CN112972475A - Application of oltipraz in preparation of product for preventing and treating osteoporosis - Google Patents

Abstract

The invention discloses application of oltipraz in preparation of a product for preventing and treating osteoporosis. The application comprises any one of the following: 1) preparing a product for preventing and treating osteoporosis caused by active vitamin D deficiency; 2) preparing a product for preventing and treating osteoporosis caused by estrogen deficiency; 3) preparing products for preventing and treating osteoporosis caused by vitamin D receptor deficiency. According to the invention, a test of supplementing a mouse model with Nrf2 agonist oltipraz reveals the function and related mechanism of oltipraz in preventing and treating osteoporosis caused by active vitamin D deficiency, and provides experimental and theoretical basis for the application of oltipraz in preventing and treating osteoporosis caused by estrogen deficiency.

Description

Application of oltipraz in preparation of product for preventing and treating osteoporosis

Technical Field

The invention relates to the technical field of medicines, and relates to application of oltipraz in preparation of a product for preventing and treating osteoporosis.

Background

Osteoporosis (osteoporotis) is a systemic bone disease in which bone fracture is easily caused by a decrease in bone density and bone mass due to various causes, a destruction of bone microarchitecture, and an increase in bone fragility. Osteoporosis is divided into primary and secondary categories. Primary osteoporosis is classified into postmenopausal osteoporosis (type i), senile osteoporosis (type ii) and idiopathic osteoporosis (including juvenile type). Postmenopausal osteoporosis generally occurs in women within 5-10 years after menopause; senile osteoporosis generally refers to osteoporosis occurring in the elderly after age 70; however, idiopathic osteoporosis mainly occurs in adolescents, and the cause of the disease is unknown. Since osteoporosis may cause various kinds of osteoporosis, it may be caused by various diseases, called secondary osteoporosis, in addition to primary osteoporosis mainly associated with menopause and old age. Common diseases that may cause osteoporosis are: endocrine diseases, chronic kidney diseases, neuromuscular diseases and the like are only painful and limited in movement if the disease is mild, and are broken if the disease is severe, so that the life quality of a patient is seriously affected, even the life of the patient is threatened, and the disease also brings heavy economic burden to individuals, families and society. However, currently, drugs are generally used for intervention treatment, and since osteoporosis may be caused by various reasons, the drug intervention needs to be purposefully implemented, and at present, no drug can effectively prevent and treat osteoporosis caused by different reasons.

Oltipraz (Oltipraz), an Nrf2 activator, promotes Nrf2 nuclear entry by disrupting the interaction between Keap1 and Nrf2, activating expression of downstream antioxidant genes. Recent studies found that oltipraz can reverse the senescence phenotype of mesenchymal stem cells derived from childhood progeria. At present, experimental researches on the aspect of treating non-alcoholic fatty liver disease, hypoxic pulmonary disease and diabetes by using oltipraz are carried out, but related researches on preventing and treating osteoporosis by using oltipraz are not reported.

Disclosure of Invention

The invention aims to provide application of oltipraz in preparation of a product for preventing and treating osteoporosis, so as to solve the problems in the prior art, and an Nrf2 agonist oltipraz test is supplemented to a mouse model, so that the effect and the related mechanism of oltipraz in preventing and treating osteoporosis caused by active vitamin D deficiency are disclosed, and experimental and theoretical bases are provided for application of oltipraz in prevention and treatment of osteoporosis caused by estrogen deficiency.

In order to achieve the purpose, the invention provides the following scheme:

the invention provides an application of oltipraz in preparing a product for preventing and treating osteoporosis.

Preferably, the application comprises any one of:

1) preparing a product for preventing and treating osteoporosis caused by active vitamin D deficiency;

2) preparing a product for preventing and treating osteoporosis caused by estrogen deficiency;

3) preparing a product for preventing and treating osteoporosis caused by Vitamin D Receptor (VDR) deficiency.

Preferably, the application described in 1) includes any one of:

a: preparing a product for correcting osteopenia of osteoporosis origin caused by active vitamin D deficiency;

b: preparing a product for promoting osteoblast bone formation and inhibiting osteoclast bone resorption to exert corrective activity, and treating osteoporosis caused by vitamin D deficiency;

c: preparing a product for inhibiting bone tissue oxidative stress and DNA damage from osteoporosis caused by active vitamin D deficiency;

d: the preparation of products for inhibiting the senescence of bone cells of osteoporotic origin caused by active vitamin D deficiency and for preparing senescence-associated secretory phenotypes.

Preferably, the application of 2) comprises:

the product is used for promoting osteoblast bone formation and inhibiting osteoclast bone absorption so as to correct osteoporosis caused by estrogen deficiency.

Preferably, the application of 3) comprises:

preparing a phenotypic product for correcting the aging of the mesenchymal stem cells caused by the deficiency of the vitamin D receptor.

Preferably, the product is a medicament.

The invention discloses the following technical effects:

the present invention is performed by administering 1 alpha (OH) ase^+/-The mouse oltipraz supplements the diet, and proves that the Nrf2 agonist oltipraz supplement can inhibit oxidative stress and DNA damage and osteocyte senescence and SASP (senescence-associated) by activating the Nrf2 antioxidant pathwaySecretory phenotype), promote osteoblast bone formation, and inhibit osteoclast bone resorption, thereby playing a role in preventing and treating osteoporosis caused by active vitamin D deficiency. Meanwhile, the invention also verifies the effect of the oltipraz in correcting the osteoporosis caused by the estrogen deficiency by giving the estrogen deficiency mouse model oltipraz supplementary diet. Through different experiments, the invention discloses the function and the related mechanism of the supplement of the Nrf2 agonist oltipraz in preventing and treating osteoporosis caused by deficiency of active vitamin D, and simultaneously provides experimental and theoretical basis for the application of the oltipraz in preventing and treating osteoporosis caused by deficiency of estrogen.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings needed to be used in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings without inventive exercise.

FIG. 1 is the results of example 1 demonstrating that oltipraz is able to correct the decrease in bone mass caused by active vitamin D deficiency; (A) 12-month-old Normal diet WT and 1 alpha (OH) ase^+/-Carrying out Micro-CT scanning and three-dimensional reconstruction on mouse vertebrae; (B) von Kossa staining microscopy images (50 ×); (C) statistical bone Density of vertebrae (BMD, mg/cm)³) (ii) a (D) Percentage of small sorghum bone volume/bone tissue volume (BV/TV,%); (E) trabecular number of bones (Tb.N, #/mm); (F) trabecular bone thickness statistics (tb.th, mm); (G) trabecular bone resolution (tb.sp, μm); **: p is a radical of<0.01, compared to normal diet WT mice; #: p is a radical of<0.05, 1 alpha (OH) ase from normal diet⁺/^-Mouse phase comparison;

fig. 2 is a result of an experiment in example 1 to verify that oltipraz promotes osteoblast bone formation and inhibits osteoclast bone resorption; (A) calcein/XO double-standard microscopic image; (B) bone mineralization deposition rate (MAR, μm/day); (C) bone formation Rate (BFR, μm)³/μm²Day); (D) HE staining microscopic image; (E) osteoblast count/bone tissue area (N.ob/T.Ar, #/mm)²) (ii) a (F) TRAP groupingStaining the microscopic image; (G) osteoclast number/bone tissue area (N.Oc/T.Ar, #/mm)²) (ii) a (H) Serum CTX levels (ng/mL); **: p is a radical of<0.01, compared to normal diet WT mice; #: p is a radical of<0.05, 1 alpha (OH) ase from normal diet^+/-Mouse phase comparison;

FIG. 3 is the results of the experiment in example 1 demonstrating that oltipraz supplementation can inhibit oxidative stress and DNA damage in bone tissue caused by active vitamin D deficiency; (A, B) SOD2 immunohistochemical staining micrographs and statistical plots of the percentage of SOD2 positive cells; (C, D)8-OHG immunohistochemical staining microscopy images and 8-OHG positive cell percentage statistics; (E, F) gamma-H2 AX immunohistochemical staining microscopy images and gamma-H2 AX positive cell percentage statistics; **: p is a radical of<0.01，*：p<0.05, compared to normal diet WT mice; #: p is a radical of<0.05, 1 alpha (OH) ase from normal diet^+/-Mouse phase comparison;

FIG. 4 is the results of example 1 demonstrating that oltipraz supplementation inhibits osteocyte senescence and SASP due to active vitamin D deficiency; (A, B) β -gal immunohistochemical staining microscopy images and statistical β -gal positive cell percentage; (C, D) p16 immunohistochemical staining microscopy images and statistical p16 positive cell percentage; (E, F) IL-6 immunohistochemical staining microscopy images and statistics for percent IL-6 positive cells; **: p is a radical of<0.01，*：p<0.05, compared to normal diet WT mice; #: p is a radical of<0.05, 1 alpha (OH) ase from normal diet^+/-Mouse phase comparison;

FIG. 5 is the test result of example 1 for verifying that oltipraz corrects the reduction of BM-MSC proliferation and clonogenic capacity caused by VDR deletion by activating Nrf 2; (A) western blot detection control (vehicle) and oltipraz (oltipraz) treatment conditions Nrf2 Total protein and nucleoprotein in VDR^-/-Expression levels in BM-MSCs; (B) a cell viability (cell viability) histogram of the MTT assay; (C) crystal violet staining and colony formation statistical plots; **: p is a radical of<0.01，*：p<0.05, compared to WT mouse BM-MSCs; #: p is a radical of<0.05, with VDR^-/-Mouse BM-MSCs comparison;

FIG. 6 is the results of the experiment in example 1 to verify that oltipraz corrects decreased BM-MSC proliferation, increased oxidative stress, DNA damage and senescence caused by VDR deletion; for three groupsCytochemical and immunochemical staining of mouse BM-MSCs: (A, B) DCFDA detection and ROS level statistical plots; (C, D) statistical plots of gamma-H2 AX immunofluorescent staining and gamma-H2 AX positive cell percentage; (E, F) senescence-associated beta galactosidase (SA-beta-gal) staining and SA-beta-gal positive cell percentage histogram; (G, H) cell proliferation index EdU staining and percentage of EdU positive cells statistical plot; **: p is a radical of<0.01，*：p<0.05, compared to WT mouse BM-MSCs; #: p is a radical of<0.05, with VDR^-/^-Mouse BM-MSCs comparison;

FIG. 7 is a graph showing the results of the experiment in example 1 to verify that oltipraz corrects the decrease in the in vivo survival and ectopic osteogenesis abilities of BM-MSCs caused by VDR deficiency; (A) administration of a stably expressed luciferase VDR^-/^-BM-MSC oltipraz or a control (vehicle) for one week, cells were transplanted to tibialis anterior muscle of nude mice, and live imaging (D, days) was performed at different time points after transplantation, respectively; (B) statistical plots of relative luciferase activity at different time points; (C) HE staining for osteoblasts and osteocytes; (D) masson Trichrome (Masson Trichrome, blue for collagen; red for the implant vehicle (gelatin sponge); (E) bone mass statistics (bone volume,%) for ectopic osteogenesis based on HE and Masson Trichrome staining;. p: p<0.01，*：p<0.05, compared to WT BM-MSCs; #: p is a radical of<0.05, with VDR^-/-BM-MSCs comparison;

fig. 8 is a graph of example 2 demonstrating the effect of oltipraz in correcting osteoporosis due to estrogen deficiency; (A) a protocol to test whether supplementation with the Nrf2 agonist oltipraz was able to correct bone mass loss following ovariectomy; (B) uterine weight statistics (g); (C) body weight (g) after ovariectomy; (D) 4-month-old normal diet SHAM and OVX mouse vertebra Micro-CT scanning and three-dimensional reconstruction; (E) von Kossa staining microscopy images (50 ×); (F) statistical bone Density of vertebrae (BMD, mg/cm)³) (ii) a (G) Percentage of small sorghum bone volume/bone tissue volume (BV/TV,%); (H) trabecular number of bones (Tb.N, #/mm); (I) trabecular bone thickness statistics (tb.th, mm); (J) trabecular bone resolution (tb.sp, μm); (K) femoral Micro-CT scanning and three-dimensional reconstruction; (L) thickness of femoral cortical bone (ct.th, mm); **: p is a radical of<0.01，***：p<0.001, with SHAM mouse phase; #: p is a radical of<0.05，##：p<0.01, compared to OVX mice;

fig. 9 is a graph of example 2 demonstrating that oltipraz plays a role in correcting osteoporosis due to estrogen deficiency by promoting osteoblast bone formation and inhibiting osteoclast bone resorption; (A) calcein/XO double-standard microscopic image; (B) bone mineralization deposition rate (MAR, μm/day); (C) bone formation Rate (BFR, μm)³/μm²Day); (D) HE staining microscopic image; (E) osteoblast count/bone tissue area (N.ob/T.Ar, #/mm)²) (ii) a (F) ALP staining microscopy images; (G) statistical plots of percentage area of ALP positive cells; (H) COL1 immunohistochemical staining microscopy images; (I) percentage of COL1 positive cell area histogram; (J) (iii) TRAP histochemical staining microscopy images; (K) osteoclast number/bone tissue area (N.Oc/T.Ar, #/mm)²) (ii) a (M) serum CTX levels (ng/mL); ***: p is a radical of<0.001, compared to the SHAM mice. #: p is a radical of<0.05，##：p<0.01, compared to OVX mice;

FIG. 10 is a typical image of agarose gel relevant to gene identification of different mice.

Detailed Description

The present invention will now be described in detail by way of examples, which should not be construed as limiting the invention but as providing more detailed descriptions of certain aspects, features and embodiments of the invention.

The test methods used in the following examples are all conventional methods unless otherwise specified; the materials, reagents and the like used are, unless otherwise specified, commercially available reagents and materials.

The cell culture conditions in the following examples were 37 ℃ and 5% CO, unless otherwise specified₂。

The quantitative experiments in the following examples were all set up in at least three replicates and the results were averaged.

Test materials and methods of detection according to the following examples

1. Oltipraz (Oltipraz): sigma, catalog No. O9389. Purity is more than or equal to 98% (HPLC), alias: 4-Methyl-5- (2-pyrazinyl) -1, 2-dithiole-3-thione; BRN 0978110;CCRIS 4048; NSC 347901; RP 35972; empirical formula (Hill representation) C₈H₆N₂S₃(ii) a Molecular weight 226.34; EINECS No. 264-736-6.

2. Model animals: 1 alpha (OH) ase^+/-Mice were introduced from the university of McGill, canada, with knockout of the VI, VII and VIII exons of the mouse Cyp27b1 gene, replaced by the neomycin resistance gene, NEO. VDR⁺/^-Mice were from the Yanchun Li laboratory, university of chicago, usa. Taking 8-week-old wild type and 1 alpha (OH) ase⁺/^-The mice are mated and female, and the genotype identification is carried out to obtain the littermate wild type (1 alpha (OH) ase)^+/+) And heterozygotes (1. alpha. (OH) ases)^+/-) A mouse. We took 8 weeks old vitamin D receptor heterozygote mice VDR⁺/^-Mating the mouse as parent mouse, determining the genotype of the child mouse, and obtaining the littermate wild type (VDR)^+/+) And homozygote (VDR)^-/^-) A mouse. WT, 1 alpha (OH) ase^+/-And VDR^+/-The mice were all housed in SPF-grade animal houses at the laboratory animal base of Nanjing medical university.

3. Mouse grouping and dietary intervention

In the following specific examples, animals were subjected to dietary or pharmaceutical and surgical intervention, as follows:

(1) after weaning, littermates wild-type and heterozygotes (1. alpha. (OH) ase) were given^+/-) Normal diet of mice: normal diet (1% calcium and 0.67% phosphorus supplement in feed).

1)

(2) Oltipraz dietary intervention: administration of 1 alpha (OH) ase^+/-The mouse diet was supplemented with the Nrf2 agonist oltipraz (0.75g/kg feed) to 12 months of age; administration of 1 alpha (OH) ase^+/-Mice were fed a normal diet as a control.

(3) After weaning, littermate wild type (VDR) was given^+/+) And homozygote (VDR)^-/-) Mouse high calcium and phosphorus diet (containing 1.25% phosphorus, 20% lactose, 2% calcium, capable of inducing VDR^-/-Calcium phosphorus and parathyroid hormone (PTH) in mice adjusted to normal levels).

(4) Ovariectomy (OVX) or Sham (Sham) was performed on WT dams aged 2 months old, mice in the Sham group were given normal diet, and mice in the OVX group were given normal diet or oltipraz supplementary diet (0.75g/kg feed), respectively, i.e., divided into three groups, and subjected to material collection analysis to 4 months old.

The mouse genotype identification method comprises the following steps: extracting DNA of mouse tail; performing PCR amplification on the sample; and (4) carrying out electrophoresis on the PCR reaction product, and recording the mouse genotype according to the band after electrophoresis. 1 alpha (OH) ase and VDR mouse gene identification PCR amplification conditions: preheating at 94 deg.C for 5min, amplifying at 94 deg.C for 45s, 55 deg.C for 45s, and 72 deg.C for 45s, and storing at 72 deg.C for 10min and 4 deg.C for 35 cycles.

1 alpha (OH) ase mouse gene identification primer sequence:

1α(OH)ase-sense-F 5’-AGACTGCACTCCACTCTGAG-3’；

1α(OH)ase-sense-R 5’-GTTTCCTACACGGATGTCTC-3’；

1α(OH)ase-neo-sense-F 5’-ACAACAGACAATCGGCTGCTC-3’；

1α(OH)ase-neo-sense-R 5’-CCATGGGTCACGACGAGATC-3’；

the primer sequence for identifying the VDR mouse gene is as follows:

VDR-sense-F 5’-CTGCCCTGCTCCACAGTCCTT-3’；

VDR-sense-R 5’-CGAGACTCTCCAATGTGAAGC-3’；

VDR-neo-sense-F 5’-GCTGCTCTGATGCCGCCGTGTTC-3’；

VDR-neo-sense-R 5’-GCACTTCGC-CCAATAGCAGCCAG-3’。

as shown in FIG. 10, the 1. alpha. (OH) ase homozygote mouse has only one 500bp band, while the heterozygote has two bands of 376bp and 500 bp. The VDR mouse homozygote has only one 294bp band, while the heterozygote has two 294bp and 750bp bands.

4. Materials for identification Material used in the following examples

4.1 mice were removed from the animal center according to the prescribed protocol at the corresponding time of material selection. In the laboratory, 2% chloral hydrate is used for intraperitoneal injection and anesthesia of mice, a special sterilized surgical instrument is used for exposing superficial tissues such as skin and the like, and vertebral tissues of the mice are taken out, or the tissues are fixed by sodium iodate-lysine-paraformaldehyde (PLP) for 48 hours, or the tissues from which protein is extracted are stored at-80 ℃.

4.2 extraction and culture of mouse bone marrow mesenchymal stem cells (BM-MSCs): collecting VDR of 8 weeks old^-/-And a same-nest WT mouse, anesthetizing by intraperitoneal injection with 2% chloral hydrate at a ratio of 0.2ml/10g mouse body weight, separating tibia and femur under aseptic condition, washing with PBS for several times, lifting cartilage end, inserting 2ml syringe containing complete culture medium into bone end to wash out bone marrow cells in bone marrow cavity, blowing bone marrow cells with 1ml syringe to prepare single cell suspension, adding appropriate amount of standard culture medium (DMEM, 10% fetal calf serum, 1% double antibody), inoculating cells into 6-well plate, and placing into 6-well plate containing 5% CO₂And 95% air at 37 ℃ in a constant temperature incubator, and after 3 days, cell exchange was performed.

5. Detection methods used in the following examples

Micro CT analysis: after the mouse vertebral tissue was fixed with PLP for 48h, it was washed with pre-chilled PBS (3-5 times, 5 min/time), subjected to Micro CT scan using the Micro-CT imaging system of the laboratory animal center of Nanjing university of medical sciences (Bruker, Germany), and subjected to 3D reconstruction analysis using Skyscan software.

And (3) morphological analysis: the fixed mouse vertebra tissue is placed in EDTA decalcification solution for decalcification for 3 days, and the shaking table is shaken at 4 ℃ and the solution is changed once a day. After decalcification is finished, performing ascending gradient alcohol dehydration, enabling dimethylbenzene to be transparent, soaking paraffin, slicing after paraffin embedding, and performing the following tissue morphology experiments after the slices are dried in an oven at 37 ℃ overnight: HE staining (tokyo founding company, staining according to the conventional method), alkaline phosphatase (ALP), tartrate-resistant acid phosphatase (TRAP) histochemical staining and immunohistochemical staining (specific steps are as follows).

Alkaline phosphatase (ALP) histochemical staining is used for detecting the osteoblast activity of bone tissue, and the method comprises the following steps: mouse vertebral tissue paraffin sections were deparaffinized and hydrated, washed with running water, incubated overnight at 4 ℃ in 1% MgCl2 solution (to reduce ALP activity), ALP staining solution [ 20mg naphthol +40mg Fast Red TR +100ml 100mM pH 9.2Tris-HCl ], filtered and used (ready-to-use, relevant reagents purchased from Sigma) incubated for 15 minutes at room temperature in the dark, washed with running water, methyl green counterstain; and taking a picture after the water-soluble sealing piece is sealed.

② the chemical staining method of tartaric acid resistant acid phosphatase tissue for identifying the osteoclast number of bone tissue, the method is as follows: mouse vertebra tissue paraffin section dewaxing hydration, running water washing, incubation for 30min at room temperature with tartrate-resistant buffer solution, incubation for 10-15min with osteoclast staining solution (25mg naphthol +1mg formamide dissolved, 5mg Fast Garnet GBC dissolved with 10mL tartrate buffer solution, filter paper filtration, ready preparation) until red product appears, running water washing for 5min, methyl green counterstaining cell nucleus for 4min, water-soluble sealing piece glue sealing piece and taking a picture.

③ immunohistochemical staining: observing the changes of oxidative stress of bone tissues, DNA damage, cell aging and aging-related secretory phenotype (SASP), type I collagen-related indexes, comprising the following steps: bone tissue slices are dewaxed and hydrated, washed by running water, incubated with antigen retrieval solution for 30min, washed by PBS (3 times 5 min/time), incubated for 20min by hydrogen peroxide immersion, washed by PBS (3 times 5 min/time), and sealed by goat serum or rabbit serum for 30min, and primary antibody is added: Anti-Superoxide dismutase (Superoxide dismutase) antibody (Anti-SOD, Abcam company, Ab16831), Anti-8-hydroxydeoxyguanosine (8-hydroxy-2deoxyguanosine, 8-OHDG) antibody (Anti-8-OHDG, Abcam company, Ab48508), Anti-phosphohistone (gamma H2A) antibody (Anti-gamma H2AX, Abcam company, Ab11174), Anti-galactosidase (beta-galactostasidase) antibody (Anti-beta-gal, Abcam company, Ab616), Anti-p16 antibody (Anti-p16, Abcam company, 211542), Anti-interleukin-6 (interleukin 6676) antibody (Anti-IL-6, Abcam company, Ab 2), 4 degrees overnight; the next day PBS was shaken (3 times, 5 min/time); adding corresponding secondary antibodies (goat anti-mouse, goat anti-rabbit, rabbit anti-goat, Sigma) marked by Biotin, and incubating for 1h at room temperature; PBS shake wash (3 times, 5 min/time), Elite AB bridge immersion incubation for 30min, DAB color development (VECTASTAIN @ ABC immunohistochemical kit, Vector Laboratories, Inc.); flushing with running water; performing hematoxylin counterstaining and flushing with running water; observing under a mirror, and drying the dehydrated transparent sealing sheet. The sections were observed under a fluorescent microscope and analyzed after photographing.

Von Kossa staining: the fixed mouse vertebral tissues were shaken (3 times, 5 min/time) with PBS, cryosectioned with the aid of cryo-resistant tape as offered by professor Liupeng, and the sections were stained with Von Kossa (relevant reagents: silver nitrate and Hydroquinone, sigma company) and analyzed by fluorescence microscopy.

Xylenol orange and calcein dual-standard: dynamic bone histomorphometry experiments, i.e. xylenol orange and calcein dual-scale experiments: groups of mice were injected with xylenol orange (90 mg/kg mouse body weight, Sigma, cat # 398187) and calcein (calcein, 10mg/kg mouse body weight, Sigma, cat # C0875) in a single injection, with 10 days between injections, and on day 5 after calcein injection, mouse vertebral tissue was fixed, washed with PBS, embedded in o.c.t, frozen sections, and analyzed by fluorescent microscopy.

Sixthly, cellular immunofluorescence staining: BM-MSCs were subcultured on chamber slides and cultured, and when cells grew to an appropriate cell density, the culture was terminated, washed 2 times with PBS, fixed 20min at room temperature with 4% paraformaldehyde, shaken (3 times, 5 min/time) with PBS, incubated for 20min at room temperature with 0.3% Triton 100-PBS, shaken (3 times, 5 min/time) with PBST, and then incubated for 60min with 10% goat serum, and then Anti-phosphohistone (gamma H2A) antibody (Anti-gamma H2AX, Abcam company, Ab11174) was added, incubated overnight at 4 ℃, shaken (3 times, 5 min/time) with PBST, and secondary antibody was added: Alexa-Fluor 488(Invitrogen, USA), incubated at room temperature for 75min, PBST shaken (3 times, 5 min/time), the slides of the chamber slides removed, mounted with anti-quenching mounting medium containing DAPI, and photographed with a fluorescent microscope.

Seventh EdU dyeing: the proliferation capacity of the cells was determined, and the relevant experimental procedures were referred to kit instructions (RiboBio).

Staining of SA-beta-gal: senescent cells were detected and relevant experimental steps were referred to kit instructions (Cell Signaling Technology).

Ninthly, DCFDA-flow cytometry detection: viable cell reactive oxygen species were stained and ROS levels were measured by flow cytometry using DCFDA available from sigma as cat # D6883.

Example 1 experimental verification that oltipraz has a preventive effect on osteoporosis induced by active vitamin D

1. Oltipraz pair 1,25(OH)₂D deficient mice (1. alpha. (OH) ase)^+/-Mouse) influence of skeletal phenotype

1.1 oltipraz corrects bone mass reduction caused by active vitamin D deficiency

Cage separation followed by WT and 1 alpha (OH) ase^+/-Normal diet of mice; administration of 1 alpha (OH) ase^+/-The mice are supplemented with diet until 12 months of age, vertebral tissues of the mice are taken, and the vertebral phenotype difference of each group of mice is compared and analyzed by utilizing a micro CT scanning and histochemical method.

As shown in the results of fig. 1: normal diet 1 alpha (OH) ase compared to normal diet WT mice^+/-The bone density, the bone mass, the thickness and the number of the trabeculae of the mouse are obviously reduced, the separation degree of the trabeculae is obviously increased, and the oltipraz dietary supplement obviously corrects the indexes in 1 alpha (OH) ase^+/-Changes in mice.

1.2 oltipraz promotes osteoblast bone formation and inhibits osteoclast bone resorption

Comparative analysis of WT and 1 alpha (OH) ase by histochemical and bone histomorphometry^+/-And 1 alpha (OH) ase^+/-The vertebral phenotype of mice in the + oltipraz 3 group is different, and the serum of the mice is taken to detect the C-terminal peptide (CTX) level of type I collagen.

As shown in the results of fig. 2: 1 alpha (OH) ase in normal diet ^+/-1 alpha (OH) ase after oltipraz supplementation in comparison with mice^+/-The number of vertebral osteoblasts, the bone mineralization deposition rate and the bone formation rate of the mice are obviously increased, and the number of osteoclasts and the level of serum CTX are obviously reduced.

1.3 oltipraz supplementation can inhibit bone tissue oxidative stress and DNA damage caused by active vitamin D deficiency

Comparative analysis of WT, 1 alpha (OH) ase by immunohistochemical method^+/-And 1 alpha (OH) ase^+/-Change in indicators related to oxidative stress and DNA damage in bone tissue of mice in group + oltipraz 3.

As shown in the results of fig. 3: 1 alpha (OH) ase in normal diet ^+/-1 alpha (OH) ase after oltipraz supplementation in comparison with mice^+/-Mouse vertebral tissue antioxidant proteinThe percentage of SOD2 positive cells is obviously increased, and the percentage of DNA damage related indexes 8-OHG and gamma H2AX positive cells is obviously reduced.

1.4 oltipraz supplementation can inhibit bone cell senescence and SASP caused by active vitamin D deficiency

Comparative analysis of WT, 1 alpha (OH) ase by immunohistochemical method^+/-And 1 alpha (OH) ase^+/-Change in cellular senescence-associated indices in vertebral tissue of mice in group + oltipraz 3.

As shown in fig. 4 results: 1 alpha (OH) ase in normal diet ^+/-1 alpha (OH) ase after oltipraz supplementation in comparison with mice^+/-The percentage of beta-gal, p16 and IL-6 positive cells, which are relevant indicators for aging of mouse vertebral tissues, was significantly reduced.

1.5 oltipraz corrects BM-MSC proliferation and clonogenic potential reduction caused by VDR deletion by activating Nrf2

Administration of VDR^-/-BM-MSCs oltipraz are treated for 7 days, and changes of indexes related to proliferation and clone formation of BM-MSCs are compared and analyzed by using Western blot, MTT experiments and a cell crystal violet staining method.

As shown in FIG. 5, Western blot results show that cells treated with oltipraz can obviously up-regulate total protein and nucleoprotein of Nrf2 in VDR^-/^-Expression levels in BM-MSCs; VDR compared to WT-BM-MSCs^-/-The proliferation activity and the cloning formation capability of BM-MSCs are obviously reduced; and VDR^-/-VDR after oltipraz treatment compared to BM-MSCs^-/-The proliferation and clonogenic capacity of BM-MSCs is significantly increased.

1.6 oltipraz corrects decreased BM-MSC proliferation, increased oxidative stress, DNA damage and aging caused by VDR deficiency

VDR treated by oltipraz is compared and analyzed by using flow cytometry, cell immunofluorescence staining and cytochemical staining methods^-/^-Changes in BM-MSCs proliferation, oxidative stress, DNA damage, and cellular senescence-associated indices.

As shown in FIG. 6, VDR compared to WT-BM-MSCs^-/-The percent of EdU positive cells of BM-MSCs is obviously reduced, and the percent of ROS level, gamma-H2 AX and SA-beta-gal positive cells is obviously reducedIncreasing; and VDR^-/-VDR after oltipraz treatment compared to BM-MSCs^-/^-The percentages of EdU-positive cells of BM-MSCs were significantly increased, and the levels of ROS, gamma-H2 AX, and SA-beta-gal-positive cells were significantly decreased.

1.7 oltipraz corrects decreased in vivo survival and ectopic osteogenesis of BM-MSCs due to VDR deficiency

In vitro administration of VDRs^-/-BM-MSCs oltipraz is transplanted to tibialis anterior muscle or subcutaneous of nude mice of 6 weeks after 1 week pretreatment, and the VDR of oltipraz is observed^-/-Effects of the in vivo survival and ectopic osteogenesis Capacity of BM-MSCs.

The BM-MSCs transplantation experiment specifically comprises the following operations:

(1) BM-MSCs implanted into tibialis anterior muscle of nude mouse

1) BM-MSCs are transferred into a vector lenti-luciferase by a lentivirus transfection method, and are treated for 7-10 days by puromycin 48 hours later, so that all BM-MSCs carry luciferase labels.

2) 50 μ L of cell suspension (1X 10) was injected with a syringe⁶Individual cell) to the tibialis anterior muscle of a 5-week-old nude mouse, injecting luciferin (luciferin) to the abdominal cavity of the mouse respectively 0, 1, 3, 5 and 7 days after transplantation, performing living body imaging by using a small animal living body visible light three-dimensional imaging system (PerkinElmer company, USA) of Nanjing medical university after 10min, and performing statistical analysis on luciferase activity, wherein the strength of luciferase signal reflects the survival capacity of the cell.

(2) BM-MSCs are implanted under the skin of the back of a nude mouse

1) Mouse BM-MSCs were passaged to 3 rd passage, followed by VDR administration^-/^-BM-MSCs oltipraz (50. mu.M) or control solvent treatment for 72 h;

2) 40 μ L of cell suspension (2X 10) was suspended with the aid of gelatin sponge⁶Individual cells) were transplanted to the back of 5-week-old nude mice, gelatin sponges were taken 6 weeks after transplantation, fixed, decalcified, sectioned and HE-stained with Masson (Masson trichrome staining kit, KeyGEN), and statistical analysis was performed on the amount of ectopically generated bones according to the staining results.

As shown in fig. 7, the in vivo imaging results show: at the comparison with the control solventVDR of Li^-/-BM-MSCs, VDR after oltipraz treatment^-/^-The in vivo survival ability of BM-MSCs is obviously increased.

HE and Masson staining showed: VDR compared to control solvent treatment^-/-BM-MSCs, oltipraz treated VDR^-/-The ectopic osteogenesis capacity of BM-MSCs is obviously increased.

Example 2 experimental verification that oltipraz has a preventive effect on osteoporosis caused by estrogen deficiency

1. Effect of oltipraz on skeletal phenotype of estrogen-deficient mouse model

1.1 effects of oltipraz in correcting osteoporosis due to estrogen deficiency

Ovariectomy (OVX) or Sham (Sham) was performed on WT mice of 2 months of age, and OVX mice were given normal diet or oltipraz supplemented diet until 4 months of age, and vertebral tissues of the mice were collected and analyzed for differences in skeletal phenotype of each group of mice by comparison using a micro CT scan and histochemical staining method.

As shown in the results of fig. 8: compared with a sham operation group, the weight of the uterus of the mice in the OVX and OVX + oltipraz groups is obviously reduced, and the weight is obviously increased; while the uterine weights and body weights of these 2 groups of mice were not statistically different.

Compared with a sham operation group, the bone density, the bone volume, the number of trabeculae and the thickness of the trabeculae of the mice in the OVX group are obviously reduced, the separation degree of the trabeculae is obviously increased, and the thickness of the cortex of the thighbone cortex is obviously reduced; compared with the OVX group under the control diet condition, the indexes of the OVX group mice under the oltipraz diet condition are well corrected.

1.2 oltipraz exerts an effect of correcting osteoporosis caused by estrogen deficiency by promoting osteoblast bone formation and inhibiting osteoclast bone resorption

The phenotypic differences of the vertebrae of each group of mice were comparatively analyzed by means of histochemical staining and bone histomorphometry. Serum from mice was taken to determine the CTX level in the serum of 3 groups of mice.

As shown by the results in fig. 9: compared with the sham operation group, the bone mineralization and deposition rate, the bone formation rate, the osteoblast number per unit area and the like of mice in the OVX group,ALP positive area and type I collagen positive area are obviously reduced, and TRAP is a unit area⁺The number of osteoclasts, the osteoclast positive area per unit area and the serum CTX level are obviously increased; compared with the OVX group under the control diet condition, the indexes of the OVX group mice under the oltipraz diet condition are well corrected.

Through the above examples 1-2, the present invention demonstrates that Nrf2 agonist oltipraz supplementation can inhibit oxidative stress and DNA damage as well as osteocyte senescence and SASP by activating Nrf2 antioxidant pathway, promote osteoblast bone formation, inhibit osteoclast bone resorption, and thus exert the effect of preventing and treating osteoporosis caused by active vitamin D deficiency. Meanwhile, the invention also verifies the function of the oltipraz in correcting the osteoporosis caused by the estrogen deficiency in an estrogen deficiency mouse model. In conclusion, the invention discloses the function and the related mechanism of the supplement of the Nrf2 agonist oltipraz in preventing and treating osteoporosis caused by active vitamin D deficiency, and provides experimental and theoretical basis for the application of the oltipraz in preventing and treating the osteoporosis caused by estrogen deficiency.

The above-described embodiments are merely illustrative of the preferred embodiments of the present invention, and do not limit the scope of the present invention, and various modifications and improvements of the technical solutions of the present invention can be made by those skilled in the art without departing from the spirit of the present invention, and the technical solutions of the present invention are within the scope of the present invention defined by the claims.

Claims (6)

1. Application of oltipraz in preparing products for preventing and treating osteoporosis.

2. Use of oltipraz according to claim 1, for the preparation of a product for the prevention and treatment of osteoporosis, comprising any one of:

1) preparing a product for preventing and treating osteoporosis caused by active vitamin D deficiency;

2) preparing a product for preventing and treating osteoporosis caused by estrogen deficiency;

3) preparing products for preventing and treating osteoporosis caused by vitamin D receptor deficiency.

3. The use of oltipraz of claim 2, for the preparation of a product for the prevention and treatment of osteoporosis, wherein said use in 1) comprises any one of:

a: preparing a product for correcting osteopenia of osteoporosis origin caused by active vitamin D deficiency;

b: preparing a product for promoting osteoblast bone formation and inhibiting osteoclast bone resorption to exert corrective activity, and treating osteoporosis caused by vitamin D deficiency;

c: preparing a product for inhibiting bone tissue oxidative stress and DNA damage from osteoporosis caused by active vitamin D deficiency;

d: the preparation of products for inhibiting the senescence of bone cells of osteoporotic origin caused by active vitamin D deficiency and for preparing senescence-associated secretory phenotypes.

4. The use of oltipraz of claim 2 in the preparation of a product for the prevention and treatment of osteoporosis, wherein said use in 2) comprises:

the product is used for promoting osteoblast bone formation and inhibiting osteoclast bone absorption so as to correct osteoporosis caused by estrogen deficiency.

5. The use of oltipraz of claim 2 for the preparation of a product for the prevention and treatment of osteoporosis, wherein said use in 3) comprises:

preparing a phenotypic product for correcting the aging of the mesenchymal stem cells caused by the deficiency of the vitamin D receptor.

6. Use of oltipraz according to any one of claims 1 to 5, for the preparation of a product for the prevention and treatment of osteoporosis, said product being a pharmaceutical.

Images