CN116870166A

CN116870166A - Shh pathway for modulating biological rhythms and related applications thereof

Info

Publication number: CN116870166A
Application number: CN202310745524.2A
Authority: CN
Inventors: 李慧艳; 涂海情; 周涛; 李爱玲; 吴敏; 胡怀斌; 何新华; 张学敏
Original assignee: Academy of Military Medical Sciences AMMS of PLA
Current assignee: Academy of Military Medical Sciences AMMS of PLA
Priority date: 2021-12-31
Filing date: 2022-09-23
Publication date: 2023-10-13
Also published as: CN115463218A; CN114668849B; CN115463218B; CN114668849A

Abstract

The application relates to the field of biotechnology, in particular to a Shh pathway-controlled biological rhythm and related application thereof. Specifically, the application provides application of a Hedgehog pathway inhibitor and an SMO inhibitor in regulating biological rhythm and treating biological rhythm related diseases; preferably, the Hedgehog pathway inhibitors include PF-5274857, mebendazole, HPI-4, SANT-1, taladegib, glasdegib, cyclopamine, itraconazole, GANT61, JK184, robotnikinin, vismodegib, purmorphamine, sonidegib Phoshate.

Description

Shh pathway for modulating biological rhythms and related applications thereof

The application is a divisional application of an application patent application with the application number of 202211162119X, the application date of 2022, 9 and 23 days and the name of 'Shh pathway regulation biological rhythm and related application'.

Technical Field

The application relates to the field of biotechnology, in particular to a method for regulating and controlling biological rhythms by using Shh pathway, which can be used for the treatment and research of rhythm related diseases.

Background

The sleeping, waking, eating and other activities of living beings and various physiological, biochemical and metabolic processes follow rhythmic changes for about 24 hours, commonly known as biological clocks, which are closely related to normal physiological functions of human bodies. Researches show that the biological rhythm is closely related to various physiological indexes such as sleep, diet, cognition, emotion, behavior, metabolism and the like of an individual, the coordination of the whole organism is orderly utilized to play the role and adapt to the environment, if the normal biological rhythm is disturbed, the abnormal psychological and physiological functions of the organism can be caused, and meanwhile, the functional disorder of each tissue and organ of the organism can be caused. For humans, changes in the biological clock can cause a number of problems such as sleep disorders, depression, metabolic disorders, aging, blood disorders, diabetes, and obesity.

The supraoptic nucleus (SCN) is a tiny region located on either side of the third brain chamber of the hypothalamus, directly above the optic intersecting nerve. SCN is used as biological clock central system and is mainly responsible for regulating and controlling circadian rhythm of organism, on one hand, it can directly receive optical signal transferred from upstream optic cross nerve, on the other hand, it can affect rhythmicity of other viscera, and can play central coordination function. The SCN area is composed of about 20000 neurons, and the coupling effect exists among different neurons, so that the rhythm of the whole neurons can be kept consistent, and the SCN area has an anti-interference function on external environment factors. At present, little is known about how SCN neurons exert a coupling effect to maintain the mechanisms of body rhythmic stability.

Three homologous genes for Hedgehog exist in mammals: sonicHedgehog (SHH) Ind Hedgehog (IHH) and Desert Hedgehog (DHH) encode Shh, ihh and Dhh proteins, respectively. In many development processes of vertebrates and invertebrates, sonic Hedgehog (Shh) signaling pathway controls proliferation and differentiation of cells, and when the signaling pathway is abnormally activated, it causes tumor generation and development, and in recent years, shh signaling pathway becomes a hot target for treating tumors.

However, there is currently no report of the Shh signaling pathway for modulating biological rhythms, and for treating rhythm-related diseases.

Disclosure of Invention

The application discloses that the Shh pathway can regulate and control the biological rhythm for the first time, and answers the scientific question of how the organism maintains the stability of the self-rhythm. Mice with the important receptor Smoothened (Smo) of the channel specifically knocked out show abnormal rhythms, particularly the adaptability to the time difference of fall is enhanced, the adaptability to the time difference of fall is obviously improved after the organism is injected with the Smo receptor inhibitor, and further, we find that related drugs acting on the Shh channel can influence the biological rhythms of brain tissues, and further provide a new application of the drugs in regulating the biological rhythms.

Application of

In one aspect, the application provides the use of Hedgehog pathway inhibitors, SMO inhibitors, for modulating biological rhythms, for treating diseases associated with biological rhythms;

preferably, the Hedgehog pathway inhibitor, SMO inhibitor includes, but is not limited to, vismodegib (GDC-0449), purmorphamine, PF-5274857, mebendazole, HPI-4 (Ciliobrevin a), SANT-1, taladegab (LY 2940680), glasdegib (PF-04449913), cyclopamine, itraconazole (R51211), gent 61, JK184, robotnikin, sonidegib Phoshate, or a pharmaceutically acceptable salt of the above.

The chemical formula of the Vismodigib is as follows:

the GANT61 has the formula:

the chemical formula of the JK184 is as follows:

the chemical formula of Purmorphamine is as follows:

the chemical formula of the PF-5274857 is as follows:

the chemical formula of the Mebendazole is as follows:

the chemical formula of HPI-4 (Ciliobrevin A) is as follows:

the chemical formula of SANT-1 is as follows:

the chemical formula of Taladegib (LY 2940680) is as follows:

the chemical formula of the Glasdigib (PF-04449913) is as follows:

the chemical formula of the Cyclopamine is as follows:

the chemical formula of the Itraconazole (R51211) is as follows:

the formula of the Robotnikin is as follows:

the chemical formula of Sonidegib Phoshate is as follows:

the Vismodygib (Vimod Ji) is a novel oral medicament with a selective Hedgehog signal path. The use of vemoroxydine has been approved by the FDA for the treatment of basal cell carcinoma. GANT61 (NSC 136476) is a GLI1 and GLI2 induced transcription inhibitor, and inhibits hedgehog signaling.

The JK184 is an effective Hedgehog pathway inhibitor and can directly inhibit the transcriptional activity of GLI1 and GLI 2.

Purmorphamine is a Smo receptor agonist. PF-5274857 of the present application is a potent, selective Smo antagonist with oral activity and blood brain barrier permeability.

The Mebendazole of the present application is a highly potent broad-spectrum anthelmintic and has also been reported as a Hedgehog inhibitor.

The HPI-4 (Ciliobrevin A) is a Hedgehog pathway inhibitor, and directly influences the stability of GLI 1.

SANT-1 of the present application is a potent Smo antagonist that inhibits the Hedgehog pathway.

Taladegib (LY 2940680) is a Smo receptor antagonist and can inhibit the Hedgehog pathway.

Glasdelb (PF-04449913) of the present application is a potent, orally active Smo inhibitor.

The Cyclopamine is a selective Smo inhibitor and can antagonize the Hedgehog pathway.

The Itraconazole (R51211) of the present application is a triazole antifungal agent and is also an effective orally active Hedgehog signaling pathway antagonist.

Robotnikin according to the application is a small molecule inhibitor capable of binding to the Shh signal upstream of Smo.

Sonidegib Phoshate (CAS number 1218778-77-8, english name: LDE-225 Diphosphorate) of the present application is a potent, selective Smo antagonist.

The "Hedgehog pathway inhibitor" of the present application, namely "Hedgehog antagonist", is the same meaning and can be used interchangeably.

The term "Smo pathway inhibitor" as used herein, i.e. "Smo antagonist", is intended to mean the same and is used interchangeably.

The "biorhythm-related diseases" of the present application include sleep disorders, jet lag adjustment, depression, metabolic disorders, aging, blood diseases, diabetes, obesity, and the like.

The specific expression "regulating biorhythms" according to the application includes a rapid adaptation of the fall time difference, for example, when the illumination period (illumination schedule) changes, the subject adapts rapidly to the new illumination period. The application can also reduce the abnormal psychological and physiological functions of the organism caused by the change of the illumination period, so that the subject can adapt to the new illumination period more smoothly under the condition of changing the illumination period.

The term "subject" as used herein 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. Preferably, the subject is a human.

The "pharmaceutically acceptable salts" of the present application are non-toxic in the amounts and concentrations to be administered. The preparation of such salts may facilitate pharmacological applications by altering the physical properties of the compound without preventing its physiological effects.

Preferably, the pharmaceutically acceptable salts further include those obtained from acids, which may be organic or inorganic; preferably, the inorganic acid comprises hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid; preferably, the organic acid is selected from formic acid, acetic acid, maleic acid, succinic acid, mandelic acid, fumaric acid, malonic acid, pyruvic acid, oxalic acid, glycolic acid, salicylic acid, pyrosidic acid (pyrosidyl acid), alpha hydroxy acids such as citric acid or tartaric acid, amino acids, aromatic acids, sulfonic acids.

Preferably, the pharmaceutically acceptable salts further include alkali addition salts, specifically alkali metal salts (e.g., sodium salt, potassium salt, etc.), alkaline earth metal salts (e.g., calcium salt, magnesium salt, etc.), and the like.

Preferably, the SMO inhibitor comprises an agent used in knocking out SMO expression by gene editing techniques such as siRNA interference techniques, CRISPR techniques, TALEN techniques, ZFN techniques, cre-loxP recombination techniques, and the like.

Preferably, the SMO inhibitor is an agent used to specifically knock out SCN region SMO.

Preferably, the Smo inhibitor is a reagent used by Cre-loxp recombination technology, wherein the reagent comprises Cre (Cre recombinase) and loxp sequences, and the full length of the coding region sequence of the Cre recombinase gene is 1029bp (EMBL database accession number X03453) and codes for 38kDa monomeric protein consisting of 343 amino acids. It has catalytic activity, and similar to restriction enzyme, can recognize specific DNA sequence, i.e. loxP site, so that the gene sequence between loxP sites can be deleted or recombined.

Preferably, the application occurs in vitro.

Preferably, the use is non-therapeutic.

Preferably, the product comprises a pharmaceutical composition.

Preferably, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier, diluent or excipient.

Preferably, the pharmaceutically acceptable carrier, diluent or excipient includes, but is not limited to, any adjuvant, carrier, excipient, glidant, sweetener, diluent, preservative, dye/colorant, flavoring agent, surfactant, wetting agent, dispersing agent, suspending agent, stabilizer, isotonic agent, solvent, surfactant or emulsifier that has been approved by the U.S. food and drug administration or the Chinese food and drug administration for use in humans or livestock.

Specific examples of substances which may be pharmaceutically acceptable carriers or components thereof according to the present application are 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 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, glycerol, sorbitol, mannitol and polyethylene glycol; alginic acid; emulsifying agents, such as wetting agents, e.g., sodium lauryl sulfate; a colorant; a flavoring agent; tabletting and stabilizing agent; an antioxidant; a preservative; non-thermal raw water; isotonic saline solution; and phosphate buffer, etc.

The composition of the present application may be formulated into various dosage forms as required, and the dosage beneficial to the patient may be determined by the physician according to the type, age, weight and general condition of the patient, the mode of administration, etc. The administration may be, for example, by injection or other therapeutic means (e.g., oral).

Preferably, the injection comprises intravenous injection, intramuscular injection, intraperitoneal injection or subcutaneous injection.

More preferably, the injection according to the present application refers to injection at a SCN specific site.

The dosage forms of the present application include tablets, pills, powders, granules, capsules, troches, syrups, liquids, emulsions, suspensions, controlled release formulations, aerosols, films, injections, intravenous drip preparations, transdermal absorption formulations, ointments, lotions, adhesive formulations, suppositories, pellets, nasal formulations, pulmonary formulations, eye drops and the like.

Method

In another aspect, the application provides a method of treating a biological rhythm-related disorder comprising administering a Hedgehog pathway inhibitor, a SMO inhibitor.

In another aspect, the application provides a pharmaceutical composition for treating a biological rhythm related disorder comprising administering a Hedgehog pathway inhibitor, a SMO inhibitor.

In another aspect, the application provides a method of modulating a biological rhythm comprising administering a Hedgehog pathway inhibitor, a SMO inhibitor.

In another aspect, the application provides a composition for modulating biological rhythms; the composition contains a Hedgehog pathway inhibitor and a SMO inhibitor.

Preferably, the method occurs in vitro or in vivo;

more preferably, the in vitro is performed on cells.

Preferably, the said may also be performed in the subject;

the term "subject" as used herein 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.

Animal model

In another aspect, the application provides a method of constructing an animal model of a biological rhythm disorder, the method comprising using a Hedgehog pathway inhibitor, a Hedgehog pathway knockout mouse, to a model organism.

Preferably, the method comprises the steps of:

1) Obtaining an animal model of specific expression Cre of the SCN region;

2) Obtaining an animal model of Smo-loxp;

3) Mating the animal models of 1) and 2).

Preferably, the method further comprises subjecting the animal model obtained in step 3) to multi-generation propagation.

The animal model of Smo-loxp has a loxp sequence at the upstream and downstream of the first exon of Smo gene; the LoxP sequence is a site in the conventional Cre-LoxP recombination technology, and the LoxP (locus of X-overlap 1) site is 34bp in length and comprises two 13bp inverted repeat sequences and an 8bp interval region. Wherein the inverted repeat is a specific recognition site for Cre recombinase and the spacer determines the orientation of the loxP site. When there is a loxP site in the genome, once there is Cre recombinase, the inverted repeat region that binds to both ends of the loxP site forms a dimer. This dimer binds to dimers of other loxP sites, thereby forming tetramers. Subsequently, the DNA between loxP sites is excised by Cre recombinase and the nicks religated by DNA ligase. The two loxP sites are positioned on the same DNA chain and have the same direction, and Cre recombinase knocks out sequences among the loxP sites.

Preferably, the terms "hypothalamic visual intersection upper core", "SCN (suprachiasmatic nucleus)", "visual intersection upper core", "thalamic area visual intersection upper core" are all used interchangeably.

On the other hand, the application provides an animal model constructed by the method for constructing the animal model with abnormal biological rhythm and application thereof.

Preferably, the application comprises studying the Shh pathway to modulate biological rhythms.

Drawings

FIG. 1 is a graph showing representative results of mouse running wheel behavior. FIG. 1A is a graph of running wheel behavior results when changing illumination periods, and FIG. 1B is a graph of phase shift 50% time statistics to the right of FIG. 1B; fig. 1C shows the phase shift statistics during the initial state of the illumination schedule, and fig. 1C shows the phase shift 50% time statistics of fig. 1C.

Fig. 2 is a graph of representative results of sleep behavior of mice. Fig. 2A is a graph of the results of the sleep behavior of mice when the illumination period is changed, and fig. 2B is a graph of the results of the real-time fluorescent quantitative PCR showing the changes of the sleep-related genes.

FIG. 3 is a graph of the detection results of Smo knockout mice, FIG. 3A is a Western Blot to detect Smo knockout effects, and FIG. 3B is a graph of the mouse physical properties and weight statistics; FIG. 3C is a graph of the mouse brain physical and statistical results of brain length and width; fig. 3D is a graph of the detection results of the microscopic nisation, and fig. 3E is a graph of the detection results of the microscopic neuronal staining.

FIG. 4 is a graph of test results of the effect of Vismodigib on biorhythms, FIG. 4A is a graph of the effect of Vismodigib on the oscillatory changes in the rhythmic genes of the SCN region at the tissue level; FIG. 4B is a graph showing mouse race behavior in the reverse-time difference model after injection of Vismodigib; FIG. 4C is a graph showing the phase shift of the mice in FIG. 4B after 8 hours in advance of the illumination schedule; FIG. 4D is a graph showing the results of monitoring the sleep behavior of mice in the time-of-fall model after injection of Vismodigib; FIG. 4E shows the process of oscillating changes in different neuronal rhythmic genes after adding Vismodigib to SCN tissue sections, wherein red and green in the upper heat map represent the highest and lowest peak of Per2 expression level, respectively; the lower phase distribution map is the statistics of different neuron phases of the upper heat map.

FIG. 5 is a graph showing the results of the effects of Hedgehog pathway inhibitors on biological rhythms, and FIG. 5A is a graph showing the effects of Purmorphamine, PF5274857 on the oscillatory changes in the rhythmic genes in the SCN region at the tissue level; FIG. 5B is a graph showing statistics of oscillation periods of FIG. 5A; FIG. 5C is a graph showing that Menndazole, ciliobrevin A, SANT-1 affect the rhythmic gene concussion changes of the SCN region at the tissue level; FIG. 5D is a graph showing statistics of oscillation periods of 5C; FIG. 5E is a graph of Taladegib, glasdegib influence on the rhythmic gene concussion changes of the SCN region at the tissue level; FIG. 5F is a graph showing statistics of oscillation periods of 5E; FIG. 5G is a graph of Cyclopamine, itraconazole influence on the rhythmic gene concussion changes of the SCN region at the tissue level; FIG. 5H is a graph showing statistics of the oscillation period of 5G; FIG. 5I is a graph of the oscillating changes in GANT61, JK184, robotnikin affecting the rhythmic genes of the SCN region at the tissue level; fig. 5J is a graph showing statistics of the oscillation period of 5I.

FIG. 6 is a graph showing the results of Sonidegib Phoshate on biological rhythms, and FIG. 6A is a graph showing Sonidegib Phoshate on the influence of the oscillating changes in the rhythmic genes in the SCN region at the tissue level; fig. 6B is a graph showing the statistics of the oscillation period of fig. 6A.

Detailed Description

The present application is further described in terms of the following examples, which are given by way of illustration only, and not by way of limitation, of the present application, and any person skilled in the art may make any modifications to the equivalent examples using the teachings disclosed above. Any simple modification or equivalent variation of the following embodiments according to the technical substance of the present application falls within the scope of the present application.

Materials and reagents for use in the present application

1. Per2:: luc mice: the method is presented for the Beijing life science research institute. Namely, the materials are described in Ju et al chemical Perturbations Reveal That RUVBL2 Regulates the Circadian Phase in Mammals. Sci. Transl. Med.12, eaba0769 (2020)' in the text, the public is available from the applicant and can be used for repeating the experiment of the present application as far as possible, but cannot be used for the other purpose; smo-loxp mice were purchased from Jackson lab under the accession number 004526; NMS-Cre mice were purchased from Jackson lab under the accession number 027205.

2. Reagent(s)

TABLE 1 reagents used in the application

	Reagent(s)	Manufacturer(s)	Goods number
				1	Smo antibodies	ABclonal	A3274
2	alpha-Tubulin antibodies	MBL Co Ltd	PM054
				3	Luciferin	Promega Co Ltd	E1602
4	DMEM medium and HBSS buffer	GIBCO Co Ltd	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	Xinbo Shengsheng (euphoria and euphoria)	AG-CR1-0069-M001
16	Mebendazole	MCE	HY-17595
				17	Purmorphamine	Alatine	P126030
18	Sonidegib Phoshate	MCE	HY-16582

Note that: 5-18 in the above table are Shh pathway related small molecule drugs.

Universal experimental method

Western blot detection knockout effect

SCN brain slices of Nms-Smo-/-mice are collected, ground into single cells and cell whole proteins are extracted for Western blot experiments to detect knockout effects.

(1) SDS-PAGE gel preparation: preparing 10% concentration glue according to the molecular weight of the protein to be detected;

(2) Loading: loading 30 μg protein per well;

(3) Electrophoresis: concentrating the gel, and keeping the constant pressure at 80V; the gel was separated and the pressure was constant at 120V. Running the front edge of bromophenol blue to the edge of the glue for glue discharging;

(4) Transfer printing: the filter paper (4 sheets per gel), fibrous pad, nitrocellulose membrane, SDS-PAGE gel required for Transfer were equilibrated in a 1 Xtransfer Buffer for 10 minutes. (PVDF, a hydrophobic membrane, requires special handling prior to use: methanol wet out for about 10 s). The prepared transfer sandwiches were placed in a transfer electrode box (note positive and negative), and transfer was started by adding transfer buffer and ice box. In the transfer printing process, the whole transfer printing groove is placed in an ice bath, and 400mA is transferred for 2 hours;

(5) Closing: after the transfer printing is finished, taking out the transfer printing sandwich, putting the transferred nitrocellulose membrane into a sealing liquid (5% skimmed milk) prepared in advance by using tweezers, and incubating and sealing for 1 hour at room temperature;

(6) An antibody: incubation with Smo, α -tubulin primary antibody, respectively, was performed overnight at 4 ℃, followed by 3 washes of TBST for 5 minutes each;

(7) And (2) secondary antibody: incubating with the secondary antibody corresponding to the primary antibody for 1 hour at room temperature, and then washing the membrane with TBST three times for 5 minutes each time;

(8) Developing: taking out the washed nitrocellulose membrane, draining TBST carried on the dry membrane as much as possible, putting the nitrocellulose membrane on a preservative film with the right side facing upwards, uniformly mixing equal amounts of ECL reagent A, B liquid taken out in advance, spreading the ECL reagent A, B liquid on the membrane drop by drop, and developing;

2. mouse SCN brain tissue section separation and culture

1. Mouse SCN brain tissue section separation

A. Anesthesia is carried out on the mice in 8-10 weeks, and when the mice lose consciousness but have not stopped breathing, the mice are rapidly broken by scissors;

B. the eyes of the mice are removed by scissors to prevent the optic nerve from activating in the subsequent process and further destroy SCN;

C. the mouse skull was cut off along both sides with scissors, and all bones were removed until visible in the olfactory bulb. Avoid squeezing the brain, prevent damage to ventral SCN;

D. cutting off the junction of the olfactory bulb and the optic nerve by using an anatomic scissors to ensure that the optic nerve is completely cut off;

E. the head was inverted, and the whole brain was allowed to fall to a full charge with pre-chilled HBSS buffer (1 x HBSS,

10mM HEPES，4.5mM NaHCO ₃ 1% Penicillin-Streptomycin) in a 10cm dish, the brain was kept at HBSS for 30-60 seconds to ensure cooling of the brain;

F. transferring the brain to a new culture dish by a spoon, and cutting off the cerebellum by a sterile surgical knife;

G. coating a strong adhesive on a cutting platform of the Vibratome vibrating slicer;

H. carefully wiping off excess HBSS at the brain incision with sterile filter paper;

I. the incision is downward, the brain tissue is fixed on a cutting platform, transferred to the cutting platform, and pre-cooled HBSS buffer solution is poured;

J. to quickly reach the SCN area, a 800 μm thick rapid cut, a decrease in speed at the beginning of reaching the hypothalamus, a decrease to 300 μm thick at the time of seeing the optic crossing nerve, and a continued cut;

K. after the SCN became visualized, the layer was cut 300 μm and brain sections were transferred with a soft brush into a petri dish with pre-chilled HBSS buffer and the SCN area was observed under a microscope;

and L, cutting SCN area under a stereoscopic microscope, removing optic cross nerve (OC), and separating out SCN brain slices with 1X 1mm tiny area.

SCN brain tissue section in vitro culture

A. Transferring the separated SCN to a Milicell plug-in cell culture dish for culture;

B. 1.2ml of DMEM medium (100. Mu.M luciferin,1% B27 serum free additive, 1% Penicillin-Streptomycin) was added and recorded in a LymiCycle apparatus.

SCN brain tissue single cell imaging

B. 1.2ml of DMEM medium was added (1mM luciferin,1%B27 without serum additives, 1% Penicillin-Streptomycin), the dishes were sealed and placed on an inverted microscope stage with a 10-fold objective (Nikon Eclipse Ti-E) in the dark, and the stage temperature was set at 36℃throughout the experiment. A CCD camera (EA 4710V-BV, raptor, uk) operating at-80 ℃ was mounted on the microscope for image capture;

C. strictly avoiding light, setting exposure time to be 60 minutes, and continuously collecting images;

D. 2. Mu.M TTX or 10. Mu.M Vismodigib was dissolved and added to the pre-heated fresh medium.

Nms-Smo-/-deficient mice time-of-fall experiment

A. Female mice of 8 weeks of age were individually placed in cages equipped with autonomous running wheels, and given sufficient food and drinking water;

b.12 hours illumination: light cycles of 12 hours darkness (07:00 on for early, 19:00 off for late) acclimate the mice for 14 days and record the movement of the mice;

C. on day 15, the lamp is turned on for 8 hours in advance, the time difference condition (23:00 on at night and 11:00 off at am) is simulated, and the time period of the mice adapting to the new photoperiod is evaluated;

D. after 14 days of recording under the new photoperiod, the photoperiod was adjusted to the original time (lamp on 07:00 early, lamp off 19:00 late) and mice were evaluated for the length of time to accommodate the original photoperiod.

Smo inhibitor Vismodigib can accelerate rapid adaptation of wild mouse fall time difference

b.12 hours illumination: light cycles of 12 hours darkness (07:00 on for early, 19:00 off for late) acclimate the mice for 7 days, and record the movement of the mice;

C. on day 8, smo inhibitor drugs were continuously delivered to SCN by a slow release pump using a brain stereotactic apparatus, the operation was resumed for 3 days, the lights were turned on for 8 hours in advance, the reverse time difference conditions were simulated (23:00 on at night, 11:00 off at am), and the time period for the mice to adapt to the new photoperiod was evaluated.

Example 1 behavioural manifestations of SCN-region specific knockout Smo mice in changes in photoperiod

Adult female mice of 8 weeks of age were individually housed in a cabinet fitted with a roller cage and roller behavior of the mice was recorded and analyzed in real time using a ClockLab. FIG. 1A is a representative result of the wheel running behavior of a corresponding mouse, wherein after the mouse is acclimated for two weeks through a standard illumination period (7:00 on in the morning and 19:00 off in the evening), the illumination schedule is advanced for 8 hours (23:00 on in the evening and 11:00 off in the morning), the illumination schedule is returned to an initial state after 15 days of observation, the wheel running behavior of the mouse is represented by black marks, and a white background and a gray background represent illumination and darkness respectively; FIG. 1B is a graph showing the phase shift statistics of the corresponding mice in the 8h advance of the illumination schedule; fig. 1C is a phase shift statistic result of a corresponding mouse during a light schedule recovery initial state.

The results show that after the illumination period is changed, the Smo condition knockout mice have an abnormal inverted time difference phenotype, and can adapt to a new illumination period more quickly.

Example 2 sleep monitoring of SCN-region specific knockout Smo mice in changes in photoperiod

In accordance with the above, the running wheel behaviors of the mice in the change of the illumination period are recorded in real time by using a ClockLab, the sleeping behaviors of the mice are analyzed by using ClockLab Analysis software (whether the mice are in a sleeping state or not is judged according to the intensity of the movement behaviors of the mice), the sleeping behaviors of the mice are represented by black marks in the figure, and a white background and a gray background represent illumination and darkness respectively; FIG. 2B shows the results of real-time fluorescence quantitative PCR corresponding to the changes of the mouse sleep-related genes.

The result shows that after the illumination period is changed, the sleep behavior of the Smo condition knockout mouse is improved, and the mouse can adapt to a new illumination period more quickly.

Example 3 identification of SCN region specific knockout Smo mice

NMS-Cre is a SCN region specific expression Cre tool mouse, smo is an important receptor of Shh signal path, and NMS-Smo-/-mice are obtained through mating NMS-Cre mice with Smo-floxp mice and multiplying for multiple generations.

FIG. 3A shows the effect of Western Blot detection on Smo knockdown (Smo is not expressed only in SCN region NMS neurons, so the results show that there is still a small amount of detection after specific Smo knockdown in SCN region; figure 3B shows Smo conditioned knockout mice body weight is unaffected; FIG. 3C shows that the whole outline of the brain of Smo conditional knockout mice is normal; FIGS. 3D and 3E show that Smo conditional knockout mice have normal neuronal differentiation in the SCN region.

The results prove that the Smo mouse with the specific nuclear SCN region knocked out on the optic cross nerve of the hypothalamus region is successfully constructed, and the whole mouse is normally developed.

Example 4, smo inhibitor Vismodegib affects mouse rhythmic behavior

The result shows that the Vismodigib can directly influence the concussion change of a rhythm gene, blocks the coupling process of SCN neurons and enables a mouse to adapt to the time difference change process more quickly. FIG. 4A shows that rhythmic genes affect the concussive changes of the rhythmic genes in the SCN region at the tissue level, per2 was freshly isolated by using Luc mouse SCN region brain chips for in vitro culture and continuously recording the Per2 expression level with lumicle; three days after recording, the medium was changed to fresh medium containing 20. Mu.M of Vismodigib, which was observed to significantly inhibit the Per2 shock change process; after three days, the drug was removed and changed to fresh medium, and Per2 concussion was found to be recovered, indicating that the effect of Vismodigib was not caused by drug toxicity. FIG. 4B is a graph showing mouse race behavior in the reverse-time difference model after injection of Vismodigib; FIG. 4C is a graph showing the phase shift of the mice in FIG. 4B after 8 hours in advance of the illumination schedule; FIG. 4D is a graph showing the results of sleep behavior analysis of mice in the time-of-fall model after injection of Vismodigib; FIG. 4E shows the process of varying the concussion of different neuronal rhythms after the addition of Vismodigib to SCN tissue sections, and the upper heat map results show that the process of varying the expression level of different neurons Per2, wherein red and green represent the highest peak and the lowest peak of the expression level of Per2 respectively, and the concussion of different neurons Per2 before and after the addition of drugs is consistent and gradually disordered; the lower phase distribution diagram is the statistics of oscillation change phases of different neurons of the upper heat diagram, and each point represents the corresponding phase of a single neuron.

The above results demonstrate that Vismodegib is able to improve mouse fall-time behavior.

Example 5 influence of Hedgehog pathway inhibitor on biological rhythms

The brain slice of the SCN region of the Luc mouse is used for in vitro culture, and the expression level of Per2 is continuously recorded by using lumicle; after about three days of recording, the medium was changed to fresh medium containing Hedgehog pathway inhibitors, and the effect of different Hedgehog pathway inhibitors on the oscillatory changes in Per2 expression was observed.

FIG. 5A is a graph of 2. Mu.M Purmorphamine, 1. Mu.M PF5274857 affecting the rhythmic gene concussive changes in SCN region at tissue level; FIG. 5B is a statistical result of the oscillation period of 5A;

FIG. 5C is a graph of the oscillating changes of 20. Mu.M Mebendazole, 60. Mu.M Ciliobrevin A, 100. Mu.M SANT-1 at the tissue level affecting the rhythmic genes of the SCN region; FIG. 5D is a statistical result of the oscillation period of 5C;

FIG. 5E is a graph of 10. Mu.M Taladegib, 20. Mu.M Glasdigib, at the tissue level affecting the rhythmic gene concussion changes in the SCN region; FIG. 5F is a statistical result of the oscillation period of 5E;

FIG. 5G is a graph of 20. Mu.M Cyclopamine, 25. Mu.M Itraconazole at tissue level affecting the rhythmic gene concussion changes in the SCN region; FIG. 5H is a statistical result of the 5G oscillation period;

FIG. 5I is a graph of 10. Mu.M GANT61, 2. Mu.M JK184, and 10. Mu.M Robotnikin affecting the rhythmic gene concussion changes of the SCN region at the tissue level; fig. 5J is a statistical result of the 5I oscillation period.

FIG. 6A is a graph showing that different concentrations Sonidegib Phoshate affect the rhythmic gene concussion changes in the SCN region at the tissue level; fig. 6B is a statistical result of the oscillation period of 6A.

The results prove that different Hedgehog pathway inhibitors can influence the Per2 gene concussion change process, and are expected to be used for regulating biological rhythms and treating rhythmic disorder diseases.

Claims

Application of shh pathway inhibitor in improving sleep behavior and preparing medicine for treating biological rhythm related diseases.
2. The use of claim 1, wherein the shh pathway inhibitor comprises PF-5274857, mebendazole, HPI-4, SANT-1, taladegib, glasdegib, cyclopamine, itraconazole, GANT61, JK184, robotnikinin, vismodegib, purmorphamine, sonidegib Phoshate, or a pharmaceutically acceptable salt of the above compound.
3. The use according to claim 1, wherein the biorhythm-related diseases include sleep disorders, jet lag adjustment, depression, metabolic disorders, aging, blood diseases, diabetes and obesity.
4. The use of claim 1, wherein the improving sleep performance comprises faster adaptation to new lighting cycles, or reducing abnormalities in mental and physiological functions of the body caused by changes in lighting cycles.
5. The use of claim 1, wherein the shh pathway inhibitor comprises an SMO inhibitor;

preferably, the SMO inhibitor comprises an agent used to knock out SMO using any one of the following gene knockout techniques: siRNA interference technology, CRISPR technology, TALEN technology, ZFN technology, cre-loxP recombination technology.
6. The use according to claim 5, wherein the Smo inhibitor is an agent used in the Cre-loxp recombination technique.
7. A method of constructing an animal model of a biorhythm abnormality, the method comprising the use of shh pathway inhibitors for model organisms, the biorhythm abnormality being manifested by improved sleep behavior, rapid adaptation to photoperiod;

preferably, the shh pathway inhibitor comprises PF-5274857, mebendazole, HPI-4, SANT-1, taladegib, glasdegib, cyclopamine, itraconazole, GANT61, JK184, robotnikinin, vismodegib, purmorphamine, sonidegib Phoshate, or a pharmaceutically acceptable salt of the above;

preferably, the shh pathway inhibitor comprises a SMO inhibitor;

preferably, the SMO inhibitor comprises an agent used to knock out SMO using any one of the following gene knockout techniques: siRNA interference technology, CRISPR technology, TALEN technology, ZFN technology, cre-loxP recombination technology.
8. The method of construction according to claim 7, wherein the method comprises the steps of:

1) Obtaining an animal model of specific expression Cre of the SCN region;

2) Obtaining an animal model expressing Smo-loxp;

3) Mating the animal models of 1) and 2).
9. The method of claim 7, wherein the model organism comprises a rat or a mouse.
10. Use of the animal model of claim 7 for studying Shh pathway modulation biorhythms.