Shh pathway regulation of biological rhythms and related applications

Abstract

The present application relates to the field of biotechnology, in particular to Shh pathway regulation biological rhythm and related application thereof. Specifically, the present application provides application of Hedgehog pathway inhibitor and SMO inhibitor in regulating biological rhythm and treating biological rhythm related diseases; preferably, the Hedgehog pathway inhibitor comprises PF-5274857, Mebendazole, HPI-4, SANT-1, Taladegib, Glasdegib, Cyclopamine, Itraconazole, GANT61, JK184, Robotnikinin, Vismodegib, Purmorphamine, Sonidegib Phoshate.

Description

[0001] This application is a divisional application of the invention patent application with application number 202211162119X, application date September 23, 2022, entitled "Shh pathway regulation of biological rhythms and related applications". Technical Field

[0002] This invention relates to the field of biotechnology, specifically to the regulation of biological rhythms by the Shh pathway, and its potential application in the treatment of rhythm-related diseases. Background Technology

[0003] Biological behaviors such as sleep, wakefulness, and eating, as well as various physiological, biochemical, and metabolic processes, follow a rhythmic pattern of approximately 24 hours, commonly known as the biological clock. The biological clock is closely related to normal human physiological functions. Studies have shown that biological rhythms are closely related to numerous physiological indicators, including sleep, diet, cognition, mood, behavior, and metabolism. They are crucial for the coordinated and orderly functioning of the entire organism and its adaptation to the environment. Disruption of normal biological rhythms can lead to abnormalities in the body's psychological and physiological functions, as well as dysfunction of various tissues and organs. For humans, changes in the biological clock can trigger many problems, such as sleep disorders, depression, metabolic disorders, aging, blood diseases, diabetes, and obesity.

[0004] The suprachiasmatic nucleus (SCN) is a small region located on either side of the third ventricle in the hypothalamus of the brain, directly above the optic chiasm. As the central system of the biological clock, the SCN is primarily responsible for regulating the body's circadian rhythm. It directly receives light signals transmitted from the upstream optic chiasm and influences the rhythms of other organs, playing a coordinating role in the central nervous system. The SCN consists of approximately 20,000 neurons, and the coupling between these neurons maintains the consistency of the overall neuronal rhythm and provides resistance to interference from external environmental factors. Currently, little is known about the mechanisms by which the neurons in the SCN exert their coupling effects to maintain the stability of the body's rhythms.

[0005] Three Hedgehog homologs exist in mammals: SonicHedgehog (SHH), IndianHedgehog (IHH), and Desert Hedgehog (DHH), encoding the proteins Shh, Ihh, and Dhh, respectively. In many developmental processes in vertebrates and invertebrates, the Sonic Hedgehog (Shh) signaling pathway controls cell proliferation and differentiation. Abnormal activation of this pathway can lead to tumor development and progression, and in recent years, the Shh signaling pathway has become a popular target for cancer treatment.

[0006] However, there are currently no reports of the Shh signaling pathway regulating biological rhythms or being used to treat rhythm-related diseases. Summary of the Invention

[0007] This invention reveals for the first time that the Shh pathway can regulate circadian rhythms, answering the scientific question of how the body maintains the stability of its own rhythms. Mice with specific knockout of the important receptor Smoothened (Smo) in this pathway exhibited rhythm abnormalities, specifically enhanced jet lag adaptation. After injection of Smo receptor inhibitors, the jet lag adaptation was significantly improved. Furthermore, we discovered that drugs acting on the Shh pathway can affect the circadian rhythms of brain tissue, thus providing a new application for these drugs in regulating circadian rhythms.

[0008] application

[0009] On the one hand, this invention provides the application of Hedgehog pathway inhibitors and SMO inhibitors in regulating circadian rhythms and treating circadian rhythm-related diseases;

[0010] Preferably, the Hedgehog pathway inhibitors and SMO inhibitors include, but are not limited to, Vismodegib (GDC-0449), Purmorphamine, PF-5274857, Mebendazole, HPI-4 (Ciliobrevin A), SANT-1, Taladegib (LY2940680), Glasdegib (PF-04449913), Cyclopamine, Itraconazole (R51211), GANT61, JK184, Robotnikinin, Sonidegib Phoshate, or pharmaceutically acceptable salts of the above compounds.

[0011]

[0012] The chemical formula of Vismodegib is as follows:

[0013] The chemical formula of GANT61 is as follows:

[0014] The chemical formula of JK184 is as follows:

[0015]

[0016]

[0017] The chemical formula of Purmorphamine is as follows:

[0018]

[0019] The chemical formula of PF-5274857 is as follows:

[0020] The chemical formula of the mebendazole is as follows:

[0021]

[0022] The chemical formula of HPI-4 (Ciliobrevin A) is as follows:

[0023]

[0024] The chemical formula of SANT-1 is as follows:

[0025]

[0026] The chemical formula of Taladegib (LY2940680) is as follows:

[0027]

[0028] The chemical formula of Glasdegib (PF-04449913) is as follows:

[0029]

[0030] The chemical formula of Cyclopamine is as follows:

[0031]

[0032] The chemical formula of the itraconazole (R 51211) is as follows:

[0033]

[0034] The chemical formula of Robotnikinin is as follows:

[0035]

[0036] The chemical formula of the Sonidegib Phoshate is as follows:

[0037]

[0038] Vismodegib, as described in this invention, is a novel oral drug that selectively targets the Hedgehog signaling pathway. Vismodegib has been approved by the FDA for the treatment of basal cell carcinoma. GANT61 (NSC136476), also described in this invention, is a GLI1 and GLI2-induced transcriptional inhibitor that inhibits the hedgehog signaling pathway.

[0039] The JK184 described in this invention is an effective Hedgehog pathway inhibitor that directly inhibits the transcriptional activity of GLI1 and GLI2.

[0040] Purmorphamine, as described in this invention, is a Smo receptor agonist. PF-5274857, also described in this invention, is a potent, selective, orally active, and blood-brain barrier-crossing Smo antagonist.

[0041] Mebendazole, as described in this invention, is a highly effective broad-spectrum anthelmintic and has also been reported as a Hedgehog inhibitor.

[0042] The HPI-4 (Ciliobrevin A) described in this invention is a Hedgehog pathway inhibitor that directly affects the stability of GLI1.

[0043] The SANT-1 described in this invention is an effective Smo antagonist that can inhibit the Hedgehog pathway.

[0044] The Taladegib (LY2940680) described in this invention is a Smo receptor antagonist that can inhibit the Hedgehog pathway.

[0045] Glasdegib (PF-04449913) described in this invention is an effective, orally active Smo inhibitor.

[0046] Cyclopamine, as described in this invention, is a selective Smo inhibitor that antagonizes the Hedgehog pathway.

[0047] Itraconazole (R 51211) described in this invention is a triazole antifungal drug and also an effective orally active Hedgehog signaling pathway antagonist.

[0048] Robotnikinin, as described in this invention, is a small molecule inhibitor capable of binding to the Shh signal upstream of Smo.

[0049] The Sonidegib Phosphate (CAS No.: 1218778-77-8, English name: LDE-225Diphosphate) described in this invention is an effective and selective Smo antagonist.

[0050] The "Hedgehog pathway inhibitor" mentioned in this invention is also known as "Hedgehog antagonist," and the two have the same meaning and can be used interchangeably.

[0051] The "Smo pathway inhibitor" mentioned in this invention is also known as "Smo antagonist," and the two have the same meaning and can be used interchangeably.

[0052] The "biological rhythm-related diseases" described in this invention include sleep disorders, jet lag, depression, metabolic disorders, aging, blood diseases, diabetes, and obesity.

[0053] The specific manifestations of "regulating biological rhythms" described in this invention include rapid adaptation to jet lag, such as the rapid adaptation of subjects to new light cycles (light schedules) when they change. "Regulating biological rhythms" as described in this invention may also include mitigating the abnormalities in psychological and physiological functions caused by changes in light cycles, enabling subjects to adapt more smoothly to new light cycles.

[0054] As used herein, the term "subject" refers to any animal (e.g., a mammal), including but not limited to humans, non-human primates, rodents, etc., that will become the recipient of a particular treatment. Preferably, the subject is a human.

[0055] The pharmaceutically acceptable salts described in this invention are non-toxic at the amounts and concentrations in which they are administered. The preparation of such salts facilitates pharmacological applications by altering the physical properties of the compounds without preventing them from exerting their physiological effects.

[0056] Preferably, the pharmaceutically acceptable salt also includes salts obtained from acids, which can be organic or inorganic acids; preferably, the inorganic acids include hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, and phosphoric acid; preferably, the organic acids include formic acid, acetic acid, maleic acid, succinic acid, mandelic acid, fumaric acid, malonic acid, pyruvic acid, oxalic acid, glycolic acid, salicylic acid, pyranosidyl acid, α-hydroxy acids such as citric acid or tartaric acid, amino acids, aromatic acids, and sulfonic acids.

[0057] Preferably, the pharmaceutically acceptable salt also includes alkali addition salts, specifically alkali metal salts (such as sodium salts, potassium salts, etc.) and alkaline earth metal salts (such as calcium salts, magnesium salts, etc.).

[0058] Preferably, the SMO inhibitor includes reagents used in gene editing technologies such as siRNA interference, CRISPR, TALEN, ZFN, and Cre-loxP recombination to knock out SMO expression.

[0059] Preferably, the SMO inhibitor is a reagent used to specifically knock out SMO in the SCN region.

[0060] Preferably, the Smo inhibitor of the present invention is a reagent used in Cre-loxp recombination technology, which contains Cre (Cre recombinase) and the loxp sequence. The full-length coding region of the Cre recombinase gene is 1029 bp (EMBL database accession number X03453), encoding a 38 kDa monomeric protein composed of 343 amino acids. It not only possesses catalytic activity but also, similar to restriction enzymes, can recognize specific DNA sequences, namely loxP sites, causing deletion or recombination of the gene sequence between loxP sites.

[0061] Preferably, the application occurs in vitro.

[0062] Preferably, the application is for non-therapeutic purposes.

[0063] Preferably, the product comprises a pharmaceutical composition.

[0064] Preferably, the pharmaceutical composition further includes a pharmaceutically acceptable carrier, diluent, or excipient.

[0065] Preferably, the pharmaceutically acceptable carrier, diluent, or excipient includes, but is not limited to, any adjuvant, carrier, excipient, gliding agent, sweetener, diluent, preservative, dye / coloring agent, flavor enhancer, surfactant, wetting agent, dispersant, suspending agent, stabilizer, isotonic agent, solvent, surfactant, or emulsifier that has been approved by the U.S. Food and Drug Administration or the China Food and Drug Administration for use in humans or livestock.

[0066] Specific examples of substances that can serve as pharmaceutically acceptable carriers or components as described in this invention include sugars such as lactose, glucose, and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium carboxymethyl cellulose, ethyl cellulose, and methyl cellulose; tragacanth gum powder; malt; gelatin; talc; solid lubricants such as stearic acid and magnesium stearate; calcium sulfate; vegetable oils such as peanut oil, cottonseed oil, sesame oil, olive oil, corn oil, and cocoa butter; polyols such as propylene glycol, glycerin, sorbitol, mannitol, and polyethylene glycol; alginic acid; emulsifiers such as wetting agents such as sodium lauryl sulfate; colorants; flavoring agents; tableting agents; stabilizers; antioxidants; preservatives; pyrogen-free water; isotonic salt solutions; and phosphate buffers, etc.

[0067] The compositions of the present invention can be formulated into various dosage forms as needed, and the dosage beneficial to the patient can be determined by a physician based on factors such as patient type, age, weight, general disease condition, and route of administration. The route of administration can be, for example, injection or other treatment methods (e.g., oral administration).

[0068] Preferably, the injection includes intravenous injection, intramuscular injection, intraperitoneal injection, or subcutaneous injection.

[0069] More preferably, the injection described in this invention refers to injection at a specific location of the SCN.

[0070] The dosage forms described in this invention include tablets, pills, powders, granules, capsules, lozenges, syrups, liquids, emulsions, suspensions, controlled-release formulations, aerosols, films, injections, intravenous infusions, transdermal absorption formulations, ointments, lotions, adhesive formulations, suppositories, small pills, nasal preparations, pulmonary preparations, eye drops, etc.

[0071] method

[0072] On the other hand, the present invention provides a method for treating circadian rhythm-related diseases, the method comprising administering a Hedgehog pathway inhibitor and an SMO inhibitor.

[0073] On the other hand, the present invention provides a pharmaceutical composition for treating circadian rhythm-related diseases, the pharmaceutical composition comprising administration of a Hedgehog pathway inhibitor and an SMO inhibitor.

[0074] On the other hand, the present invention provides a method for regulating biological rhythms, the method comprising administering Hedgehog pathway inhibitors and SMO inhibitors.

[0075] On the other hand, the present invention provides a composition for regulating biological rhythms; the composition contains a Hedgehog pathway inhibitor and an SMO inhibitor.

[0076] Preferably, the method occurs in vitro or in vivo;

[0077] More preferably, the procedure is performed in vitro on cells.

[0078] Preferably, the procedure can also be performed inside a subject;

[0079] As used in this article, the term "subject" refers to any animal (e.g., mammal), including but not limited to humans, non-human primates, rodents, etc., that will become the recipient of a particular treatment.

[0080] animal models

[0081] On the other hand, the present invention provides a method for constructing an animal model of abnormal biological rhythms, the method comprising using Hedgehog pathway inhibitors and Hedgehog pathway knockout mice on model organisms.

[0082] Preferably, the method includes the following steps:

[0083] 1) Obtain an animal model of SCN-specific Cre expression;

[0084] 2) Obtain an animal model of Smo-loxp;

[0085] 3) Mating the animal models from 1) and 2).

[0086] Preferably, the method further includes multi-generation breeding of the animal model obtained in step 3).

[0087] The Smo-loxp animal model has a loxp sequence upstream and downstream of the first exon of the Smo gene. The loxp sequence described in this invention is the site in conventional Cre-loxp recombination technology. The LoxP (locus of X-overP1) site is 34 bp long, consisting of two 13 bp inverted repeat sequences and an 8 bp spacer region. The inverted repeat sequences are specific recognition sites for Cre recombinase, while the spacer region determines the orientation of the loxP site. When a loxP site is present in the genome, Cre recombinase binds to the inverted repeat sequence regions at both ends of the loxP site to form a dimer. This dimer binds to dimers at other loxP sites, forming a tetramer. Subsequently, the DNA between the loxP sites is cleaved by Cre recombinase, and the cleavage is reconnected by DNA ligase. When two loxP sites are located on the same DNA strand and oriented in the same direction, Cre recombinase knocks out the sequence between the loxP sites.

[0088] Preferably, the terms "suprachiasmatic nucleus of hypothalamus", "SCN (suprachiasmatic nucleus)", "suprachiasmatic nucleus" and "suprachiasmatic nucleus of thalamus" all have the same meaning and can be used interchangeably.

[0089] On the other hand, the present invention provides an animal model constructed by the above-mentioned method for constructing an animal model of abnormal biological rhythms, and its application.

[0090] Preferably, the application includes studying the regulation of biological rhythms by the Shh pathway. Attached Figure Description

[0091] Figure 1 This is a representative result of the mouse running on the wheel. Figure 1 A is a graph showing the behavior of the running wheel when the light cycle is changed. Figure 1 B is right Figure 1 Statistical results of phase shift time of left B (B); Figure 1 C on the left represents the phase shift statistics during the process of restoring the illumination schedule to its initial state. Figure 1 C is right Figure 1 Statistical results of phase shift of C left by 50% time.

[0092] Figure 2This is a representative result of mouse sleep behavior. Figure 2 Figure A shows the results of mouse sleep behavior when the light cycle was changed. Figure 2 B shows the results of real-time quantitative PCR indicating changes in sleep-related genes.

[0093] Figure 3 This is a graph showing the test results for Smo knockout mice. Figure 3 A represents the effect of Smo knockout detected by Western blotting. Figure 3 B shows a photograph of the mouse and its weight statistics. Figure 3 C represents a physical image of the mouse brain and the statistical results of its length and width; Figure 3 D is the image showing the results of Nissell staining under a microscope. Figure 3 E is a diagram showing the results of neuronal staining under a microscope.

[0094] Figure 4 This is a graph showing the test results of the effects of Vismodegib on biological rhythms. Figure 4 A represents the effect of Vismodegib on the oscillatory changes of rhythmic genes in the SCN region at the tissue level; Figure 4 B represents the wheel-running behavior of mice in a reverse jet lag model after injection of Vismodegib. Figure 4 C represents the phase shift in mice in 4B 8 hours before the scheduled light exposure; Figure 4 D represents the results of monitoring mouse sleep behavior in a reverse jet lag model after injection of Vismodegib. Figure 4 E represents the oscillatory changes in rhythmic genes of different neurons after Vismodegib was added to SCN tissue slices. In the upper heatmap, red and green represent the highest and lowest peaks of Per2 expression, respectively. The lower phase distribution map shows the statistical results of different neuron phases in the upper heatmap.

[0095] Figure 5 This is a graph showing the effects of Hedgehog pathway inhibitors on circadian rhythms. Figure 5 A represents the effect of Purmorphamine and PF5274857 on the oscillatory changes of rhythmic genes in the SCN region at the tissue level. Figure 5 B represents the statistical results of the 5A oscillation cycle; Figure 5 C represents the effects of Mebendazole, Cilibrevin A, and SANT-1 on the oscillatory changes of rhythmic genes in the SCN region at the tissue level. Figure 5 D represents the statistical result of the 5C oscillation cycle; Figure 5 E represents the effect of Taladegib and Glasdegib on the oscillatory changes of rhythmic genes in the SCN region at the tissue level. Figure 5 F represents the statistical results of the 5E oscillation cycle; Figure 5G represents the effect of Cyclopamine and Itraconazole on the oscillatory changes of rhythmic genes in the SCN region at the tissue level; Figure 5 H represents the statistical results of the 5G oscillation cycle; Figure 5 I is the effect of GANT61, JK184, and Robotnikinin on the oscillatory changes of rhythmic genes in the SCN region at the tissue level; Figure 5 J represents the statistical result of the 5I oscillation cycle.

[0096] Figure 6 This is a graph showing the effects of Sonidegib Phoshate on circadian rhythms. Figure 6 A represents the effect of Sonidegib Phosphate on oscillatory changes in rhythmic genes in the SCN region at the tissue level; Figure 6 B represents the statistical results of the 6A oscillation cycle. Detailed Implementation

[0097] The present invention will be further described below with reference to embodiments. The following description is merely a preferred embodiment of the present invention and is not intended to limit the invention in any other way. Any person skilled in the art may make equivalent modifications to the disclosed technical content to create equivalent embodiments. Any simple modifications or equivalent changes made to the following embodiments based on the technical essence of the present invention without departing from the scope of the invention are all within the protection scope of the present invention.

[0098] Materials and reagents used in this invention

[0099] 1. Per2::Luc mice: donated by Professor Zhang Erquan of the Beijing Institute of Life Sciences. These are the mice described in the paper "Ju et al. Chemical Perturbations Reveal That RUVBL2 Regulates the Circadian Phase in Mammals. Sci. Transl. Med. 12, eaba0769 (2020)". They are available to the public from the applicant and may only be used to repeat the experiments of this invention; no other use is permitted. Smo-loxp mice were purchased from Jackson Lab, catalog number 004526; NMS-Cre mice were purchased from Jackson Lab, catalog number 027205.

[0100] 2. Reagents

[0101] Table 1. Reagents used in this invention

[0102] 1 Smo antibody ABclonal A3274 2 α-Tubulin antibody MBL Company PM054 3 Luciferin Promega E1602 4 DMEM medium, HBSS buffer GIBCO 21063029、15630080 5 PF-5274857 Selleck S2777 6 HPI-4 (Ciliobrevin A) Selleck S8249 7 Itraconazole (R 51211) Selleck S2476 8 GANT61 Selleck S8075 9 Vismodegib (GDC-0449) Selleck S1082 10 JK184 Selleck S6565 11 Taladegib (LY2940680) Selleck S2157 12 Cyclopamine Selleck S1146 13 SANT-1 Selleck S7092 14 Glasdegib (PF-04449913) Selleck S7160 15 Robotnikinin Xinbosheng AG-CR1-0069-M001 16 Mebendazole MCE HY-17595 17 Purmorphamine Aladdin P126030 18 Sonidegib Phoshate MCE HY-16582

[0103] Note: 5-18 in the table above are small molecule drugs related to the Shh pathway.

[0104] General experimental methods

[0105] 1. Western blot analysis of knockout effect

[0106] SCN brain slices from Nms-Smo- / - mice were collected, ground into single cells, and total cellular protein was extracted for Western blot analysis to detect the knockout effect.

[0107] (1) SDS-PAGE gel preparation: Prepare a 10% concentration gel according to the molecular weight of the protein to be detected.

[0108] (2) Sample loading: Load 30 μg of protein into each well;

[0109] (3) Electrophoresis: Stacking gel, constant voltage 80V; separating gel, constant voltage 120V. The bromophenol blue front is run to the edge of the gel before discharging.

[0110] (4) Transfer: Place the filter paper (4 sheets per gel), fiber pad, nitrocellulose membrane, and SDS-PAGE gel into 1×Transfer Buffer and equilibrate for 10 minutes. (PVDF is a hydrophobic membrane and requires special treatment before use: soak in methanol for about 10 seconds). Place the prepared transfer sandwich into the transfer electrode box (note the positive and negative electrodes), add the transfer buffer and ice pack to begin the transfer. During the transfer process, the entire transfer tank is placed in an ice bath, and the transfer is performed at 400mA for 2 hours.

[0111] (5) Sealing: After the transfer is completed, take out the transfer sandwich and use tweezers to put the transferred nitrocellulose membrane into the pre-prepared sealing solution (5% skim milk) and incubate at room temperature for 1 hour.

[0112] (6) Primary antibody: Incubate with Smo and α-tubulin primary antibodies overnight at 4°C, then wash the membrane three times with TBST for 5 minutes each time;

[0113] (7) Secondary antibody: Incubate with the secondary antibody corresponding to the primary antibody at room temperature for 1 hour, then wash the membrane three times with TBST for 5 minutes each time;

[0114] (8) Development: Take out the washed nitrocellulose membrane, drain as much TBST as possible from the membrane, place it face up on plastic wrap, mix equal amounts of ECL reagent A and B solutions that were taken out beforehand, and spread them drop by drop on the membrane for development.

[0115] 2. Isolation and culture of mouse SCN brain tissue sections

[0116] 1. Isolation of mouse SCN brain tissue sections

[0117] A. Anesthetize 8-10 week old mice. When the mice lose consciousness but have not yet stopped breathing, quickly decapitate them with scissors.

[0118] B. Remove the mouse's eyes with scissors to prevent optic nerve activation and further damage to the SCN in the subsequent process;

[0119] C. Use scissors to cut open the mouse's skull along both sides, removing all bones until the olfactory bulb is visible. Avoid compressing the brain to prevent damage to the ventral SCN;

[0120] D. Use dissecting scissors to cut off the connection between the olfactory bulb and the optic nerve, ensuring that the optic nerve is completely severed;

[0121] E. Invert the head, allowing the entire brain to fall into a container filled with pre-cooled HBSS buffer (1×HBSS).

[0122] In a 10cm culture dish containing 10mM HEPES, 4.5mM NaHCO3, and 1% Penicillin-Streptomycin, keep the brain in HBSS for 30-60 seconds to ensure the brain is cooled.

[0123] F. Transfer the brain to a new petri dish using a spoon, and remove the cerebellum with a sterile scalpel blade;

[0124] G. Apply strong adhesive to the cutting platform of the Vibratome vibratory slicer;

[0125] H. Carefully wipe away any excess HBSS at the brain incision site using sterile filter paper;

[0126] I. With the incision facing down, fix the brain tissue on the cutting platform, transfer it to the cutting platform, and pour in pre-cooled HBSS buffer;

[0127] J. In order to quickly reach the SCN region, the cutting speed was increased to 800μm, the speed was reduced when the hypothalamus was first reached, and the cutting speed was reduced to 300μm when the optic chiasm was seen.

[0128] K. Once the SCN becomes visible, the layer is cut at 300 μm, and the brain slices are transferred to a culture dish containing pre-cooled HBSS buffer using a soft brush for microscopic observation of the SCN region.

[0129] L. The SCN region was cut under a stereomicroscope, the optic chiasm (OC) was removed, and a 1×1 mm microscale SCN brain slice was separated.

[0130] 2. In vitro culture of SCN brain tissue sections

[0131] A. The isolated SCN was transferred to a Millicell insert cell culture dish for culture;

[0132] B. Add 1.2 ml of DMEM medium (with 100 μM luciferin, 1% B27 serum-free additive, and 1% Penicillin-Streptomycin) and record it in a LymiCycle instrument.

[0133] 3. Single-cell imaging of SCN brain tissue

[0134] A. The isolated SCN was transferred to a Millicell insert cell culture dish for culture;

[0135] B. Add 1.2 ml of DMEM medium (containing 1 mM luciferin, 1% B27 serum-free additive, and 1% Penicillin-Streptomycin), seal the culture dish, and place it in a darkroom on an inverted microscope stage with a 10x objective lens (Nikon Eclipse Ti-E). The stage temperature was set to 36°C throughout the experiment. A CCD camera (EA4710V-BV, Raptor, UK) operating at -80°C was mounted on the microscope for image capture.

[0136] C. Strictly avoid light, set the exposure time to 60 minutes, and continuously acquire images;

[0137] D. Dissolve 2 μM TTX or 10 μM Vismodegib and add to preheated fresh culture medium.

[0138] 3. Reverse time difference experiment in Nms-Smo- / - defective mice

[0139] A. Place 8-week-old female mice alone in cages equipped with autonomous running wheels, and provide them with ample food and drinking water;

[0140] B. 12-hour light: Mice were trained for 14 days with a 12-hour darkness cycle (lights on at 07:00 AM and off at 19:00 PM), and their movement was recorded.

[0141] C. On day 15, turn on the lights 8 hours earlier to simulate time zone adjustment conditions (turn on the lights at 23:00 at night and turn off the lights at 11:00 in the morning) and evaluate the time it takes for the mice to adapt to the new photocycle.

[0142] D. After recording for 14 days under the new photoperiod, adjust the photoperiod back to the original time (lights on at 07:00 AM and off at 19:00 PM) and evaluate the time it takes for the mice to adapt to the original photoperiod.

[0143] 4. The Smo inhibitor Vismodegib can accelerate the rapid adaptation to jet lag in wild-type mice.

[0144] A. Place 8-week-old female mice alone in cages equipped with autonomous running wheels, and provide them with ample food and drinking water;

[0145] B. 12-hour light: Mice were trained for 7 days with a 12-hour darkness cycle (lights on at 07:00 AM and off at 19:00 PM), and their movement was recorded.

[0146] C. On day 8, the Smo inhibitor drug was continuously delivered to the SCN area via a slow-release pump using a stereotaxic device. After 3 days of postoperative recovery, the lights were turned on 8 hours in advance to simulate jet lag conditions (lights were turned on at 23:00 and turned off at 11:00 in the morning) to evaluate the time it took for the mice to adapt to the new photocycle.

[0147] Example 1: Behavioral performance of SCN-specific knockout Smo mice under photoperiod alterations

[0148] Eight-week-old adult female mice were housed individually in a cabinet equipped with a running wheel cage, and their running wheel behavior was recorded and analyzed in real time using ClockLab. Figure 1 A represents the representative results of the mouse wheel-running behavior. After two weeks of training with a standard light cycle (lights on at 7:00 AM and off at 7:00 PM), the light cycle was advanced by 8 hours (lights on at 11:00 PM and off at 11:00 AM). After 15 days of observation, the light cycle was returned to its initial state. The mouse wheel-running behavior in the figure is indicated by black marks, and white and gray backgrounds represent light and darkness, respectively. Figure 1 B represents the phase shift statistics of the corresponding mice during the process of advancing the light schedule by 8 hours; Figure 1 C represents the phase shift statistics of the corresponding mouse during the process of restoring its initial state in the light cycle.

[0149] The results show that after the photoperiod is changed, the Smo conditional knockout mice exhibit a time-reversed abnormal phenotype and can adapt to the new photoperiod more quickly.

[0150] Example 2: Sleep monitoring of SCN-specific knockout Smo mice during photoperiod changes

[0151] Consistent with the above, ClockLab was used to record the running behavior of mice in real time during changes in the light cycle. ClockLab Analysis software was used to analyze the sleep behavior of mice (the strength of the mouse's movement behavior was used to determine whether the mouse was in a sleep state). In the figure, the mouse sleep behavior is marked with black, and the white and gray backgrounds represent light and darkness, respectively. Figure 2 B shows the results of real-time quantitative PCR analysis of changes in sleep-related genes in the corresponding mice.

[0152] The results show that after the photoperiod was changed, the sleep behavior of Smo knockout mice was improved and they were able to adapt to the new photoperiod more quickly.

[0153] Example 3: Identification of SCN-specific knockout Smo mice

[0154] NMS-Cre is a tool mouse that specifically expresses Cre in the SCN region. Smo is an important receptor in the Shh signaling pathway. NMS-Smo– / – mice are obtained by mating NMS-Cre mice with Smo-floxp mice and breeding them for multiple generations.

[0155] Figure 3 A shows the effect of Smo knockout detected by Western Blot (Smo is not only expressed in NMS neurons in the SCN region, so the results show that a small amount of Smo was still detected after SCN-specific knockout). Figure 3 B shows that the body weight of Smo conditional knockout mice is not affected; Figure 3 C shows that the overall brain contour of the Smo conditional knockout mouse is normal; Figure 3 D and 3E show that the SCN region neurons in Smo conditional knockout mice are differentiated normally.

[0156] The above results demonstrate that a Smo mouse with a specific knockout of the suprachiasmatic nucleus (SCN) region of the hypothalamus was successfully constructed, and the mouse exhibits normal overall development.

[0157] Example 4: The Smo inhibitor Vismodegib affects the rhythmic behavior of mice.

[0158] Vismodegib is a novel oral drug that selectively targets the Shh signaling pathway. The results show that Vismodegib can directly affect the oscillatory changes in rhythm genes and block the coupling process of SCN neurons, enabling mice to adapt to time zone changes more quickly. Figure 4 A. To investigate the effect of rhythm genes on the oscillatory changes of rhythm genes in the SCN region at the tissue level, freshly isolated brain slices from the SCN region of Per2::Luc mice were cultured in vitro, and Per2 expression levels were continuously recorded using lumicycle. After three days of recording, the culture medium was replaced with fresh medium containing 20 μM Vismodegib. It was observed that Vismodegib could significantly inhibit the oscillatory changes of Per2. After three days, the drug was removed and replaced with fresh medium, and the oscillatory changes of Per2 were restored, indicating that the effect of Vismodegib was not due to drug toxicity. Figure 4 B represents the wheel-running behavior of mice in a reverse jet lag model after injection of Vismodegib. Figure 4 C represents the phase shift in mice in 4B 8 hours before the scheduled light exposure; Figure 4 D represents the results of sleep behavior analysis in mice in a jet lag model after injection of Vismodegib. Figure 4E shows the oscillatory changes of different neuronal rhythm genes after Vismodegib was added to SCN tissue sections. The heatmap above shows the changes in Per2 expression levels in different neurons. Red and green represent the highest and lowest peaks of Per2 expression, respectively. It can be seen that the Per2 oscillations of different neurons were consistent before drug addition, but the oscillations of different neurons gradually became disordered after drug addition. The phase distribution map below shows the phase statistics of the oscillatory changes of different neurons in the heatmap above. Each point represents the phase corresponding to a single neuron.

[0159] The above results demonstrate that Vismodegib can improve jet lag behavior in mice.

[0160] Example 5: Effects of Hedgehog pathway inhibitors on circadian rhythms

[0161] Freshly isolated brain slices from the SCN region of Per2::Luc mice were cultured in vitro, and Per2 expression levels were continuously recorded using a lumicycle. After about three days of recording, the culture medium was replaced with fresh culture medium containing Hedgehog pathway inhibitors to observe the effects of different Hedgehog pathway inhibitors on the oscillatory changes in Per2 expression.

[0162] Figure 5 A represents the effect of 2 μM Purmorphamine and 1 μM PF5274857 on the oscillatory changes of rhythmic genes in the SCN region at the tissue level; Figure 5 B represents the statistical result of the 5A oscillation cycle;

[0163] Figure 5 C represents the effect of 20 μM Mebendazole, 60 μM Ciliobrevin A, and 100 μM SANT-1 on the oscillatory changes of rhythmic genes in the SCN region at the tissue level. Figure 5 D is the statistical result of the 5C oscillation cycle;

[0164] Figure 5 E represents the effect of 10 μM Taladegib and 20 μM Glasdegib on the oscillatory changes of rhythmic genes in the SCN region at the tissue level. Figure 5 F represents the statistical result of the 5E oscillation cycle;

[0165] Figure 5 G represents the effect of 20 μM Cyclopamine and 25 μM Itraconazole on the oscillatory changes of rhythmic genes in the SCN region at the tissue level. Figure 5 H represents the statistical result of the 5G oscillation cycle;

[0166] Figure 5I. The effects of 10 μM GANT61, 2 μM JK184, and 10 μM Robotnikin on the oscillatory changes of rhythmic genes in the SCN region at the tissue level; Figure 5 J is the statistical result of the 5I oscillation cycle.

[0167] Figure 6 A represents the effect of different concentrations of Sonidegib Phoshate on the oscillatory changes of rhythmic genes in the SCN region at the tissue level; Figure 6 B represents the statistical results of the 6A oscillation cycle.

[0168] The above results demonstrate that different Hedgehog pathway inhibitors can affect the oscillatory changes in the Per2 gene, and may be used to regulate biological rhythms and treat rhythm disorders.

Claims

1. The use of Vismodegib and its pharmaceutically acceptable salts in the preparation of drugs for improving sleep disorders.

2. The application as described in claim 1, characterized in that, The improvement of sleep disorders specifically includes adapting to new light cycles more quickly, or reducing the abnormalities in the body's psychological and physiological functions caused by changes in light cycles.

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