Department of Endocrinology, First Affiliated Hospital, Sun Yat-sen University, Guangzhou 510080, China.
Acta Pharmacol Sin. 2011 May;32(5):591-600. doi: 10.1038/aps.2011.15. Epub 2011 Apr 18.
To investigate the effects of bezafibrate on the proliferation and differentiation of osteoblastic MC3T3-E1 cells, and to determine the signaling pathway underlying the effects.
MC3T3-E1 cells, a mouse osteoblastic cell line, were used. Cell viability and proliferation were examined using MTT assay and colorimetric BrdU incorporation assay, respectively. NO production was evaluated using the Griess reagent. The mRNA expression of ALP, collagen I, osteocalcin, BMP-2, and Runx-2 was measured using real-time PCR. Western blot analysis was used to detect the expression of AMPK and eNOS proteins.
Bezafibrate increased the viability and proliferation of MC3T3-E1 cells in a dose- and time-dependent manner. Bezafibrate (100 μmol/L) significantly enhanced osteoblastic mineralization and expression of the differentiation markers ALP, collagen I and osteocalcin. Bezafibrate (100 μmol/L) increased phosphorylation of AMPK and eNOS, which led to an increase of NO production by 4.08-fold, and upregulating BMP-2 and Runx-2 mRNA expression. These effects could be blocked by AMPK inhibitor compound C (5 μmol/L), or the PPARβ inhibitor GSK0660 (0.5 μmol/L), but not by the PPARα inhibitor MK886 (10 μmol/L). Furthermore, GSK0660, compound C, or N(G)-nitro-L-arginine methyl ester hydrochloride (L-NAME, 1 mmol/L) could reverse the stimulatory effects of bezafibrate (100 μmol/L) on osteoblast proliferation and differentiation, whereas MK886 only inhibited bezafibrate-induced osteoblast proliferation.
Bezafibrate stimulates proliferation and differentiation of MC3T3-E1 cells, mainly via a PPARβ-dependent mechanism. The drug might be beneficial for osteoporosis by promoting bone formation.
研究苯扎贝特对小鼠成骨细胞 MC3T3-E1 增殖和分化的影响，并确定其作用的信号通路。
采用小鼠成骨细胞系 MC3T3-E1 细胞。分别用 MTT 法和比色 BrdU 掺入法检测细胞活力和增殖。用 Griess 试剂检测 NO 产生。用实时 PCR 法检测碱性磷酸酶（ALP）、Ⅰ型胶原、骨钙素、骨形态发生蛋白 2（BMP-2）和 runt 相关转录因子 2（Runx-2）的 mRNA 表达。用 Western blot 分析检测 AMPK 和 eNOS 蛋白的表达。
苯扎贝特呈剂量和时间依赖性地增加 MC3T3-E1 细胞的活力和增殖。苯扎贝特（100 μmol/L）显著增强了成骨细胞的矿化和分化标志物 ALP、Ⅰ型胶原和骨钙素的表达。苯扎贝特（100 μmol/L）增加了 AMPK 和 eNOS 的磷酸化，使 NO 产生增加了 4.08 倍，并上调了 BMP-2 和 Runx-2 的 mRNA 表达。这些作用可被 AMPK 抑制剂化合物 C（5 μmol/L）或 PPARβ 抑制剂 GSK0660（0.5 μmol/L）阻断，但不能被 PPARα 抑制剂 MK886（10 μmol/L）阻断。此外，GSK0660、化合物 C 或 N(G)-硝基-L-精氨酸甲酯盐酸盐（L-NAME，1 mmol/L）可逆转苯扎贝特（100 μmol/L）对成骨细胞增殖和分化的刺激作用，而 MK886 仅抑制苯扎贝特诱导的成骨细胞增殖。
苯扎贝特主要通过 PPARβ 依赖的机制刺激 MC3T3-E1 细胞的增殖和分化。该药物可能通过促进骨形成对骨质疏松症有益。
