The role of a novel marine protein LeAlkB in BMSCs osteogenic differentiation with mechanical stress

The role of a novel marine protein LeAlkB in BMSCs osteogenic differentiation with mechanical stress | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article The role of a novel marine protein LeAlkB in BMSCs osteogenic differentiation with mechanical stress Qihang Zhao, Zhiheng Ren, Zhengxuan Hu, Zhenggang Chen, Xiaomeng Zhao, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5335118/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Le AlkB is a marine protein derived from the Lysobacter enzymogenes . It has a dependence on Fe 2+ and α- Ketoglutaric acid. The ALKBH family of methylases, which are functionally conserved for ketoglutarate, have a similar structure. FTO（Fat Mass and Obesity-associated Protein） in the ALKBH family is a key promoter of osteogenic differentiation under mechanical stress and has an upregulation effect on inducing osteogenic differentiation phenotype markers. Research has found that ALKBH5 also has osteogenic ability, and the application of marine drugs in bone disease and bone regeneration research is gradually increasing. The structure similarly to the ALKBH family enables Le AlkB to have the same osteogenic differentiation ability. To explore the potential application of the novel marine protein Le AlkB in bone reconstruction, this study investigated the bone regeneration characteristics induced by Le AlkB. It has been demonstrated that mechanical stress encourages bone marrow mesenchymal stem cells (BMSCs) to differentiate into osteogenic tissue. This study discovered that BMSCs' expression of osteogenic differentiation markers in Le AlkB dramatically increased under mechanical stress settings. Furthermore, Le AlkB significantly raises the expression of FTO. This suggests that BMSCs' osteogenic differentiation can be stimulated by the marine protein Le AlkB. Le AlkB is a marine new protein that exhibits much greater osteogenic ability and minimal cytotoxicity when compared to FTO and ALKBH5. This suggests that Le AlkB has better therapeutic potential. Le AlkB is therefore anticipated to be used in the clinical treatment of orthodontics in the future. Our study's conclusions, however, offer fresh concepts for the potential uses of marine naturally occurring bioactive compounds in the future. Biological sciences/Biochemistry/Proteins Biological sciences/Biochemistry/Structural biology Bone marrow mesenchymal stem cells osteogenic differentiation marine protein osteogenic related factors mechanical stress Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Highlights The structural similarity between the novel marine protein Le AlkB and ALKBH5 and FTO, both of which possess osteogenic properties, is highly pronounced. The novel marine protein Le AlkB exhibits no discernible impact on the viability of bone marrow mesenchymal stem cells and demonstrates negligible cytotoxicity towards cellular systems. When the novel marine protein Le AlkB enters bone marrow mesenchymal stem cells, it stimulates the production of osteogenic active factors. New marine protein Le AlkB as a potential strategy for treating osteoporosis. Introduction The human skeleton is a dynamic, self-repairing organ[1]. Continuous bone remodeling ensures that bone structure and function gradually recover over time. However, the self-healing capability of bones is limited and cannot repair large-scale injuries[2]. Traditional osteogenic methods, such as autologous bone transplantation, allogeneic bone transplantation, distraction osteogenesis, and artificial bone grafting materials, have significant drawbacks, including extensive damage and lengthy recovery periods[3]. As a result, the development of new osteogenic methods has become a research hotspot[4]. In clinical practice, orthodontic tooth movement is facilitated by the alveolar bone under mechanical force[5]. However, larger mechanical forces can sometimes lead to abnormal absorption of the alveolar bone, resulting in defects. Currently, no mature osteogenesis method can completely address this issue[6]. Finding a new osteogenic promoter and studying its molecular mechanism for promoting osteogenic differentiation is of great clinical significance. It can help reduce the side effects of mechanical force and improve the stability of orthodontic treatment[7]. As is well known, marine microbiota is one of the largest known microbial communities[8]. Natural active substances from marine microorganisms have been used to develop low-toxicity and environmentally friendly bone regeneration materials[9]. These substances offer high biocompatibility, good biodegradability, and non-toxicity. In recent years, active substances such as collagen, chitosan, alginate, and oyster shell have been made into osteogenic polymer, hydrogels, and micro/nanospheres for bone tissue engineering and osteogenesis research[2]. Bone implantation is currently a key technology for addressing clinical problems in orthopedics, oral surgery, neurosurgery, and maxillofacial surgery[10]. However, the regeneration of bone defects hinders the rapid development of bone transplantation technology, which has become a significant clinical health issue[11]. Research has shown that existing active substances and polymers with osteogenic potential are mainly limited to traditional terrestrial sources, such as bone meal from cadaver donors[12]. Our current research has found that the marine protein Le AlkB may induce osteogenic differentiation, presenting a promising new avenue for bone regeneration[13]. Le AlkB is a demethylating protein expressed by a marine lysozyme gene isolated from the coast of Qingdao. The enzymatic bacteria belong to the family Xanthomonadaceae, a significant class of plant biocontrol bacteria with multiple drug resistances. These bacteria produce various extracellular enzymes and antibacterial secondary metabolites[14]. Literature reports indicate that Escherichia coli and its AlkB homologs significantly impact human diseases. Research has shown that Escherichia coli AlkB, homologous to the ALKBH superfamily, shares the same structure as ALKBH2 and ALKBH3 and exhibits functions similar to ALKBH2[15]. Based on these findings, we speculate that the AlkB protein derived from YC36 may have similar functions, leading us to hypothesize that it could become an effective drug target for treating bone defects[16]. Our research found that the protein sequence of Le AlkB is highly similar to that of the mammalian ALKBH protein family. The ALKBH gene family encodes nine homologous demethylases (ALKBH1-8 and FTO)[17]. Among them, ALKBH2-4 has been associated with the onset of cancer, while ALKBH6-7 has been linked to protein repair in humans[18]. Additionally, ALKBH1, ALKBH5, and FTO have been confirmed to be associated with osteogenic differentiation. Compared with other enzymes in the family, ALKBH1 can degrade various substrates and has a wide range of substrate specificity[19]. Liu et al. demonstrated that knocking down ALKBH1 inhibits the osteogenic differentiation of human mesenchymal stem cells[19]. Furthermore, Sun et al. found that FTO promotes the osteogenic differentiation of rat BMSCs under mechanical force conditions, with osteogenic differentiation performance decreasing in the absence of mechanical force[16]. Research has shown that ALKBH5 and FTO are also involved in regulating osteogenic differentiation, although there is controversy over whether ALKBH5 and FTO play positive or negative roles in this process[20]. Given the high similarity between Le AlkB and the ALKBH family, it is plausible that Le AlkB may have a similar role in promoting osteogenic differentiation. Therefore, this study explores the role of the novel marine protein Le AlkB in osteogenesis under mechanical force. We overexpressed Le AlkB in BMSCs to observe the expression of osteogenic factors. Our research aims to contribute to the development of osteogenic drugs and the application of marine resources in medical science. Results 3.1. Protein Le AlkB 3.1.1 The CDS (coding sequences) of Le alkB is: ATGCCGCCGACCCGCCTGCCGCTGCAAGACGCCGACCTGGCCTACGACCCGCACTGGCTCGACGCCGACGCGGCCGATGCGCTGTACGCG GCGGTGCTGGCGCAGGTCGCGTGGGAAGTGCACCGCATCCGCCTGTTCGGGCGCGAGCACGATTCGCCGCGGCTGAGCAGTTGGATCGGC GACCCCGACGCGCGCTATCGCTATTCCGGCGCCGAGTTCCGCCCGCAGCCGTGGCCGCCGGCGCTGCGGCCGGTGCGCGAACGGCTCGC GCGCGAACTCGGCGCGGCGTTCAACAGCGTGCTGGCCAACCGCTACCGCGACGGCCGCGACGCGATGGGCTGGCACAGCGACGACGAACC CGAGCTCGGCCCGGCGCCGGTCATCGCCTCGCTGAGCCTGGGCGCGCGGCGGCGCTTCGCGCTCAAGCACCGGCGCGATCCGTCGTTGAA GGCGGCGCTGGAACTGGGCCATGGCAGCTTGTTGGTGATGTCCGGGCCGACCCAGGCGAATTACCGACATGCGCTGCCGCGGACCGCGCGG CCGGTGGGGGAGCGGATCAATCTGACGTTCCGGGTGATTGCGCCGCGGCGGTAA The amino acid sequence of Le AlkB protein is: MPPTRLPLQDADLAYDPHWLDADALYAAVAQVAWEVHRIRLFGREHDSPRLSSWIGDPDARYSGAEFRPQPWPPALRPVRERLARELGAAFNSV LANRYRDGRDAMGWHSDDEPELGPAPVIASLSLGARRRFALKHRRDPSLKAALELGHGSLLVMPTQANYRHALPRTARPVGERINLTFRVIAPRR An annotated AlkB protein was found in the genome of the marine-derived strain Le YC36. This protein was sequentially compared in the UniProtKB reference proteomes plus Swiss-Prot database (E-Threshold=0.0001). The results show that it has a relatively lower similarity to other prokaryotic proteins of the AlkB superfamily that have been studied. The most similar prokaryotic AlkB protein reported is from E. coli (Fig 1C). In order to distinguish this AlkB protein from other proteins in the AlkB superfamily, we named it Le AlkB. Of all the reported proteins, the top eight proteins with the highest sequence similarity to Le AlkB belong to the mammalian ALKBH family. It has been proved that ALKBH1, ALKBH5 and FTO in the ALKBH family can promote osteogenic differentiation (Fig 1E). Pymol was used to fit the protein structure. The fitting results show that Le AlkB has a high spatial structure fitting degree with the proteins E. coli-alkB (Fig 1C), ALKBH2 (Fig 1A), ALKBH5(Fig 1B) and FTO (Fig 1D). This may have given Le AlkB a similar function to the three proteins above in promoting osteogenic differentiation. Therefore, we heterogeneously expressed Le AlkB in BMSCs and explored its osteogenic function. Comparing the protein Le AlkB and E . coli alkB (PDB id: 4rfr) in PyMOL, with an RMSD of 4.919, they have similar three-dimensional structures, except for the two reversed ones near the 51st amino acid position ( Le alkB) β Layer by layer, E.coli alkb is a circular structure. Comparing the structure of Le AlkB and ALKBH2 (PDB id: 3rzh) in PyMOL, RMSD=0.728, and ALKBH2 is the protein with the most similar structure to Le AlkB among the studied proteins. Structural comparison of Le AlkB and ALKBH5 (PDB id: 4nrm) was performed in PyMOL, with RMSD=5.537. The two structures are relatively similar, except that the N-terminus of ALKBH5 is one α Spiral, while Le Alkb has no obvious secondary structure. Comparing the structures of Le AlkB and FTO (PDB id: 3lfm) in PyMOL, the RMSD is 2.970. The main structural domains of the two are similar, but the C-terminus of FTO has an additional one composed of multiple domains α Spiral domain. 3.2 Heterologous expression of Le AlkB in BMSCs We used the overexpression technique of Le alkB MBSC mechanical stress group genes to investigate the effect of ALKBH on osteogenic differentiation. Three groups were established for control experiments, including untreated wild-type BMSC (WT) group, BMSC carrying empty plasmids (MS-EP) group, and BMSC overexpressing Le AlkB group (MS- Le AlkB). After in vitro culture and incubation, the morphology and quantity of the four groups of cells were uniform (Figure 2A, B). To more clearly demonstrate the effect of Le AlkB on osteogenic differentiation of BMSCs, we used heterologous overexpression technique to verify whether it is stably expressed in BMSCs. Firstly, the q-PCR absolute quantitative experimental results also confirmed the expression of Le alkB in BMSCs (Figure 2C). Then, the Le alkB gene and fluorescent marker gene were connected together, inserted into the pCDNA3.1 plasmid, and transfected into mouse bone marrow stromal stem cells. If the protein is successfully expressed in BMSC cells, the cells will display green fluorescence under excitation light irradiation. The fluorescence staining results indicate that Le alkB can be stably expressed in bone marrow stromal stem cells (Figure 2D, E). Fig1: A: 3D protein structure alignment of ALKBH2 and Le AlkB; B: 3D protein structure alignment of ALKBH5 and Le AlkB; C : 3D protein structure alignment of AlkB- E .coli and Le AlkB; D : 3D protein structure alignment of FTO and Le AlkB; E : Neighbor-joining phylogenetic tree based on amino acid sequences showing the phylogenetic position of the Le alkB and other alkB family proteins. Bootstrap values (>50%) after 1000 simulations are shown at branch nodes. Fig 2: A : Experimental grouping: From top left to bottom right, there are WT group, MS group, MS-EP group, and MS- Le AlkB group; B : Observe whether the four groups have osteogenic ability; C : PCR absolute quantification experiment of Le AlkB D : Fluorescent labeling control group(MS-EP) (Magnification×20); E : Fluorescence labeling experimental group(MS- Le AlkB) (Magnification×20) Proteins are successfully expressed in BMSC cells, and cells exhibit green fluorescence under excitation light irradiation. 3.3 Cell apoptosis and survival rate experiment In order to investigate the effect of Le AlkB on the normal activity of BMSCs, we conducted survival rate experiments and CCK-8 cytotoxicity experiments. The initial cells and culture conditions of the three groups remained consistent. The apoptosis results of flow cytometry showed that overexpression of Le AlkB did not increase the apoptosis rate of BMSCs, indicating that the presence of Le AlkB does not affect the normal life of cells. The CCK-8 results showed that the cell survival rate of the Le AlkB overexpression group was slightly lower than that of the empty plasmid group (Figure 3 A, B, C ). In this experiment, the cell viability of the Le AlkB overexpression group was greater than 70% of the control group. Therefore, Le AlkB does not affect the normal life cycle of cells and is non-toxic to cells [25]. Fig 3: A : Cell survival rate measurement. (The cell viability of the MS-LeAlkB group was 70% higher than that of the control group).ns: No statistical significance; *: P ≤ 0.05; **: P ≤ 0.01; ***: P ≤ 0.001; ****: P ≤ 0.0001 B : Detection of cell growth rate using flow cytometry; ns: No statistical significance; *: P ≤ 0.05; **: P ≤ 0.01; ***: P ≤ 0.001; ****: P ≤ 0.0001 C : Scatter plot of relative cell viability 3.4 Le AlkB promotes mechanical stress-induced osteogenic differentiation of BMSCs After BMSCs entered the differentiation stage, the cell shape changed from irregular fusiform to a regular elongated shape. After in vitro culture, the cell morphology and number of 4 groups were uniform and showed a long spindle shape. Initial cell numbers and culture conditions were the same for all groups, except for mechanical stress. Mechanical stress was applied for three days except for the WT group. In addition to directly observing the changes in ALP activity, the ALP staining experiment can also roughly observe the changes in cell shape. The stress caused the cells to become more regular than the WT group, and the MS- Le AlkB group had obvious changes in cell shape, accompanied by a tight arrangement of cells (Fig4 A, B, C, D). ALP activity represents the osteogenic activity of BMSCs. In ALP staining experiments, darker colors indicate more naphthol produced by ALP catalysis and higher phosphatase activity. In Fig 4D, the color of the MS- Le AlkB group was clearly darker than that of the other three groups, indicating that the overexpression of Le AlkB might increase the ALP activity of the cells. To verify this hypothesis, we used an alkaline phosphatase (AKP/ALP) activity assay kit to detect ALP activity quantitatively. The results showed that the ALP activity of the MS- Le AlkB group was 2-fold higher than that of the MS and MS-EP groups, it was about 5-fold higher than that of the WT group. These indicated that the ALP activity of BMSCs was increased under mechanical stress, but the presence of Le AlkB could enhance the ALP activity to a greater extent. ALP activity is only one of the markers of BMSC osteogenic differentiation. To explore whether Le AlkB could actually catalyze osteogenic differentiation of BMSC, cDNA was extracted from four groups of cells for qPCR. The expression levels of osteogenic differentiation markers, RUNX2, BMP2, and ALP, were analyzed. Primers for qPCR of each gene are detailed in Supplementary Table 1. The results showed that the expression of the three markers in wild-type BMSCs was increased to varying degrees under mechanical stress. Compared to MS and MS-EP groups, the presence of Le AlkB resulted in upregulating these genes expression levels 2-3 times (Fig 4 E, F, G, J), the expression level of FTO has increased by 150 times(Fig 4 H). Taken together, it can be concluded that Le AlkB significantly promotes the osteogenic activity of BMSCs under stress conditions. Fig 4 : A : WT group ALP staining( multiple ✕ 20 ) ; B : MS group ALP staining ; C : MS-EP group ALP staining ; D : MS- Le AlkB group ALP staining (The group was uniformly stained with red, and the ALP activity was significantly enhanced, The arrow represents a significant increase in nuclear staining) ; E :ALP expression Level ; F : BMP2 expression Level ; G: RUNX2 expression Level ; H : FTO expression Level ; I : ALP enzyme activity test ; * : P ≤0.05;** : P ≤0.01; *** : P ≤0.001 Discussion Bone regeneration is a complex physiological process regulated by different factors. The proliferation, differentiation, survival and function of osteoblasts are regulated by a variety of extracellular factors, such as growth factors, cytokines and hormones[21]. The generation of osteoblasts results from the differentiation of mesenchymal stem cells (MSCs) into bone, and mechanical forces play an important role in regulating the behavior and function of MSCs[22]. For example, the mechanical load generated by exercise favors the development of bone density and strength. BMSCs can convert external mechanical stimuli into intracellular biochemical signals to promote osteogenic differentiation[23]. In other words, mechanical stimuli can be applied locally in the absence of specific biochemical stimuli, which provides the possibility of selectively altering cell differentiation[24]. Previous studies have found that cyclic tensile stress applied to rat BMSCs promotes osteogenic differentiation and found the optimal stress phase for bone formation. It is an established fact that BMSCs tend to differentiate into osteocytes under mechanical stimuli[25]. The mechanical stress induction technology for stem cells has become mature worldwide and can simulate the process of orthodontic tooth movement in clinical practice[23]. Therefore, a low-cost and effective drug can be sought to catalyze and accelerate the osteogenic differentiation process of stem cells under stress conditions. Mechanical stress refers to the internal reaction force caused by external forces applied to an object, while orthodontic force refers to the force used to correct the alignment and position of teeth. Orthodontic force is a specific type of mechanical stress applied to teeth and their surrounding tissues to cause tooth movement. In our study, we reproduced the role of orthodontic force in teeth and periodontal tissue by simulating mechanical stress. Cells sense this stress through mechanical receptors on the membrane, such as integrins and ion channels, and activate related signaling pathways, such as MAPK and Wnt. The activation of these signaling pathways leads to changes in intracellular gene expression, regulating cell behavior and function[26]. Therefore, mechanical stress is transformed into orthodontic forces that can promote osteogenic differentiation through cellular perception and signal transduction, explaining how mechanical stimuli guide biological responses and tissue reconstruction during orthodontic processes[27]. Previous studies have shown that certain proteins in the mammalian ALKBH protein family can promote osteogenic differentiation of stem cells[20]. We found a Le AlkB protein belonging to the AlkB superfamily from marine strain Le YC36. We loaded the gene sequence onto the plasmid pCDNA3.1 and transformed the recombinant into BMSC through liposome transfection. Results of qPCR and fluorescence staining indicated that Le AlkB could be expressed in BMSCs. Phenotypic markers of osteoblasts including collagen matrix accumulation, ALP expression and bone nodule mineralization were observed during in vitro differentiation[19]. Our results showed that overexpression of Le AlkB led to deepening of ALP staining, increased ALP enzyme activity, and increased expression of osteogenic marker genes RUNX2, BMP2, and ALP. Therefore, these results indicated that the marine protein Le AlkB stimulated osteogenic differentiation under mechanical stress. In addition, the presence of Le AlkB also increased the expression level of FTO in cells. FTO is a controversial protein in the ALKBH family involved in osteogenic differentiation[16]. Feng et al. have demonstrated that FTO promoted osteogenic differentiation by reducing the inhibitory effect of glucocorticoids and improving the RNA stability of osteogenic marker genes[28]. Meanwhile, Son et al. have also confirmed the promoting effect of FTO on osteogenic differentiation and indicated that overexpression of FTO could increase the expression level of osteogenic marker genes such as Runx2[29]. However, Wang et al. believed that overexpression of FTO could reduce the mRNA methylation level of Runx2, thereby inhibiting osteogenic differentiation, which is inevitably accompanied by a decrease in Runx2 gene expression[29]. We are of the opinion that the different functions exhibited by FTO in osteogenic differentiation may be because FTO, as a gene inherent in BMSCs, is situated within a vast eukaryotic gene regulatory network. In this network, FTO is involved in multiple regulatory pathways with different effects on osteogenic differentiation[30]. However, in various studies, both positive and negative regulatory relationships have been discussed regarding the relationship between FTO and Runx2 expression levels[31]. Moreover, Runx2 positively regulates osteogenic differentiation. In our study, overexpression of Le AlkB increased FTO expression, accompanied by a significant increase in Runx2 and other marker genes’ expression. It indicates that Le AlkB can promote the expression of FTO genes and activate the FTO-mediated regulatory pathway for promoting osteogenic differentiation, maximizing the promotion of osteogenic differentiation[28]. The above results may prove that Le AlkB is a potential peptide drug promoting osteogenesis. In previous drug research, some peptides have also been used as candidate drugs. The first peptide drug, insulin, can be traced back a century[32]. At present, various diseases such as cancer, multiple sclerosis, and even osteoporosis is treated with peptide drugs as the first choice in clinical practice[33]. Chen et al. also reported in 2021 that terlipide, a parathyroid hormone (PTH) analog containing the first 34 amino acids of endogenous hormones, has shown significant efficacy in the treatment of osteoporosis[34]. Le AlkB, as a prokaryotic peptide derived from the ocean, has the advantages of low toxicity and low cost, making it a highly feasible candidate drug for osteoporosis. In addition, autologous bone marrow transplantation contains substances that promote bone formation such as growth factors, cytokines, and stem cells, which can promote new bone formation and repair defects[35]. Autologous bone marrow transplantation can be combined with marine-derived peptides to further accelerate the repair process of bone defects[36]. Based on the above facts, we propose a vision for the future. This article demonstrates that the marine protein Le AlkB has strong osteogenic potential. Therefore, it is worth exploring and researching whether the protein can be optimized and mutated to form better marine drugs for the clinical treatment of osteoporosis or orthodontic processes. The pharmaceutical potential of Le AlkB is technically feasible. According to ISO 10993-5:2009[17], in the cytotoxicity test, a cell survival rate higher than 70% in the control group can be considered non-toxic to cells (Figure 2C, D).The experiments on cell apoptosis and survival rate have shown that Le AlkB has no toxic effect on BMSCs, which provides possibilities for the medicinal research of Le AlkB[37]. Protein sequence alignment and three-dimensional structural analysis revealed that Le AlkB shares the same conserved residues (such as H115, D117, 172R, 177) with human ALKBH family proteins, and their spatial positions are similar. It will help optimize the activity of Le AlkB through amino acid-directed mutagenesis in the future, making it more suitable for clinical osteogenic research. Walker et al. demonstrated that site-specific mutations in the ALKBH family can improve or even inhibit cancer development[18]. Optimizing the amino acid sites and protein structure of Le AlkB may also give it potential in the treatment of other diseases. In addition, the technical methods required to study Le AlkB, derived from marine prokaryotic microorganisms, are relatively mature. Compared with eukaryotic expression, prokaryotic expression systems have bright application prospects which have been used for the large-scale production of recombinant proteins, such as drugs, vaccines, and antibodies. It has the advantages of high expression level, low cost, simple operation, and environmental protection, providing a new approach for future clinical development[17]. Therefore, compared to the ALKBH family proteins from eukaryotic cells that can catalyze osteogenesis, the prokaryotic protein Le AlkB is more suitable for research on pharmacology and new materials. Finally, our study on the marine protein Le AlkB provides a new research formula for the application research of other similar marine proteins. We appeal to researchers to actively search for bioactive drugs more suitable for osteogenic differentiation from the ocean treasure trove and are looking forward to truly solving the problem of osteogenic drug deficiency in orthodontic treatment or diseases such as osteoporosis in the future. Materials And Methods 5.1 Bacterial strains, plasmids, and general methods The strain of Le YC36, which produces enzymatic lytic bacteria, originated from the coastal area of Qingdao, China. The Le YC36 used in this experiment was donated by the Marine Life Science Laboratory of Ocean University of China, and the samples were stored in the laboratory of Ocean University of China. Cultivate Le YC36 strain in 40% trypsin soy broth (TSB) medium. DH5a cells were purchased from AngYu Biotechnology Co., Ltd (Shanghai, China) for plasmid amplification and protein purification. The PcDNA3.1 (Supplementary Material Figure S3) plasmid was purchased from Yunnan Luoyu Biotechnology Co., Ltd(Yunnan, China).Primary rat bone marrow stromal stem cells were purchased from Procter&Gamble CP-M131. 5.2. Structural comparison and biological prediction of Le AlkB In this experiment, computer-based structural prediction is essential for inferring the biological function of the Le AlkB protein. To solve the prediction issue, we employed the discovered structures in the protein database (PDB) as templates to construct structural models. The alignment of Le AlkB protein sequences in the Uniprot database was shown, and protein sequences with over 35% similarity (approximately 1000 sequences) were selected for detailed analysis. The Le AlkB protein sequence was indexed and compared in the UniProtKB reference protein plus Swiss protein database (E-Threshold=0.0001). We obtained 1000 similar sequences with a similarity greater than 35%, but they have not been studied yet. Therefore, 54 representative sequences (similarity ≥ 55%) were selected as the proximal sequences of Le AlkB for systematic analysis (in the green part of the evolutionary tree). At the same time, the AlkB protein was searched in the protein database (PDB), and homology modeling was performed using AlphaFold software. Select the PDB file from the AlphaFol database for RMSD calculation. We imported the PDB files of Le AlkB, FTO, ALKBH2, ALKBH5, and Escherichia coli ALKB proteins into Pymol and selected Plugin. → Align, parameters (Method: Align; Period: 5; Deadline: 2.0). Then, the truncated 56 sequences were aligned using clusterX software, and an evolutionary tree was established using adjacency method. It is known that the human self-produced enzyme ALKBH5 is numbered 4NRM in the protein database (PDB), and after analysis, it is found that the protein sequence similarity with Le AlkB is 16.27%. Then, the PDB file of ALKBH5 was imported into PyMOL software for comparison with FTO and Le AlkB. 5.3 Construction of plasmid pCDNA3.1 -eGFP-MCS- Le AlkB and heat shock conversion method Amplify the target fragment using AlkB-F/R primers containing cleavage sites (Supplementary Material S1) and obtain the PCR product of the Le alkB gene. Restrictive endonucleases Hind III and BamH I are used to digest amplified products and plasmids pCDNA3.1-eGFP-MCS. In the experiment, the plasmid pCDNA3.1-e GFP-MCS (Supplementary Material Figure S4) was added to a culture dish containing sensing cells and subjected to heat shock treatment for plasmid transfection. Take out 200 μ L suspension from the freezer at -70 ° C and immediately place it on ice after thawing. Add plasmid DNA solution (content not exceeding 50ng, volume not exceeding 10 μ L). Place it on ice and let it stand for 30 minutes. Add 1mL of LB liquid culture medium (excluding Amp) to the test tube. Mix thoroughly and shake at 37 ° C for 1 hour to restore normal bacterial growth and express the plasmid encoded antibiotic resistance gene (Ampr). Shake the bacterial solution thoroughly and place 100 μ L of Amp containing solution facing upwards on the filter plate for half an hour. The plasmid pCDNA3.1-eGFP-MCS-LealkB was introduced into Escherichia coli DH5 using heat shock transformation method. Leave the bacterial solution overnight for 16 hours, then centrifuge at 8000rpm for 10 minutes. Collect bacterial cells and resuspend with binding buffer. Then use an ultrasonic crusher (300W, crush for 3 seconds, pause for 5 seconds) to crush the cells until the bacterial solution is clear and transparent. Centrifuge the broken bacterial solution at 10000rpm for 10 minutes and filter the supernatant using a 0.22 μ m filter membrane. Subsequently, protein purification was performed using nickel columns. Obtain purified protein Le AlkB (Supplementary Materials Figure S1, S2). 5.4 Liposome transfected cells BMSC cells were obtained from Cytogen Biosciences Co., Ltd. (Yunnan, China). Subsequently, the cells were seeded onto a 6-well stress plate (flex, project number BF-3001C) and allowed to adhere to the surface for approximately 48 hours, reaching a logarithmic growth stage with a fusion degree of approximately 70%. Afterwards, replace the medium with Opti MEM medium (2mL per well) and incubate for 3 hours to allow adaptation. Once adaptation is complete, replace the culture medium again and add 1.99mL of Opti MEM medium per well. Add 0.8μg plasmid and 1.6μL lipo3000 to Opti MEM, shake well, and let stand for 5 minutes. Granular CDNA3.1 and transfection agent lipo3000. After adding each plate, gently stir 2-3 times by shaking, and then place it in a CO2 incubator for further cultivation for 72 hours. Subsequently, discard the culture medium, wash the wells twice with PBS solution, and then add 200 μL of Trizol reagent to each well. Collect cells using Calcium chloride reagents for subsequent analysis, including q-PCR based detection of alkb mRNA expression levels and evaluation of transfection efficiency and stress load treatment based on experimental grouping. 5.5 Cell survival rate experiment A cell counting kit (CCK-8) was used to test the survival rate of cells. Bone marrow mesenchymal stem cells of different groups were placed into a 24-well plate. 10 5 cells were inoculated into each well and cultured for 1, 3 and 5days. The culture medium was removed and 300 µL of CCK-8 working solution was added. It was incubated at a constant temperature of 37°C in darknes for 2h. The supernatant was transferred to a 96-well plate. An enzyme-linked immunosorbent assay was used to measure its absorbance at 450 nm. 5.6 Flow cytometry apoptosis experiment When the cells revive and grow up to 80%-90% in the 25T culture bottle, the supernatant was discard and gently washed twice with PBS. And then, 2 mL trypsin was added for digestion, and another 2 mL of complete culture medium was added. The mixture was blowed evenly and transfered to a 15 mL centrifuge tube, incubating at room temperature and dark for 5 min. After that, 5 µL of propidium iodide solution (PI) and 400 µL of PBS were added into it, and then performed the flow cytometry detection at once. Finally, To reach the results, the flow cytometry (Beckman Kurt International Trade (Shanghai) Co. Ltd, Shanghai, China) was utilized for detection and data preservation. 5.7 Application of cyclic mechanical stress Cells were collected and inoculated onto six wells of wear-resistant silicone rubber Bio Flex ™ Coating plate with type I rat tail collagen (Flexcell International Corp, Hillsborough, USA), then initial density of 5×10 5 good growth medium was replaced by basic medium without serum until reaching 70% consistency. After 24 h, the 1Hz sine curve of cells subjected to cyclic mechanical strain which set to 5% elongation was obtained from FX-4000 tons™ Flexcell Tension Plus™ Unit (Flexcell Inter-national Corp), 6 h per day for a total of 3 days. 5.8 ALP vitality determination Using alkaline phosphatase (ALP) activity assay kit (Nanjing Jiancheng Biotechnology Research Institute, A059-2, Nanjing, China). 5.9 ALP staining experiment After gently rinsing bone cells three times by using the ALP staining kit (P0321S, Shanghai Beyotime Biotechnology Co., Ltd.), fix the cells with 4% paraformaldehyde, then stain the cells with the ALP staining kit, and finally scan the stained cells with a scanner to obtain ALP staining images. 5.10 Real time quantitative q-PCR Query target gene mRNA sequences of corresponding species on NCBI（ https://www.ncbi.nlm.nih.gov/ ）And design primers using CDS sequences (Supplementary Material Table 1). Extract total RNA, measure RNA concentration, and calculate the volume of total RNA required in RT using the following formula: volume of total RNA required in reverse transcription=2 µ g/measured RNA concentration. Follow the instructions of the FastKing RT Kit (With gDNase) to synthesize the first strand of cDNA. After obtaining the first strand, the target gene was amplified by Q-PCR. Taq Pro Universal SYBR qPCR Master Mix 5 µ l, upstream primer 0.25 µ l, downstream primer 0.25 µ l, cDNA template 1.0 µ l, Nuclease Free Water 3.5 µ l were added to each well in the order of loading, until the final reaction volume was 10 µ l. Put it into the LightCycle instrument, set the PCR cycle reaction conditions, complete the reaction, collect information, and perform Ct value analysis. 5.11 Statistical analysis The data is presented as mean ± SD and analyzed using GraphPad Prism9 (GraphPad Software, USA). Use ANONA to evaluate differences between groups. P -value less than 0.05 is considered statistically significant. Conclusion In summary, this study indicates that the marine protein LeAlkB is safe for BMSCs. LeYC36, which has a wide range of sources, is an enzyme producing bacterium isolated from the coast of Qingdao, with simple processing and low cost. Through bioinformatics analysis, the novel demethylated protein LeAlkB in this study has a similar structure and function to FTO protein, and its osteogenic performance is superior. This indicates that the marine protein LeAlkB may become a new source of future bone formation. The specific process of this study is shown in the following figure (Figure 5). Other potentially valuable components need to be studied in future research. For example, further research and improvement are needed on how to apply it to clinical practice to bring this sustainable osteogenic product to the market. Abbreviations Le AlkB, newly discovered marine bacterial proteins; BMSC, bone marrow mesenchymal stem cells; FTO, Obesity related genes; ALKBH5, AlkB Homolog 5,RNA Demethylase; ALKBH2, AlkB Homolog 2,RNA Demethylase; ALP, Alkaline Phosphatase; BMP2, Bone morphogenetic protein 2; RUNX2, Runt Related Transcripyion Factor 2. Declarations Funding: This study was funded by the General Project of the Natural Science Foundation of Shandong Province, China (ZR2023MH155), the Natural Science Foundation of Qingdao City, Shandong Province, China (23-2-198-zyyd-jch), and the Science and Technology Plan of Shinan District,Qingdao Shandong Province, China (2022-4-003-YY) Grant funding Author Contribution This study was designed by Q.G. The isolation, extraction, and genome sequencing of bacteria were carried out by Z.R. Bioinformatics methods, data analysis, and numbering were designed and implemented by Q.Z, while cell staining experiments were designed and implemented by F.Y and Z.H; The cytotoxicity experiment was conducted by Z.C, The cell PCR experiment was conducted by R.Y and X.Z. The initial draft was written by Q.Z. This study was funded and/or supervised by Q.G. All authors have read and agreed to the published version of the manuscript. Data Availability Data is provided within the manuscript or supplementary information files.The datasets used and/or analysed during the current study available from the corresponding author on reasonable request References Huang, H., et al., Modulation of T Cell Responses by Fucoidan to Inhibit Osteogenesis. Front Immunol, 2022. 13 : p. 911390. Carson, M.A. and S.A. Clarke, Bioactive Compounds from Marine Organisms: Potential for Bone Growth and Healing. Mar Drugs, 2018. 16 (9). Guo, Q., et al., Mechanical stimulation induced osteogenic differentiation of BMSCs through TWIST/E2A/p21 axis. Biosci Rep, 2020. 40 (5). Hardy, S., et al., Manzamine-A Alters In Vitro Calvarial Osteoblast Function. Mar Drugs, 2022. 20 (10). Yang, G.J., et al., The emerging role of KDM5A in human cancer. J Hematol Oncol, 2021. 14 (1): p. 30. Zhu, L., et al., T6SS translocates a micropeptide to suppress STING-mediated innate immunity by sequestering manganese. Proceedings of the National Academy of Sciences, 2021. 118 (42). Makarewich, C.A. and E.N. Olson, Mining for Micropeptides. Trends Cell Biol, 2017. 27 (9): p. 685-696. Baek, J.W., et al., Marine Plankton-Derived Whitlockite Powder-Based 3D-Printed Porous Scaffold for Bone Tissue Engineering. Materials (Basel), 2022. 15 (10). Bravo, A. and M. Soberon, Mining versus in vitro evolution for the selection of novel microbial insecticidal proteins. Microb Biotechnol, 2022. 15 (10): p. 2518-2520. Carnovali, M., et al., Aerophobin-1 from the Marine Sponge Aplysina aerophoba Modulates Osteogenesis in Zebrafish Larvae. Mar Drugs, 2022. 20 (2). Chen, X., et al., Regulatory Role of RNA N6-Methyladenosine Modification in Bone Biology and Osteoporosis. Frontiers in Endocrinology, 2020. 10 . Chen, X., et al., Enhanced bone regeneration via spatiotemporal and controlled delivery of a genetically engineered BMP-2 in a composite Hydrogel. Biomaterials, 2021. 277 : p. 121117. Cherigo, L., D. Lopez, and S. Martinez-Luis, Marine natural products as breast cancer resistance protein inhibitors. Mar Drugs, 2015. 13 (4): p. 2010-29. Derkach, S.R., et al., Properties of Protein Isolates from Marine Hydrobionts Obtained by Isoelectric Solubilisation/Precipitation: Influence of Temperature and Processing Time. Int J Mol Sci, 2022. 23 (22). Pilzys, T., et al., ALKBH overexpression in head and neck cancer: potential target for novel anticancer therapy. Sci Rep, 2019. 9 (1): p. 13249. Sun, R., et al., Demethylase FTO promotes mechanical stress induced osteogenic differentiation of BMSCs with up-regulation of HIF-1alpha. Mol Biol Rep, 2022. 49 (4): p. 2777-2784. Wang, S.W., et al., Current applications and future perspective of CRISPR/Cas9 gene editing in cancer. Mol Cancer, 2022. 21 (1): p. 57. Walker, A.R., et al., ALKBH7 Variant Related to Prostate Cancer Exhibits Altered Substrate Binding. PLoS Comput Biol, 2017. 13 (2): p. e1005345. Ouyang, L., et al., ALKBH1-demethylated DNA N6-methyladenine modification triggers vascular calcification via osteogenic reprogramming in chronic kidney disease. J Clin Invest, 2021. 131 (14). Li, Z., et al., The N(6)-methyladenosine demethylase ALKBH5 negatively regulates the osteogenic differentiation of mesenchymal stem cells through PRMT6. Cell Death Dis, 2021. 12 (6): p. 578. Ponzetti, M. and N. Rucci, Osteoblast Differentiation and Signaling: Established Concepts and Emerging Topics. Int J Mol Sci, 2021. 22 (13). Lv, S., et al., Expression of HMGB1 in the periodontal tissue subjected to orthodontic force application by Waldo's method in mice. J Mol Histol, 2015. 46 (1): p. 107-14. Yu, H., et al., Expression of HIF ‑1alpha in cycling stretch ‑induced osteogenic differentiation of bone mesenchymal stem cells. Mol Med Rep, 2019. 20 (5): p. 4489-4498. Childs, P.G., et al., Use of nanoscale mechanical stimulation for control and manipulation of cell behaviour. Acta Biomater, 2016. 34 : p. 159-168. Zhang, X., et al., Local delivery of insulin/IGF-1 for bone regeneration: carriers, strategies, and effects. Nanotheranostics, 2020. 4 (4): p. 242-255. Zhong, J., et al., Developing a new treatment for superficial fungal infection using antifungal Collagen-HSAF dressing. Bioeng Transl Med, 2022. 7 (3): p. e10304. Zhou, J., et al., Advanced glycation end products impair bone marrow mesenchymal stem cells osteogenesis in periodontitis with diabetes via FTO-mediated N(6)-methyladenosine modification of sclerostin. J Transl Med, 2023. 21 (1): p. 781. Feng, L., et al., RNA N6-methyladenosine demethylase FTO inhibits glucocorticoid-induced osteoblast differentiation and function in bone marrow mesenchymal stem cells. J Cell Biochem, 2023. 124 (11): p. 1835-1847. Son, H.E., et al., Fat Mass and Obesity-Associated (FTO) Stimulates Osteogenic Differentiation of C3H10T1/2 Cells by Inducing Mild Endoplasmic Reticulum Stress via a Positive Feedback Loop with p-AMPK. Mol Cells, 2020. 43 (1): p. 58-65. Huang, M., et al., m6A demethylase FTO and osteoporosis: potential therapeutic interventions. Front Cell Dev Biol, 2023. 11 : p. 1275475. . Lewis, G.F. and P.L. Brubaker, The discovery of insulin revisited: lessons for the modern era. J Clin Invest, 2021. 131 (1). Lee, Y.S., et al., Antiosteoporosis effects of a marine antimicrobial peptide pardaxin via regulation of the osteogenesis pathway. Peptides, 2022. 148 : p. 170686. Chen, T., et al., Parathyroid hormone and its related peptides in bone metabolism. Biochem Pharmacol, 2021. 192 : p. 114669. Hu, S., et al., Structural Characterization and Anti-Osteoporosis Effects of a Novel Sialoglycopeptide from Tuna Eggs. Mar Drugs, 2023. 21 (11). Chen, X., et al., Mesoporous Silica Promotes Osteogenesis of Human Adipose-Derived Stem Cells Identified by a High-Throughput Microfluidic Chip Assay. Pharmaceutics, 2022. 14 (12). Kim, J.T., et al., Safety evaluation and consideration of 4 Pin Multi-needle for meso-therapy. Technol Health Care, 2018. 26 (S1): p. 291-306. Additional Declarations No competing interests reported. Supplementary Files Supplementarymaterials.docx SupplementaryMaterialFlowApoptosis.xlsx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5335118","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":384989359,"identity":"93f724a0-7e80-41d5-872f-6a36fdac90d4","order_by":0,"name":"Qihang Zhao","email":"","orcid":"","institution":"Qingdao Municipal Hospital","correspondingAuthor":false,"prefix":"","firstName":"Qihang","middleName":"","lastName":"Zhao","suffix":""},{"id":384989361,"identity":"5a1f9b3c-287c-4164-b0e7-7af4927db8c8","order_by":1,"name":"Zhiheng Ren","email":"","orcid":"","institution":"Qingdao Municipal Hospital","correspondingAuthor":false,"prefix":"","firstName":"Zhiheng","middleName":"","lastName":"Ren","suffix":""},{"id":384989364,"identity":"cd671f02-9d39-4dc0-8772-75fb707159b9","order_by":2,"name":"Zhengxuan Hu","email":"","orcid":"","institution":"Qingdao Municipal Hospital","correspondingAuthor":false,"prefix":"","firstName":"Zhengxuan","middleName":"","lastName":"Hu","suffix":""},{"id":384989366,"identity":"03459d3a-fdb2-4844-a9cf-b1695742eba0","order_by":3,"name":"Zhenggang Chen","email":"","orcid":"","institution":"Qingdao Municipal Hospital","correspondingAuthor":false,"prefix":"","firstName":"Zhenggang","middleName":"","lastName":"Chen","suffix":""},{"id":384989368,"identity":"8d7f4659-5555-42d7-bc08-f9111a8ad658","order_by":4,"name":"Xiaomeng Zhao","email":"","orcid":"","institution":"Qingdao University","correspondingAuthor":false,"prefix":"","firstName":"Xiaomeng","middleName":"","lastName":"Zhao","suffix":""},{"id":384989369,"identity":"4cec41ee-1646-4407-9574-7e737767934d","order_by":5,"name":"Fang Yang","email":"","orcid":"","institution":"Qingdao Municipal Hospital","correspondingAuthor":false,"prefix":"","firstName":"Fang","middleName":"","lastName":"Yang","suffix":""},{"id":384989371,"identity":"baa828f7-4092-434e-9f95-d94552bfaf44","order_by":6,"name":"Rongtao Yuan","email":"","orcid":"","institution":"Qingdao Municipal Hospital","correspondingAuthor":false,"prefix":"","firstName":"Rongtao","middleName":"","lastName":"Yuan","suffix":""},{"id":384989372,"identity":"c9b34742-c326-49d7-9515-18d0a7890ad1","order_by":7,"name":"Qingyuan Guo","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA6UlEQVRIiWNgGAWjYLCCBBiDp0JCTp5ELWcsjA0bSLKOt60ikeEAAUXyEckHbzzcUZu44fjZgzfezpNIYGxgfvjoBh4thjfSki0SzxxP3HAmL9ly7jaJPHYGNmPjHHxaZuSYSSS2HUvccCDHTJp3m0QxYwMPmzRxWs6/AWqZI5HYcICAFnkJsJaaxA03QLY0EKHFgOcZ0C9tB4xn3nhjbDnnmISxYTMBv8i3Jx+8+bOtTrbvfI7hjTc1dXLy7M0PH+O15QADgwQDw2HHBgYwAwiY8SgH29IAVllnzwDXMgpGwSgYBaMADQAAj5xQvzkHzxkAAAAASUVORK5CYII=","orcid":"","institution":"Qingdao Municipal Hospital","correspondingAuthor":true,"prefix":"","firstName":"Qingyuan","middleName":"","lastName":"Guo","suffix":""}],"badges":[],"createdAt":"2024-10-26 02:23:06","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5335118/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5335118/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":70698044,"identity":"abdcdb01-2059-4bc4-8b02-efa396105710","added_by":"auto","created_at":"2024-12-05 18:01:44","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":193615,"visible":true,"origin":"","legend":"\u003cp\u003eA: 3D protein structure alignment of ALKBH2 and\u003cem\u003e Le\u003c/em\u003eAlkB; B: 3D protein structure alignment of ALKBH5 and\u003cem\u003e Le\u003c/em\u003eAlkB; C : 3D protein structure alignment of AlkB-\u003cem\u003eE\u003c/em\u003e.coli and \u003cem\u003eLe\u003c/em\u003eAlkB; D : 3D protein structure alignment of FTO and \u003cem\u003eLe\u003c/em\u003eAlkB; E : Neighbor-joining phylogenetic tree based on amino acid sequences showing the phylogenetic position of the \u003cem\u003eLe\u003c/em\u003ealkB and other alkB family proteins. Bootstrap values (\u0026gt;50%) after 1000 simulations are shown at branch nodes.\u003c/p\u003e","description":"","filename":"Picture1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5335118/v1/f1e7d0a48bc836f85edc35bc.jpg"},{"id":70697623,"identity":"85a67521-2f28-474a-b0ac-7123d7c506bc","added_by":"auto","created_at":"2024-12-05 17:53:52","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":56349,"visible":true,"origin":"","legend":"\u003cp\u003eA : Experimental grouping: From top left to bottom right, there are WT group, MS group, MS-EP group, and MS-\u003cem\u003eLe\u003c/em\u003eAlkB group; B : Observe whether the four groups have osteogenic ability; C : PCR absolute quantification experiment of\u003cem\u003e Le\u003c/em\u003eAlkB D : Fluorescent labeling control group(MS-EP) (Magnification×20); E : Fluorescence labeling experimental group(MS-\u003cem\u003eLe\u003c/em\u003eAlkB) (Magnification×20) Proteins are successfully expressed in BMSC cells, and cells exhibit green fluorescence under excitation light irradiation.\u003c/p\u003e","description":"","filename":"Picture2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5335118/v1/2430be9c3a18e6dd1b0ecd15.jpg"},{"id":70698048,"identity":"56bfa27e-622c-4c6e-b398-b3389bd4189d","added_by":"auto","created_at":"2024-12-05 18:01:46","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":75654,"visible":true,"origin":"","legend":"\u003cp\u003eA : Cell survival rate measurement. (The cell viability of the MS-LeAlkB group was 70% higher than that of the control group).ns: No statistical significance; *: P ≤ 0.05; **: P ≤ 0.01; ***: P ≤ 0.001; ****: P ≤ 0.0001 B : Detection of cell growth rate using flow cytometry; ns: No statistical significance; *: P ≤ 0.05; **: P ≤ 0.01; ***: P ≤ 0.001; ****: P ≤ 0.0001 C : Scatter plot of relative cell viability\u003c/p\u003e","description":"","filename":"Picture3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5335118/v1/898766eb9ab77cfed6e9973b.jpg"},{"id":70697542,"identity":"f4a7999e-613c-4b92-9548-ce6c36c77f4f","added_by":"auto","created_at":"2024-12-05 17:53:40","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":40592,"visible":true,"origin":"","legend":"\u003cp\u003eA : WT group ALP staining( multiple ✕ 20 ) ; B : MS group ALP staining ; C : MS-EP group ALP staining ; D : MS-\u003cem\u003eLe\u003c/em\u003eAlkB group ALP staining (The group was uniformly stained with red, and the ALP activity was significantly enhanced, The arrow represents a significant increase in nuclear staining) ; E :ALP expression Level ; F : BMP2 expression Level ; G: RUNX2 expression Level ; H : FTO expression Level ; I : ALP enzyme activity test ; * : \u003cem\u003eP\u003c/em\u003e≤0.05;** : \u003cem\u003eP\u003c/em\u003e≤0.01;\u003cem\u003e*** \u003c/em\u003e: \u003cem\u003eP\u003c/em\u003e≤0.001\u003c/p\u003e","description":"","filename":"Picture4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5335118/v1/4772bf833d4710de42fdb893.jpg"},{"id":70697546,"identity":"dc6636d6-e9d6-49f1-8330-d47149a16644","added_by":"auto","created_at":"2024-12-05 17:53:41","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":101981,"visible":true,"origin":"","legend":"\u003cp\u003eMechanism diagram\u003c/p\u003e","description":"","filename":"Picture5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5335118/v1/6c27b8ecd631939d6cb7e072.jpg"},{"id":87787767,"identity":"5e0df19e-dc85-4237-9416-91289e869fa3","added_by":"auto","created_at":"2025-07-29 04:32:22","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1193309,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5335118/v1/5247fc8a-95b2-4b4e-b968-1d9d0b455579.pdf"},{"id":70698386,"identity":"e983541b-5f20-4ead-af07-f2beb5983c9a","added_by":"auto","created_at":"2024-12-05 18:09:48","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":431243,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementarymaterials.docx","url":"https://assets-eu.researchsquare.com/files/rs-5335118/v1/2c9ef638fa5b049111c9bff9.docx"},{"id":70697557,"identity":"2111dd40-1570-4a3d-a6e7-6b275662f0f8","added_by":"auto","created_at":"2024-12-05 17:53:42","extension":"xlsx","order_by":12,"title":"","display":"","copyAsset":false,"role":"supplement","size":10119,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryMaterialFlowApoptosis.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-5335118/v1/960d408877a1b219e8ceeec7.xlsx"}],"financialInterests":"No competing interests reported.","formattedTitle":"The role of a novel marine protein LeAlkB in BMSCs osteogenic differentiation with mechanical stress","fulltext":[{"header":"Highlights","content":"\u003cul start=\"50\"\u003e\n \u003cli\u003eThe structural similarity between the novel marine protein \u003cem\u003eLe\u003c/em\u003eAlkB and ALKBH5 and FTO, both of which possess osteogenic properties, is highly pronounced.\u003c/li\u003e\n \u003cli\u003eThe novel marine protein \u003cem\u003eLe\u003c/em\u003eAlkB exhibits no discernible impact on the viability of bone marrow mesenchymal stem cells and demonstrates negligible cytotoxicity towards cellular systems.\u003c/li\u003e\n \u003cli\u003eWhen the novel marine protein \u003cem\u003eLe\u003c/em\u003eAlkB enters bone marrow mesenchymal stem cells, it stimulates the production of osteogenic active factors.\u003c/li\u003e\n \u003cli\u003eNew marine protein \u003cem\u003eLe\u003c/em\u003eAlkB as a potential strategy for treating osteoporosis.\u003c/li\u003e\n\u003c/ul\u003e"},{"header":"Introduction","content":"\u003cp\u003eThe human skeleton is a dynamic, self-repairing organ[1]. Continuous bone remodeling ensures that bone structure and function gradually recover over time. However, the self-healing capability of bones is limited and cannot repair large-scale injuries[2]. Traditional osteogenic methods, such as autologous bone transplantation, allogeneic bone transplantation, distraction osteogenesis, and artificial bone grafting materials, have significant drawbacks, including extensive damage and lengthy recovery periods[3]. As a result, the development of new osteogenic methods has become a research hotspot[4]. In clinical practice, orthodontic tooth movement is facilitated by the alveolar bone under mechanical force[5]. However, larger mechanical forces can sometimes lead to abnormal absorption of the alveolar bone, resulting in defects. Currently, no mature osteogenesis method can completely address this issue[6]. Finding a new osteogenic promoter and studying its molecular mechanism for promoting osteogenic differentiation is of great clinical significance. It can help reduce the side effects of mechanical force and improve the stability of orthodontic treatment[7].\u003c/p\u003e\n\u003cp\u003eAs is well known, marine microbiota is one of the largest known microbial communities[8]. Natural active substances from marine microorganisms have been used to develop low-toxicity and environmentally friendly bone regeneration materials[9]. These substances offer high biocompatibility, good biodegradability, and non-toxicity. In recent years, active substances such as collagen, chitosan, alginate, and oyster shell have been made into osteogenic polymer, hydrogels, and micro/nanospheres for bone tissue engineering and osteogenesis research[2]. Bone implantation is currently a key technology for addressing clinical problems in orthopedics, oral surgery, neurosurgery, and maxillofacial surgery[10]. However, the regeneration of bone defects hinders the rapid development of bone transplantation technology, which has become a significant clinical health issue[11]. Research has shown that existing active substances and polymers with osteogenic potential are mainly limited to traditional terrestrial sources, such as bone meal from cadaver donors[12]. Our current research has found that the marine protein \u003cem\u003eLe\u003c/em\u003eAlkB may induce osteogenic differentiation, presenting a promising new avenue for bone regeneration[13].\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eLe\u003c/em\u003eAlkB is a demethylating protein expressed by a marine lysozyme gene isolated from the coast of Qingdao. The enzymatic bacteria belong to the family Xanthomonadaceae, a significant class of plant biocontrol bacteria with multiple drug resistances. These bacteria produce various extracellular enzymes and antibacterial secondary metabolites[14]. Literature reports indicate that Escherichia coli and its AlkB homologs significantly impact human diseases. Research has shown that Escherichia coli AlkB, homologous to the ALKBH superfamily, shares the same structure as ALKBH2 and ALKBH3 and exhibits functions similar to ALKBH2[15]. Based on these findings, we speculate that the AlkB protein derived from YC36 may have similar functions, leading us to hypothesize that it could become an effective drug target for treating bone defects[16]. Our research found that the protein sequence of \u003cem\u003eLe\u003c/em\u003eAlkB is highly similar to that of the mammalian ALKBH protein family. The ALKBH gene family encodes nine homologous demethylases (ALKBH1-8 and FTO)[17]. Among them, ALKBH2-4 has been associated with the onset of cancer, while ALKBH6-7 has been linked to protein repair in humans[18]. Additionally, ALKBH1, ALKBH5, and FTO have been confirmed to be associated with osteogenic differentiation. Compared with other enzymes in the family, ALKBH1 can degrade various substrates and has a wide range of substrate specificity[19]. Liu et al. demonstrated that knocking down ALKBH1 inhibits the osteogenic differentiation of human mesenchymal stem cells[19]. Furthermore, Sun et al. found that FTO promotes the osteogenic differentiation of rat BMSCs under mechanical force conditions, with osteogenic differentiation performance decreasing in the absence of mechanical force[16]. Research has shown that ALKBH5 and FTO are also involved in regulating osteogenic differentiation, although there is controversy over whether ALKBH5 and FTO play positive or negative roles in this process[20]. Given the high similarity between\u003cem\u003e\u0026nbsp;Le\u003c/em\u003eAlkB and the ALKBH family, it is plausible that \u003cem\u003eLe\u003c/em\u003eAlkB may have a similar role in promoting osteogenic differentiation.\u003c/p\u003e\n\u003cp\u003eTherefore, this study explores the role of the novel marine protein \u003cem\u003eLe\u003c/em\u003eAlkB in osteogenesis under mechanical force. We overexpressed \u003cem\u003eLe\u003c/em\u003eAlkB in BMSCs to observe the expression of osteogenic factors. Our research aims to contribute to the development of osteogenic drugs and the application of marine resources in medical science.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e3.1. Protein \u003cem\u003eLe\u003c/em\u003eAlkB\u003c/p\u003e\n\u003cp\u003e3.1.1 The CDS (coding sequences) of \u003cem\u003eLe\u003c/em\u003ealkB is:\u003c/p\u003e\n\u003cp\u003eATGCCGCCGACCCGCCTGCCGCTGCAAGACGCCGACCTGGCCTACGACCCGCACTGGCTCGACGCCGACGCGGCCGATGCGCTGTACGCG GCGGTGCTGGCGCAGGTCGCGTGGGAAGTGCACCGCATCCGCCTGTTCGGGCGCGAGCACGATTCGCCGCGGCTGAGCAGTTGGATCGGC GACCCCGACGCGCGCTATCGCTATTCCGGCGCCGAGTTCCGCCCGCAGCCGTGGCCGCCGGCGCTGCGGCCGGTGCGCGAACGGCTCGC GCGCGAACTCGGCGCGGCGTTCAACAGCGTGCTGGCCAACCGCTACCGCGACGGCCGCGACGCGATGGGCTGGCACAGCGACGACGAACC CGAGCTCGGCCCGGCGCCGGTCATCGCCTCGCTGAGCCTGGGCGCGCGGCGGCGCTTCGCGCTCAAGCACCGGCGCGATCCGTCGTTGAA GGCGGCGCTGGAACTGGGCCATGGCAGCTTGTTGGTGATGTCCGGGCCGACCCAGGCGAATTACCGACATGCGCTGCCGCGGACCGCGCGG CCGGTGGGGGAGCGGATCAATCTGACGTTCCGGGTGATTGCGCCGCGGCGGTAA\u003c/p\u003e\n\u003cp\u003eThe amino acid sequence of \u003cem\u003eLe\u003c/em\u003eAlkB protein is:\u003c/p\u003e\n\u003cp\u003eMPPTRLPLQDADLAYDPHWLDADALYAAVAQVAWEVHRIRLFGREHDSPRLSSWIGDPDARYSGAEFRPQPWPPALRPVRERLARELGAAFNSV LANRYRDGRDAMGWHSDDEPELGPAPVIASLSLGARRRFALKHRRDPSLKAALELGHGSLLVMPTQANYRHALPRTARPVGERINLTFRVIAPRR\u003c/p\u003e\n\u003cp\u003eAn annotated AlkB protein was found in the genome of the marine-derived strain \u003cem\u003eLe\u003c/em\u003eYC36. This protein was sequentially compared in the UniProtKB reference proteomes plus Swiss-Prot database (E-Threshold=0.0001). The results show that it has a relatively lower similarity to other prokaryotic proteins of the AlkB superfamily that have been studied. The most similar prokaryotic AlkB protein reported is from \u003cem\u003eE. coli\u003c/em\u003e (Fig 1C). In order to distinguish this AlkB protein from other proteins in the AlkB superfamily, we named it \u003cem\u003eLe\u003c/em\u003eAlkB. Of all the reported proteins, the top eight proteins with the highest sequence similarity to \u003cem\u003eLe\u003c/em\u003eAlkB belong to the mammalian ALKBH family. It has been proved that ALKBH1, ALKBH5 and FTO in the ALKBH family can promote osteogenic differentiation (Fig 1E). Pymol was used to fit the protein structure. The fitting results show that \u003cem\u003eLe\u003c/em\u003eAlkB has a high spatial structure fitting degree with the proteins \u003cem\u003eE. coli-alkB\u003c/em\u003e (Fig 1C), ALKBH2 (Fig 1A), ALKBH5(Fig 1B) and FTO (Fig 1D). This may have given \u003cem\u003eLe\u003c/em\u003eAlkB a similar function to the three proteins above in promoting osteogenic differentiation. Therefore, we heterogeneously expressed \u003cem\u003eLe\u003c/em\u003eAlkB in BMSCs and explored its osteogenic function.\u003c/p\u003e\n\u003cp\u003eComparing the protein\u003cem\u003e Le\u003c/em\u003eAlkB and \u003cem\u003eE\u003c/em\u003e. coli alkB (PDB id: 4rfr) in PyMOL, with an RMSD of 4.919, they have similar three-dimensional structures, except for the two reversed ones near the 51st amino acid position (\u003cem\u003eLe\u003c/em\u003ealkB) \u0026beta; Layer by layer, \u003cem\u003eE.coli\u003c/em\u003e alkb is a circular structure. Comparing the structure of\u003cem\u003e Le\u003c/em\u003eAlkB and ALKBH2 (PDB id: 3rzh) in PyMOL, RMSD=0.728, and ALKBH2 is the protein with the most similar structure to \u003cem\u003eLe\u003c/em\u003eAlkB among the studied proteins. Structural comparison of \u003cem\u003eLe\u003c/em\u003eAlkB and ALKBH5 (PDB id: 4nrm) was performed in PyMOL, with RMSD=5.537. The two structures are relatively similar, except that the N-terminus of ALKBH5 is one \u0026alpha; Spiral, while \u003cem\u003eLe\u003c/em\u003eAlkb has no obvious secondary structure. Comparing the structures of\u003cem\u003e Le\u003c/em\u003eAlkB and FTO (PDB id: 3lfm) in PyMOL, the RMSD is 2.970. The main structural domains of the two are similar, but the C-terminus of FTO has an additional one composed of multiple domains \u0026alpha; Spiral domain.\u003c/p\u003e\n\u003cp\u003e3.2 Heterologous expression of \u003cem\u003eLe\u003c/em\u003eAlkB in BMSCs\u003c/p\u003e\n\u003cp\u003eWe used the overexpression technique of \u003cem\u003eLe\u003c/em\u003ealkB MBSC mechanical stress group genes to investigate the effect of ALKBH on osteogenic differentiation. Three groups were established for control experiments, including untreated wild-type BMSC (WT) group, BMSC carrying empty plasmids (MS-EP) group, and BMSC overexpressing \u003cem\u003eLe\u003c/em\u003eAlkB group (MS-\u003cem\u003eLe\u003c/em\u003eAlkB). After in vitro culture and incubation, the morphology and quantity of the four groups of cells were uniform (Figure 2A, B). To more clearly demonstrate the effect of \u003cem\u003eLe\u003c/em\u003eAlkB on osteogenic differentiation of BMSCs, we used heterologous overexpression technique to verify whether it is stably expressed in BMSCs. Firstly, the q-PCR absolute quantitative experimental results also confirmed the expression of \u003cem\u003eLe\u003c/em\u003ealkB in BMSCs (Figure 2C). Then, the \u003cem\u003eLe\u003c/em\u003ealkB gene and fluorescent marker gene were connected together, inserted into the pCDNA3.1 plasmid, and transfected into mouse bone marrow stromal stem cells. If the protein is successfully expressed in BMSC cells, the cells will display green fluorescence under excitation light irradiation. The fluorescence staining results indicate that \u003cem\u003eLe\u003c/em\u003ealkB can be stably expressed in bone marrow stromal stem cells (Figure 2D, E).\u003c/p\u003e\n\u003cp\u003eFig1: A: 3D protein structure alignment of ALKBH2 and\u003cem\u003e Le\u003c/em\u003eAlkB; B: 3D protein structure alignment of ALKBH5 and\u003cem\u003e Le\u003c/em\u003eAlkB; C : 3D protein structure alignment of AlkB-\u003cem\u003eE\u003c/em\u003e.coli and \u003cem\u003eLe\u003c/em\u003eAlkB; D : 3D protein structure alignment of FTO and \u003cem\u003eLe\u003c/em\u003eAlkB; E : Neighbor-joining phylogenetic tree based on amino acid sequences showing the phylogenetic position of the \u003cem\u003eLe\u003c/em\u003ealkB and other alkB family proteins. Bootstrap values (\u0026gt;50%) after 1000 simulations are shown at branch nodes.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFig 2: \u003c/strong\u003eA : Experimental grouping: From top left to bottom right, there are WT group, MS group, MS-EP group, and MS-\u003cem\u003eLe\u003c/em\u003eAlkB group; B : Observe whether the four groups have osteogenic ability; C : PCR absolute quantification experiment of\u003cem\u003e Le\u003c/em\u003eAlkB D : Fluorescent labeling control group(MS-EP) (Magnification\u0026times;20); E : Fluorescence labeling experimental group(MS-\u003cem\u003eLe\u003c/em\u003eAlkB) (Magnification\u0026times;20) Proteins are successfully expressed in BMSC cells, and cells exhibit green fluorescence under excitation light irradiation.\u003c/p\u003e\n\u003cp\u003e3.3 Cell apoptosis and survival rate experiment\u003c/p\u003e\n\u003cp\u003eIn order to investigate the effect of\u003cem\u003e Le\u003c/em\u003eAlkB on the normal activity of BMSCs, we conducted survival rate experiments and CCK-8 cytotoxicity experiments. The initial cells and culture conditions of the three groups remained consistent. The apoptosis results of flow cytometry showed that overexpression of \u003cem\u003eLe\u003c/em\u003eAlkB did not increase the apoptosis rate of BMSCs, indicating that the presence of \u003cem\u003eLe\u003c/em\u003eAlkB does not affect the normal life of cells. The CCK-8 results showed that the cell survival rate of the \u003cem\u003eLe\u003c/em\u003eAlkB overexpression group was slightly lower than that of the empty plasmid group (Figure 3 A, B, C ). In this experiment, the cell viability of the \u003cem\u003eLe\u003c/em\u003eAlkB overexpression group was greater than 70% of the control group. Therefore, \u003cem\u003eLe\u003c/em\u003eAlkB does not affect the normal life cycle of cells and is non-toxic to cells [25].\u003c/p\u003e\n\u003cp\u003eFig 3: A : Cell survival rate measurement. (The cell viability of the MS-LeAlkB group was 70% higher than that of the control group).ns: No statistical significance; *: P \u0026le; 0.05; **: P \u0026le; 0.01; ***: P \u0026le; 0.001; ****: P \u0026le; 0.0001 B : Detection of cell growth rate using flow cytometry; ns: No statistical significance; *: P \u0026le; 0.05; **: P \u0026le; 0.01; ***: P \u0026le; 0.001; ****: P \u0026le; 0.0001 C : Scatter plot of relative cell viability\u003c/p\u003e\n\u003cp\u003e3.4 \u003cem\u003eLe\u003c/em\u003eAlkB promotes mechanical stress-induced osteogenic differentiation of BMSCs\u003c/p\u003e\n\u003cp\u003eAfter BMSCs entered the differentiation stage, the cell shape changed from irregular fusiform to a regular elongated shape. After in vitro culture, the cell morphology and number of 4 groups were uniform and showed a long spindle shape. Initial cell numbers and culture conditions were the same for all groups, except for mechanical stress. Mechanical stress was applied for three days except for the WT group. In addition to directly observing the changes in ALP activity, the ALP staining experiment can also roughly observe the changes in cell shape. The stress caused the cells to become more regular than the WT group, and the MS-\u003cem\u003eLe\u003c/em\u003eAlkB group had obvious changes in cell shape, accompanied by a tight arrangement of cells (Fig4 A, B, C, D). ALP activity represents the osteogenic activity of BMSCs. In ALP staining experiments, darker colors indicate more naphthol produced by ALP catalysis and higher phosphatase activity. In Fig 4D, the color of the MS-\u003cem\u003eLe\u003c/em\u003eAlkB group was clearly darker than that of the other three groups, indicating that the overexpression of\u003cem\u003e Le\u003c/em\u003eAlkB might increase the ALP activity of the cells. To verify this hypothesis, we used an alkaline phosphatase (AKP/ALP) activity assay kit to detect ALP activity quantitatively. The results showed that the ALP activity of the MS-\u003cem\u003eLe\u003c/em\u003eAlkB group was 2-fold higher than that of the MS and MS-EP groups, it was about 5-fold higher than that of the WT group. These indicated that the ALP activity of BMSCs was increased under mechanical stress, but the presence of \u003cem\u003eLe\u003c/em\u003eAlkB could enhance the ALP activity to a greater extent. ALP activity is only one of the markers of BMSC osteogenic differentiation. To explore whether \u003cem\u003eLe\u003c/em\u003eAlkB could actually catalyze osteogenic differentiation of BMSC, cDNA was extracted from four groups of cells for qPCR. The expression levels of osteogenic differentiation markers, RUNX2, BMP2, and ALP, were analyzed. Primers for qPCR of each gene are detailed in Supplementary Table 1. The results showed that the expression of the three markers in wild-type BMSCs was increased to varying degrees under mechanical stress. Compared to MS and MS-EP groups, the presence of \u003cem\u003eLe\u003c/em\u003eAlkB resulted in upregulating these genes expression levels 2-3 times (Fig 4 E, F, G, J), the expression level of FTO has increased by 150 times(Fig 4 H). Taken together, it can be concluded that \u003cem\u003eLe\u003c/em\u003eAlkB significantly promotes the osteogenic activity of BMSCs under stress conditions.\u003c/p\u003e\n\u003cp\u003eFig 4 : A : WT group ALP staining( multiple ✕ 20 ) ; B : MS group ALP staining ; C : MS-EP group ALP staining ; D : MS-\u003cem\u003eLe\u003c/em\u003eAlkB group ALP staining (The group was uniformly stained with red, and the ALP activity was significantly enhanced, The arrow represents a significant increase in nuclear staining) ; E :ALP expression Level ; F : BMP2 expression Level ; G: RUNX2 expression Level ; H : FTO expression Level ; I : ALP enzyme activity test ; * : \u003cem\u003eP\u003c/em\u003e\u0026le;0.05;** : \u003cem\u003eP\u003c/em\u003e\u0026le;0.01;\u003cem\u003e*** \u003c/em\u003e: \u003cem\u003eP\u003c/em\u003e\u0026le;0.001\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eBone regeneration is a complex physiological process regulated by different factors. The proliferation, differentiation, survival and function of osteoblasts are regulated by a variety of extracellular factors, such as growth factors, cytokines and hormones[21]. The generation of osteoblasts results from the differentiation of mesenchymal stem cells (MSCs) into bone, and mechanical forces play an important role in regulating the behavior and function of MSCs[22]. For example, the mechanical load generated by exercise favors the development of bone density and strength. BMSCs can convert external mechanical stimuli into intracellular biochemical signals to promote osteogenic differentiation[23]. In other words, mechanical stimuli can be applied locally in the absence of specific biochemical stimuli, which provides the possibility of selectively altering cell differentiation[24]. Previous studies have found that cyclic tensile stress applied to rat BMSCs promotes osteogenic differentiation and found the optimal stress phase for bone formation. It is an established fact that BMSCs tend to differentiate into osteocytes under mechanical stimuli[25]. The mechanical stress induction technology for stem cells has become mature worldwide and can simulate the process of orthodontic tooth movement in clinical practice[23]. Therefore, a low-cost and effective drug can be sought to catalyze and accelerate the osteogenic differentiation process of stem cells under stress conditions. Mechanical stress refers to the internal reaction force caused by external forces applied to an object, while orthodontic force refers to the force used to correct the alignment and position of teeth. Orthodontic force is a specific type of mechanical stress applied to teeth and their surrounding tissues to cause tooth movement. In our study, we reproduced the role of orthodontic force in teeth and periodontal tissue by simulating mechanical stress. Cells sense this stress through mechanical receptors on the membrane, such as integrins and ion channels, and activate related signaling pathways, such as MAPK and Wnt. The activation of these signaling pathways leads to changes in intracellular gene expression, regulating cell behavior and function[26]. Therefore, mechanical stress is transformed into orthodontic forces that can promote osteogenic differentiation through cellular perception and signal transduction, explaining how mechanical stimuli guide biological responses and tissue reconstruction during orthodontic processes[27].\u003c/p\u003e\n\u003cp\u003ePrevious studies have shown that certain proteins in the mammalian ALKBH protein family can promote osteogenic differentiation of stem cells[20]. We found a \u003cem\u003eLe\u003c/em\u003eAlkB protein belonging to the AlkB superfamily from marine strain \u003cem\u003eLe\u003c/em\u003eYC36. We loaded the gene sequence onto the plasmid pCDNA3.1 and transformed the recombinant into BMSC through liposome transfection. Results of qPCR and fluorescence staining indicated that \u003cem\u003eLe\u003c/em\u003eAlkB could be expressed in BMSCs. Phenotypic markers of osteoblasts including collagen matrix accumulation, ALP expression and bone nodule mineralization were observed during in vitro differentiation[19]. Our results showed that overexpression of \u003cem\u003eLe\u003c/em\u003eAlkB led to deepening of ALP staining, increased ALP enzyme activity, and increased expression of osteogenic marker genes RUNX2, BMP2, and ALP. Therefore, these results indicated that the marine protein \u003cem\u003eLe\u003c/em\u003eAlkB stimulated osteogenic differentiation under mechanical stress. In addition, the presence of \u003cem\u003eLe\u003c/em\u003eAlkB also increased the expression level of FTO in cells. FTO is a controversial protein in the ALKBH family involved in osteogenic differentiation[16]. Feng \u003cem\u003eet al.\u003c/em\u003e have demonstrated that FTO promoted osteogenic differentiation by reducing the inhibitory effect of glucocorticoids and improving the RNA stability of osteogenic marker genes[28]. Meanwhile, Son\u003cem\u003e et al.\u003c/em\u003e have also confirmed the promoting effect of FTO on osteogenic differentiation and indicated that overexpression of FTO could increase the expression level of osteogenic marker genes such as Runx2[29]. However, Wang \u003cem\u003eet al.\u003c/em\u003e believed that overexpression of FTO could reduce the mRNA methylation level of Runx2, thereby inhibiting osteogenic differentiation, which is inevitably accompanied by a decrease in Runx2 gene expression[29]. We are of the opinion that the different functions exhibited by FTO in osteogenic differentiation may be because FTO, as a gene inherent in BMSCs, is situated within a vast eukaryotic gene regulatory network. In this network, FTO is involved in multiple regulatory pathways with different effects on osteogenic differentiation[30]. However, in various studies, both positive and negative regulatory relationships have been discussed regarding the relationship between FTO and Runx2 expression levels[31]. Moreover, Runx2 positively regulates osteogenic differentiation. In our study, overexpression of \u003cem\u003eLe\u003c/em\u003eAlkB increased FTO expression, accompanied by a significant increase in Runx2 and other marker genes\u0026rsquo; expression. It indicates that \u003cem\u003eLe\u003c/em\u003eAlkB can promote the expression of FTO genes and activate the FTO-mediated regulatory pathway for promoting osteogenic differentiation, maximizing the promotion of osteogenic differentiation[28].\u003c/p\u003e\n\u003cp\u003eThe above results may prove that \u003cem\u003eLe\u003c/em\u003eAlkB is a potential peptide drug promoting osteogenesis. In previous drug research, some peptides have also been used as candidate drugs. The first peptide drug, insulin, can be traced back a century[32]. At present, various diseases such as cancer, multiple sclerosis, and even osteoporosis is treated with peptide drugs as the first choice in clinical practice[33]. Chen\u003cem\u003e et al.\u003c/em\u003e also reported in 2021 that terlipide, a parathyroid hormone (PTH) analog containing the first 34 amino acids of endogenous hormones, has shown significant efficacy in the treatment of osteoporosis[34]. \u003cem\u003eLe\u003c/em\u003eAlkB, as a prokaryotic peptide derived from the ocean, has the advantages of low toxicity and low cost, making it a highly feasible candidate drug for osteoporosis. In addition, autologous bone marrow transplantation contains substances that promote bone formation such as growth factors, cytokines, and stem cells, which can promote new bone formation and repair defects[35]. Autologous bone marrow transplantation can be combined with marine-derived peptides to further accelerate the repair process of bone defects[36]. Based on the above facts, we propose a vision for the future. This article demonstrates that the marine protein \u003cem\u003eLe\u003c/em\u003eAlkB has strong osteogenic potential. Therefore, it is worth exploring and researching whether the protein can be optimized and mutated to form better marine drugs for the clinical treatment of osteoporosis or orthodontic processes.\u003c/p\u003e\n\u003cp\u003eThe pharmaceutical potential of \u003cem\u003eLe\u003c/em\u003eAlkB is technically feasible. According to ISO 10993-5:2009[17], in the cytotoxicity test, a cell survival rate higher than 70% in the control group can be considered non-toxic to cells (Figure 2C, D).The experiments on cell apoptosis and survival rate have shown that\u003cem\u003e Le\u003c/em\u003eAlkB has no toxic effect on BMSCs, which provides possibilities for the medicinal research of \u003cem\u003eLe\u003c/em\u003eAlkB[37]. Protein sequence alignment and three-dimensional structural analysis revealed that \u003cem\u003eLe\u003c/em\u003eAlkB shares the same conserved residues (such as H115, D117, 172R, 177) with human ALKBH family proteins, and their spatial positions are similar. It will help optimize the activity of \u003cem\u003eLe\u003c/em\u003eAlkB through amino acid-directed mutagenesis in the future, making it more suitable for clinical osteogenic research. Walker \u003cem\u003eet al.\u003c/em\u003e demonstrated that site-specific mutations in the ALKBH family can improve or even inhibit cancer development[18]. Optimizing the amino acid sites and protein structure of \u003cem\u003eLe\u003c/em\u003eAlkB may also give it potential in the treatment of other diseases. In addition, the technical methods required to study \u003cem\u003eLe\u003c/em\u003eAlkB, derived from marine prokaryotic microorganisms, are relatively mature. Compared with eukaryotic expression, prokaryotic expression systems have bright application prospects which have been used for the large-scale production of recombinant proteins, such as drugs, vaccines, and antibodies. It has the advantages of high expression level, low cost, simple operation, and environmental protection, providing a new approach for future clinical development[17]. Therefore, compared to the ALKBH family proteins from eukaryotic cells that can catalyze osteogenesis, the prokaryotic protein \u003cem\u003eLe\u003c/em\u003eAlkB is more suitable for research on pharmacology and new materials. Finally, our study on the marine protein \u003cem\u003eLe\u003c/em\u003eAlkB provides a new research formula for the application research of other similar marine proteins. We appeal to researchers to actively search for bioactive drugs more suitable for osteogenic differentiation from the ocean treasure trove and are looking forward to truly solving the problem of osteogenic drug deficiency in orthodontic treatment or diseases such as osteoporosis in the future.\u003c/p\u003e"},{"header":"Materials And Methods","content":"\u003cp\u003e\u003cstrong\u003e5.1 Bacterial strains, plasmids, and general methods\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe strain of \u003cem\u003eLe\u003c/em\u003eYC36, which produces enzymatic lytic bacteria, originated from the coastal area of Qingdao, China. The \u003cem\u003eLe\u003c/em\u003eYC36 used in this experiment was donated by the Marine Life Science Laboratory of Ocean University of China, and the samples were stored in the laboratory of Ocean University of China. Cultivate \u003cem\u003eLe\u003c/em\u003eYC36 strain in 40% trypsin soy broth (TSB) medium. DH5a cells were purchased from AngYu Biotechnology Co., Ltd (Shanghai, China) for plasmid amplification and protein purification. The PcDNA3.1 (Supplementary Material Figure S3) plasmid was purchased from Yunnan Luoyu Biotechnology Co., Ltd(Yunnan, China).Primary rat bone marrow stromal stem cells were purchased from Procter\u0026amp;Gamble CP-M131.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e5.2. Structural comparison and biological prediction of \u003cem\u003eLe\u003c/em\u003eAlkB\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn this experiment, computer-based structural prediction is essential for inferring the biological function of the \u003cem\u003eLe\u003c/em\u003eAlkB protein. To solve the prediction issue, we employed the discovered structures in the protein database (PDB) as templates to construct structural models. The alignment of\u003cem\u003e\u0026nbsp;Le\u003c/em\u003eAlkB protein sequences in the Uniprot database was shown, and protein sequences with over 35% similarity (approximately 1000 sequences) were selected for detailed analysis. The \u003cem\u003eLe\u003c/em\u003eAlkB protein sequence was indexed and compared in the UniProtKB reference protein plus Swiss protein database (E-Threshold=0.0001). We obtained 1000 similar sequences with a similarity greater than 35%, but they have not been studied yet. Therefore, 54 representative sequences (similarity\u0026nbsp;\u0026ge;\u0026nbsp;55%) were selected as the proximal sequences of \u003cem\u003eLe\u003c/em\u003eAlkB for systematic analysis (in the green part of the evolutionary tree). At the same time, the AlkB protein was searched in the protein database (PDB), and homology modeling was performed using AlphaFold software. Select the PDB file from the AlphaFol database for RMSD calculation. We imported the PDB files of \u003cem\u003eLe\u003c/em\u003eAlkB, FTO, ALKBH2, ALKBH5, and Escherichia coli ALKB proteins into Pymol and selected Plugin. \u0026rarr; Align, parameters (Method: Align; Period: 5; Deadline: 2.0). Then, the truncated 56 sequences were aligned using clusterX software, and an evolutionary tree was established using adjacency method. It is known that the human self-produced enzyme ALKBH5 is numbered 4NRM in the protein database (PDB), and after analysis, it is found that the protein sequence similarity with \u003cem\u003eLe\u003c/em\u003eAlkB is 16.27%. Then, the PDB file of ALKBH5 was imported into PyMOL software for comparison with FTO and \u003cem\u003eLe\u003c/em\u003eAlkB.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e5.3\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eConstruction of plasmid pCDNA3.1\u003c/strong\u003e\u003cstrong\u003e-eGFP-MCS-\u003cem\u003eLe\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003eAlkB\u003c/strong\u003e\u003cstrong\u003eand heat shock conversion method\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAmplify the target fragment using AlkB-F/R primers containing cleavage sites (Supplementary Material S1) and obtain the PCR product of the \u003cem\u003eLe\u003c/em\u003ealkB gene. Restrictive endonucleases Hind III and BamH I are used to digest amplified products and plasmids pCDNA3.1-eGFP-MCS. In the experiment, the plasmid pCDNA3.1-e GFP-MCS (Supplementary Material Figure S4) was added to a culture dish containing sensing cells and subjected to heat shock treatment for plasmid transfection. Take out 200 \u0026mu; L suspension from the freezer at -70 \u0026deg; C and immediately place it on ice after thawing. Add plasmid DNA solution (content not exceeding 50ng, volume not exceeding 10 \u0026mu; L). Place it on ice and let it stand for 30 minutes. Add 1mL of LB liquid culture medium (excluding Amp) to the test tube. Mix thoroughly and shake at 37 \u0026deg; C for 1 hour to restore normal bacterial growth and express the plasmid encoded antibiotic resistance gene (Ampr). Shake the bacterial solution thoroughly and place 100 \u0026mu; L of Amp containing solution facing upwards on the filter plate for half an hour. The plasmid pCDNA3.1-eGFP-MCS-LealkB was introduced into Escherichia coli DH5 using heat shock transformation method. Leave the bacterial solution overnight for 16 hours, then centrifuge at 8000rpm for 10 minutes. Collect bacterial cells and resuspend with binding buffer. Then use an ultrasonic crusher (300W, crush for 3 seconds, pause for 5 seconds) to crush the cells until the bacterial solution is clear and transparent. Centrifuge the broken bacterial solution at 10000rpm for 10 minutes and filter the supernatant using a 0.22 \u0026mu; m filter membrane. Subsequently, protein purification was performed using nickel columns. Obtain purified protein \u003cem\u003eLe\u003c/em\u003eAlkB (Supplementary Materials Figure S1, S2).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e5.4 Liposome transfected cells\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBMSC cells were obtained from Cytogen Biosciences Co., Ltd. (Yunnan, China). Subsequently, the cells were seeded onto a 6-well stress plate (flex, project number BF-3001C) and allowed to adhere to the surface for approximately 48 hours, reaching a logarithmic growth stage with a fusion degree of approximately 70%. Afterwards, replace the medium with Opti MEM medium (2mL per well) and incubate for 3 hours to allow adaptation. Once adaptation is complete, replace the culture medium again and add 1.99mL of Opti MEM medium per well. Add 0.8\u0026mu;g plasmid and 1.6\u0026mu;L lipo3000 to Opti MEM, shake well, and let stand for 5 minutes. Granular CDNA3.1 and transfection agent lipo3000. After adding each plate, gently stir 2-3 times by shaking, and then place it in a CO2 incubator for further cultivation for 72 hours. Subsequently, discard the culture medium, wash the wells twice with PBS solution, and then add 200 \u0026mu;L of Trizol reagent to each well. Collect cells using Calcium chloride reagents for subsequent analysis, including q-PCR based detection of alkb mRNA expression levels and evaluation of transfection efficiency and stress load treatment based on experimental grouping.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e5.5 Cell survival rate experiment\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA cell counting kit (CCK-8) was used to test the survival rate of cells. Bone marrow mesenchymal stem cells of different groups were placed into a 24-well plate. 10\u003csup\u003e5\u003c/sup\u003e cells were inoculated into each well and cultured for 1, 3 and 5days. The culture medium was removed and 300 \u0026micro;L of CCK-8 working solution was added. It was incubated at a constant temperature of 37\u0026deg;C in darknes for 2h. The supernatant was transferred to a 96-well plate. An enzyme-linked immunosorbent assay was used to measure its absorbance at 450 nm.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e5.6 Flow cytometry apoptosis experiment\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWhen the cells revive and grow up to 80%-90% in the 25T culture bottle, the\u0026nbsp;supernatant was discard and gently washed twice with PBS. And then, 2 mL trypsin was added for digestion, and another 2 mL of complete culture medium was added. The mixture was blowed evenly and transfered to a 15 mL centrifuge tube, incubating\u0026nbsp;at room temperature and dark for 5 min.\u0026nbsp;After that, 5 \u0026micro;L of propidium iodide solution (PI) and 400 \u0026micro;L of PBS were added into it, and then\u0026nbsp;performed the flow cytometry detection at once.\u0026nbsp;Finally, To reach the results, the flow cytometry (Beckman Kurt International Trade (Shanghai) Co. Ltd, Shanghai, China) was utilized for detection and data preservation.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e5.7 Application of cyclic mechanical stress\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCells were collected and inoculated onto six wells of wear-resistant silicone rubber Bio Flex \u0026trade; Coating plate with type I rat tail collagen (Flexcell International Corp, Hillsborough, USA), then initial density of 5\u0026times;10\u003csup\u003e5\u0026nbsp;\u003c/sup\u003egood growth medium was replaced by basic medium without serum until reaching 70% consistency. After 24 h, the 1Hz sine curve of cells subjected to cyclic mechanical strain which set to 5% elongation was obtained from FX-4000 tons\u0026trade; Flexcell Tension Plus\u0026trade; Unit (Flexcell Inter-national Corp), 6 h per day for a total of 3 days.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e5.8 ALP vitality determination\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eUsing alkaline phosphatase (ALP) activity assay kit (Nanjing Jiancheng Biotechnology Research Institute, A059-2, Nanjing, China).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e5.9\u003c/strong\u003e \u003cstrong\u003eALP staining experiment\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAfter gently rinsing bone cells three times by using the ALP staining kit (P0321S, Shanghai Beyotime Biotechnology Co., Ltd.), fix the cells with 4% paraformaldehyde, then stain the cells with the ALP staining kit, and finally scan the stained cells with a scanner to obtain ALP staining images.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e5.10 Real time quantitative q-PCR\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eQuery target gene mRNA sequences of corresponding species on NCBI（\u0026nbsp;https://www.ncbi.nlm.nih.gov/\u0026nbsp;）And design primers using CDS sequences (Supplementary Material Table 1). Extract total RNA, measure RNA concentration, and calculate the volume of total RNA required in RT using the following formula: volume of total RNA required in reverse transcription=2 \u0026micro; g/measured RNA concentration. Follow the instructions of the FastKing RT Kit (With gDNase) to synthesize the first strand of cDNA. After obtaining the first strand, the target gene was amplified by Q-PCR. Taq Pro Universal SYBR qPCR Master Mix 5 \u0026micro; l, upstream primer 0.25 \u0026micro; l, downstream primer 0.25 \u0026micro; l, cDNA template 1.0 \u0026micro; l, Nuclease Free Water 3.5 \u0026micro; l were added to each well in the order of loading, until the final reaction volume was 10 \u0026micro; l. Put it into the LightCycle instrument, set the PCR cycle reaction conditions, complete the reaction, collect information, and perform Ct value analysis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e5.11 Statistical analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data is presented as mean \u0026plusmn; SD and analyzed using GraphPad Prism9 (GraphPad Software, USA). Use ANONA to evaluate differences between groups. \u003cem\u003eP\u003c/em\u003e-value less than 0.05 is considered statistically significant.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn summary, this study indicates that the marine protein LeAlkB is safe for BMSCs. LeYC36, which has a wide range of sources, is an enzyme producing bacterium isolated from the coast of Qingdao, with simple processing and low cost. Through bioinformatics analysis, the novel demethylated protein LeAlkB in this study has a similar structure and function to FTO protein, and its osteogenic performance is superior. This indicates that the marine protein LeAlkB may become a new source of future bone formation. The specific process of this study is shown in the following figure (Figure 5). Other potentially valuable components need to be studied in future research. For example, further research and improvement are needed on how to apply it to clinical practice to bring this sustainable osteogenic product to the market.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003e\u003cem\u003eLe\u003c/em\u003eAlkB, newly discovered marine bacterial proteins; BMSC, bone marrow mesenchymal stem cells; FTO, Obesity related genes; ALKBH5, AlkB Homolog 5,RNA Demethylase; ALKBH2, AlkB Homolog 2,RNA Demethylase; ALP, Alkaline Phosphatase; BMP2, Bone morphogenetic protein 2; RUNX2, Runt Related Transcripyion Factor 2.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eFunding:\u003c/h2\u003e \u003cp\u003eThis study was funded by the General Project of the Natural Science Foundation of Shandong Province, China (ZR2023MH155), the Natural Science Foundation of Qingdao City, Shandong Province, China (23-2-198-zyyd-jch), and the Science and Technology Plan of Shinan District,Qingdao Shandong Province, China (2022-4-003-YY) Grant funding\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eThis study was designed by Q.G. The isolation, extraction, and genome sequencing of bacteria were carried out by Z.R. Bioinformatics methods, data analysis, and numbering were designed and implemented by Q.Z, while cell staining experiments were designed and implemented by F.Y and Z.H; The cytotoxicity experiment was conducted by Z.C, The cell PCR experiment was conducted by R.Y and X.Z. The initial draft was written by Q.Z. This study was funded and/or supervised by Q.G. All authors have read and agreed to the published version of the manuscript.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eData is provided within the manuscript or supplementary information files.The datasets used and/or analysed during the current study available from the corresponding author on reasonable request\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eHuang, H., et al., \u003cem\u003eModulation of T Cell Responses by Fucoidan to Inhibit Osteogenesis.\u003c/em\u003e Front Immunol, 2022. \u003cstrong\u003e13\u003c/strong\u003e: p. 911390.\u003c/li\u003e\n\u003cli\u003eCarson, M.A. and S.A. Clarke, \u003cem\u003eBioactive Compounds from Marine Organisms: Potential for Bone Growth and Healing.\u003c/em\u003e Mar Drugs, 2018. \u003cstrong\u003e16\u003c/strong\u003e(9).\u003c/li\u003e\n\u003cli\u003eGuo, Q., et al., \u003cem\u003eMechanical stimulation induced osteogenic differentiation of BMSCs through TWIST/E2A/p21 axis.\u003c/em\u003e Biosci Rep, 2020. \u003cstrong\u003e40\u003c/strong\u003e(5).\u003c/li\u003e\n\u003cli\u003eHardy, S., et al., \u003cem\u003eManzamine-A Alters In Vitro Calvarial Osteoblast Function.\u003c/em\u003e Mar Drugs, 2022. \u003cstrong\u003e20\u003c/strong\u003e(10).\u003c/li\u003e\n\u003cli\u003eYang, G.J., et al., \u003cem\u003eThe emerging role of KDM5A in human cancer.\u003c/em\u003e J Hematol Oncol, 2021. \u003cstrong\u003e14\u003c/strong\u003e(1): p. 30.\u003c/li\u003e\n\u003cli\u003eZhu, L., et al., \u003cem\u003eT6SS translocates a micropeptide to suppress STING-mediated innate immunity by sequestering manganese.\u003c/em\u003e Proceedings of the National Academy of Sciences, 2021. \u003cstrong\u003e118\u003c/strong\u003e(42).\u003c/li\u003e\n\u003cli\u003eMakarewich, C.A. and E.N. Olson, \u003cem\u003eMining for Micropeptides.\u003c/em\u003e Trends Cell Biol, 2017. \u003cstrong\u003e27\u003c/strong\u003e(9): p. 685-696.\u003c/li\u003e\n\u003cli\u003eBaek, J.W., et al., \u003cem\u003eMarine Plankton-Derived Whitlockite Powder-Based 3D-Printed Porous Scaffold for Bone Tissue Engineering.\u003c/em\u003e Materials (Basel), 2022. \u003cstrong\u003e15\u003c/strong\u003e(10).\u003c/li\u003e\n\u003cli\u003eBravo, A. and M. Soberon, \u003cem\u003eMining versus in vitro evolution for the selection of novel microbial insecticidal proteins.\u003c/em\u003e Microb Biotechnol, 2022. \u003cstrong\u003e15\u003c/strong\u003e(10): p. 2518-2520.\u003c/li\u003e\n\u003cli\u003eCarnovali, M., et al., \u003cem\u003eAerophobin-1 from the Marine Sponge Aplysina\u0026amp;nbsp;aerophoba Modulates Osteogenesis in Zebrafish Larvae.\u003c/em\u003e Mar Drugs, 2022. \u003cstrong\u003e20\u003c/strong\u003e(2).\u003c/li\u003e\n\u003cli\u003eChen, X., et al., \u003cem\u003eRegulatory Role of RNA N6-Methyladenosine Modification in Bone Biology and Osteoporosis.\u003c/em\u003e Frontiers in Endocrinology, 2020. \u003cstrong\u003e10\u003c/strong\u003e.\u003c/li\u003e\n\u003cli\u003eChen, X., et al., \u003cem\u003eEnhanced bone regeneration via spatiotemporal and controlled delivery of a genetically engineered BMP-2 in a composite Hydrogel.\u003c/em\u003e Biomaterials, 2021. \u003cstrong\u003e277\u003c/strong\u003e: p. 121117.\u003c/li\u003e\n\u003cli\u003eCherigo, L., D. Lopez, and S. Martinez-Luis, \u003cem\u003eMarine natural products as breast cancer resistance protein inhibitors.\u003c/em\u003e Mar Drugs, 2015. \u003cstrong\u003e13\u003c/strong\u003e(4): p. 2010-29.\u003c/li\u003e\n\u003cli\u003eDerkach, S.R., et al., \u003cem\u003eProperties of Protein Isolates from Marine Hydrobionts Obtained by Isoelectric Solubilisation/Precipitation: Influence of Temperature and Processing Time.\u003c/em\u003e Int J Mol Sci, 2022. \u003cstrong\u003e23\u003c/strong\u003e(22).\u003c/li\u003e\n\u003cli\u003ePilzys, T., et al., \u003cem\u003eALKBH overexpression in head and neck cancer: potential target for novel anticancer therapy.\u003c/em\u003e Sci Rep, 2019. \u003cstrong\u003e9\u003c/strong\u003e(1): p. 13249.\u003c/li\u003e\n\u003cli\u003eSun, R., et al., \u003cem\u003eDemethylase FTO promotes mechanical stress induced osteogenic differentiation of BMSCs with up-regulation of HIF-1alpha.\u003c/em\u003e Mol Biol Rep, 2022. \u003cstrong\u003e49\u003c/strong\u003e(4): p. 2777-2784.\u003c/li\u003e\n\u003cli\u003eWang, S.W., et al., \u003cem\u003eCurrent applications and future perspective of CRISPR/Cas9 gene editing in cancer.\u003c/em\u003e Mol Cancer, 2022. \u003cstrong\u003e21\u003c/strong\u003e(1): p. 57.\u003c/li\u003e\n\u003cli\u003eWalker, A.R., et al., \u003cem\u003eALKBH7 Variant Related to Prostate Cancer Exhibits Altered Substrate Binding.\u003c/em\u003e PLoS Comput Biol, 2017. \u003cstrong\u003e13\u003c/strong\u003e(2): p. e1005345.\u003c/li\u003e\n\u003cli\u003eOuyang, L., et al., \u003cem\u003eALKBH1-demethylated DNA N6-methyladenine modification triggers vascular calcification via osteogenic reprogramming in chronic kidney disease.\u003c/em\u003e J Clin Invest, 2021. \u003cstrong\u003e131\u003c/strong\u003e(14).\u003c/li\u003e\n\u003cli\u003eLi, Z., et al., \u003cem\u003eThe N(6)-methyladenosine demethylase ALKBH5 negatively regulates the osteogenic differentiation of mesenchymal stem cells through PRMT6.\u003c/em\u003e Cell Death Dis, 2021. \u003cstrong\u003e12\u003c/strong\u003e(6): p. 578.\u003c/li\u003e\n\u003cli\u003ePonzetti, M. and N. Rucci, \u003cem\u003eOsteoblast Differentiation and Signaling: Established Concepts and Emerging Topics.\u003c/em\u003e Int J Mol Sci, 2021. \u003cstrong\u003e22\u003c/strong\u003e(13).\u003c/li\u003e\n\u003cli\u003eLv, S., et al., \u003cem\u003eExpression of HMGB1 in the periodontal tissue subjected to orthodontic force application by Waldo's method in mice.\u003c/em\u003e J Mol Histol, 2015. \u003cstrong\u003e46\u003c/strong\u003e(1): p. 107-14.\u003c/li\u003e\n\u003cli\u003eYu, H., et al., \u003cem\u003eExpression of HIF\u003c/em\u003e\u003cem\u003e‑1alpha in cycling stretch\u003c/em\u003e\u003cem\u003e‑induced osteogenic differentiation of bone mesenchymal stem cells.\u003c/em\u003e Mol Med Rep, 2019. \u003cstrong\u003e20\u003c/strong\u003e(5): p. 4489-4498.\u003c/li\u003e\n\u003cli\u003eChilds, P.G., et al., \u003cem\u003eUse of nanoscale mechanical stimulation for control and manipulation of cell behaviour.\u003c/em\u003e Acta Biomater, 2016. \u003cstrong\u003e34\u003c/strong\u003e: p. 159-168.\u003c/li\u003e\n\u003cli\u003eZhang, X., et al., \u003cem\u003eLocal delivery of insulin/IGF-1 for bone regeneration: carriers, strategies, and effects.\u003c/em\u003e Nanotheranostics, 2020. \u003cstrong\u003e4\u003c/strong\u003e(4): p. 242-255.\u003c/li\u003e\n\u003cli\u003eZhong, J., et al., \u003cem\u003eDeveloping a new treatment for superficial fungal infection using antifungal Collagen-HSAF dressing.\u003c/em\u003e Bioeng Transl Med, 2022. \u003cstrong\u003e7\u003c/strong\u003e(3): p. e10304.\u003c/li\u003e\n\u003cli\u003eZhou, J., et al., \u003cem\u003eAdvanced glycation end products impair bone marrow mesenchymal stem cells osteogenesis in periodontitis with diabetes via FTO-mediated N(6)-methyladenosine modification of sclerostin.\u003c/em\u003e J Transl Med, 2023. \u003cstrong\u003e21\u003c/strong\u003e(1): p. 781.\u003c/li\u003e\n\u003cli\u003eFeng, L., et al., \u003cem\u003eRNA N6-methyladenosine demethylase FTO inhibits glucocorticoid-induced osteoblast differentiation and function in bone marrow mesenchymal stem cells.\u003c/em\u003e J Cell Biochem, 2023. \u003cstrong\u003e124\u003c/strong\u003e(11): p. 1835-1847.\u003c/li\u003e\n\u003cli\u003eSon, H.E., et al., \u003cem\u003eFat Mass and Obesity-Associated (FTO) Stimulates Osteogenic Differentiation of C3H10T1/2 Cells by Inducing Mild Endoplasmic Reticulum Stress via a Positive Feedback Loop with p-AMPK.\u003c/em\u003e Mol Cells, 2020. \u003cstrong\u003e43\u003c/strong\u003e(1): p. 58-65.\u003c/li\u003e\n\u003cli\u003eHuang, M., et al., \u003cem\u003em6A demethylase FTO and osteoporosis: potential therapeutic interventions.\u003c/em\u003e Front Cell Dev Biol, 2023. \u003cstrong\u003e11\u003c/strong\u003e: p. 1275475.\u003c/li\u003e\n\u003cli\u003e\u003cem\u003e\u0026lt;FTO\u003c/em\u003e\u003cem\u003e与RUNX2.pdf\u0026gt;.\u003c/em\u003e\u003c/li\u003e\n\u003cli\u003eLewis, G.F. and P.L. Brubaker, \u003cem\u003eThe discovery of insulin revisited: lessons for the modern era.\u003c/em\u003e J Clin Invest, 2021. \u003cstrong\u003e131\u003c/strong\u003e(1).\u003c/li\u003e\n\u003cli\u003eLee, Y.S., et al., \u003cem\u003eAntiosteoporosis effects of a marine antimicrobial peptide pardaxin via regulation of the osteogenesis pathway.\u003c/em\u003e Peptides, 2022. \u003cstrong\u003e148\u003c/strong\u003e: p. 170686.\u003c/li\u003e\n\u003cli\u003eChen, T., et al., \u003cem\u003eParathyroid hormone and its related peptides in bone metabolism.\u003c/em\u003e Biochem Pharmacol, 2021. \u003cstrong\u003e192\u003c/strong\u003e: p. 114669.\u003c/li\u003e\n\u003cli\u003eHu, S., et al., \u003cem\u003eStructural Characterization and Anti-Osteoporosis Effects of a Novel Sialoglycopeptide from Tuna Eggs.\u003c/em\u003e Mar Drugs, 2023. \u003cstrong\u003e21\u003c/strong\u003e(11).\u003c/li\u003e\n\u003cli\u003eChen, X., et al., \u003cem\u003eMesoporous Silica Promotes Osteogenesis of Human Adipose-Derived Stem Cells Identified by a High-Throughput Microfluidic Chip Assay.\u003c/em\u003e Pharmaceutics, 2022. \u003cstrong\u003e14\u003c/strong\u003e(12).\u003c/li\u003e\n\u003cli\u003eKim, J.T., et al., \u003cem\u003eSafety evaluation and consideration of 4 Pin Multi-needle for meso-therapy.\u003c/em\u003e Technol Health Care, 2018. \u003cstrong\u003e26\u003c/strong\u003e(S1): p. 291-306.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Bone marrow mesenchymal stem cells, osteogenic differentiation, marine protein, osteogenic related factors, mechanical stress","lastPublishedDoi":"10.21203/rs.3.rs-5335118/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5335118/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cem\u003eLe\u003c/em\u003eAlkB is a marine protein derived from the \u003cem\u003eLysobacter enzymogenes\u003c/em\u003e. It has a dependence on Fe\u003csup\u003e2+\u003c/sup\u003eand α- Ketoglutaric acid. The ALKBH family of methylases, which are functionally conserved for ketoglutarate, have a similar structure. FTO（Fat Mass and Obesity-associated Protein） in the ALKBH family is a key promoter of osteogenic differentiation under mechanical stress and has an upregulation effect on inducing osteogenic differentiation phenotype markers. Research has found that ALKBH5 also has osteogenic ability, and the application of marine drugs in bone disease and bone regeneration research is gradually increasing. The structure similarly to the ALKBH family enables \u003cem\u003eLe\u003c/em\u003eAlkB to have the same osteogenic differentiation ability. To explore the potential application of the novel marine protein \u003cem\u003eLe\u003c/em\u003eAlkB in bone reconstruction, this study investigated the bone regeneration characteristics induced by \u003cem\u003eLe\u003c/em\u003eAlkB. It has been demonstrated that mechanical stress encourages bone marrow mesenchymal stem cells (BMSCs) to differentiate into osteogenic tissue. This study discovered that BMSCs' expression of osteogenic differentiation markers in \u003cem\u003eLe\u003c/em\u003eAlkB dramatically increased under mechanical stress settings. Furthermore, \u003cem\u003eLe\u003c/em\u003eAlkB significantly raises the expression of FTO. This suggests that BMSCs' osteogenic differentiation can be stimulated by the marine protein \u003cem\u003eLe\u003c/em\u003eAlkB. \u003cem\u003eLe\u003c/em\u003eAlkB is a marine new protein that exhibits much greater osteogenic ability and minimal cytotoxicity when compared to FTO and ALKBH5. This suggests that \u003cem\u003eLe\u003c/em\u003eAlkB has better therapeutic potential. \u003cem\u003eLe\u003c/em\u003eAlkB is therefore anticipated to be used in the clinical treatment of orthodontics in the future. Our study's conclusions, however, offer fresh concepts for the potential uses of marine naturally occurring bioactive compounds in the future.\u003c/p\u003e","manuscriptTitle":"The role of a novel marine protein LeAlkB in BMSCs osteogenic differentiation with mechanical stress","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-12-05 17:53:08","doi":"10.21203/rs.3.rs-5335118/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"066afd12-62b2-4c9f-88c8-85e2a733d17d","owner":[],"postedDate":"December 5th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":41008096,"name":"Biological sciences/Biochemistry/Proteins"},{"id":41008097,"name":"Biological sciences/Biochemistry/Structural biology"}],"tags":[],"updatedAt":"2025-07-29T04:24:10+00:00","versionOfRecord":[],"versionCreatedAt":"2024-12-05 17:53:08","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-5335118","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5335118","identity":"rs-5335118","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}