scholarly journals Gujin Dan is A Chinese Medicine Formulation That Stimulates Cell Proliferation And Differentiation By Controlling Multiple Genes Involved In MC3T3-E1 Cells

2021 ◽  
Author(s):  
Yu Zhou ◽  
Chaozong Liu ◽  
Zhenwei Zhou ◽  
Xin Li ◽  
Songchuan Su ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Chinese Medicine ◽  
Expression Patterns ◽  
Osteoclast Formation ◽  
Alizarin Red ◽  
Gene Markers ◽  
Expression Levels ◽  
Chinese Herbal ◽  
Alkaline Phosphatase Staining ◽  
Proliferation And Differentiation

Abstract Background: The development of Chinese medicine has been practised in China over a long period of time, and China has long used single medicines in various forms of decoction to treat illnesses, and later learned to combine several medicines to form formulas to enhance the effects of the medicines. The use of Chinese herbal medicines and formulas has played a pivotal role in the prevention and treatment of diseases in China since ancient times. The application of Chinese herbal preparations in the field of osteoporosis treatment has received widespread attention, and Gujin Dan（GJD） is one of the representative herbal formulas, however, the exact minute mechanism of its treatment of osteoporosis remains to be elucidated.Methods: In the study, we prepared an aqueous extract of GJD and measured the effect of different administration concentrations of GJD on cell proliferation by CCK-8 assay, and the effect of GJD on cell differentiation ability by Alizarin Red S Staining, Alkaline Phosphatase Staining and quantitative assay. Changes in gene expression patterns of MC3T3-E1 cells under GJD treatment were investigated by RNA-seq analysis and validation methods.Results: We demonstrate that GJD promotes the proliferation and differentiation of Mc3t3-e1 cells through the regulation of multiple functional genes. This was mainly achieved by regulating the expression levels of four categories of genes that promote the proliferation of Mc3t3-e1 cells or osteoblasts, inhibit apoptosis and autophagy, inhibit osteoclast formation and differentiation, and promote osteoblast differentiation. In addition, GJD slightly increased the expression levels of gene markers in osteoblasts. Conclusions: Our findings suggest that GJD promotes proliferation and differentiation of MC3T3-E1 cells and inhibits osteoclastogenesis and differentiation, as well as apoptosis and autophagy, through the synergistic interaction of various herbs and their active components in GJD. This study has significantly improved the current understanding of the molecular effects of GJD on MC3T3-E1 cells. This study also provides new ideas for possible strategies to further prevent and treat bone metabolism-related diseases using traditional Chinese medicinal preparations.

scholarly journals The enzymatic hydrolysates from deer sinew promote MC3T3-E1 cell proliferation and extracellular matrix synthesis by regulating multiple functional genes

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Zhenwei Zhou ◽  
Daqing Zhao ◽  
Pengcheng Zhang ◽  
Mei Zhang ◽  
Xiangyang Leng ◽  
...  
Keyword(s):  
Extracellular Matrix ◽  
Cell Proliferation ◽  
Molecular Mechanism ◽  
Dose Effect ◽  
Functional Genes ◽  
Matrix Synthesis ◽  
Alizarin Red ◽  
Gene Markers ◽  
Expression Levels ◽  
Enzymatic Hydrolysates

Abstract Background Deer Sinew serves as a medicinal food, and has been used for treating skeletal diseases, especially bone diseases in a long history. Thus, it could become an alternative option for the prevention and therapeutic remedy of bone-related diseases. In our previous study, we established an optimal extraction process of the enzymatic hydrolysates from Chinese Sika deer sinews (DSEH), and we demonstrated that DSEH significantly promoted the proliferation of MC3T3-E1 cells (an osteoblast-like cell line) with a certain dose-effect relationship. However, the precise molecular mechanism of deer sinew in regulating bone strength is still largely unknown. The aim of this study was to explore the underlying molecular mechanism of DSEH on MC3T3-E1 cells proliferation and extracellular matrix synthesis. Methods Preparation and quality control were performed as previously described. The effect of DSEH at different administrated concentrations on cell proliferation was measured using both CCK-8 and MTT assays, and the capacity of DSEH on extracellular matrix synthesis was detected by Alizarin red staining and quantification. The gene expression pattern change of MC3T3-E1 cells under the treatment of DSEH was investigated by RNA-seq analysis accompanied with validation methods. Results We demonstrated that DSEH promoted MC3T3-E1 cell proliferation and extracellular matrix synthesis by regulating multiple functional genes. DSEH significantly increased the expression levels of genes that promoted cell proliferation such as Gstp1, Timp1, Serpine1, Cyr61, Crlf1, Thbs1, Ctgf, P4ha2, Sod3 and Nqo1. However, DSEH significantly decreased the expression levels of genes that inhibited cell proliferation such as Mt1, Cdc20, Gas1, Nrp2, Cmtm3, Dlk2, Sema3a, Rbm25 and Hspb6. Furthermore, DSEH mildly increased the expression levels of osteoblast gene markers. Conclusions Our findings suggest that DSEH facilitate MC3T3-E1 cell proliferation and extracellular matrix synthesis to consolidate bone formation and stability, but prevent MC3T3-E1 cells from oxidative stress-induced damage, apoptosis and further differentiation. These findings deepened the current understanding of DSEH on regulating bone development, and provided theoretical support for the discovery of optional prevention and treatment for bone-related diseases.

scholarly journals Influences of Compressive Force and Zoledronic Acid on Osteoblast Proliferation and Differentiation: An In Vitro Study

2021 ◽  
Vol 11 (23) ◽  
pp. 11273
Author(s):  
Kazuyuki Yusa ◽  
Shigeo Ishikawa ◽  
Tomoharu Hemmi ◽  
Hiroshi Takano ◽  
Masayuki Fukuda ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Zoledronic Acid ◽  
Osteoblast Differentiation ◽  
Compressive Force ◽  
In Vitro Study ◽  
Osteonecrosis Of The Jaw ◽  
Alizarin Red ◽  
Matrix Mineralization ◽  
Proliferation And Differentiation

This study investigates the effects of zoledronic acid (ZA) and compressive force on osteoblast functions, to elucidate the pathogenesis of medication-related osteonecrosis of the jaw (MRONJ). MC3T3-E1 cells were exposed to ZA (1, 10 and 100 µM) to evaluate the effects of ZA on cell proliferation. Furthermore, to investigate the influence of ZA with or without compressive force on osteoblast differentiation, real-time polymerase chain reaction and Alizarin Red S staining were performed. ZA concentrations > 10 μM were highly cytotoxic to MC3T3-E1 cells. Combining 1-μM ZA with compressive force influenced expression levels of osteoblast-related genes and matrix mineralization. The inhibitory effects of ZA on cell proliferation and the combination of ZA and compressive force on osteoblast differentiation may contribute to the pathogenesis of MRONJ.

scholarly journals Dysregulation of ANKRD11 Influenced Hematopoisis By Histone Acetylation-Mediated Gene Expression in Myelodysplastic Syndrome

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 4292-4292
Author(s):  
Youshan Zhao ◽  
Feng Xu ◽  
Juan Guo ◽  
Sida Zhao ◽  
Chunkang Chang ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Proliferation ◽  
Histone Acetylation ◽  
Mrna Expression ◽  
Cell Line ◽  
Expression Levels ◽  
Target Sequencing ◽  
Cell Proliferation And Differentiation ◽  
Proliferation And Differentiation ◽  
Mrna Expression Levels

Abstract Background and Object In addition to histone deacetylation, the importance of histone over-acetylation induced oncogene transcription in initiation and progression of myelodysplastic syndrome (MDS) has been proposed recently. Our previous whole-exome sequencing identified a new somatic mutation, ANKRD11, an important factor in histone acetylation regulation. Its roles in MDS pathophysiology need to be clarified. Methods The next generation target sequencing (Including ANKRD11) was carried out in 320 patients with MDS using the MiSeq Benchtop Sequencer. ANKRD11 mRNA expression in bone marrow of MDS was measured by real-time PCR. Loss and gain of function assay were carried out in myeloid cell lines K562, MEG-01£¬or SKM-1 to observe the influence on cell proliferation and differentiation . The levels of histone acetylation at H3 and H4 were detected by Western blot. Results Target sequencing in a cohort of 320 MDS patients identified 14 of ANKRD11 mutations (4.38%, Fig.1), which were confirmed by Sanger sequencing. Meanwhile, no ANKRD11 mutations in 100 normal controls were defined. ANKRD11 mutations occurred frequently in exons 10 and 9. The mRNA expression levels of ANKRD11 were significantly decreased in MDS patients, especially in ANKRD11mutant patients (Fig.2). ANKRD11 knockdown in K562 and MEG-1 resulted in growth inhibition, cell cycle arrest and erythroid/megakaryocytic differentiation retardant. In MDS cell line SKM-1, the arrested differentiation was rescued by over-expression of ANKRD11. Consistent with a role for ANKRD11 in histone acetylation, ANKRD11 KD increased acetylation of histones H3 and H4 at H3K14 and H4K5 and resulted in the upregulation of genes involved in differentiation inhibilation (SOX6, P21, et al). Finally, the ANKRD11 KD-mediated influence on cell proliferation and differentiation were reversed by inhibiting histone acetyltransferase activity. Conclusion Our assay defined that ANKRD11 was a crucial chromatin regulator that suppress histone acetylation and then decrease gene expression during myeloid differentiation, providing a likely explanation for its role in MDS pathogenesis. This study further support histone acetylase inhibitor as a potential treatment in MDS. Figure ANKRD11mutation distribution (a) and coexist with other mutations (b). Figure. ANKRD11mutation distribution (a) and coexist with other mutations (b). Figure The mRNA expression levels of ANKRD11in our MDS (A, C) subset and GEO data (B). Figure. The mRNA expression levels of ANKRD11in our MDS (A, C) subset and GEO data (B). Changes of histone acetylation in ANKRD11-KD cell line (MEG-01). ANKRD11 KD significantly increased acetylation of histones H3 and H4 at H3K14 and H4K5. Changes of histone acetylation in ANKRD11-KD cell line (MEG-01). ANKRD11 KD significantly increased acetylation of histones H3 and H4 at H3K14 and H4K5. Disclosures No relevant conflicts of interest to declare.

MicroRNAs in Normal Human Terminal Granulocytic Differentiation.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 1463-1463
Author(s):  
Su Ming Sun ◽  
Menno K Dijkstra ◽  
André C Bijkerk ◽  
Rik Brooijmans ◽  
Peter J Valk ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Mrna Expression ◽  
Expression Pattern ◽  
Mirna Expression ◽  
Expression Patterns ◽  
Granulocytic Differentiation ◽  
Expression Levels ◽  
Mirna Expression Pattern ◽  
Normal Human

Abstract Abstract 1463 Poster Board I-486 Normal human myelopoiesis is a complex biological process, where the balance between cell proliferation, differentiation and apoptosis is tightly regulated by a transcriptional program that results in the production of appropriate numbers of circulating mature myeloid cells. MicroRNAs (miRNAs) are small non-coding RNAs of 18∼25 nt that can affect cellular protein levels. Several studies show specific miRNA expression patterns in different subtypes of myeloid malignancies, however only limited data is available on miRNA expression patterns during normal myeloid differentiation of primary human cells. We set out to characterize miRNA expression patterns in the different stages of granulocytic differentiation in two models. First myeloblast, promyelocytes, metamyelocytes and granulocytes from normal human bone marrow were cell-sorted with flow cytometry using the markers CD10, CD11, CD34, CD36, CD45 and CD117. Second, CD34+ cells from primary human fetal livers were differentiated in vitro towards neutrophils. MiRNA expression levels were determined at different time points (day 0, 3 and 10), representing different stages of granulocytic differentiation. MiRNA expression was measured using the qPCR platform, containing 365 miRNAs, from Applied Biosystems. To identify potential miRNA target genes, we performed mRNA expression profiling in the latter in vitro differentiation. The negative correlations between miRNA and mRNA expression were identified and integrated with a target prediction database (Targetscan). The miRNA profiling showed that approximately 70% of the 365 miRNAs analyzed, were expressed during granulocytic differentiation and that the miRNA expression pattern during this process change significantly in both models. Principal component analysis showed clear separation of the different subsets of granulopoiesis based on the miRNA expression. We determined the differentially expressed miRNAs between the various subsets using ANOVA with a P value <0.05, after correction for multiple testing. We found 24 miRNAs to be differentially upregulated in the both models. The top 5 upregulated miRNA, with the highest fold change in granulocytes as compared to myeloblasts, were miR-223, miR-145, miR-148, miR-24 and miR-23a. We identified 27 miRNAs that were downregulated, the top 5 were of miR-10a, miR-196a, miR-130a, miR-135a and miR-125b. Concomitant miRNA and mRNA expression analysis of the in vitro model with the Targetscan database, demonstrates a potential regulatory role for these miRNAs in various processes, such as cell proliferation, apoptosis and cell cycle regulation. For example, miR-130a, miR-20b and miR-191, miR-301 expression levels were negatively correlated with E2F2 and SOX4 respectively. Furthermore, MAPK1 levels correlated inversely with miR-17-5p, miR-130a, miR-181b, miR-181d and miR-20b. We observed potential regulation of BCL2L11 by miR-10a, miR-10b and CDK6 by miR-148a, miR-148b, miR-191 and miR-21, as well as CHEK1 by the miR-15a and miR-16, LATS2 by miR-142-3p and CCND3 by miR-133a. In addition we also identified myeloid specific genes to be potentially regulated by miRNAs such as CEBPA by miR-181b, KIT by miR-148a, miR-148b and miR-301 and RUNX3 by miR-301. This is the first comprehensive study of miRNA expression in normal human granulocytic differentiation. We show in two models that the miRNA expression pattern changes during granulocytic differentiation. miRNA-mRNA analyses suggest involvement of miRNAs in regulation of important cellular processes during granulocytic differentiation. Experimental validations of several candidate targets as well as functional studies are currently ongoing. Disclosures No relevant conflicts of interest to declare.

The Clinical and Biological Characterization of De Novo Acute Myeloid Leukemia (AML) with GATA2 Mutation

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 3822-3822
Author(s):  
Feng-Ming Tien ◽  
Wen-Chien Chou ◽  
Hsin-An Hou ◽  
Yu-Chiao Chiu ◽  
Eric Y. Chuang ◽  
...  
Keyword(s):  
Amino Acids ◽  
Cell Proliferation ◽  
De Novo ◽  
Point Mutations ◽  
Gene Set Enrichment Analysis ◽  
Mutation Status ◽  
Negatively Associated ◽  
Expression Levels ◽  
Cell Proliferation And Differentiation ◽  
Proliferation And Differentiation

Abstract Introduction GATA-binding factor 2 (GATA2) contributes to the regulation of hematopoietic stem cell proliferation and differentiation. Mutations of GATA2 are identified in AML patients, but their clinical and biological correlations in AML remain to be delineated. Methods and Materials We determined GATA2 mutations by Sanger sequencing and next generation sequencing in consecutively enrolled 766 newly diagnosed de novo AML patients who had cryopreserved cells for analysis. The expression of GATA2 was also analyzed by microarray in 335 patients. We then looked for the clinical and biological correlations of the expression levels and mutation status of GATA2. Computational network analysis of GATA2 mutations was conducted by Ingenuity Pathway Analysis (IPA) and Gene set enrichment analysis (GSEA). Result GATA2 mutations were detected in 58 patients (7.5%) and were associated with FAB M1 subtype, and intermediate-risk cytogenetics, but negatively associated with M4 subtype and favorable-risk cytogenetics. There was no difference in other clinical parameters such as age, hemogram and LDH levels between the patients with and without GATA2 mutations. GATA2 mutations were closely associated with CEBPA double mutations (44.8% vs. 10.2%, P<0.001), but negatively associated with WT1 (0% vs. 7.4%, P=0.003) and IDH1 mutations (0% vs. 6.4%, P<0.0001). The mutation hot spots were between amino acids 315 to 361. The mutant burden ranged from 4.89% to 52% with a median of 35.31%. Only two patients harbored 2 mutations; mutations in the others were heterozygous. Most were point mutations except for 3 (5.5%) with indel. With a median follow-up time of 25 months (ranges 0 to 160), patients with GATA2 mutations had a trend of better overall survival (OS) than those without GATA2 mutations (median 40.7 vs. 24.9 months P=0.123). Among patients with CEBPA double mutations, there was also a trend toward better OS in patients concomitant with GATA2 mutations (P=0.199). By comparing the mRNA expression profiles between patients with and without GATA2 mutations, we found GATA2 expression levels were higher in those with GATA2 mutations (P=0.003). GATA2 mutations were also associated with a significant change in genes related to cell proliferation and differentiation by IPA. We also performed GSEA analysis to identify modest functional changes related to GATA2 mutation status. Leukemogenesis-related genes were significantly enriched in the GATA2-mutated subgroup (nominal P value= 0.006; Normalized enrichment score= 1.62), whereas gene signatures associated with myeloid differentiation, apoptosis, leukemia cell death, CEBPA and WT1 pathways were enriched in GATA2 -wild type patients. Conclusion GATA2 mutations, which are commonly heterozygous point mutations between amino acids 315 to 361, occur in 7.5% of AML patients and are associated with certain clinical features, a trend of better treatment outcome, and higher GATA2 expression. The GATA2 mutation-associated expression signatures suggest the effects on leukemogenesis by GATA2 mutations. Figure 1. Figure 1. Disclosures Tang: Novartis: Consultancy, Honoraria.

scholarly journals Depletion of SNRNP200 inhibits the osteo−/dentinogenic differentiation and cell proliferation potential of stem cells from the apical papilla

2020 ◽  
Vol 20 (1) ◽  
Author(s):  
Xiaomin Su ◽  
Haoqing Yang ◽  
Ruitang Shi ◽  
Chen Zhang ◽  
Huina Liu ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Stem Cells ◽  
Cell Proliferation ◽  
S Phase ◽  
Alizarin Red ◽  
Differentiation Potential ◽  
Alp Activity ◽  
Proliferation And Differentiation ◽  
Apical Papilla ◽  
Dentinogenic Differentiation

Abstract Background Tissue regeneration mediated by mesenchymal stem cells (MSCs) is deemed a desirable way to repair teeth and craniomaxillofacial tissue defects. Nevertheless, the molecular mechanisms about cell proliferation and committed differentiation of MSCs remain obscure. Previous researches have proved that lysine demethylase 2A (KDM2A) performed significant function in the regulation of MSC proliferation and differentiation. SNRNP200, as a co-binding factor of KDM2A, its potential effect in regulating MSCs’ function is still unclear. Therefore, stem cells from the apical papilla (SCAPs) were used to investigate the function of SNRNP200 in this research. Methods The alkaline phosphatase (ALP) activity assay, Alizarin Red staining, and osteogenesis-related gene expressions were used to examine osteo−/dentinogenic differentiation potential. Carboxyfluorescein diacetate, succinimidyl ester (CFSE) and cell cycle analysis were applied to detect the cell proliferation. Western blot analysis was used to evaluate the expressions of cell cycle-related proteins. Results Depletion of SNRNP200 caused an obvious decrease of ALP activity, mineralization formation and the expressions of osteo−/dentinogenic genes including RUNX2, DSPP, DMP1 and BSP. Meanwhile, CFSE and cell cycle assays revealed that knock-down of SNRNP200 inhibited the cell proliferation and blocked cell cycle at the G2/M and S phase in SCAPs. In addition, it was found that depletion of SNRNP200 up-regulated p21 and p53, and down-regulated the CDK1, CyclinB, CyclinE and CDK2. Conclusions Depletion of SNRNP200 repressed osteo−/dentinogenic differentiation potentials and restrained cell proliferation through blocking cell cycle progression at the G2/M and S phase, further revealing that SNRNP200 has crucial effects on preserving the proliferation and differentiation potentials of dental tissue-derived MSCs.

scholarly journals Characterization and expression analysis of four members genes of flavanone 3-hydroxylase families from Chamaemelum nobile

2020 ◽  
Vol 48 (1) ◽  
pp. 102-115
Author(s):  
Shuiyuan CHENG ◽  
Qiling SONG ◽  
Tian YU ◽  
Xiaomeng LIU ◽  
Lanlan WANG ◽  
...  
Keyword(s):  
Binding Sites ◽  
Bioinformatics Analysis ◽  
Expression Patterns ◽  
Secondary Structures ◽  
High Similarity ◽  
Expression Levels ◽  
Traditional Chinese Herbal Medicine ◽  
Chinese Herbal ◽  
Significant Difference ◽  
Chamaemelum Nobile

Chamaemelum nobile is a traditional Chinese herbal medicine, whose secondary metabolites used in the pharmacology of Chinese medicine. Among them, the flavonoids have great research value. Flavanone 3-hydroxylase (F3H) is one of the core enzymes in the early steps of flavonoid biosynthesis. This study aimed to elucidate the structures, functions, and expression levels of F3H families from C. nobile. Four members of the F3H family were screened from C. nobile transcriptome data and performed bioinformatics analysis. Results showed that CnF3H1~4 had a high similarity with the other F3H plants, and all genes contained two conserved isopenicillin N synthase-like and oxoglutarate/iron-dependent dioxygenase domains. Further analysis revealed that the four CnF3H proteins contained some differences in binding sites. The results of secondary and 3-D structures displayed that the composition and proportion of the four CnF3H secondary structures were basically the same, and their 3D structures were consistent with the secondary structures. The phylogenetic tree displayed that CnF3H2, CnF3H3, and CnF3H4 were grouped with Asteraceae. The expression patterns of CnF3Hs in the roots, stems, leaves, and flowers of C. nobile were evaluated using the value of RPKM. The results indicated that CnF3Hs had significant difference in the expression of different tissues. Especially, CnF3H1~3 and CnF3H4 had the highest expression levels in the flowers and roots, respectively. Hence, CnF3Hs played a significant role in the flavonoid metabolism.

scholarly journals The Aqueous Extract of Eucommia Leaves Promotes Proliferation, Differentiation, and Mineralization of Osteoblast-Like MC3T3-E1 Cells

2021 ◽  
Vol 2021 ◽  
pp. 1-12
Author(s):  
Mengqi Guan ◽  
Daian Pan ◽  
Mei Zhang ◽  
Xiangyang Leng ◽  
Baojin Yao
Keyword(s):  
Cell Proliferation ◽  
Aqueous Extract ◽  
High Performance ◽  
Bone Development ◽  
Expression Level ◽  
Functional Health ◽  
Health Food ◽  
Promoting Effect ◽  
Alizarin Red ◽  
Gene Markers

Eucommia leaves are dry leaves of Eucommia ulmoides which have long been considered as a functional health food for the treatment of hypertension, hypercholesterolemia, fatty liver, and osteoporosis. With the recent development of Chinese medicine, Eucommia leaves are widely used for tonifying the kidneys and strengthening bone. However, the specific molecular mechanism of Eucommia leaves for strengthening bone remains largely unknown. Osteoblasts are the main functional cells of bone formation; thus, it is essential to study the effect of Eucommia leaves on osteoblasts to better understand their mechanism of action. In the present study, we prepared an aqueous extract of Eucommia leaves (ELAE) and determined its content by high-performance liquid chromatography (HPLC). The effects of ELAE on MC3T3-E1 cells were investigated by CCK-8 assay, alkaline phosphatase (ALP), and Alizarin red S staining assays, combined with RNA sequencing (RNA-seq) and qRT-PCR validation. We demonstrated that ELAE had a significant promoting effect on the proliferation of MC3T3-E1 cells and significantly enhanced extracellular matrix synthesis and mineralization, which were achieved by regulating various functional genes and related signaling pathways. ELAE significantly increased the expression level of genes promoting cell proliferation, such as Rpl10a, Adnp, Pex1, Inpp4a, Frat2, and Pcdhga1, and reduced the expression level of genes inhibiting cell proliferation, such as Npm1, Eif3e, Cbx3, Psmc6, Fgf7, Fxr1, Ddx3x, Mbnl1, and Cdc27. In addition, ELAE increased the expression level of gene markers in osteoblasts, such as Col5a2, Ubap2l, Dkk3, Foxm1, Col16a1, Col12a1, Usp7, Col4a6, Runx2, Sox4, and Bmp4. Taken together, our results suggest that ELAE could promote osteoblast proliferation, differentiation, and mineralization and prevent osteoblast apoptosis. These findings not only increase our understanding of ELAE on the regulation of bone development but also provide a possible strategy to further study the prevention and treatment of osteogenic related diseases by ELAE.

Inhibition of the Mammalian Target of Rapamycin Pathway Stimulates Osteoblast Proliferation and Differentiation Through Autophagy in MC3T3-E1 Cells

2020 ◽  
Author(s):  
Xining Li ◽  
Xi-Ning Li ◽  
Yu Zhao ◽  
Zhi-Gang Zhou ◽  
Hong-Chang Zhou ◽  
...  
Keyword(s):  
Alkaline Phosphatase ◽  
Mammalian Target Of Rapamycin ◽  
Mtor Pathway ◽  
Target Of Rapamycin ◽  
Differentiation Markers ◽  
Alizarin Red ◽  
Sequestosome 1 ◽  
Alkaline Phosphatase Staining ◽  
Proliferation And Differentiation ◽  
Ribosomal S6 Kinase

Abstract Background: OP(Osteoporosis) is a common bone metabolic disorder in the elderly characterized by loss of bone mass and a tendency to fracture. The mammalian target of rapamycin (mTOR) pathway in autophagy plays an indispensable role in maintaining the stability of the intracellular environment and ensuring the normal physiological functions of cells. Methods: In this study, different concentrations(20, 40, 60, 80, 100, 120, 140, 160, 180 and 200nM) of rapamycin were used to act on MC3T3-E1 osteoblasts for different time lengths(6, 12, 24, 36 and 48 hours). CCK8 was used to detect the proliferative activity of cells and screen suitable rapamycin concentration for subsequent experiments. Western blot and real-time quantitative PCR were used to detect the expression changes of phosphorylated mTOR, upstream and downstream mTOR pathway, autophagy and osteogenic differentiation markers. The expression of LC3 was observed by immunofluorescence. The differentiation ability of osteoblasts was observed by alizarin red and alkaline phosphatase staining.Results: The results showed that the induction of proliferation activity of osteoblasts from 20 nM to 200 nM presented a parabolic feature. After the action time of 50 μM rapamycin exceeded 12 hours, the proportion of S stage cells was significantly increased. The results of gene and protein analysis showed that rapamycin significantly inhibited the phosphorylation of mTOR, and the phosphorylation of the downstream factors of mTOR, 4E-BP1(eIF4E-binding protein 1) and S6K1(p70 ribosomal S6 kinase 1) also decreased. Rapamycin significantly increased the expression of LC3 II (microtubule associated protein 1 light chain 3-α), significantly increased the ratio of LC3II/LC3I, and significantly decreased the expression of p62(sequestosome-1). Rapamycin significantly induced the expression of ALP(Alkaline phosphatase), Runx2(Runt-related transcription factor 2) and osterix. Conclusions: This study confirmed that rapamycin stimulates the autophagy of osteoblasts by inhibiting mTOR and promotes their proliferation and differentiation, suggesting that mTOR may be a potential therapeutic target for osteoporosis.

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