| Abstract:Objective To investigate the effect of Gubi Tongxiao Granules on osteogenesis and lipogenic differentiation of human bone marrow mesenchymal stem cell (hBMMSC) in steroid-induced avascular necrosis of the femoral head. Methods The hBMMSC were cultured in vitro from the bone marrow of patients with steroid-induced avascular necrosis of the femoral head. The hBMMSC were identified by morphological observation, osteogenesis and lipogenic differentiation potential. The effect of 1%, 2%, 5%, 8%, 10% volume fraction of Gubi Tongxiao drug-containing serum group A on cell proliferation after 24 h was measured by CCK-8 method with the complete culture group (basic medium +10% fetal bovine serum) as control. In the process of cell culture in 96-well plates, 0.52 μl Dexamethasone solution was added to each well, and the effect of 2%, 5%, 8% volume fraction of Gubi Tongxiao drug-containing serum group B on cell viability in hormonal environment was measured by CCK-8 method with the complete culture group (basic medium +10% fetal bovine serum) and the injured group (basic medium+ 10% fetal bovine serum + 0.52 μl Dexamethasone solution) as control. Non-hormone-induced cells were used as control group (base medium +10% fetal bovine serum). The hBMMSC cells induced by hormones in vitro were divided into model group (basic medium +10% fetal bovine serum +10-5 mol/L Dexamethasone solution) and experimental group (basic medium +5% Gubi Tongxiao drug-containing serum +10-5 mol/L Dexamethasone solution). The formation of mineralized nodules after osteogenic differentiation was determined by alizarin red staining kit. The formation of lipid droplets after differentiation was determined by oil red O staining kit. The mRNA expressions of PPARγ, C/EBP-α, and Fabp4 were determined by RT-qPCR, and the protein expressions of BMP-2, Runx2, and β-catenin were detected by Western blot. Results After seven days of primary cell culture, fusiform, fusiform or polygonal adherent cells could be seen. The third generation cells grew evenly, arranged in parallel, and had good light transmittance. After 21 days of osteogenic induction, the cells were changed, local cells were aggregated, and there were mineralized nodules, alizarin red staining was positive. After 21 days of lipid induction, lipid droplets combined with oil red O staining solution turned red, and oil red O staining was positive. Compared with the complete culture group, the cell viability of 10% volume fraction of Gubi Tongxiao drug-containing serum group A decreased (P＜0.05). Compared with the complete culture group, the cell viability of the injured group was decreased (P＜0.05). Compared with the injured group, the cell viability of 2%, 5%, and 8% volume fraction of Gubi Tongxiao drug-containing serum group B was increased (P＜0.05). Compared with the control group, the staining area of the model group was decreased (P＜0.05). Compared with the model group, the mineralized nodules and deposits in the experimental group were increased, and the staining range was also wider (P＜0.05). Compared with control group, intracellular lipid accumulation increased in model group(P＜0.05). Compared with model group, intracellular lipid accumulation in experimental group was decreased (P＜0.05). Compared with control group, the expressions levels of PPARγ, C/EBP-α, and Fabp4 in model group were increased (P＜0.01). Compared with model group, the expression of PPARγ, C/EBP-α, and Fabp4 in experimental group were decreased (P＜0.05). Compared with the control group, the protein expression contents of BMP-2, Runx2, and β-catenin in the model group were increased (P＜0.01), while the protein expression contents of BMP-2, Runx2, and β-catenin in the experimental group were decreased (P＜0.05). Conclusion Gubi Tongxiao Granules can promote the proliferation and osteogenic differentiation of hBMMSC in steroid-induced avascular necrosis of the femoral head and inhibit lipogenic differentiation, which provides a theoretical basis for clinical prevention and treatment of steroid-induced avascular necrosis of the femoral head. |
