A method for promoting osteogenic differentiation of mesenchymal stem cells

Abstract

The present invention belongs to the field of biomedical technology. More specifically, it relates to a method for promoting osteogenic differentiation of mesenchymal stem cells. The present invention has found through research that Panax notoginseng polysaccharide can significantly improve the imbalance of osteogenic-adipogenic differentiation of BMSCs caused by CGs, that is, promote osteogenic differentiation of BMSCs and inhibit adipogenic differentiation, suggesting that Panax notoginseng polysaccharide can be used to prevent and / or treat SANFH.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology and more specifically relates to a method for promoting osteogenic differentiation of mesenchymal stem cells. Background Art

[0002] Steroid-induced avascular necrosis of the femoral head (SANFH) is a metabolic orthopedic disease caused by long-term excessive use of glucocorticoids, which leads to osteoblast death and impaired blood supply in the femoral head, eventually leading to femoral head collapse and joint dysfunction. At present, the specific pathogenesis of steroid-induced avascular necrosis of the femoral head is still unclear, and there is no effective treatment. [1] .

[0003] Bone marrow mesenchymal stem cells (BMSCs) are a type of adult stem cells that can self-renew and differentiate into mesoderm cells in the body and have multidirectional differentiation potential. The lineage-specific differentiation of BMSCs into osteoblasts or adipocytes is regulated by many paracrine factors, including cytokines, growth factors, and hormones, which induce intercellular signaling and subsequently activate key transcription factors, thereby regulating the direction of lineage differentiation. [2] . One of the main mechanisms of SANFH is that glucocorticoids break the dynamic balance between osteogenic and adipogenic differentiation of BMSCs. Therefore, finding key factors that regulate osteogenic and adipogenic differentiation of BMSCs to intervene in the balance of osteogenic-adipogenic differentiation, promote osteogenic differentiation and inhibit adipogenic differentiation is the key to the treatment of SANFH.

[0004] Panax japonicus poly saccharide (PJPS) is a polysaccharide compound extracted from Panax japonicus CAMey, which has multiple pharmacological effects such as anti-inflammatory and anti-acute liver damage. Studies have shown that Panax japonicus polysaccharide has the function of reducing serum liver indicators, reducing hepatocyte lipid accumulation, and improving mouse obesity (Chinese patent CN117257828A). However, there are few reports on the regulation of osteogenic-adipogenic differentiation balance of mesenchymal stem cells by Panax japonicus polysaccharide, and the related mechanism is still unclear. Summary of the invention

[0005] In order to overcome the deficiencies of the prior art, the present invention proposes the use of Panax notoginseng polysaccharide in regulating the imbalance of osteogenic-adipogenic differentiation of mesenchymal stem cells, that is, promoting the osteogenic differentiation ability of bone marrow mesenchymal stem cells and inhibiting their adipogenic differentiation ability.

[0006] In order to achieve the above-mentioned invention object, the technical solution adopted by the present invention is:

[0007] In one aspect, the present invention provides the use of Panax notoginseng polysaccharide in promoting osteogenic differentiation of mesenchymal stem cells.

[0008] Preferably, the concentration of the Panax japonicus polysaccharide is 1-100 μg / mL, more preferably 1 μg / mL, 10 μg / mL or 100 μg / mL, more preferably 10 μg / mL.

[0009] Preferably, the preparation of Panax japonicus polysaccharide in the present invention is prepared by referring to the method of Example 1 in Chinese patent application CN 107929308 A.

[0010] On the other hand, the mesenchymal stem cells are bone marrow mesenchymal stem cells treated with glucocorticoids. Long-term excessive use of glucocorticoids (CGs) is the main cause of SANFH. BMSCs can differentiate into a variety of cells in a specific microenvironment, which plays a key role in the repair of bone necrosis. When bone necrosis occurs, fibrous tissue carrying undifferentiated BMSCs migrates to the necrotic area, and BMSCs differentiate into osteoblasts, promoting new bone formation, thereby repairing the necrotic area. [3] Studies have shown that the osteogenic and adipogenic differentiation of BMSCs in SANFH patients is abnormal, the number of BMSCs in the proximal femur of patients is reduced, and the proliferation capacity is reduced. [4] High-dose CGs can induce apoptosis of BMSCs and cause BMSCs differentiation dysfunction by downregulating osteogenic transcription factors such as RUNX2 and Cbfα1 and upregulating adipogenic transcription factors such as PPARγ and C / EBP family members. Therefore, if a substance can improve the imbalance of BMSCs osteogenic-adipogenic differentiation caused by CGs in vitro, it will be beneficial for the prevention and treatment of SANFH.

[0011] The results showed that after DEX treatment of BMSCs, the mRNA expression levels of bone-related genes ALP and Runx2 in the DEX group decreased significantly, and the mRNA expression levels of adipogenic genes FABP4 and PPARγ increased significantly. However, the above situation can be significantly improved after the addition of 1-100 μg / mL PJPS intervention. After the addition of different concentrations of PJPS intervention, the mRNA expression levels of bone-related genes ALP and Runx2 were significantly increased, and compared with the DEX group, the differences were statistically significant. This suggests that PJPS can inhibit the imbalance of osteogenic-adipogenic differentiation of BMSCs caused by hormones, promote its osteogenic differentiation, and inhibit adipogenic differentiation, so it is expected to play a role in the treatment of femoral head necrosis, especially steroid-induced avascular necrosis of the femoral head (SANFH).

[0012] In another aspect, the present invention provides a method for promoting osteogenic differentiation of bone marrow mesenchymal stem cells, comprising the following steps:

[0013] Step (1): obtaining bone marrow mesenchymal stem cells;

[0014] Step (2): treating the bone marrow mesenchymal stem cells with Panax notoginseng polysaccharide for a certain period of time;

[0015] Step (3): Inducing the above-mentioned bone marrow mesenchymal stem cells using conventional osteogenic differentiation induction fluid.

[0016] Preferably, in step (2), the concentration is 1 to 100 μg / mL.

[0017] Preferably, in the step (2), the bone marrow mesenchymal stem cells 12 are treated with Panax notoginseng polysaccharide.

[0018] Preferably, in step (3), the induction time is 5 to 7 days.

[0019] On the other hand, the present invention claims protection for the bone marrow mesenchymal stem cell population prepared by the method.

[0020] On the other hand, the present invention claims to protect the use of Panax notoginseng polysaccharide in the preparation of drugs for treating femoral head necrosis.

[0021] On the other hand, the present invention also claims a drug for treating hormone-induced femoral head necrosis, wherein the drug contains Panax notoginseng polysaccharide.

[0022] The present invention has the following beneficial effects:

[0023] The present invention has found through research that Panax notoginseng polysaccharide can significantly improve the imbalance of osteogenic-adipogenic differentiation of BMSCs caused by CGs, that is, promote osteogenic differentiation of BMSCs and inhibit adipogenic differentiation. Therefore, Panax notoginseng polysaccharide is expected to be used for preventing and / or treating SANFH. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 It is a bar graph of mRNA expression levels of bone-related genes in each group;

[0025] Compared with the normal control group, ** P<0.01; compared with the DEX group, # P<0.05, ## P<0.01.

[0026] Figure 2 It is a bar graph of mRNA expression levels of lipid-related genes in each group;

[0027] Compared with the normal control group, **P<0.01; compared with the DEX group, # P<0.05, ## P<0.01. DETAILED DESCRIPTION

[0028] In order to make the technical means, creative features, objectives and effects achieved by the present invention easy to understand, the present invention is further explained below in conjunction with specific implementation methods.

[0029] The preparation of Panax notoginseng polysaccharide in the present invention is prepared by referring to the method of Example 1 in Chinese patent application CN 107929308 A.

[0030] 1. Experimental research

[0031] 1. Test methods

[0032] 1.1 Isolation and culture of bone marrow mesenchymal stem cells Bone marrow fluid was diluted with complete culture medium (DMEM medium containing 10% fetal bovine serum and 1% penicillin-streptomycin) and plated in 6-well plates. After culturing for 48 hours at 37°C and 5% CO2, the culture medium was replaced and cultured for another 6-8 days. The cells were then digested with trypsin and passaged.

[0033] 1.2 CCK-8 assay for cytotoxicity

[0034] The third generation BMSCs in good condition were collected and resuspended in complete culture medium to obtain a cell concentration of 1×10 4 180 μL of cell suspension with a concentration of 1 μg / mL was inoculated into a 96-well plate per well. After adherence, 20 μL of PJPS at 0, 1, 10, and 100 μg / mL were added respectively. Six replicate wells were set up in each group. After culturing for 24 h, the medium was replaced with DMEM medium containing 10% CCK-8. After incubation for 4 h, the A value at a wavelength of 450 nm was measured with an enzyme reader.

[0035] 1.3 Dexamethasone (DEX) intervention to establish BMSCs hormone environment model

[0036] The third generation BMSCs in good condition were collected and resuspended in complete culture medium to obtain a cell concentration of 1×10 4 100 μL of cell suspension was inoculated into each well of a 96-well plate, and dexamethasone (DEX) was added to make the final concentration of 1×10 -6 mol / L and cultured at 37°C, 5% CO2 for 48 h for subsequent experiments.

[0037] 1.4 Osteogenic differentiation culture

[0038] The DEX-treated BMSCs prepared by the method of 1.3 were collected, and a normal control group (BMSCs not treated with DEX) was set up. Six duplicate wells were set up in each group. Except for the normal control group, the rest were treated with complete medium containing 0, 1, 10, and 100 μg / mL PJPS for 12 hours. When the cell confluence reached more than 80%, osteogenic differentiation medium was used for induction for 7 days. The osteogenic differentiation medium was α-MEM complete medium containing 50 μg / mL vitamin C and 10 mmol / L sodium β-glycerophosphate. ALP staining was performed 7 days after induction, and the area of ​​the staining positive area was observed under an optical microscope (scale bar = 50 μm). ImageJ was used to perform statistical analysis of the area of ​​the staining positive area (the percentage of the positive area to the total area, the positive area of ​​the normal control group was set as 100, and the relative positive area of ​​the remaining groups was calculated). The data are expressed as mean ± SD.

[0039] 1.5 Adipogenic differentiation culture

[0040] The DEX-treated BMSCs prepared by the method of 1.3 were collected, and a normal control group (BMSCs not treated with DEX) was set up. Each group was set up with 6 replicates. Except for the normal control group, the rest were treated with complete medium containing 0, 1, 10, and 100 μg / mL PJPS for 12 hours. When the cell confluence reached more than 80%, the adipogenic differentiation medium was used to induce differentiation for 72 hours. The adipogenic medium was based on the H-DMEM medium containing 10% FBS and 1% penicillin / streptomycin, and the formula was different: 10 mg / mL insulin, 1 μmol / L dexamethasone, 62.5 mmol / L 3-isobutyl-1-methylxanthine, 1 mmol / L rosiglitazone on the first day; 10 mg / mL insulin, 1 mmol / L rosiglitazone on the fourth day; 10 mg / mL insulin on the seventh day. Oil red O staining was performed 7 days after induction and observed under an optical microscope (scale bar = 50 μm). The area of ​​the positive staining region was statistically analyzed by ImageJ (the percentage of the positive area to the total area, and the positive area of ​​the normal control group was set as 100, and the relative positive area of ​​the remaining groups was calculated). The data are expressed as mean ± SD.

[0041] 1.6 RT-qPCR detection of osteogenic-adipogenic related gene expression

[0042] Cells from groups 1.4 and 1.5 were collected, and the cell RNA was extracted by column method using RNA extraction kit according to the kit instructions. After reverse transcription to obtain cDNA, the expression of osteoblast-related genes ALP and Runx2 and adipocyte-related genes fatty acid binding protein 4 (FABP4) and PPARγ was detected. -ΔΔCtThe relative expression of target genes was calculated by the primer sequence shown in Table 1.

[0043] Table 1: Primer sequences

[0044]

[0045] Table 2: PCR reaction system

[0046]

[0047]

[0048] Table 3: PCR amplification program

[0049] Cycle steps temperature time Number of cycles Pre-denaturation 95℃ 5min 1 transsexual 95℃ 10s annealing 55～60℃ 20s 40 extend 72℃ 20s Melting curve 95℃ 15s 1

[0050] 1.7 Statistical analysis The experimental data were analyzed using SPSS software.

[0051] 2. Results

[0052] 2.1 Effect of PJPS on the activity of BMSCs: As shown in Table 4 below, there was no statistically significant difference in cell survival rate between the 0-10 μg / mL concentration groups (P>0.05), and the cell survival rate of BMSCs after treatment with 100 μg / mL PJPS was less than 95%.

[0053] Table 4: Effects of PJPS on BMSCs activity

[0054] PJPS concentration (μg / mL) Survival rate (%) 0 100.00±2.35 1 100.27±1.48 10 98.69±1.67 100 92.38±3.54

[0055] 2.2 ALP staining results: As shown in Table 5 below: After DEX intervention, the ALP staining positive area of ​​each group was smaller than that of the normal control group, and the most obvious was the DEX group (0μg / mL PJPS+DEX group). In addition, compared with the DEX group, the area of ​​the staining positive area increased significantly after 1-100μg / mL PJPS intervention, especially the 10μg / mL PJPS group, indicating that PJPS can enhance the osteogenic differentiation ability of DEX-treated BMSCs.

[0056] Table 5: Comparison of relative positive area of ​​ALP staining

[0057] Group ALP staining relative positive area (%) Normal control group 100.00 DEX Group <![CDATA[43.21±3.65 ** ]]> 1μg / mL PJPS+DEX group <![CDATA[71.15±5.90 △△ ]]> 10 μg / mL PJPS+DEX group <![CDATA[88.67±6.32 △△ ]]> 100 μg / mL PJPS+DEX group <![CDATA[59.44±5.16 △ ]]>

[0058] Note: Compared with the normal control group, ** P<0.01; compared with the DEX group, △ P<0.05, △△ P<0.01.

[0059] 2.3 Oil Red O staining results: As shown in Table 6 below, compared with the normal control group, the Oil Red O staining-positive area of ​​BMSCs in the DEX group (0 μg / mL PJPS+DEX group) was significantly increased. After the addition of PJPS intervention, the Oil Red O staining-positive area of ​​BMSCs in each group was significantly reduced, and the difference was statistically significant compared with the DEX group (P < 0.01).

[0060] Table 6: Comparison of relative areas of Oil Red O staining positive regions

[0061] Group Relative area of ​​Oil Red O staining positive area (%) Normal control group 100.00 DEX Group <![CDATA[189.34±12.41 ** ]]> 1μg / mL PJPS+DEX group <![CDATA[137.05±8.33 △△ ]]> 10 μg / mL PJPS+DEX group <![CDATA[119.41±9.19 △△ ]]> 100 μg / mL PJPS+DEX group <![CDATA[166.63±5.24 △ ]]>

[0062] Note: Compared with the normal control group, ** P<0.01; compared with the DEX group, △ P<0.05, △△ P<0.01.

[0063] 2.4 Comparison of relative expression levels of osteogenesis-related gene mRNA: Figure 1 As shown in the figure, compared with the normal control group, the mRNA expression levels of bone-related genes ALP and Runx2 in the DEX group were significantly decreased, and there was a significant difference with the normal control group (P < 0.01), indicating that the osteogenic differentiation ability of BMSCs was significantly weakened after DEX treatment. However, after adding different concentrations of PJPS intervention, the mRNA expression levels of bone-related genes ALP and Runx2 were significantly upregulated, and compared with the DEX group, the differences were statistically significant (P < 0.01).

[0064] 2.5 Comparison of relative expression levels of adipogenesis-related gene mRNA: Figure 2 As shown in the figure, analysis of the results of each group showed that after DEX intervention, the mRNA expression levels of BMSCs adipogenesis-related genes FABP4 and PPARγ were significantly upregulated, which was significantly different from the normal control group (P < 0.01). On this basis, after adding 1-100 μg / mL PJPS intervention, the mRNA expression levels of adipogenesis-related genes FABP4 and PPARγ were significantly lower than those in the DEX group.

[0065] In summary, DEX intervention in BMSCs inhibited the osteogenic differentiation of BMSCs and promoted their adipogenic differentiation, but the addition of 1-100 μg / mL PJPS intervention could significantly improve the above situation, which suggests that PJPS can inhibit the imbalance of osteogenic-adipogenic differentiation of BMSCs caused by hormones, promote their osteogenic differentiation, and inhibit adipogenic differentiation. Therefore, it is expected to play a role in the treatment of femoral head necrosis, especially hormone-induced avascular necrosis of the femoral head (SANFH).

[0066] References:

[0067] [1] Li Xiangzhou. Effect of Zigan Bushen Decoction on the expression level of cytokines related to osteogenic and adipogenic differentiation of bone marrow mesenchymal stem cells[D]. Gansu. Gansu University of Traditional Chinese Medicine. 2023-03-01.

[0068] [2] Wang Cui, Li Jiao, Lu Lingyun, et al. Effect of lithocholic acid on the balance of osteogenic and adipogenic differentiation of BMSCs[J]. Stem Cells and Tissue Engineering, 2024, 38(1):82-90.

[0069] [3]Zhou D, Chen YX, Yin JH, et al. Valproic acid prevents glucocorticoid-induced osteonecrosis of the femoral head of rats[J]. Int J Mol Med. 2018; 41(6): 3433-3447.

[0070] [4]Zhou W, Lin J, Zhao K, et al. Single-cell profiles and clinically useful properties of human mesenchymal stem cells of adipose and bone ma rroworigin[J]. Am J Sports Med. 2019; 47(7):1722-1733.

[0071] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. Use of Panax notoginseng polysaccharide in preparing a drug for treating steroid-induced femoral head necrosis caused by glucocorticoids, characterized in that: The concentration of the Panax notoginseng polysaccharide is 1-100 μg / mL, and the Panax notoginseng polysaccharide is prepared by the following steps: Take the dried Panax notoginseng powder, add 95% ethanol (V / V), reflux at 60°C for 3h to defat; then add 80% ethanol, reflux at 60°C for 2h to remove monosaccharides and oligosaccharides; after the drug residue is placed in an oven for drying, reflux extraction is performed with purified water as a solvent for 3 times, each time for 2h, the filtrate is combined and placed in a rotary evaporator to be concentrated to a fluid extract state, ethanol is added to adjust the ethanol concentration to 80%, 4°C overnight, filtered the next day, precipitated and dried to obtain the crude Panax notoginseng; take the obtained crude Panax notoginseng, add an appropriate amount of 60°C water to dissolve, after removing protein, dialyze in running water for 48h, freeze-dry the dialyzate to obtain the crude polysaccharide of Panax notoginseng; the crude polysaccharide of Panax notoginseng is purified 3 times by DEAE-Sepharose FF column; then separated and purified by Sephadex G-75 column to obtain a main peak, the peak eluate is dialyzed for desalting and then dried to obtain the Panax notoginseng polysaccharide.

2. The use according to claim 1, characterized in that The concentration of the Panax japonicus polysaccharide is 10 μg / mL.

3. A method for promoting osteogenic differentiation of bone marrow mesenchymal stem cells treated with glucocorticoids in vitro, characterized in that: The following steps are involved: Step (1): obtaining bone marrow mesenchymal stem cells; Step (2): using Panax notoginseng polysaccharide to treat the bone marrow mesenchymal stem cells for a certain period of time, wherein the concentration of Panax notoginseng polysaccharide is 1-100 μg / mL; the Panax notoginseng polysaccharide is prepared by the following steps: taking dried Panax notoginseng medicinal material powder, adding 95% ethanol (V / V), refluxing at 60°C for 3h, and defatting; then adding 80% ethanol, refluxing at 60°C for 2h, and removing monosaccharides and oligosaccharides; placing the drug residue in an oven for drying, and then using purified water as a solvent for reflux extraction for 3 times, each time for 2h, combining the filtrate and placing it in a rotary evaporator to concentrate it to a fluid extract state, adding ethanol, adjusting the ethanol concentration to 80%, 4°C overnight, filtering the next day, precipitating and drying, and obtaining a crude Panax notoginseng product; taking the obtained crude Panax notoginseng product, adding an appropriate amount of 60°C water to dissolve it, removing protein, dialyzing it in running water for 48h, and freeze-vacuum drying the dialyzate to obtain a crude Panax notoginseng polysaccharide; the crude Panax notoginseng polysaccharide is subjected to DEAE-Sepharose chromatography After FF column purification for 3 times, it was separated and purified by Sephadex G-75 column to obtain a main peak, and the peak eluate was dialyzed for desalination and then dried to obtain Panax japonicus polysaccharide; Step (3): Inducing the bone marrow mesenchymal stem cells using conventional osteogenic differentiation induction fluid.

4. The method according to claim 3, characterized in that In the step (2), the bone marrow mesenchymal stem cells are treated with the Panax notoginseng polysaccharide for 12 to 24 hours.

5. The method according to claim 3, characterized in that: In the step (3), the induction time is 5 to 7 days.

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