PI3K/AKT通路在促红细胞生成素调控钙磷陶瓷表面成骨中的作用

doi:10.3969/j.issn.1006-5725.2026.13.017

Abstract

Abstract:

Objective To explore the osteogenic efficacy and molecular mechanism of the EPO/pDA/BCP composite scaffold， this study focuses on verifying whether it promotes the osteogenic differentiation of bone marrow mesenchymal stem cells （BMSCs） and the repair of critical bone defects in vivo by activating the PI3K/AKT pathway and up-regulating MTSS1 and CYTL1. Methods The EPO/pDA/BCP composite material was fabricated by grafting dopamine and erythropoietin onto a biphasic calcium phosphate scaffold. In in-vitro experiments， four groups were established： the control group， the BCP group， the EPO/pDA/BCP group， and the EPO/pDA/BCP + LY294002 group with the addition of a PI3K/AKT pathway inhibitor. The cell proliferation and osteogenic differentiation capacity were assessed using the CCK-8 method， alkaline phosphatase activity assay， RT-qPCR， and Western blotting. The expression levels of the PI3K/AKT pathway and its downstream genes MTSS1 and CYTL1 were determined by RT-qPCR and Western blotting. In in-vivo experiments， a critical bone defect model of the femoral condyle in SD rats was created and randomly assigned to the blank group， the BCP group， and the EPO/pDA/BCP group for intervention. At 4 and 12 weeks after surgery， Micro-CT， hematoxylin-eosin staining， Masson staining， and immunohistochemical analysis were carried out to evaluate bone regeneration. Results The CCK-8 experiment verified that the EPO/pDA/BCP composite material exhibited excellent biocompatibility. The outcomes of the alkaline phosphatase activity assay， RT-qPCR， and Western blot demonstrated that， in comparison with the BCP group and the control group， the EPO/pDA/BCP group could notably promote the osteogenic differentiation of BMSCs （P 0.05）. Following the addition of the inhibitor LY294002， the osteogenic effect was markedly weakened， and the expression of molecules associated with the PI3K/AKT signaling pathway （p-PI3K， p-AKT， CYTL1， and MTSS1） was down-regulated （P 0.05）. Micro-CT scanning revealed that the new bone volume fraction and bone density in the bone defect area of the EPO/pDA/BCP group were significantly higher than those of the blank group and the BCP group （P 0.05）. Histological staining results suggested that the bone tissue regeneration in the EPO/pDA/BCP group was more comprehensive and mature than that in the other two groups. Immunohistochemical staining further indicated that the positive staining intensities of the osteogenic markers bone morphogenetic protein-2 and osteocalcin in the bone defect area of the EPO/pDA/BCP group were significantly higher than those of the BCP group and the blank control group. Conclusion The EPO/pDA/BCP bioceramic exhibits excellent osteogenic properties both in vitro and in vivo， and this effect might be associated with the activation of the expression of PI3K/AKT signaling pathway proteins.

Key words: biphasic calcium phosphate, erythropoietin, bone marrow mesenchymal stem cells, osteogenic differentiation, PI3K/AKT signaling pathway

CLC Number:

R318

Xuefei XIANG,Heyou GAO,Zhiyun CHEN,Qingchuang HU,Liu HE,Yu WANG. Study on the role of the PI3K/AKT pathway in the regulation of osteogenesis on the surface of calcium phosphate ceramics by erythropoietin[J]. The Journal of Practical Medicine, 2026, 42(13): 2404-2418.

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基因	引物序列（5′-3′）
Runx2	正义链：AAG GAG CAG ACC GTC AAA GG 反义链：CCC AGA GTC CAC CTC TCT GA
Osterix	正义链：GCA TCT GAA AGC CCA CTT GC 反义链：AGT GGT CGC TTC GGG TAA AG
OPN	正义链：GAT GAC GAC GAC GAT GAC GA 反义链：GCT GGC AGT GAA GGA CTC AT
CYTL1	正义链：CAC CGT CCT CCA GAG TGA AC 反义链：CGG ACC CAG TTT GGA TGG AA
MTSS1	正义链；GCG GTT TTC TCT GAC CCA AG 反义链：CCC CAA GTC CCA ATC CGA AA
GAPDH	正义链：GAC ATG CCG CCT GGA GAA AC 反义链：AGC CCA GGA TGC CCT TTA GT

组别	第1天	第3天	第7天
空白组	0.28 ± 0.03	0.47 ± 0.06	1.02 ± 0.07
BCP组	0.32 ± 0.01	0.47 ± 0.06	0.99 ± 0.06
EPO/pDA/BCP组	0.27 ± 0.01	0.41 ± 0.01	1.04 ± 0.04

组别	第3天	第7天
空白组	0.02 ± 0.00	0.05 ± 0.00
BCP组	0.04 ± 0.01^*	0.07 ± 0.00^*
EPO/pDA/BCP组	0.06 ± 0.01^*#	0.08 ± 0.00^*#
EPO/pDA/BCP+LY组	0.04 ± 0.00^△	0.05 ± 0.01^△

组别	p-PI3K/PI3K	p-AKT/AKT	CYTL1	MTSS1
空白组	0.29 ± 0.13	0.24 ± 0.07	0.46 ± 0.03	0.41 ± 0.08
BCP组	0.47 ± 0.07	0.48 ± 0.06	0.48 ± 0.04	0.51 ± 0.10
EPO/pDA/BCP组	0.85 ± 0.09^*#	0.80 ± 0.03^*#	0.61 ± 0.05^*#	0.71 ± 0.05^*#
EPO/pDA/BCP+LY组	0.49 ± 0.13^△	0.39 ± 0.12^△	0.39 ± 0.05^△	0.40 ± 0.06^△

组别	CYTL1	MTSS1
空白组	1.01 ± 0.17	1.00 ± 0.15
BCP组	2.50 ± 0.55	2.56 ± 0.41
EPO/pDA/BCP组	4.10 ± 0.75^*#	3.34 ± 0.12^*#
EPO/pDA/BCP+LY组	2.11 ± 0.12^△	2.21 ± 0.11^△

Study on the role of the PI3K/AKT pathway in the regulation of osteogenesis on the surface of calcium phosphate ceramics by erythropoietin

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组别	Runx2	Osterix	OPN
空白组	0.27 ± 0.07	0.38 ± 0.01	0.41 ± 0.05
BCP组	0.49 ± 0.08*	0.58 ± 0.06^*	0.51 ± 0.02
EPO/pDA/BCP组	0.83 ± 0.12^*#	0.80 ± 0.11^*#	0.75 ± 0.08^*#
EPO/pDA/BCP+LY组	0.45 ± 0.02^△	0.54 ± 0.07^△	0.49 ± 0.13^△

组别	Runx2	Osterix	OPN
空白组	1.01 ± 0.18	1.00 ± 0.09	1.01 ± 0.15
BCP组	3.36 ± 0.26^*	3.05 ± 0.11^*	1.35 ± 0.11^*
EPO/pDA/BCP组	4.87 ± 1.02^*#	4.15 ± 0.33^*#	1.84 ± 0.16^*#
EPO/pDA/BCP+LY组	2.34 ± 0.38^△	1.34 ± 0.19^△	1.05 ± 0.03^△

组别	骨体积分数/%	骨小梁厚度/mm	骨密度/（mg/cm³）	骨小梁数量/（1/mm）
空白组（4周）	25.15 ± 8.02	0.30 ± 0.06	162.0 ± 60.67	0.42 ± 0.03
BCP组（4周）	45.90 ± 1.06^*	0.49 ± 0.03^*	312.72 ± 25.24^*	0.59 ± 0.04^*
EPO/pDA/BCP组（4周）	56.09 ± 3.55^*#	0.69 ± 0.07^*#	423.73 ± 11.54^*#	0.75 ± 0.09^*#
空白组（12周）	30.20 ± 3.31	0.40 ± 0.06	238.85 ± 29.61	0.54 ± 0.03
BCP组（12周）	53.31 ± 1.57^*	0.55 ± 0.02^*	389.68 ± 28.29^*	0.80 ± 0.06^*
EPO/pDA/BCP组（12周）	72.83 ± 1.70^*#	0.80 ± 0.07^*#	631.11 ± 51.07^*#	0.96 ± 0.06^*#

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