Effect of autophagy gene DRAM on proliferation, cell cycle, apoptosis, and autophagy of osteoblast in osteoporosis rats.

OBJECTIVE

The research aimed at detecting the autophagy level of osteoblast in osteoporosis rat, and investigating the effect of autophagy gene damage-regulated autophagy modulator (DRAM) on osteoblast proliferation, cell cycle, apoptosis, and autophagy.

METHODS

The level of osteocalcin (OCN) and C-telopeptide (CTX) in serum of ovariectomized (OVX) rats was detected by enzyme-linked immunosorbent assay (ELISA). The Oil Red-O staining was used to observing bone histological changes. The messenger RNA level and protein expression level of Runt-related transcription factor 2 (Runx2; osteoblast markers) and other autophagy-related genes were revealed using quantitative real-time polymerase chain reaction (qRT-PCR) and western blot. The changes of autophagy in osteoblasts were detected by immunofluorescence staining. The following experiments were performed in osteoblasts of OVX rats through transfected with silencing DRAM to detecting cell proliferation, cell cycle, and apoptosis by Cell Counting Kit-8 assays and flow cytometry.

RESULTS

The result of ELISA showed a significantly elevated of OCN and CTX in OVX rats as well a high fat content compared with sham-operated rats. The expression of Runx2 in bone of proximal tibia was higher by qRT-PCR and western blot analysis. The immunofluorescence staining and transmission electron microscope observe revealed that pcDNA3-DRAM could promote the autophagy in OVX rats. Besides that, overexpression of DRAM inhibited cell proliferation, promoted apoptosis, and enhanced autophagy in osteoblasts. The results of Oil Red-O staining indicated that overexpression of DRAM enhanced lipid accumulation in osteoporosis rats.

CONCLUSIONS

The autophagy level of OVX rats was weakened, but overexpressed DRAM could increase the autophagy level of osteoblast, suppress proliferation, and induce apoptosis of osteoblast.