Psoralen Inhibited Apoptosis of Osteoporotic Osteoblasts by Modulating IRE1-ASK1-JNK Pathway

Abstract

Osteoporosis is a common disease causing fracture in older populations. Abnormal apoptosis of osteoblasts contributes to the genesis of osteoporosis. Inhibiting apoptosis of osteoblasts provides a promising strategy to prevent osteoporosis. The proliferation of osteoblasts isolated from osteoporotic patients or healthy subjects was determined by MTT assay. Apoptosis was determined by Annexin V/PI assay. Protein expression was measured by western blot. The proliferation of osteoblasts isolated from osteoporotic patients was inhibited and the apoptosis level of these cells was higher than the osteoblasts from healthy subjects. Incubation with psoralen or estradiol significantly enhanced the proliferation and decreased the apoptosis level of osteoporotic osteoblasts. Western blot demonstrated that psoralen or estradiol treatment downregulated the expression of IRE1, p-ASK, p-JNK, and Bax. Meanwhile, expression of Bcl-2 was upregulated. Pretreatment by IRE1 agonist tunicamycin or JNK agonist anisomycin attenuated the effect of psoralen on osteoporotic osteoblasts. Psoralen inhibited apoptosis of osteoporotic osteoblasts by regulating IRE1-ASK1-JNK pathway.

Figure 1

Proliferation of primary osteoblasts isolated from osteoporotic patients was inhibited. (a) Optical microscopic observation of osteoblasts from normal and osteoporotic subjects after 72 h incubation. (b) Proliferation of osteoblasts from osteoporotic patients was inhibited. Osteoporotic and normal osteoblasts were incubated for 72 h. The cell viability at different time points was determined by MTT assay. (c) The EDU stain also performed in cells treated as described above with a magnification of 200. (d) Osteoporotic osteoblasts exhibited higher apoptosis level. Apoptosis was determined by Annexin V/PI assay. ^∗∗P < 0.01 compared with normal group.

Figure 2

Psoralen increased the proliferation and inhibited the apoptosis of osteoporotic osteoblasts. (a) Psoralen increased the proliferation of osteoporotic osteoblasts. Osteoporotic osteoblasts treated by DMSO, psoralen, and E2 were incubated for 72 h. The cell viability at different time points was determined by MTT assay. (b) The EDU stain also performed in cells treated as described above with a magnification of 200. (c) Psoralen inhibited the apoptosis of osteoporotic osteoblasts. Osteoporotic osteoblasts were coincubated with DMSO, psoralen, or E2 for 48 h. Then cell apoptosis was determined by Annexin V/PI assay. ^∗P < 0.05, ^∗∗P < 0.01 compared with control.

Figure 3

Psoralen regulated the mRNA expression of IRE1, Bax, and Bcl-2. (a, b, and c) Osteoporotic osteoblasts were pretreated by psoralen or E2 for 48 h, and then the mRNA expressions of IRE1, Bax, and Bcl-2 were assayed by qPCR. ^∗P < 0.05 versus control group.

Figure 4

Psoralen regulated the IRE1-ASK1-JNK pathway in osteoporotic osteoblasts. (a, e, and g) Osteoporotic osteoblasts were pretreated by TM, AM, or DMSO for 1 h, and then osteoblasts were incubated with psoralen or E2 for 48 h. Then protein expression was determined by western blot, and (d and f) mRNA expression was determined by qPCR. (b and c) Osteoporotic osteoblasts were pretreated by TM or AM for 1 h. Then the cells were incubated with psoralen for 48 h. The cell viability was determined by MTT assay. ^∗P < 0.05, ^∗∗P < 0.01, ^∗∗∗P < 0.001.