Composition and application thereof

Abstract

The invention relates to the field of medicines. The invention particularly relates to a composition and application thereof. The present invention provides a composition comprising: 1-deoxynojirimycin (DNJ) and a nicotinamide mononucleotide (NMN). The invention provides a new application of DNJ combined with NMN in promoting osteoblast differentiation, preliminarily proves the promoting effect and potential mechanism of DNJ and NMN combined nutrition intervention on osteoblast differentiation, and is expected to provide an important scientific basis for developing anti-inflammatory factor and anti-skeletal disease functional drugs.

Description

Technical Field

[0001] The present invention relates to the field of medicine, and in particular to a composition and its application. Background Art

[0002] Osteoporosis is a bone disease that causes a decrease in bone mass and an increased risk of fractures. It is well known that osteoporosis can be caused by various endocrine, metabolic, and mechanical factors, but there is also a view that inflammation can be a causative factor for osteoporosis. Epidemiological studies have found that people with inflammatory diseases (such as ankylosing spondylitis, rheumatoid arthritis, and systemic lupus erythematosus) have a higher incidence of osteoporosis compared with other populations. Clinically, the two have also been found to be associated, with osteoporosis and inflammation being comparable in degree. If the inflammation is systemic, bone loss occurs in all skeletal sites, while if the inflammation is limited to a certain site, bone loss only occurs locally at the site of inflammation. Elderly patients are more susceptible to osteoporosis due to the increased production of proinflammatory cytokines with aging.

[0003] DNJ is a glucose analog with an imine group, which has multiple pharmacological effects, including hypoglycemic, anti-tumor, anti-viral and anti-inflammatory activities. Studies have shown that DNJ, as a new anti-inflammatory molecule, plays a protective role in various diseases and can protect the liver by downregulating the expression of inflammatory cytokines such as IL-1β, IL-6 and TNF-α.

[0004] Nicotinamide mononucleotide (NMN), a key intermediate of NAD, has gradually attracted attention in related fields. NAD is an important coenzyme involved in multiple energy metabolism pathways such as glycolysis, β-oxidation, and oxidative phosphorylation. It is a necessary cofactor for post-translational modification of multiple enzymes, such as ribosylation and deacetylation of poly (ADP-ribose) polymerase. It regulates energy metabolism, DNA damage repair, gene expression, and stress response through these enzymes. A large number of studies have shown that NAD levels decrease with age and are closely related to the development of various metabolic disorders, including diseases such as osteoporosis. Many studies have shown that the administration of NAD precursors, such as nicotinamide mononucleotide (NMN), can effectively increase NAD levels in various tissues and prevent such metabolic diseases. As a key intermediate in the biosynthesis of NAD, NMN can reverse mitochondrial homeostasis, redox state, cell survival, and defects in DNA repair. Studies have shown that NMN administration can not only inhibit LPS-induced cellular proinflammatory responses, but also inhibit proinflammatory responses in vivo.

[0005] DNJ and NMN each play an important role in promoting osteoblast differentiation and improving osteoporosis. At present, there are no reports on the effects of DNJ and NMN combined on osteoblasts, so further research and discussion on the specific relationship between them is of great significance. Summary of the invention

[0006] In view of this, the present invention provides a composition and its application. The present invention proposes a new use of DNJ combined with NMN to promote osteoblast differentiation, preliminarily proves the promoting effect and potential mechanism of DNJ and NMN combined with nutritional intervention on osteoblast differentiation, and is expected to provide an important scientific basis for the development of anti-inflammatory factors and anti-bone disease functional drugs.

[0007] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0008] The present invention provides a composition comprising: 1-deoxynojirimycin and nicotinamide mononucleotide.

[0009] In some embodiments of the present invention, the molar ratio of the 1-deoxynojirimycin to the nicotinamide mononucleotide in the above composition is 1:10.

[0010] In some embodiments of the present invention, the concentration of 1-deoxynojirimycin in the above composition is 10 μM.

[0011] In some embodiments of the present invention, the concentration of the nicotinamide mononucleotide in the above composition is 100 μM.

[0012] The present invention also provides application of the above composition in preparing a product for treating osteoporosis.

[0013] In some embodiments of the present invention, in the above-mentioned use, the composition relieves inflammatory stimulation.

[0014] In some embodiments of the present invention, in the above use, the composition promotes osteogenic differentiation.

[0015] In some embodiments of the present invention, in the above-mentioned use, the treatment comprises the following step: administering the composition.

[0016] The invention also provides a product, comprising: the above composition.

[0017] In some embodiments of the present invention, the above-mentioned product includes: one or more of food, health food and pharmaceutical preparation.

[0018] The present invention provides a composition comprising: 1-deoxynojirimycin and nicotinamide mononucleotide.

[0019] The present invention uses a certain concentration of 1-deoxynojirimycin (DNJ) and nicotinamide mononucleotide (NMN) combined reagents to perform nutritional intervention on osteoblasts that induce inflammation after hydrogen peroxide stimulation. The results show that the differentiation degree, markers and other indicators of osteoblasts after nutritional intervention have obvious improvement effects compared with the aging group, DNJ group and NMN group and the DNJ and NMN combination group, and may promote osteoblast differentiation by inhibiting the phosphorylation of S6K1 downstream of mTOR, thereby causing the upregulation of osteoblast markers OCN, OSX, ALP, and RUNX2.

[0020] The present invention proposes a new use of DNJ combined with NMN to promote osteoblast differentiation, and preliminarily proves the promoting effect and potential mechanism of DNJ and NMN combined nutritional intervention on osteoblast differentiation, which is expected to provide an important scientific basis for the development of anti-inflammatory factors and anti-bone disease functional drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] 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 are briefly introduced below.

[0022] Figure 1 Shows the isolation and extraction process of primary osteoblasts and experimental grouping;

[0023] Figure 2 Shown are the results of Alizarin Red (ARS) staining in each group after 14 days of differentiation in osteoblasts;

[0024] Figure 3 Shown are the mRNA expression levels of OCN in each group after 14 days of differentiation in osteoblasts;

[0025] Figure 4 Shows the mRNA expression levels of OSX in each group after 14 days of differentiation in osteoblasts;

[0026] Figure 5 Shows the mRNA expression levels of ALP in each group after 14 days of differentiation in osteoblasts;

[0027] Figure 6 Shows the mRNA expression levels of RUNX2 in each group after 14 days of differentiation in osteoblasts;

[0028] Figure 7 Shows the mRNA expression levels of IL-6 in osteoblasts after 48 h of treatment in each group;

[0029] Figure 8 Shows the protein expression levels of OSX in osteoblasts after 24 h of treatment in each group;

[0030] Fig. 9Shows the protein expression levels of S6K1 and P-S6K1 in osteoblasts after treatment for 24 h in each group;

[0031] Table 1 shows the amplification system of the TAKARA RR037A kit;

[0032] Table 2 shows the ReaL time PCR system;

[0033] Table 3 shows the primer sequences used in ReaL time PCR;

[0034] Table 4 shows the raw data of OCN mRNA expression in each group after 14 days of differentiation in osteoblasts;

[0035] Table 5 shows the raw data of OSX mRNA expression in osteoblasts of each group after 14 days of differentiation;

[0036] Table 6 shows the raw data of ALP mRNA expression in osteoblasts of each group after 14 days of differentiation;

[0037] Table 7 shows the raw data of RUNX2 mRNA expression in osteoblasts after 14 days of differentiation in each group;

[0038] Table 8 shows the original data of IL-6 mRNA expression in osteoblasts of each group after 48h of treatment. DETAILED DESCRIPTION

[0039] The invention discloses a composition and application thereof.

[0040] It should be understood that the expression "one or more of..." includes each of the items recited after the expression individually and in various different combinations of two or more of the recited items, unless otherwise understood from the context and usage. The expression "and / or" in combination with three or more recited items should be understood to have the same meaning, unless otherwise understood from the context.

[0041] The use of the terms "comprising", "having" or "containing", including their grammatical synonyms, should generally be understood as open and non-restrictive, for example not excluding other unrecited elements or steps, unless otherwise specifically stated or otherwise understood from the context.

[0042] It should be understood that the order of steps or the order in which certain actions are performed is not important as long as the present invention remains operable. In addition, two or more steps or actions may be performed simultaneously.

[0043] The use of any and all examples or exemplary language, such as "for example" or "including", herein is intended only to better illustrate the invention and does not limit the scope of the invention unless otherwise claimed. No language in this specification should be construed as indicating that any non-claimed element is essential to the practice of the invention.

[0044] In addition, the numerical ranges and parameters used to define the present invention are approximate values, and the relevant values ​​in the specific embodiments have been presented as accurately as possible. However, any numerical value inherently inevitably contains standard deviations due to individual test methods. Therefore, unless otherwise expressly stated, it should be understood that all ranges, quantities, values ​​and percentages used in this disclosure are modified by "about". Here, "about" generally means that the actual value is within plus or minus 10%, 5%, 1% or 0.5% of a specific value or range.

[0045] The present invention provides a composition for alleviating inflammatory stimulation and promoting osteogenic differentiation, wherein the composition comprises DNJ and NMN, wherein the dosage ratio of the DNJ to the NMN is 1:10, the concentration of the DNJ is 10 μM, and the concentration of the NMN is 100 μM.

[0046] The application of the composition in promoting osteoblast differentiation in an aging state.

[0047] A food or health food or medicine, comprising the above-mentioned composition, is used for promoting osteoblast differentiation.

[0048] Experimental drug preparation:

[0049] 1-Deoxynojirimycin (DNJ): Accurately weigh 1-deoxynojirimycin powder (Selleck, S3839) and prepare it to 10 mM, which is 1000 times the required concentration for subsequent experiments. Carry out cell experiments at a dose of 10 μM and dissolve it in sterile water.

[0050] Nicotinamide mononucleotide (NMN): Accurately weigh nicotinamide mononucleotide powder (Selleck, S5259) and prepare it to 100 mM, which is 1000 times the required concentration for subsequent experiments. Carry out cell experiments at a dose of 100 μM and dissolve the powder in sterile water.

[0051] Hydrogen peroxide (H2O2): Take 30% hydrogen peroxide solution (Sigma, 95294), dilute 1 μL of hydrogen peroxide stock solution with 23.5 μL of sterile water to 1000 times, and the solvent concentration is 400 μM.

[0052] 1-deoxynojirimycin (DNJ) and nicotinamide mononucleotide (NMN) mixed solution: accurately weigh a certain amount of 1-deoxynojirimycin (DNJ) and nicotinamide mononucleotide (NMN) powder respectively, and the dosage ratio of 1-deoxynojirimycin (DNJ) to nicotinamide mononucleotide (NMN) is 1:10.

[0053] Cell Grouping:

[0054] They were the control group (NC group), inflammation model group (H2O2 group), 1-deoxynojirimycin group (H2O2+DNJ group), nicotinamide mononucleotide group (H2O2+NMN group), and 1-deoxynojirimycin group and nicotinamide mononucleotide combined intervention group (H2O2+DNJ+NMN group).

[0055] In Examples 1 to 4 and the effect examples of the present invention, the raw materials and reagents used can be obtained from the market.

[0056] The present invention will be further described below in conjunction with embodiments:

[0057] Example 1 Cell experiment

[0058] Osteoblast isolation, culture and passaging

[0059] 1) Take 0-3 day old newborn mice, disinfect them with 70% ethanol, and place them in a large culture dish.

[0060] 2) Cut the skin on the head, remove the skull and place it in a 10 cm culture dish filled with sterile PBS. Cut the skull into small pieces, place them in a centrifuge tube and wash them with PBS.

[0061] 3) Digest the skull twice in collagenase type I, incubating at 37°C with shaking for 10 min each time. Remove and discard the supernatant.

[0062] 4) Digest the cells three times in collagenase type I, incubate at 37°C with shaking for 15 min each time, and pipette the digestion solution through a 70 μM cell sieve into a centrifuge tube.

[0063] 5) Centrifuge the mixture collected three times at 1000 rpm for 5 min, discard the supernatant and resuspend the cell pellet in complete osteoblast culture medium. Combine the cell suspension, pipette evenly and seed into the culture dish.

[0064] 6) Place the culture dish in a 37°C / 5% CO2 incubator, observe the cells and change the medium on the next day, and then change the medium every 2 days until subculture.

[0065] 7) When subculturing, first aspirate the culture medium, then wash the cells once with PBS to ensure that the culture medium is washed clean, add 1.5 mL of trypsin to digest for 2 min, and observe under a microscope to see if the cells are completely digested. If not, a second digestion is required. After adding culture medium to terminate digestion, collect the cells and centrifuge at 1000 rpm for 5 min.

[0066] 8) Add culture medium to resuspend the cells and inoculate them into culture dishes or 12-well plates.

[0067] Example 2 Cell differentiation and drug treatment

[0068] 1) Prepare complete osteogenic differentiation medium (α-MEM, 10% FBS, 1% P / S, 0.05 mM ascorbic acid, 10 mM sodium β-glycerophosphate).

[0069] 2) Take OB cells with cell fusion of about 85%, digest them with trypsin, add complete culture medium to stop digestion after cell dissociation, and centrifuge at 800 rpm for 5 min.

[0070] 3) Adjust the cell density to 5×10 3 cells / cm 2 The cells were inoculated into the treated 12-well plates, and 1 mL of complete culture medium was added to each well. When the cell confluence reached 100%, the complete culture medium was replaced with the complete culture medium for inducing osteogenic differentiation to start osteogenic differentiation.

[0071] 4) The cells were cultured in a 37°C, 5% CO2 saturated humidity incubator for 2 weeks. The osteogenic medium was changed every two days: the control group was a normal differentiation medium; the inflammation model group was added with 400 μM H2O2 only at the first medium change, and the medium change was normal differentiation medium thereafter; the 1-deoxynojirimycin group was added with 400 μM H2O2 and 1 μM DNJ only at the first medium change, and only 1 μM DNJ was added thereafter; the nicotinamide mononucleotide group was added with 400 μM H2O2 and 10 μM NMN only at the first medium change, and only 10 μM NMN was added thereafter; the 1-deoxynojirimycin group and nicotinamide mononucleotide combined intervention group was added with 400 μM H2O2, 1 μM DNJ, and 10 μM NMN only at the first medium change, and only 1 μM DNJ and 10 μM NMN were added thereafter.

[0072] Alizarin red staining: On the 14th day after osteogenic induction, discard the complete medium for inducing osteogenic differentiation in the well plate, wash once with PBS, add 100% anhydrous ethanol (to cover the cell surface), fix the cells for 10 min, discard the fixative, wash once with PBS, add an appropriate amount of Alizarin red working solution (to cover the cell surface), stain on a shaker at room temperature for 30 min, discard the staining solution, wash 1-2 times with distilled water, observe the osteogenic staining effect, mainly stain the mineralized nodule material in the cells, and observe the color depth after staining to judge the difference in mineralization degree.

[0073] Example 3 Expression of osteogenic differentiation marker molecule signals in cells

[0074] 1) Aspirate the complete medium in the 24-well plate, wash once with PBS, add 1 mL of TrizoL to all samples, let stand on ice for about 10 min, and then collect the liquid into the corresponding sterile enzyme-free centrifuge tube.

[0075] 2) Freeze in -80℃ freezer and thaw on ice.

[0076] 3) Add 500 μL of chloroform to the centrifuge tube, cover it with two centrifuge tube racks, shake it up and down, and let it stand at room temperature for 3 min.

[0077] 4) Centrifuge at 2000 rpm, 4°C for 15 min (make sure all tubes are placed in the same direction during centrifugation to facilitate subsequent supernatant collection).

[0078] 5) Prepare a new set of 1.5 mL centrifuge tubes and transfer as much of the supernatant as possible into them.

[0079] 6) Add 0.6-1 times the volume of isopropanol to the supernatant, shake well, and let stand at room temperature for 15 min or on ice for 30 min.

[0080] 7) Centrifuge at 12,000 rpm, 4°C for 15 min.

[0081] 8) Pour the supernatant into the waste tank (be careful not to let the centrifuge tube touch the waste tank or anywhere else). After all the supernatant has been poured out, add 500 μL of 75% ethanol to each tube and turn the centrifuge tube upside down to clean the precipitate.

[0082] 9) Centrifuge at 7500 rpm, 4°C for 5 min.

[0083] 10) Take a new clean paper towel, pour the supernatant into the waste tank, open the centrifuge tube, place the tube mouth downward on the paper towel, and air dry for 10-15 minutes. If there is still liquid on the top of the centrifuge tube that has not dried after 15 minutes, use a 10 μL gun to suck it away.

[0084] 11) Add 20 μL of DEPC water to the centrifuge tube and tap the tube to dissolve the RNA.

[0085] 12) Nanodrop concentration measurement: Before measuring the concentration, add 1 μL DEPC water to clean the Nanodrop, dry it with a soft paper towel, then add 1 μL DEPC water as a blank, then take 1 μL of each sample, measure the concentration, and record the concentration and 260 / 280 of each sample.

[0086] 13) Using the TAKARA RR037A kit, 10 μL system, calculate the concentration and the amplification system is as follows:

[0087] Table 1

[0088]

[0089] 14) After inversion, the sample can be directly frozen at -20℃, or diluted 10-15 times with water in a 500 μL centrifuge tube and frozen at -20℃.

[0090] 15) β-actin was selected as the internal reference gene, and the levels of osteocalcin (OCN), alkaline phosphatase (ALP), Osterix (Osx), and Runt-related transcription factor 2 (Runx2) were analyzed.

[0091] The ReaL time PCR system is as follows:

[0092] Table 2

[0093]

[0094] The primers used for ReaL time PCR are as follows:

[0095] Table 3

[0096]

[0097] Example 4 Western blot experiment

[0098] 1. Cell Lysis

[0099] 1) The cultured cells were rinsed once with pre-cooled PBS;

[0100] 2) Aspirate PBS and add pre-cooled lysis buffer (add protease and phosphatase inhibitors to the lysis buffer before use) and lyse for 10 minutes;

[0101] 3) Use a cell scraper to scrape the adherent cells, gently transfer the cells and lysate into a pre-cooled centrifuge tube, and freeze in a -80℃ refrigerator;

[0102] 4) After thawing on ice, centrifuge at 13,000 rpm for 15 min at 4°C;

[0103] 5) Gently aspirate the supernatant and transfer it to a newly pre-cooled centrifuge tube and place it on ice. This is the protein sample and discard the precipitate.

[0104] 2. Protein quantification and denaturation

[0105] 1) According to the number of samples, prepare an appropriate amount of BCA working solution by adding 50 volumes of BCA reagent A to 1 volume of BCA reagent B (50:1) and mix thoroughly;

[0106] 2) Add 100 μL BCA working solution and 2 μL protein sample to each well, incubate at 37°C with shaking for 30 min, and add protein standards to several wells at the same time;

[0107] 3) Colorimetric determination at 562 nm. Draw a standard curve with protein content as the horizontal axis and absorbance value as the vertical axis;

[0108] 4) Calculate the corresponding protein concentration based on the absorbance value of the measured sample;

[0109] 5) Level the tube, add loading buffer, mix well, and boil at 100℃ for 10 min.

[0110] 3. SDS-PAGE gel electrophoresis

[0111] 1) Align the glass plate and clamp it in the clamp. Then clamp it vertically on the rack and prepare for gel pouring. Select the appropriate separation gel concentration according to the instructions of the gel preparation kit, and finally add APS and TEMED, then shake well and pour the gel.

[0112] 2) After preparing the lower layer of glue, add a layer of isopropyl alcohol to make the gelling faster after liquid sealing.

[0113] 3) When there is a refraction line between the water and the glue, it means the glue has solidified. Wait a few minutes and then remove the isopropyl alcohol on the glue and use absorbent paper to absorb the water.

[0114] 4) Fill the remaining space with concentrated glue and then insert the comb into the concentrated glue. When pouring glue, make sure the glue flows down along the glass plate to avoid bubbles in the glue. When inserting the comb, keep the comb horizontal.

[0115] 5) After the gel is ready, install it together with the glass plate on the electrophoresis tank. After adding the electrophoresis buffer, start preparing to load the sample. The electrophoresis solution should at least cover the small glass plate inside. Do not pour too much electrophoresis solution under the electrophoresis tank). Use a micropipette to absorb the sample from the wall. Do not absorb bubbles. Insert it into the sample well and slowly add the sample.

[0116] 4. Transfer

[0117] 1) Immerse the PVDF membrane in anhydrous methanol for 1 min, then immerse it in transfer buffer along with two 3 mm filter papers and a sponge.

[0118] 2) Cut the gel according to the marker and the position of the target band (make sure to mark the cutting corners).

[0119] 3) Prepare the "sandwich": follow the following order: filter paper-PVDF membrane-gel-filter paper-sponge-fiber pad.

[0120] 5. Blocking and Antibody Incubation

[0121] 1) After transfer, incubate with 10 mL of 10% milk powder blocking solution at room temperature on a shaker for 1 h.

[0122] 2) Wash the membrane three times with TBST, 5 min each time.

[0123] 3) Add 5 mL of primary antibody dilution buffer and shake gently at 4°C overnight.

[0124] 4) Wash the membrane three times with TBST, 10 min each time.

[0125] 5) Add secondary antibody (usually 1:2000 dilution) and gently shake at room temperature for 1 h.

[0126] 6) Wash the membrane three times with TBST, 10 min each time.

[0127] 6. Development and analysis

[0128] The present invention has completed cell level verification.

[0129] Effect example

[0130] 1. Figure 2 Alizarin red staining was used to observe the formation of osteoblast mineralized nodules in each group. The results showed that compared with the control group (NC group), the mineralized nodules in the aging group (H2O2 group) were significantly reduced, and the calcium salt crystals were reduced, indicating that the experimental model was successful. After the combination of 1-deoxynojirimi and nicotinamide mononucleotide (H2O2+DNJ+NMN group), the mineralized nodules were significantly higher than those in the 1-deoxynojirimi group (H2O2+DNJ group) and the nicotinamide mononucleotide group (H2O2+NMN group), indicating that the combined intervention promoted the differentiation of inflammatory osteoblasts and could restore the differentiation function inhibited by H2O2.

[0131] 2. Osteoblasts undergo four stages during bone formation: osteoblast proliferation, extracellular matrix maturation, extracellular matrix mineralization, and osteoblast apoptosis. Osteocalcin (OCN) appears at the end of osteoblast differentiation and can react with Ca 2+Binds to regulate calcium homeostasis and bone mineralization. Osterix (OSX) is an essential transcription factor in osteoblast differentiation and bone formation, and is highly specific to osteoblasts. ALP is the most commonly used marker for early osteoblast differentiation, and it is also an important enzyme that catalyzes and upregulates the expression of the osteoblast differentiation gene OPN. Runx2 belongs to the Runx family of transcription factors and is one of the most critical transcription factors for osteoblast differentiation and bone formation. It can effectively initiate the expression of major bone matrix protein genes. Figure 3 , Figure 4 , Figure 5 , Figure 6 The results in Tables 4 to 7 show that compared with the control group (NC group), the gene expressions of osteoblast differentiation-related transcription factors OCN, OSX, ALP, and Runx2 in the inflammation group (H2O2 group) were significantly reduced. After the combination of 1-deoxynojirimi and nicotinamide mononucleotide (H2O2+DNJ+NMN group), the osteoblast markers were significantly higher than those in the 1-deoxynojirimi group (H2O2+DNJ group) and the nicotinamide mononucleotide group (H2O2+NMN group), indicating that the combined intervention significantly promoted osteoblast differentiation.

[0132] Table 4

[0133]

[0134] Table 4 corresponds to Figure 3 data;

[0135] Table 5

[0136]

[0137] Table 5 corresponds to Figure 4 data;

[0138] Table 6

[0139]

[0140] Table 6 corresponds to Figure 5 data;

[0141] Table 7

[0142]

[0143] Table 7 corresponds to Figure 6 data.

[0144] 3. IL-6 (interleukin-6) is a multifunctional cytokine, a type of cytokine. It is involved in a variety of physiological processes such as immune response, acute phase response and hematopoiesis. In inflammatory response, increased levels of IL-6 can be used as an indicator of the presence of inflammation. Figure 7The results in Table 8 show that the gene expression of IL-6 in the inflammation group (H2O2 group) was significantly increased compared with the control group (NC group). The expression level of IL-6 in the group treated with 1-deoxynojirimi and nicotinamide mononucleotide (H2O2+DNJ+NMN group) was significantly lower than that in the 1-deoxynojirimi group (H2O2+DNJ group) and the nicotinamide mononucleotide group (H2O2+NMN group), indicating that the combined intervention significantly reduced the occurrence of inflammatory response, thereby promoting osteoblast differentiation.

[0145] Table 8

[0146]

[0147] Table 8 corresponds to Figure 7 data.

[0148] 4. Figure 8 The results showed that the expression level of osteoblast marker OSX protein in the group treated with 1-deoxynojirimi and nicotinamide mononucleotide (H2O2+DNJ+NMN group) was significantly higher than that in the 1-deoxynojirimi group (H2O2+DNJ group) and the nicotinamide mononucleotide group (H2O2+NMN group), indicating that the combined intervention significantly promoted osteoblast differentiation.

[0149] 5. Fig. 9 The results showed that compared with the control group (NC group), the protein expression level of S6K1 phosphorylation downstream of mTOR in the aging group (H2O2 group) was significantly increased, and after administration of 1-deoxynojirima and nicotinamide mononucleotide combined group (H2O2+DNJ+NMN group), it was significantly lower than that in the 1-deoxynojirima group (H2O2+DNJ group) and the nicotinamide mononucleotide group (H2O2+NMN group), indicating that the combined intervention inhibited the activation of the mTOR signaling pathway in osteoblasts.

[0150] The above experiments verified that the combination of DNJ and NMN can promote osteoblast differentiation by inhibiting the upregulation of osteoblast markers caused by S6K1 phosphorylation downstream of mTOR.

[0151] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A composition, characterized in that include: 1-Deoxynojirimycin and nicotinamide mononucleotide.

2. The composition according to claim 1, characterized in that The molar ratio of the 1-deoxynojirimycin to the nicotinamide mononucleotide is 1:

10.

3. Use of the composition as claimed in claim 1 or 2 in the preparation of a product for treating osteoporosis.

4. The use according to claim 3, characterized in that The composition relieves inflammatory irritation.

5. The use according to claim 3 or 4, characterized in that: The composition promotes osteogenic differentiation.

6. The use according to any one of claims 3 to 5, characterized in that The treatment comprises the step of administering the composition.

7. The product, characterized in that include: The composition according to claim 1 or 2.

8. The product according to claim 7, characterized in that The products include: one or more of food, health food and pharmaceutical preparations.

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