CN114432431B - Application of reticulin-1 in inhibiting bone morphogenetic protein - Google Patents

Application of reticulin-1 in inhibiting bone morphogenetic protein Download PDF

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CN114432431B
CN114432431B CN202210110147.0A CN202210110147A CN114432431B CN 114432431 B CN114432431 B CN 114432431B CN 202210110147 A CN202210110147 A CN 202210110147A CN 114432431 B CN114432431 B CN 114432431B
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bmp7
bone morphogenetic
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morphogenetic protein
omentin
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CN114432431A (en
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聂宇
羊慧君
张国刚
柏勇平
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Fuwai Hospital of CAMS and PUMC
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Abstract

The invention provides an application of secretory glycoprotein in inhibiting bone morphogenetic protein activity. The invention finds that the reticulin-1 is applied to inhibiting the bone morphogenetic protein, not only can inhibit the combination of the bone morphogenetic protein and a receptor thereof, but also solves the uncertainty and the risk caused by the change of the expression level of the existing medicament because the reticulin-1 is not regulated and controlled by the bone morphogenetic protein. Following the pre-treatment with reticulin-1, BMP7 reduced activation of downstream signaling pathways.

Description

Application of reticulin-1 in inhibiting bone morphogenetic protein
Technical Field
The invention belongs to the technical field of biological medicines, and particularly relates to application of reticulin-1 in inhibition of bone morphogenetic protein.
Background
Bone Morphogenetic Protein (BMP) is the largest group of members in the transforming growth factor superfamily, is widely distributed in various cells and tissues of human bodies, participates in development processes of mesoderm formation, nervous system, skeleton development, kidney, stomach and intestine, lung, teeth and the like, regulates cell proliferation, differentiation, migration and apoptosis and participates in tumorigenesis, development and metastasis.
Bone morphogenic protein antagonists have been reported to date as: noggin, follistatin, myostatin, etc., wherein noggin, follistatin inhibit initiation of bone morphogenetic protein signaling by binding to bone morphogenetic protein and affecting its binding to receptor, and myostatin antagonizes receptor by competitively inhibiting its receptor due to its structural similarity to bone morphogenetic protein. The bone morphogenetic protein inhibitor on one hand antagonizes the bone morphogenetic protein activity, and on the other hand, the expression of the bone morphogenetic protein inhibitor is regulated by the bone morphogenetic protein to form positive and negative feedback regulation. Therefore, these inhibitors may have uncertainty and risk due to the change in expression level of themselves while regulating the activity of bone morphogenetic proteins.
The reticulin is highly expressed in vascular stromal cells of omental adipose tissue, is less expressed in intestinal tract and heart, and is hardly expressed in kidney and muscle tissue. Reticulin-1 and omentum-2 are two major subtypes thereof, each encoded by two independent genes on chromosome 1q22.23. Much current research is directed to seroretin-1, which is present in the blood circulation. The research shows that the annexin-1 is closely related to the occurrence and development of type 2 diabetes, hypertension, atherosclerosis and the like.
Methods for diagnosing a disease or predicting an increased risk of a disease, such as obesity, obesity-dependent subacute inflammation, atherosclerosis, cardiovascular disease and metabolic disease, by determining the level of reticulin-1 in a subject are disclosed in US20100021899 A1.
Japanese patent JP2015151386A discloses a partial peptide of human omentum-1 having physiological activity and a drug containing the peptide as an active ingredient, and provides its use as a drug for treating heart disease. The disclosed therapeutic principle is that omentum-1 inhibits cardiac apoptosis.
At present, no document discloses the application of the reticulin-1 as a medicament for inhibiting bone morphogenetic protein.
Disclosure of Invention
In order to address the potential risks of bone morphogenetic protein inhibitors, there is a need to find new inhibitors that are safer and more effective. The invention provides a novel activity inhibitor of bone morphogenetic protein, the secretory glycoprotein has similar action mode with the prior inhibitor and is a potential good inhibitor target of the bone morphogenetic protein, and experiments show that the expression level of the secretory glycoprotein is not influenced by the expression of the bone morphogenetic protein, and the inhibition effect is more stable.
Through computer protein docking technology, the novel secretory glycoprotein provided by the invention is verified to be very close to bone morphogenetic protein receptor in shape energy. The computer simulation theoretically verifies the influence of the secreted glycoprotein on the bone morphogenetic protein, verifies that the secreted glycoprotein can be combined with the bone morphogenetic protein, inhibits the combination of the bone morphogenetic protein and a receptor, inhibits the downstream signal transduction of the bone morphogenetic protein, and has similar effects with the existing inhibitors noggin and follistatin. Furthermore, the secreted glycoprotein can actually influence the activity of the bone morphogenetic protein through means of co-immunoprecipitation, proximity ligation, transcriptome sequencing and the like. Meanwhile, the expression level of the secretory glycoprotein is not affected by bone morphogenetic proteins, compared with conventional inhibitors.
The invention provides an application of secretory glycoprotein in inhibiting bone morphogenetic protein activity.
The secretory glycoprotein is omentum-1, adiponectin (adiponectin), visfatin (Visfatin) or Chemerin.
The bone morphogenetic protein is bone morphogenetic protein-2, bone morphogenetic protein-4, bone morphogenetic protein-6, bone morphogenetic protein-7 or bone morphogenetic protein-9.
The application is non-disease therapeutic application of secretory glycoprotein in inhibiting bone morphogenetic protein activity.
The application is the application of secretory glycoprotein in preparing bone morphogenetic protein inhibitor.
The application is the application of secretory glycoprotein in preparing medicines for inhibiting bone morphogenetic protein.
Preferably, the use is the use of the secreted glycoprotein in the manufacture of a medicament for the treatment of heart maturation disorders, osteoporosis, autosomal dominant polycystic kidney disease, tumors, anemia, iron-refractory iron deficiency anemia (IRIDA), ectopic ossification, non-hereditary myositis ossificans, traumatic myositis ossificans, localized myositis ossificans, skin fibrotic diseases and/or thrombosis.
The tumor includes, but is not limited to, breast cancer, lung cancer, liver cancer, ovarian cancer, gastric cancer, esophageal cancer, pancreatic cancer, colorectal cancer, sporadic colorectal cancer, acute myelogenous leukemia, chronic myelogenous leukemia, non-small cell lung cancer (NSCLC), serous ovarian cancer, epithelial ovarian cancer, melanoma or Head and Neck Squamous Cell Carcinoma (HNSCC) or central nervous system cancer; such central system cancers include, but are not limited to: glioma, astrocytoma, diffuse endogenous pontine glioma (DIPG), high differentiated glioma (HGG), blastoma, glioblastoma multiforme (GBM), oligodendroglioma, pituitary tumor or ependymoma.
The secretory glycoprotein interferes with the binding of a bone morphogenetic protein to its receptor by binding to the bone morphogenetic protein, thereby inhibiting its activity.
The secretory glycoprotein inhibits the activity of the downstream target molecule of the bone morphogenetic protein by antagonizing the downstream target molecule; such downstream target molecules include, but are not limited to, SMAD, YAP, and/or MAPK.
Preferably, when the application is the application in preparing a medicament for inhibiting bone morphogenetic protein, the bone morphogenetic protein is bone morphogenetic protein-7, and the medicament comprises reticulin-1.
Further preferably, the content of the medicine containing the reticulin-1 is 100-500ng/mL, and more preferably 300ng/mL.
The medicine also comprises other pharmaceutically acceptable salts or carriers or excipients.
The invention has the beneficial effects that:
the secretory glycoprotein is applied to the medicines for inhibiting the bone morphogenetic protein, can inhibit the combination of the bone morphogenetic protein and a receptor thereof, is not regulated and controlled by the bone morphogenetic protein, and solves the uncertainty and risk caused by the change of the expression level of the existing medicines. In particular, following Omentin-1 pretreatment, BMP7 activation of downstream signaling pathways was reduced.
The terms:
secreted glycoproteins: refers to a protein secreted outside the cell membrane. The secreted glycoproteins in the present application can be numerous cytokines, complements, degrading enzymes, peptide hormones, immunoglobulins, etc. in the blood and extracellular matrix.
Bone morphogenetic protein: bone Morphogenic Protein (BMP), also known in the art as bone morphogenic protein, BMP-1, BMP-7, have equivalent and/or identical meanings in this application, and materials having equivalent meanings in certain documents are also within the scope of the present application. The invention of the present application does not aim at proposing a novel bone morphogenetic protein, but at the inhibitory effect of reticulin on bone morphogenetic protein, and therefore variants of bone morphogenetic protein should not be taken as a basis for limiting the scope of protection claimed in the present application. That is, the type of bone morphogenetic protein present in the bone and other structural substances that are found in the future to have substantially equivalent functions to the bone morphogenetic protein present in the bone are all within the scope of the present application.
Omentin-1 and omeretin-1: in the present application, omentin-1 is equivalent to reticulin-1, both of which have the same meaning, and it should be noted that substances having equivalent meaning in some documents, also referred to as reticulin 1 or Omentin1 or equivalent expressions, are intended to be within the scope of the present application. It should also be noted that the object of the invention of the present application is not to propose a new omentum substance, but to propose a new use of the omentum substance, and therefore variants of the omentum substance should not be taken as a basis for limiting the scope of the claims of the present application, that is to say that other structural omentum substances having an equivalent basic function to omentum-1 or variants of omentum-1 should also be within the scope of the present application, for example mutants of omentum-1 should also be within the scope of the present application. To achieve the technical effects of the present invention, the source of omentum-1 may be a legal commercial source or may be a self-synthesized one by any protein synthesis means. As an example, IN the illustrative examples presented herein, reticulin-1 was used as a commercial recombinant human reticulin 1 protein (R & D System,9137-IN-050, USA) with a protein purity of greater than 95%.
BMP7: bone morphogenetic protein 7, bone morphogenetic protein-7. In order to achieve the technical effects of the present invention, the present invention is exemplified by bone morphogenetic protein-7 as a typical example.
Inhibition: inhibition of a molecule or a signaling pathway described herein has its conventional meaning in the art, including but not limited to being capable of reducing, slowing, interrupting, attenuating and/or eliminating the activity and/or signaling capacity of an assay and/or signaling pathway.
Combining: the association of two molecules as described herein has its conventional meaning in the art, including but not limited to aggregation, cross-linking, interaction between two molecules by chemical, physical, biological factors, and the like.
Receptor: receptors for a molecule described herein have the conventional meaning in the art, including but not limited to any biological macromolecule capable of binding to the molecule and of causing a change in the function of the molecule, the cell/tissue/organ/organism in which the molecule is located. The bone morphogenic protein receptors can be, for example, the type I and type II BMP receptors which have been found at present, or, in particular, the biomacromolecules which will be found in the future and which have the meaning indicated above.
Downstream targeting molecule: the downstream target molecules described in this application have the conventional meaning in the art and may also be referred to as downstream molecules, generally referring to the target molecules downstream of the signaling pathway. In some cases, a target molecule downstream of a signaling pathway refers to a molecule in anabolism that is at the rear of an enzymatic reaction.
Previous studies, stubborn, yang Lianjia, progress in the study of bone morphogenetic proteins in relation to bone disorders [ J ] journal of orthopedic surgery, china, 2000,7 (2): 4 ] showed that BMP acts as a local inducing factor, and can induce myoblast to differentiate into osteoblasts, and thus, new bone formation. Ectopic ossification refers to a pathological change of bone formation in soft tissues, and is often seen in head and spinal cord injuries, elbow trauma, acetabular fractures, complete hip arthroplasty and the like. The 3 requirements for ectopic ossification are: osteogenic precursor cells, induction of stimulating factors and a suitable environment for bone formation. In the case of traumatic ectopic ossification, bone fragments residing in muscle may be the source of BMP. The skilled person will be able to combine this with the techniques of the present application to apply the present application to the treatment of ectopic ossification and will therefore be able to unambiguously determine that reticulin-1 can be applied to the preparation of a drug for ectopic ossification.
The previous chinese patent CN201880017935.7 suggests that fused heterocyclic compounds as selective BMP inhibitors may be used in cancers such as colorectal cancer, sporadic colorectal cancer, acute myeloid leukemia, chronic myeloid leukemia, non-small cell lung cancer (NSCLC), pancreatic cancer, ovarian cancer, serous ovarian cancer, epithelial ovarian cancer, melanoma or Head and Neck Squamous Cell Carcinoma (HNSCC). In particular, cancers of the central nervous system such as gliomas, astrocytomas, diffuse endogenous pontine gliomas (DIPGs), highly differentiated gliomas (HGGs), blastomas, glioblastoma multiforme (GBMs), oligodendrogliomas, pituitary tumors or ependymomas. Can also be used for treating anemia, iron-refractory iron deficiency anemia (IRIDA), heterotopic ossification, non-hereditary myositis ossificans, traumatic myositis ossificans, and local myositis ossificans. The skilled person can combine this with the technology of the present application to apply the present application to the aforementioned diseases and can then unambiguously determine that reticulin-1 can be applied to drugs for the treatment of the aforementioned diseases.
Drawings
FIG. 1 is a graphical representation of the overlay comparison of BMP7/ActRIIB docking results with crystal structures. The crystal structure and the red butt joint result are basically combined together, so that the butt joint parameters are feasible, and the interaction mechanism can be reproduced by the model.
FIG. 2 is a schematic diagram of the binding hydrogen bond interaction of BMP7/Omentin-1 protein, hydrogen bond on Omentin-1 protein: TYR297, GLU274, LYS198; corresponding hydrogen bonds on BMP7: GLU-42, ALA63, GLU60.
FIG. 3 is a graph of molecular docking calculation of intermolecular interaction energies, wherein A is the BMP7 receptor ActrRIIB and Omentin-1 shape energies; and B shows that the binding of Omentin-1 and ActRIIB protein and BMP7 has competition relationship.
FIG. 4 shows the interaction between BMP7 and Omentin-1 calculated by molecular simulation. FIG. A shows that the BMP7 protein, BMP7& ActRIIB, BMP7& Omentin-1 protein 3 individual lines reach equilibrium in a molecular mimic 30ns time. B, calculating the structural change difference (RMSD) by using a graph, and finding that the framework structure change difference of the BMP7 protein and the BMP7& ActRIIB protein complex is not large, but the RMSD fluctuation range of the BMP7& Omentin-1 protein complex is obviously lower than that of the BMP7& ActRIIB, which shows that the structure of the BMP7 is greatly influenced after the Omentin-1 is combined with the BMP 7.
FIG. 5 shows the calculation of the amino acids affected by Omentin-1 on BMP7 by molecular modeling. Graph A suggests that during 30ns molecular dynamics simulation, the range of motion of individual amino acid residues is large, the corresponding residues at the peak position are mostly amino acid residues in the hinge region, and RMSF is greatly reduced after binding of Omentin-1 to BMP 7. B Panel A calculation results are visualized, and the strongly fluctuating amino acids on BMP7 were found to be the amino acids of the ActRIIB binding region of BMP7 and its receptor, LYS-126, ILE-124, ASN-122, ASP119, TRP55.
FIG. 6 shows co-immunoprecipitation to verify that Omentin-1 and BMP7 can interact. The left panel uses Flag tag antibody to pull down protein, the right panel uses BMP7 antibody to pull down protein, and it can be seen that Omentin-1 and BMP7 can interact.
FIG. 7 is a graph demonstrating that Omentin-1 interferes with BMP7 and its receptor Act RIIB binding by ortholigation. The binding of BMP7 and its receptor ActRIIB in cardiomyocytes in the group treated simultaneously with Omentin-1+ BMP7 was reduced compared to the BMP7 treated group, suggesting that Omentin-1 interferes with BMP7 and its receptor ActRIIB binding.
FIG. 8 is the result of transcriptome sequencing. Indicating that Omentin-1 antagonizes the function change caused by BMP 7. The BMP7 (100 ng/mL) is used for treating the myocardial cells, and the BMP7 (100 ng/mL) + Omentin-1 (300 ng/mL) is used for treating the myocardial cells for 48 hours, so that the trend of the vast majority of gene changes caused by the BMP7 treatment is opposite to that of the BMP7+ Omentin-1 group, and the gene sets are shown in a left panel a, a left panel d and a left panel e. The right panel shows that the a gene set is mainly enriched in energy metabolism and myocardial cell contraction, and the e gene set is mainly enriched in cell proliferation.
FIG. 9 shows the Western blot results of downstream pathways of BMP 7. It is suggested that Omentin-1 antagonizes BMP 7-induced pathway changes. SMAD-YAP and P38 MAPK are the most classical pathways downstream of BMP7, and the BMP 7-induced pathway changes can be antagonized by Omentin-1.
Detailed Description
The present invention will be further illustrated in detail with reference to the following specific examples, which are not intended to limit the present invention but are merely illustrative thereof. The experimental methods used in the following examples are not specifically described, and the materials, reagents and the like used in the following examples are generally commercially available under the usual conditions without specific descriptions.
Example 1 prediction of protein molecular docking and molecular modeling techniques
Protein docking technique calculates the energy of binding of Omentin-1 to bone morphogenetic proteins and their receptors. Bone morphogenetic proteins and their receptor interacting protein crystals have been reported to be PDB ID:1LX5 (1,2). Based on the crystal structure data development, structures in the crystal are respectively extracted, and model parameters and subsequent Omentin-1 docking are verified through protein-protein docking software. The integrity of the Omentin-1, the bone morphogenetic protein and three protein monomers of a receptor thereof is checked by using Chimera software, then the bone morphogenetic protein is set as the receptor, and the other two proteins are respectively butted to the bone morphogenetic protein by using HEX DOCK software. The sampling method (sampling method) is a 15angstrom acceptor box (receptor box), 3D fourier transform (FFT), and shape + charge (shape and electro) correction method. Other parameters not mentioned all adopt software default values, and the docking result retains the analysis of the maximum binding energy (most stable binding mode).
Firstly, in order to ensure the correctness of the docking model, the BMP7 receptor ActRIIB is docked with BMP7, then the docking model is compared with the crystal structures PDB ID:1LX5 (1,2) of the two known interaction complex, the RMS superposed by PyMol v1.7 software is 1.04, and the difference is very small as can be seen in figure 1, which indicates that the parameter model adopted by the protein-protein docking model is reasonable.
Based on the aforementioned model, by inoculating crystals of Omentin-1 protein, it was found from FIG. 2 that Omentin-1 and BMP7 can interact through hydrogen bonds (Omentin-1 hydrogen bond: TYR297, GLU274, LYS198; corresponding hydrogen bonds on BMP7: GLU-42, ALA63, GLU 60). Further calculation of the energy required for protein-protein docking suggests that the binding energy of Omentin-1 to BMP7 is slightly less than that of BMP7 and its receptor, actRIIB, but that the shape energies of Omentin-1 and ActRIIB are very close (see visual results in fig. 3), suggesting that the shape of Omentin-1 and ActRIIB is similar and that binding of Omentin-1 to BMP7 may competitively inhibit binding of BMP7 to its receptor, actRIIB.
In order to accurately describe the protein-protein interaction mechanism, next molecular dynamics simulation is carried out, the flexible change of the protein structure and the change of the solvated (aqueous solution) protein structure are calculated, and the protein-protein interaction under the real system environment is simulated.
The molecular dynamics simulation is completed by YASARA V14 software, and 30ns dynamics simulation is respectively carried out on the bone morphogenetic protein, the bone morphogenetic protein and the receptor protein interaction complex thereof, and the bone morphogenetic protein and the secreted glycoprotein interaction complex. Adding a virtual box, wherein the minimum distance from the edge of the box to the protein is 1nm, adding TIP3 model water molecules, and adding reasonable ions to enable the system to be electrically neutral (neutral); amber99sb force field, PME method, cutoff is 12; the other parameters all use software default parameters.
After the molecular dynamics simulation is carried out for 30ns, the system quickly reaches the dynamic equilibrium, the total energy fluctuates in a small range after being reduced to a value, and the molecular dynamics simulation time is long enough to reach a stable equilibrium state. Respectively calculating the structural change difference (RMSD) of three model systems of BMP7, BMP7& ActRIIB and BMP7& Omentin-1, and finding that the skeleton structure change difference of the BMP7 protein and the BMP7& ActRIIB protein compound is small, namely the influence difference on the structure before and after the BMP7 is combined with the ActRIIB is small; however, the RMSD fluctuation range of the BMP7& omnitin-1 protein complex was significantly lower than that of BMP7& ActRIIB, indicating that the structure of BMP7 was greatly affected by binding of omnitin-1 to BMP7 (fig. 4). Further calculation of the amino acids with the largest ascending and descending changes of BMP7 protein revealed that after Omentin-1 binds BMP7, the fluctuation of the amino acid sites (LYS-126, ILE-124, ASN-122, ASP119, TRP 55) in the binding region of BMP7 to ActRIIB as a receptor thereof was large (FIG. 5), and that Omentin-1 binds BMP7 and affects the binding of BMP7 to ActRIIB as a receptor thereof was demonstrated from the amino acid change level.
Example 2 Co-immunoprecipitation (CO-IP) and Proximity Ligation Assay (PLA)
(1) To verify the above-described simulation results, we performed co-immunoprecipitation experiments to verify the interaction of Omentin-1 with BMP7 in vitro by first extracting cardiomyocytes (primary Cardiomyocyte extraction using Kit method, which is Neonal Cardiomyocyte Isolation Kit, rat, milenti biotec,130-105-420, according to the instruction)After the cells are dissociated from the organ tissues, the cells which are not attached to the wall are placed in a culture dish again for culturing for 24 hours by a differential adherence method according to slow adherence, fast adherence and 70 minutes of differential adherence of the myocardial cells to obtain the myocardial cells, and the cells are transfected with Omentin-1-Flag lentivirus (Ji Kai Gen. Company, with the titer of 1 multiplied by 10 and customized by the company of Ji Kai) 9 Per mL, used in an amount of 1X 10 6 4 μ L for each cardiomyocyte), transfection method is provided by Ji Kai: the lentivirus and the infection-assisting agent polybrene (10. Mu.g/mL, sigma) were added to the medium 10% FBS (fetal bovine serum, super grade, heat inactivated, australia, gibico, 10100147) + DMEM (DMEM, high sugar, 11965092), mixed and added to the cardiomyocytes for culturing. After 6 hours of transfection, the virus-containing medium was discarded, and fresh medium (10% FBS + DMEM) was added for further culture for 48 hours, thereby carrying out the subsequent experiments. After transfection for 48 hours, cellular proteins were collected, and the binding of the interacting protein to the cells was pulled down with Flag antibody and BMP7 antibody, respectively, and the interacting protein was verified using Western blot. The specific operation is as follows:
secreted glycoproteins were overexpressed in primary cardiomyocytes for 48 hours, cellular proteins were lysed using RIPA lysate (Beyotime, P0013D) and the protease inhibitor PMSF (Beyotime, ST 505) (100. The next day with Flag Co-IP kit (sigma,
Figure BDA0003494851500000081
immunopropractination Kit, cat: FLAGIPT 1), washing Flag gel magnetic beads, adding the Flag gel magnetic beads into antibody protein liquid according to protein concentration, and incubating overnight by shaking at 4 degrees again. On the third day, the supernatant was centrifuged, the beads were washed, 60. Mu.l of 2 XSDS was added to the beads, and the beads were boiled for denaturation and subjected to a Western blotting experiment.
The results show that Omentin-1 can indeed interact with BMP7 (FIG. 6).
(2) To further observe the effect of the interaction of Omentin-1 with BMP7 on BMP7 and its receptor ActRIIB, it was observed by the proximity ligation technique (PLA) whether the treatment with Omentin-1 affected the binding of BMP7 to its receptor ActRIIB. PLA signals are generated when a pair of PLA probes are close enough (< 40 nm) that they directly reflect changes in the amount of protein complex expression.
In primary myocardial cells, cells are treated for 48 hours by bone morphogenetic proteins or bone morphogenetic proteins and Omentin-1, and the specific conditions are as follows: seeding the isolated cardiomyocytes into a confocal chamber (millicell EZ slide R9EA 07847), 1X 10 5 Well, cultured using 10% fbs (fetal bovine serum, grade, heat inactivated, australia, gibico, 10100147) + DMEM (DMEM, high sugar, 11965092); the dose of Omentin-1 was 300ng/mL and the dose of BMP7 was 100ng/mL. Commercial recombinant human reticulin 1 protein (R)&DSystem,9137-IN-050, USA), the protein purity is more than 95%. Recombinant human BMP7 protein, concentration 100ng/mL (R)&D System,354-BP-010/CF, USA), protein purity greater than 95%.
The experiment was continued with reference to the PLA kit (sigma) instructions. The method comprises the following specific steps: (1) fixing the cells by using 4% paraformaldehyde for 20 minutes, and washing the cells by using a PBS (phosphate buffer solution) solution after fixing; (2) a pair of antibodies, rabbit anti-bone morphogenetic protein (1, proteintech, 12221-1-AP) and murine anti-bone morphogenetic protein receptor (1, 100, santa Cruz, sc-376593) were added and incubated overnight at 4 degrees; (3) the next day, the antibodies were washed and PLUS the PLUS and MINUS probes for 1 hour; (4) after cleaning, adding a Ligation solution for 30 minutes; (5) after cleaning, adding amplification solution; (6) imaging was then performed using a confocal microscope.
The results indicate that at the cellular level, myocardial cell BMP7 and its receptor ActRIIB were reduced in the Omentin-1+ BMP7 concurrently treated group compared to the BMP7 treated group, suggesting that Omentin-1 interferes with BMP7 and its receptor ActRIIB binding (FIG. 7).
Example 3 in vitro Experimental transcriptome sequencing
To further explore the effect of Omentin-1 inhibiting BMP7 on its function, we further validated the inhibitory effect of cell-level Omentin-1 on BMP7 function by transcriptome sequencing as follows:
primary Cardiomyocyte extraction was performed using the Kit method (Neonatal myocardial Isolation Kit, rat, milenyi Biotec, 130-105-420), according to the Kit instructions. After the heart tissue is dissociated into cells, the cells are attached to the wall in a differential speed way for 70 minutes, and the non-attached cells are put into the culture dish again for 24 hours. The next day, the cardiomyocytes were changed and divided into 3 groups, respectively a control group, an Omentin-1+ BMP7 simultaneous treatment group, and a BMP7 treatment group, each of which was repeated 3 times. The dose of addition of Omentin-1 was 300ng/mL and the dose of BMP7 was 100ng/mL. Commercial recombinant human omentum-1 protein (R & D System,9137-IN-050, USA) with protein purity greater than 95%. Recombinant human BMP7 protein, concentration 100ng/mL (R & D System,354-BP-010/CF, USA), protein purity greater than 95%.
After 48 hours of culture, the supernatant was discarded, the cells were washed with PBS (balanced salt solution, gibico, 20012-050), and the cells were lysed by adding tirzol (ABI, 15596018) every 10 hours 6 Adding 1 ml of tirzol, collecting a tirzol cell mixed solution, quickly freezing, and then conveying to a Nozao kinawa company for RNA extraction and sequencing.
After sequencing result analysis, 1381 genes caused by BMP7 treatment (100 mu g/mL) of the myocardial cells after 48 hours are changed (P value after correction is less than 0.05), and the myocardial cell proliferation and the myocardial cell energy metabolism are mainly enriched. However, after addition of Omentin-1 treatment (300. Mu.g/mL) to the BMP7 treated group, thermographic analysis suggested that the majority of the BMP 7-induced changes could be antagonized by Omentin-1 (1090 genes), as shown in FIG. 8 for the panel of genes a, c, d.
To verify whether Omentin-1 antagonizes BMP7 activity at the protein level, we tested the BMP7 downstream pathway:
primary Cardiomyocyte extraction was performed using the Kit method (Neonatal myocardial Isolation Kit, rat, milenyi Biotec, 130-105-420), according to the Kit instructions. After the heart tissue dissociates the cells, the cells are attached to the wall by a differential wall attaching method for 70 minutes at a differential speed, and the non-attached cells are placed into the culture dish again for culturing for 24 hours. The next day, the cardiomyocytes were changed and divided into 3 groups, respectively a control group, an Omentin-1+ BMP7 simultaneous treatment group, and a BMP7 treatment group, each of which was repeated 3 times. The dose of addition of Omentin-1 was 300ng/mL and the dose of BMP7 was 100ng/mL. The commercial recombinant human omentum-1 protein (R & D System,9137-IN-050, USA) has a protein purity of more than 95%. The recombinant human BMP7 protein, the concentration was 100ng/mL (R & D System,354-BP-010/CF, USA), the protein purity was greater than 95%.
After 48 hours of culture, the supernatant was discarded, and the cells were washed with PBS (balanced salt solution, gibico, 20012-050). RIPA lysate and protease inhibitor PMSF (100 TM Protein denaturation was performed with SDS-PAGE protein staining and loading buffer (5 ×) (beyond time, P0280). Subsequently, western blots were run and antibodies anti-YAP (CST, 14074, 1.
As a result, it was found that the activation of downstream signaling pathway by BMP7 was reduced by Omentin-1 pretreatment (FIG. 9).
From examples 1-3, it is seen that Omentin-1 interacts with BMP7, antagonizes the binding of BMP7 to its receptor ActRIIB, and thereby inhibits the activity of BMP 7.

Claims (3)

1. Application of reticulin-1 in preparing bone morphogenetic protein-7 inhibitor.
2. The use of claim 1, wherein omentum-1 is used in the manufacture of a medicament for inhibiting bone morphogenetic protein-7.
3. The use of any one of claims 1-2, wherein omeretin-1 inhibits the activity of bone morphogenetic protein-7 by interfering with its receptor binding by binding to bone morphogenetic protein-7.
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