Effect of Erxian Decoction on proteomics and PI3K signaling pathway of osteoblasts stimulated by H2O2 / 中国中药杂志

ABSTRACT

The present study aimed to explore the effect of Erxian Decoction on proteomics of osteoblasts stimulated by hydrogen peroxide(H_2O_2) and its protective mechanism with the H_2O_2-induced cell model of oxidative stress. The primary osteoblasts were cultured from the skulls of newborn rats(within 24 hours) and divided into a control group, a model group, a Fosamax group, and an Erxian Decoction group. Blank serum was added in the control group and model group, and the drug-containing serum was added correspondingly to the remaining two groups. After 45 hours, H_2O_(2 )stimulation was conducted for three hours except for the control group, followed by protein extraction. Nano-LC-LTQ-Orbitrap system was used for protein detection, Protein Discovery for protein identification, and SIEVE for quantitative and qualitative analysis. Furthermore, following the blocking of PI3 K signaling pathway by LY294002(10 μmol·L~(-1)), a control group, a model group, an LY294002 group, an Erxian Decoction group, and an Erxian Decoction + LY294002 group were set up to observe the effect of Erxian Decoction on cell proliferation, alkaline phosphatase(ALP) activity, and the relative expression of BMP-2, OPG, p-Akt, p-FoxO1 of osteoblasts stimulated by H_2O_2 under LY294002 intervention. The results revealed that 78 differential proteins were discovered between the Erxian Decoction group and model group, which were involved in the regulation of PI3 K/Akt, glucagon, estrogen, insulin, and other signaling pathways. LY294002 blunted the promoting effect of Erxian Decoction on osteoblast proliferation and significantly down-regulated the expression of OPG and p-FoxO1, whereas its down-regulation on the expression of BMP-2 and p-Akt was not significant. Both LY294002 and Erxian Decoction increased the ALP activity of osteoblasts, which may be related to the cell state and the cell differentiation. The above results suggest that Erxian Decoction can protect osteoblasts stimulated by H_2O_2, with the PI3 K/Akt signaling pathway as one of the internal mechanisms.