人参皂苷CK对PDGF-BB诱导的肺动脉平滑肌细胞增殖和表型转换的影响及机制

刘桃; 谢亮; 刘瀚旻; 刘斌

doi:10.12182/20210760101

人参皂苷CK对PDGF-BB诱导的肺动脉平滑肌细胞增殖和表型转换的影响及机制

The Effects of Ginsenoside Compound K on PDGF-BB-Induced PASMCs Proliferation and Phenotypic Conversion of Pulmonary Arterial Smooth Muscle Cells

摘要

摘要:
目的探讨人参皂苷CK（ginsenoside compound K, CK）对体外培养肺动脉平滑肌细胞（pulmonary arterial smooth muscle cells, PASMCs）增殖、表型转换的影响和相关机制。
方法以体外培养的PASMCs为研究对象，血小板衍生生长因子（platelet-derived growth factor-BB, PDGF-BB）为诱导剂，CK为干预剂。实验分为对照组（不做处理）、模型组（给予20 ng/mL PDGF-BB）和干预组（同时给予20 ng/mL PDGF-BB+5 μmol/L CK）。CCK-8法检测细胞增殖情况〔在上述分组的基础上，将干预组CK浓度设为1、3、5 μmol/L，并增设药物组（分别予以1、3、5 μmol/L CK）〕，流式细胞术检测细胞周期及凋亡情况，实时荧光定量PCR和Western blot检测各组细胞表型转换标志基因α-肌动蛋白（α-smooth muscle actin, α-SMA）和平滑肌22α蛋白（smooth muscle 22α, SM22α）mRNA和蛋白的表达，Western blot检测Wnt/β-连环蛋白（β-catenin）信号通路相关蛋白表达。
结果与模型组比较，CK以剂量依赖性的方式抑制PDGF-BB诱导的PASMCs增殖，5 μmol/L CK干预时结果与对照组无明显差异（P>0.05），故后续实验CK浓度选用5 μmol/L；CK以1、3、5 μmol/L剂量单独与PASMCs孵育，未发现细胞毒性作用（P>0.05）；CK将细胞周期阻滞于G₀/G₁期，促进PASMCs的凋亡，并逆转α-SMA、SM22α的mRNA和蛋白表达（P<0.01）；同时CK下调β-catenin蛋白和细胞周期蛋白D1（cyclinD1）的表达，并上调磷酸化糖原合酶激酶3β（phosphorylated glycogen synthase kinase-3β, pGSK-3β）/糖原合酶激酶3β（glycogen synthase kinase-3β, GSK-3β）蛋白表达（P<0.01）。
结论 CK抑制异常的PASMCs增殖并逆转了表型转换，其作用机制可能与Wnt/β-catenin信号通路有关。提示CK可能具有控制肺动脉高压的治疗潜力。

Abstract:
Objective To explore the inhibitory effects of ginsenoside compound K (CK) on pulmonary arterial smooth muscle cells (PASMCs) proliferation and phenotypic conversion in vitro and investigate its related mechanisms.
Methods PASMCs cultured in vitro were examined in the study. They were induced with platelet-derived growth factor-BB (PDGF-BB) and then treated with CK. The cells were randomly assigned to the control group (receiving no treatment), the model group (PDGF-BB, 20 ng/mL), and the intervention group (20 ng/mL PDGF-BB+5 μmol/L CK). The cell proliferation was measured by CCK-8 assay (on the basis of the above group assignment, concentrations of CK was set at 1, 3, and 5 μmol/L in the intervention group, and the drug group was added, receiving 1, 3, and 5 μmol/L CK, respectively). Cell cycle and apoptosis were examined by flow cytometry. The levels of mRNA and proteins of α-smooth muscle actin (α-SMA) and smooth muscle 22α (SM22α), markers of phenotypic conversion, were detected by quantitative real-time PCR and Western blot. The levels of protein expression related to Wnt/β-catenin signaling pathway were examined by Western blot.
Results Compared with the model group, CK significantly inhibited PDGF-BB-induced proliferation of PASMCs in a dose-dependent way. The results of 5 μmol/L CK intervention were not significantly different from that of the control group (P>0.05). Hence, 5 μmol/L CK was chosen for subsequent experiments. Separate treatment of PASMCs with CK at doses of 1, 3, and 5 μmol/L did not reveal any cytotoxicity to PASMCs (P>0.05). CK also arrested the cell cycle of PASMCs at the G₀/G₁ phase, promoted the apoptosis of PASMCs, and reversed the mRNA and protein expression of α-SMA and SM22α (P<0.01). In addition, CK down-regulated the expressions of cyclin D1 and β-catenin, while it up-regulated the protein expressions of phosphorylated glycogen synthase kinase-3β (pGSK-3β)/glycogen synthase kinase-3β (GSK-3β) (P<0.01).
Conclusion CK was capable of inhibiting the abnormal proliferation of PASMCs and reversing the phenotypic conversion, and its acting mechanism may be related to the Wnt/β-catenin signaling pathway, suggesting the therapeutic potential of CK in controlling pulmonary arterial hypertension.

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