After the transplantation of BMSCs in the GIOP rats, the growth rate of a large proportion of adipocytes and primary cells at three, four, five, and eight days was slower than the growth rate of primary cells from normal rats. Moreover, BMSCs from GIOP rats were worse than the BMSCs from normal rats with poor refraction. Approximately 10.10% of BMSCs in the normal rats were arrested at S + G2 + M but only 5.82% of the BMSCs from the GIOP rats were arrested at S + G2 + M. Thus, the proliferation of BMSCs from GIOP rats was worse than the proliferation of normal cells.
Serum osteogenic differentiation ALP is an endocellular enzyme that can be used to evaluate the degrees of osteogenic differentiation of various cells and OB functions. Moreover, ALP serves as the relative specific index of the activity of OBs and the capacity of bone tissue calcification[10].
RANKL and OPG are recently discovered members of a superfamily of tumor necrosis factor receptors and ligands. They form the RANKL–RANK–OPG axis, which is the final pathway in the regulation of bone absorption and in vitro calcium ion balance, and plays a key role in bone formation and bone resorption coupling[11, 12]. A variety of bone metabolism factors regulate bone formation through this axis. Studies demonstrated that OPGs isolated from humans and rats were 94% homologous and that OPG was mainly produced by OBs in bone tissues[3]. Further studies showed that OPG could inhibit osteoclast formation, differentiation, and survival and could induce OB apoptosis[4]. Patients with bone metabolic diseases were observed with RANK and OPG mutations[13]. GCs at physiological concentrations enhance osteoclast function by increasing the expression of parathyroid hormone receptors, whereas excessive physiological doses of GCs promote an increase in the RANKL/OPG ratio, thus accelerating osteoclast generation and aggravating osteoclasia[14].
In this study, we measured ALP, OPG, and RANKL expressions in BMSCs from normal and GIOP rats in the presence or absence of osteogenic induction to compare the osteogenic differentiation capacity in the different groups.
The ALP-positive expression rate results showed that the ALP-positive rates at 7, 14, and 21 days were significantly higher in the normal induction group than in the other groups (P < 0.01). The ALP-positive expression rates at 7, 14, and 21 days were significantly higher in the GIOP induction group than in the normal and GIOP groups (P < 0.01). Moreover, at 14 and 21 days, the ALP-positive rate was significantly higher in the normal group than in the GIOP group (P < 0.05). These results indicated that in the presence of osteogenic inductor, ALP-positive expression increased in BMSCs from both normal and GIOP rats. BMSCs from normal rats showed stronger reactive activity toward the inductor than BMSCs from GIOP rats.
OPG expression in the normal induction group was significantly higher than in the normal, GIOP (P < 0.01), and GIOP induction groups (P < 0.05). Moreover, OPG expression in the GIOP induction group was significantly higher than in the GIOP (P < 0.01) and normal groups (P < 0.05). RANKL expression was significantly higher in the normal induction group compared with the other groups (P < 0.01). RANKL expression was significantly higher in the normal group compared than in the GIOP and GIOP induction groups (P < 0.01). These findings indicated that the osteogenic inductor could stimulate OPG and RANKL expression.
KL is associated with aging and was discovered by Kuro-o et al.[15]. The Kl gene in humans and mice are located in chromosome 13 (13 q12) and exhibits high homology (83%)[16]. KL is mainly distributed in the kidneys and brain choroid. KL gene mutation or deletion can result in various phenotypes similar to human aging, such as shortened life span, arteriosclerosis, reduced immune function, and osteoporosis[17]. However, excessive KL expression or exogenous KL supplementation for KL-knockout mice can delay or improve aging symptoms[18]. In the experiments of the current study, renal KL mRNA expression was significantly reduced in the GIOP group compared with the normal group (P < 0.01), thus indicating that KL mRNA expression significantly decreased during GIOP. The GC-induced reduction of KL mRNA expression may be involved in the molecular mechanism of bone metabolism.
The results of this study were consistent with previously reported results. In future studies, we will prepare drug-containing sera intervened by kidney reinforcing and marrow-benefiting Chinese medicine to investigate the effects of different blood collecting times and drug-containing sera at different concentrations on the proliferative activity of rat BMSCs based on ALP. We will also determine the optimal BMSC proliferation-promoting dose and bone-directed differentiation concentration of drug-containing sera. We will use the optimal concentration to explore the possible mechanism of kidney-reinforcing Chinese medicine during the osteogenic differentiation of BMSCs.