚组（CTR+Administration组）4组。通过冯库萨染色观察主动脉中膜钙沉积情况，使用HE染色法观察小鼠主动脉中膜的厚度。结果 观察LC3的荧光强度和Western blot提示：细辛脂素（Asarinim）、补骨脂甲素（Bavachin）、灯心草酚（Juncusol）、桑黄酮（Mullberrin）、地榆皂苷2（Ziyuglycoside 2）、补骨脂酚（Bakuchiol）、补骨乙素（Corylifolinin）、五味子甲素（Schisandrin A）、五味子乙素（Schisandrin B）可以显著增加自噬小体的表达（P<0.05）。Western blot结果提示，在钙化的血管平滑肌细胞中，BMP2、Runx2以及LC3的表达显著增加（P<0.05）。使用补骨脂酚干预后Western blot结果提示BMP2和Runx2表达降低（P<0.05），LC3-Ⅱ表达显增加（P<0.05）。茜素红结果显示钙结节沉积也显著降低（P<0.05）。动物实验中，补骨脂酚干预后HE结果显示中膜厚度较钙化组显著增厚，冯库萨染色结果提示补骨脂酚可以显著减轻钙沉积。结论 补骨脂酚可以激活VSMCs的自噬，并减缓小鼠主动脉钙化的进程。

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.