Gu Ling Pian Regulates the Function of Osteoblasts via p38 MAPK Pathway in Vitro
|School||First Military Medical University|
|Course||Traditional Chinese Medicine|
|Keywords||Gu Ling Pian human osteoblasts p38 osteoprotegerin receptor activator of nuclear factor kappa B ligand|
Backgrounds:Osteoporosis is a common disease that affects both men and women, which makes bones prone to fracture. Osteoporosis is caused by the imbalance of bone formation and bone absorption impaired, makes the loss exceed the formation of bone. Either bone absorption is excessive, or bone formation is diminished. Bone matrix is manufactured by the osteoblast cells, while bone absorption is accomplished by osteoclast cells.Many studies have carefully investigated the interaction between osteoblasts and osteoclasts. 0steoprotegerin （OPG）, receptor activator of NF-κB （RANK） and RANK ligand （RANKL） play a critical role in bone remodeling by regulating the function of osteoclasts. OPG is a member of the TNFR family and a soluble decoy receptor competitive against RANKL and soluble RANKL （sRANKL）. OPG, produced by osteoblasts and other cells, has been found to be a key factor in the inhibition of differentiation and activation of osteoclasts. On the other hand, RANKL, a member of the TNF family, induces osteoclasts differentiation, maturation and activates mature osteoclasts though binding with their receptor RANK expressed on the surface, while sRANKL cleaved from RANKL behaves similarly to RANKL as a soluble factor. The increase of RANKL expression and sRANKL release leads to bone absorption and loss. The expression of OPG and RANKL regulate activation of osteoclasts, and thereforedeeply affects bone remodeling.Osteoblasts growth and differentiation mediated by receptor tyrosine kinases are regulated by many extracellular signals, most of which activate the Ras-MAPK kinase cascade. Three distinct subgroups within the MAPK family have been extensively investigated and described to have profound effects on osteoblasts function. These include: （1） the extracellular signal-regulated kinase （ERK1/2） pathway; （2） the c-Jun N-terminal kinase/stress activated protein kinase （JNK/SAPK） pathway; and （3） the p38 MAPK pathway. Recent studies have illustrated that ERK1/2 pathway is responsible for the signal transduction of growth factors and mitogens, which is predominantly involved in the osteoblasts differentiation. JNK, weakly activated by growth factors, while p38 MAPK activated in cellular responses to various environmental stresses and factors regulates expression and gene transcription of OPG and/or RANKL in osteoblasts.Traditional Chinese Medicine has its own ascendancy on preventing and curing OP. Our previous studies have demonstrated that Gu Ling Wan, pill dosage form with the same composition of GLP, is able to prevent from primary osteoporosis animal model, moreover in patients with osteoporosis, regulate the factors related to bone formation and absorption and increase bone mineral density （BMD）. And also the GLP may increase Ca2+ and stimulate proliferation and differentiation of adult osteoblasts in vitro. Provided the pharmacological mechanism of GLP in preventing osteoporosis has not been disclosed, we have utilized a human osteoblast model to test in vitro the effects of GLP. Our results, for the first revealed the working mechanism of GLP on the prevention and therapy of osteoporosis and supply for an efficient reagent as a potential. Objective1. To observe the the effects of GLP on OB differentiation, mineralized bone nodule formation, and OPG/RANKL expression.2. To exploer the effects of GLP on regulating the OPG/RANKL expression via p38 MAPK pathway in vitro.Methods1. In experiment A, human osteoblast were cultured with serum from rat fed with media, low dose GLP, high dose GLP respectively.2. In experiment B, to study the effect of p38 inhibitor SB203580, two groups of human osteoblast were treated with 10μM SB203580 for 2 h prior to high dose rat serum treatment. And the other one group of human osteoblast was cultured with serum from rat fed with high dose GLP without SB203580.3. The expression of the OPG and RANKL mRNA, the formation of mineralized bone nodule, the total protein of p38 and phosphorylated p38, were observed.4. All data are expressed as means±SD, and differences among groups were analyzed by one-way ANOVA followed by LSD test or Dunnett test. A P value of＜0.05 was considered statistically significant.Results1. In experiment A, GLP serum promoted osteoblasts differentiation, increased the formation of mineralized bone nodule, and also inhanced OPG expression while RANKL expression decreased and increased the total protein of phosphorylated p38.（1） There is significant difference in total about the osteoblasts differentiation （F=24.211 P＜0.001）. The rate of osteoblasts differentiation: （152.06±14.35）% （HD）、（143.42±8.46）%（MD）、（118.96±15.68）%（LD）and（100.32±14.46）%（control）. MD and HD groups increase notably compared with control group （P＜0.01）. （2） There is significant difference in total about the osteoblasts mineralized bone nodule formation （F=315.247 P＜0.001）. Mineralized bone nodule formation was significantly increased in three level dose GLP groups. The data are （201.60±11.25）（HD）、（181.10±6.76）（MD）、（118.02±8.97）（LD） and （86.97±6.17）（control）. LD、MD and HD groups increase notably compared with control group （P＜0.01）.（3） There is significant difference in total about the osteoblasts OPG mRNA’s expression （F=100.971 P＜0.001）. OPG mRNA’s expression result: the data are （1.84±0.16）（LD）、（3.58±0.47） （MD）、（4.13±0.54）（HD） and （1.004±0.14）（control）. MD and HD groups increase significantly compared with control group （P＜0.01）.（4） There is significant difference in total about the osteoblasts RANKL mRNA’s expression （F=161.360 P＜0.001）. RANKL mRNA’s expression result: the data are （0.89±0.07）（LD）、（0.47±0.06） （MD）、（0.25±0.07）（HD） and （1.01±0.08）（control）. MD and HD groups decrease significantly compared with control group （P＜0.01）. The rate of OPG/RANKL was increase significantly compared with control group （P＜0.01）.（5） The western-blot test shows that significant increase was found in phosphorylation p38 in OB treated with all dose of GLP serum, compared to the control group.2. In experiment B, we evaluated cell proliferation and differentiation, synthesis of OPG/RANKL in hunan osteoblasts by using inhibitor specific to p38.（1） There is significant difference in total about the osteoblasts differentiation （F=7.971 P＜0.001）. The rate of osteoblasts differentiation:（99.75±16.12）（SB）、（100.91±16.12）（SB+GLP）、（101.31±13.21）（control） and （136.17±16.01）（HD）. The result of SB, （SB+GLP）, control has no significant difference（P＞0.05）. Compared to the control group, the result of HD group was significantly increased （P＜0.05）.（2） There is significant difference in total about mineralized bone nodule formation （F=79.683 P＜0.001）. Mineralized bone nodule formation was significantly increased in HD group, contrasted to the （SB+GLP） one （P＜0.01）. And in the other two groups, mineralized bone nodule formation was not significantly increased.（3） There is significant difference in total about OPG mRNA’s expression （F=112.374 P＜0.001）. OPG mRNA’s expression result: the data are （0.96±0.11）（SB）、（1.07±0.07） （SB+GLP）、（1.01±0.10） （control） and （2.93±0.42）（HD）. In the HD group, the expression was significantly upgraded contrasted to the other three groups（P＜0.01）.（4） There is significant difference in total about RANKL mRNA’s expression （F=38.333 P＜0.001）. Lower expression of RANKL mRNA was found in cells treated with high dose rat serum containing GLP only, which is in contrast to cells exposed to SB203580（P＜0.05）. And the rate of OPG/RANKL was increased in the GLP group only which is in contrast to cells exposed to SB203580（P＜0.01）.（5） Total p38 was not altered in human osteoblasts whether they were treated with GLP or not. But significant increase was found in phosphorylation p38 in human OB treated with rat serum containing GLP.The expression of p38 induced by GLP was effectively inhibited by SB203580.ConclusionWe conclude that GLP effectively induces differentiation and maturation of osteoblasts and regulates the expression of OPG and RANKL via p38 MAPK pathway, and therefore, suggest that GLP may be beneficial in stimulating the osteoblastic activity resulting inbone formation.