Application of propranolol in preparation of functional medicine

Abstract

The invention relates to the technical field of biological medicines, in particular to application of propranolol in preparation of functional medicines. The invention provides an application of propranolol in preparation of a functional medicine. Researches show that propranolol can block a beta2-AR signal and reverse a negative effect activated and induced by a receptor, and is beneficial to early spreading, migration, proliferation and adhesion of HaCaT / HSF on the surface of a titanium sheet; the expression of half-desmosome related LAMA3 and Integrin beta 4 genes and proteins can be promoted, so that the capacity of further enhancing the soft tissue closure around implantation is achieved; migration, proliferation and adhesion of HaCaT cells on the surface of the titanium sheet and expression of related proteins can be promoted through an MAPK / ERK signal channel.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and particularly to the application of propranolol in the preparation of functional drugs. Background Art

[0002] Implant rehabilitation is currently the most commonly used method for restoring partially or completely missing teeth. The implant penetrates through the gingiva and is implanted into the alveolar bone, establishing two parts of connection with the body: the implant-bone interface and the implant / abutment-soft tissue interface. However, the stability and long-term survival rate of the implant also depend on the long-term existing biological barrier formed by high-quality and firm soft tissue integration. Therefore, in recent years, researchers have begun to turn their attention to the relationship between the transmucosal area of the implant and the soft tissue.

[0003] Propranolol, as a non-selective adrenergic receptor (AR) blocker, its main function is to effectively block AR and its related downstream signaling pathways, thereby exerting pharmacological effects. AR is an important member of the G protein-coupled transmembrane receptor family (GPCRs), which plays a crucial role in the process of signal transduction inside and outside the cell. Catecholamines, as ligands of this receptor, include adrenaline and noradrenaline. β-AR is considered a key functional regulator in the heart, lungs, blood vessels, endocrine and central nervous systems, and is mainly expressed by cardiovascular cells and respiratory cells. According to different pharmacological effects, protein sequences and responses to catecholamines, it is divided into β1, β2 and β3 receptors. Among them, only the β2-AR subtype is expressed in cells closely related to soft tissue healing, such as keratinocytes, fibroblasts, endothelial cells, etc. The peri-implant soft tissue seal is composed of an epithelial seal formed by surrounding epithelial cells and a connective tissue seal formed by fibroblasts. However, there are few reports on the effect of β2-AR on the biological behavior of cells related to peri-implant soft tissue healing.

[0004] In view of this, the present invention is specifically proposed. Summary of the Invention

[0005] To solve the above technical problems, the present invention provides an application of propranolol in the preparation of functional drugs.

[0006] Specifically, the technical solution of the present invention is as follows: In a first aspect, the present invention provides an application of propranolol in the preparation of functional drugs, and the functional drugs have the function of promoting the binding of peri-implant soft tissue.

[0007] Preferably, the functional drugs have the function of promoting the expression of hemidesmosome-related genes and proteins.

[0008] Preferably, the hemidesmosome-related genes and proteins include: LAMA3 gene and LAMA3 protein.

[0009] Preferably, the hemidesmosome-related genes and proteins include: Integrinβ4 gene and Integrin β4 protein.

[0010] Preferably, the functional drug has the functions of promoting cell spreading, migration, proliferation and adhesion on the titanium sheet surface.

[0011] Preferably, the functional drug has the function of interfering with the MAPK / ERK signaling pathway.

[0012] Preferably, the functional drug promotes the migration, proliferation, adhesion and expression of related proteins of HaCaT cells on the titanium sheet surface through the MAPK / ERK signaling pathway.

[0013] Preferably, the functional drug has the functions of blocking the β2-AR signal and reversing the negative effects induced by receptor activation.

[0014] Preferably, the functional drug promotes the early spreading, migration, proliferation and adhesion of HaCaT / HSF on the titanium sheet surface by blocking the β2-AR signal and reversing the negative effects induced by receptor activation.

[0015] Beneficial effects: The present invention provides an application of propranolol in the preparation of a functional drug. The research of the present invention shows that propranolol can block the β2-AR signal and reverse the negative effects induced by receptor activation, which is beneficial to the early spreading, migration, proliferation and adhesion of HaCaT / HSF on the titanium sheet surface; it can promote the expression of hemidesmosome-related LAMA3, Integrin β4 genes and proteins, thereby further enhancing the ability of the per-implant soft tissue seal; it can promote the migration, proliferation, adhesion and expression of related proteins of HaCaT cells on the titanium sheet surface through the MAPK / ERK signaling pathway. Description of the Drawings

[0016] In order to more clearly illustrate the technical solutions in the present invention or the prior art, the following will describe the drawings required for use in the embodiments or the description of the prior art.

[0017] Figure 1 Relative cell viability (%) of epithelial cells (A, C) and fibroblasts (B, D) after treatment with different concentrations of epinephrine / propranolol for 36 h in the embodiments of the present invention. Relative cell viability (%) of epithelial cells (E, G) and fibroblasts (F, H) after treatment with 1 μM epinephrine / 10 μM propranolol for 0, 12, 24, 36 h. n = 4, compared with the control group, * P< 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001。

[0018] Figure 2 For the SEM detection in the embodiments of the present invention, the morphology of HaCaT (upper row, scale bar is 10 μm) and HSF (lower row, scale bar is 10 μm) adhered to the surface of titanium sheets in different drug treatment groups (A), and the morphology of HaCaT (upper row, scale bar is 40 μm) and HSF (lower row, scale bar is 100 μm) adhered to the surface of titanium sheets in different drug treatment groups detected by immunofluorescence staining (B).

[0019] Figure 3 For the 24 / 48h proliferation of epithelial cells (A) and fibroblasts (B) on the surface of titanium sheets in different drug treatment groups in the embodiments of the present invention. n = 4, * P < 0.05 indicates a significant difference compared with the control group, # P < 0.05 indicates a significant difference compared with the adrenaline group, & P < 0.05 indicates a significant difference compared with the propranolol group.

[0020] Figure 4 For the early adhesion numbers of epithelial cells (A) and fibroblasts (B) on the surface of titanium sheets in different drug treatment groups at 6 / 12h in the embodiments of the present invention. n = 4, scale bar is 200 μm. * P < 0.05 indicates a significant difference compared with the control group cells, # P < 0.05 indicates a significant difference compared with the adrenaline group, & P < 0.05 indicates a significant difference compared with the propranolol group.

[0021] Figure 5 For the scratch healing rate (%) of epithelial cells (A) and fibroblasts (B) on the surface of titanium sheets in different drug treatment groups at 24 / 48h in the embodiments of the present invention. n = 4, scale bar is 200 μm. * P < 0.05 indicates a significant difference compared with the control group, # P < 0.05 indicates a significant difference compared with the adrenaline group, & P < 0.05 indicates a significant difference compared with the propranolol group.

[0022] Figure 6In the embodiments of the present invention, the expression levels of migration and adhesion-related genes of epithelial cells and fibroblasts cultured on titanium sheets in different treatment groups were detected by qRT-PCR for 3 days and 7 days. Epithelial cells include LAMA3, ITGB4, and Plectin (A, B, C), and fibroblasts include Fn, Vcl, and Fak (D, E, F). n = 4, * P <0.05 indicates a significant difference compared with the control group, # P <0.05 indicates a significant difference compared with the adrenaline group, & P <0.05 indicates a significant difference compared with the propranolol group.

[0023] Figure 7 In the embodiments of the present invention, the expression levels of adhesion-related proteins LAMA3 and ITGB4 of epithelial cells cultured on titanium sheets in different treatment groups were analyzed by Western blot for 3 days and 7 days. n = 3, * P <0.05 indicates a significant difference compared with the control group, # P <0.05 indicates a significant difference compared with the adrenaline group, & P <0.05 indicates a significant difference compared with the propranolol group.

[0024] Figure 8 In the embodiments of the present invention, the expression levels of adhesion-related proteins LAMA3 (A) and ITGB4 (B) in epithelial cells on the surface of titanium sheets in different treatment groups were analyzed by fluorescence quantification. n = 3, and the scale bar is 20 μm. * P <0.05 indicates a significant difference compared with the control group, # P <0.05 indicates a significant difference compared with the adrenaline group, & P <0.05 indicates a significant difference compared with the propranolol group.

[0025] Figure 9 In the embodiments of the present invention, the expression levels of p-MAPK / MAPK and p-ERK / ERK proteins in epithelial cells on the surface of titanium sheets in different treatment groups were analyzed by WB. n = 3, * P <0.05 indicates a significant difference compared with the control group, # P <0.05 indicates a significant difference compared with the adrenaline group, & P <0.05 indicates a significant difference compared with the propranolol group.

[0026] Figure 10 In the embodiments of the present invention, the expression levels of LAMA3, ITGB4, p-MAPK / MAPK, and p-ERK / ERK proteins in epithelial cells on titanium sheets treated with propranolol or propranolol + SCH772984. n = 3, there is a significant difference compared with the propranolol group, **P <0.01,*** P <0.001,**** P <0.0001。

[0027] Figure 11 Morphology of epithelial cells on the surface of titanium sheets treated with propranolol with / without SCH772984 under scanning electron microscopy (upper row, scale bar: 10 μm) and immunofluorescence (lower row, scale bar: 50 μm) in the embodiments of the present invention.

[0028] Figure 12 Cell proliferation activity on the surface of titanium sheets treated with propranolol or propranolol + SCH772984 at 24 / 48 h in the embodiments of the present invention. n = 4, * P <0.05,** P <0.01。

[0029] Figure 13 Immunofluorescence results of cells on the surface of titanium sheets and quantitative analysis of cell adhesion number after treatment with propranolol or propranolol + SCH772984 for 3 h in the embodiments of the present invention. n = 4, scale bar: 200 μm. **** P <0.0001。

[0030] Figure 14 Wound healing rate of epithelial cells on the surface of titanium sheets treated with propranolol with / without SCH772984 for 24 h in the embodiments of the present invention. n = 4, scale bar: 200 μm. ** P <0.01。 Detailed implementation manners

[0031] Early studies have confirmed that β2-AR activation delays the healing of salamander skin wounds. Subsequent studies have successively confirmed that β2-AR activation inhibits keratinocyte proliferation and migration, hinders fibroblast proliferation, and delays the migration and repair of corneal and airway epithelial cells. Local or systemic application of receptor-specific antagonists to block β2-AR reverses these adverse effects. Notably, even in the absence of exogenous agonist addition, wound healing can be accelerated in in vitro and in vivo wound models. Therefore, AR antagonists, especially β2-AR antagonists such as propranolol and timolol, have been proposed for promoting the healing of refractory or burn wounds. Oral mucosal soft tissues show significant similarities with the skin during the process of wound healing. Both rely on similar cell-cell / extracellular matrix (ECM) interactions and require the participation of similar cytokines or growth factors to jointly promote tissue regeneration and repair. Although these in vivo or clinical studies have confirmed the beneficial effects of propranolol on soft tissue healing, previous studies by our research group and other scholars have also shown that the β-AR blocker propranolol has the effect of regulating bone metabolism and promoting osteogenesis. However, the effect of propranolol on the cells related to the healing of peri-implant soft tissues has not been elucidated.

[0032] Saliva is rich in adrenaline, and more adrenaline is secreted in the oral cavity under the stress condition of implant surgery trauma. It is hypothesized that the "stress hormone" adrenaline will interfere with its biological behavior by activating β2-AR on the surface of epithelial and fibroblast cells. In addition, propranolol has been shown to simultaneously activate the MAPK / ERK pathway downstream of β2-AR, which is closely related to epithelial and fibroblast cells. Based on this speculation: adrenaline activation of β2-AR on the surface of peri-implant epithelial cells and fibroblast cells may inhibit the formation of peri-implant soft tissue seal. Propranolol may have the potential to promote the biological behavior of epithelial and fibroblast cells on the surface of titanium sheets, thereby enhancing the peri-implant soft tissue seal. The present invention selects the main cells participating in the healing of peri-implant soft tissues, namely epithelial cells and fibroblast cells, as the research objects, and activates or blocks β2-AR in epithelial / fibroblast cells on the surface of titanium sheets by adrenaline to explore the effect of this signal on the biological behavior of cells related to the healing of peri-implant soft tissues and the related mechanism, so as to provide a basis for enhancing the peri-implant soft tissue seal.

[0033] Although it has been elucidated that the β-AR blocker propranolol accelerates skin wound healing, the effect of interfering with receptor-mediated signaling by the blocker on cells related to the healing of the per-implant soft tissue has not been explored. The present invention has confirmed that adrenaline activates β2-AR to inhibit the proliferation, migration and adhesion of epithelial cells and fibroblasts on the titanium sheet surface. The β-AR blocker propranolol can reverse these adverse effects, enhance the spreading and adhesion of epithelial / fibroblasts on the titanium sheet surface, increase the scratch healing rate and cell proliferation rate, promote epithelial cell adhesion and the expression of related genes and proteins, suggesting that propranolol may have the potential to enhance the soft tissue seal around the implant.

[0034] Establishing effective soft tissue integration between titanium metal and soft tissue is one of the current problems to be solved. After the titanium metal implant is implanted into the oral cavity, an effective soft tissue barrier needs to be established between the internal and external environments to prevent pathogen invasion, tissue inflammation and implant movement, and to ensure stable osseointegration between the submucosal alveolar bone and the implant. Insufficient soft tissue integration around the transmucosal dental implant leads to peri-implant mucositis and peri-implantitis, which may ultimately result in implant failure. Some scholars' research has shown that stress hormones may induce DNA damage in oral keratinocytes and may lead to the occurrence of oral cancer or delay wound healing. These adverse effects can be reversed by β-AR antagonists, indicating that the migratory effect is mediated by β-AR. Since β2-AR is the only β-AR expressed in epithelial and fibroblast cells, the present invention has also confirmed that β2-AR is expressed in both epithelial cells and fibroblasts, and this effect is most likely mediated by β2-AR.

[0035] The migration and adhesion of epithelial cells and fibroblasts involve the temporal and spatial reorganization of the actin cytoskeleton. The research results of this invention show that compared with the control group, the epithelial cells and fibroblasts in the adrenaline group exhibited a round "resting phenotype", while the propranolol group retained the migratory epithelial cell's pro-motility phenotype; the cells were crescent-shaped, mostly with filopodia / lamellipodia extended, which are characteristics of the migratory phenotype. A large amount of actin is contained in the filopodia / lamellipodia, which can bind to ligands in the ECM, thus completing the action of cell migration. More pseudopod structures confirmed the pro-migratory effect of propranolol. In the research related to epithelial cells, whether using the culture medium containing propranolol alone or treating with the adrenaline-containing culture medium after pre-treating with propranolol for 30 minutes had a positive effect on the cells. However, in the research related to fibroblasts, only using the culture medium containing propranolol alone could produce a positive effect. When adrenaline was added after pre-treating with propranolol for 30 minutes, although the value showed a small upward trend compared with the control group, this change was not statistically significant. The reason for this result may be that epithelial cells can secrete adrenaline by themselves, forming a local hormone regulation network for epithelial cells that express both receptors and ligands, while fibroblasts cannot. Compared with the control group, pre-treatment with propranolol prevented the binding of autocrine adrenaline in epithelial cells to AR on the cell membrane, thus promoting their biological behavior.

[0036] The soft tissue seal around the implant is a complex and coordinated biological process that requires the coordination of epithelial cells, fibroblasts, and other types of cells. After receiving the injury signal, the cells proliferate and migrate directionally to the implant surface to initiate the repair process to restore epithelial integrity. The cells participating in wound healing are not only the structural basis for tissue reconstruction but also an important source of secreting healing-related cytokines. Therefore, the proliferation ability, scratch healing rate, and adhesion number of epithelial / fibroblasts directly determine the efficiency of soft tissue repair. The research results of this invention show that adrenaline activates β2-AR, thereby inhibiting the proliferation, migration, and adhesion of epithelial cells and fibroblasts. On the contrary, propranolol increased the adhesion and spreading of attached cells, with the largest cell number, projected area, and perimeter, showing a high degree of adhesion and spreading. This indicates that propranolol promotes the attachment and spreading of epithelial cells and fibroblasts. Competitive binding to β2-AR can block the negative effect of endogenous catecholamines on the migration of epithelial and fibroblast cells, thus enhancing the motility and promoting wound healing. Some studies have confirmed the weak proliferation ability of peri-implant epithelial cells by detecting the expression of proliferating cell nuclear antigen in the junctional epithelium and peri-implant epithelial cells. The level of catecholamines in saliva under the stimulation of implant surgery stress is about 10 times higher than the normal level. Therefore, improving the bioreactivity of epithelial / fibroblasts on the titanium surface by treating with propranolol can lay a foundation for the strong adhesion of epithelial and fibroblast cells and the tight soft tissue seal around the titanium abutment clinically.

[0037] Hemidesmosome structure is a key adhesion structure that mediates the adhesion between natural teeth and junctional epithelium, and between titanium implants and peri-implant epithelial cells. It is mainly composed of laminin 332, integrin α6β4, and Plectin. Epithelial cells receiving trauma signals secrete laminin 332 and bind to each other through integrin α6β4 to enhance the affinity for the titanium substrate. Laminin α3 (LAMA3) is the core part of this structure, which binds to integrin receptors to regulate a series of cell behaviors. Integrin α6β4 is a key structure that forms hemidesmosomes, but in regulating cell-cell or cell-ECM interactions, β4 plays a greater role. In this invention, the expression levels of genes LAMA3, Integrin β4, and Plectin related to epithelial cell migration and adhesion were detected by PCR in each treatment group. Propranolol enhanced the mRNA levels of LAMA3, Integrin β4, and Plectin, thereby enhancing epithelial cell migration and adhesion.

[0038] The contact between fibroblasts and the material surface mainly relies on focal adhesions (FAs). FAs not only transmit forces at the adhesion sites to maintain strong adhesion to the extracellular matrix but also act as signal centers from which many intracellular pathways start to regulate cell behavior. During the assembly of FAs, its signaling molecules such as non-receptor tyrosine kinase FAK and structural proteins such as Vcl form supramolecular complexes, where FAK regulates cell adhesion signals and mechanosensation, and Vcl regulates adhesion strength and cell migration. Fak achieves receptor-ligand binding with Fn through its transmembrane protein to regulate cell adhesion. In this invention, the expression levels of Fn, Vcl, and Fak were detected by PCR in each treatment group. However, the effects on epithelial cells were different. Only Fn was significantly increased after propranolol treatment, and there was no statistical difference in Vcl and Fak compared with the control group. The reason may be that during wound healing, epithelial cells can participate in the formation of the connective tissue around the implant by secreting ECM factors such as laminin 332, and Fn mediates cell adhesion by binding to cell membrane integrins, thereby regulating tissue repair and wound healing. This may be the clue for the enhanced migration and adhesion of the aforementioned fibroblasts.

[0039] Activation of β-AR triggers the phosphorylation of multiple intracellular signaling molecules including MAPK / ERK to coordinate the biological behaviors of cells. ERK1 / 2 is an important part of the MAPK family, and the phosphorylation of ERK1 / 2 is crucial for biological behaviors such as cell migration, proliferation, and adhesion. Propranolol has been shown to activate the MAPK pathway downstream of β-AR. To further investigate the possible signaling mechanisms by which propranolol effectively increases the migration, proliferation, and adhesion of epithelial cells on the titanium sheet surface, this invention focused on the phosphorylation of MAPK and ERK.

[0040] It has been reported that the growth regulation of propranolol on keratinocytes depends on the MAPK / ERK signaling pathway. Therefore, propranolol may promote soft tissue healing by blocking the negative effect of β2-AR activation on MAPK / ERK phosphorylation. The protein expression levels of MAPK, ERK1 / 2, p-MAPK, and p-ERK1 / 2 in epithelial cells on the surface of titanium sheets treated with or without adrenaline and / or propranolol were detected by immunoblotting. The results showed that adrenaline activation of β2-AR reduced the expression of p-MAPK and p-ERK1 / 2 proteins. Treatment with propranolol significantly increased the phosphorylation of MAPK and ERK in epithelial cells by 4.95-fold and 2.41-fold compared with the adrenaline group and the control group. Thus, the present invention speculates that MAPK and ERK phosphorylation are necessary conditions for propranolol-mediated increased epithelial cell proliferation, migration, and adhesion, and may accelerate the soft tissue closure around implants. The results of the present invention confirm that propranolol positively regulates the biological behavior of epithelial cells through the MAPK / ERK signaling pathway, which is consistent with the results observed in other cell types previously.

[0041] The present invention further verified that propranolol regulates the biological behavior of epithelial cells through the ERK signaling pathway by adding SCH772984, which is a specific MAPK / ERK inhibitor. Compared with the addition of SCH772984, the phosphorylation levels of ITGB4, LAMA3, MAPK, and ERK were higher, and the cell proliferation rate, scratch healing, and the number of cell adhesions were also more when propranolol was treated alone, demonstrating that the functional connection between the MAPK / ERK signaling pathway and epithelial cells can be blocked by the inhibitor SCH772984. Nevertheless, the possibility of the activation of the ERK pathway by growth factor receptors secreted by cells cannot be excluded. In particular, the phosphorylation of downstream ERK activated by EGFR has been reported in epithelial cells. Therefore, further studies are needed to clarify the role of β-AR or EGFR as upstream signaling molecules in the ERK pathway in epithelial cell behavior.

[0042] To make the objectives, technical solutions, and advantages of the present invention clearer, the technical solutions in the present invention will be clearly and completely described below. Obviously, the described embodiments are some, but not all, of the embodiments of the present invention. All other embodiments obtained by those of ordinary skill in the art based on the embodiments in the present invention without creative efforts shall fall within the protection scope of the present invention.

[0043] Neither the endpoints nor any value in the ranges disclosed in this specification are limited to the exact range or value. These ranges or values should be understood to include values close to these ranges or values. For numerical ranges, the endpoint values of each range, between the endpoint values of each range, between the endpoint values of each range and individual point values, and between individual point values can be combined with each other to obtain one or more new numerical ranges, and these numerical ranges should be regarded as specifically disclosed herein.

[0044] In the description of this specification, the descriptions referring to terms such as "one embodiment", "some embodiments", "specific implementation manners", or "some specific implementation manners" etc. mean that the specific features, structures, materials, or characteristics described in connection with the embodiment or example are included in at least one embodiment or example of the embodiments of the present invention. In this specification, the schematic expressions of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials, or characteristics described can be combined in a suitable manner in any one or more embodiments or examples. In addition, without contradiction, those skilled in the art can combine and combine the different embodiments or examples described in this specification and the features of different embodiments or examples.

[0045] In the embodiments provided in this specification, where no specific technology or conditions are indicated, they shall be in accordance with the technologies or conditions described in the literature in this field or in accordance with the product specifications. Where the manufacturers of the reagents or instruments used are not indicated, they are all conventional products that can be obtained through regular channels.

[0046] Example 1 (1) Detect the effects of propranolol on cell spreading, migration, proliferation, and adhesion on the surface of titanium sheets.

[0047] First, use the CCK8 method to detect the cell viability on the surface of titanium sheets and select appropriate concentrations of adrenaline / propranolol for subsequent experiments. Treat the cells on the surface of titanium sheets with different drugs (control group: DMEM complete medium; experimental groups: DMEM containing adrenaline, DMEM containing propranolol, DMEM with propranolol pretreated for 30 min followed by adding adrenaline), and detect the effects of different drug treatments on cell morphology, proliferation, adhesion, and migration by scanning electron microscopy, immunofluorescence staining, CCK8 method, cell counting, and scratch assay. The results are shown in Figures 1-5 .

[0048] The detection results showed that when the concentration of propranolol was greater than 50 μM, cell viability was inhibited, while when the concentration of adrenaline was greater than 1 μM, cell activity decreased in a dose-dependent manner (P < 0.05). Considering the activity characteristics of the two types of cells and the cytotoxicity of the two drugs, 10 μM of propranolol and 1 μM of adrenaline were selected for subsequent studies. After adrenaline activated β2-AR, the cells shrank into a round shape, did not show the typical cell morphology, inhibited cell proliferation activity, reduced the number of cell adhesions, and hindered cell migration ( P <0.05). In contrast, the cells in the propranolol group showed good spreading. Fine and dense filamentous or lamellar pseudopodia were radially arranged around the epithelial cells. The fibroblasts showed a typical spindle or star shape. The cell scratch healing rate, proliferation activity, and adhesion number were all significantly increased ( P <0.05).

[0049] (2) Detect the effects of propranolol on the migration and adhesion-related markers of cells on the titanium surface at the gene and protein levels.

[0050] Real-time fluorescence quantitative PCR (qRT-PCR) was used to detect the mRNA expression levels of migration and adhesion-related genes of HaCaT on the titanium surface in different drug treatment groups (the same grouping) at 3 / 7 days, including laminin 332 (LAMA3), integrin β4 (ITGB4), plectin, and the migration and adhesion-related genes of human skin fibroblasts (HSF), including fibronectin (Fn), vinculin (Vcl), and focal adhesion kinase (Fak). Western Blot (WB) and immunofluorescence were used to detect the expression of migration and adhesion-related proteins LAMA3 and ITGB4 on the titanium surface. The results are shown in Figures 6-8 .

[0051] The detection results showed that the mRNA expression levels of LAMA3, ITGB4, plectin, and Fn in the propranolol alone or combined with adrenaline treatment groups increased, showing statistical differences compared with the other two groups, and the difference in the propranolol alone treatment group was more significant ( P <0.05). Except for the difference in the expression of the Vcl gene between the adrenaline group and the propranolol group after incubation for 3 days ( P <0.05), the expression of Vcl and Fak genes was similar in all treatment groups ( P > 0.05). The WB and immunofluorescence results showed that the protein expression of ITGB4 and LAMA3 in the propranolol group was higher than that in the control group and the adrenaline group ( P <0.05).

[0052] (3) Explore and verify the mechanism by which propranolol regulates the biological behavior of HaCaT on the titanium surface.

[0053] After 24 hours of treatment with different drugs (the same grouping), the expression levels of total MAPK, ERK1 / 2, p-MAPK, and p-ERK1 / 2 proteins of HaCaT on the titanium surface were detected by WB. The group treated with propranolol alone was used as the control group, and the group treated with the specific MAPK / ERK pathway inhibitor SCH772984 combined with propranolol was used as the experimental group. The morphology, proliferation, adhesion, migration, and protein expression of HaCaT were detected to verify that the regulation of the biological behavior of HaCaT on the titanium surface by propranolol was mediated by the MAPK / ERK signaling pathway. The results are shown in Figures 9-14 .

[0054] The results showed that treatment with propranolol alone or in combination with epinephrine significantly increased the phosphorylation of MAPK and ERK1 / 2 proteins ( P <0.05), especially in the group treated with propranolol alone. The expression levels of p-MAPK (P<0.01) and p-ERK ( P <0.001) in the group treated with SCH772984 plus propranolol were significantly decreased, indicating that SCH772984 successfully inhibited this pathway. Under the action of SCH772984, the scratch healing rate and the number of adhesions decreased by 61.63% ( P <0.01) and 38.28% ( P <0.0001), respectively, the proliferation activity was weakened ( P <0.05), and the protein expression levels of LAMA3 and Integrin β4 decreased ( P <0.01).

[0055] In summary, blocking the β2-AR signal with propranolol can reverse the negative effects induced by receptor activation, which is beneficial to the early spreading, migration, proliferation, and adhesion of HaCaT / HSF on the titanium surface. Propranolol promotes the expression of genes and proteins related to hemidesmosomes such as LAMA3 and Integrin β4, suggesting that propranolol has the potential to further enhance the soft tissue seal around implants. Propranolol promotes the migration, proliferation, adhesion, and expression of related proteins of HaCaT cells on the titanium surface through the MAPK / ERK signaling pathway.

[0056] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that they can still modify the technical solutions recorded in the foregoing embodiments, or perform equivalent replacements for some of the technical features; and these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. The use of propranolol in the preparation of functional drugs, characterized in that: The functional medicine has the function of promoting the integration of peri-implantation soft tissues.

2. The application according to claim 1, characterized in that: The functional medicine has the function of promoting the expression of hemidesmosome-related genes and proteins.

3. The application according to claim 2, characterized in that: The hemidesmosome-related genes and proteins include: LAMA3 gene and LAMA3 protein.

4. The application according to claim 2, characterized in that: The hemidesmosome-related genes and proteins include: Integrin β4 Gene and Integrin β4 protein.

5. The use according to claim 1, characterized in that: The functional medicine has the function of promoting the spreading, migration, proliferation and adhesion of cells on the surface of the titanium sheet.

6. The use according to claim 5, characterized in that: The functional drug has the function of interfering with the MAPK / ERK signaling pathway.

7. The use according to claim 6, characterized in that: The functional drug promotes the migration, proliferation, adhesion and expression of related proteins of HaCaT cells on the surface of the titanium sheet through the MAPK / ERK signaling pathway.

8. The use according to claim 5, characterized in that: The functional drug has the function of blocking β2-AR signals and reversing the negative effects induced by receptor activation.

9. The use according to claim 8, characterized in that: The functional drug promotes the early spreading, migration, proliferation and adhesion of HaCaT / HSF on the titanium sheet surface by blocking β2-AR signaling and reversing the negative effects induced by receptor activation.

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