Icariin promotes the proliferation and differentiation of osteoblasts from the rat mandible by the Wnt/β‑catenin signalling pathway

Aims This study aimed to investigate the effect of solute carrier family 20 member 2 ( SLC20A2) gene mutation (identified from a hereditary multiple exostoses family) on chondrocyte proliferation and differentiation. Methods ATDC5 chondrocytes were cultured in insulin-transferrin-selenium medium to induce differentiation. Cells were transfected with pcDNA3.0 plasmids with either a wild-type (WT) or mutated (MUT) SLC20A2 gene. The inorganic phosphate (Pi) concentration in the medium of cells was determined. The expression of markers of chondrocyte proliferation and differentiation, the Indian hedgehog (Ihh), and parathyroid hormone-related protein (PTHrP) pathway were evaluated by quantitative real-time polymerase chain reaction (qRT-PCR) and western blotting. Results The expression of SLC20A2 in MUT group was similar to WT group. The Pi concentration in the medium of cells in MUT group was significantly higher than WT group, which meant the SLC20A2 mutation inhibited Pi uptake in ATDC5 chondrocytes. The proliferation rate of ATDC5 chondrocytes in MUT group was greater than WT group. The expression of aggrecan (Acan), α-1 chain of type II collagen (COL2A1), and SRY-box transcription factor 9 (SOX9) were higher in MUT group than WT group. However, the expression of Runt-related transcription factor 2 (Runx2), α-1 chain of type X collagen (COL10A1), and matrix metallopeptidase 13 (MMP13) was significantly decreased in the MUT group. Similar results were obtained by Alcian blue and Alizarin red staining. The expression of Ihh and PTHrP in MUT group was higher than WT group. An inhibitor (cyclopamine) of Ihh/PTHrP signalling pathway inhibited the proliferation and restored the differentiation of chondrocytes in MUT group. Conclusion A mutation in SLC20A2 (c.C1948T) decreases Pi uptake in ATDC5 chondrocytes. SLC20A2 mutation promotes chondrocyte proliferation while inhibiting chondrocyte differentiation. The Ihh/PTHrP signalling pathway may play an important role in this process. Cite this article: Bone Joint Res 2020;9(11):751–760.

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Neonatal Anesthesia by Ketamine in Rats Interferes with Proliferation and Differentiation of Hippocampal Neural Stem Cells and Neurocognitive Function in Adulthood via Inhibition of Notch1 Signalling Pathway

10.21203/rs.3.rs-178207/v1 ◽

2021 ◽

Author(s):

He Huang ◽

Chao Zhao ◽

Qian Hu ◽

Qiang Liu ◽

Yi-Man Sun ◽

...

Keyword(s):

Stem Cells ◽

Water Maze ◽

Notch Signalling ◽

Signalling Pathway ◽

Morris Water Maze Test ◽

Notch Signalling Pathway ◽

Maze Test ◽

Proliferation And Differentiation ◽

Old Rats ◽

Related Proteins

Abstract Objective: The proliferation and differentiation of developing neural stem cells (NSCs) have been particularly interesting targets to study ketamine-induced neurotoxicity. Our previous findings have shown that neonatal ketamine exposure inhibits the proliferation of NSCs in the hippocampal dentate gyrus (DG) and promotes neuronal differentiation. However, the potential mechanisms are poorly understood. Notch signalling pathway plays an important role in the regulation of neurogenesis. The objective of this study was to investigate whether Notch signalling pathway was involved in neurogenesis impairment and long-term neurocognitive dysfunction caused by neonatal ketamine exposure. Methods: Postnatal day 7 (PND-7) male Sprague-Dawley (SD) rats were intraperitoneally injected with normal saline (NS) or 40 mg/kg ketamine for four consecutive times (40 mg/kg×4) at an interval of 1 h. Notch ligand Jagged1 (0.5 mg/kg) was micro-injected into the hippocampal DG with the stereotactic apparatus at 1 h before NS or ketamine administration. Lentivirus over-expressing Notch1 intracellular domain (LV-NICD1) was micro-injected into the hippocampal DG 4 days before NS or ketamine administration. Western blot was used to detect the changes of Notch1 signalling pathway related proteins in the hippocampal DG at 24 h after administration. The S-phase marker 5-bromodeoxyuridine (BrdU) was administered immediately after the treatment, then the proliferation and differentiation of NSCs in hippocampal DG were detected by using double-immunofluorescence staining at 24 h after treatment. Moreover, the changes of hippocampus-dependent spatial memory in the adult rats were tested by Morris water maze test in 2-month-old rats. Results: Ketamine anesthesia in neonatal rats decreased the expression levels of Jagged1, Notch1, Notch1 intracellular domain (NICD1) and hairy enhancer of split 1 (Hes1), and inhibited the proliferation and astrocytic differentiation of NSCs and promoted neuronal differentiation. Neonatal ketamine exposure induced the deficit in hippocampus-dependent spatial reference memory tasks in 2-month-old rats. The micro-injection of Jagged1 or LV-NICD1 reversed the inhibitory effect of ketamine on the expression of Notch1 related proteins in the hippocampal DG, and attenuated the ketamine-induced interference of NSCs proliferation and differentiation. In addition, Morris water maze test suggested that the administration of Jagged1 or LV-NICD1 could improve cognitive function in 2-month-old rats after neonatal ketamine exposure. Conclusions: These results suggest that neonatal exposure to ketamine in rats interferes with the proliferation and differentiation of hippocampal NSCs and impairs neurocognitive function in adulthood via inhibition of Notch1 signalling pathway. These findings contribute to further understanding of the neonatal neurotoxicity induced by ketamine and its underlying mechanisms.

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Use of an Osteoblast Overload Damage Model to Probe the Effect of Icariin on the Proliferation, Differentiation and Mineralization of MC3T3-E1 Cells through the Wnt/β-Catenin Signalling Pathway

Cellular Physiology and Biochemistry ◽

10.1159/000470896 ◽

2017 ◽

Vol 41 (4) ◽

pp. 1605-1615 ◽

Cited By ~ 19

Author(s):

Yingjie Liu ◽

Lulu Huang ◽

Baohui Hao ◽

Hao Li ◽

Shuanglong Zhu ◽

...

Keyword(s):

Bone Cells ◽

Physiological Stress ◽

Damage Model ◽

Osteoblastic Differentiation ◽

Signalling Pathway ◽

Alizarin Red ◽

Protein Levels ◽

Bone Injury ◽

Gene And Protein Expression ◽

Proliferation And Differentiation

Background/Aims: Mechanical loading plays an important role in the regulation of bone mass. However, bone cells are not always under physiological stress. In some cases, bone tissue is subjected to an overloaded mechanical environment. For example, a person who is weight training and a stevedore often experience bone pain, inflammation and other bone fatigue damage symptoms. Icariin is the major ingredient of Herba epimedii, which has been widely used for the treatment of bone injury in traditional Chinese medicine, but its mechanism remains unknown. The aim of this study was to probe the effect of icariin on the proliferation and differentiation of osteoblasts exposed to overload and to determine whether the Wnt/β-catenin signalling pathway is involved in the drug response in osteoblasts. Methods: Mouse MC3T3-E1 cells were exposed to mechanical tensile strain using a four- point bending device to create an overload damage model. An MTT assay was performed to determine the effects of icariin on MC3T3-E1 cell proliferation. The mRNA and protein levels of ALP, COL-I, OCN, RUNX2 and β-catenin were assessed using RT-PCR and immunoblotting. The protein levels of β-catenin in the MC3T3-E1 cells were also determined using fluorescence microscopy. The mineralization of osteoblasts was assessed using Alizarin Red S staining. Results: We found that icariin enhanced the proliferation of osteoblasts exposed to overload and promoted MC3T3-E1 cell differentiation and mineralization. Furthermore, the gene and protein expression levels of β-catenin and RUNX2 all increased with icariin treatment compared with those in the damage group. Conclusion: Our study suggested that icariin promotes proliferation and differentiation in osteoblasts exposed to overload. The effect of icariin on osteoblastic differentiation acted by activating the RUNX2 promoter and the Wnt/β- catenin pathway.

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Effects of Acupuncture on Cortical Expression of Wnt3A, β-Catenin and Sox2 in a Rat Model of Traumatic Brain Injury

Acupuncture in Medicine ◽

10.1136/acupmed-2014-010742 ◽

2016 ◽

Vol 34 (1) ◽

pp. 48-54 ◽

Cited By ~ 10

Author(s):

Yi-min Zhang ◽

Qiu-fu Dai ◽

Wei-hao Chen ◽

Shu-ting Jiang ◽

Sheng-xin Chen ◽

...

Keyword(s):

Traumatic Brain Injury ◽

Brain Injury ◽

Signalling Pathway ◽

Mrna Levels ◽

Sprague Dawley ◽

Western Blots ◽

Sox2 Expression ◽

Sprague Dawley Rats ◽

Proliferation And Differentiation ◽

Free Drop

Objective To observe the effects of acupuncture treatment on the expression of Wnt/β-catenin signalling pathway-related genes (Wnt3a, β-catenin and Sox2) in the injured cerebral cortex of rats with traumatic brain injury (TBI). Methods A controlled impact model of TBI was established using Feeney's free-drop method. Seventy-eight Sprague-Dawley rats were randomly divided into the following three groups: a normal group (n=18) that was left untreated; a model group (n=30) that received no treatment after TBI; and an acupuncture group (n=30) that received acupuncture (at LI4, GV20, GV26 and GV16) after TBI. Rats in each group were randomly and equally divided into 3-day, 7-day and 14-day subgroups according to the duration of therapy. Real-time fluorescence quantitative PCR (RT-qPCR) was used to measure mRNA expression of Wnt3a, β-catenin and Sox2. Western blots were performed to determine the expression levels of WNT3a, β-Catenin and SOX2. Results Wnt3a mRNA was upregulated in the 7-day and 14-day acupuncture subgroups compared with the corresponding model subgroups (p<0.05). β-catenin expression was significantly increased in the 7-day and 14-day acupuncture subgroups compared with the corresponding model subgroups (p<0.01). In the 3-day and 7-day acupuncture subgroups, Sox2 expression was significantly higher than that in the normal and model groups (p<0.01 each). The levels of WNT3a, β-catenin and SOX2 were generally consistent with the corresponding mRNA levels. Conclusions Acupuncture exerts a regulatory effect on the Wnt/β-catenin signalling pathway, which may in turn influence the proliferation and differentiation of endogenous neural stem cells.

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