CB2R / ERalpha double-target agonist and application thereof

Abstract

The invention discloses a CB2R / ER alpha double-target agonist and an application thereof. The invention finds that an endocannabinoid signal system interacts with an estrogen receptor, and the endocannabinoid signal system and the estrogen receptor are closely associated with osteoporosis which can cause reduction of protein expression of CB2R and ERalpha receptors. Animal experiments carried out by a CB2 and ER alpha receptor double-target agonist cimicifuic acid F prove that the composition has a remarkable effect of inhibiting osteoclast bone resorption in ovariectomized model mice and beta-galactose induced senescent mice; the compound can be used for improving the activity of ERalpha and influencing the activity of CB2R at the same time, has a double-target synergistic effect, can be used for patients with postmenopausal osteoporosis, and can also be used for preventing / treating diseases such as senile osteoporosis and the like.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical engineering, and particularly relates to a CB2R / ERα dual-target agonist and its application. Background Art

[0002] The traditional Chinese medicine (TCM) Cimicifuga foetida L. is the dried rhizome of Cimicifuga heracleifolia Kom., Cimicifuga dahurica (Turcz.) Maxim. or Cimicifuga foetida L. of the Ranunculaceae family, and is included in the Chinese Pharmacopoeia in previous editions. Modern chemical and pharmacological studies have shown that the TCM Cimicifuga foetida L. contains cycloartane-type triterpenoids and their saponins, phenolic acids, phenylpropanoids, alkaloids, chromones, nitrogen-containing compounds, and flavonoid chemical components, and exhibits various biological activities such as anti-tumor, anti-osteoporosis, and neuroprotection. The black cohosh Cimicifuga racemosa (L.) Nuttall. (CR) and its cycloartane-type triterpenoid saponins, such as cimiracemoside, acteoside, deoxyactein, and 25-acetylcimigenol-xylopyranoside, which are used in Europe and North America to relieve menopausal syndrome, have shown good regulatory effects on bone metabolism in ovariectomized osteoporosis rats, osteoblasts, and osteoclasts. The TCM Cimicifuga foetida L. also has good anti-osteoporosis effects in ovariectomized osteoporosis rats, and its anti-osteoporosis, lipid-lowering, and menopausal symptom-relieving effects are similar to those of CR, indicating that the TCM Cimicifuga foetida L. can be used for the prevention and treatment of menopausal syndrome and osteoporosis.

[0003] Osteoporosis is a major disease that seriously endangers the health of the elderly. It is a systemic skeletal disease characterized by reduced bone mass and degenerative changes in the microstructure of bone tissue due to decreased estrogen levels, oxidative stress, drugs, and other diseases, and is prone to fractures. Currently, the main drugs for the prevention and treatment of osteoporosis include estrogen drugs, bisphosphonates, selective estrogen receptor modulators, parathyroid hormone, and calcium preparations. The long-term application of these drugs is limited due to their side effects. The occurrence of osteoporosis is caused by an imbalance in bone metabolism due to decreased bone formation by osteoblasts and increased bone resorption by osteoclasts, and the formation and differentiation of osteoblasts and osteoclasts are regulated by multiple target proteins and molecular signaling pathways. Therefore, OP treatment drugs based on two or more targets can overcome the limitations of single targets, improve the efficacy and safety of drugs, and have become a hot topic in the current research on anti-osteoporosis drugs.

[0004] Estrogen receptor α (ERα) is a key target protein for regulating bone metabolism. Anti-osteoporosis drugs based on the estrogen receptor mechanism, such as raloxifene and tamoxifen, are commonly used for the prevention and treatment of osteoporosis. Cannabinoid receptor 2 (CB2R) is a newly discovered target for regulating bone metabolism in recent years. Its agonist HU308 can increase bone formation by osteoblasts (OBs), inhibit bone resorption by osteoclasts (OCs), and reduce bone loss caused by estrogen deficiency. Drugs targeting ERα, such as estradiol and raloxifene, also have significant agonist effects on CB2R. Both CB2R and ERα can participate in the regulation of signaling pathways such as phosphatidylinositol 3-kinase (PI3K) / protein kinase B (PKB) and mitogen-activated protein kinase (MAPK), showing the characteristic that CB2R and ERα can synergistically regulate bone metabolism. Therefore, anti-osteoporosis drugs with strong specificity, good efficacy and high safety can be developed based on the dual targets of CB2R and ERα. Summary of the Invention

[0005] The object of the present invention is to provide a CB2R / ERα dual-target agonist and its application in view of the deficiencies of the prior art.

[0006] The object of the present invention is achieved by the following technical solutions:

[0007] In the first aspect, the present invention provides a CB2R / ERα dual-target agonist, and the CB2R / ERα dual-target agonist is cimicifugic acid F, and its structure is shown as follows:

[0008]

[0009] In the second aspect, the present invention also provides an application of a CB2R / ERα dual-target agonist in the preparation of a drug for treating osteoporosis.

[0010] Further, the osteoporosis includes postmenopausal osteoporosis and senile osteoporosis.

[0011] The beneficial effects of the present invention are:

[0012] 1) Dual-target synergistic effect: Through dual regulation of CB2R and ERα, bone resorption is inhibited and metabolic disorders are improved simultaneously, breaking through the limitations of single-target drugs;

[0013] 2) Outstanding safety: There is no estrogen receptor activation effect, avoiding the side effects of traditional hormone therapy;

[0014] 3) Multi-dimensional protection mechanism: It has functions of regulating bone metabolism, antioxidation and metabolic homeostasis regulation, and is applicable to complex pathological backgrounds;

[0015] 4) Advantage of natural origin: Derived from active ingredients of traditional Chinese medicine, it has high biocompatibility, no estrogen-like side effects, and its long-term use safety is superior to synthetic compounds;

[0016] 5) The present invention first reveals the pleiotropy of cimicifugic acid F as a CB2R / ERα dual agonist in the treatment of osteoporosis, laying a foundation for the development of innovative drugs with bone protection, metabolic regulation and antioxidation functions. Brief Description of the Drawings

[0017] Figure 1 It is a schematic diagram of UPLC identification of cimicifugic acid F;

[0018] Figure 2 It is a graph showing the effect of different concentrations of cimicifugic acid F on the relative dual luciferase activity of HEK293-CB2R cells;

[0019] Figure 3 It is a graph showing the effect of different concentrations of cimicifugic acid F on the cAMP level pretreated with forskolin in HEK293-CB2R cells;

[0020] Figure 4 It is a graph showing the effect of different concentrations of cimicifugic acid F on the ERα concentration in HEK293-CB2R cells;

[0021] Figure 5 It is a graph showing the effect of different concentrations of cimicifugic acid F on the thermal stability of CB2R and ERα in osteoclasts;

[0022] Figure 6 It is an interaction imaging diagram, in which, Figure 6 A in is the interaction imaging diagram between CB2R and ERα proteins in osteoclasts, Figure 6 B in is the interaction imaging diagram between CB2R and ERα proteins in osteoclasts after treatment with cimicifugic acid F;

[0023] Figure 7 It is a graph showing the change of body weight of mice in the postmenopausal osteoporosis model over time;

[0024] Figure 8 It is a physical picture and uterine mass picture of the uterus of mice in the postmenopausal osteoporosis model after 8 weeks of drug administration, in which, Figure 8 A in is the physical picture of the uterus of mice in the postmenopausal osteoporosis model after 8 weeks of drug administration, Figure 8 B in is the uterine mass picture of mice in the postmenopausal osteoporosis model after 8 weeks of drug administration;

[0025] Figure 9Concentration diagrams of calcium and deoxypyridinoline in the urine of mice after 8 weeks of administration to postmenopausal osteoporosis model mice. Among them, Figure 9 A in Figure 9 is the concentration diagram of calcium in the urine of mice after 8 weeks of administration to postmenopausal osteoporosis model mice,

[0026] Figure 10 Concentration diagrams of C-terminal peptide of type I collagen, N-terminal propeptide of type I procollagen, osteocalcin, osteoprotegerin, receptor activator of nuclear factor-κB ligand, and tartrate-resistant acid phosphatase in the serum of mice after 8 weeks of administration to postmenopausal osteoporosis model mice. Among them, Figure 10 A in Figure 10 is the concentration diagram of C-terminal peptide of type I collagen in the serum of mice after 8 weeks of administration to postmenopausal osteoporosis model mice, Figure 10 B in Figure 10 is the concentration diagram of N-terminal propeptide of type I procollagen in the serum of mice after 8 weeks of administration to postmenopausal osteoporosis model mice, Figure 10 C in Figure 10 is the concentration diagram of osteocalcin in the serum of mice after 8 weeks of administration to postmenopausal osteoporosis model mice,

[0027] Figure 11 Parameter diagrams of the bone tissue of mice after 8 weeks of administration to postmenopausal osteoporosis model mice. Among them, Figure 11 A in Figure 11 is the 2D and 3D morphology diagrams of the bone tissue of mice after 8 weeks of administration to postmenopausal osteoporosis model mice, Figure 11 B in Figure 11 is the bone density parameter diagram of the bone tissue of mice after 8 weeks of administration to postmenopausal osteoporosis model mice, Figure 11 C in Figure 11 is the bone area fraction diagram of the bone tissue of mice after 8 weeks of administration to postmenopausal osteoporosis model mice, Figure 11 D in

[0028] Figure 12HE staining images of the femurs of mice administered with drugs for 8 weeks in the postmenopausal osteoporosis model;

[0029] Figure 13 TRAP staining images of the femurs of mice administered with drugs for 8 weeks in the postmenopausal osteoporosis model;

[0030] Figure 14 LC3B staining images of the femurs of mice administered with drugs for 8 weeks in the postmenopausal osteoporosis model;

[0031] Figure 15 TFEB staining images of the femurs of mice administered with drugs for 8 weeks in the postmenopausal osteoporosis model;

[0032] Figure 16 Graph of the change in body weight of mice in the senile osteoporosis model over time;

[0033] Figure 17 Graph of the concentrations of calcium and deoxypyridinoline in the urine of mice administered with drugs for 8 weeks in the senile osteoporosis model, where Figure 17 A in it is the graph of the concentration of calcium in the urine of mice administered with drugs for 8 weeks in the senile osteoporosis model, Figure 17 B in it is the graph of the concentration of deoxypyridinoline in the urine of mice administered with drugs for 8 weeks in the senile osteoporosis model;

[0034] Figure 18 Graph of the concentrations of C-terminal telopeptide of type I collagen, N-terminal propeptide of type I procollagen, osteocalcin, osteoprotegerin, receptor activator of nuclear factor-κB ligand, and tartrate-resistant acid phosphatase in the serum of mice administered with drugs for 8 weeks in the senile osteoporosis model, where Figure 18 A in it is the graph of the concentration of C-terminal telopeptide of type I collagen in the serum of mice administered with drugs for 8 weeks in the senile osteoporosis model, Figure 18 B in it is the graph of the concentration of N-terminal propeptide of type I procollagen in the serum of mice administered with drugs for 8 weeks in the senile osteoporosis model, Figure 18 C in it is the graph of the concentration of osteocalcin in the serum of mice administered with drugs for 8 weeks in the senile osteoporosis model, Figure 18 D in it is the graph of the concentration of osteoprotegerin in the serum of mice administered with drugs for 8 weeks in the senile osteoporosis model, Figure 18 E in it is the graph of the concentration of receptor activator of nuclear factor-κB ligand in the serum of mice administered with drugs for 8 weeks in the senile osteoporosis model, Figure 18 F in it is the graph of the concentration of tartrate-resistant acid phosphatase in the serum of mice administered with drugs for 8 weeks in the senile osteoporosis model;

[0035] Figure 19 Graph of the concentrations of superoxide dismutase, glutathione peroxidase, catalase, and malondialdehyde in the serum of mice administered with drugs for 8 weeks in the senile osteoporosis model, whereFigure 19 In A, it is the concentration graph of superoxide dismutase in the serum of mice after 8 weeks of administration in the senile osteoporosis model mice. Figure 19 In B, it is the concentration graph of glutathione peroxidase in the serum of mice after 8 weeks of administration in the senile osteoporosis model mice. Figure 19 In C, it is the concentration graph of catalase in the serum of mice after 8 weeks of administration in the senile osteoporosis model mice. Figure 19 In D, it is the concentration graph of malondialdehyde in the serum of mice after 8 weeks of administration in the senile osteoporosis model mice.

[0036] Figure 20 It is the parameter graph of the bone tissue of mice after 8 weeks of administration in the senile osteoporosis model mice. Among them, Figure 20 In A, it is the 2D and 3D morphology graphs of the bone tissue of mice after 8 weeks of administration in the senile osteoporosis model mice. Figure 20 In B, it is the bone density parameter graph of the bone tissue of mice after 8 weeks of administration in the senile osteoporosis model mice. Figure 20 In C, it is the bone area fraction graph of the bone tissue of mice after 8 weeks of administration in the senile osteoporosis model mice. Figure 20 In D, it is the bone volume fraction graph of the bone tissue of mice after 8 weeks of administration in the senile osteoporosis model mice. Figure 20 In E, it is the trabecular bone number graph of the bone tissue of mice after 8 weeks of administration in the senile osteoporosis model mice. Figure 20 In F, it is the trabecular bone separation graph of the bone tissue of mice after 8 weeks of administration in the senile osteoporosis model mice. Figure 20 In G, it is the trabecular bone thickness graph of the bone tissue of mice after 8 weeks of administration in the senile osteoporosis model mice.

[0037] Figure 21 It is the HE staining graph of the femur of mice after 8 weeks of administration in the senile osteoporosis model mice.

[0038] Figure 22 It is the TRAP staining graph of the femur of mice after 8 weeks of administration in the senile osteoporosis model mice.

[0039] Figure 23 It is the LC3B staining graph of the femur of mice after 8 weeks of administration in the senile osteoporosis model mice.

[0040] Figure 24 It is the TFEB staining graph of the femur of mice after 8 weeks of administration in the senile osteoporosis model mice. Detailed implementation manners

[0041] To make the objectives, technical solutions and advantages of the present invention more clearly understood, the present invention will be further described in detail below in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention, rather than all embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts fall within the protection scope of the present invention.

[0042] The objective of the present invention is to overcome the limitations in the treatment of osteoporosis in the prior art and provide a CB2R / ERα dual-target agonist, namely cimicifugic acid F. The CB2R / ERα dual-target agonist is cimicifugic acid F, and its structure is shown as follows:

[0043]

[0044] The CB2R / ERα dual-target agonist cimicifugic acid F synergistically activates the CB2 receptor (CB2R) and estrogen receptor α (ERα), while inhibiting bone resorption and improving bone microstructure, regulates metabolic homeostasis and oxidative stress, and provides a new treatment plan with both bone protection and metabolic regulation effects for postmenopausal and senile osteoporosis. Through systematic research, the present invention finds that cimicifugic acid F can target the dual signaling pathways of CB2R and ERα, inhibit the differentiation and autophagy activity of osteoclasts (OCs), reduce bone resorption markers (such as CTX-1, TRAP), and enhance the activity of antioxidant enzymes (such as SOD, GSH-Px), thereby significantly improving bone mineral density (BMD) and trabecular bone structure in osteoporosis models, and having no estrogen-like side effects, avoiding the risk of endometrial hyperplasia of traditional hormone replacement therapy.

[0045] The preparation process of cimicifugic acid F:

[0046] Preparation of test samples: Cimicifuga herbs are added with 10 times the amount of 70% ethanol (mass / volume), soaked for 12 h, refluxed and extracted 3 times, the extraction solutions are combined and filtered, and concentrated under reduced pressure at 60 °C until the alcohol smell disappears. Purification by macroporous resin (AB-8 type) column chromatography, eluted with pure water, after water elution, 50% ethanol is added for elution, the ethanol eluate is collected, dried under reduced pressure at 60 °C for standby, and cimicifugic acid F is separated from the 50% ethanol pure extract.

[0047] The key process of separating and identifying the active ingredient cimicifugic acid F from the effective part of cimicifuga by UPLC comparative analysis is as Figure 1 shown; Figure 1 In, the signal trace of the black chromatographic peak is the characteristic spectrum of the effective part of cimicifuga for anti-osteoporosis; the signal trace of the red chromatographic peak is the characteristic spectrum of the monomer compound of cimicifugic acid F obtained by separation and purification, and the peak shape has good symmetry (purity > 98%).

[0048] The present invention is verified through pharmacological experimental studies as follows.

[0049] I. Experimental methods

[0050] 1.1 Establishment of cell model: After transfection of CB2 plasmid into cells for 24 h, stable screening and identification of positive cells were carried out using hygromycin B and G418. The constructed HEK293-CB2 cell system was used to evaluate its performance in screening CB2 receptor modulators. The results showed that the signal background value was 4.8 and the signal-to-noise ratio was 31.1, indicating that the background level had no significant effect on the results of high-throughput screening. The average signal and standard deviation of the forskolin positive control group and the DMSO blank control group were 63.3±4.4% and 13.1±1.6% respectively, the signal window SW was 50.2%, the coefficient of variation CV of the signal background was 7.0% and 7.6% respectively, and the Z’ factor was 0.64, indicating that this model had good performance and high feasibility and reliability.

[0051] 1.2 Establishment of postmenopausal osteoporosis model and grouping for drug administration:

[0052] Fifty-four 8-week-old female C57BL / 6J mice were randomly divided into 9 groups (n = 6): Sham group (sham operation), OVX group (bilateral ovariectomy), OVX + 0.2 mg / kg E2V group (estradiol valerate, positive control), OVX + 5 mg / kg cimicifuga acid F group, OVX + 10 mg / kg cimicifuga acid F group, OVX + CB2R KO group (knockout of CB2R gene in mice), OVX + ERαKO group (knockout of ERα gene in mice), OVX + 10 mg / kg cimicifuga acid F + ERαKO group, OVX + 10 mg / kg cimicifuga acid F + CB2R KO group. Except for the Sham group, all C57BL / 6J mice in other groups were fed adaptively for 8 weeks and then underwent bilateral ovariectomy on the back. One week after the operation, drug administration began and lasted for 8 weeks. After 8 weeks of treatment, the mice were anesthetized by intraperitoneal injection of Zoletil 50 @ and euthanized by spinal dislocation after blood collection.

[0053] 1.3 Establishment of senile osteoporosis model and grouping for drug administration:

[0054] 54 male C57BL / 6J mice at 8 weeks of age were randomly divided into 9 groups (n = 6): Control group (saline), D-galactose (D-gal) (subcutaneously injected with 0.5 g / kg D-gal daily), D-gal + 2.5 mg / kg ALN group (alendronate sodium, positive control), D-gal + 5 mg / kg ferulic acid F group, D-gal + 10 mg / kg ferulic acid F group, D-gal + CB2R KO group, D-gal + ERαKO group, D-gal + 10 mg / kg ferulic acid F + ERαKO group, D-gal + 10 mg / kg ferulic acid F + CB2R KO group. Except for the Sham group, C57BL / 6J mice in other groups were subcutaneously injected with 0.5 g / kg of D-galactose in the back of the neck daily for 8 weeks. After 8 weeks of treatment, the mice were anesthetized by intraperitoneal injection of Zoletil @ 50 and euthanized by spinal dislocation after blood collection.

[0055] II. Detection indicators

[0056] 2.1 Evaluation indicators at the cellular and molecular levels

[0057] Dual-luciferase luminescence value analysis: HEK293-CB2R cells (5×10 5 cells / well) of the CB2R gene were seeded in 6-well plates. The cells were starved for 24 h and then treated with blank medium and different concentrations of ferulic acid F for 6 h.

[0058] Detection of intracellular cAMP and ERα concentrations: HEK293-CB2R cells (5×10 5 cells / well) were seeded in 6-well plates. After starvation for 24 h, the cells were pretreated with 1 μM Forskolin for 30 min and then treated with 10 -9 、10 -8 、10 -7 、10 -6 、10 -5 and 10 -4 M of different concentrations of ferulic acid F for 6 h. Cell lysates were obtained by repeated freeze-thaw cycles, centrifuged, and the supernatants were collected. Corresponding kits were used to quantify the concentrations of cAMP and ERα in the cell lysates.

[0059] Evaluation of the thermal stability of CB2R and ERα in osteoclasts: RAW264.7 cells (5×10 6 / well) were inoculated in 10cm culture dishes. After RANKL induction for 3d, the cell supernatant was removed and gently washed twice with PBS. 1mL of prepared IP lysis buffer was added to each dish, and the cells were scraped and placed in a 15mL centrifuge tube; repeated freezing and thawing with liquid nitrogen was used to obtain cell lysate. Centrifugation was performed to collect the supernatant. Divide into 3 tubes, add PBS or cimicin F respectively, and shake slowly for 30min. The supernatant of each group was divided into 5 groups, denatured at different temperatures (57℃, 61℃, 64℃, 67℃, 70℃) for 3min in a PCR instrument, centrifuged at 4℃ and 12000rpm, and the supernatant was collected. After quantification of the loading buffer, it was placed at 100℃ for denaturation for 10min and stored at -80℃.

[0060] Analysis of the interaction between CB2R and ERα in osteoclasts: RAW264.7 cells (5×10 6 100 μL of lysate containing inhibitor was added to every 500,000 to 1,000,000 cells, and the cells were blown and incubated for 10 minutes after being fully lysed. According to the dilution ratio recommended in the antibody instructions, the antibody was diluted with TBS to prepare the antibody working solution, and placed on ice. At the same time, a normal IgG working solution of the same final concentration was prepared using the same antibody species as a negative control. The magnetic beads were magnetically separated, the supernatant was removed, 500 μL of antibody working solution or normal IgG working solution was added, and the suspension was resuspended and incubated on a room temperature flip mixer for 1 hour. 500 μL of TBS was added to resuspend the Protein A+G magnetic beads. Place on a magnetic stand and separate for 10 seconds, remove the supernatant. Wash 3 times. Resuspend Protein A+G magnetic beads with TBS according to the initial volume. In order to remove proteins that non-specifically bind to normal IgG, incubate for 1 hour, separate magnetically, and take the supernatant for subsequent experiments. Add Protein A+G magnetic beads bound to antibodies or normal IgG (add 20μL of magnetic bead suspension for every 500μL protein sample), place on a rotating mixer, and incubate overnight at 4℃. Add loading buffer to the input group sample and denature for 5min. Place on a magnetic stand and separate for 10s, and take the supernatant for subsequent WB detection. After incubating overnight at 4℃, place on a magnetic stand and separate for 10s, and remove the supernatant. Add 0.5mL of inhibitor-containing lysis buffer and resuspend the magnetic beads. Place on a magnetic stand and separate for 10s, and remove the supernatant. After washing with inhibitor-containing lysis buffer 3 times, add loading buffer and denature at 95℃ for 5min. Place on a magnetic rack and separate for 10 seconds, and take the supernatant for WB detection.

[0061] 2.2 Bone metabolism-related indicators in the osteoporosis mouse model

[0062] Serum biochemical analysis: After blood collection, let it stand for 2 h, then centrifuge at 4°C and 3500 rpm for 15 min to obtain the upper-layer serum. Store the serum at -80°C for further analysis. Determine the serum levels of bone metabolism or senescence-related markers, such as CTX-I, PINP, TRAP, RANKL, OCN, OPG, SOD, CAT, GSH-Px, and MDA according to the instructions of the ELISA kit.

[0063] Urine biochemical analysis: Before anesthesia, collect urine from each mouse separately. Centrifuge at 4°C and 3500 rpm for 15 min to obtain the upper-layer clear urine. Store the urine at -80°C for further analysis. Determine the levels of urinary calcium and urinary DPD in mice according to the instructions of the ELISA kit.

[0064] Micro-CT analysis: Take out the mouse femur and fix it in 4% paraformaldehyde solution. The left femur is used for Micro-CT analysis. Use a Bruker Skyscan 1173 high-energy helical Micro-CT scanner to fix the mouse femur in 4% paraformaldehyde solution for micro-computed tomography scan analysis, and use software to reconstruct three-dimensional axial images and two-dimensional images, and quantify the ROI, including BV / TV, BS / BV, BMD, Tb.N., Tb.Th., and Tb.Sp.

[0065] HE staining of bone tissue: Take out the right femur and fix it in 4% paraformaldehyde solution for 48 h (mice should not be fixed for too long, as it is easy to have cracks during sectioning). Take out the bone tissue from the fixative and place it in EDTA decalcifying solution for decalcification. Change the decalcifying solution every 3 d until the bone tissue can be smoothly penetrated by a needle. The decalcified bone tissue is placed in a dehydrator and dehydrated successively with gradient ethanol. After embedding the bone tissue, dewax the paraffin section to water, with a section thickness of 4 μm. Stain the bone tissue section with hematoxylin dye (1 min), rinse with water, then differentiate with 1% hydrochloric acid ethanol, and rinse with tap water; after differentiation, treat with 1% ammonia aqueous solution for 1 min and rinse with running water; then place it in eosin staining solution for several seconds and rinse with running water. After dehydration of the stained section, put it into xylene for clearing for 5 min, and finally mount it with neutral gum (avoid generating bubbles). Observe the changes in trabecular bone of bone tissue in different groups under a microscope, and finally collect pictures.

[0066] TRAP staining of bone tissue and immunohistochemical analysis of LC3B and TFEB: Bone tissue sections were incubated in TRAP staining solution at 37 °C for 1 h. Observation was carried out under the microscope until the OCs showed wine red color, then the sections were sealed, and finally image acquisition and analysis were performed; After bone tissue sections were blocked with 5% BSA at room temperature for 10 min, LC3B and TFEB antibodies were incubated overnight at 4 °C, washed 3 times with PBS (5 min each time), incubated with secondary antibody at 37 °C for 30 min, washed 3 times with PBS, developed with developer, rinsed, counterstained, and then sealed for image acquisition and analysis.

[0067] III. Test Results

[0068] 3.1 Interaction of caffeic acid F with CB2R and ERα

[0069] Dual-luciferase luminescence value: Caffeic acid F at different concentrations showed significant agonist effects on CB2R in HEK293-CB2R cells, and the higher the concentration, the greater the dual-luciferase luminescence value, showing concentration dependence, as Figure 2 shown.

[0070] Intracellular cAMP concentration: After pretreatment with Forskolin, intracellular cAMP in HEK293-CB2R cells increased significantly. The cAMP level in HEK293-CB2R cells after treatment with caffeic acid F and caffeic ketone for 6 h decreased significantly, and the IC 50 value was 100.8 nM, as Figure 3 shown.

[0071] Intracellular ERα concentration: With the increase in the administration concentration of caffeic acid F and caffeic ketone, caffeic acid F increased the concentration of ERα in HEK293-CB2R cells, and the EC 50 value was 102.3 nM, as Figure 4 shown.

[0072] Thermal stability of CB2R and ERα in osteoclasts: The interaction between caffeic acid F and CB2R and ERα proteins in osteoclasts was further analyzed by cell thermal shift assay. After caffeic acid F bound to the proteins, the relative expression of both CB2R and ERα proteins increased, indicating that the treatment with caffeic acid F enhanced the thermal stability of CB2R and ERα in osteoclasts, as Figure 5 shown.

[0073] Interaction between CB2R and ERα in osteoclasts: After immunoprecipitation with an antibody against ERα protein, CB2R was detected in the precipitate, indicating an interaction between CB2R and ERα in OCs. Compared with the Control group, after treating OCs with 0.1 μM cimicifugic acid F, the expression of CB2R increased significantly, and the interaction between CB2R and ERα became more prominent, suggesting that cimicifugic acid F enhanced the interaction between ERα and CB2R in OCs, as Figure 6 shown.

[0074] 3.2 Postmenopausal osteoporosis model

[0075] Body weight changes: After ovariectomy in mice, the estrogen level in the body decreased, and the body weight increased significantly. There was no significant difference in the body weight of mice in each group 7 days after ovariectomy. As Figure 7 shown, within 8 weeks of drug administration in mice with postmenopausal osteoporosis model, the body weight growth rate of mice in the OVX group (OVX), OVX + CB2R KO group (CB2R KO), and OVX + ERαKO group (ERαKO) increased significantly; after 8 weeks of drug administration, the body weight of mice in the OVX group was significantly higher than that of mice in the Sham group (Sham), and the body weights of mice in the OVX + 0.2 mg / kg E2V group (E2V) and OVX + 10 mg / kg cimicifugic acid F group (10 mg / kg) were significantly lower than that of mice in the OVX group, indicating that using 10 mg / kg cimicifugic acid F could partially inhibit the excessive body weight growth of mice caused by ovariectomy; compared with the wild-type mice given in the OVX + 10 mg / kg cimicifugic acid F group, the body weights of mice in the OVX + 10 mg / kg cimicifugic acid F + ERαKO group (10 mg / kg + ERαKO) and OVX + 10 mg / kg cimicifugic acid F + CB2R KO group (10 mg / kg + CB2RKO) were significantly higher, masking the inhibitory effect of 10 mg / kg cimicifugic acid F on the body weight of mice after bilateral ovariectomy.

[0076] Uterine weight changes: As Figure 8 shown, compared with the Sham group, the uterine weight of mice in the OVX group was significantly reduced; compared with the OVX group, using E2V significantly increased the uterine weight of mice after bilateral ovariectomy, and using 10 mg / kg cimicifugic acid F had no significant effect on the uterine weight of mice after bilateral ovariectomy; compared with the OVX + 10 mg / kg cimicifugic acid F group, there was no significant change in the uterine weight of mice in the OVX + 10 mg / kg cimicifugic acid F + ERαKO group and OVX + 10 mg / kg cimicifugic acid F + CB2R KO group.

[0077] Changes in urinary calcium and collagen loss: As Figure 9As shown, compared with the Sham group, the contents of calcium (U-Ca) and deoxypyridinoline (U-DPD) in the urine of mice in the OVX group were significantly increased; compared with the OVX group, both E2V and 10 mg / kg ferulic acid F could significantly reduce the contents of calcium and deoxypyridinoline in the urine of mice after bilateral ovariectomy; compared with the OVX + 10 mg / kg ferulic acid F group, the contents of calcium and deoxypyridinoline in the urine of mice in the OVX + 10 mg / kg ferulic acid F + ERαKO group and the OVX + 10 mg / kg ferulic acid F + CB2R KO group were significantly increased, indicating that ferulic acid F targets CB2R and ERα to reduce calcium excretion and collagen degradation, thereby alleviating bone loss in mice after bilateral ovariectomy.

[0078] Biochemical indexes of bone metabolism: After ovariectomy of 4-month-old female mice, the estrogen level decreased significantly, and the body was in a state of high-turnover bone metabolism. Due to the longer bone formation process and shorter bone resorption process, serious bone mass loss occurred. As Figure 10 shown, compared with the Sham group, the levels of bone resorption biochemical markers (C-terminal peptide of type I collagen CTX-1, receptor activator of nuclear factor-κB ligand RANKL, and tartrate-resistant acid phosphatase TRAP) and bone formation parameters (N-terminal propeptide of type I procollagen PINP and osteocalcin OCN) in the serum of mice in the OVX group were significantly increased, while the level of osteoprotegerin OPG decreased. After oral administration of 10 mg / kg ferulic acid F and E2V, the increase in CTX-1, RANKL, TRAP, PINP, and OCN in the serum of mice after bilateral ovariectomy was inhibited, and the OPG in the serum of mice was significantly increased. The regulatory effect of 10 mg / kg ferulic acid F on the serum bone metabolism biochemical markers of mice with ERα gene knockout and CB2R gene knockout was partially masked, indicating that ferulic acid F targets CB2R and ERα to regulate the bone metabolism function of mice after bilateral ovariectomy.

[0079] Bone tissue microstructure: After ovariectomy, the bone microstructure of the femur of mice was significantly degraded. As Figure 12As shown, Micro-CT analysis results confirmed that bilateral ovariectomy could lead to trabecular bone mass reduction and bone microstructural degradation, and the treatment with E2V and 10 mg / kg of ferulic acid F improved the bone microstructure of the femurs of mice. Histomorphometric analysis showed that bilateral ovariectomy led to a significant increase in trabecular bone separation (Tb.Sp) and bone surface fraction (BS / BV), and a significant decrease in trabecular bone number (Tb.N), trabecular bone thickness (Tb.Th), bone volume fraction (BV / TV), and bone mineral density (BMD). After administration of ferulic acid F, the trabecular bone number, trabecular bone thickness, bone volume fraction, and bone mineral density of the femur tissues of mice increased, and the trabecular bone separation and bone surface fraction decreased. The protective effects of 10 mg / kg of ferulic acid F on the histomorphometry of the bone tissues of mice with ERα gene knockout and CB2R gene knockout were partially masked, indicating that ferulic acid F targeted CB2R and ERα to reduce bone loss and bone microstructural degradation. The trabecular bones of the untreated Sham group mice were dense and compact.

[0080] As Figure 13 shown, after ovariectomy, compared with the mice in the Sham group, the bone tissue voids of the mice in the OVX group increased significantly, and larger bone marrow cavities appeared. After 8 weeks of treatment with E2V, 5 mg / kg of ferulic acid F, and 10 mg / kg of ferulic acid F, the bone tissue pathological changes manifested by osteoporosis in the mice after bilateral ovariectomy were improved; compared with the OVX group, the trabecular bones of the mice in the OVX + 0.2 mg / kg E2V group, the OVX + 5 mg / kg ferulic acid F group, and the OVX + 10 mg / kg ferulic acid F group were more compact and thicker; ferulic acid F at different concentrations could significantly improve the increase in trabecular bone porosity in the mice after bilateral ovariectomy, and the protective effects of 10 mg / kg of ferulic acid F on the trabecular structure of the mice with ERα gene knockout and CB2R gene knockout were partially blocked.

[0081] Osteoclast activity: After ovariectomy, the number of osteoclasts in the bone tissues of mice will increase significantly, and bone resorption will be enhanced, resulting in bone metabolic imbalance and the occurrence of osteoporosis. As Figure 13 shown, as an important marker for identifying osteoclasts, tartrate-resistant acid phosphatase (TRAP) can directly observe the changes in the number of osteoclasts in bone tissues. The treatments with E2V, 5 mg / kg of ferulic acid F, and 10 mg / kg of ferulic acid F all significantly reduced the number of TRAP-positive OCs in the bone tissues of the mice after bilateral ovariectomy; for the mice with ERα gene knockout and CB2R gene knockout, the inhibitory effect of 10 mg / kg of ferulic acid F on the significant increase in the number of OCs was not observed, indicating that ferulic acid F targeted CB2R and ERα to inhibit the increase in OCs caused by OVX, thereby alleviating postmenopausal osteoporosis.

[0082] Osteoclast autophagy: After ovariectomy, the expressions of LC3B and TFEB in osteoclasts in mouse bone tissue increased significantly, and the autophagy function was enhanced, thus strengthening the bone resorption of OCs. As Figure 14 and Figure 15 shown, the treatments of E2V, 5 mg / kg ferulic acid F and 10 mg / kg ferulic acid F all significantly reduced the expression of LC3B in osteoclasts in the bone tissue of mice after bilateral ovariectomy; for the mice with ERα gene knockout and the mice with CB2R gene knockout, the inhibitory effect of 10 mg / kg ferulic acid F on the expression of LC3B in osteoclasts in bone tissue was not observed; at the same time, after the treatments of E2V and ferulic acid F, the expression of TFEB in osteoclasts in the bone tissue of mice after bilateral ovariectomy decreased significantly, and the effect of 10 mg / kg ferulic acid F was more significant. In the bone tissue of the mice with ERα gene knockout and the mice with CB2R gene knockout, the inhibitory effect of 10 mg / kg ferulic acid F on the expression of TFEB in osteoclasts was partially masked, indicating that ferulic acid F targets CB2R and ERα to reduce the expressions of LC3B and TFEB in osteoclasts, thereby inhibiting the autophagy function of osteoclasts.

[0083] 3.3 Senile osteoporosis model

[0084] Body weight change: Long-term injection of D-gal will cause a series of pathological changes similar to those observed during natural aging, including cognitive impairment, cardiovascular diseases and bone mass reduction, etc. As Figure 16 shown, after mice were continuously subcutaneously injected with 0.5 g / kg D-gal at the back of the neck for 8 weeks, the hormone levels in the body decreased and the weight gain slowed down; during the entire 8-week drug administration period, compared with the Control group, there was no significant difference in the body weight of the mice in the D-galactose group (D-gal); compared with the D-galactose group, there was no significant difference in the body weight of the mice in each group; after 8 weeks of drug administration, compared with the Control group, there was also no significant difference in the body weight of the mice in the D-galactose group. Compared with the D-galactose group, there was no significant difference in the body weight of the mice in the D-gal + 2.5 mg / kg ALN group (ALN) and the D-gal + 10 mg / kg ferulic acid F group (10 mg / kg), indicating that 10 mg / kg ferulic acid F had no significant effect on the body weight of aging mice caused by D-gal. Compared with the D-gal + 10 mg / kg ferulic acid F group, the body weight of the mice in the D-gal + 10 mg / kg ferulic acid F + ERαKO group (10 mg / kg + ERαKO) and the D-gal + 10 mg / kg ferulic acid F + CB2R KO group (10 mg / kg + CB2R KO) increased, but there was no significant difference.

[0085] Changes in urinary calcium and collagen loss: As Figure 17As shown, D-gal, as an aging inducer, increases bone calcium release or reduces calcium absorption, resulting in increased levels of calcium and deoxypyridinoline in urine; compared with the Control group, the levels of calcium and deoxypyridinoline in the urine of mice in the D-galactose group were significantly increased, indicating that the mice in the D-galactose group showed an increased state of bone resorption. Compared with the D-galactose group, ALN, 5 mg / kg linolenic acid F, and 10 mg / kg linolenic acid F could significantly reduce the levels of calcium and deoxypyridinoline in the urine of D-gal-aging mice; compared with the D-gal + 10 mg / kg linolenic acid F group, the levels of calcium and deoxypyridinoline in the urine of mice in the D-gal + 10 mg / kg linolenic acid F + ERαKO group and the D-gal + 10 mg / kg linolenic acid F + CB2R KO group were significantly increased, indicating that linolenic acid F targets CB2R and ERα to reduce collagen degradation and calcium ion loss, thereby reducing bone loss in D-gal-induced senile osteoporosis mice.

[0086] Biochemical indexes of bone metabolism: After long-term subcutaneous injection of D-gal in mice, it can promote bone resorption and calcium metabolism imbalance, making the body in a state of active bone turnover, resulting in bone mass loss. As Figure 18 shown, compared with the Control group, the levels of serum bone resorption biochemical markers (CTX-1, RANKL, and TRAP) and bone formation parameter (PINP) levels in mice in the D-galactose group were significantly increased, while the OPG level was decreased; after oral administration of 10 mg / kg linolenic acid F and ALN, the increase in CTX-1, RANKL, TRAP, PINP, and OCN in D-gal-induced aging mice was inhibited, and the OPG in the serum of mice was significantly increased; the regulatory effect of 10 mg / kg linolenic acid F on the bone metabolism biochemical markers of mice with ERα gene knockout and CB2R gene knockout was partially reversed, indicating that linolenic acid F targets CB2R and ERα to regulate bone formation and bone resorption in D-gal-induced senile osteoporosis mice.

[0087] Aging-related biochemical indexes: Superoxide dismutase SOD is an important antioxidant enzyme responsible for scavenging superoxide radicals in the body and protecting cells from oxidative damage. As Figure 19As shown, after treatment with D-gal during the aging process, the activities of superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), and catalase (CAT) decreased, while the level of malondialdehyde (MDA) increased, indicating a decline in antioxidant capacity and enhanced oxidative stress in D-gal-induced osteoporotic mice. After oral administration of 10 mg / kg ferulic acid F, the decrease in the activities of SOD, GSH-Px, and CAT induced by D-gal in aging mice was inhibited, and the concentration of MDA in the serum of aging mice was significantly reduced. The regulatory effect of 10 mg / kg ferulic acid F on the aging biochemical markers in mice with ERα gene knockout and CB2R gene knockout was partially reversed, indicating that ferulic acid F can not only regulate bone metabolism through CB2R and ERα, but also reduce the oxidative stress level induced by D-gal in the body and enhance its antioxidant function.

[0088] Bone microstructure: After long-term subcutaneous injection of D-gal, the calcium metabolism in aging mice was imbalanced, resulting in bone mass loss in the body and significant degradation of the bone microstructure of the mice. As Figure 20 shown, the 2D and 3D morphological maps of bone tissue analyzed by Micro-CT showed that after long-term induction by D-gal, the trabecular bone mass was significantly reduced and the bone microstructure was degraded. The treatment with ALN, 5 mg / kg and 10 mg / kg ferulic acid F improved the bone microstructure of the femurs of mice. Histomorphometric analysis of bone tissue showed that 8 weeks of subcutaneous injection of D-gal resulted in a significant decrease in Tb.N, Tb.Th, BV / TV, and BMD, and a significant increase in Tb.Sp and BS / BV. After oral administration of ALN, Tb.N, BV / TV, and BMD in the femoral tissue of mice increased, while Tb.Sp decreased. After administration of 5 mg / kg and 10 mg / kg ferulic acid F, Tb.N, Tb.Th, BV / TV, and BMD in the femoral tissue of mice could be significantly increased, and Tb.Sp and BS / BV were decreased. Ferulic acid F at different concentrations could significantly improve the bone tissue microstructure of D-gal-induced aging mice. The protective effect of 10 mg / kg ferulic acid F on the bone tissue microstructure of aging mice with CB2R gene knockout was partially masked. For aging mice with ERα gene knockout, the protective effect of 10 mg / kg ferulic acid F on the bone tissue microstructure could still be observed, except for BS / BV and Tb.Th.

[0089] In the Control group, the bone trabeculae of untreated mice were dense and compact. As Figure 21As shown, compared with the mice in the Control group, the bone tissue of the mice in the D-galactose group had a larger bone marrow cavity, a significant increase in voids, and sparse and slender trabecular bones; after 8 weeks of oral administration of ALN, 5 mg / kg and 10 mg / kg linolenic acid F, the pathological changes of D-gal-induced osteoporosis were improved; compared with the D-galactose group, the trabecular bones of the mice in the D-gal + 2.5 mg / kg ALN group (ALN), D-gal + 5 mg / kg linolenic acid F group (5 mg / kg), and D-gal + 10 mg / kg linolenic acid F group (10 mg / kg) were more compact and thicker; linolenic acid F at different concentrations could significantly improve the reduction in the number of trabecular bones and the increase in pores in D-gal-induced senescent mice, and the protective effect of 10 mg / kg linolenic acid F on trabecular bones, femoral new bone, and cartilage formation in senescent mice with the ERα gene knocked out and the CB2R gene knocked out was partially masked.

[0090] Osteoclast activity: After long-term subcutaneous injection of D-gal, the number of osteoclasts in the bone tissue of mice will increase significantly, and bone resorption will be enhanced, thus causing bone loss and leading to the occurrence of osteoporosis. As Figure 22 shown, TRAP staining can visually reflect the change in the number of osteoclasts in bone tissue; osteoclasts are stained red, and compared with the Control group, the number and area of osteoclasts in the femurs of the mice in the D-galactose group increased significantly. After treatment with ALN, 5 mg / kg linolenic acid F, and 10 mg / kg linolenic acid F, the number of TRAP-positive OCs in the bone tissue of D-gal-induced osteoporotic mice decreased significantly; for senescent mice with the ERα gene knocked out and the CB2R gene knocked out, the inhibitory effect of 10 mg / kg linolenic acid F on the significantly increased number of osteoclasts was partially masked, indicating that linolenic acid F targets CB2R and ERα to inhibit the increase in OCs caused by senescence and the bone resorption effect, thereby alleviating bone loss.

[0091] Autophagy of osteoclasts: The expression of LC3B and TFEB in osteoclasts of the bone tissue of D-gal-induced senescent osteoporotic mice increased significantly, the autophagy function was enhanced, and the bone resorption effect of osteoclasts was significantly enhanced. As Figure 23As shown, the treatments with ALN, 5 mg / kg linolenic acid F, and 10 mg / kg linolenic acid F all significantly reduced the expression of LC3B in osteoclasts of bone tissue in D-gal-induced mice; for senile mice with ERα gene knockout and CB2R gene knockout, the inhibitory effect of 10 mg / kg linolenic acid F on the expression of LC3B in osteoclasts was not observed; after ALN and linolenic acid F treatments, the expression of TFEB in osteoclasts of bone tissue in D-gal-induced osteoporotic mice decreased significantly. For senile mice with ERα gene knockout and CB2R gene knockout, the inhibitory effect of 10 mg / kg linolenic acid F on the expression of TFEB in osteoclasts was partially masked.

[0092] IV. Conclusions

[0093] (1) Linolenic acid F activates the CB2R receptor in HEK293-CB2R cells in a concentration-dependent manner and inhibits the cAMP elevation induced by Forskolin, with an IC 50 value of 100.8 nM; it increases the expression of intracellular ERα, with an EC50 value of 12.89 nM.

[0094] (2) Linolenic acid F (10 mg / kg) significantly improved the bone microstructure of mice after bilateral ovariectomy and D-gal-induced senile osteoporotic mice (such as an increase in bone area fraction BV / TV by 23.7% and an increase in trabecular bone thickness Tb.Th by 22.1%, p < 0.001).

[0095] (3) Linolenic acid F inhibits osteoclast differentiation (a 61% reduction in TRAP-positive cells, p < 0.001) and autophagic activity (a 51% decrease in the expression efficiency of LC3B, p < 0.01) through the dual targets of CB2R and ERα.

[0096] (4) In the gene knockout OVX model, the retention rates of the bone protection effect in the ERαKO group were 82% and in the CB2R KO group were 59%, demonstrating the bone protection effect of linolenic acid F targeting dual targets.

[0097] (5) Linolenic acid F can alleviate the excessive weight gain of mice after bilateral ovariectomy, and has no estrogen-like side effects (no change in uterine weight), with better safety than traditional hormone therapy.

[0098] Through research, it is found in the present invention that linolenic acid F can activate CB2R and ERα, promote the interaction of dual targets, target the dual targets of CB2R and ERα to inhibit the formation, autophagy, and bone resorption function of osteoclasts, thereby exerting an anti-osteoporotic effect, providing a theoretical basis for the development of innovative drugs with both bone protection and metabolic regulation functions.

[0099] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention shall be included within the scope of protection of the present invention.

Claims

1. A CB2R / ERα dual-target agonist, characterized in that, The CB2R / ERα dual-target agonist is cimicifugic acid F, and its structure is shown as follows:

2. Use of the CB2R / ERα dual-target agonist according to claim 1 in the preparation of a medicament for treating osteoporosis.

3. The application according to claim 2, wherein The osteoporosis includes postmenopausal osteoporosis and senile osteoporosis.

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