New target for regulating and controlling balance of osteoclast and osteoblast and application of new target

Abstract

The invention discloses a new target for regulating and controlling the balance of osteoclasts and osteoblasts and application of the new target, and belongs to the technical field of biomedicine.The new target for regulating and controlling the balance of the osteoclasts and the osteoblasts is AQP9 and belongs to members of aquaporin Aquaporin and AQPs subpopulations. Therefore, the progress of rheumatoid arthritis (RA) is relieved by adjusting the balance of osteoclasts and osteoblasts, and a new target is provided for the treatment of RA.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to a new target for regulating the balance between osteoclasts and osteoblasts and its application. Background Art

[0002] Rheumatoid arthritis (RA) is a common chronic autoimmune disease with a global prevalence of 0.5-1% in adults, posing a significant burden and challenge to global public health. RA is characterized by persistent synovial inflammation and cartilage and bone destruction. During disease progression, cartilage damage and bone erosion subsequently occur, during which an imbalance between osteoblasts and osteoclasts plays a major role. In short, synovial inflammation and related immune disorders promote the inactivation of osteoblasts and activation of osteoclasts in RA, leading to damage.

[0003] Bone erosion is a progressive characteristic of RA and is caused by an imbalance between bone-forming osteoblasts and bone-resorbing osteoclasts. Osteoclasts are specialized for bone resorption, which involves the breakdown of bone matrix and the release of stored minerals into the blood. In contrast, osteoblasts, derived from mesenchymal stem cells, are responsible for bone formation through the synthesis and mineralization of bone matrix. Osteoclasts, derived from hematopoietic stem cells and belonging to the macrophage / monocyte lineage, are unique in their ability to break down mineralized bone. Physiological osteoclastogenesis is driven by the interaction between receptor activator of nuclear factor κB (NF-κB) (RANK) and its ligand (RANKL), as well as macrophage colony-stimulating factor (M-CSF). Binding of M-CSF to its receptor, c-fms, provides the signals required for the proliferation and survival of osteoclast precursors, while binding of RANKL to RANK mimics the signals required for osteoclast differentiation, survival, and resorptive activity. Osteoblasts are primarily derived from mesenchymal stem cells (MSCs), which are multipotent stem cells found in various tissues, including bone marrow, fat, muscle, and dental pulp. These stem cells have the ability to self-renew and can differentiate into various cell types, including osteoblasts, chondrocytes, myocytes, and adipocytes. During osteoblast differentiation, MSCs transform into preosteoblasts under the regulation of various cytokines and signaling pathways, such as bone morphogenetic protein (BMP), transforming growth factor-β (TGF-β), and the Wnt / β-catenin signaling pathway. Preosteoblasts then mature and differentiate into osteoblasts, which synthesize and mineralize bone matrix to form new bone tissue. Some osteoblasts may further differentiate into osteocytes, a major cell type in bone tissue responsible for the long-term maintenance and remodeling of bone.

[0004] Aquaporins (AQPs) are a family of water channel proteins that facilitate the transport of water and other small molecules across cell membranes. AQP9 is widely distributed throughout the body, including in the nervous, digestive, and reproductive systems. Although its molecular structure and water permeability closely resemble those of other aquaporins, little is known about its specific physiological functions. AQP9 is most highly expressed in the liver. Due to its aquaporin and peroxidase activities, it facilitates the transmembrane diffusion of glycerol and hydrogen peroxide (H2O2), thereby influencing general metabolism and redox homeostasis. AQP9 has been implicated in numerous pathophysiological conditions, including edema, glaucoma, nephrogenic diabetes insipidus, oxidative stress, sepsis, cancer, and metabolic dysfunction. Furthermore, AQP9 is one of the most highly expressed AQPs in immune cells (lymphocytes, neutrophils, and monocytes). Numerous studies have shown that reduced AQP9 expression reduces NF-κB activation and NLRP3 inflammasome expression in the heart and kidneys, making it a novel genetic target for polymicrobial sepsis. Rheumatoid arthritis (RA) is an immune-related disease. Due to its elusive pathogenesis, clinical management of RA remains unmet. Current drug treatments include nonsteroidal anti-inflammatory drugs (NSAIDs), disease-modifying antirheumatic drugs (DMARDs), glucocorticoids, and biologics (such as adalimumab and rituximab). This invention discloses a novel target for the treatment of RA, AQP9, which can effectively alleviate RA by regulating the balance between osteoclasts and osteoblasts. Summary of the Invention

[0005] Technical issues solved:

[0006] This application addresses the shortcomings of the existing technology and solves technical problems such as the lack of effective treatment methods for the balance of osteoclasts and osteoblasts. It provides a new target for regulating the balance of osteoclasts and osteoblasts and its application. By regulating the balance of osteoclasts and osteoblasts, the alleviating effect of AQP9 is verified from in vitro experiments.

[0007] Technical solution:

[0008] To achieve the above objectives, this application is implemented through the following technical solutions:

[0009] A new target for regulating the balance between osteoclasts and osteoblasts, wherein the new target for regulating the balance between osteoclasts and osteoblasts is

[0010] AQP9.

[0011] Furthermore, the AQP9 is a member of the aquaporin subgroup.

[0012] The present application also discloses the use of a new target for regulating the balance between osteoclasts and osteoblasts in the preparation of a drug for treating rheumatoid arthritis.

[0013] Furthermore, the drug for treating rheumatoid arthritis is in an injectable or non-injectable form.

[0014] Furthermore, the drug for treating rheumatoid arthritis is an injection, tablet, capsule, powder, pill, granule, solution, suspension, syrup, suppository or inhalant.

[0015] Principle explanation: Bixin has antioxidant, anti-tumor and anti-inflammatory effects. When rheumatoid arthritis occurs, the patient's osteoclasts and osteoblasts become unbalanced, and inhibiting the function of AQP9 can significantly alleviate this imbalance, thereby inhibiting the progression of rheumatoid arthritis.

[0016] Beneficial effects:

[0017] This application provides a new target for regulating the balance between osteoclasts and osteoblasts and its application, which has the following beneficial effects compared with the existing technology:

[0018] 1. AQP9, a member of the aquaporin (AQPs) subgroup, can alleviate rheumatoid arthritis by regulating the balance between osteoclasts and osteoblasts, further providing new possibilities for the treatment of rheumatoid arthritis;

[0019] 2. Reducing the expression level of AQP9 can effectively regulate the induction of mouse bone marrow macrophages (BMDM) into osteoclasts. After lentiviral infection, all indicators of osteoclast differentiation were inhibited.

[0020] 3. After the sh-AQP9 lentivirus was used to infect mouse bone marrow mesenchymal stem cells (BMSCs), the generation rate of osteoblasts was effectively increased, and the levels of various osteoblast markers increased;

[0021] 4. RNAseq results showed that AQP9 inhibited osteoclast differentiation through the TCA cycle, thereby alleviating rheumatoid arthritis. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 Figure 1 shows a mouse CIA rheumatoid arthritis model established for this application; a is a comparison of DBA mouse WT and CIA models, b is a micro CT analysis of the degree of disease in the ankle joint of the mouse, c is a quantification of bone mineral, d is a quantification of bone volume, e is a quantification of bone surface area, f is a quantification of trabecular number, g is a quantification of trabecular separation, h is a quantification of trabecular thickness, and i is a comparison of TRAP staining of the ankle joints of WT and CIA mice;

[0023] Figure 2 This is a graph showing changes in AQP9 content in a mouse CIA rheumatoid arthritis model detected by qPCR;

[0024] Figure 3 This application uses western blot to detect the increase in AQP9 protein level after primary mouse bone marrow macrophages BMDM are induced into osteoclasts;

[0025] Figure 4 This application shows the increase in AQP9 mRNA level after osteoclasts were induced by primary mouse bone marrow macrophages BMDM detected by qPCR;

[0026] Figure 5 This is a graph showing the decrease in AQP9 protein level after sh-AQP9 lentivirus infection of BMDM cells verified by western blot;

[0027] Figure 6 This is a graph verifying by qPCR that the mRNA level of AQP9 is reduced after sh-AQP9 lentivirus infection of BMDM cells;

[0028] Figure 7 This is a diagram of the present application detecting the effective inhibition of osteoclast differentiation after sh-AQP9 lentivirus infection of BMDM cells by TRAP staining, wherein the left figure is a TRAP staining image of BMDM cells inducing osteoclast differentiation, and the right figure is a TRAP staining image of BMDM cells inducing osteoclast differentiation in which AQP9 is knocked down;

[0029] Figure 8 This application uses western blot to detect that sh-AQP9 lentivirus effectively inhibits osteoclast differentiation in BMDM cells;

[0030] Figure 9 Figure 1 is a graph showing that sh-AQP9 lentiviral infection of BMDM cells effectively inhibited osteoclast differentiation by qPCR. From left to right, the first graph shows the inhibitory effect of sh-AQP9 on the expression of Trap secreted by osteoclasts; the second graph shows the inhibitory effect of sh-AQP9 on the expression of TRAF6 secreted by osteoclasts; the third graph shows the inhibitory effect of sh-AQP9 on the expression of Nfatc1 secreted by osteoclasts; the fourth graph shows the inhibitory effect of sh-AQP9 on the expression of Atp6v0d2 secreted by osteoclasts; and the fifth graph shows the inhibitory effect of sh-AQP9 on the expression of CTSK secreted by osteoclasts.

[0031] Figure 10 This is a graph showing the reduction of AQP9 protein levels after sh-AQP9 lentivirus infection of BMSC cells verified by western blot;

[0032] Figure 11This is a diagram of the application detecting the increase in osteoblast generation after sh-AQP9 lentivirus infection of BMSC cells by alkaline phosphatase ALP staining, wherein the left figure is an ALP staining image of osteoblasts differentiated from BMSC cells, and the right figure is an ALP staining image of osteoblasts differentiated from BMSC cells after sh-AQP9 lentivirus infection;

[0033] Figure 12 This is a diagram of the present application detecting the increase in osteoblast generation after sh-AQP9 lentivirus infection of BMSC cells by Alizarin Red ARS staining, wherein the left figure is an ARS staining image of osteoblasts differentiated from BMSC cells, and the right figure is an ARS staining image of osteoblasts differentiated from BMSC cells after sh-AQP9 lentivirus infection;

[0034] Figure 13 This application uses western blot to detect that sh-AQP9 lentivirus-infected BMSC cells effectively promote the generation of osteoblasts;

[0035] Figure 14 The present application detects by qPCR that sh-AQP9 lentivirus effectively promotes the generation of osteoblasts after infecting BMSC cells, wherein the first figure from left to right shows that sh-AQP9 promotes the expression of OPG secreted by osteoblasts; the second figure shows that sh-AQP9 promotes the expression of RUNX2 secreted by osteoblasts; the third figure shows that sh-AQP9 promotes the expression of SP7 secreted by osteoblasts; the fourth figure shows that sh-AQP9 promotes the expression of APLP secreted by osteoblasts; the fifth figure shows that sh-AQP9 promotes the expression of COL0A1 secreted by osteoblasts; the sixth figure shows that sh-AQP9 promotes the expression of IBSP secreted by osteoblasts;

[0036] Figure 15 This is a heat map of the differences in expression of osteoclast differentiation-specific indicators CTSK, nfatc1, Traf6, Nfkbia, Oscar, Fos, Acp5, and Fosl2 among the BMDM group, osteoclast group, and sh-AQP9-osteoclast group;

[0037] Figure 16 The middle left figure is an intersection Venn diagram of genes that were elevated in the osteoclast group (compared with the BMDM group) and decreased in the sh-AQP9-osteoclast group (compared with the osteoclast group) infected with sh-AQP9. The right figure is a KEGG pathway analysis based on 1324 intersection genes, suggesting that AQP9 regulates osteoclast differentiation through the TCA cycle.

[0038] Figure 17 The most likely target molecule map of AQP9 involved in the TCA cycle was obtained by performing protein interaction analysis on genes involved in the TCA cycle in the KEGG pathway map. DETAILED DESCRIPTION

[0039] The present invention will be described in detail below with reference to specific embodiments. The following embodiments will help those skilled in the art to further understand the present invention, but are not intended to limit the present invention in any form. It should be noted that, for those skilled in the art, several variations and improvements can be made without departing from the scope of the present invention. These all fall within the scope of protection of the present invention.

[0040] Example 1:

[0041] A new target for regulating the balance between osteoclasts and osteoblasts, wherein the new target for regulating the balance between osteoclasts and osteoblasts is

[0042] AQP9; Application of a new target for regulating the balance between osteoclasts and osteoblasts in the preparation of drugs for the treatment of rheumatoid arthritis,

[0043] AQP9 expression is elevated in the CIA mouse rheumatoid arthritis model:

[0044] 1. Experimental process:

[0045] Six 8-week-old DBA male mice were divided into a control group and a CIA group (n=3). The control group was immunized with normal saline, while the CIA group was first immunized with bovine type II collagen (2 mg / ml) emulsified in complete Freund's adjuvant, with each mouse receiving 100 μl. Twenty-one days later, a booster immunization was performed with bovine type II collagen (2 mg / ml) emulsified in incomplete Freund's adjuvant. One week later, ankle thickness was measured and clinical scores were performed. Forty-two days after the first immunization, the mice were euthanized by anesthesia and cervical dislocation. Ankle joint tissue was collected for micro-CT, TRAP staining, and qPCR to detect changes in AQP9 expression in the CIA model.

[0046] 2. Experimental Results

[0047] In a mouse model of rheumatoid arthritis, AQP9 expression in the ankle joint is elevated.

[0048] like Figure 1 As shown, Figure 1 For this application, a CIA rheumatoid arthritis model in mice was established, and micro-CT and TRAP staining were used to verify that the occurrence of rheumatoid arthritis was accompanied by the aggravation of osteoclast distribution.

[0049] like Figure 2 As shown, Figure 2This is a graph showing changes in AQP9 content in a mouse CIA rheumatoid arthritis model detected by qPCR, demonstrating that AQP9 shows an increasing trend during the pathogenesis of rheumatoid arthritis.

[0050] Example 2:

[0051] The mRNA expression level of AQP9 was increased in osteoclasts.

[0052] 1. Experimental process:

[0053] Preparation of primary mouse bone marrow macrophages (BMDM): Bone marrow cells were isolated from the femur and tibia of 8-week-old C57BL / 6J mice and then cultured in IMDM medium containing 10% FBS, 1% penicillin-streptomycin, and 10 ng / mL M-CSF at 37°C in an environment of 5% CO2 for 7 days to prepare BMDM cells.

[0054] Induction of osteoclasts: BMDM cells were digested, resuspended and counted at 1*10 per well. 5 Cells were plated in 24-well plates. A control group and an osteoclast group were set up. In the osteoclast group, 40 ng / ml RANKL and 40 ng / ml m-CSF were added to each well. The induction medium was changed every three days. On the sixth day, a large number of osteoclasts were observed to be fused.

[0055] RNA extraction: RNA was extracted using the Yishan Bio RNA Extraction Kit. After absorbing the culture medium, wash once with PBS, add 500μL Lysis Buffer, pipette vigorously 10 times, transfer to an EP tube, and vortex for 10 seconds to fully lyse the cells. Add 500μL of anhydrous ethanol to the lysed cells, mix thoroughly, and transfer to a centrifuge column. Centrifuge at 4000× for 1 minute and discard the waste liquid. Add 500μL of Wash Buffer to the RNA column, centrifuge at 12000×g for 1 minute, and discard the waste liquid. After centrifuging the empty tube at 12000×g for 1 minute, transfer the column to a clean RNase-free 1.5ml centrifuge tube, open the lid and let it dry for 2 minutes. Add 20-50μL of Elution Buffer to the center of the membrane of the RNA column and let it stand at room temperature for 2 minutes. Centrifuge at 12000×g for 1 minute, add the eluted RNA solution back to the column, let it stand on ice for 5 minutes, and centrifuge again to measure the concentration of the eluted RNA;

[0056] Reverse transcription: Reverse transcription was performed using the Novozymes kit. 1 μg RNA was added to 4 μL of 4× gDNA wiper mix, which was made up to 16 μL with enzyme-free water and incubated at 42°C for 2 minutes. Then, 4 μL of 5× qRT SuperMix II was added and incubated at 37°C for 15 minutes, followed by 85°C for 5 seconds.

[0057] Experimental results: qPCR detection of BMDM cells and osteoclasts showed that in rheumatoid arthritis, AQP9 expression increased when osteoclast differentiation increased. Figure 3 As shown in FIG, the present application detected the increase in AQP9 mRNA level after inducing osteoclasts from primary mouse bone marrow macrophages BMDM by qPCR. From the mRNA level, the expression level of AQP9 in osteoclasts increased.

[0058] Example 3:

[0059] The protein expression level of AQP9 is increased in osteoclasts.

[0060] Experimental process:

[0061] Preparation of primary mouse bone marrow macrophages (BMDM): Bone marrow cells were isolated from the femur and tibia of 8-week-old C57BL / 6J mice and then cultured in IMDM medium containing 10% FBS, 1% penicillin-streptomycin, and 10 ng / mL M-CSF at 37°C in an environment of 5% CO2 for 7 days to prepare BMDM cells.

[0062] Induction of osteoclasts: BMDM cells were digested and resuspended in a-MEM medium and counted at 1*10 per well. 5 Cells were plated in 24-well plates. A control group and an osteoclast group were set up. In the osteoclast group, 40 ng / ml RANKL and 40 ng / ml m-CSF were added to each well. The induction medium was changed every three days. On the sixth day, a large number of osteoclasts were observed to be fused.

[0063] Protein extraction: Aspirate the culture medium, wash once with PBS, and add 150 μL of protein lysis buffer to each well. Incubate on ice for 5 minutes. Scrape the lysed cells with a clean scraper and transfer to a clean 1.5 ml microcentrifuge tube. Vortex for 10 seconds to fully lyse the cells. Incubate on ice for 15 minutes. Centrifuge at 13,000 rpm at 4°C for 10 minutes. Mix 40 μL of the supernatant with 40 μL of 2× loading buffer and boil at 100°C for 10 minutes to denature the proteins.

[0064] Experimental results: Western blot analysis of BMDM cells and osteoclasts showed that AQP9 expression increased when osteoclast differentiation increased in rheumatoid arthritis

[0065] like Figure 4 As shown, the present application detected the increase in AQP9 protein level after primary mouse bone marrow macrophages BMDM induced osteoclasts by western blot. From the protein level, the expression level of AQP9 in osteoclasts increased.

[0066] Example 4

[0067] After sh-AQP9 lentivirus infection of BMDM cells, AQP9 expression level was reduced

[0068] Experimental process:

[0069] Preparation of primary mouse bone marrow macrophages (BMDM): Bone marrow cells were isolated from the femur and tibia of 8-week-old C57BL / 6J mice and then cultured in IMDM medium containing 10% FBS, 1% penicillin-streptomycin, and 10 ng / mL M-CSF at 37°C in an environment of 5% CO2 for 7 days to prepare BMDM cells.

[0070] Lentivirus infection of BMDM cells: digest the prepared BMDM cells and inoculate 5*10 cells per well. 5 Plate the cells in a 12-well plate and incubate in an incubator for 24 hours. Infect the sh-NC and sh-AQP9 groups with the sh-NC and sh-AQP9 viruses, respectively. After 8 hours, discard the supernatant and replace with fresh culture medium. Analyze mRNA and protein levels 24 and 48 hours later.

[0071] RNA extraction: RNA was extracted using the Yishan Bio RNA Extraction Kit. After absorbing the culture medium, wash once with PBS, add 500μL Lysis Buffer, pipette vigorously 10 times, transfer to an EP tube, and vortex for 10 seconds to fully lyse the cells. Add 500μL of anhydrous ethanol to the lysed cells, mix thoroughly, and transfer to a centrifuge column. Centrifuge at 4000× for 1 minute and discard the waste liquid. Add 500μL of Wash Buffer to the RNA column, centrifuge at 12000×g for 1 minute, and discard the waste liquid. After centrifuging the empty tube at 12000×g for 1 minute, transfer the column to a clean RNase-free 1.5ml centrifuge tube, open the lid and let it dry for 2 minutes. Add 20-50μL of Elution Buffer to the center of the membrane of the RNA column and let it stand at room temperature for 2 minutes. Centrifuge at 12000×g for 1 minute, add the eluted RNA solution back to the column, let it stand on ice for 5 minutes, and centrifuge again to measure the concentration of the eluted RNA;

[0072] Reverse transcription: Reverse transcription was performed using the Novozymes kit. 1 μg RNA was added to 4 μL of 4× gDNA wiper mix, which was then made up to 16 μL with enzyme-free water and incubated at 42°C for 2 minutes. Then, 4 μL of 5× qRT SuperMix II was added and incubated at 37°C for 15 minutes, followed by 85°C for 5 seconds.

[0073] Protein extraction: Aspirate the culture medium, wash once with PBS, and add 150 μL of protein lysis buffer to each well. Incubate on ice for 5 minutes. Scrape the lysed cells with a clean scraper and transfer to a clean 1.5 ml microcentrifuge tube. Vortex for 10 seconds to fully lyse the cells. Incubate on ice for 15 minutes. Centrifuge at 13,000 rpm at 4°C for 10 minutes. Mix 40 μL of the supernatant with 40 μL of 2× loading buffer and boil at 100°C for 10 minutes to denature the proteins.

[0074] Experimental results: sh-AQP9 lentivirus can effectively inhibit the expression of AQP9 in BMDM cells.

[0075] like Figure 5 and Figure 6 As shown in the results, the sh-AQP9 lentivirus used can inhibit AQP9 expression in BMDM cells at both protein and mRNA levels.

[0076] Example 5

[0077] Inhibiting the expression of AQP9 can regulate osteoclast differentiation.

[0078] Experimental process:

[0079] Preparation of primary mouse bone marrow macrophages (BMDM): Bone marrow cells were isolated from the femur and tibia of 8-week-old C57BL / 6J mice and then cultured in IMDM medium containing 10% FBS, 1% penicillin-streptomycin, and 10 ng / mL M-CSF at 37°C in an environment of 5% CO2 for 7 days to prepare BMDM cells.

[0080] Induction of osteoclasts: BMDM cells were digested and resuspended in a-MEM medium and counted at 1*10 per well. 5 Cells were plated in 24-well plates and incubated in an incubator for 24 hours. Six hours after infection with either sh-NC or sh-AQP9 lentivirus, the supernatant was discarded and fresh culture medium was replaced. 24 hours later, 40 ng / ml RANKL and 40 ng / ml m-CSF were added to each well. The medium was changed every 3 days.

[0081] TRAP staining: Discard the supernatant, wash three times with PBS, and fix with 4% paraformaldehyde for 30 minutes. Discard the fixative, wash three times with PBS, and permeabilize with 0.5% Triton 100 for 30 minutes. Discard the supernatant, wash three times with PBS, and add the prepared TRAP staining solution. Incubate at 37°C for 1 hour.

[0082] Protein extraction: Aspirate the culture medium, wash once with PBS, and add 150 μL of protein lysis buffer to each well. Incubate on ice for 5 minutes. Scrape the lysed cells with a clean scraper and transfer to a clean 1.5 ml microcentrifuge tube. Vortex for 10 seconds to fully lyse the cells. Incubate on ice for 15 minutes. Centrifuge at 13,000 rpm at 4°C for 10 minutes. Mix 40 μL of the supernatant with 40 μL of 2× loading buffer and boil at 100°C for 10 minutes to denature the proteins.

[0083] RNA extraction: RNA was extracted using the Yishan Bio RNA Extraction Kit. After absorbing the culture medium, wash once with PBS, add 500μL Lysis Buffer, pipette vigorously 10 times, transfer to an EP tube, and vortex for 10 seconds to fully lyse the cells. Add 500μL of anhydrous ethanol to the lysed cells, mix thoroughly, and transfer to a centrifuge column. Centrifuge at 4000× for 1 minute and discard the waste liquid. Add 500μL of Wash Buffer to the RNA column, centrifuge at 12000×g for 1 minute, and discard the waste liquid. After centrifuging the empty tube at 12000×g for 1 minute, transfer the column to a clean RNase-free 1.5ml centrifuge tube, open the lid and let it dry for 2 minutes. Add 20-50μL of Elution Buffer to the center of the membrane of the RNA column and let it stand at room temperature for 2 minutes. Centrifuge at 12000×g for 1 minute, add the eluted RNA solution back to the column, let it stand on ice for 5 minutes, and centrifuge again to measure the concentration of the eluted RNA;

[0084] Reverse transcription: Reverse transcription was performed using the Novozymes kit. 1 μg RNA was added to 4 μL of 4× gDNA wiper mix, which was made up to 16 μL with enzyme-free water and incubated at 42°C for 2 minutes. Then, 4 μL of 5× qRT SuperMix II was added and incubated at 37°C for 15 minutes, followed by 85°C for 5 seconds.

[0085] Experimental results: TRAP staining, western blot and qPCR detection were performed on osteoclasts induced by sh-NC and sh-AQP9 infection, indicating that after knocking down AQP9 in BMDM, osteoclast differentiation was reduced and the levels of various indicators of osteoclast differentiation were reduced.

[0086] like Figure 7 、 Figure 8 and Figure 9 As shown, Figure 7 This is a diagram of the present application detecting the effective inhibition of osteoclast differentiation after sh-AQP9 lentivirus infection of BMDM cells by TRAP staining, wherein the left figure is a TRAP staining image of BMDM cells inducing osteoclast differentiation, and the right figure is a TRAP staining image of BMDM cells inducing osteoclast differentiation in which AQP9 is knocked down;

[0087] Figure 8 The present application detects by western blot that sh-AQP9 lentivirus effectively inhibits osteoclast differentiation after infecting BMDM cells. The first figure from top to bottom shows that sh-AQP9 has an inhibitory effect on the expression of c-Fos secreted by osteoclasts; the second figure shows that sh-AQP9 has an inhibitory effect on the expression of MMP9 secreted by osteoclasts; the third figure shows that sh-AQP9 has an inhibitory effect on the expression of CTSK secreted by osteoclasts; the fourth figure is an internal reference, representing the homogeneity of protein content. AQP9 can regulate osteoclast differentiation and inhibit the expression of osteoclast indicators such as CTSK, MMP9, and c-Fos; Figure 9 The present application detects by qPCR that sh-AQP9 lentivirus effectively inhibits osteoclast differentiation after infection of BMDM cells. The first figure from left to right shows the inhibitory effect of sh-AQP9 on the expression of Trap secreted by osteoclasts; the second figure shows the inhibitory effect of sh-AQP9 on the expression of CTSK secreted by osteoclasts; the third figure shows the inhibitory effect of sh-AQP9 on the expression of Nfatc1 secreted by osteoclasts; and the fourth figure shows the inhibitory effect of sh-AQP9 on the expression of Atp6v0d2 secreted by osteoclasts.

[0088] Example 6

[0089] After BMDM cells were infected with sh-AQP9 lentivirus, the expression level of AQP9 was reduced.

[0090] Experimental process:

[0091] Preparation of primary mouse bone marrow mesenchymal stem (BMSC): Bone marrow cells were isolated from the femur and tibia of 4-week-old C57BL / 6J mice. The cells were then cultured in a-MEM medium containing 10% FBS and 1% penicillin-streptomycin at 37°C and 5% CO₂ for 48 hours before the medium was replaced. These were primary BMSCs.

[0092] Infect BMSC cells with lentivirus: digest the prepared primary BMSC cells and inoculate them at 5*10 5 Plate the cells in a 12-well plate and place them in an incubator for 24 hours. Infect the sh-NC and sh-AQP9 groups with sh-NC and sh-AQP9 viruses, respectively. After 8 hours, discard the supernatant and replace with fresh culture medium.

[0093] Protein extraction: Aspirate the culture medium, wash once with PBS, and add 150 μL of protein lysis buffer to each well. Incubate on ice for 5 minutes. Scrape the lysed cells with a clean scraper and transfer to a clean 1.5 ml microcentrifuge tube. Vortex for 10 seconds to fully lyse the cells. Incubate on ice for 15 minutes. Centrifuge at 13,000 rpm at 4°C for 10 minutes. Mix 40 μL of the supernatant with 40 μL of 2× loading buffer and boil at 100°C for 10 minutes to denature the proteins.

[0094] Experimental results: PCR detection of bone marrow mesenchymal stem cells (BMSCs) treated with sh-NC and sh-AQP9 lentiviruses showed that the sh-AQP9 lentivirus used could effectively inhibit the expression of AQP9 in BMSC cells.

[0095] like Figure 10 As shown, BMSC cells infected with sh-AQP9 lentivirus had a lower expression level of AQP9.

[0096] Example 7

[0097] sh-AQP9 lentivirus promotes osteoblastogenesis induced by bone marrow mesenchymal stem cells (BMSCs).

[0098] Experimental process:

[0099] Preparation of primary mouse bone marrow mesenchymal stem (BMSC): Bone marrow cells were isolated from the femur and tibia of 4-week-old C57BL / 6J mice. The cells were then cultured in a-MEM medium containing 10% FBS and 1% penicillin-streptomycin at 37°C and 5% CO₂ for 48 hours before the medium was replaced. These were primary BMSCs.

[0100] Inducing osteoblasts: Take 2nd-3rd generation bone marrow mesenchymal stem cells (BMSCs), digest with trypsin, resuspend and count, 5*10 per well 5 Cells were plated in a 6-well plate and allowed to rest in an incubator for 6 hours. After the cells attached, sh-NC or sh-AQP9 virus was added. After 8 hours, the medium was changed and the plate was allowed to rest for 24 hours. The supernatant was discarded and Saiye Bio's mouse osteoblast differentiation induction medium was added. The medium was changed every 3 days.

[0101] ALP staining: After 7 days of osteoblast induction stimulation, discard the supernatant and wash once with PBS. Add 4% paraformaldehyde and let stand for 30 minutes. Wash once with PBS and incubate with the prepared Biyuntian alkaline phosphatase staining solution in a 37°C incubator for 30 minutes.

[0102] ARS Alizarin Red Staining: After 21 days of stimulation with osteoblast induction medium, discard the supernatant and wash once with PBS. Add 4% paraformaldehyde and let stand for 30 minutes. Wash once with PBS and incubate with Saiye Bio Alizarin Red Staining Solution in a 37°C incubator for 30 minutes.

[0103] Protein extraction: Aspirate the culture medium, wash once with PBS, and add 150 μL of protein lysis buffer to each well. Incubate on ice for 5 minutes. Scrape the lysed cells with a clean scraper and transfer to a clean 1.5 ml microcentrifuge tube. Vortex for 10 seconds to fully lyse the cells. Incubate on ice for 15 minutes. Centrifuge at 13,000 rpm at 4°C for 10 minutes. Mix 40 μL of the supernatant with 40 μL of 2× loading buffer and boil at 100°C for 10 minutes to denature the proteins.

[0104] RNA extraction: RNA was extracted using the Yishan Bio RNA Extraction Kit. After absorbing the culture medium, wash once with PBS, add 500μL Lysis Buffer, pipette vigorously 10 times, transfer to an EP tube, and vortex for 10 seconds to fully lyse the cells. Add 500μL of anhydrous ethanol to the lysed cells, mix thoroughly, and transfer to a centrifuge column. Centrifuge at 4000× for 1 minute and discard the waste liquid. Add 500μL of Wash Buffer to the RNA column, centrifuge at 12000×g for 1 minute, and discard the waste liquid. After centrifuging the empty tube at 12000×g for 1 minute, transfer the column to a clean RNase-free 1.5ml centrifuge tube, open the lid and let it dry for 2 minutes. Add 20-50μL of Elution Buffer to the center of the membrane of the RNA column and let it stand at room temperature for 2 minutes. Centrifuge at 12000×g for 1 minute, add the eluted RNA solution back to the column, let it stand on ice for 5 minutes, and centrifuge again to measure the concentration of the eluted RNA;

[0105] Reverse transcription: Reverse transcription was performed using the Novozymes kit. 1 μg RNA was added to 4 μL of 4× gDNA wiper mix, which was made up to 16 μL with enzyme-free water and incubated at 42°C for 2 minutes. Then, 4 μL of 5× qRT SuperMix II was added and incubated at 37°C for 15 minutes, followed by 85°C for 5 seconds.

[0106] Experimental Results: Alkaline phosphatase (ALP) / Alizarin Red (ARS) staining of osteoblasts induced by sh-NC and sh-AQP9 treatment showed that reducing AQP9 expression in BMSCs increased osteoblast production. Western blot analysis revealed elevated levels of the osteoblast-specific markers RUNX2, SP7, and OPN, indicating that sh-AQP9 inhibited AQP9 expression in BMSCs and promoted osteoblast formation. qPCR analysis also revealed increased expression of the osteoblast-specific markers OPG, RUNX2, SP7, APLP, and IBSP following sh-AQP9 infection, indicating enhanced osteoblast formation.

[0107] like Figure 11 、 Figure 12 、 Figure 13 、 Figure 14 As shown, Figure 11 This is a diagram of the application detecting the increase in osteoblast generation after sh-AQP9 lentivirus infection of BMSC cells by alkaline phosphatase ALP staining, wherein the left figure is an ALP staining image of osteoblasts differentiated from BMSC cells, and the right figure is an ALP staining image of osteoblasts differentiated from BMSC cells after sh-AQP9 lentivirus infection; Figure 12 This is a diagram of the present application detecting the increase in osteoblast generation after sh-AQP9 lentivirus infection of BMSC cells by Alizarin Red ARS staining, wherein the left figure is an ARS staining image of osteoblasts differentiated from BMSC cells, and the right figure is an ARS staining image of osteoblasts differentiated from BMSC cells after sh-AQP9 lentivirus infection; Figure 13 The present application uses western blot to detect that sh-AQP9 lentivirus effectively promotes the generation of osteoblasts after infecting BMSC cells. The first figure from top to bottom shows that sh-AQP9 promotes the expression of RUNX2 secreted by osteoblasts; the second figure shows that sh-AQP9 promotes the expression of OPN secreted by osteoblasts; the third figure shows that sh-AQP9 promotes the expression of SP7 secreted by osteoblasts; the fourth figure is an internal control, representing the homogeneity of protein content, and inhibiting AQP9 expression promotes osteoblast formation; Figure 14The present application detects by qPCR that sh-AQP9 lentivirus effectively promotes the generation of osteoblasts after infecting BMSC cells, wherein the first figure from left to right shows that sh-AQP9 promotes the expression of OPG secreted by osteoblasts; the second figure shows that sh-AQP9 promotes the expression of RUNX2 secreted by osteoblasts; the third figure shows that sh-AQP9 promotes the expression of SP7 secreted by osteoblasts; the fourth figure shows that sh-AQP9 promotes the expression of APLP secreted by osteoblasts; the fifth figure shows that sh-AQP9 promotes the expression of COL0A1 secreted by osteoblasts; and the sixth figure shows that sh-AQP9 promotes the expression of IBSP secreted by osteoblasts.

[0108] Example 8

[0109] AQP9 may regulate osteoclast differentiation through the glycolysis pathway

[0110] Experimental process:

[0111] Preparation of primary mouse bone marrow macrophages (BMDM): Bone marrow cells were isolated from the femur and tibia of 8-week-old C57BL / 6J mice and then cultured in IMDM medium containing 10% FBS, 1% penicillin-streptomycin, and 10 ng / mL M-CSF at 37°C in an environment of 5% CO2 for 7 days to prepare BMDM cells.

[0112] Induction of osteoclasts: BMDM cells were digested and resuspended in a-MEM medium and counted at 1*10 per well. 5 Cells were plated in 24-well plates and incubated in an incubator for 24 hours. Six hours after infection with either sh-NC or sh-AQP9 lentivirus, the supernatant was discarded and fresh culture medium was replaced. 24 hours later, 40 ng / ml RANKL and 40 ng / ml m-CSF were added to each well. The medium was changed every 3 days.

[0113] RNA extraction with Trizol: 1 ml of Trizol was added to each of the BMDM group, osteoclast group, and sh-AQP9 lentivirus-infected osteoclast sh-AQP9-osteoclast group and frozen in a 1.5 ml EP tube. Three samples were collected for each group. RNA sequencing was performed.

[0114] Experimental results: Through RNA sequencing analysis, it was speculated that AQP9 may affect osteoclast differentiation through the TCA cycle, thereby promoting the occurrence and development of rheumatoid arthritis.

[0115] like Figure 15 、 Figure 16 and Figure 17As shown, the present application analyzed AQP9 by RNA sequencing to regulate osteoclast differentiation through TCA cycle, wherein Figure 15 This is a heat map showing the differences in expression of osteoclast differentiation-specific indicators CTSK, nfatc1, Traf6, Nfkbia, Oscar, Fos, Acp5, and Fosl2 among the BMDM group, osteoclast group, and sh-AQP9-osteoclast group, which once again confirmed that reducing AQP9 expression can inhibit osteoclast differentiation;

[0116] Figure 16 The middle left figure is an intersection Venn diagram of genes that were elevated in the osteoclast group (compared with the BMDM group) and decreased in the sh-AQP9-osteoclast group (compared with the osteoclast group) infected with sh-AQP9. The right figure is a KEGG pathway analysis using 1324 intersection genes, suggesting that AQP9 regulates osteoclast differentiation through the TCA cycle, thereby alleviating the development of rheumatoid arthritis.

[0117] Figure 17 The most likely target molecule map of AQP9 involved in the TCA cycle was obtained by performing protein interaction analysis on genes involved in the TCA cycle in the KEGG pathway map.

[0118] The examples selected in the above materials are intended to facilitate understanding and are not intended to limit the process. Those skilled in the art may readily modify the process or transfer it to other cases without inventive change. If such modifications fall within the same scope of claims or similar technologies as the present invention, the invention is intended to encompass such modifications.

Claims

1. A novel target for regulating the balance between osteoclasts and osteoblasts, characterized by: The new target for regulating the balance between osteoclasts and osteoblasts is AQP9.

2. A novel target for regulating the balance between osteoclasts and osteoblasts according to claim 1, characterized in that: The AQP9 is a member of the aquaporin subgroup.

3. Use of the new target for regulating the balance between osteoclasts and osteoblasts according to claim 1 or 2 in the preparation of a drug for treating rheumatoid arthritis.

4. The use according to claim 3, characterized in that: The medicine for treating rheumatoid arthritis is in the form of an injection or a non-injection dosage form.

5. The use according to claim 3, characterized in that: The medicine for treating rheumatoid arthritis is an injection, tablet, capsule, powder, pill, granule, solution, suspension, syrup, suppository or inhalant.

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