Application of AANAT in preparation of medicine for regulating and controlling synthesis and regeneration potential of endogenous melatonin of inflammatory microenvironment stem cells and AANAT-targeted agonist

Abstract

The invention relates to application of AANAT in preparation of a medicine for regulating and controlling synthesis and regeneration potential of endogenous melatonin of inflammatory microenvironment stem cells, and an AANAT-targeted agonist, and relates to the technical field of biological medicine. The molecular target AANAT protein closely related to periodontal ligament stem cell endogenous melatonin synthesis and cementum differentiation in a targeted activation inflammation microenvironment is found for the first time, experimental results show that expression of AANAT in the inflammation microenvironment is reduced on the gene level and the protein level, and after the AANA level is overexpressed, the expression of the AANAT protein in the inflammation microenvironment is reduced on the protein level. The PDLSC cementum differentiation potential of inflammation injury is reversed. On the basis, the screened target molecule AANAT is subjected to drug virtual screening, and the application of the AANAT targeted activator in synthesis of stem cell endogenous melatonin and regulation and control of cementum differentiation in the inflammatory microenvironment is verified and disclosed.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and in particular to the use of AANAT in the preparation of a drug for regulating the synthesis and regeneration potential of endogenous melatonin in stem cells in an inflammatory microenvironment, and an agonist targeting AANAT. Background Art

[0002] Melatonin (MT) is one of the hormones secreted by the pineal gland in the brain. Melatonin belongs to the class of indole heterocyclic compounds, and its chemical name is N-acetyl-5-methoxytryptamine, also known as pinealogen, melatonin, and melatonin. In the human body, it is mainly secreted by the pineal gland. Other tissues, organs, and cells such as the gastrointestinal tract, retina, placenta, specific brain regions, skin, and immune cells can also synthesize melatonin. The latest research results show that almost every cell in the human body has the ability to synthesize melatonin. Melatonin has a variety of physiological and pharmacological functions, including improving sleep, regulating biological rhythms, regulating the immune system, regulating reproductive activities, etc., and is widely expressed in the human body.

[0003] In previous studies, exogenous melatonin has been taken as the main research object. However, it cannot be ignored that most sources of exogenous melatonin are chemically synthesized, so its metabolic decomposition rate is fast and its residence time in the body is short, which cannot have a significant effect on serum melatonin levels. It can also cause drowsiness, irritability, hangover effects, and even increase the risk of fractures, and can also lead to hypogonadotropic dysfunction. Therefore, exogenous melatonin supplementation cannot replace endogenous melatonin.

[0004] The synthesis of endogenous melatonin is controlled by the nervous system, and the nervous system pathway is roughly as follows: originating from the paraventricular nucleus of the hypothalamus, directly or indirectly projecting to the preganglionic sympathetic neurons of the first thoracic segment of the spinal cord. Then, through the projection of the postganglionic sympathetic neurons of the superior cervical ganglion, the nerve fibers of the pyramidal nerves are formed to reach the pineal gland to regulate melatonin synthesis. In this synthesis pathway, two melatonin synthases, arylalkylamine N-acetyltransferase (AANAT) and acetylserotonin methyltransferase (ASMT), play an important role. The expression level of their genes and enzyme activity play a decisive role in the level of melatonin synthesis; in particular, AANAT is the rate-limiting enzyme for the synthesis of endogenous melatonin.

[0005] Progressive destruction of periodontal supporting tissues caused by periodontitis is the main cause of loose or falling teeth in adults, and tissue engineering methods based on stem cell transplantation are an important strategy to achieve regeneration in the environment of periodontitis. Periodontal ligament stem cells (PDLSC) are key cells for repairing periodontal defects, but unfortunately, the periodontal inflammatory microenvironment can cause impaired function of PDLSC, especially inflammatory cytokines damage the cementum differentiation of PDLSC, which ultimately leads to difficulty in cementum regeneration. However, there is still a lack of relevant research on whether PDLSC has endogenous melatonin synthesis and whether endogenous melatonin synthesis affects the differentiation potential of stem cells in an inflammatory environment. Summary of the invention

[0006] In order to solve the above problems, the present invention provides the use of AANAT in the preparation of a drug for regulating the synthesis of endogenous melatonin and the regenerative potential of stem cells in an inflammatory microenvironment, and an agonist targeting AANAT.

[0007] In a first aspect, the present invention provides the use of AANAT in the preparation of a drug for regulating the synthesis and regenerative potential of endogenous melatonin in stem cells of an inflammatory microenvironment.

[0008] Furthermore, the expression of AANAT was downregulated at both the gene level and the protein level under the inflammatory microenvironment. Overexpression of AANAT reversed the cementum differentiation potential of PDLSC damaged by inflammation.

[0009] In a second aspect, the present invention provides a molecular target for preparing a drug for regulating the synthesis and regenerative potential of endogenous melatonin in stem cells in an inflammatory microenvironment, wherein the molecular target is AANAT.

[0010] In a third aspect, the present invention provides a method for screening drugs that regulate the synthesis and regenerative potential of endogenous melatonin in stem cells in an inflammatory microenvironment, wherein the method uses AANAT as a target.

[0011] In a fourth aspect, the present invention provides an agonist targeting ANAT, wherein the agonist targeting ANAT comprises at least one of isofelianthrin and LCKLSL.

[0012] Furthermore, the agonist targeting AANAT is isofysiaside.

[0013] In a fifth aspect, the present invention provides a use of an agonist targeting AANAT as described in any one of the fourth aspects in the preparation of a drug for regulating the synthesis and regenerative potential of endogenous melatonin in stem cells of an inflammatory microenvironment.

[0014] In a sixth aspect, the present invention provides a drug for regulating the synthesis and regenerative potential of endogenous melatonin in inflammatory microenvironment stem cells, wherein the drug for regulating the synthesis and regenerative potential of endogenous melatonin in inflammatory microenvironment stem cells comprises: 1) an agonist targeting AANAT as described in any one of the fourth aspects; 2) a pharmaceutically acceptable carrier or excipient.

[0015] The above technical solution provided by the embodiment of the present invention has at least the following advantages compared with the prior art:

[0016] The embodiments of the present invention provide the use of AANAT in the preparation of drugs for regulating the synthesis and regeneration potential of endogenous melatonin in stem cells in an inflammatory microenvironment, and agonists targeting AANAT. The present invention is the first to discover the molecular target AANAT protein that is closely related to the synthesis and cementoblastic differentiation of endogenous melatonin in periodontal ligament stem cells in an inflammatory microenvironment. The experimental results show that the expression of the AANAT in an inflammatory microenvironment is downregulated at both the gene level and the protein level. After overexpressing the AANAT level, the cementoblastic differentiation potential of PDLSC damaged by inflammation is reversed. On this basis, the present invention conducts virtual drug screening on the selected target molecule AANAT, verifies and discloses the use of an AANAT targeted activator in regulating the synthesis and cementoblastic differentiation of endogenous melatonin in stem cells in an inflammatory microenvironment. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate embodiments consistent with the invention and, together with the description, serve to explain the principles of the invention.

[0018] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, for ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative labor.

[0019] Figure 1 This is a graph showing the results of the inflammatory microenvironment inhibiting the cementoblast differentiation potential of stem cells and the synthesis of endogenous melatonin in an embodiment of the present invention.

[0020] Figure 2 This is a graph showing the results of the key molecules that interfere with melatonin synthesis damaging the cementum differentiation potential of PDLSC in an embodiment of the present invention.

[0021] Figure 3 This is a graph showing the results of PDLSC cementoblastic differentiation that reverses inflammatory damage by overexpressing key molecules for melatonin synthesis in an example of the present invention.

[0022] Figure 4Schematic diagram of the predicted active site distribution and scoring of the Human AANAT protein and the virtual screening of small molecule compounds targeting the Human AANAT protein in the embodiments of the present invention.

[0023] Figure 5 This is a diagram showing the evaluation results of the cytotoxicity of small molecule compounds in the examples of the present invention.

[0024] Figure 6 This is a graph showing the effect of small molecule compounds on the expression level of stem cell AANAT protein in an example of the present invention.

[0025] Figure 7 This is a graph showing the effect of small molecule compounds on the cementum differentiation of PDLSC in an example of the present invention.

[0026] Figure 8 2D and 3D schematic diagrams of the binding mode between the drug and AANAT in the embodiments of the present invention.

[0027] Fig. 9 This is a surface plasmon resonance (SPR) result diagram in an embodiment of the present invention.

[0028] Fig.10 The molecular dynamics simulation results of the present invention verify the interaction between forsythiaside and AANAT protein Figure 1 .

[0029] Fig.11 The molecular dynamics simulation results of the present invention verify the interaction between forsythiaside and AANAT protein Figure 2 . DETAILED DESCRIPTION

[0030] In order to make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the technical solution in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0031] Unless otherwise specified, various raw materials, reagents, instruments and equipment used in the present invention can be purchased from the market or prepared by existing methods.

[0032] Melatonin is a neurohormone synthesized in the pineal gland and various organs and cells, and has the function of regulating sleep and circadian rhythm. As a strong antioxidant, melatonin can alleviate cell damage caused by oxidative stress, such as cell regeneration potential damage caused by inflammatory microenvironment. Arylalkylamine N-acetyltransferase (AANAT) and acetyl serotonin methyltransferase (ASMT) are important enzymes in the synthesis of melatonin. Their expression level and activity determine the synthesis level of melatonin, especially AANAT as the rate-limiting enzyme for the synthesis of endogenous melatonin. The maintenance of its level and activity is of great significance to the synthesis of melatonin and the maintenance of cell function. The present invention selects endogenous melatonin as the research object, explores the effect of changes in endogenous melatonin on cells under the inflammatory microenvironment, and uses its key enzymes as targets for drug screening, in order to provide a solid foundation for the future targeted regulation of endogenous melatonin to reverse cell damage.

[0033] In order to make up for the shortcomings of the existing technology, the inventors combined the previous experimental basis and confirmed that under the in vitro inflammatory microenvironment, the regeneration potential of human periodontal ligament stem cells (PDLSC) was inhibited, the synthesis level of cementoblast differentiation markers such as CEMP-1, BSP, and CAP was reduced, and the secretion of alkaline phosphatase was reduced; the expression of melatonin and its related synthases AANAT and ASMT were all downregulated. This shows that the inflammatory microenvironment inhibits the biosynthesis of endogenous melatonin in PDLSC.

[0034] To further verify whether the biosynthesis of endogenous melatonin plays an important role in the regeneration of stem cells, the present invention knocked down and overexpressed AANAT and ASMT to observe the changes in the level of cementoblastic differentiation of stem cells after melatonin synthesis was restricted. Finally, the results of qRT-PCR and Western Blot showed that interfering with the key molecules of melatonin synthesis can significantly reduce the expression of stem cell cementoblastic differentiation markers, that is, the regeneration potential of stem cells is impaired after melatonin synthesis is impaired.

[0035] Improving the level and activity of AANAT is crucial to reversing the stem cell regeneration potential damaged by the inflammatory microenvironment. However, there are currently no recognized agonists and related drugs targeting AANAT. Through high-throughput virtual screening of small molecule compounds targeting AANAT, a small molecule compound that can target AANAT and increase its protein expression level is identified. The present invention predicts the three-dimensional structure of AANAT by AlphaFold, and uses the sitemap module in the Schrödinger software to predict and score the active sites in the AANAT structure, and selects the target with the highest score for subsequent molecular screening. Then, 18,700 compounds in the compound library are output in the form of 3D structures and molecularly docked with AANAT. Through three rounds of screening in high-throughput screening mode, standard mode, and high-precision mode, the top 47 compounds with molecular docking scores greater than 10 are initially obtained, and then cytotoxicity tests and stem cell regeneration potential impact tests are performed on them, as well as a series of molecular dynamics simulation experiments, and finally it is determined that isoflavone glycosides are the best targeted drug.

[0036] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are intended only to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods in the following examples that do not specify specific conditions are usually measured according to national standards. If there is no corresponding national standard, then the conditions recommended by the manufacturer are followed.

[0037] The information of the main reagents and equipment involved in the following examples is as follows:

[0038] enamel matrix protein derivative EMD (Straumann, USA);

[0039] TNF-α (Novoprotein, China);

[0040] IL-1β (Novoprotein, China);

[0041] Micropoly TransfecterTM Cell Reagent (Micropoly, China);

[0042] Ⅱ1st Strand cDNA Synthesis SuperMix (Yisheng, China);

[0043] qPCR SYBR Green Master Mix (Yisheng, China);

[0044] CFX96 real-time fluorescence quantitative PCR system (Bio-Rad, USA);

[0045] Rabbit anti-BSPII(#5468, Cell Signaling Technology);

[0046] Rabbit anti-CEMP-1 (ab134231, Abcam);

[0047] Mouse anti-CAP (sc-53947, Santa Cruz Biotechnolog);

[0048] Mouseanti-β-actin (BM0627, Boster);

[0049] HRP-Goat Anti-Mouse IgG (H+L) (SA00001-1, ProteinTech);

[0050] HRP-Goat Anti-Rabbit IgG (H+L) (SA00001-2, ProteinTech);

[0051] Rabbit Anti-AANAT (PA5-26631, Proteintech);

[0052] Rabbit Anti-AMST (NBP2-92663, Novus);

[0053] Alexa 488-labeled donkey anti-rabbit IgG (Yisheng, China);

[0054] BCIP / NBT alkaline phosphorylation staining kit (Biyuntian, China);

[0055] BCA protein concentration assay kit (Heter, China);

[0056] CCK-8 cell proliferation / toxicity detection kit (Dojindo, China);

[0057] Melatonin ELISA test kit (Jianglai Biotechnology, China);

[0058] Mito Red CMXRos (Invitrogen, USA);

[0059] Puromycin (Diyi Biologicals, China);

[0060] AANAT overexpression lentivirus (TSINGKE, China);

[0061] ASMT overexpression lentivirus (TSINGKE, China);

[0062] recombinant AANAT protein (TSINGKE, China);

[0063] BPA (MedChemExpress, USA);

[0064] Calcium chloride solution (Beyotime, China);

[0065] Dithiothreitol (Beyotime, China);

[0066] Tri-HCl buffer (Beyotime, China);

[0067] Protein tracer loading buffer (Zhonghui Hecai, China);

[0068] Horseradish peroxidase-labeled Streptavidin (Beyotime, China);

[0069] Methacryloyl gelatin-60 (EngineeringForLife, China);

[0070] Nikon confocal laser microscope (Nikon, Japan);

[0071] The primers were ordered from Qingke Biotechnology Co., Ltd. and the sequences are as follows:

[0072] β-actin (Forward), 5'-TGGCACCCAGCACAATGAA-3';

[0073] β-actin (Reverse), 5'-CTAAGTCATAGTCCGCCTAGAAGCA-3';

[0074] CEMP-1(Forward), 5'-CTGAGGATGGCACATCAAG-3';

[0075] CEMP-1(Reverse), 5'-GAGGTTCTCAGCCGATGTGTTAG-3';

[0076] CAP (Forward), 5'-CGCGTGGACTGTGACAGAGA-3';

[0077] CAP (Reverse), 5'-GGCAAGGCAGCGTATATTGTAAGA-3';

[0078] BSP (Forward), 5'-GAACCACTTCCCCACCTTTT-3';

[0079] BSP (Reverse), 5'-ATTCTGACCATCATAGCCATCG-3';

[0080] AANAT(Forward),5'-CTTCATCTCCGTCTTGGGCG-3';

[0081] AANAT(Reverse),5'-ATGATGAAGGCCACAAGGCA-3';

[0082] ASMT (Forward), 5'-CTGGGCGTTGTTTGACCTTCT-3';

[0083] ASMT(Reverse),5'-TCCCCTCGTCTCCACTTTCA-3';

[0084] si-AANAT,5'-CUGGUACCCUUCUAUGAGATT-3';

[0085] si-ASMT, 5'-GCAGCAUGCUGAAGUACAUTT-3';

[0086] si-NC, 5'-UUCUCCGAACGUGUCACGUTT-3'.

[0087] Example

[0088] This example provides the use of AANAT in the preparation of a drug for regulating the synthesis and regenerative potential of endogenous melatonin in inflammatory microenvironment stem cells and an agonist targeting AANAT, which specifically includes the following steps:

[0089] 1. Effects of inflammatory microenvironment on endogenous melatonin biosynthesis and differentiation potential of PDLSCs

[0090] The present invention simulates the periodontal inflammatory microenvironment by inflammatory cytokines (TNF-α plus IL-1β), and induces the differentiation of stem cells into cementum by adding cementoblastic induction solution (α-MEM complete medium with 100 μg / mL enamel matrix protein derivative EMD). After culturing the stem cells (normal group) induced by simple cementoblastic differentiation and the stem cells (inflammatory group) co-treated with inflammatory factors + cementoblastic differentiation induction solution for 7 days, samples were collected to detect the expression of stem cell cementoblastic differentiation markers, and it was found that the expression of BSP and CEMP-1 was reduced at the gene level, and all three were reduced at the protein level. Then, by alkaline phosphatase staining experiment, compared with the normal cell inflammatory microenvironment, the secretion of alkaline phosphatase was reduced, and the differentiation potential of PDLSC was significantly weakened. Therefore, the inflammatory microenvironment will inhibit the differentiation potential of PDLSC. At the same time, the present invention detected that the endogenous melatonin secretion of cells under the inflammatory microenvironment was reduced, and the expression of melatonin-related synthases AANAT and ASMT was downregulated at both the gene level and the protein level. In particular, the present invention extracted melatonin from mitochondria in two states: normal and after inflammation treatment, and found that the inflammatory microenvironment inhibited the secretion of mitochondrial melatonin. Further cell immunofluorescence experiments also showed that the secretion of mitochondrial melatonin in PDLSC under the inflammatory microenvironment was reduced. Endogenous melatonin secretion decreased under the inflammatory microenvironment and was accompanied by a significant weakening of the differentiation potential of PDLSC. Therefore, the present invention preliminarily determined that the inflammatory microenvironment inhibited the biosynthesis of endogenous melatonin in PDLSC mitochondria, thereby inhibiting the differentiation of PDLSC.

[0091] 2. Functional verification of the effect of endogenous melatonin synthesis on the cementogenic differentiation of PDLSCs

[0092] In order to determine whether endogenous melatonin synthesis is involved in the cementoblastic differentiation of PDLSC in an inflammatory microenvironment, the present invention transfected PDLSC with siRNA of the target molecule, silenced the expression of the key enzymes AANAT and ASMT molecules in endogenous melatonin synthesis, and observed its effect on the cementoblastic differentiation of stem cells.

[0093] 2.1 siRNA transfection into PDLSCs and evaluation of transfection efficiency

[0094] ① In order to silence the expression of AANAT and ASMT molecules, siRNA targeting the target molecules and negative control (si-NC) were ordered from Qingke Bio in advance. The sequences of siRNA were si-AANAT, 5'-CUGGUACCCUUCUAUGAGATT-3'; si-ASMT, 5'-GCAGCAUGCUGAAGUACAUTT-3'. After centrifugation of the centrifuge tube containing siRNA lyophilized powder, sterile RNase-free ddH 2 O, prepare a 20μM working solution. ② Use 5×104 / well, inoculate PDLSC in a 6-well plate. When PDLSC covers 50% of the bottom of the culture plate, replace it with fresh 1200μL culture medium and use the transfection reagent MicropolyTransfecterTM Cell Reagent for transient transfection. ③ Prepare transfection complex: First, directly mix 8μL transfection reagent with 8μL si-NC or specific siRNA (both at a concentration of 20μM) in a 1:1 ratio, and pipette 15 times to mix. Let the mixture stand at room temperature for 10 minutes. ④ Add the prepared 16μL transfection complex to 1200μL culture medium in a 6-well plate and place it at 37°C, 5% CO 2 ⑤ Evaluation of transfection efficiency: After 24-72h, the silencing efficiency of each RNA in PDLSC was evaluated by qRT-PCR. The results showed that siRNA can successfully transfect PDLSC and silence the expression of target molecules, that is, compared with the control group (si-NC group) cells, siRNA (si-AANAT and si-ASMT) can significantly reduce the expression levels of AANAT and ASMT in PDLSC after 48h of transfection.

[0095] 2.2 Effects of silencing endogenous melatonin key molecules on cementogenic differentiation of PDLSCs

[0096] After successfully verifying the interference effect of siRNA, si-AANAT and si-ASMT were used to interfere with PDLSC after cementoblast differentiation induction (normal group). Since transfection is a transient transfection, the culture medium was replaced every 3 days and re-transfected. After 7 days, qRT-PCR and protein immunoblotting (Western blot) were used to detect the effect of silencing target molecules on the expression of PDLSC cementoblast differentiation-related genes and protein markers (BSP, CEMP-1 and CAP). The results showed that compared with the control group (si-NC), silencing of AANAT and ASMT (si-AANAT and si-ASMT) significantly reduced the expression levels of cementoblast-related genes and proteins in PDLSC, and the effect of AANAT was more significant.

[0097] 3. Further verify the role of endogenous melatonin key molecules in regulating the cementoblastic differentiation of PDLSC through functional studies.

[0098] Previous studies have confirmed that silencing of endogenous melatonin key molecules AANAT and ASMT will inhibit the cementogenic differentiation of PDLSC. Subsequently, the present invention will further study the effect of overexpression of AANAT and ASMT on PDLSC in the inflammatory microenvironment treated with melatonin.

[0099] 3.1 PDLSCs infected with lentivirus overexpressing AANAT and ASMT

[0100] ① In order to overexpress the target molecules AANAT and ASMT, the sequences of AANAT and ASMT were cloned into the lentiviral vector pLVX-IRES-Puro, and then the lentivirus was used to infect PDLSC according to the instructions. The non-specific lentiviral vector was used as a negative control vector (ov-NC). ② After determining the appropriate infection multiplicity MOI through preliminary experiments, the 3rd-5th generation PDLSCs were digested with trypsin, resuspended, and counted on a cell counting plate. 2×10 5 ③ When PDLSCs cover 50% of the bottom of the culture plate, replace the culture medium with 2 mL of fresh α-MEM complete medium, and infect PDLSCs with the control lentivirus and the target lentivirus according to the selected MOI value (MOI = 10), and place them at 37°C, 5% CO 2 incubator overnight. ④Replace with fresh culture medium (2mL α-MEM complete medium) and continue culturing for 48-72h. ⑤Use 5μg / mL of puromycin (Polybrene) to screen the virus-infected cells for stable strains. After 48h of puromycin treatment, collect the surviving PDLSCs and re-inoculate them in a 60mm diameter culture dish for passaging. ⑥After 2-4 generations, evaluate the transfection efficiency of AANAT and ASMT overexpressing lentivirus in PDLSCs by qRT-PCR.

[0101] The identification results of qRT-PCR showed that compared with the control group (ov-NC group) cells, the monoclonal stable cell lines overexpressing AANAT and ASMT (ov-AANAT and ov-ASMT) could significantly increase the expression levels of AANAT and ASMT in PDLSCs.

[0102] 3.2 Effects of overexpression of AANAT and ASMT on melatonin-reversed cementogenic differentiation of PDLSCs in an inflammatory environment

[0103] After successfully verifying the overexpression effect of AANAT and ASMT overexpression lentivirus, PDLSCs were infected according to the above steps to construct monoclonal stable cell lines overexpressing AANAT and ASMT (ov-AANAT and ov-ASMT) to evaluate the changes in their cementoblastic differentiation levels. After the constructed monoclonal stable cell lines overexpressing AANAT and ASMT (ov-AANAT and ov-ASMT) and the control group cells (ov-NC) were incubated in the inflammation treatment group for 7 days, the effects of overexpression of AANAT and ASMT (ov-AANAT and ov-ASMT) on the expression of PDLSC cementoblastic differentiation-related genes and protein markers (BSP, CEMP-1 and CAP) were detected. The results showed that compared with the control group (ov-NC), the overexpression of AANAT and ASMT reversed the damage of the inflammatory environment to the cementoblastic differentiation-related genes and proteins of PDLSC.

[0104] 4. High-throughput virtual screening of small molecule compounds targeting AANAT proteins

[0105] The present invention first predicts the three-dimensional structure of AANAT through AlphaFold, uses the sitemap module in the Schrödinger software to predict and score the active sites in the AANAT structure, and selects site1 with a higher score as the target for subsequent screening. After determining the target, the present invention outputs 18,700 compounds in the compound library in the form of 3D structures and performs molecular docking with AANAT. The Virtual Screening Workflow module is used for virtual screening, the prepared compounds are imported, and the Glide module is used for molecular docking, that is, the receptor and ligand molecules are docked with each other through geometric matching and energy matching.

[0106] First, the prepared molecules were screened using the high-throughput screening (HTVS) mode in the Glide module, and the molecules with the top 15% of the scores were selected for the second round of screening using the standard (SP) mode; then the molecules with the top 15% of the scores were selected for the third round of screening using the high-precision (XP) mode, and all screening results were output. The binding force between the target and the compound, the compound structure, etc. were manually reviewed. Thus, through three rounds of screening in high-throughput screening mode, standard mode, and high-precision mode, the top 47 compounds with molecular docking scores greater than 10 were initially obtained as screening results.

[0107] 5. Validation and identification of small molecule compounds targeting AANAT proteins

[0108] In order to verify whether the small molecule compounds screened out in the above experiments can target and activate AANAT in cells, the present invention selects the top 47 small molecule compounds with the absolute value of docking score for subsequent research.

[0109] 5.1 Evaluation of cytotoxicity of small molecule compounds

[0110] ① Drug screening first requires confirmation that the drug has no obvious toxicity to cells. The present invention uses CCK-8 to detect the effects of different concentrations of drugs on cell toxicity. In order to detect the toxicity of small molecule compounds to cells, the above 47 small molecule compounds (concentration of 10mM) were ordered from MCE Company, and after standing and melting at room temperature, cells were treated with final concentrations of 10μM and 100μM. ② 3×10 3 PDLSCs were seeded into 96-well plates at a density of 1 / well, and 100 μL of complete medium was added to each well. After the cells adhered to the wall, small molecule compounds were added. ③ 0.1 μL and 1 μL of different small molecule compounds were added respectively, mixed thoroughly with the culture medium, and placed at 37°C and 5% CO 2 ④ Cytotoxicity evaluation: After 72 hours, the cytotoxicity of different small molecule compounds was evaluated by using CCK-8 reagent. Small molecule compounds with obvious cytotoxicity were excluded, and the remaining small molecule compounds were subjected to subsequent studies. 26 drugs were screened out that had no significant toxicity to cells at concentrations of 10μM and 100μM.

[0111] 5.2 Effects of small molecule compounds on the expression of AANAT protein in stem cells

[0112] ① In order to detect the effects of the remaining small molecule compounds in the above experiment on the expression level of AANAT protein in stem cells, 1×10 5 PDLSCs were seeded in 6-well plates at a density of 100 μg / well, and 2 mL of complete medium was added to each well. When the PDLSC fusion reached 80%, 2 μL of different small molecule compounds were added. ② After 72 hours, the effects of different small molecule compounds on the expression level of AANAT protein in PDLSC were evaluated by Western blot. Six small molecule compounds that can significantly increase the expression level of AANAT protein were screened for subsequent research.

[0113] 5.3 Further verification of the effect of small molecule compounds on cementogenic differentiation of PDLSCs

[0114] ① In order to detect the effect of the small molecule compounds screened in the above experiment on the differentiation and regeneration potential of stem cells into cementum, 1×10 5PDLSCs were seeded in 6-well plates at a density of 100 μg / well. ② When the PDLSC fusion reached 80%, it was replaced with 2 mL of cementum induction solution, and 2 μL of different small molecule compounds were added respectively. ③ After replacing the new cementum induction solution every 3 days, small molecule compounds need to be added again. ④ After 7 days, the effects of different small molecule compounds on the expression of genes and protein markers related to PDLSC cementum differentiation were detected by qRT-PCR and Western blot. It was found that the two drugs, forsythiaside and LCKLSL, had significant effects on stem cell differentiation. Among them, forsythiaside had the most significant effect on the AANAT protein level and stem cell differentiation potential. Therefore, forsythiaside was selected as the best drug for subsequent drug-level verification.

[0115] 6. Surface plasmon resonance technology and molecular dynamics simulation to verify the interaction between forsythiaside and AANAT protein

[0116] The binding mode of the drug and AANAT was mapped in 2D and 3D, and then the affinity constant of the drug and AANAT was determined to be 7.41×10 -6 M, indicating that there is a strong bond between the two.

[0117] Gromacs 2021.7 software was used to perform molecular dynamics simulations on the complex of AANAT protein and ligand as well as AANAT pure protein. The Amber99SB all-atom force field and TIP3P water model were selected. During the MD simulation, all bonds involving hydrogen atoms were constrained using the LINCS algorithm with an integration step of 2 fs. The electrostatic interaction was calculated using the (Particle-mesh Ewald) PME method. The non-bonded interaction cutoff value was set to Updated every 50 steps. The V-rescale temperature coupling method was used to control the simulation temperature to 298.15K, and the Parrinello-Rahman method was used to control the pressure to 1 bar. First, the steepest descent method was used to minimize the energy of the two systems to eliminate the close contact between atoms; then, 100ps of NVT and NPT equilibrium simulations were performed at 298.15K; finally, the system was simulated by MD for 100ns, and the conformation was saved every 50ps. The visualization of the simulation results was completed using the Gromacs embedded program and VMD. Finally, the present invention analyzes the root mean square deviation (RMSD), root mean square fluctuation (RMSF), radius of gyration (Rg), solvent accessible surface area (SASA), protein secondary structure statistics in the molecular dynamics simulation trajectories of the prot-lig system and the prot-only system, as well as the number of hydrogen bonds, salt bridges, Pi-Pi interactions between the protein and the ligand in the complex, the relative free energy distribution, and the comparison of the complex structure at six moments of 0, 20, 40, 60, 80, and 100 ns, as well as the comparison of the small molecule conformations. In addition, the present invention uses the MM / GBSA method to calculate the average binding free energy between the protein and the small molecule.

[0118] RMSD and RMSF analysis found that the curve after AANAT binds to the drug is lower and smoother, proving that the binding of drug molecules contributes to the stability of the protein as a whole, that is, the complex system is more stable. Rg analysis, SASA analysis, and free energy landscape diagrams show that after drug binding, the stability of AANAT protein is enhanced and the expression level increases. Secondly, observation of the conformation of the complex at six moments found that the conformation of the drug molecule has obvious changes, but the drug and AANAT are always combined in the same position, indicating that the drug has good adaptability to AANAT and the complex formed by the two has good stability. Therefore, the present invention confirms through a series of molecular dynamics experiments that the drug has good affinity with AANAT and strong stability after binding.

[0119] 7. The experimental results are as follows:

[0120] Figure 1 This is the result of the inflammatory microenvironment inhibiting the differentiation potential of stem cells into cementum and the synthesis of endogenous melatonin. Figure 1Middle: Figure A shows that inflammation treatment reduces the gene level of stem cell cementoblast differentiation markers; Figure B shows that inflammation treatment reduces stem cell alkaline phosphatase activity (i.e., mineralization level decreases); Figure C shows that inflammation treatment reduces the protein level of stem cell cementoblast differentiation markers; Figure D shows that inflammation treatment reduces the protein level of stem cell cementoblast differentiation markers; Figure E shows that inflammation treatment reduces the level of melatonin secreted by stem cells on the third day; Figure F shows that inflammation treatment reduces the gene level of stem cell melatonin; Figure G shows that inflammation treatment reduces the protein level of stem cell melatonin; Figure H shows that inflammation treatment reduces the protein level of stem cell melatonin; Figure I shows that inflammation treatment reduces the level of melatonin in stem cell mitochondria; Figure J shows that the level of melatonin in stem cell mitochondria decreased after inflammation treatment by immunofluorescence detection, and the co-localization of mitochondrial markers and melatonin synthase markers was used for judgment. After 7 days of culture of stem cells induced by simple cementoblast differentiation (normal group) and stem cells co-treated with inflammatory factors + cementoblast differentiation induction solution (inflammatory group), the cementoblast differentiation potential of PDLSC in the inflammatory microenvironment was significantly weakened compared with normal cells ( Figure 1 AD). Therefore, the inflammatory microenvironment will inhibit the regeneration potential of PDLSC. At the same time, the present invention detected that the secretion of endogenous melatonin was reduced under the inflammatory microenvironment ( Figure 1 E), the expression of melatonin-related synthases AANAT and ASMT was downregulated at both the gene and protein levels ( Figure 1 FH). In addition, the inflammatory microenvironment inhibits the secretion of mitochondrial melatonin ( Figure 1 IJ), therefore, the present invention determines that the inflammatory microenvironment inhibits the biosynthesis of endogenous melatonin in the mitochondria of periodontal ligament stem cells, thereby inhibiting the differentiation of PDLSC.

[0121] Figure 2 The results of interfering with the key molecules of melatonin synthesis and damaging the cementum differentiation potential of PDLSC are shown in the figure. Figure 2Middle: Figure A shows that the key molecules AANAT and ASMT that interfere with melatonin synthesis (si-AANAT and si-ASMT groups) affect the level of melatonin synthesis in stem cells; Figure B shows the interference efficiency gene identification of the key molecules AANAT and ASMT that interfere with melatonin synthesis (si-AANAT and si-ASMT groups); Figure C shows the gene level of the key molecules AANAT and ASMT that interfere with melatonin synthesis (si-AANAT and si-ASMT groups) that affect the cementoblastic differentiation markers of stem cells; Figure D shows the protein level of the key molecules AANAT and ASMT that interfere with melatonin synthesis (si-AANAT and si-ASMT groups) that affect the cementoblastic differentiation markers of stem cells; Figure E shows the protein level identification of the interference efficiency protein level of the key molecules AANAT and ASMT that interfere with melatonin synthesis (si-AANAT and si-ASMT groups); Figure F shows the protein level of the key molecules AANAT and ASMT that interfere with melatonin synthesis (si-AANAT and si-ASMT groups) that affect the cementoblastic differentiation markers of stem cells. After siRNA of AANAT and ASMT was transfected into PDLSC, siRNA could successfully transfect PDLSC and silence the expression of AANAT and ASMT, and reduce the secretion level of melatonin ( Figure 2 AB). After si-AANAT and si-ASMT interfered with the induction of cementoblast differentiation in PDLSC, compared with the control group (si-NC), the silencing of AANAT and ASMT (si-AANAT and si-ASMT groups) significantly inhibited the expression levels of cementoblast-related genes and proteins in PDLSC ( Figure 2 CF). Therefore, the regenerative potential of stem cells is impaired after impaired melatonin synthesis.

[0122] Figure 3 The results of PDLSC differentiation into cementum by overexpressing key molecules for melatonin synthesis reversed inflammatory damage. Figure 3Middle: Figure A shows the overexpression efficiency gene identification of overexpression of AANAT, a key molecule in melatonin synthesis (ov-AANAT); Figure B shows the overexpression efficiency gene identification of overexpression of ASMT, a key molecule in melatonin synthesis (ov-ASMT group); Figure C shows the effect of overexpression of AANAT and ASMT, key molecules in melatonin synthesis (ov-AANAT and ov-ASMT groups), on the gene level of BSP, a marker for cementoblastic differentiation of stem cells; Figure D shows the effect of overexpression of AANAT and ASMT, key molecules in melatonin synthesis (ov-AANAT and ov-ASMT groups), on the gene level of BSP, a marker for cementoblastic differentiation of stem cells Figure 1 shows the CAP gene level of stem cells after overexpression of key molecules of melatonin synthesis, AANAT and ASMT (ov-AANAT and ov-ASMT groups), which affect the gene level of CEMP-1, a marker of cementoblastic differentiation of stem cells; Figure 2 shows the protein level of cementoblastic differentiation markers after overexpression of key molecules of melatonin synthesis, AANAT and ASMT (ov-AANAT and ov-ASMT groups); Figure 3 shows the quantitative protein level of cementoblastic differentiation markers after overexpression of key molecules of melatonin synthesis, AANAT and ASMT (ov-AANAT and ov-ASMT groups). Overexpression of AANAT and ASMT significantly increased the levels of cementoblastic differentiation markers, confirming that increasing the synthesis levels of AANAT and ASMT can restore the regenerative potential of stem cells damaged by inflammation.

[0123] Figure 4 The predicted active site distribution and scoring of Human AANAT protein and the schematic diagram of virtual screening of small molecule compounds targeting Human AANAT protein. Figure 4 In: Figure A is the predicted active site distribution of Human AANAT protein; Figure B is a schematic diagram of the virtual screening process of drugs targeting Human AANAT protein; Figure C shows 6 drugs with molecular docking scores greater than 10 and good effects on regulating AANAT protein that were screened out after virtual drug screening. The three-dimensional structure of AANAT was predicted by AlphaFold, and the sitemap module in the Schrödinger software was used to predict and score the active sites in the AANAT structure, and site1 with a higher score was selected as the target for subsequent screening. After determining the target, the present invention outputs 18,700 compounds in the compound library in the form of 3D structures and performs molecular docking with AANAT. After three rounds of screening in high-throughput screening mode, standard mode, and high-precision mode, the top 47 compounds with molecular docking scores greater than 10 were initially obtained ( Figure 4 ).

[0124] Figure 5 This is the evaluation result of the cytotoxicity of small molecule compounds. Figure 5Middle: Figures AH show the effects of different drugs on cell proliferation detected by CCK8 after 47 compounds were screened and treated with compounds at 10μM and 100μM concentrations for 3 days. The cytotoxicity of different small molecule compounds was detected using CCK-8 reagent, and small molecule compounds with significant inhibitory effects on PDLSC proliferation were excluded. The effects of different concentrations of drugs on cytotoxicity were detected by CCK-8, and it was found that 26 drugs had no significant toxicity to cells at concentrations of 10μM and 100μM ( Figure 5 ).

[0125] Figure 6 This is the result diagram of the effect of small molecule compounds on the expression level of stem cell AANAT protein. Figure 6 Middle: Figure A shows the effect of the first batch of 8 compounds (at 10μM) on the expression level of stem cell AANAT protein; Figure B shows the effect of the second batch of 9 compounds (at 10μM) on the expression level of stem cell AANAT protein; Figure C shows the effect of the third batch of 9 compounds (at 10μM) on the expression level of stem cell AANAT protein. Western blot was used to verify the effect of different small molecule compounds on the expression level of stem cell AANAT protein after treatment, and small molecule compounds that can significantly increase the expression level of AANAT protein were screened out. Finally, 6 drugs were confirmed to significantly increase the expression level of AANAT protein ( Figure 6 ).

[0126] Figure 7 This is the result diagram of the effect of small molecule compounds on the cementum differentiation of PDLSC. Figure 7 Middle: Figure A shows the effect of Auxinole (at 10μM) on stem cell cementoblastic differentiation markers; Figure B shows the effect of Isoforsythiaside (at 10μM) on stem cell cementoblastic differentiation markers; Figure C shows the effect of LCKLSL (at 10μM) on stem cell cementoblastic differentiation markers; Figure D shows the effect of Forsythoside E (at 10μM) on stem cell cementoblastic differentiation markers; Figure E shows the effect of Stachyose (at 10μM) on stem cell cementoblastic differentiation markers; Figure F shows the effect of Maltopentaose (at 10μM) on stem cell cementoblastic differentiation markers. Through the detection of stem cell differentiation markers, it was found that the two drugs, Isoforsythiaside and LCKLSL, had a significant effect on stem cell differentiation ( Figure 7 ), among which isofuscin was the drug with the most significant effect on AANAT protein level and stem cell differentiation potential, so isofuscin was selected as the best drug for subsequent drug-level verification.

[0127] Figure 8 2D and 3D schematic diagrams of the binding mode of drugs and AANAT; Fig. 9 Schematic diagram of molecular dynamics simulation of surface plasmon resonance (SPR): strong binding of drug to protein (affinity 7.41×10-6M). Fig.10 and Fig.11 This is the result of molecular dynamics simulation to verify the interaction between forsythiaside and AANAT protein. Fig.10 Middle: Figure I shows that the curve after AANAT and drug binding is lower and more stable according to RMSD analysis; Figure J shows that the curve after AANAT and drug binding is lower and more stable according to RMSF analysis; Fig.11 Middle: K shows that after drug binding, the stability of AANAT protein is enhanced and the expression level is increased through Rg analysis; L shows that after drug binding, the stability of AANAT protein is enhanced and the expression level is increased through SASA analysis; M shows that after drug binding, the stability of AANAT protein is enhanced and the expression level is increased through free energy landscape diagram; N shows that after drug binding, the stability of AANAT protein is enhanced and the expression level is increased through protein secondary structure statistics; O shows the number of hydrogen bonds, salt bridges, Pi-Pi and other interactions between protein and ligand in the complex; P shows that after drug binding, the stability of AANAT protein is enhanced and the expression level is increased through relative free energy distribution; Q shows drug binding through the contribution of amino acid residues of protein; R shows the small molecule conformation comparison at six times: 0, 20, 40, 60, 80, and 100 ns; S shows the complex structure comparison at six times: 0, 20, 40, 60, 80, and 100 ns. 2D and 3D mapping of the binding mode of drug and AANAT ( Figure 8 ), and then the affinity constant between the drug and AANAT was determined by surface plasmon resonance technology to be 7.41×10 -6 M, indicating that there is a strong bond between the two ( Fig. 9). The two were subjected to molecular dynamics simulation, and RMSD and RMSF analysis revealed that the curve after AANAT and the drug were bound was lower and smoother, proving that the binding of drug molecules contributes to the overall stability of the protein, that is, the complex system is more stable. Through Rg analysis, SASA analysis, free energy landscape diagram, protein secondary structure statistics, the number of hydrogen bonds, salt bridges, Pi-Pi and other interactions between the protein and the ligand in the complex, relative free energy distribution, and the comparison of complex structures at six moments of 0, 20, 40, 60, 80, and 100 ns, as well as small molecule conformation comparison, it was shown that after drug binding, the stability of the AANAT protein was enhanced and the expression level increased. Secondly, observation of the conformation of the complex at six moments revealed that the conformation of the drug molecule had obvious changes, but the drug and AANAT were always bound to the same position, indicating that the drug has good adaptability to AANAT and the stability of the complex formed by the two is good. Therefore, the present invention confirms through a series of molecular dynamics experiments that the drug has a good affinity with AANAT and strong stability after binding ( Fig.10 ).

[0128] In summary, the present invention provides a drug that can target and activate the rate-limiting enzyme (AANAT protein) of endogenous melatonin synthesis. First, a molecule AANAT that is closely related to the regulation of cementoblast differentiation in an inflammatory microenvironment was confirmed, and it was found that endogenous melatonin synthesis and the level of synthase AANAT protein were significantly reduced under inflammatory conditions, thereby affecting the cementoblast differentiation potential of stem cells. Further virtual drug screening was performed on the screened target molecule AANAT, and the use of an AANAT targeted activator (isoforsythiaside) in the regulation of endogenous melatonin synthesis and cementoblast differentiation of stem cells in an inflammatory microenvironment was verified and disclosed. It provides a theoretical basis for the future targeted regulation of endogenous melatonin to reverse cell damage, and provides a new clinical transformation path for the treatment of bone and periodontal defects in patients with periodontitis.

[0129] Various embodiments of the present invention may be presented in the form of a range; it should be understood that the description in the form of a range is only for convenience and brevity, and should not be understood as a rigid limitation on the scope of the present invention; therefore, the range description should be considered to have specifically disclosed all possible sub-ranges and single numerical values ​​within the range. For example, the range description from 1 to 6 should be considered to have specifically disclosed sub-ranges, such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., as well as single numbers within the range, such as 1, 2, 3, 4, 5 and 6, which apply regardless of the range. In addition, whenever a numerical range is indicated herein, it is meant to include any cited number (fractional or integer) within the indicated range.

[0130] The foregoing is merely a specific embodiment of the present invention, which enables those skilled in the art to understand or implement the present invention. Various modifications to these embodiments will be apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but rather to the widest scope consistent with the principles and novel features claimed herein.

Claims

1. Application of AANAT in the preparation of drugs for regulating endogenous melatonin synthesis and regenerative potential of stem cells in the inflammatory microenvironment.

2. The use according to claim 1, characterized in that: The expression of AANAT was downregulated at both the gene level and the protein level under the inflammatory microenvironment. Overexpression of AANAT reversed the cementum differentiation potential of PDLSC damaged by inflammation.

3. A molecular target for preparing a drug for regulating the synthesis and regeneration potential of endogenous melatonin in inflammatory microenvironment stem cells, characterized in that: The molecular target is AANAT.

4. A method for screening drugs for regulating endogenous melatonin synthesis and regeneration potential of inflammatory microenvironment stem cells, characterized in that: The method employs AANAT as a target.

5. An agonist targeting AANAT, characterized in that The agonist targeting ANAT includes at least one of isofelianthrin and LCKLSL.

6. The agonist targeting AANAT according to claim 5, characterized in that The agonist targeting AANAT is isofelianthrin.

7. Use of an agonist targeting AANAT according to any one of claims 3 to 5 in the preparation of a drug for regulating the synthesis and regenerative potential of endogenous melatonin in stem cells in an inflammatory microenvironment.

8. A drug for regulating the synthesis and regeneration potential of endogenous melatonin in inflammatory microenvironment stem cells, characterized in that: The drug for regulating the synthesis and regeneration potential of endogenous melatonin in inflammatory microenvironment stem cells comprises: 1) an agonist targeting AANAT according to any one of claims 3 to 5; and 2) a pharmaceutically acceptable carrier or excipient.

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