Polypeptide for preventing and / or treating osteoporosis and application thereof
By using a combination of green LED light source and GnIH peptide, GnIH release is promoted and osteoclast activity is inhibited, thus solving the problems of treatment side effects and safety hazards for osteoporosis and providing a safe and effective treatment solution.
Patent Information
- Application Number
- CN202410685429.2
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-05-30
- Publication Date
- 2025-12-02
AI Technical Summary
Existing osteoporosis medications have serious side effects with long-term use, and traditional physical therapy methods pose safety risks. There is an urgent need to develop safer and more effective treatment methods.
Using a green LED light source with a wavelength of 492-577nm combined with the HPG axis-related upstream key reproductive hormone GnIH peptide, the synthesis and release of GnIH in the hypothalamus is promoted, osteoclast activity is inhibited, and osteoporosis is alleviated.
It achieves safe, time-saving, and effective prevention and treatment of osteoporosis, reduces drug side effects and the risks of physical therapy, and increases bone density and inhibits osteoclast activity.
Smart Images
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Abstract
Description
Technical Field
[0001] This invention relates to therapeutic drugs and methods for treating and preventing osteoporosis in mice, and particularly to a polypeptide for the prevention and / or treatment of osteoporosis and its application. Background Technology
[0003] Osteoporosis (OP) is a common age-related disease that leads to decreased bone mass, deterioration of bone microstructure, and increased bone fragility, causing osteoporotic fractures and significantly increasing the probability of disability and death. While many medications are available for treating osteoporosis, long-term use can lead to various serious side effects. For example, long-term use of bisphosphonates increases the risk of fractures; long-term use of hormonal drugs can lead to tumors, thrombosis, and other diseases; denosumab treatment is expensive, and long-term use can cause severe joint pain and other complications. Furthermore, while osteoporosis treatment guidelines recommend physical therapy, such as ultraviolet and infrared light therapy, prolonged ultraviolet light exposure can induce cellular aging and gene mutations, increasing the risk of skin cancer; infrared therapy also carries the risk of high-temperature burns and eye damage, posing significant safety concerns. Therefore, it is essential to identify novel drug targets for treating osteoporosis and develop safer and more effective drugs and treatments.
[0004] GnIH (gonadotropin-inhibiting hormone), a key upstream reproductive hormone associated with the hypothalamic-pituitary-gonadal axis (HPG), has been shown to play an important role in regulating reproductive development, and light exposure can promote the synthesis and release of GnIH. Furthermore, some studies suggest that elucidating the function of key upstream reproductive hormones related to the HPG axis may provide new drugs and methods for treating orthopedic diseases; however, whether these can be applied to the treatment of orthopedic diseases remains unclear and requires further investigation. Summary of the Invention
[0005] To address the problems of long-term medication and severe side effects in treating osteoporosis, and the numerous hazards associated with traditional physical therapy, this invention provides a polypeptide and its applications for the treatment and prevention of osteoporosis. This invention is the first to propose that the upstream key reproductive hormone GnIH, associated with the HPG axis, has a therapeutic function for osteoporosis. Based on targeted GnIH release conditions and physical therapy, treatment of mice with osteoporosis using a green LED light source with a wavelength of 492-577 nm promotes the synthesis and release of GnIH in the hypothalamus, thus treating osteoporosis. Furthermore, treatment of subjects with a green LED light source with a wavelength of 492-577 nm can alleviate serum bone resorption marker levels. This invention offers advantages such as greater safety, cost-effectiveness, and time-saving, facilitating the clinical prevention and / or treatment of osteoporosis.
[0006] This invention proposes a polypeptide for the prevention and / or treatment of osteoporosis, wherein the polypeptide is a gonadotropin-inhibiting hormone GnIH polypeptide with the sequence: Phe-Pro-Ser-Leu-Pro-Gln-Arg-Phe-NH2 (SEQ ID NO.1).
[0007] The present invention also proposes the use of the described polypeptide in the preparation of medicaments for the prevention and / or treatment of osteoporosis.
[0008] In this invention, the drug is a drug for rescuing bone loss;
[0009] In this invention, the drug is a drug that inhibits the activity of osteoclasts in vivo;
[0010] In this invention, the drug is a drug that inhibits osteoclast formation;
[0011] In this invention, the drug can be used alone or in combination with green light source illumination.
[0012] In this application, an LED green light source is selected for phototherapy, with the wavelength of the green light being 492-577nm.
[0013] In this application, green LED light sources are used to treat osteoporotic mice with light irradiation. The wavelength of the green light is 492-577nm, and the treatment time needs to be at least 30 days.
[0014] In this application, healthy adult subjects undergo green light therapy with a wavelength of 492-577nm, and the treatment time needs to be at least 5 days.
[0015] The present invention also proposes a nucleotide encoding a polypeptide as described above, wherein the nucleotide encoding the polypeptide comprises one of the following:
[0016] (a) A nucleotide encoding a polypeptide having the amino acid sequence described above;
[0017] (b) A nucleotide complementary to the nucleotide described in (a);
[0018] (c) A nucleotide that has ≥75% identity with the nucleotide described in (a) or (b).
[0019] The present invention also proposes an expression vector comprising the nucleotides described above.
[0020] The present invention also proposes a host cell comprising the expression vector described above.
[0021] The present invention also proposes a drug / drug composition comprising the polypeptide as described above.
[0022] In this invention, the drug / drug composition can be used alone.
[0023] In this invention, the drug / drug composition further includes excipients, diluents, adjuvants, mediators, or combinations thereof.
[0024] The present invention also provides a method for treating osteoporosis by administering the polypeptide, or drug / drug composition as described above, to a subject individual, or by administering green light therapy to the subject individual.
[0025] In the method described, the treatment method uses an LED green light source with a wavelength of 492-577nm.
[0026] In this application, green LED light sources are used to treat osteoporotic mice with light irradiation. The wavelength of the green light is 492-577nm, and the treatment time needs to be at least 30 days.
[0027] In this application, healthy adult subjects undergo green light therapy with a wavelength of 492-577nm, and the treatment time needs to be at least 5 days.
[0028] The polypeptide was dissolved in PBS and stored at -80 to 0°C. The daily treatment dose for osteoporotic mice was 0.08-0.12 mg / kg for 20-40 days.
[0029] In one specific implementation scheme, the experimental protocol for treating osteoporosis in mice proposed by this invention is as follows:
[0030] (1) Obtaining osteoporosis mice: Osteoporosis-stricken mice were obtained by ovariectomy (OVX).
[0031] a. Ovariectomy (OVX) of mice: Female C57BL / 6J mice aged 80-90 days were used. Each mouse was anesthetized with 0.4-0.6 ml of anesthetic (each 250 ml of 1X PBS contains 3.10-3.14 g of 2,2,2-tribromoethanol and 5.8-6.2 ml of tert-amyl alcohol, and incubated overnight in a water bath at 58-62 degrees Celsius). Both ovaries were removed using sterile surgical instruments. After suturing the wounds, the mice were placed on a heating plate at 35-39 degrees Celsius until they regained consciousness and were then transferred to their rearing cages.
[0032] b. Animal husbandry and handling were carried out in the SPF animal room. All mice were kept under complete dark light cycles (white light level of 200 lux) and constant temperature and humidity levels (20-26°C) for 12 hours.
[0033] (2) Treatment of OVX mice with GnIH peptide:
[0034] a. One week after ovariectomy (OVX) surgery, osteoporotic mice were treated daily with GnIH peptide (dissolved in PBS).
[0035] b. The administration method is intraperitoneal injection, the dosage is 0.08-0.12 mg / kg, and the treatment time is 20-40 days.
[0036] (3) Green light therapy was used to treat OVX mice with green LED light source:
[0037] a. One week after the mice underwent OVX surgery, they were given light therapy during the daytime each day.
[0038] b. A green LED light source is fixed to the top of a specially designed cabinet. OVX mice receive green LED light inside the cabinet while having unrestricted access to food and water. The wavelength of the green light is 492-577nm.
[0039] c. OVX mice that did not receive green light therapy were exposed to normal ambient light intensity of 190-210 lux.
[0040] d. After daily green light therapy, the mice were then returned to their standard animal housing.
[0041] The methods for detecting femoral bone mass and bone mineral density in OVX mice that received green light therapy are as follows:
[0042] a. OVX mice treated with GnIH peptides for 20-40 days, and OVX mice treated continuously for at least 30 days and exposed to green light during the day, were euthanized by carbon dioxide asphyxiation. The mice were sprayed with 75% alcohol 3-7 times. Using sterile surgical scissors, the right leg of the mouse was completely separated from the femoral head, ensuring the integrity of the femur was not damaged. Excess skin and muscle tissue were removed, leaving only the femur.
[0043] b. Fix the femoral tissue with a 3.8-4.2% paraformaldehyde solution for 22-26 hours, then replace with a fresh 3.8-4.2% paraformaldehyde solution and continue fixing for another 22-26 hours. The entire process should be performed in a 4°C refrigerator.
[0044] c. Discard the paraformaldehyde solution, rinse with running water for 18-22 minutes, and then transfer to 75% alcohol for storage for 22-26 hours.
[0045] d. Mouse femoral tissue was scanned using Bruker's Mic-CT scanner at a resolution of 9 μm. After scanning and analysis, the images were processed using the accompanying image processing tools, and longitudinal sections of the femur were plotted. The locations of growth plates were identified, and the corresponding images were marked. Images 98-102 were selected from the original sequence, and bone mineral density and bone mass-related parameters of the reconstructed images (98-102) were analyzed. Images of the cancellous bone were then plotted.
[0046] e. Use GraphPad Prism 8 plotting software to plot histograms of bone mass-related parameters for the sham group (sham group), OVX mouse group, OVX mouse group after GnIH peptide treatment, and OVX mouse group after green light irradiation treatment.
[0047] The method for detecting osteoclast activity in the femoral bone of OVX mice treated with GnIH peptide or green light is as follows:
[0048] a. After the CT analysis data is completed, treat the femoral tissue with 0.5M EDTA (decalcification solution) in a refrigerator shaker at 4℃ for 18-22 days.
[0049] Environmental conditions for processing The solution being treated Processing time room temperature 75% ethanol 0.8-1.2h room temperature 85% ethanol 0.8-1.2h room temperature 95% ethanol 0.8-1.2h 4℃ refrigerator 100% Anhydrous Ethanol 10-14h room temperature 100% anhydrous ethanol:xylene (1:1) 30-40min room temperature xylene 30-40min room temperature xylene 30-40min 65℃ water bath paraffin 1-2h 65℃ water bath paraffin 10-14h 65℃ water bath paraffin 1.5-2.5h
[0050] b. After waiting for further processing, the femoral tissue is embedded in paraffin. The processing procedure is shown in the table below. The embedded tissue is then sectioned in paraffin to a thickness of 5-7 μm.
[0051] c. The obtained paraffin sections of mouse femoral tissue were first treated on a heating plate at 63-67℃ for 4-6 minutes, and then the tissue was dewaxed according to the table below.
[0052]
[0053]
[0054] d. Treat tissue sections with 0.1% Triton X-100 PBS solution for 8-12 minutes, followed by TRAP staining using a TRAP staining kit (Sigma-Aldrich). The entire process is performed in an incubator at 35-39 degrees Celsius for 40-50 minutes. Then, treat with methyl green solution (Sigma-Aldrich) for 3-7 seconds, wash with PBS, air dry, and mount with resin. Observe and photograph under a microscope. Figure 3 As shown.
[0055] e. Observe and photograph under a microscope.
[0056] f. Quantitative statistical analysis of TRAP staining in the femur was performed using the OsteoMeasure Analysis System (Osteometrics) to analyze osteoclast-related activities in the mouse femur.
[0057] j. Use GraphPad Prism 8 plotting software to plot histograms of osteoclast activity values in the sham-operated group, the OVX mouse group, the OVX mouse group after GnIH peptide treatment, and the OVX mouse group after green light irradiation.
[0058] The method for detecting GnIH in the serum of OVX mice that received green light therapy is as follows:
[0059] a. Blood is drawn from the orbital venous plexus.
[0060] b. Centrifuge the collected venous blood samples at 800-1200g / 3-7min.
[0061] c. Only collect serum, ensuring that the lower plasma layer is not drawn during the collection process.
[0062] d. The OD value of the bone resorption marker GnIH in mouse serum was analyzed using an enzyme-linked immunosorbent assay kit (Kanglang), and the corresponding value was calculated based on the standard curve.
[0063] e. Use GraphPad Prism 8 plotting software to draw histograms of GnIH values before and after green light therapy.
[0064] In one specific implementation scheme, the present invention proposes the following application method for preventing osteoporosis:
[0065] a. Healthy adult subjects underwent green light therapy.
[0066] b. In human green light therapy, subjects use LED green light sources with wavelengths between 492-577nm for phototherapy.
[0067] c. To ensure personal comfort, subjects can flexibly adjust the distance between the light source and their eyes within a specified range. There are no other light sources in the subject's room besides the treatment environment. During phototherapy, subjects are specifically instructed to remain awake and avoid falling asleep. Each subject wears a hospital gown made of the same material while receiving green light irradiation. The applicant encourages them to engage in activities that do not require additional light sources, such as talking and listening to music. Activities involving external light sources, such as watching television or using screen devices, are prohibited. To optimize the treatment process, subjects are advised to keep their eyes open, maintain a normal blinking frequency, and avoid staring directly at the light source.
[0068] d. Peripheral venous blood samples were collected from the subjects before and after green light therapy.
[0069] The methods for detecting serum bone resorption markers in people receiving green light therapy are as follows:
[0070] a. Centrifuge the collected venous blood samples at 800-1200g / 3-7min.
[0071] b. Only collect serum, ensuring that the lower plasma layer is not drawn during the collection process.
[0072] c. Use an enzyme-linked immunosorbent assay kit (Jingmei) to analyze the OD values of bone resorption markers TRACP and CTX in human serum, and calculate the corresponding values based on the standard curve.
[0073] d. Use GraphPad Prism 8 plotting software to draw histograms of TRACP and CTX values before and after green light therapy.
[0074] The beneficial effects of this invention include: This invention proposes a polypeptide for the prevention and / or treatment of osteoporosis. This invention achieves the purpose of treating osteoporosis in mice, rescuing bone loss, and inhibiting osteoclast activity in vivo by using a gonadotropin-inhibiting hormone (GnIH) polypeptide. In addition to direct treatment with the GnIH polypeptide, this invention also utilizes green light therapy to promote the synthesis and release of GnIH in the hypothalamus, achieving the purpose of preventing and / or treating osteoporosis. This invention not only provides a new pharmaceutical basis for the treatment of osteoporosis, but also, compared with traditional osteoporosis treatment methods, the green light therapy proposed in this invention has the advantages of being simpler, safer, more effective, and time-saving, providing a new and feasible method for the clinical prevention and treatment of osteoporosis. Attached Figure Description
[0075] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.
[0076] In the attached diagram, in the experiment of treating osteoporosis with GnIH peptide, mice in the sham-operated group were labeled as Sham+PBS, mice in the OVX group were labeled as OVX+PBS, and mice in the OVX group treated with GnIH peptide were labeled as OVX+GnIH.
[0077] In the green light therapy experiment, OVX mice were designated as OVX. They were treated with 400 lux green light for 8 hours daily. After 60 days of treatment, the OVX mice were designated as OVX+Green light. Subjects before green light therapy were designated as Ctrl, and subjects after green light therapy were designated as Green light.
[0078] Figure 1 Representative images of the fallopian tubes of Sham mice, OVX mice, and OVX mice treated with GnIH peptide.
[0079] Figure 2 These are representative Micro-CT reconstruction images of the femur and trabeculae in Sham mice, OVX mice, and OVX mice treated with GnIH peptide. The top image represents the femur, and the bottom image represents the trabeculae.
[0080] Figure 3 Trabecular bone parameters of the femur in Sham mice, OVX mice, and OVX mice treated with GnIH peptides are given. BMD is bone mineral density, BV / TV is bone volume fraction, BS / TV is bone area fraction, TB.TH is trabecular thickness, TB.N is the number of trabeculae, and TB.SP is the width between trabeculae.
[0081] Figure 4 This image represents TRAP enzyme activity staining in paraffin sections of the femur from Sham mice, OVX mice, and OVX mice treated with GnIH peptide. The purple-red signal represents osteoclast activity.
[0082] Figure 5 This section presents the statistical results of TRAP enzyme activity staining in paraffin sections of the femurs of Sham mice, OVX mice, and OVX mice treated with GnIH peptide. N.Oc / B.Pm represents the number of osteoclasts, Oc.S / BS represents the osteoclast area, and ES / BS represents the bone erosion area.
[0083] Figure 6These are representative Micro-CT reconstruction images of the femur and trabeculae in OVX mice and OVX mice after green light therapy. The top image is a representative image of the femur, and the bottom image is a representative image of the trabeculae.
[0084] Figure 7 The data represent the trabecular bone parameters of the femur in OVX mice and OVX mice after green light therapy. BMD is bone mineral density, BV / TV is bone volume fraction, BS / TV is bone area fraction, TB.TH is trabecular thickness, TB.N is the number of trabeculae, and TB.SP is the width between trabeculae.
[0085] Figure 8 This image shows representative staining of TRAP enzyme activity in paraffin sections of the femur from OVX mice and OVX mice after green light therapy. The purple-red signal represents osteoclast activity.
[0086] Figure 9 The results of TRAP enzyme activity staining in paraffin sections of the femur from OVX mice and OVX mice after green light phototherapy are statistically analyzed. N.Oc / B.Pm represents the number of osteoclasts, Oc.S / BS represents the osteoclast area, and ES / BS represents the bone erosion area.
[0087] Figure 10 The concentration of GnIH in the serum of OVX mice and OVX mice after green light therapy.
[0088] Figure 11 The study investigated the changes in serum levels of TRACP and CTX, markers of bone resorption, in subjects before (Ctrl) and after (Green light) phototherapy. Detailed Implementation
[0089] The present invention will be further described in detail below with reference to the specific embodiments and accompanying drawings. Except for the contents specifically mentioned below, the processes, conditions, and experimental methods for implementing the present invention are all common knowledge and general knowledge in the art, and the present invention does not have any particular limitations.
[0090] Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. The following description of at least one exemplary embodiment is merely illustrative and is in no way intended to limit the invention or its application or use.
[0091] In all examples shown and discussed herein, any specific values should be interpreted as merely exemplary and not as limitations. Therefore, other examples of exemplary embodiments may have different values.
[0092] Unless otherwise specified, the experimental materials used in the examples are all conventional biochemical reagents and routine procedures.
[0093] Example 1
[0094] (1) Obtaining osteoporosis mice: Osteoporosis-affected mice were obtained through ovariectomy (OVX).
[0095] a. Ovariectomy (OVX) of mice: 84-day-old female C57BL / 6J mice were used. Each mouse was anesthetized with 0.5 ml of anesthetic (each 250 ml of 1X PBS contained 3.12 g of 2,2,2-tribromoethanol and 6 ml of tert-amyl alcohol, and was incubated overnight in a 60°C water bath). The ovaries were removed bilaterally using sterile surgical instruments. After the wounds were sutured, the mice were placed on a 37°C heating plate until they regained consciousness and were then transferred to their rearing cages.
[0096] b. Animal husbandry and handling were carried out in the SPF animal room. All mice were kept under complete dark light cycles (white light level of 200 lux) and constant temperature and humidity levels (20-26°C) for 12 hours.
[0097] (2) Treatment of OVX mice with GnIH peptide:
[0098] a. One week after ovariectomy (OVX) surgery, osteoporotic mice were treated daily with GnIH peptide (dissolved in PBS).
[0099] b. The administration method is intraperitoneal injection, the dosage is 0.1 mg / kg, and the treatment period is 30 days.
[0100] (3) Green light therapy was used to treat OVX mice with green LED light source:
[0101] a. One week after the mice underwent OVX surgery, they were given green light therapy from 8:00 AM to 4:00 PM every day for 8 hours, for a total of 60 days.
[0102] b. A green LED light source is fixed to the top of a specially designed cabinet. OVX mice receive green LED light inside the cabinet while having unrestricted access to food and water. The green light intensity is 400 lux, and the wavelength is 520-525 nm.
[0103] c. OVX mice that did not receive green light therapy were exposed to normal ambient light intensity of 200 lux.
[0104] d. After daily green light therapy, the mice were then returned to their standard animal housing.
[0105] The methods for detecting femoral bone mass and bone mineral density in OVX mice that received green light therapy are as follows:
[0106] a. OVX mice that received GnIH peptide treatment for 30 days (e.g. Figure 1 As shown, the OVX surgery was successful. Mice in the OVX+PBS group (with significant fallopian tube atrophy) and mice treated with green light (520-525nm, 400lux) for 8 hours over 60 consecutive days were euthanized by carbon dioxide asphyxiation. The mice were sprayed with 75% alcohol five times, and their right legs were completely separated from the femoral head using sterile surgical scissors, ensuring the integrity of the femur was not compromised. Excess skin and muscle tissue were removed, leaving only the femur.
[0107] b. After fixing the femoral tissue with a 4% paraformaldehyde solution for 24 hours, replace with a fresh 4% paraformaldehyde solution and continue fixing for another 24 hours. The entire process should be carried out in a refrigerator at 4°C.
[0108] c. Discard the paraformaldehyde solution, rinse with running water for 20 minutes, and then transfer to 75% alcohol for storage for 24 hours.
[0109] d. Mouse femoral tissue was scanned using Bruker's Mic-CT scanner at a resolution of 9 μm. After scanning and analysis, the images were processed using the accompanying image processing tools, and longitudinal sections of the femur were plotted. This invention locates the growth plates, marks the corresponding images, and selects 100 images from the original sequence. Based on these 100 reconstructed images, bone mineral density and bone mass-related parameters of the cancellous bone are analyzed, and images of the cancellous bone are plotted. (See attached image.) Figure 2 and Figure 6 As shown, the transverse sections and trabecular bone images of mice reconstructed by Mic-CT analysis revealed that OVX mice treated with GnIH peptides and those treated with green light had more bone mass.
[0110] e. Using GraphPad Prism 8 plotting software, plot histograms of osteoclast activity values in the sham group (sham group), OVX mouse group, OVX mouse group treated with GnIH peptide, and OVX mouse group treated with green light. (See attached image.) Figure 3 and Figure 7 As shown in the figure. Statistical results showed that, compared with the control group OVX mice, the OVX mice treated with GnIH peptide and green light therapy had higher values of BMD, BV / TV, BS / TV, TB.TH, and TB.N, and lower values of TB.SP. This indicates that OVX mice treated with GnIH peptide or green light therapy had higher bone mass and bone density. GnIH peptide treatment or green light therapy can treat osteoporosis in OVX mice and salvage bone loss.
[0111] The method for detecting osteoclast activity in the femoral bone of OVX mice treated with GnIH peptide or green light is as follows:
[0112] a. After the CT analysis data is completed, treat the femoral tissue with 0.5M EDTA (decalcification solution) for 20 days in a refrigerator shaker at 4℃.
[0113]
[0114] b. After waiting for further processing, the femoral tissue is embedded in paraffin. The processing procedure is shown in the table below. The embedded tissue is then sectioned in paraffin to a thickness of 6 μm.
[0115] c. The obtained paraffin sections of mouse femoral tissue were first treated on a heating plate at 65°C for 5 minutes, and then the tissue was dewaxed according to the table below.
[0116] Environmental conditions for processing The solution being treated Processing time room temperature xylene 7min room temperature xylene 7min room temperature 100% anhydrous ethanol:xylene (1:1) 7min room temperature 100% Anhydrous Ethanol 7min room temperature 95% ethanol 7min room temperature 85% ethanol 7min room temperature 75% ethanol 7min room temperature H2O 7min
[0117] d. Treat tissue sections with 0.1% Triton X-100 PBS solution for 10 minutes, then perform TRAP staining using a TRAP staining kit (Sigma-Aldrich). The entire process is carried out in a 37°C incubator for 40-50 minutes. After treatment with methyl green solution (Sigma-Aldrich) for 5 seconds, wash with PBS, air dry with a hairdryer, and then mount with resin.
[0118] e. Observe and photograph under a microscope. For example... Figure 4 and Figure 8 As shown in the figure. TRAP staining results showed that the TRAP staining signal in the femur of OVX mice treated with GnIH peptide and green light therapy, respectively, was significantly lower than that in the control OVX group mice.
[0119] f. Quantitative statistical analysis of TRAP staining in the femur was performed using the OsteoMeasure Analysis System (Osteometrics) to analyze osteoclast-related activities in the mouse femur.
[0120] j. Use GraphPad Prism 8 plotting software to create histograms of osteoclast activity in OVX mice and OVX mice after green light therapy. For example... Figure 5 and Figure 9 As shown in the figure. Statistical results showed that, compared with the control group OVX mice, the OVX mice treated with GnIH peptide and green light irradiation had significantly lower N.Oc / B.Pm, Oc.S / BS, and ES / BS values, indicating that the osteoclast activity in the femur of OVX mice treated with GnIH peptide and green light irradiation was lower.
[0121] The method for detecting GnIH in the serum of OVX mice that received green light therapy is as follows:
[0122] a. Blood is drawn from the orbital venous plexus.
[0123] b. Centrifuge the collected venous blood samples at 1000g / 5min.
[0124] c. Only collect serum, ensuring that the lower plasma layer is not drawn during the collection process.
[0125] d. The OD value of the bone resorption marker GnIH in mouse serum was analyzed using an enzyme-linked immunosorbent assay kit (Kanglang), and the corresponding value was calculated based on the standard curve.
[0126] e. Use GraphPad Prism 8 plotting software to draw histograms of GnIH values before and after green light therapy. For example... Figure 10 As shown, after treatment with green light, the secretion of GnIH in the serum of mice increased.
[0127] Example 2: An application method for preventing osteoporosis is as follows:
[0128] a. Individuals receiving green light exposure should be between 20 and 40 years old, in good health, without visual impairment or eye diseases, have no history of taking hormones or osteoporosis medications within the past 3 months, have no chronic diseases or other medical history, and have normal blood routine tests.
[0129] b. In human green light therapy, subjects undergo phototherapy using LED green light sources with wavelengths between 520-525nm. Treatment is conducted daily from 9:00 AM to 11:00 AM in a room designated by the subject. The distance between the LED green light source and the subject's eyes is 1-2 meters, and the light intensity is 400-1000 lux. The treatment cycle is one week.
[0130] c. To ensure personal comfort, subjects can flexibly adjust the distance between the light source and their eyes within a specified range. There are no other light sources in the subject's room besides the treatment environment. During phototherapy, subjects are specifically instructed to remain awake and avoid falling asleep. Each subject wears a hospital gown made of the same material while receiving green light irradiation. This invention encourages them to engage in activities that do not require additional light sources, such as talking and listening to music. Activities involving external light sources, such as watching television or using screen devices, are prohibited. To optimize the treatment process, subjects are advised to keep their eyes open, maintain a normal blinking frequency, and avoid staring directly at the light source.
[0131] d. Peripheral venous blood samples were collected from the subjects before and one week after green light therapy.
[0132] The methods for detecting serum bone resorption markers in people receiving green light therapy are as follows:
[0133] a. Centrifuge the collected venous blood samples at 1000g / 5min.
[0134] b. Only collect serum, ensuring that the lower plasma layer is not drawn during the collection process.
[0135] c. Use an enzyme-linked immunosorbent assay kit (Jingmei) to analyze the OD values of bone resorption markers TRACP and CTX in human serum, and calculate the corresponding values based on the standard curve.
[0136] d. Use GraphPad Prism 8 plotting software to draw histograms of TRACP and CTX values before and after green light therapy. For example... Figure 11 As shown in the figure, the serum bone resorption marker levels of the subjects were compared before and after treatment. Statistical results showed that the serum bone resorption marker (TRACP, CTX) levels of the subjects before green light phototherapy were significantly higher than those after green light phototherapy, indicating that green light phototherapy can alleviate the levels of bone resorption markers in human serum.
[0137] The above examples and results show that there was no significant difference in bone mass between the GnIH peptide treatment group and the sham group, indicating that the present invention can achieve the purpose of treating osteoporosis in mice, rescuing bone loss, and inhibiting osteoclast activity in vivo by using gonadotropin-inhibiting hormone (GnIH) peptide.
[0138] Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used in this specification is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.
[0139] As used in this invention, the term "comprising" is an open-ended expression, meaning it includes the contents specified in this invention but does not exclude other aspects.
[0140] As used in this invention, the term "and / or" includes any one or more of the related listed items and all combinations thereof.
[0141] The scope of protection of this invention is not limited to the above embodiments. Any variations and advantages that can be conceived by those skilled in the art without departing from the spirit and scope of the inventive concept are included in this invention and are protected by the appended claims.
Claims
1. Application of GnIH peptide in the preparation of drugs for the prevention and / or treatment of osteoporosis.
2. The application as described in claim 1, characterized in that, The sequence of the GnIH polypeptide is: Phe-Pro-Ser-Leu-Pro-Gln-Arg-Phe-NH2.
3. The application as described in claim 1, characterized in that, The drug in question is a medication that helps to recover from bone loss.
4. The application as described in claim 1, characterized in that, The drug is a drug that inhibits the activity of osteoclasts in the body.
5. The application as described in claim 1, characterized in that, The drug can be used alone or in combination with green light source illumination.
6. The application as described in claim 5, characterized in that, The wavelength of the green light is 492-577nm.
7. The application as described in claim 5, characterized in that, The green light source is an LED light source.
8. A method for treating osteoporosis, characterized in that, The subject is administered the GnIH polypeptide as described in claim 1, or is exposed to green light.
9. The method as described in claim 8, characterized in that, The dosage of the polypeptide is 0.08-0.12 mg / kg / day; and / or the wavelength of the green light is 492-577 nm.