The Effect Of Tonifying Kidney Recipe On Proliferation, Differentiation And Expression Of OPG And RANKL MRNA In Osteoblasts In Vitro

Objective: Osteoporosis includes primary osteoporosis and secondary osteoporosis, with high incidence in middle and old crowd. Because of related fracture complication, it threatens health and life of middle and old people severely. With the increasing proportion of social old age population, it is a hard task for us to study osteoporosis and prevention and therapy of fracture. Chinese traditional medical theories believe that osteoporosis belongs to scope of bone paralysis and originates from deficit of kidney essence. According to this theory, most doctors use tonifying kidney method as usual principle for treatment of osteoporosis. In previous study of our division, tonifying kidney recipe—anti-osteoporosis liquid showed good anti-osteoporosis effect in both osteoporosis patients and animal models. Its action mechanism included improving generation of destradiol and 1,25-(OH)₂-D₃, inhibiting production of prostaglandin E2 and ameliorating bone matrix metabolism. Osteoblasts are main functional cells of bone formation, in charge of synthesis, secretion and mineralization of bone matrix. They also express many bone related genes and secrete a lot of proteins and polypeptides. In recent years, osteoprotegerin (OPG) and receptor activator of NF-κB ligand (RANKL) are new discovery of coupling molecular mechanism between osteoblasts and osteoclasts, are also great progress in molecular biological research of bone cells. Binding berween RANKL secreted by osteoblasts and RANK existing in osteoclasts and precursors is the last action part of many factors in regulating activation of osteoclasts. Another cytokine secreted by osteoblasts, OPG, can oppose this action. Many physiological and pathologic agents influence differentiation and function of osteoclasts through this system. Experiments show OPG/RANKL/RANK system has close relation with osteoporosis. OPG inhibits bone resorption physiologically, inhibits hypercalcemia and bone loss caused by IL-1α, TNF-α, PTH and ovary excision and also inhibits bone resorption in menopausal women obviously. OPG gene knocking out mouse can lead to osteoporosis. In a word, OPG opposes osteoporosis. In this experiment, many techniques and methods such as osteoblasts culture, serum pharmacology, molecular biology and so on was adopted to study whether anti-osteoporosis liquid suppresses differentiation and maturation of osteoclasts indirectly by changing expression of OPG and RANKL mRNA in osteoblasts in cellular and molecular level. It does good to widening our thoughts in studying drugs of prevention andtherapy of osteoporosis. Methods 1 Make serum containing Chinese traditional medicine 36 SD rats weigh 250g, divided into two groups at random. The number of female equals to male. In one group, every rats was administered with 3ml distilled water, twice per day, successive three days. Blood was colleted by femoral artery at 1h, 2h and 4h respectively after last administration. Another group was administered with 3ml recipe, amounting to 8.31g herbs. Other treatment was as above. Collect serum after centrifugation, inactivation and filtration sterilization, preserve in -20℃. 2 Experiment groups division According to different time getting blood after administration of herbs, serum was divided into 1h, 2h, 4h groups. Serum get respectively at 1h, 2h, 4h after administration of distilled water was used as control groups. The concentration of serum in culture medium was 10%. Additionally, according to concentration of herbs serum and distilled water serum get at 4h, medium was divided into control, low dose, middle dose, high dose groups: control group, 20% distilled water serum; low dose group, 10% distilled water serum and 10% herbs serum; middle dose group, 5% distilled water serum and 15% herbs serum; high dose group, 20% herbs serum.3 Osteoblasts culture 4 Cell morphological observation Observe cells form, growth condition and difference between control and herbs serum groups by phase contrast microscope every day. 5 Detecting proliferation of osteoblasts in control and various herbs serum groups by MTT method 6 Alkaline phosphatase activity assay Velocity method was used in detecting activity of alkaline phosphatase. Lowry method was used in detecting protein. Enzyme activity was indicated by U/g protein. 7 Extraction of total RNA in osteoblasts Guanidium thiocyanate-phenol-chloroform method of Chomczynski and Sacchi. 8 The expression of mRNA assay The relative mRNA content was measured by RT-PCR with β-actin used as inner standard. Calculate proportion between OPG and RANKL. Result 1 Cell morphological observation In various herbs serum groups, osteoblasts were denser and tighter than that in various control groups. There was no obvious difference in cell shape. 2 Effect of herbs serum on proliferation of osteoblasts 2.1Effect of herbs serum get at different timeCompared with control group, there was apparent difference in OD value at 4h group, p<0.01. It showed herbs serum get at 4h contained more valid components and improved proliferation of osteoblasts significantly. Effects of 1h group and 2h group were light. 2.2 Effect of herbs serum of different dose Low dose group, middle dose group, high dose group improved proliferation of osteoblasts in statistical sense, p<0.001. Higher the content of herbs serum was, stronger the effect was. 3 Effect of herbs serum on differentiation of osteoblasts 3.1 Effect of herbs serum get at different time Compared with control group, enzyme activity of alkaline phosphatase increased in 1h group, 2h group and 4h group. But there was no statistical difference. 3.2 Effect of herbs serum of different dose In middle dose group and high dose group, enzyme activity of alkaline phosphatase was increased obviously, compared with control group, p<0.01. It showed the recipe improved differentiation of osteoblasts. Higher the content of herbs serum was, higher enzyme activity of alkaline phosphatase was. 4 Effect of herbs serum get at different time on expression of OPG and RANKL mRNA. 4.1 Effect on OPG mRNA Expression of OPG mRNA was up-regulated in 1h group,2h group and 4h group, but there was no statistical significance. 4.2 Effect on RANKL mRNA Expression of RANKL mRNA was down-regulated in 1h group, 2h group and 4h group, but there was no statistical significance. 4.3 Effect on OPG/RANKL In 2h group and 4h group, compared with respective control groups, proportion between OPG and RANKL was increased. Difference in 4h group (p<0.05) was more remarkable than that in 2h group (p<0.01). Effects of 1h groups were light. 5 Effect of herbs serum with different dose on expression of OPG and RANKL mRNA. 5.1 Effect on OPG mRNA Herbs serum of middle dose and high dose improved expression of OPG mRNA. Compared with control group, there was obvious difference, p<0.05 and p<0.01. 5.2 Effect on RANKL mRNA Herbs serum of middle dose and high dose inhibited expression of RANKL mRNA. Compared with control group, there was obvious difference, p<0.01 and p<0.001. 5.3 Effect on OPG/RANKL Compared with control group, OPG/RANKL was increased in low dose group (p<0.05), middle dose group (p<0.05) and high dose group (p<0.001). Effect increased with increasing...