Huang Qin, Wang Yue, Li Xiao-Rong, Xiang Yi, Shi Qiong
Key Laboratory of Clinical Laboratory Diagnostics, Ministry of Education, College of Laboratory Medicine,Chongqing Medical University Chongqing 400016, China.
Zhongguo Zhong Yao Za Zhi. 2022 Jun;47(11):3059-3065. doi: 10.19540/j.cnki.cjcmm.20211203.702.
This research focused on the effect and mechanism of berberine on osteogenic differentiation of valve interstitial cells(VICs) induced by osteogenic induction medium, in order to provide new insights into the clinical treatment of calcified aortic valve disease. The expression of osteogenic and fibrotic makers in three cases of calcified valve tissues and one case of normal control was assayed by Western blot. After the porcine aortic VICs were isolated, the effects of different concentrations of berberine on their viability were examined by MTT assay for determining the optimal concentration range. VICs were cultured in osteogenic induction medium and treated with different concentrations of berberine. Western blot and q-PCR were conducted to detect the effects of berberine on the expression of osteogenic and fibrotic makers in VICs. The effects of berberine on osteogenic differentiation of VICs in the early and late stages were separately measured by ALP staining and alizarin red S staining. The effects of berberine on the phosphorylation of ERK1/2 at different time points were assayed by Western blot. And PD98059, an inhibitor of ERK1/2, was added for verification. The results suggested that related osteogenic and fibrotic makers were significantly up-regulated in calcified valve tissues as compared with those in the normal control. The up-regulated fibrosis and osteogenic makers of VICs under osteogenic conditions were reversed by berberine and the ALP activity and calcium deposition in VICs were also reduced obviously. The level of ERK1/2 phosphorylation was decreased. Similarly, the osteogenic and fibrotic makers of VICs induced by osteogenic induction medium were lowered by PD98059. This study has confirmed that berberine is able to inhibit the differentiation of VICs into myofibroblasts or osteoblast-like cells, which may be associated with the inhibition of ERK1/2 signaling pathway.
本研究聚焦于黄连素对成骨诱导培养基诱导的瓣膜间质细胞（VICs）成骨分化的影响及机制，旨在为钙化性主动脉瓣疾病的临床治疗提供新的见解。通过蛋白质免疫印迹法检测3例钙化瓣膜组织和1例正常对照中骨生成和纤维化标志物的表达。分离猪主动脉VICs后，采用MTT法检测不同浓度黄连素对其活力的影响，以确定最佳浓度范围。将VICs培养于成骨诱导培养基中，并用不同浓度的黄连素处理。采用蛋白质免疫印迹法和q-PCR检测黄连素对VICs中骨生成和纤维化标志物表达的影响。通过碱性磷酸酶（ALP）染色和茜素红S染色分别测定黄连素对VICs早期和晚期成骨分化的影响。采用蛋白质免疫印迹法检测黄连素在不同时间点对细胞外信号调节激酶1/2（ERK1/2）磷酸化的影响。并加入ERK1/2抑制剂PD98059进行验证。结果表明，与正常对照相比，钙化瓣膜组织中相关的骨生成和纤维化标志物显著上调。黄连素可逆转成骨条件下VICs中上调的纤维化和骨生成标志物，同时VICs中的ALP活性和钙沉积也明显降低。ERK1/2磷酸化水平降低。同样，PD98059可降低成骨诱导培养基诱导的VICs的骨生成和纤维化标志物。本研究证实，黄连素能够抑制VICs向肌成纤维细胞或成骨样细胞的分化，这可能与抑制ERK1/2信号通路有关。
