The Mechanism Research Of PDK1 Targeting AKT Regulates The Differentiation Of Mouse Bone Marrow Macrophages Into Osteoclast

Background/objective: Osteoporosis is a common and frequently occurring disease in China.It is a serious threat to the health of the middle-aged and the elderly,especially the postmenopausal women.Complications such as fracture caused by osteoporosis not only cause great pain to patients themselves,but also bring heavy life and financial burden to families and society.The stability of bone metabolism requires the dynamic balance between osteoclast mediated bone resorption and osteoblast mediated bone formation.In healthy individuals,the two maintain functional balance,and once the balance is broken,it can lead to bone disease including osteoporosis,osteoarthritis,rheumatoid arthritis and so on.Osteoclasts are the only cells responsible for bone resorption in vivo.Targeted inhibition of osteoclast activation is considered to be the most effective and direct treatment strategy to reverse bone destruction,and has become a research hotspot in the field.Studies have shown that osteoclast activation and inhibition of osteoclast active drugs play a crucial role in the development and treatment of osteoporosis.However,there are many deficiencies in the current treatment of osteoporosis drugs.Therefore,it is necessary to further strengthen and improve the study of the molecular mechanism of signal transduction pathway in the process of osteoclast differentiation,the development of new targeted drugs to improve the therapeutic effect on osteoporosis,which will have a very important clinical value,basic research value and social economic value.In the field of Department of orthopedics,the research on PDK1 is still shallow,and it has not been paid much attention and systematic research.Therefore,this study will preliminarily explore the effect of PDK1 on the differentiation of bone marrow macrophages into osteoclast induced by RANKL and MCSF,exploring a new regulation pathway of osteoclast differentiation will help us further understand the molecular biological regulation mechanism of osteoclast related osteoporosis.Method:(1)extraction and culture of C57BL/6 mouse bone marrow macrophages,the mouse bone marrow macrophages were cultured in the induction solution for 6 days.The growth and morphological changes of osteoclasts were observed under microscope every day.Osteoclasts were identified by TRAP staining and counted;(2)The mouse bone marrow macrophages were cultured in the induction liquid for 0,2,4 and 6 days respectively,and no RANKL in the culture medium was used as the control group,the total RNA and total protein of the cells were extracted at 0,2,4 and 6 days.qRT-PCR was used to detect the expression of PDK1 mRNA.Determination of protein concentration by BCA Kit.Detection of PDK1,AKT protein and its phosphorylation by Western blot;(3)Negative Control siRNA transfected mouse bone marrow macrophages and searching for the optimal concentration of siRNA in transfected mouse bone marrow macrophages according to the proportion of 1:0.01-1:0.1(siRNA : lipofectamin2000)and calculation of transfection efficiency;(4)three PDK1-siRNA were designed and transfected into mouse bone marrow macrophages respectively,after transfection of 48 h,the total RNA and total protein of cells were extracted from each group(blank group,Negative Control siRNA group,PDK1-si RNA-1 group,PDK1-siRNA-2 group and PDK1-si RNA-3 group),the expression of PDK1 mRNA in each group was detected by qRT-PCR,and the expression of PDK1 protein in each group was detected by Western blot;(5)The effect of PDK1-siRNA on the proliferation of mouse bone marrow macrophages by Cell Counting Kit-8;(6)After transfection of PDK1-siRNA into mouse bone marrow macrophages,two groups(Negative Control siRNA group and PDK1-siRNA group)of mouse bone marrow macrophages were cultured for 0,2,4 and 6 days,respectively,the total RNA and total protein of cells were extracted for 0,2,4 and 6 days,respectively,qRT-PCR was used to detect the gene expression of PDK1,western blot detection of the expression of PDK1 and AKT protein and its phosphorylation;Two groups of cells were cultured in the induction solution for 6 days,the expression of osteoclast related genes(TRAP,MMP-9,CtsK and NFATc1)was detected by qRT-PCR and then TRAP staining was carried out and the number of osteoclast formation was counted;(7)Cell Counting Kit-8 was used to detect the effect of PDK1 specific inhibitors BX912 on the proliferation of bone marrow macrophages;(8)Different concentrations of PDK1 specific inhibitors(BX912:0-0.312uM)were combined with induction fluid to culture bone marrow macrophages for 6 days.The expression of osteoclast related genes(TRAP,MMP-9,CtsK and NFATc1)was detected by qRT-PCR,and then TRAP staining was performed,and the number of osteoclasts was counted.Result: Mouse bone marrow macrophages are adherent growth,the growth-condition of cells was good;6 days after RANKL induced differentiation of bone marrow macrophages,a large number of osteoclasts were found,with large cell volume and deep red granules in the cytoplasm,containing 3 or more nuclei;(2)compared with the control group,in the differentiation of mouse bone marrow macrophages into osteoclasts,the mRNA and protein phosphorylation level of PDK1 gradually increased,and promoted the phosphorylation of AKT protein,which was statistically significant(P<0.05);(3)The number of fluorescent cells was observed under the fluorescence microscope.It was found that the transfection efficiency of siRNA was the highest when the ratio of siRNA to lipofectamin2000 was 1 pmol:0.05 UL in the transfected bone marrow macrophages;(4)Compared with the blank group,Negative Control si RNA had no effect on the expression of PDK1 mRNA and its protein in the cells,and there was no statistical difference between the two groups(P>0.05);Compared with the blank group,the expression level of PDK1 mRNA and protein in PDK1-siRNA group decreased significantly,the difference was statistically significant(P<0.05),especially in PDK1-siRNA-1 group.(5)CCK-8 assay showed no significant difference between the OD value of the three groups(P>0.05).The results show that PDK1-siRNA has no effect on cell proliferation;(6)Compared with Negative Control si RNA group,the expression of PDK1 mRNA in PDK1-siRNA-1 group was significantly decreased,and the phosphorylation level of PDK1 and AKT protein was significantly decreased,the difference was statistically significant(P<0.05),the total PDK1 and AKT protein expression had no significant change(P>0.05);The results of qRT-PCR detection showed that compared with the blank group,there was no significant difference in the expression of osteoclast related genes in Negative Control siRNA group(P>0.05),while the expression of osteoclast related genes in PDK1-siRNA-1 group was significantly decreased(P<0.05);The results of TRAP staining showed that the number of osteoclasts in PDK1-siRNA-1 group decreased significantly compared with that of Negative Control siRNA group,and there was a statistical difference(P<0.05);(7)CCK-8 assay results showed that compared with the blank group(BX912:0uM),after 144 h intervention,the PDK1 specific inhibitor concentration below 0.625 uM had no obvious effect on the proliferation of bone marrow macrophages(P>0.05);(8)Compared with the blank group(0u M group),the mRNA expression level of osteoclast related genes in the BX912 group of 0.078-0.312 u M concentration decreased to varying degrees,indicating that BX912 inhibited the differentiation of bone marrow macrophages into osteoclasts in vitro;TRAP staining showed that compared with the control group,different concentrations of PDK1 specific inhibitors BX912(0-0.312uM)inhibited the differentiation of osteoclasts(P<0.05),and the greater the concentration of BX912,the greater the inhibitory effect on osteoclasts.When BX912 concentration was 0.312 uM,the inhibitory effect was the most obvious and the number of osteoclasts was the least.Conclusion:(1)In the differentiation process of mouse bone marrow macrophages to osteoclasts,PDK1 gene expression and protein phosphorylation level gradually increased,and promoted the phosphorylation of AKT protein.It indicates that PDK1/AKT signaling pathway is involved in the process of osteoclast differentiation and maturation.(2)PDK1-siRNA can effectively silence the expression of PDK1 mRNA and protein in bone marrow macrophages,and silence PDK1 gene can effectively prevent bone marrow macrophages from differentiating and mature into osteoclasts.(3)PDK1 specific inhibitors BX912 can significantly inhibit the differentiation and maturation of osteoclasts.