Traditional Chinese medicine monomer composition for delaying osteoblast senescence

Abstract

The invention discloses a traditional Chinese medicine monomer composition for delaying osteoblast senescence. The provided traditional Chinese medicine monomer composition is prepared from the following raw materials in parts by mass: 3 parts of icariin and 5 parts of oleanolic acid. According to the invention, by controlling the ratio of ICA to OA to be 3: 5, ICA and OA synergistically play a role (CIlt; 1) ICA and OA are combined for synergistic interaction, osteoblast aging can be delayed, the effect of preventing and treating SOP is improved, bone repair and regeneration are promoted, new bone generation is accelerated, and the composition is expected to play an important role in bone injury or bone defect treatment.

Description

Technical Field

[0001] The present invention belongs to the field of natural active ingredients, and particularly relates to a traditional Chinese medicine monomer composition for delaying the senescence of osteoblasts. Background Art

[0002] Senile osteoporosis (SOP) specifically refers to a type of low-turnover osteoporosis (OP) that occurs in people aged 65 or older in women or 70 or older in men. It is mainly characterized by bone mass reduction and the degradation of the microscopic structure of bones, accompanied by increased bone fragility and an increased susceptibility to fractures. It is a systemic skeletal disease. With the acceleration of the aging process of the population, the incidence of SOP is gradually increasing, seriously affecting the physical and mental health of the elderly and imposing a huge medical and economic burden on society.

[0003] According to the theory of traditional Chinese medicine that "the kidney stores essence, dominates bones, and produces marrow", the core pathogenesis of SOP is "kidney deficiency and essence depletion", and drugs with the main efficacy of "tonifying the kidney and replenishing essence" have unique advantages in the treatment of SOP. The combination of Epimedii Folium and Ligustri Lucidi Fructus (EL) is a pair of herbs that tonifies both yin and yang in the kidney selected by Professor Li Shizeng, a national famous veteran Chinese medicine doctor, based on decades of clinical experience. A large number of studies have confirmed the significant effects of single Epimedii Folium, Ligustri Lucidi Fructus, or related compound prescriptions containing Epimedii Folium and Ligustri Lucidi Fructus in the prevention and treatment of SOP.

[0004] Although EL has the advantages of a refined formula and significant clinical efficacy, it still has the following limitations: The traditional decoction process has problems such as being cumbersome and complex, a large volume of the medicinal liquid, and a bitter and unpalatable taste, resulting in reduced patient compliance; at the same time, there are physical property defects such as poor portability, easy mildew, and fermentation and deterioration, making it difficult to store stably for a long time and unable to meet the requirements of large-scale production. In addition, although Epimedii Folium and Ligustri Lucidi Fructus can prevent and treat SOP, how to further improve the curative effect remains a difficult problem. Summary of the Invention

[0005] The purpose of the present invention is to provide a traditional Chinese medicine monomer composition for delaying the senescence of osteoblasts. The traditional Chinese medicine monomer composition is composed of icariin (ICA) and oleanolic acid (OA); the mass ratio of icariin to oleanolic acid is 3:5.

[0006] The present invention discovers through experiments that when the mass ratio of icariin to oleanolic acid is 3:5, the effect is optimal. ICA and OA play a synergistic role (CI < 1) in delaying the senescence of osteoblasts, so it can be used in the preparation of drugs for delaying the senescence of osteoblasts.

[0007] Preferably, the drug of the present invention further comprises an adjuvant, which is a solubilizer for dissolving ICA and OA.

[0008] The delay of osteoblast senescence by the Chinese medicine monomer composition of the present invention is specifically reflected in at least one of the following 1)-11):

[0009] 1) Improving the cell viability of osteoblasts;

[0010] 2) Reducing the expression of osteoblast β-galactosidase (β-Gal);

[0011] 3) Improving the total antioxidant capacity (T-AOC) and superoxide dismutase (SOD) activity of osteoblasts, and reducing the contents of osteoblast malondialdehyde (MDA) and nitric oxide (NO);

[0012] 4) Reducing the expression of osteoblast cyclin-dependent kinase inhibitor 2A (p16), cyclin-dependent kinase inhibitor 1A (p21), interleukin (IL) 6, IL8, matrix metalloproteinase 3 (MMP3), tumor necrosis factor (TNF) α, γH2A.X, and increasing the expression of Lamin B1;

[0013] 5) Increasing the expression of osteoblast alkaline phosphatase (ALP);

[0014] 6) Improving the proliferation activity of osteoblasts;

[0015] 7) Increasing the expression of osteoblast transforming growth factor (TGF)-β1, bone morphogenetic protein (BMP) 2, Wnt family member 5A (Wnt5a), osteoprotegerin (OPG), and insulin-like growth factor (IGF)-1;

[0016] 8) Improving the bone mineral density (BMD), bone volume percentage (BV / TV), bone surface area to tissue volume ratio (BS / TV), trabecular bone number (Tb.N), trabecular bone area (Tb.Ar), trabecular bone thickness (Tb.Th) of mammals, reducing the trabecular bone separation (Tb.Sp), and improving the microscopic structure of bone;

[0017] 9) Improving the gait function and coordination of mammals;

[0018] 10) Improving the expression of ALP in mammals and promoting bone formation;

[0019] 11) Reducing the expression of MDA, p16, and p21 in mammals and increasing the expression of SOD;

[0020] The mammals include humans.

[0021] The traditional Chinese medicine monomer composition provided by the present invention can delay the senescence of osteoblasts, which not only improves the effect of preventing and treating SOP, but also is beneficial to promoting bone repair and regeneration and accelerating new bone formation. Therefore, the traditional Chinese medicine monomer composition is expected to be applied in the preparation of drugs for treating bone injuries or bone defects.

[0022] When preparing the drug of the present invention, each raw material drug is formulated according to parts by mass, and can be increased or decreased according to the corresponding ratio during production. For example, in large-scale production, it can be in kilograms or tons, and in small-scale production, it can also be in grams. The weight can be increased or decreased, but the mass ratio of the raw material drugs between the components remains unchanged.

[0023] Compared with the prior art, the present invention has the following advantages:

[0024] (1) The traditional Chinese medicine monomer composition of the present invention combines ICA and OA, and by controlling the ratio of ICA to OA to be 3:5, enables ICA and OA to play a synergistic role (CI < 1). The combination of icariin and oleanolic acid (IO) has a synergistic effect, can delay the senescence of osteoblasts, not only improves the effect of preventing and treating SOP, but also is beneficial to promoting bone repair and regeneration and accelerating new bone formation, and is expected to play an important role in the treatment of bone injuries or bone defects.

[0025] (2) The active ingredients of the traditional Chinese medicine monomer composition provided by the present invention are derived from the medicinal pair EL (the compatibility of Epimedium and Ligustrum lucidum), which not only continues the treatment concept of "tonifying the kidney and replenishing essence, tonifying both yin and yang", but also further clarifies the effective ingredients of the drug for delaying the senescence of osteoblasts.

[0026] (3) The raw material drugs of the traditional Chinese medicine monomer composition of the present invention have a wide distribution of resources, low toxicity and side effects, and can be taken for a long time.

[0027] (4) The traditional Chinese medicine monomer composition of the present invention has a simple formula, clear active ingredients, definite curative effect, controllable quality, and does not require the decocting process of traditional Chinese medicine, and is convenient to take. Specific embodiments

[0028] The method of the present invention will be described below through specific implementation examples, but the present invention is not limited thereto. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

[0029] The experimental methods used in the following examples are all conventional methods unless otherwise specified.

[0030] The materials, reagents, etc. used in the following examples can be obtained from commercial channels unless otherwise specified.

[0031] In the quantitative tests in the following examples, three repeated experiments were set up, and the results were averaged.

[0032] Mouse calvarial pre-osteoblast subclone 14 (MC3T3-E1 Subclone 14) cells were purchased from Wuhan Punosai Life Science Co., Ltd., product number: CL-0378.

[0033] SPF-grade 6-month-old (8 mice) and 12-month-old (16 mice) healthy male C57BL / 6J mice were provided by Beijing Vital River Laboratory Animal Technology Co., Ltd., certificate number: SCXK(Jing) 2022-0049.

[0034] Example 1: Preparation of ICA and OA and Preparation of IO

[0035] I. Preparation of ICA solution:

[0036] Weigh 1 g of ICA (icariin) powder, add 100 μl of DMSO to dissolve it, dilute it stepwise to 50 μg / ml, and filter it through a 0.22 μm filter membrane. Dilute and configure it with αMEM complete medium (containing 100 U / ml of penicillin and 10 mg / ml of streptomycin) into ICA solutions with concentrations of 50000, 10000, 5000, 1000, 500, 100, 50, 10, 5 ng / ml.

[0037] II. Preparation of OA solution:

[0038] Weigh 1 g of OA (oleanolic acid) powder, add 100 μl of DMSO to dissolve it, dilute it stepwise to 50 μg / ml, and filter it through a 0.22 μm filter membrane. Dilute and configure it with αMEM complete medium into OA solutions with concentrations of 50000, 10000, 5000, 1000, 500, 100, 50, 10, 5 ng / ml.

[0039] III. Preparation of IO solution:

[0040] Set the compatibility ratios of ICA and OA as 0 (D-galactose (D-Gal)), 1:1, 4:5, 3:5, 2:5, 1:5, 1:10, 1:15, 1:20, 1:25 ratio gradients, and intervene for 48 h respectively.

[0041] On the basis of determining the optimal compatibility ratio of ICA and OA, set the concentration gradients of their combination, including 0 (D-Gal), 200, 400, 800, 1600, 3200 ng / ml, and intervene for 48 h respectively.

[0042] The experimental data were expressed as mean ± standard error It is indicated that the homogeneity of variance test and one-way analysis of variance were used for inter-group comparison. The results are as follows:

[0043] (I) Intervention concentrations of ICA and OA

[0044] Compared with the D-Gal group, after OBs were intervened with ICA at 50 and 100 ng / ml for 48 h, the cell viability was significantly up-regulated (P<0.05 or 0.01), and the effect of 100 ng / ml was the best.

[0045] Compared with the D-Gal group, after OBs were intervened with OA at 10, 50, 500, 1000, and 5000 ng / ml for 48 h, the cell viability was significantly up-regulated (P<0.05 or 0.01), and the effect of 500 ng / ml was the best. Therefore, 48 h of intervention with 100 ng / ml of ICA and 500 ng / ml of OA was used as the basis for screening the compatibility ratio and concentration of the following two monomers.

[0046] Table 1 Effects of ICA and OA on the cell viability of OBs intervened by D-Gal( n = 3)

[0047]

[0048] Note: Compared with the D-Gal group, * P<0.05, ** P<0.01.

[0049] (II) Compatibility ratio of ICA combined with OA

[0050] Table 2 Effects of ICA:OA on the cell viability of OBs intervened by D-Gal( n = 3)

[0051]

[0052] Note: Compared with the D-Gal group, * P<0.05, ** P<0.01.

[0053] After OBs were intervened with different ratios of ICA and OA for 48 h, compared with the control group, when the ratio of ICA to OA was 4:5, 3:5, 1:10, 1:15, and 1:20, the cell viability was significantly up-regulated (P<0.05 or 0.01), and the effect was the best when the ratio of ICA to OA was 3:5 (CI<1). Therefore, the compatibility ratio of ICA combined with OA was selected as 3:5 as the basis for screening the compatibility concentration, that is, 300 ng / ml of ICA and 500 ng / ml of OA, for subsequent screening of the compatibility concentration.

[0054] (III) Intervention concentration of IO

[0055] Table 3 Effects of IO at different concentrations on the viability of OBs cells intervened by D-Gal( n = 3)

[0056]

[0057] Note: Compared with the D-Gal group, * P < 0.05.

[0058] After OBs were intervened with IO at different combined concentrations for 48 h and compared with the D-Gal group, ICA and OA at 800 ng / ml could significantly up-regulate cell viability (P < 0.05). Therefore, OBs cells were cultured with 300 ng / ml ICA and 500 ng / ml OA for 48 h as the intervention conditions.

[0059] Example 2: Effects of IO on the senescent state and osteogenic function of D-Gal-induced OBs

[0060] I. Select OBs cells in good condition at about the 15th passage. After inducing with 5 mg / ml D-Gal for 4 days, they were randomly divided into a D-Gal group and an IO group. Another normal control group was set without D-Gal induction. The IO group was intervened with 800 ng / ml IO (ICA:OA = 3:5) for 48 h.

[0061] Second, after the corresponding drug intervention was given to OBs, β-galactosidase (β-Gal) staining was performed to evaluate the degree of senescence; the colorimetric method was used to evaluate the activity of alkaline phosphatase (ALP) and total antioxidant capacity (T-AOC); the WST-1 method was used to detect the activity of superoxide dismutase (SOD); the TBA method was used to detect the content of malondialdehyde (MDA); the microplate method was used to detect the content of nitric oxide (NO); the EdU method was used to detect the proliferative activity of OBs; the immunofluorescence (IF) method combined with real-time quantitative reverse transcription polymerase chain reaction (qPCR) was used to detect the expression of cyclin-dependent kinase inhibitor 2A (p16), cyclin-dependent kinase inhibitor 1A (p21), interleukin (IL) 6, IL8, matrix metalloproteinase 3 (MMP3), tumor necrosis factor (TNF) α, Lamin B1, transforming growth factor (TGF)-β1, bone morphogenetic protein (BMP) 2, Wnt family member 5A (Wnt5a), Osteoprotegerin (OPG), and insulin-like growth factor (IGF)-1; the IF method was used to detect the protein expression of γH2A.X. The results are as follows:

[0062] (1) Effect of IO on the expression of β-Gal in D-Gal-induced OBs

[0063] Table 4 Comparison of β-Gal expression in OBs of each group ( n = 3)

[0064]

[0065] Note: Compared with the normal group, **P < 0.01; compared with the D-Gal group, # P < 0.05.

[0066] Compared with the normal group, the IOD of β-Gal positive in the D-Gal group was significantly up-regulated (P < 0.01), indicating that D-Gal successfully induced the senescence of OBs. Compared with the D-Gal group, the positive expression of β-Gal in the IO group was significantly down-regulated (P < 0.05), indicating that IO could alleviate the senescence of OBs caused by D-Gal.

[0067] (2) Effects of IO on free radical damage of OBs induced by D-Gal

[0068] Table 5 Comparison of free radical damage of OBs in each group ( n = 3)

[0069]

[0070] Note: Compared with the normal group, * P < 0.05, ** P < 0.01; compared with the D-Gal group, # P < 0.05, ## P < 0.01.

[0071] Compared with the normal group, the levels of T-AOC and SOD in OBs of the D-Gal group were significantly decreased (P < 0.01), while the levels of MDA and NO were significantly increased (P < 0.05), indicating that free radical damage occurred in the OBs senescence model induced by D-Gal and the oxidative stress level increased. Compared with the D-Gal group, the levels of T-AOC and SOD in OBs of the IO group were significantly increased (P < 0.05 or 0.01), while the levels of MDA and NO were significantly down-regulated (P < 0.05 or 0.01), indicating that IO could alleviate the oxidative stress state of OBs caused by D-Gal.

[0072] (3) Effects of IO on the expression of p16 and p21 in OBs induced by D-Gal

[0073] Table 6 Comparison of the expression of p16 and p21 in OBs in each group ( n = 3)

[0074]

[0075] Note: Compared with the normal group, * P < 0.05, ** P < 0.01; compared with the D-Gal group, # P < 0.05, ## P < 0.01.

[0076] Compared with the normal group, the expressions of cell cycle-related factors p16 and p21 proteins and mRNAs were significantly up-regulated in the D-Gal group (P<0.05 or 0.01), suggesting that D-Gal induced cell cycle arrest in OBs. Compared with the D-Gal group, the expressions of p16 and p21 proteins and mRNAs were significantly down-regulated in the IO group (P<0.05 or 0.01), suggesting that IO could down-regulate the expressions of cell cycle-related factors p16 and p21, relieve cell cycle arrest caused by D-Gal, and thus delay aging.

[0077] (IV) Effects of IO on the expressions of IL6 and IL8 in D-Gal-induced OBs

[0078] Table 7 Comparison of the expressions of IL6 and IL8 in OBs of each group ( n = 3)

[0079]

[0080] Note: Compared with the normal group, * P<0.05, ** P<0.01; compared with the D-Gal group, # P<0.05, ## P<0.01.

[0081] Compared with the normal group, the expressions of SASP indexes IL6 and IL8 proteins and mRNAs were significantly up-regulated in the D-Gal group (P<0.05 or 0.01), suggesting that the expression of SASP increased during D-Gal-induced aging. Compared with the D-Gal group, the expressions of IL6 and IL8 proteins and mRNAs were significantly down-regulated in the IO group (P<0.05 or 0.01), suggesting that IO could inhibit the expressions of IL6 and IL8, down-regulate SASP, and may delay aging by adjusting the cell microenvironment.

[0082] (V) Effects of IO on the expressions of MMP3 and TNFα in D-Gal-induced OBs

[0083] Table 8 Comparison of the expressions of MMP3 and TNFα in cells of each group ( n = 3)

[0084]

[0085] Note: Compared with the normal group, ** P<0.01; compared with the D-Gal group, # P<0.05, ## P<0.01.

[0086] Compared with the normal group, the expressions of SASP indicators MMP3, TNFα protein and mRNA in the D-Gal group were significantly up-regulated (P<0.01), suggesting that the expression of SASP increased during D-Gal-induced senescence. Compared with the D-Gal group, the expressions of MMP3, TNFα protein and mRNA in the IO group were significantly down-regulated (P<0.05 or 0.01), suggesting that IO could inhibit the expressions of MMP3 and TNFα, down-regulate SASP, and might delay senescence by adjusting the cellular microenvironment.

[0087] (VI) Effect of IO on the expression of Lamin B1 in D-Gal-induced OBs

[0088] Table 9 Comparison of the expressions of Lamin B1 in OBs of each group ( n = 3)

[0089]

[0090] Note: Compared with the normal group, * P<0.05, ** P<0.01; compared with the D-Gal group, ## P<0.01.

[0091] Compared with the normal group, the expressions of the nuclear morphological marker Lamin B1 protein and mRNA in the D-Gal group were significantly down-regulated (P<0.05 or 0.01), suggesting that after 4 days of D-Gal induction in OBs, typical nuclear biological characteristics of senescence appeared. Compared with the D-Gal group, the expressions of Lamin B1 protein and mRNA in the IO group were significantly up-regulated (P<0.01), suggesting that IO could delay cellular senescence and up-regulate the expression of the nuclear morphological marker Lamin B1.

[0092] (VII) Effect of IO on the expression of γH2A.X in D-Gal-induced OBs

[0093] Table 10 Comparison of the expressions of γH2A.X protein in cells of each group ( n = 3)

[0094]

[0095] Note: Compared with the normal group, ** P<0.01; compared with the D-Gal group, ## P<0.01.

[0096] Compared with the normal group, the expression of the DNA damage response marker γH2A.X protein was significantly upregulated in the D-Gal group (P<0.01), indicating that DNA damage characteristics appeared during senescence after OBs were induced by D-Gal for 4 days. Compared with the D-Gal group, the expression of γH2A.X protein was significantly downregulated in the IO group (P<0.01). It is suggested that IO delays senescence and improves the DNA damage response.

[0097] (VIII) Effects of IO on the proliferative activity and ALP level of D-Gal-induced OBs

[0098] Table 11 Comparison of the proliferative activity and ALP level of OBs in each group ( n = 3)

[0099]

[0100] Note: Compared with the normal group, ** P<0.01; compared with the D-Gal group, ## P<0.01.

[0101] Compared with the normal group, while the proliferative activity of OBs in the D-Gal group was significantly downregulated, the ALP level decreased (both P<0.01), indicating that D-Gal inhibits the proliferation of OBs and reduces the osteogenic ability. Compared with the D-Gal group, the cell proliferative activity and ALP activity in the IO group were significantly upregulated (P<0.01), suggesting that IO can increase the proliferative activity of D-Gal-inhibited OBs and improve the osteogenic function.

[0102] (IX) Effects of IO on the TGF-β1 expression of D-Gal-induced OBs

[0103] Table 12 Comparison of the protein expression of bone formation factors in OBs in each group ( n = 3)

[0104]

[0105] Note: Compared with the normal group, * P<0.05, ** P<0.01; compared with the D-Gal group, ## P<0.01.

[0106] Table 13 Comparison of the mRNA expression of bone formation factors in OBs in each group ( n = 3)

[0107]

[0108] Note: Compared with the normal group, * P<0.05, ** P<0.01; compared with the D-Gal group,# P < 0.05, ## P < 0.01.

[0109] Compared with the normal group, the expressions of bone formation factors TGF-β1, BMP2, Wnt5a, OPG, IGF-1 proteins and mRNAs in the D-Gal group were significantly down-regulated (P < 0.05 or 0.01), suggesting that D-Gal inhibits osteoblast (OBs) bone formation. Compared with the D-Gal group, the expressions of bone formation factors TGF-β1, BMP2, Wnt5a, OPG, IGF-1 proteins and mRNAs in the IO group were significantly up-regulated (P < 0.05 or 0.01), suggesting that IO can improve the osteogenic function of OBs inhibited by D-Gal.

[0110] Example 3: Effects of IO on SOP mice

[0111] I. Eight 6-month-old SPF-grade healthy male C57BL / 6J mice (control group) and sixteen 15-month-old mice. The 15-month-old mice were randomly divided into 2 groups: SOP group and IO group. The IO group was intragastrically administered with an IO suspension at a dose of 160 mg / kg (ICA:OA = 3:5) body weight once a day for a total of 28 days. The intragastric volume was 15 ml / kg, and the SOP group was given an equal volume of distilled water by gavage.

[0112] II. The normal group consisted of purchased 6-month-old mice, which were sacrificed after 1 week of environmental adaptation; the remaining groups were sacrificed at 16 months. After intraperitoneal anesthesia, blood was collected by eye socket puncture, and the femurs and tibias on both sides were taken and fixed with 4% paraformaldehyde solution for micro-CT detection and staining. Micro-CT and H&E staining were used to evaluate the changes in the bone microstructure of mice; the DigiGaitTM gait imaging analysis system was used to analyze the gait of mice; ELISA and tissue staining methods were used to detect the expressions of ALP and TRACP in serum and bone tissues; IF method was used to detect the expressions of p16 and p21 proteins in bone tissues; colorimetric method was used to detect the levels of SOD and MDA in serum. The results are as follows:

[0113] (I) Effects of IO on the bone tissue microstructure of SOP mice

[0114] 1. Effects of IO on the micro-CT parameters of the bone tissue of SOP mice

[0115] Table 14 Comparison of micro-CT parameters of the bone tissue of mice in each group ( n = 5)

[0116]

[0117] Note: Compared with the normal group, * P < 0.05, ** P < 0.01; compared with the SOP group, ##P < 0.01.

[0118] Compared with the normal group, the bone mineral density (BMD), bone volume / total volume (BV / TV), bone surface / total volume (BS / TV), and trabecular number (Tb.N) of the femurs of mice in the SOP group were significantly downregulated (P < 0.05 or 0.01), the trabecular separation (Tb.Sp) was significantly upregulated (P < 0.01), and the trabecular thickness (Tb.Th) showed no significant change, suggesting that the microstructure of the femurs of SOP mice was damaged, presenting typical pathological changes of osteoporosis.

[0119] Compared with the SOP group, the BMD, BV / TV, BS / TV, and Tb.N of the IO group were significantly upregulated (P < 0.01), and the Tb.Sp was significantly downregulated (P < 0.01), suggesting that IO could improve the degenerative changes in the microstructure of bone tissue.

[0120] 2. Effects of IO on bone histopathology in SOP mice

[0121] Table 15 Comparison of bone histopathological parameters in mice of each group ( n = 5)

[0122]

[0123] Note: Compared with the normal group, ** P < 0.01; compared with the SOP group, ## P < 0.01.

[0124] Microscopically, it could be seen that in the normal group, the trabeculae were thick, arranged tightly and regularly, forming a continuous reticular structure with uniform gaps; in the SOP model group, the trabeculae were thin and fractured, arranged sparsely and with poor connectivity, the reticular structure was damaged, and the gaps were widened; the bone histomorphology of the IO group was improved.

[0125] Compared with the normal group, the trabecular area (trabecular perimeter, Tb.Ar), Tb.N, and Tb.Th of the femurs of mice in the SOP group were significantly downregulated (P < 0.01), suggesting that the femurs of SOP mice presented typical pathological changes of osteoporosis. Compared with the SOP group, the Tb.Ar and Tb.Th of the IO group were significantly upregulated (P < 0.01), suggesting that IO could improve the pathological morphology of the femur.

[0126] (2) Effects of IO on the gait of SOP mice

[0127] 1. Effects of IO on the swing duration of SOP mice

[0128] Table 16 Comparison of swing duration of mice in each group( n = 7)

[0129]

[0130] Note: Compared with the normal group, ** P < 0.01; compared with the SOP group, # P < 0.05.

[0131] Compared with the normal group, the swing duration of the bilateral forelimbs of SOP mice was significantly increased (P < 0.01); compared with the SOP group, the swing duration of the left forelimb of the IO group was significantly decreased (P < 0.05).

[0132] 2. Effects of IO on the braking duration of SOP mice

[0133] Table 17 Comparison of braking duration of mice in each group( n = 7)

[0134]

[0135] There were no significant differences in the braking duration of the limbs of mice in each group.

[0136] 3. Effects of IO on the propulsion duration of SOP mice

[0137] Table 18 Comparison of propulsion duration of mice in each group( n = 7)

[0138]

[0139]

[0140] Note: Compared with the normal group, * P < 0.05, ** P < 0.01; compared with the SOP group, # P < 0.05, ## P < 0.01.

[0141] Compared with the normal group, the propulsion duration of the left forelimb and bilateral hindlimbs of SOP mice was significantly increased (P < 0.05 or 0.01); compared with the SOP group, the propulsion duration of the right forelimb and bilateral hindlimbs of the IO group was significantly decreased (P < 0.05 or 0.01).

[0142] 4. Effects of IO on the ground contact duration of SOP mice

[0143] Table 19 Comparison of ground contact duration of mice in each group( n = 7)

[0144]

[0145] Note: Compared with the normal group, * P < 0.05, ** P < 0.01; compared with the SOP group, # P < 0.05, ## P < 0.01.

[0146] Compared with the normal group, the contact time of the SOP group mice's four limbs on the ground increased significantly (P < 0.05 or 0.01); compared with the SOP group, the contact time of the IO group mice's four limbs on the ground decreased significantly (P < 0.05 or 0.01).

[0147] 5. Effect of IO on the stride duration of SOP mice

[0148] Table 20 Comparison of the stride duration of mice in each group ( n = 7)

[0149]

[0150] Note: Compared with the normal group, ** P < 0.01; compared with the SOP group, # P < 0.05, ## P < 0.01.

[0151] Compared with the normal group, the stride duration of the SOP group mice's four limbs increased significantly (P < 0.01); compared with the SOP group, the stride duration of the IO group mice's four limbs decreased significantly (P < 0.05 or 0.01).

[0152] 6. Effect of IO on the stride length of SOP mice

[0153] Table 21 Comparison of the stride length of mice in each group ( n = 7)

[0154]

[0155]

[0156] Note: Compared with the normal group, * P < 0.05, ** P < 0.01; compared with the SOP group, # P < 0.05, ## P < 0.01.

[0157] Compared with the normal group, the stride lengths of the bilateral forelimbs and the left hindlimb of the mice in the SOP group were significantly reduced (P<0.05 or 0.01); compared with the SOP group, the stride lengths of the four limbs of the mice in the IO group were significantly increased (P<0.05 or 0.01).

[0158] 7. Effect of IO on the stride frequency of SOP mice

[0159] Table 22 Comparison of the stride frequencies of mice in each group ( n = 7)

[0160]

[0161] Note: Compared with the SOP group, # P<0.05, ## P<0.01.

[0162] Compared with the normal group, there were no significant changes in the stride frequencies of the four limbs of the mice in the SOP group; compared with the SOP group, the stride frequencies of the left forelimb and the bilateral hindlimbs of the mice in the IO group were significantly reduced (P<0.05 or 0.01).

[0163] 8. Effect of IO on the paw area of SOP mice

[0164] Table 23 Comparison of the paw areas of mice in each group ( n = 7)

[0165]

[0166] Note: Compared with the normal group, ** P<0.01; compared with the SOP group, # P<0.05, ## P<0.01.

[0167] Compared with the normal group, the paw areas of the four limbs of the mice in the SOP group were significantly reduced (P<0.01); compared with the SOP group, the paw areas of the four limbs of the mice in the IO group were significantly increased (P<0.05).

[0168] (3) Effect of IO on serum ALP and TRACP of SOP mice

[0169] Table 24 Comparison of serum ALP and TRACP of mice in each group ( n = 5)

[0170]

[0171] Note: Compared with the normal group, ** P<0.01; compared with the SOP group, # P<0.05.

[0172] Compared with the normal group, the serum ALP concentration of mice in the SOP group was significantly decreased (P<0.01); compared with the SOP group, the serum ALP concentration of mice in the IO group was significantly increased (P<0.05). There was no significant change in the serum TRACP concentration among all groups.

[0173] (IV) Effects of IO on ALP and TRACP in the bone tissue of SOP mice

[0174] Table 25 Comparison of ALP and TRACP in the bone tissue of mice in each group ( n = 5)

[0175]

[0176] Note: Compared with the normal group, * P<0.05, ** P<0.01; compared with the SOP group, # P<0.05.

[0177] Compared with the normal group, the positive expressions of ALP and TRACP in the bone tissue of mice in the SOP group were significantly decreased (P<0.05 or 0.01), suggesting a decrease in bone remodeling in SOP mice. Compared with the SOP group, the positive expression of ALP in the IO group was significantly increased (P<0.05), and there was no significant change in the positive expression of TRACP, suggesting that IO can promote the osteogenic function of SOP mice and improve the osteogenic function.

[0178] (V) Effects of IO on SOD and MDA in the serum of SOP mice

[0179] Table 26 Comparison of SOD and MDA in the serum of mice in each group ( n = 5)

[0180]

[0181] Note: Compared with the normal group, ** P<0.01; compared with the SOP group, ## P<0.01.

[0182] Compared with the normal group, the expression of SOD in the serum of mice in the SOP group was significantly decreased (P<0.01), and the expression of MDA was significantly increased (P<0.01), suggesting an increase in the serum oxidative stress level in SOP mice. Compared with the SOP group, the expression of SOD in the IO group was significantly increased (P<0.01), and the expression of MDA was significantly decreased (P<0.01), suggesting that IO can relieve the oxidative stress level in SOP mice.

[0183] (VI) Effects of IO on p16 and p21 in the bone tissue of SOP mice

[0184] Table 27 Comparison of the protein expressions of p16 and p21 in the bone tissue of mice in each group ( n = 5)

[0185]

[0186] Note: Compared with the normal group, * P < 0.05, ** P < 0.01; compared with the SOP group, ## P < 0.01.

[0187] Compared with the normal group, the expressions of p16 and p21 proteins in the bone tissues of mice in the SOP group were significantly increased (P < 0.05 or 0.01), suggesting that the expressions of cell cycle-related factors p16 and p21 in SOP mice were elevated and the cells presented a senescent state. Compared with the SOP group, the expressions of p16 and p21 proteins in the bone tissues of the IO group were significantly decreased (P < 0.01), suggesting that IO could down-regulate the expressions of p16 and p21 in SOP mice and relieve the cell cycle arrest caused by senescence.

[0188] The above are the specific embodiments of the present invention, but the protection scope of the present invention is not limited thereto. Any person skilled in the art within the technical scope disclosed by the present invention can easily think of changes or substitutions, which should all be covered by the protection scope of the present invention. Therefore, the protection scope of the present invention should be subject to the protection scope of the claims.

Claims

1. A traditional Chinese medicine monomer composition for delaying the senescence of osteoblasts, characterized in that, The monomeric traditional Chinese medicine composition is composed of icariin and oleanolic acid, and the mass ratio of icariin to oleanolic acid is 3:

5.

2. Use of the monomeric traditional Chinese medicine composition according to claim 1 in the preparation of a drug for delaying the senescence of osteoblasts.

3. The application according to claim 2, characterized in that: The delay in osteoblast senescence is specifically manifested in at least one of the following 1)-11): 1) Improving the cell viability of osteoblasts; 2) Reducing the expression of β-galactosidase in osteoblasts; 3) Improving the total antioxidant capacity and superoxide dismutase activity of osteoblasts, and reducing the contents of malondialdehyde and nitric oxide in osteoblasts; 4) Reducing the expression of cyclin-dependent kinase inhibitor 2A, cyclin-dependent kinase inhibitor 1A, interleukin 6, interleukin 8, matrix metalloproteinase 3, tumor necrosis factor α, and γH2A.X in osteoblasts, and increasing the expression of Lamin B1; 5) Increasing the expression of alkaline phosphatase in osteoblasts; 6) Improving the proliferation activity of osteoblasts; 7) Increasing the expression of transforming growth factor-β1, bone morphogenetic protein 2, Wnt family member 5A, osteoprotegerin, and insulin-like growth factor-1 in osteoblasts; 8) Increasing the bone density, bone volume percentage, bone surface area to tissue volume ratio, trabecular bone number, trabecular bone area, and trabecular bone thickness of mammals, reducing the trabecular bone separation, and improving the microscopic structure of bone; 9) Improving the gait function and coordination of mammals; 10) Increasing the expression of alkaline phosphatase in mammals and promoting bone formation; 11) Reducing the expression of malondialdehyde, cyclin-dependent kinase inhibitor 2A, and cyclin-dependent kinase inhibitor 1A in mammals and increasing the superoxide dismutase activity.

4. Use of the monomeric traditional Chinese medicine composition according to claim 1 in the preparation of a drug for treating bone injury or bone defect.

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