Effect Of Osteoclast Regulated By PDK1 On Bone Mineral Density In Osteoporotic Mice

BACKGROUND:Bone tissue is a complex organ composed of cells,fibrous connective tissue and matrix.Bone cell matrix is composed of a large amount of calcium salts,which forms a hard tissue and constitutes the human skeletal system.The function of bone includes supporting and protecting the body.Because 99% of calcium in the body is concentrated in the bone tissue,it ensures the appropriate blood calcium concentration for the heart to work.It is an organ to maintain blood calcium balance,and also a necessary mineral repository for calcium homeostasis.At the same time,there are a large number of bone marrow stem cells in bone marrow,which can be differentiated into a variety of blood cells into the blood,ensuring the environment and niche needed for hematopoiesis.In mammals,the metabolism of bone tissue is in a dynamic balance.Its principle is the complex interaction between the successful bone formation of osteoblasts and the bone resorption function of osteoclasts.The normal human body continuously carries out bone resorption and bone formation,and the absorption and formation will maintain a steady state,which ensures the healthy and orderly state of bone tissue.When osteoclasts are activated by time,hormone or some diseases,the function of bone resorption is stronger than that of bone formation,which can lead to osteoporosis,osteomalacia and other diseases.When the activity of osteoclasts is decreased and bone formation is stronger than bone resorption,bone sclerosis,such as osteopetrosis,may occur.In view of the above problems,it can be seen that the regulation of osteoclast differentiation is of great clinical significance.Objective:Starting from the direction of regulation of osteoclast differentiation,to ex plore the preventive effect of conditional knockout mice of 3-phosphoinosi tide-dependent protein kinase 1(PDK1)gene on osteoporosis caused by m imicking female postmenopausal estrogen-deficient mice and to study its molecular mechanism,providing new ideas for the basic research of osteo porosis.METHODS:1.construct PDK1 knockout mice in conditional osteoclasts using the Cre-Lox P system driven by the RANK promoter.2.skeleton radiography to compare the developmental phenotypes of conditional knockout PDK1 gene mice and wild-type mice.3.microcomputed tomography(Micro-CT scan),Micro-CT scan of the femoral secondary ossification center to compare whether the physical development of conditional knockout PDK1 gene mice and wild-type mice is delayed.4.osteoclast differentiation assay,the number and area of osteoclasts were counted by tartrate-resistant acid phosphatase staining(TRAP staining)assay to observe the difference in osteoclast differentiation between conditional PDK1 knockout mice and wild-type mice.5.pseudopodial body assay to study the effect of conditional PDK1 knockout mice on osteoclast actin rings.6.bone resorption assay,To evaluate whether the bone resorption ability of osteoclasts in PDK1 knockout mice is weakened.7.real-time PCR(real-time PCR)was used to detect the effect of conditional knockout PDK1 gene mice on the expression of osteoclast-specific genes CTSK,MMP9,NFATc1,and TRAP.8.Western blotting was used to detect the expression of PDK1/AKT/NF-κB pathway-specific protein in conditional knockout PDK1 gene mice.9.A mouse model of osteoporosis was constructed.To compare the preventive and therapeutic effects of conditional knockout PDK1 gene mice and wild-type mice on osteoporosis.10.TRAP staining analysis was performed to detect changes in the number of osteoclasts in the bone tissue of conditional PDK1 gene mice compared with wild-type mice.11.Micro-CT scanning and VK staining(Von Kossa staining)staining were used to compare the preventive effect of conditional knockout PDK1 gene mice and wild-type mice on osteoporosis after ovariectomy osteoporosis model was constructed.Results:1.Conditional knockout PDK1 gene mice showed macroscopically smaller body length compared with wild-type mice,but no malformation manifestations.2.conditional knockout PDK1 gene mice showed differences in body size compared with wild-type mice,but the time of secondary ossification center appearance was not delayed.3.RANKL-induced in vitro differentiation experiments demonstrated that PDK1 gene reduction inhibited osteoclasts compared with wild-type mice,and the number and area of osteoclasts were reduced.4.pseudopodia formation experiments demonstrated that conditional PDK1 gene knockout inhibited pseudopodia band formation in osteoclasts,i.e.And the inhibition was significant.5.Bone resorption assay demonstrated that conditional PDK1 gene knockout had the ability to inhibit osteoclast bone resorption.6.Real-time PCR demonstrated that conditional knockout of PDK1 gene could significantly inhibit the expression of osteoclast-related genes CTSK,MMP9,NFATc1,and TRAP.7.Westernblotting detected that the expression of PDK1,AKT,p-AKT,p-IKKα and other proteins in the PDK1/AKT/NF-κB signaling pathway was inhibited.8.VK staining showed that conditional knockout PDK1 gene mice had lumbar spine bone volume fraction(Bonevolume/Tissuume,BV/TV)and bone number(Trabecular Number,Tb.N),bony thickness(Trabecular Thickness,Tb.Th)increased,bone separation(Trabecular Separation,Tb.Sp)was significantly reduced,and the degree of osteoporosis was significantly reduced.9.TRAP staining analysis showed that osteoclasts in the lumbar spine of conditional knockout PDK1 gene mice were significantly reduced.10.Micro-CT scanning of conditional knockout PDK1 gene mice and wild-type mice to construct ovariectomized osteoporosis models revealed that compared with wild-type mice,conditional knockout PDK1 gene mice had BV/TV and Tb in the third lumbar spine.N,and Tb.Significant increase in Th,Tb.Sp was significantly decreased,which was consistent with the results of VK staining and could effectively protect the bone loss of osteoporosis model mice.CONCLUSION:1.PDK1 gene can positively regulate bone resorption by regulating the differentiation of osteoclasts in vivo,and significantly increase systemic bone mass and bone mineral density.2.Conditional knockout of PDK1 gene inhibits the differentiation and bone resorption of osteoclasts by inhibiting the PDK1/AKT/NF-κB pathway.3.Conditional knockout of PDK1 gene mice compared with wild-type mouse osteoporosis model,PDK1 knockout can positively regulate bone mass and prevent osteoporosis.