New application of salidroside in preventing premature senescence of bone marrow mesenchymal stem cells induced by oxidative stress

Abstract

The present application relates to a new use of salidroside for preventing premature aging of bone marrow mesenchymal stem cells induced by oxidative stress. The implementation process of prevention includes materials, instruments and implementation methods; materials and instruments include reagents, antibodies, primers and instruments; implementation methods include bone marrow mesenchymal stem cell recovery and culture and generation age calculation, CCK8 method for detecting cell viability, free radical inducer-induced premature aging model construction of bone marrow mesenchymal stem cells, flow cytometry detection of cell reactive oxygen free radical levels, Western blot detection of aging-related protein expression, qRT-PCR detection of aging-related gene expression, β-galactosidase staining and statistical analysis. The present application has the effect of significantly reducing the increase in the level of intracellular reactive oxygen free radicals in rat bone marrow mesenchymal stem cells caused by the oxidant AAPH and improving its premature cell aging characteristics, and has important application prospects for improving the quality of stem cells.

Description

Technical Field

[0001] The present application relates to the field of medical technology, and in particular to a new use of salidroside in preventing premature aging of bone marrow mesenchymal stem cells induced by oxidative stress. Background Art

[0002] Bone marrow mesenchymal stem cells are non-hematopoietic pluripotent stem cells with differentiation ability derived from bone marrow. They have become commonly used seed cells in the field of stem cells and regenerative medicine due to their low immunogenicity, easy access to materials, and stable biological properties. However, bone marrow mesenchymal stem cells are very prone to aging during in vitro culture and transplantation, which is a more difficult problem that limits their clinical application and is also one of the reasons for the high cost of clinical use of cells.

[0003] With the increase of in vitro passage times, mesenchymal stem cells can age rapidly: cell morphology changes, proliferation ability weakens, stemness gradually disappears, etc. Studies have shown that compared with the 5th generation of mesenchymal stem cells, the proliferation ability of the 15th generation cells is reduced, the number of aging-related β-galactosidase-positive cells increases, and the expression of aging-related genes is upregulated. The aging of bone marrow mesenchymal stem cells leads to problems such as low transplantation migration rate and survival rate, poor differentiation function, etc., which seriously affects the effect of transplantation treatment.

[0004] Therefore, finding a method that can effectively delay the aging of bone marrow mesenchymal stem cells has important application value in breaking the bottleneck of their clinical transplantation for disease treatment.

[0005] Oxidative stress-induced premature aging is caused by excessive free radical production and imbalance of the antioxidant defense system in the body, leading to premature aging of cells and tissues, involving multiple cellular and molecular mechanisms. In recent years, research on premature aging induced by oxidative stress has made significant progress. Many studies have confirmed that oxidative stress not only plays an important role in the natural aging process, but also aggravates cell and tissue damage in the early stages of many diseases. The research and development of anti-aging drugs based on the oxidative stress mechanism has also made certain progress. For example, the mTOR inhibitor rapamycin and the AMPK activator metformin have been preliminarily confirmed to have the potential to delay aging. In addition, the anti-aging effects of natural products such as resveratrol and curcumin are also being verified in clinical trials. In the future, the combination of modern scientific and technological means such as precision medicine, systems biology and regenerative medicine is expected to delay the premature aging process and improve human quality of life and healthy life span.

[0006] Rhodiola rosea belongs to the genus Rhodiola of the Crassulaceae family. It has the effects of strengthening the body and replenishing qi, promoting blood circulation and removing blood stasis, resisting hypoxia, enhancing immune function and bidirectional regulation. It is mainly used for qi deficiency and blood stasis, chest pain, hemiplegia due to stroke, fatigue and asthma. The chemical components of Rhodiola rosea include phenylpropanoid compounds, flavonoid compounds, polysaccharide compounds, alkaloids, linalool compounds, volatile oils and organic acids. Salidroside is one of the representative active ingredients of Rhodiola rosea. The chemical name of Salidroside is: 2-(4-Hydroxyphenyl)-3,4-dihydroxybutanoic acid-1-O-β-D-glucopyranoside, and the chemical structure is (2R,3R)-2-(4-hydroxyphenyl)-3,4-dihydroxybutyronitrile-1-O-β-D-glucoside. Whether Rhodiola rosea can prevent the premature aging of bone marrow mesenchymal stem cells induced by oxidative stress needs further confirmation.

[0007] In view of the above-mentioned related technologies, the inventors believe that further experiments are needed to confirm the new use of salidroside in preventing premature aging of bone marrow mesenchymal stem cells induced by oxidative stress. Summary of the invention

[0008] In order to further confirm the new use of salidroside in preventing premature aging of bone marrow mesenchymal stem cells induced by oxidative stress, the present application provides a new use of salidroside in preventing premature aging of bone marrow mesenchymal stem cells induced by oxidative stress.

[0009] The application provides a new use of salidroside in preventing premature aging of bone marrow mesenchymal stem cells induced by oxidative stress, which adopts the following technical solution:

[0010] A new use of salidroside for preventing premature aging of bone marrow mesenchymal stem cells induced by oxidative stress, wherein the prevention implementation process includes materials, instruments and implementation methods;

[0011] The materials and instruments include reagents, antibodies, primers and instruments;

[0012] The implementation method includes bone marrow mesenchymal stem cell recovery and culture and generation age calculation, CCK8 method to detect cell viability, free radical inducer to induce premature aging model of bone marrow mesenchymal stem cells, flow cytometry to detect the level of cell active oxygen free radicals, Western blot to detect the expression of aging-related proteins, qRT-PCR to detect the expression of aging-related genes, β-galactosidase staining and statistical analysis.

[0013] By adopting the above technical scheme, by using salidroside to prevent premature aging of bone marrow mesenchymal stem cells induced by oxidative stress, the aging process of bone marrow mesenchymal stem cells can be effectively inhibited, their biological activity in an aging-induced environment can be improved, the aging state of stem cells can be delayed, and the regeneration ability of stem cells can be enhanced, providing a new theoretical basis and technical support for the clinical application of bone marrow mesenchymal stem cells. During the implementation process, salidroside was used in combination with an oxidative stress inducer, and the cell viability was detected by the CCK8 method to evaluate the survival rate and functional state of stem cells; flow cytometry was used to detect the level of intracellular reactive oxygen free radicals to further reveal the effect of oxidative stress on stem cells; Western blot and qRT-PCR were used to analyze the expression levels of aging-related proteins and genes, respectively, to reveal the anti-aging mechanism of salidroside at the molecular level; β-galactosidase staining was used to detect the appearance of senescent cells, and combined with statistical analysis to comprehensively evaluate the preventive effect of salidroside on premature aging of bone marrow mesenchymal stem cells.

[0014] Preferably, the Western blot detection of aging-related protein expression includes extracting total protein, determining protein concentration, protein denaturation treatment, SDS-PAGE protein electrophoresis and immunoblotting.

[0015] By adopting the above technical scheme and the method of using Western blot to detect the expression of aging-related proteins, it is possible to accurately analyze the expression changes of related proteins in the aging process of bone marrow mesenchymal stem cells induced by oxidative stress, and then reveal the mechanism of action of salidroside in preventing aging. The specific technical functions include: first, extracting total protein and determining its concentration to ensure the accuracy of the protein content in the sample; then separating the protein by protein denaturation treatment and SDS-PAGE protein electrophoresis, and further quantitatively analyzing the target protein; immunoblotting technology recognizes the target protein through specific antibodies, and quantitatively determines its expression level in different experimental groups, which can sensitively detect aging-related proteins, such as p16, p21, etc., revealing the changes in protein expression caused by oxidative stress, and the regulatory effect of salidroside on these changes, thereby providing molecular-level evidence for salidroside to delay stem cell aging and improve stem cell function, achieving in-depth analysis of the aging mechanism of bone marrow mesenchymal stem cells, and verifying the preventive effect of salidroside, laying a foundation for clinical application in related fields.

[0016] Preferably, the reagents include salidroside, rat bone marrow mesenchymal stem cells, complete medium for rat bone marrow mesenchymal stem cells, trypsin-EDTA digestion solution, Cell Counting Kit-8, phosphatase inhibitors, protease inhibitors, protein markers, 2×SYBR Green, 10×

[0017] Tris-Glycine SDS electrophoresis buffer, 10× blotting membrane transfer buffer, RNA rapid extraction kit, dimethyl sulfoxide, polyethylene glycol dehydrated sorbitan monolaurate,

[0018] 2,2'-azobis-2-methyl-propanimidamide,dihydrochlori, tetramethylethylenediamine solution, ammonium persulfate, Western primary antibody, secondary antibody diluent, Western primary antibody and secondary antibody removal buffer, 5× protein loading buffer, RIPA lysis buffer, acrylamide / methylenebisacrylamide solution, 4× Tris-HCl / SDS buffer, 4× Tris-HCl / SDS buffer, senescence-associated β-galactosidase staining.

[0019] By adopting the above technical scheme, the preventive effect of salidroside on premature aging of bone marrow mesenchymal stem cells induced by oxidative stress was comprehensively analyzed to reveal its anti-aging mechanism. First, rat bone marrow mesenchymal stem cells and their complete culture medium were used to resuscitate and culture cells, and the cells were digested with trypsin-EDTA digestion solution to ensure that the cell state was appropriate. Cell Counting Kit-8 (CCK-8) was used to evaluate cell viability and detect the effect of salidroside; phosphatase inhibitors and protease inhibitors were used to protect proteins in cells to ensure the accuracy of protein expression in Western blot analysis. SDS-PAGE protein electrophoresis and immunoblotting techniques were used to analyze the expression of aging-related proteins, and Western primary antibodies, secondary antibodies and corresponding dilutions were combined for immune reaction, and protein markers were combined for quantitative detection. RNA was extracted using an RNA rapid extraction kit, and 2×SYBR Green and 10×Tris-Glycine SDS electrophoresis buffer were used for qRT-PCR analysis of the expression of aging-related genes. In addition, senescence-related β-galactosidase staining was used to further verify the changes in senescent cells, and the role of salidroside in preventing premature senescence of bone marrow mesenchymal stem cells was evaluated, providing a scientific basis for the study of stem cell aging mechanisms and clinical applications.

[0020] Preferably, the concentration of the pancreatin-EDTA digestion solution is 0.25%;

[0021] The pH value of the 10×Tris-Glycine SDS electrophoresis buffer is pH 8.3;

[0022] The Western primary and secondary antibody removal solution is strongly alkaline;

[0023] The 5× protein loading buffer contains DTT;

[0024] The concentration of the acrylamide / methylenebisacrylamide solution is 40%;

[0025] The ratio of acrylamide to methylene bisacrylamide is 29:1;

[0026] The pH value of the 4×Tris-HCl / SDS buffer is pH 6.8.

[0027] By adopting the above technical scheme, the experimental treatment and protein analysis of bone marrow mesenchymal stem cells can be accurately carried out to ensure the stability and reliability of each experimental step. The concentration of trypsin-EDTA digestion solution is 0.25%, which can effectively digest cells while maintaining cell activity; the pH 8.3 of 10×Tris-Glycine SDS electrophoresis buffer can optimize the separation effect of proteins and improve the accuracy of SDS-PAGE electrophoresis; the strong alkalinity of Western primary and secondary antibody removal solution ensures the removal efficiency of antibodies during immunoblotting and avoids interference with subsequent detection; 5× protein loading buffer contains DTT, which can effectively reduce the disulfide bonds in proteins, ensure complete denaturation of proteins, and contribute to accurate Western blot analysis; the concentration of acrylamide / methylenebisacrylamide solution is 40% and the ratio is 29:1, which ensures the appropriate hardness and resolution of SDS-PAGE gel, so that proteins can be clearly separated. The pH 6.8 of 4×Tris-HCl / SDS buffer helps to maintain a stable protein separation environment during electrophoresis.

[0028] Preferably, the antibodies include GAPDH antibody, p21 antibody, p16 antibody, p53 antibody and pRb antibody.

[0029] By adopting the above technical scheme and using GAPDH, p21, p16, p53 and pRb antibodies, we can accurately detect the expression changes of aging-related proteins in Western blot experiments, evaluate the intervention effect of salidroside on oxidative stress-induced aging of bone marrow mesenchymal stem cells, reveal its molecular mechanism in regulating the cell cycle and inhibiting the aging process, and provide experimental support for the potential application of salidroside in the prevention of stem cell aging.

[0030] Preferably, the primers include p53, p16INK4a, p21CIP1 and β-actin.

[0031] By adopting the above technical scheme, by using p53, p16INK4a, p21CIP1 and β-actin primers, the expression changes of related genes in the aging process of bone marrow mesenchymal stem cells induced by oxidative stress can be accurately analyzed in the qRT-PCR experiment, revealing the anti-aging mechanism of salidroside. p53, p16INK4a and p21CIP1 play a key role in cell response to DNA damage, regulation of cell cycle and induction of aging. By detecting the expression levels of these genes, the regulatory effect of salidroside on cell aging is evaluated, and it is determined that stem cell aging can be delayed by inhibiting these aging pathways; β-actin is used as an internal reference gene to ensure the standardization and accuracy of the experimental results and eliminate possible errors in the experimental process. Combined with the use of these primers, the regulatory mechanism of salidroside in the anti-aging process is comprehensively analyzed at the molecular level, providing a scientific basis for the potential application of salidroside in stem cell therapy and the prevention and treatment of aging-related diseases. At the same time, qRT-PCR, as a sensitive gene expression detection method, can efficiently and accurately quantify the expression changes of target genes, helping to deeply understand the biological effects of salidroside.

[0032] Preferably, the primer p53 includes forward5'-GCAGCACAGGAACCTGGAACTG-3', reverse5'-AGAAGGGACGGAAGATGACAGAGG-3';

[0033] The primer p16INK4a includes forward5'-CAGCTCTCCTGCTCTCCTATGGTG-3'

[0034] and

[0035] reverse5'-CAGGCATCGCGCACATCCAG-3';

[0036] The primer p21CIP1 includes forward 5-CAGTAGACACGAAACAGGCTCAGG-3' and reverse 5'-CTGTCTTGTCTTCGCTGAGGTCTC-3;

[0037] The primer β-actin includes forward 5'-GTAAAGACCTCTATGCCAACA-3' and reverse 5'-GGACTCATCGTACTCCTGCT-3.

[0038] By adopting the above technical scheme, by using p53, p16INK4a, p21CIP1 and β-actin primers, the expression changes of relevant genes in the aging process of bone marrow mesenchymal stem cells induced by oxidative stress can be accurately quantitatively analyzed by qRT-PCR technology, further revealing the mechanism of action of salidroside in preventing aging; p53, p16INK4a and p21CIP1 are important genes related to cell cycle regulation, DNA damage repair and aging, and upregulation of expression is usually accompanied by the initiation of the cell aging process; by detecting the expression levels of these genes, it is evaluated whether salidroside delays stem cell aging by regulating these aging pathways. β-actin is used as an internal reference gene to ensure the standardization of the experiment and the reliability of the results, eliminate exogenous errors in the experiment, and use optimized primers to efficiently and accurately quantify the expression changes of the target genes, providing reliable data support for in-depth analysis of the molecular mechanism of salidroside in aging prevention.

[0039] Preferably, the instrument includes an analytical balance, a biological safety cabinet, a carbon dioxide constant temperature incubator, a three-level pure water system, an ELISA reader, an ultra-micro spectrophotometer, a fluorescence quantitative PCR instrument, a chemiluminescence imaging system, a vertical electrophoresis instrument, and a fluorescence microscope.

[0040] By adopting the above technical solutions, accurate and efficient experimental operations such as cell culture, protein and gene analysis can be achieved to ensure the scientificity and accuracy of bone marrow mesenchymal stem cell aging research; analytical balances are used to accurately weigh experimental reagents to ensure the accurate dosage of reagents; biological safety cabinets ensure a sterile environment for cell operations to prevent contamination; carbon dioxide constant temperature incubators provide ideal growth conditions for cells to maintain the physiological state of cells; three-level pure water systems provide high-purity water sources to ensure the quality of reagents in experiments; microplate readers and ultra-micro spectrophotometers are used to detect cell activity and the absorbance of related substances to accurately determine experimental data; fluorescent quantitative PCR instruments are used to efficiently perform qRT-PCR experiments and quantitatively analyze the expression of aging-related genes; chemiluminescence imaging systems can sensitively detect target proteins in Western blots and evaluate the effects of salidroside on protein expression; vertical electrophoresis instruments provide a stable separation environment for SDS-PAGE protein electrophoresis; and fluorescence microscopes are used to observe cell morphological changes and identify aging cells.

[0041] In summary, the present application includes at least one of the following beneficial technical effects:

[0042] 1. The combined use of salidroside and oxidative stress inducers can effectively slow down the aging process of bone marrow mesenchymal stem cells under oxidative stress, delay the occurrence of cell aging, and effectively inhibit the aging process of bone marrow mesenchymal stem cells;

[0043] 2. Salidroside can enhance the biological activity of bone marrow mesenchymal stem cells in an aging-induced environment, maintain cell survival rate and functional state, enhance the regenerative ability of stem cells, and improve the biological activity of stem cells;

[0044] 3.: Use CCK-8 method to detect cell viability and flow cytometry to detect the level of reactive oxygen free radicals, accurately evaluate the aging state, and evaluate the protective effect of salidroside on stem cell aging in real time;

[0045] 4. Analyze the expression changes of aging-related proteins and genes through Western blot and qRT-PCR technology, reveal the anti-aging mechanism, and provide molecular mechanism evidence for the anti-aging effect of salidroside;

[0046] 5. β-galactosidase staining technology can visually detect the changes in senescent cells, confirm the appearance of senescent cells, and further verify the anti-aging effect of salidroside.

[0047] 6. Through a variety of precision instruments and optimized experimental methods, ensure the stability of the experimental process and the accuracy of the data, provide a scientific basis for the application of salidroside in the prevention of stem cell aging, and improve the accuracy and reliability of experimental data;

[0048] 7. Provide theoretical and technical support for clinical applications: This technical solution provides a theoretical basis and technical support for the potential application of salidroside in stem cell aging prevention and regenerative medicine, and promotes its application prospects in the clinical field. BRIEF DESCRIPTION OF THE DRAWINGS

[0049] Figure 1 This is a flow chart showing that salidroside prevents oxidative stress-induced premature senescence of bone marrow mesenchymal stem cells.

[0050] Figure 2 This is a schematic diagram of the CCK8 method for determining the effect of salidroside on the cell viability of bone marrow mesenchymal stem cells.

[0051] Figure 3 This is a schematic diagram of the experimental results showing the effects of different concentrations of salidroside pretreatment on the ROS level of rat bone marrow mesenchymal stem cells induced by AAPH.

[0052] Figure 4 This is a schematic diagram of the experimental results of the effects of different concentrations of salidroside pretreatment on the expression of aging-related proteins in rat bone marrow mesenchymal stem cells induced by AAPH.

[0053] Figure 5 This is a schematic diagram of the experimental results of the effects of different concentrations of salidroside pretreatment on AAPH-induced aging-related mRNA changes in rat bone marrow mesenchymal stem cells.

[0054] Figure 6 It is a schematic diagram of the experimental results of normal cell culture;

[0055] Figure 7 Roche real-time fluorescence quantitative PCR instrument was used to detect the expression of genes related to the aging pathway. DETAILED DESCRIPTION

[0056] The following is combined with Figure 1-6 This application is described in further detail.

[0057] The present application discloses a novel use of salidroside for preventing premature aging of bone marrow mesenchymal stem cells induced by oxidative stress. Figure 1 A new use of salidroside for preventing premature aging of bone marrow mesenchymal stem cells induced by oxidative stress, the implementation process of prevention includes materials, instruments and implementation methods; materials and instruments include reagents, antibodies, primers and instruments; implementation methods include bone marrow mesenchymal stem cell recovery and culture and generation age calculation, CCK8 method for detecting cell viability, free radical inducer-induced premature aging model of bone marrow mesenchymal stem cells, flow cytometry detection of cell reactive oxygen free radical levels, Western Blot was used to detect the expression of aging-related proteins, qRT-PCR was used to detect the expression of aging-related genes, β-galactosidase staining and statistical analysis were performed, bone marrow mesenchymal stem cells were isolated and cultured in vitro, and bone marrow mesenchymal stem cells were treated with free radical inducers. AAPH produces free radicals by thermal decomposition at physiological temperature. These free radicals can attack proteins or lipids to induce peroxidation reactions and ultimately lead to oxidative damage, inducing premature cellular aging. Different doses of Rhodiola rosea extract were used for intervention to explore the intervention effect of Rhodiola rosea extract on free radical-induced premature aging. Salidroside was used to improve premature aging of bone marrow mesenchymal stem cells induced by oxidative stress through the detection of intracellular reactive oxygen free radicals, aging-related β-galactosidase staining and other aging-related signaling pathway molecules.

[0058] Materials and instruments: Reference Figure 1 The reagents included salidroside, rat bone marrow mesenchymal stem cells, complete medium for rat bone marrow mesenchymal stem cells, trypsin-EDTA digestion solution, Cell Counting Kit-8, phosphatase inhibitors, protease inhibitors, protein marker, 2×SYBR Green, 10×Tris-Glycine SDS electrophoresis buffer, 10×blotting membrane transfer buffer, RNA rapid extraction kit, dimethyl sulfoxide, polyethylene glycol dehydrated sorbitan monolaurate,

[0059] 2,2'-azobis-2-methyl-propanimidamide, dihydrochlori, tetramethylethylenediamine solution, ammonium persulfate, Western primary antibody, secondary antibody diluent, Western primary antibody and secondary antibody removal solution, 5× protein loading buffer, RIPA lysis buffer, acrylamide / methylenebisacrylamide solution, 4× Tris-HCl / SDS buffer, 4× Tris-HCl / SDS buffer, senescence-associated β-galactosidase staining;

[0060] The concentration of trypsin-EDTA digestion solution is 0.25%; the pH value of 10×Tris-Glycine SDS electrophoresis buffer is pH 8.3; the Western primary and secondary antibody removal solution is strongly alkaline; the 5× protein loading buffer contains DTT; the concentration of acrylamide / methylenebisacrylamide solution is 40%; the ratio of acrylamide / methylenebisacrylamide is 29:1; the pH value of 4×Tris-HCl / SDS buffer is pH 6.8;

[0061] The reagent system optimizes experimental steps such as cell culture, protein extraction, gene quantification, immunoblotting and cell aging detection. Through precise configuration and scientific selection of various key reagents, the reliability, repeatability and stability of the experiment are effectively improved. The concentration of trypsin-EDTA digestion solution is 0.25%, which effectively digests and disperses cells, promotes the separation of rat bone marrow mesenchymal stem cells, does not cause excessive damage to cells, and ensures cell activity. The RIPA lysis buffer used can quickly lyse cells and efficiently extract proteins. Combined with the addition of phosphatase inhibitors and protease inhibitors, protein degradation and loss of phosphorylation modifications are avoided, providing high-quality protein and RNA samples for subsequent Western blot and qRT-PCR analysis. The reagent system includes an efficient 5× protein loading buffer, and DTT reducing agent is added to destroy disulfide bonds, ensuring complete denaturation and separation of proteins and avoiding interference from polymers. The concentration and ratio of acrylamide / methylenebisacrylamide solution are 40% and 29:1, providing a suitable gel concentration, which helps to separate proteins of different molecular weights and ensure efficient separation and clear bands during electrophoresis. The 10×

[0062] The pH value of Tris-Glycine SDS electrophoresis buffer is pH 8.3, which ensures the stability of the protein and good electrophoresis effect. The 10× blotting membrane transfer buffer used in the transfer process provides a guarantee for efficient transfer, and the strong alkaline Western primary and secondary antibody removal solution effectively removes excess antibodies, reduces background signals, and ensures specific detection of target proteins. The RNA rapid extraction kit is used to ensure efficient extraction of RNA, and the reasonable ratio of 2×SYBR Green and 10×Tris-HCl / SDS buffer ensures high sensitivity and accuracy of qRT-PCR. The use of aging-related β-galactosidase staining reagent makes the detection of cell aging status more intuitive, and positive cells appear blue-green, which helps to evaluate changes in cell aging under different treatment conditions.

[0063] Reference Figure 1 The antibodies include GAPDH antibody, p21 antibody, p16 antibody, p53 antibody and pRb antibody, and the primers include p53, p16INK4a, p21CIP1 and β-actin.

[0064] Primers p53 included forward5′-GCAGCACAGGAACCTGGAACTG-3′, reverse5′-AGAAGGGACGGAAGATGACAGAGG-3′;

[0065] Primers p16INK4a included forward5′-CAGCTCTCCTGCTCTCCTATGGTG-3′ and reverse5′-CAGGCATCGCGCACATCCAG-3′;

[0066] Primer p21CIP1 included forward 5-CAGTAGACACGAAACAGGCTCAGG-3′ and reverse5′-CTGTCTTGTCTTCGCTGAGGTCTC-3;

[0067] Primers for β-actin included forward 5'-GTAAAGACCTCTATGCCAACA-3' and reverse 5'-GGACTCATCGTACTCCTGCT-3'.

[0068] The antibody and primer system is precisely selected for the detection of aging-related genes and proteins, covering multiple key aging pathways, ensuring efficient and specific detection of aging-related proteins and genes such as p53, p16INK4a, and p21CIP1. Combined with β-actin as an internal reference gene, it can effectively standardize experimental data and eliminate differences between samples. The primers for p53, p16INK4a, and p21CIP1 have high specificity and good amplification efficiency, and can accurately detect the transcription levels of these genes, helping to reveal the main signaling pathways involved in the process of cell aging. The use of antibodies such as GAPDH, p21, p16, p53, and pRb makes the quantitative analysis of aging-related proteins by Western blot more accurate, which can clearly reflect the changes in protein levels and reveal important molecular mechanisms in the aging process.

[0069] Reference Figure 1 The instruments include analytical balance, biological safety cabinet, carbon dioxide constant temperature incubator, three-level pure water system, microplate reader, ultra-micro spectrophotometer, fluorescence quantitative PCR instrument, chemiluminescence imaging system, vertical electrophoresis instrument, fluorescence microscope. By combining high-precision analytical balance, biological safety cabinet, carbon dioxide constant temperature incubator and other infrastructure, the sterility and accuracy of cell culture and processing are ensured, and the three-level pure water system provides high-quality water source for the experiment to ensure the stability of experimental conditions; the cooperation of microplate reader and ultra-micro spectrophotometer makes it possible to measure the absorbance of samples with high precision, meeting the analysis requirements of protein, RNA, DNA quantification, etc. The combination of fluorescence quantitative PCR instrument and chemiluminescence imaging system provides a highly sensitive detection method for quantitative analysis of gene expression and protein quantification, ensuring the accuracy and reliability of data; the application of vertical electrophoresis instrument makes protein separation clearer and more efficient, providing accurate data for subsequent immunoblotting analysis; in addition, the fluorescence microscope provides high-resolution images of cell observation, which can effectively analyze the state of aging cells and related markers, comprehensively improve the operation accuracy and data reliability of the experiment, and help the mechanism analysis of aging research and related diseases.

[0070] Reference Figure 1 In the Western blot, the expression of aging-related proteins was detected, including total protein extraction, protein concentration determination, protein denaturation treatment, SDS-PAGE protein electrophoresis and immunoblotting;

[0071] The method for extracting total protein is to take bone marrow mesenchymal stem cells in the logarithmic growth phase and inoculate them in a 12-well plate at 5×104 / well. After 24 hours, the old culture medium is discarded, and cells are pretreated with drugs containing different concentrations for 12 hours. After that, the cells are treated with AAPH for 24 hours, and the medium containing drugs is replaced and cultured for 72 hours. The cell culture medium is removed, and the cells are washed with 1mL PBS and then the PBS is removed. 100μL RIPA lysis buffer is added to each well, and the cells are allowed to stand at 4℃ for 30 minutes to complete cell lysis. Use a cell scraper to gently scrape the cells, transfer them to a 1.5mL centrifuge tube, centrifuge at 12000rpm at 4℃ for 15 minutes, and then transfer the supernatant to a new 1.5mL centrifuge tube, which is the total protein sample obtained;

[0072] The method for determining protein concentration is to prepare a working solution by mixing BCA reagent and Cu reagent in a ratio of 50:1 according to the instructions of the BCA protein quantification kit, taking a 5 mg / mL BSA standard solution, diluting it to 0.5 mg / mL with PBS, adding different volumes of standard protein to a 96-well plate to prepare a standard curve, adding 2 μL of extracted protein sample to each well to be tested, filling it up to a total volume of 20 μL with PBS, adding 200 μL of BCA working solution and incubating at 37°C for 30 min, measuring the absorbance at a wavelength of 562 nm, calculating the sample protein concentration according to the BSA standard curve, and unifying the samples to the same concentration for later use;

[0073] The protein denaturation treatment method is to mix the protein sample with 5× Loading Buffer in a volume ratio of 4:1, then heat it in a 100°C metal bath for 8 min for denaturation, centrifuge it at low speed after mixing, and store it in a -20°C refrigerator;

[0074] The method of SDS-PAGE protein electrophoresis is to prepare separation gel and concentration gel in sequence. Select a comb with the appropriate number of holes according to the experimental requirements. The prepared gel should be stored in electrophoresis buffer at 4°C or used directly. After thawing the protein sample, gently shake and mix it and centrifuge it at a low speed. After adding the protein sample, run it at 80V for 30 minutes, then adjust it to 120V and continue electrophoresis for 60 minutes;

[0075] The immunoblotting method is:

[0076] 1. According to the size of the gel, cut 2 layers of filter paper and 1 PVDF membrane in advance. Soak the PVDF membrane in methanol for 2 minutes, and then soak the filter paper in transfer buffer. After the electrophoresis is completed, carefully remove the gel and place it in transfer buffer, and then perform semi-dry or wet transfer.

[0077] 2. The semi-dry method is as follows: Place a layer of filter paper on the anode of the semi-dry transfer apparatus, place the PVDF membrane on it, place the glue on the membrane, and then place the filter paper. Align the layers strictly, remove bubbles, and finally cover the cathode of the transfer apparatus. Perform electrotransfer at 0.65-0.80 mA / cm2 for 2 hours. Use semi-dry or wet transfer methods to ensure that the protein can be effectively transferred to the PVDF membrane. Strict control of the transfer process improves the transfer efficiency of the protein on the membrane and ensures the sensitivity of subsequent antibody detection.

[0078] 3. Wet transfer is as follows: Pre-cool the transfer buffer in a 4°C refrigerator. Lay out a "sandwich" according to the transfer instructions: Place the gray electrotransfer box downward, align the pad, filter paper, glue, and PVDF membrane soaked in transfer buffer in sequence, and then cover with filter paper and pad, and use a glass rod to gently remove bubbles. After the electrotransfer box is installed, put it into the electrotransfer tank, install the cooling box, add about 500ml of transfer buffer, and transfer the membrane at 320mA for 1 hour;

[0079] 4. After the transfer, remove the membrane, soak it in methanol for a while to fully fix the protein on the PVDF membrane, and then put it in TBST containing 5% skim milk powder and shake for 1 hour to block. The blocked membrane is placed in a hybridization bag and reacted with the primary antibody solution at room temperature for 1 hour or at 4°C overnight, and then washed 3 times with TBST containing 0.1% Tween 20 for 5 minutes each time to wash away the unbound antibody;

[0080] 5. In a new hybridization bag, react with horseradish peroxidase-labeled secondary antibody solution at room temperature for 1 hour, then wash 3 times with TBST 5 minutes each time;

[0081] 6. Finally, the protein was detected by chemiluminescence, and the color was developed according to the instructions of the ECLPLUS immunoblotting kit. The image was captured by the Chemi Imager 5500 gel imaging system. The immunoblot image was captured using the Chemi Imager 5500 gel imaging system to ensure high image quality and enable clear quantitative analysis of protein bands, providing an accurate basis for experimental data.

[0082] Western blot detection of aging-related protein expression. RIPA lysis buffer was used to effectively lyse the cells, and the proteins were separated by centrifugation to ensure that the total protein sample obtained had high purity and integrity. The total protein was accurately extracted to provide a reliable basis for subsequent protein analysis. The BCA protein quantification kit was used to quantify the protein concentration to accurately determine the protein content in the sample, ensuring that the same amount of protein was used for analysis in subsequent experiments, improving the reliability and repeatability of the data, and comparing the protein sample with the Loading Buffer was mixed and heated to 100℃ for denaturation, which ensured the destruction of protein structure, facilitated electrophoretic separation and immunoblotting detection of proteins, and improved the comparability of experimental results. High-resolution SDS-PAGE protein electrophoresis: protein separation by electrophoresis, effectively separated different proteins according to molecular weight differences, provided clear band patterns, achieved high-resolution SDS-PAGE protein electrophoresis, and facilitated subsequent immunoblotting and quantitative analysis. By blocking nonspecific binding sites and using secondary antibodies labeled with horseradish peroxidase, the specificity of immunoblotting was significantly improved, the background signal was reduced, and the accurate detection of target proteins was ensured. Using the ECLPLUS immunoblotting kit, protein detection by chemiluminescence can significantly improve the signal intensity and detection sensitivity of protein bands, and low-abundance target proteins can also be effectively detected. Combining the protein quantification, denaturation, electrophoresis, membrane transfer, and immunodetection links of Western blot technology, the expression level of aging-related proteins can be accurately and reliably quantified and analyzed, which is suitable for a variety of biological studies, especially the dynamic change analysis of aging-related proteins, making Western The blot protocol for detecting aging-related proteins has high sensitivity, high accuracy and high reliability.

[0083] Reference Figure 1 In the qRT-PCR test of aging-related gene expression, the total RNA of bone marrow mesenchymal stem cells in different treatment groups was obtained by RNA rapid extraction kit, and cDNA was obtained by reverse transcription; cDNA was used as template, and the expression of aging-related genes was detected by qRT-PCR. The PCR reaction system was prepared according to the instructions of the PremixEx TaqTM Fluorescence Quantification Kit. The transcription levels of genes related to the aging pathway (p16, p21, and p53) were detected using a Roche real-time fluorescence quantitative PCR instrument. The RNA rapid extraction kit was used to extract high-purity total RNA in a short time, reducing the risk of RNA degradation and providing a stable and reliable RNA sample for subsequent experiments. The RNA was transcribed into cDNA using a high-efficiency reverse transcriptase to ensure that the generated cDNA had high fidelity and retained the original mRNA sequence information, providing an accurate template for subsequent gene expression detection. Premix Ex TaqTM fluorescence quantitative kit can quickly detect the transcription level of low-abundance genes with high sensitivity. It is suitable for the analysis of expression changes of aging-related genes. It uses efficient fluorescence quantitative PCR technology to optimize primer design and reaction conditions to ensure high amplification specificity of target genes such as p16, p21, and p53, significantly reduce the impact of nonspecific amplification, and detect the changes in fluorescence intensity during gene amplification by real-time fluorescence signal, which can accurately reflect the expression level of the target gene and avoid data deviations that may be caused by endpoint detection. For classic aging pathway genes such as p16, p21 and p53, qRT-PCR can efficiently and quantitatively detect their transcription levels, directly reflecting the dynamic changes of cell aging status. The Ct value data generated by the fluorescence quantitative PCR instrument can be used to calculate the relative expression of the target gene. Combined with reference genes such as GAPDH or β-actin, the difference in gene expression can be intuitively displayed.

[0084] Reference Figure 1 Instructions for β-galactosidase staining are as follows:

[0085] 1. Prepare the dyeing working solution according to the proportion of each dyeing liquid component;

[0086] 2. Discard the old culture medium, add 500 μL PBS to each well to wash once, discard the PBS, add 500 μL fixative, and incubate at room temperature for 15 min; 3. Aspirate the fixative, add 500 μL PBS to each well to wash the cells, 3 min each time, a total of three times, and remove the PBS after washing;

[0087] 3. Add 500 μL of staining working solution to each well for staining. Use plastic wrap to wrap the cell culture plate to prevent the liquid from evaporating and causing changes in the concentration of the staining solution;

[0088] 4. Incubate at 37℃ for 3-16 hours, count and observe under a microscope. Positive cells will appear blue-green after staining.

[0089] The β-galactosidase staining method optimizes the fixation, washing and staining steps, adopts low-evaporation staining technology and controllable incubation conditions, and can efficiently and accurately display the activity of β-galactosidase in senescent cells. Positive cells appear blue-green, providing a reliable and intuitive detection method for evaluating the cellular aging status.

[0090] Reference Figure 1 Statistical analysis: The experimental results used in this section were obtained by three independent parallel experiments or three parallel samples in one experiment. All data were analyzed and plotted using Graphpad9.5. One-way analysis of variance was used to compare the means of multiple groups, and independent sample t-test was used to compare between two groups. Differences were considered statistically significant when P<0.05.

[0091] Experimental methods:

[0092] 1. Recovery and culture of bone marrow mesenchymal stem cells and calculation of generation age (multiplication times): After taking out the cells from the liquid nitrogen tank, quickly place them in a 37°C water bath and shake them to melt them quickly; when only a small piece of ice crystal is left, take them out of the water bath, spray them with 75% alcohol and wipe them dry, then transfer them to a safety cabinet; transfer the melted cells to a preheated 10-fold volume of low-glucose culture medium, centrifuge at 800 rpm for 5 minutes; discard the supernatant, resuspend the cells in the medium of bone marrow mesenchymal stem cells, and then inoculate them in a cell culture dish of appropriate size and shake them evenly, and place them in an incubator at 37°C and 5% CO2 culture; change the medium after 24 hours and continue to culture; when the cells grow to about 80-90% confluence, subculture the cells at a ratio of 1:2 or 1:4; count the cells before and after subculture, and sample and count the cells 20 hours after subculture to calculate the cell adhesion rate R; the cell generation age, i.e. the cumulative doubling times, is calculated according to ∑log2(D / D0 / R), where D and D0 are the cell densities at harvest and inoculation, respectively, and R is the cell adhesion rate.

[0093] 2. CCK8 method to determine the effect of salidroside on the cell viability of bone marrow mesenchymal stem cells: Figure 2 The figure shows the effect of salidroside on the cell viability of bone marrow mesenchymal stem cells determined by CCK8 method.

[0094] Among them: vertical axis - cell viability (%), the control group is set as 100,

[0095] Horizontal axis: 1-DMSO treatment for 3 days (Control),

[0096] 2-Salidroside (1 μM) was treated alone for 3 days.

[0097] 3-Salidroside (2 μM) was treated alone for 3 days.

[0098] 4-Salidroside (5 μM) was treated alone for 3 days.

[0099] 5-Salidroside (10 μM) was treated alone for 3 days.

[0100] 6-Salidroside (20 μM) was treated alone for 3 days.

[0101] 7-Salidroside (50 μM) was treated alone for 3 days.

[0102] 8-Salidroside (100 μM) was treated alone for 3 days.

[0103] Bone marrow mesenchymal stem cells in the logarithmic growth phase were collected at a rate of 1×10 4Cells were inoculated in 96-well plates at 100 μL / well. After culturing for 24 h, gradient concentrations of test drugs were added. Six parallel wells were set for each concentration, and a solvent control group without drugs and a blank group without cells were set. After culturing for 3 days, 10 μL of CCK-8 solution was added to each well and mixed by shaking. The cells were then placed in a constant temperature incubator and cultured for 1-2 hours. The absorbance was detected at 450 nm using an ELISA reader, and then the cell viability after treatment was calculated. The relative viability was calculated with the blank control group with only cells as 100%. Cell viability = (OD of drug-treated group - OD of blank group / OD of control group - OD of blank group) * 100%.

[0104] 3. Construction of AAPH-induced premature aging model of bone marrow mesenchymal stem cells: The cells were treated with different concentrations of free radical inducer AAPH (0-20mmol / L) for 24 hours. The optimal AAPH concentration was selected based on Western Blot, reactive oxygen free radical (ROS) level detection and other experiments. Bone marrow mesenchymal stem cells were treated to construct a free radical-induced premature aging cell model.

[0105] IV. Flow cytometry to detect the level of cellular reactive oxygen free radicals: Figure 3 As shown in Figure 3 This is a schematic diagram of the experimental results of the effects of different concentrations of salidroside pretreatment on the ROS level of rat bone marrow mesenchymal stem cells induced by AAPH.

[0106] Where: ordinate - relative fluorescence intensity, set the solvent control group to 1

[0107] Horizontal axis: 1-normal cell growth for 4.5 days (Control)

[0108] 2- After 1.5 days of normal cell growth, cells were treated with AAPH (1 mM) alone for 1 day, and then returned to normal culture medium for another 2 days.

[0109] 3- After 1 day of normal cell growth, cells were treated with DMSO alone for 12 hours and then replaced with AAPH (1mM)

[0110] Treat alone for 1 day, then switch back to normal culture medium and continue culturing for 2 days. (Solvent control group)

[0111] 4- After one day of normal cell growth, NAC (2 mM) was treated alone for 12 h, and then AAPH was used.

[0112] (1mM) for 1 day, then switch back to normal medium and continue culturing for 2 days

[0113] 5- After the cells grew normally for 1 day, they were treated with SAL (5 μM) alone for 12 h, then with AAPH (1 mM) alone for 1 day, and then returned to normal culture medium for another 2 days.

[0114] 6- After the cells grew normally for 1 day, they were treated with SAL (10 μM) alone for 12 h, then changed to AAPH (1 mM) alone for 1 day, and then returned to normal culture medium for 2 days. After the cells grew normally for 1 day, they were treated with SAL (20 μM) alone for 12 h, then changed to AAPH (1 mM) alone for 1 day, and then returned to normal culture medium for 2 days.

[0115] The experimental process is as follows: bone marrow mesenchymal stem cells in the logarithmic growth phase were taken at a rate of 5×10 4 100 cells / well were inoculated in a 12-well plate. After 24 hours, the old culture medium was discarded, and cells were pretreated with drugs containing different concentrations for 12 hours. The cells were treated with AAPH for 24 hours, and the medium containing drugs was replaced and cultured for 72 hours. ROS levels were detected, the old culture medium was aspirated, and the cells were washed with PBS for 3 times. 1 mL of reactive oxygen free radicals and free radical fluorescent probes with a molar concentration of 10 μM were added to each well, and the cells were incubated in a cell culture incubator for 20 minutes; the probes were discarded, and the cells were washed with DMEM basal culture medium for 3 times; the cells were digested with trypsin, collected and centrifuged, and washed once with PBS, and the cells were collected by centrifugation again, and the cells were resuspended with 500 μL PBS, and transferred to a flow tube for detection; 10,000 cells were collected from each group, and the cell fluorescence intensity (FL1) was analyzed. The stronger the fluorescence, the higher the level of reactive oxygen free radicals.

[0116] 5. Western blot detection of aging-related protein expression

[0117] (1) Extraction of total protein

[0118] Take the bone marrow mesenchymal stem cells in the logarithmic growth phase and inoculate them in a 12-well plate at 5×104 / well. After 24 hours, discard the old culture medium, add cells pretreated with drugs at different concentrations for 12 hours, treat the cells with AAPH for 24 hours, return to the medium containing drugs and continue to culture for 72 hours, remove the cell culture medium, wash the cells with 1mL PBS, remove PBS, add 100μLRIPA lysis solution to each well, and let stand at 4℃ for 30min to complete cell lysis. Use a cell scraper to gently scrape the cells, transfer to a 1.5mL centrifuge tube, centrifuge at 12000rpm at 4℃ for 15min, and then transfer the supernatant to a new 1.5mL centrifuge tube, which is the total protein sample obtained;

[0119] (2) Determination of protein concentration

[0120] According to the instructions of the BCA protein quantification kit, mix the BCA reagent and the Cu reagent in a ratio of 50:1 to prepare a working solution. Take a 5mg / mL BSA standard solution and dilute it to 0.5mg / mL with PBS. Add different volumes of standard protein to a 96-well plate to prepare a standard curve. Add 2μL of extracted protein sample to each well to be tested, fill it up to a total volume of 20μL with PBS, add 200μL BCA working solution and incubate at 37°C for 30min, measure the absorbance at a wavelength of 562nm, calculate the sample protein concentration according to the BSA standard curve, and unify the samples to the same concentration for later use;

[0121] (3) Protein denaturation treatment

[0122] The protein sample was mixed with 5× Loading Buffer at a volume ratio of 4:1, and then heated in a 100°C metal bath for 8 min for denaturation. After mixing, the mixture was centrifuged at low speed and stored in a -20°C refrigerator;

[0123] (4) SDS-PAGE protein electrophoresis

[0124] Prepare separation gel and concentration gel in order. Select a comb with the appropriate number of holes according to the experimental requirements. After the gel is prepared, store it in electrophoresis buffer at 4°C or use it directly. After thawing the protein sample, gently shake it to mix it and centrifuge it at a low speed. After adding the protein sample, run it at 80V for 30 minutes, then adjust it to 120V and continue electrophoresis for 60 minutes;

[0125] (5) Immuno-imprinting

[0126] Experimental results refer to Figure 4 As shown in the figure, according to the size of the gel, cut 2 layers of filter paper and 1 PVDF membrane that are consistent with the gel in advance, first soak the PVDF membrane in methanol for 2 minutes, and then soak it in the transfer buffer together with the filter paper. After the electrophoresis is completed, carefully remove the gel and put it in the transfer buffer as well, and then perform the semi-dry or wet transfer operation. The semi-dry operation steps are as follows: Place a layer of filter paper on the anode of the semi-dry transfer instrument, place the PVDF membrane on it, place the gel on the membrane, and then place the filter paper. The layers are strictly aligned, and bubbles are removed. Finally, the cathode of the transfer instrument is covered, and the transfer is performed at 0.65-0.80mA / cm2 for 2 hours. The wet transfer operation is as follows: Pre-cool the transfer buffer in a 4℃ refrigerator and lay out the "sandwich" according to the transfer instructions: With the gray electrotransfer box facing down, lay out the pad, filter paper, gel, and PVDF membrane soaked in transfer buffer in advance, and then cover them with filter paper and pads, and pay attention to gently remove bubbles with a glass rod. After the electroporation box is installed, put it into the electroporation tank, install the cooling box, add about 500ml of transfer buffer, and transfer the membrane at 320mA current for 1 hour.

[0127] After the transfer, remove the membrane, soak it in methanol for a while to fully fix the protein on the PVDF membrane, and then put it in TBST containing 5% skimmed milk powder and shake it for 1 hour. The blocked membrane is placed in a hybridization bag and reacted with the primary antibody solution at room temperature for 1 hour or at 4°C overnight, and then washed 3 times with TBST containing 0.1% Tween 20 for 5 minutes / time to wash away the unbound antibody. In a new hybridization bag, react with the horseradish peroxidase-labeled secondary antibody solution at room temperature for 1 hour, and then wash 3 times with TBST 5 minutes / time. Finally, the protein is detected by chemiluminescence, and the color is developed according to the instructions of the ECLPLUS immunoblotting kit, and the image is captured by the Chemi Imager 5500 gel imaging system;

[0128] (6) qRT-PCR detection of aging-related gene expression

[0129] Experimental results refer to Figure 5 , Figure 7 As shown in the figure, the total RNA of bone marrow mesenchymal stem cells in different treatment groups was obtained by RNA rapid extraction kit, and cDNA was obtained by reverse transcription; cDNA was used as a template, and the The PCR reaction system was prepared according to the instructions of the Premix Ex TaqTM fluorescence quantitative kit, and Roche real-time fluorescence quantitative PCR instrument was used to detect genes related to the aging pathway, such as p16, p21 and p53.

[0130] (7) β-galactosidase staining

[0131] Experimental results refer to Figure 6 As shown in the figure, follow the instructions of the kit: prepare each staining solution according to the proportion to form a staining working solution. Discard the old culture medium, add 500μL PBS to each well for washing once, discard the PBS, add 500μL fixative, and incubate at room temperature for 15min. Aspirate the fixative, add 500μL PBS to each well to wash the cells, 3min each time, three times in total, and remove the PBS after washing. Add 500μL staining working solution to each well for staining. Use plastic wrap to wrap the cell culture plate to prevent the liquid from evaporating and causing changes in the concentration of the staining solution. Incubate the staining at 37℃ for 3-16h, count and observe under a microscope after completion, and the positive cells will appear blue-green after staining.

[0132] The implementation principle of a new use of salidroside in the present application embodiment for preventing premature aging of bone marrow mesenchymal stem cells induced by oxidative stress is as follows: oxidative stress refers to the accumulation of free radicals such as reactive oxygen species (ROS), which causes oxidation reactions in molecules within cells, damages cell structure and function, and then induces aging. In bone marrow mesenchymal stem cells (BMSCs), oxidative stress accelerates cell aging, causes decreased stem cell function, and affects tissue repair and regeneration. As a natural antioxidant ingredient, salidroside can scavenge reactive oxygen species and free radicals, slow down or prevent oxidative stress damage to stem cells, and thereby inhibit premature aging of stem cells. It can reduce the damage of free radicals to cells through direct or indirect mechanisms, and reduce the level of reactive oxygen species and oxidative damage by increasing the activity of intracellular antioxidant enzymes (such as SOD, GPX, etc.). It can scavenge free radicals, inhibit The cell aging and dysfunction caused by oxidative stress are alleviated by inhibiting lipid peroxidation and other pathways. Free radicals are generated at physiological temperature by free radical inducers (such as AAPH) to simulate oxidative stress conditions and establish a premature aging model of bone marrow mesenchymal stem cells. The accumulation of free radicals in cells will lead to oxidative damage, causing peroxidation of DNA, proteins and lipids, thereby accelerating the cell aging process. Under oxidative stress conditions, salidroside is used to observe its effect in reducing free radicals and inhibiting the expression of aging-related proteins and genes. The expression of aging markers (such as β-galactosidase, p53, p16, p21, etc.) is reduced through antioxidant effects, thereby delaying or reversing the cell aging process. The total protein and RNA of the cells are extracted by using reagents such as trypsin-EDTA digestion solution and RIPA lysis solution to ensure the accuracy and reliability of the experimental data, and the Western blot is used to detect the expression of salidroside. The expression levels of senescence-related proteins were detected by blot, and the transcription levels of senescence-related genes were detected by qRT-PCR. The molecular mechanism of cell senescence after salidroside intervention was analyzed. Senescent cells usually show enhanced β-galactosidase activity. After salidroside intervention, the proportion of senescent cells can be detected by staining to evaluate the anti-aging effect of salidroside. Salidroside can directly remove free radicals or activate the intracellular antioxidant enzyme system. Salidroside reduces oxidative stress response, inhibits oxidative damage, and inhibits the expression of senescence-related genes such as p16INK4a, p21CIP1 and p53, reduces cell cycle arrest and cell death, and improves cell activity. Salidroside prevents and delays premature senescence of bone marrow mesenchymal stem cells induced by oxidative stress through antioxidant effects.

[0133] The technical scheme adopted in this application is as follows: bone marrow mesenchymal stem cells are isolated and cultured in vitro, and the bone marrow mesenchymal stem cells are treated with a free radical inducer AAPH. AAPH generates free radicals by thermal decomposition at physiological temperature. These free radicals can attack proteins or lipids to induce peroxidation reactions and ultimately lead to oxidative damage, inducing premature aging of cells. Different doses of Rhodiola rosea extract are used for intervention to explore the intervention effect of Rhodiola rosea extract on free radical-induced premature aging. Through the detection of intracellular reactive oxygen free radical levels, aging-related β-galactosidase staining, and aging-related signaling pathway molecules such as p53 / p21 and p16 / Rb, it is found that salidroside has the effect of improving premature aging of bone marrow mesenchymal stem cells induced by oxidative stress.

[0134] The results of biological experiments show that:

[0135] 1. Salidroside alone treated BM-MSCs 72 and showed no obvious cytotoxicity at 0-100 μM.

[0136] 2. Salidroside can improve the premature aging characteristics of bone marrow mesenchymal stem cells induced by oxidative stress. Specifically, at 5-20 μM, it can significantly reduce the increase in the level of reactive oxygen species in BM-MSCs induced by AAPH, significantly inhibit the expression of p53, p21, and p16, and reduce the positive rate of aging-related β-galactosidase.

[0137] The technical advantages and new uses of a new use of salidroside in the present application embodiment for preventing premature aging of bone marrow mesenchymal stem cells induced by oxidative stress are as follows:

[0138] 1. Prevent premature aging of bone marrow mesenchymal stem cells: Salidroside significantly reduces free radical-induced aging of bone marrow mesenchymal stem cells by effectively intervening in the oxidative stress process, and delays their premature aging process. This effect was confirmed by the detection of intracellular reactive oxygen free radical levels, β-galactosidase staining, and aging-related genes and proteins;

[0139] 2. Improve cell activity: Salidroside can significantly improve the survival rate of bone marrow mesenchymal stem cells under oxidative stress, reduce the oxidative damage of free radicals to cells, and promote cell proliferation and function maintenance. The cell activity was detected by CCK-8 method, and the experimental results showed that Salidroside has a good cell protection effect within a certain dosage range;

[0140] 3. Inhibit activation of aging-related signaling pathways: Salidroside can regulate key aging-related signaling pathways, including p53, p16INK4a, and p21CIP1. Western blot and qRT-PCR test results showed that Salidroside can significantly reduce the expression levels of these aging-related genes and proteins, thereby effectively inhibiting the aging process;

[0141] 4. Improve the reliability and repeatability of the experiment: By optimizing the reagent system and experimental steps, the stability, reliability and high repeatability of the experiment are ensured. The optimization of key steps such as cell culture, protein extraction, gene quantification, and immunoblotting ensures the accuracy and consistency of the experimental data;

[0142] 5. Improvement of cell senescence detection methods: Using cell senescence-related detection methods, such as β-galactosidase staining, flow cytometry, Western blot, and qRT-PCR technology, the assessment of cell senescence is more intuitive and accurate, providing a reliable technical means for aging research;

[0143] 6. Dose-dependent effect of salidroside: Through intervention experiments with different doses of salidroside, its protective effect at different concentrations was revealed, providing an optimal dose range and providing a basis for subsequent clinical application or further research;

[0144] 7. Innovative aging models and intervention strategies: The oxidative stress-induced premature aging model of bone marrow mesenchymal stem cells provides a new experimental model for aging research, and the strategy of intervention with salidroside demonstrates actual anti-aging effects at the cellular level, which has high potential for clinical transformation.

[0145] 8. Through a series of biological experiments, it was found and proved that salidroside has a significant effect in preventing premature aging of bone marrow mesenchymal stem cells induced by oxidative stress at a dose without obvious cytotoxicity. It is expected to be developed into a drug that effectively delays the aging of bone marrow mesenchymal stem cells, which has important application value in breaking the bottleneck of clinical transplantation for disease treatment.

[0146] The above are all preferred embodiments of the present application, and the protection scope of the present application is not limited thereto. Therefore, any equivalent changes made according to the structure, shape, and principle of the present application should be included in the protection scope of the present application.

Claims

1. A new use of salidroside for preventing premature aging of bone marrow mesenchymal stem cells induced by oxidative stress, characterized in that: The implementation process of prevention includes materials, instruments and implementation methods; The materials and instruments include reagents, antibodies, primers and instruments; The implementation method includes bone marrow mesenchymal stem cell recovery and culture and generation age calculation, CCK8 method to detect cell viability, free radical inducer to induce premature aging model of bone marrow mesenchymal stem cells, flow cytometry to detect the level of cell active oxygen free radicals, Western blot to detect the expression of aging-related proteins, qRT-PCR to detect the expression of aging-related genes, β-galactosidase staining and statistical analysis.

2. The novel use of salidroside for preventing premature aging of bone marrow mesenchymal stem cells induced by oxidative stress according to claim 1, characterized in that: The Western blot method for detecting the expression of aging-related proteins includes extracting total protein, determining protein concentration, protein denaturation, SDS-PAGE protein electrophoresis and immunoblotting.

3. The novel use of salidroside for preventing premature aging of bone marrow mesenchymal stem cells induced by oxidative stress according to claim 1, characterized in that: The reagents include salidroside, rat bone marrow mesenchymal stem cells, rat bone marrow mesenchymal stem cell complete culture medium, trypsin-EDTA digestion solution, Cell Counting Kit-8, phosphatase inhibitors, protease inhibitors, protein markers, 2×SYBR Green, 10×Tris-Glycine SDS electrophoresis buffer, 10×blotting membrane transfer buffer, RNA rapid extraction kit, dimethyl sulfoxide, polyethylene glycol dehydrated sorbitan monolaurate, 2,2'-azobis-2-methyl-propanimidamide, dihydrochlori, tetramethylethylenediamine solution, ammonium persulfate, Western primary antibody, secondary antibody diluent, Western primary antibody secondary antibody removal solution, 5× protein loading buffer, RIPA lysis buffer, acrylamide / methylenebisacrylamide solution, 4×Tris-HCl / SDS buffer, 4×Tris-HCl / SDS buffer, and aging-related β-galactosidase staining.

4. The novel use of salidroside for preventing premature aging of bone marrow mesenchymal stem cells induced by oxidative stress according to claim 3, characterized in that: The concentration of the pancreatin-EDTA digestion solution is 0.25%; The pH value of the 10×Tris-Glycine SDS electrophoresis buffer is pH 8.3; The Western primary and secondary antibody removal solution is strongly alkaline; The 5× protein loading buffer contains DTT; The concentration of the acrylamide / methylenebisacrylamide solution is 40%; The ratio of acrylamide to methylene bisacrylamide is 29:1; The pH value of the 4×Tris-HCl / SDS buffer is pH 6.

8.

5. The novel use of salidroside for preventing premature aging of bone marrow mesenchymal stem cells induced by oxidative stress according to claim 1, characterized in that: The antibodies include GAPDH antibody, p21 antibody, p16 antibody, p53 antibody and pRb antibody.

6. The novel use of salidroside for preventing premature aging of bone marrow mesenchymal stem cells induced by oxidative stress according to claim 1, characterized in that: The primers include p53, p16INK4a, p21CIP1 and β-actin.

7. The novel use of salidroside for preventing premature aging of bone marrow mesenchymal stem cells induced by oxidative stress according to claim 6, characterized in that: The primer p53 includes forward5'-GCAGCACAGGAACCTGGAACTG-3', reverse5'-AGAAGGGACGGAAGATGACAGAGG-3'; The primers p16INK4a include forward5'-CAGCTCTCCTGCTCTCCTATGGTG-3' and reverse5'-CAGGCATCGCGCACATCCAG-3'; The primer p21CIP1 includes forward 5-CAGTAGACACGAAACAGGCTCAGG-3' and reverse 5'-CTGTCTTGTCTTCGCTGAGGTCTC-3; The primer β-actin includes forward 5'-GTAAAGACCTCTATGCCAACA-3' and reverse 5'-GGACTCATCGTACTCCTGCT-3.

8. The novel use of salidroside for preventing premature aging of bone marrow mesenchymal stem cells induced by oxidative stress according to claim 1, characterized in that: The instruments include an analytical balance, a biological safety cabinet, a carbon dioxide constant temperature incubator, a three-level pure water system, an enzyme marker, an ultra-micro spectrophotometer, a fluorescence quantitative PCR instrument, a chemiluminescence imaging system, a vertical electrophoresis instrument, and a fluorescence microscope.

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