CN118662636B

CN118662636B - Application of G protein-coupled receptor ADGRA3 and hesperetin in the preparation of drugs promoting fat browning

Info

Publication number: CN118662636B
Application number: CN202410610150.8A
Authority: CN
Inventors: 杨中汉; 赵泽伟; 李锦�; 江涛; 李肖肖; 胡龙云; 宋必贵
Original assignee: Sun Yat Sen University; Sun Yat Sen University Shenzhen Campus
Current assignee: Sun Yat Sen University; Sun Yat Sen University Shenzhen Campus
Priority date: 2024-05-16
Filing date: 2024-05-16
Publication date: 2025-09-30
Anticipated expiration: 2044-05-16
Also published as: CN118662636A

Abstract

The invention discloses an application of a G protein coupled receptor ADGRA and hesperetin in preparing a medicament for promoting white fat browning, and the research of the invention shows that the hesperetin and the over-expression ADGRA have the effect of promoting the white fat browning, so the invention firstly provides an application of the G protein coupled receptor ADGRA3 serving as a medicament target in preparing the medicament for promoting the white fat browning. Further, the invention further provides the application of the hesperetin in preparing the medicine for promoting the white fat browning, and provides more directions for the medicine development for treating metabolic diseases such as obesity and the like.

Description

Application of G protein coupled receptor ADGRA and hesperetin in preparation of medicine for promoting fat browning

Technical Field

The invention belongs to the technical field of biological medicines, and particularly relates to an application of a G protein coupled receptor ADGRA and hesperetin in preparation of a medicine for promoting white fat browning.

Background

The metabolic syndrome such as obesity, hyperglycemia and the like is a group of clinical syndrome characterized by insulin resistance and abnormal multi-metabolic components, the syndrome is easy to cause the incidence risk of metabolic diseases such as obesity, type II diabetes and the like, and the obesity is one of the most serious public health problems worldwide at present. The main cause of obesity is an imbalance in energy intake and expenditure. In the current clinical treatment, the effects of reducing weight and improving metabolic homeostasis are realized mainly through intervention of dietary structures and life styles of patients and treatment of medicines and operations, however, the treatment method has longer time consumption and limited effects, and other adverse reactions of the body are easily caused by using medicines and operations, so that the prior art has popularization and application limitations on the treatment of obesity.

Past epidemiological studies have shown that excessive fat mass is closely related to a higher incidence of metabolic diseases. Thus, people have previously ascribed excessive fat accumulation and ectopic deposition as the only causes of obesity and related metabolic diseases. However, in fact, there are significant differences between individuals, and some obese people remain metabolically healthy, and some people with normal body weight exhibit significant metabolic disturbances. In addition, malnourished patients have a low fat content but suffer from a number of metabolic diseases similar to those of severely obese patients. With the recent intensive research into adipose tissue, it has been found that adipose tissue is an endocrine and immune organ having high heterogeneity and plasticity. Adipose tissue has different cellular compositions and structures at different sites, which also results in different metabolic functions at each site. Adipocytes mainly comprise white and brown adipocytes, white adipose tissue can be transformed into beige adipose tissue after cold stimulation, with heat and energy generating effects resembling brown adipose tissue, a process known as lipobrowning. Whereas uncoupling protein 1 (UCP 1) is one of the accepted molecular markers of lipofuscation, its up-regulation of expression is considered to be a classical manifestation of induction of lipofuscation. The method searches more markers which can induce or promote the brown formation of the fat cells, and can provide a new idea for the treatment of metabolic diseases such as obesity and the like.

Disclosure of Invention

The invention aims to provide an application of G protein coupled receptor ADGRA and hesperetin in preparing a medicine for promoting white fat browning.

The above object of the present invention is achieved by the following technical solutions:

The invention firstly provides application of G protein coupled receptor ADGRA as a target in preparation of a medicament for promoting white fat browning.

In the experiment at the cellular level, the invention researches the action mechanism of the hesperetin for inducing the beige of the adipocytes by activating the PKA signal path, namely, the hesperetin activates the PKA signal path and up-regulates the UCP1 expression level in the adipocytes, and the PKA signal path inhibitor (PKAi) or the knock-down ADGRA3 can block the effects of the hesperetin on activating the PKA signal path and up-regulating the UCP1 expression level of the adipocytes. In addition, in experiments at animal level and cell level, the invention also researches and obtains the action mechanism of over-expression ADGRA3 for inducing the beige of fat cells by activating PKA signal channels, namely over-expression ADGRA3 activates PKA signal channels and up-regulates UCP1 expression levels in fat cells, and PKA signal channel inhibitor (PKAi) can block the effect of over-expression ADGRA3 for activating PKA signal channels and up-regulating UCP1 expression levels of fat cells.

In conclusion, through a large number of experiments, the invention proves that the hesperetin and the over-expression ADGRA have the effect of promoting the brown white fat, and the hesperetin plays the effect of promoting the brown white fat through the G protein coupled receptor ADGRA 3.

Thus, the present invention provides the use of a formulation that promotes ADGRA expression in the manufacture of a medicament that promotes browning of white fat.

Preferably, the agent that promotes ADGRA3 expression comprises a Adgra3 gene expression promoter.

Preferably, the Adgra gene expression promoter comprises a Adgra3 overexpression vector.

Preferably, the vector is a plasmid vector or a viral vector.

Preferably, the Adgra3 over-expression vector is a pLV3-CMV-Adgra3-3×FLAG-CopGFP-Puro over-expression plasmid.

Preferably, the preparation method of the pLV3-CMV-Adgra3-3 xFLAG-CopGFP-Puro over-expression plasmid comprises the steps of cloning a CDS sequence of a Adgra3 gene onto a pLV3-CMV-3 xFLAG-CopGFP-Puro plasmid skeleton, constructing and obtaining the pLV3-CMV-Adgra3-3 xFLAG-CopGFP-Puro over-expression plasmid, wherein the CDS sequence of the Adgra3 gene is shown as SEQ ID No. 1.

The invention also provides the use of hesperetin in the preparation of an agonist of the PKA signaling pathway.

Preferably, the hesperetin acts as an agonist of the PKA signaling pathway which activates the PKA-CREB signal to exert an effect of promoting browning of white fat.

The invention also provides application of the hesperetin in preparation of a preparation for promoting UPC1 expression.

The invention also provides application of the hesperetin in preparation of a medicament for promoting white fat browning.

A medicament for promoting browning of white fat, the medicament comprising an effective amount of a formulation that promotes ADGRA expression and/or hesperetin.

The medicament may be a single medicament, a pharmaceutical composition or a kit/kit containing an effective amount of a formulation that promotes ADGRA expression and/or hesperetin, together with a pharmaceutically acceptable carrier. By "effective amount" is meant "an amount of the disclosed drug that is effective at the dosages and for periods of time necessary to achieve the desired result or therapeutic result. The effective amount may vary depending upon factors known in the art, such as the disease condition, age, sex, and weight of the human or animal being treated. It will be appreciated by those skilled in the art that the dosage regimen may be varied to provide the optimal therapeutic response. For example, several separate doses may be administered daily or the dose may be proportionally reduced as indicated by the urgency of the treatment situation. In addition, the disclosed medicaments may be administered at a desired frequency to achieve a therapeutically effective amount. The medicament may be for use in a patient or other animal receiving a medicament of the application for the treatment, prevention, alleviation and/or relief of a disease or a condition of the present application, and in a preferred embodiment the medicament or nutraceutical is for use in a mammal. Including but not limited to humans, cows, horses, sheep, pigs, goats, rabbits, cats, dogs, mice, and any other mammal having a liver and which may be damaged.

In a preferred embodiment, the medicament is for use in humans.

The pharmaceutically acceptable carrier refers to a carrier for administration of the therapeutic agent, and includes various excipients and diluents. Including, but not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, starches, gum acacia, calcium phosphate, alginates, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, mineral oil and the like. The medicine of the present application may contain, in addition to the above-mentioned components, a lubricant, a wetting agent, a sweetener, a flavoring agent, an emulsifier, a suspending agent, a preservative, and the like.

The medicament may be administered in unit dosage form by the enteral or parenteral route, such as oral, intramuscular, subcutaneous, nasal, oral mucosal, dermal, peritoneal or rectal etc. For example, tablets, capsules, dripping pills, aerosols, pills, powders, solutions, suspensions, emulsions, granules, liposomes, transdermal agents, buccal tablets, suppositories, freeze-dried powder injection and the like. Can be common preparation, slow release preparation, controlled release preparation and various microparticle administration systems.

The Adgra over-expression vector refers to cloning Adgra gene coding region onto corresponding vector, and utilizing regulatory elements constructed on vector skeleton to make gene realize mass transcription and translation under artificial control condition, thus realizing over-expression of target gene, the vector includes but is not limited to plasmid vector, virus vector (mammal) and siRNA expression vector.

Preferably, the vector is a plasmid vector or a viral vector.

Compared with the prior art, the invention has the following beneficial effects:

the study of the invention shows that the hesperetin and the over-expression ADGRA < 3 > have the effect of promoting the brown of white fat, so the invention firstly provides the application of the G protein coupled receptor ADGRA < 3 > serving as a drug target in the preparation of the drug for promoting the brown of white fat. Further, the invention further provides the application of the hesperetin in preparing the medicine for promoting the white fat browning, and provides more directions for the medicine development for treating metabolic diseases such as obesity and the like.

Drawings

FIG. 1 is a graph showing that hesperetin Hes induces white fat browning through ADGRA-PKA signaling axis. The method comprises the steps of (A) inducing intracellular cAMP levels of mature adipocytes under different treatment conditions to form 3T3-L1, (B) inducing mRNA levels of Ucp1 of mature adipocytes under different treatment conditions to form 3T3-L1, (C) inducing protein levels of UCP1, p-CREB and CREB of mature adipocytes under different treatment conditions to form 3T3-L1, (D) inducing mRNA levels of Adgra and Ucp1 of mature adipocytes under different treatment conditions to form 3T3-L1, (E) inducing protein levels of ADGRA, UCP1, p-CREB and CREB of mature adipocytes under different treatment conditions to form 3T3-L1, and (F) inducing mRNA levels of Ucp1 of mature adipocytes under different treatment conditions to form 3T3-L1, (G) inducing protein levels of UCP1, p-CREB and CREB of mature adipocytes.

FIG. 2 is ADGRA inducing white fat browning. The method comprises the steps of (A) changing mRNA levels of Adgra3 and Ucp1 after the adipocytes are overexpressed Adgra3, (B) changing protein levels of ADGRA-3 xFLAG and UCP1 of mature adipocytes induced by 3T3-L1 under different treatment conditions, (C) changing the body temperature of a ADGRA3 overexpressed mouse, (D) performing thermal imaging and brown adipose tissue temperature on a ADGRA3 overexpressed mouse, (E) changing the protein levels of ADGRA-3 xFLAG and UCP1 of a ADGRA3 overexpressed mouse inguinal white adipose tissue, F) changing the protein levels of ADGRA-3 xFLAG and UCP1 of a ADGRA overexpressed mouse brown adipose tissue, G performing hematoxylin-eosin staining of a ADGRA3 overexpressed mouse inguinal white adipose tissue and a brown adipose tissue, and (H) performing UCP1 immunohistochemical staining of a ADGRA overexpressed mouse inguinal white adipose tissue and a brown adipose tissue.

FIG. 3 is a schematic diagram of the mechanism by which hesperetin Hes and ADGRA induce fat browning.

Detailed Description

The invention is further illustrated in the following drawings and specific examples, which are not intended to limit the invention in any way. Unless specifically stated otherwise, the reagents, methods and apparatus employed in the present invention are those conventional in the art.

Unless otherwise specified, reagents and materials used in the following examples were commercially available, with hesperetin being purchased from Shanghai Meilin Biochemical technologies Co., ltd.

English name HESPERETIN

CAS number 520-33-2

Molecular formula C ₁₆H₁₄O₆

Molecular structure: Example 1 validation of hesperetin Hes induces white fat browning through ADGRA-PKA signaling axis

To verify whether hesperetin exerts an effect of promoting browning of white fat, we treated mature adipocytes induced by 3T3-L1 with 10 μm hesperetin for 24h and examined the same. The specific experimental method is as follows:

(1) ELISA detects intracellular cAMP levels of mature adipocytes induced by 3T 3-L1:

Samples were collected by diluting the cell suspension with PBS to achieve a cell concentration of 100 tens of thousands/mL. Further disrupting the cells by sonication to destroy and release intracellular components, centrifuging at 3000rpm for 20 minutes at 4 ℃, and carefully collecting the supernatant;

preparing solution, namely preparing cAMP standard substance solutions of 0nM,2nM,4nM,8nM,16nM and 32nM, respectively adding standard substance solutions with different concentrations and a sample to be detected into the bottom of an enzyme-labeled plate hole, and slightly shaking and uniformly mixing;

Incubation, namely placing a 37 ℃ constant temperature incubator at the rear of a sealing plate membrane sealing plate for 30 minutes;

carefully tearing the sealing plate membrane, discarding the liquid, spin-drying, filling PBS in each hole, standing for 30 seconds, discarding, repeating the steps for 5 times, and beating;

adding enzyme, namely adding 50 mu L of enzyme-labeled reagent into each hole;

Color development, namely adding 50 mu L of the color developing agent A into each hole, adding 50 mu L of the color developing agent B, gently shaking and uniformly mixing, and developing for 10 minutes at 37 ℃ in a dark place;

stop, adding 50 mu L of stop solution into each hole;

The absorbance of each well was measured sequentially at a wavelength of Kong Diaoling, 450nM for a 0nM standard, and the cAMP concentration of the sample to be measured was calculated from the concentrations of each standard.

(2) MRNA level detection of mature adipocytes:

RNA extraction, namely extracting RNA of the mature adipocytes after treatment by using a total RNA extraction reagent (BS 258A), wherein the extraction method refers to the instruction of the extraction reagent;

Reverse transcription, namely reverse transcribing the obtained RNA into cDNA by using a tubular reverse transcription kit (BL 696A), wherein the specific experimental method is shown in the specification of the kit;

mRNA level detection, namely, carrying out related detection on cDNA obtained by reverse transcription by using a universal fluorescent quantitative PCR kit (BL 697A), wherein a specific experimental method is shown in a kit instruction, and finally calculating the mRNA level of mature adipocytes under different treatment conditions.

(3) Protein level detection of mature adipocytes:

The treated 6-well plate was placed on ice, washed 3 times with PBS, and then added with an appropriate amount of 1 XRIPA lysis buffer (CST). Placing at room temperature for 2min, scraping the wall of a dish with a cell scraper, collecting lysis buffer in a 1.5ml centrifuge tube, boiling at 95 ℃ for 10min to thoroughly denature the Protein, quantifying the collected Protein concentration with a Kevlar BCA Protein quantification kit, preparing a Protein upper sample according to the proportion of adding 1 mu L of Protein buffer (beta-mercaptoethanol, 0.4% bromophenol blue) into each 9 mu L of denatured Protein sample, boiling at 95 ℃ for 10min, taking a prepared SDS-PAGE gel (prepared by 10% separating gel and 5% concentrating gel), adding 20 mu g/hole of the upper sample, adding Protein LADDER MARKER, carrying out 80V electrophoresis on the concentrated gel part, changing the voltage to 120V when the bromophenol blue is run to the separating gel, and stopping electrophoresis when the bromophenol blue is run to a proper position. Placing PVDF film on the surface of PAGE gel, transferring protein on the PAGE gel to PVDF film by 300mA constant flow transfer film for 80min, closing PVDF film by 7% milk for 2h after the end of the electrotransfer, reducing non-specific background on PVDF film, adding target protein antibody and placing on 4 deg.C shaking table for shaking overnight, adding corresponding secondary antibody and placing on 4 deg.C shaking table for shaking 4h. Washing the membrane with TBST for 3 times and 10 min/time, and adding ECL hypersensitive chemiluminescence liquid onto PVDF membrane for development.

Analysis of results:

As a result, as shown in FIG. 1, FIG. 1 (A) shows intracellular cAMP levels of mature adipocytes induced by 3T3-L1 under different treatment conditions, and it can be seen that hesperetin promotes the production of intracellular cAMP in adipocytes. FIGS. 1 (B) and (C) show that hesperetin increases the expression levels of UCP1 and p-CREB, respectively, as a result of the mRNA level of Ucp1 and the protein levels of UCP1, p-CREB and CREB of mature adipocytes induced by 3T3-L1 under different treatment conditions. The above results demonstrate that hesperetin is capable of inducing a white fat browning.

Example 2 verifies that hesperetin exerts an effect of promoting browning of white fat by activation of PKA-CREB signal by ADGRA3

To verify whether hesperetin exerts an effect of promoting white fat browning by ADGRA activation of PKA-CREB signal, we knockdown (sh Adgra 3) mature adipocytes with ADGRA3 by treatment with 10 μm hesperetin for 24 h. The specific experimental method is as follows:

(1) ADGRA3 knocking down mature adipocytes:

Constructing knock-down plasmids, namely constructing knock-down plasmids with shNC sequences and sh Adgra sequences respectively by using PCR by using a plKO.1-U6-scramble-EF1 a-copGGFP-T2A-puro plasmid skeleton;

Transfected cells knockdown ADGRA. The above knockdown plasmid was transfected into mature adipocytes using Lipo8000 transfection reagent (purchased from Shanghai Biyun Tian Biotechnology Co.) for the purpose of knockdown ADGRA3, specific transfection steps were referenced in Lipo8000 transfection reagent specifications.

The shNC sequence and sh Adgra3 sequence are as follows:

shNC sequence:

CCTAAGGTTAAGTCGCCCTCGCTCGAGCGAGGGCGACTTAACCTTAG G;

sh Adgra3 sequence:

AGGAGTAGGAGAGCTTATTTACTCGAGTAAATAAGCTCTCCTACTCCT。

(2) mRNA level detection of mature adipocytes:

(3) Protein level detection of mature adipocytes:

Analysis of results:

The mRNA levels of Adgra and Ucp1 and protein levels of UCP1, p-CREB and CREB were found in mature adipocytes induced from 3T3-L1 under different treatment conditions (FIGS. 1D-E), and when Adgra3 was knocked down, the induction of UCP1 and p-CREB by hesperetin was eliminated. The above results demonstrate that hesperetin promotes white fat browning through ADGRA.

Example 3 demonstration that the promotion of white fat browning by hesperetin is dependent on PKA-CREB signaling

We used PKAi (PKA signaling pathway inhibitor) to verify whether the white fat browning promoting effect of hesperetin depends on PKA-CREB signal, treated mature adipocytes with H-89 at PKAi treatment condition of 10. Mu.M for 24H, treated mature adipocytes with hesperetin at 10. Mu.M for 24H, and tested mRNA and protein levels of mature adipocytes as follows:

(1) mRNA level detection of mature adipocytes:

(2) Protein level detection of mature adipocytes:

Analysis of results:

From the results of the protein levels of UCP1, p-CREB and CREB in Ucp's 1 mRNA levels of mature adipocytes induced by 3T3-L1 under different treatment conditions (FIG. 1F-G), hesperetin up-regulates UCP1 and p-CREB protein expression. However, when treated simultaneously with PKAi, the effect of hesperetin in up-regulating UCP1 and p-CREB protein expression was lost, suggesting that the effect of hesperetin in promoting white fat browning was dependent on PKA-CREB signal.

In combination with examples 1-3, the results indicated that hesperetin Hes induced a white fat browning through ADGRA-PKA-CREB signaling axis.

Example 4 verifies ADGRA that 3 has an effect of inducing browning of white fat

To verify whether ADGRA3 exerts an effect of promoting white fat browning, we overexpressed ADGRA3 (Adgra OE) on 3T3-L1 induced mature adipocytes and ADGRA3 (Adgra 3 OE) in mice and related assays. The specific experimental method is as follows:

(1) Adgra3 overexpression (Adgra 3 OE):

1) Cloning CDS sequence of Adgra gene of mouse to pLV3-CMV-3 xFLAG-CopGFP-Puro plasmid skeleton by using codon optimization technique and PCR technique to obtain pLV3-CMV-Adgra3-3 xFLAG-CopGFP-Puro over-expression plasmid;

2) Transfection the above-mentioned overexpression plasmid is used for transfecting mice or mature adipocytes by using Lipo8000 transfection reagent (purchased from Shanghai Biyun biotechnology company), and the aim of overexpression ADGRA is fulfilled, and specific transfection steps are referred to the Lipo8000 transfection reagent specification.

3) The CDS sequence of the Adgra gene after optimization is shown as SEQ ID No.1 (the optimized part is shown in capital letters):

ATGGAACCACCTCCACCGCTGCTTTTGTTGCCACTGGCTCTTTTGGCACTGTTGTGGGGCGGTGAAAGGGGAGCAGCTGCTTTGCCAGCAGGCTGCAAGCATGATGGACGGGCAAGAGGAACCGGAAGAGCTGCTGCTGCTGCTGAAGGAAAGGTGGTGtgcagcagcctggagctcgcgcaggttctgcccccggacacgctgcccaaccgcacggtcaccctgattttaagcaacaacaagatctccgagctgaagaatggttcattttctggcttaagtctcctcgaaagactggacctccggaacaaccttattagtaggatagccccaggtgccttttggggactgtcttcactgaagagattggacctgacgaacaaccgaataggttgtctgaatgcagatgtatttcgaggactcaccaatctggttcggctaaacctttcagggaatctgtttacttcactgtctcaaggaacttttgattatcttggctcgttgcggtctttagaatttcagactgagtaccttctgtgtgactgtaacattctgtggatgcatcgctgggtaaaggagagaaacatcactgtgcgagacaccaggtgtgtttatcccaagtcactgcaggcccagcctgtcacgggggtgaaacaggagctcctcacttgcgatcctccccttgaactgccgtccttctacatgactccgtcgcaccgccaggttgtgtttgaaggagacagccttcccttccagtgcatggcttcctatatagatcaggacatgcaagtgctgtggtatcaggatgggcgcattgttgagaccgatgagtcccaagggatctttgtggagaaaagcatgattcacaattgctccttgatcgccagtgccctaaccatttctaatattcaagctggatctactggaaattggggctgtcatgttcagacgaaacgtgggaataacacaagaactgttgacattgtggtattagaaagctccgcccaatactgtccaccagagagggttgtgaacaacaaaggtgatttcagatggcccaggacactggcgggcatcacagcatatctccagtgtacccggaacacccacagcagtgggatctaccctggaagcgcacaggatgaaaggaaggcgtggcgccgatgcgacagaggtggcttttgggcagatgatgattattctaggtgccagtatgcaaatgacgtcactagagtcctgtatatgtttaatcagatgcccctcaaccttacaaatgcggtcgctacagctcggcagctgctggcttacacagtggaggccgccaacttctctgacaaaatggacgttatatttgtggctgaaatgatagaaaagtttggaagatttaccagagaggaaaaatcaaaagagcttggtgatgtaatggtcgatgtggcaagcaacatcatgttggctgatgagcgggtcctgtggctggcacagagggaagcaaaggcctgcagtcggattgtccagtgcctgcagcgcattgccacacatcgcctggccagtggggcccacgtgtactccacgtactcgcccaacattgctctggaggcttacgtcatcaaggctgctggcttcacaggaatgacctgctccgtgttccagaaggtggctgcctccgaccgtgcaggtctttctgactatgggcgaagggacccggatggaaacctggataagcagctgagcttcaaatgcaatgtctccagcaccttctcaagcctggccctgaagaacaccatcatggaggcctccattcagcttccttcctcccttttgtcaccaaaacacaagcgagaagcccgagcggcggatgacgccctctataagctccagctcattgccttccgcaacggaaagctttttccagccacaggaaattcaacaaagttggcagacgatggcaagcggcggacagtagtgacccctgtgatcctcacgaaaatagatggtgcaaccgtagatacccaccacatccctgttaatgtgacgctgcgccgaattgcccacggagcagatgcggttgcagcgcagtgggactttgatttgctgaacggacaaggaggctggaagtcagatgggtgctgtatactctactcggatgagaacatcaccaccattcagtgcggctccctgggcaactatgctgtgctaatggatctgactgggacagagttgtacaccccagcagccagtctcctgcaccctgtggtttacaccactgccatcactctcctcttgtgtctcttggctgttatcatcagttacatgtaccaccacagcttgatccgaatcagtctcaagagctggcacatgcttgtgaacctgtgctttcacattctcctgacctgcgtggtgtttgtgggaggaataacccagaccagaaatgccagcgtctgtcaagcagttgggatcattcttcattattctacccttgccacggtattgtgggttggagtcactgctagaaatatctataaacaagtcaccaagaaagccaagagatgccaggatccagatgagccacccgctcctccacgaccgatgctgaggttctacctgattggtggtgggatccccatcatagtgtgtggtatcaccgcggcagcaaacatcaagaactatggcagtcggcccagtgcaccgtattgctggatggcctgggaaccgtccttgggagccttctacggacctgccagcttcatcacttttgtaaactgtatgtattttctaagcatatttattcagttgaaaagacaccctgagcgcaaatatgagctcaaggagccgacagaagagcagcagagattggcagccaatgaaaatggtgaaatcaaccatcaggactccatgtctctgtctctcatctctacgtccacgttggagaacgagcacagttttcagtctcagcttctgggcgccagccttactttgcttttgtatgtcatcttgtggatgtttggggccatggctgtttctctgtattaccctctggacttggtttttagcttcttcttcggagccacttgtttaagcttcagtgctttcatgatggtgcaccactgcatcaacagggaggacgtgagacttgcgtggatcatgatgtgctgcccagggcggagctcgtactccgtgcaagtcaacgtccaacctcccaactcaagcgccactaatggagaggctccaaagtgcaccaatagcagcgcagagtcttcgtgcacgaacaaaagcgcatcgagcttcaaaaactcttcccagggctgcaagctgacaaacttgcaggctgctgcggcacagtaccacagcaatgccctacctgtgaatgccacgccgcagcttgataacagtctgaccgaacactcgatggacaacgatattaaaatgcatgtggctcctttagacgtgcagtttcgaacaaacgtgcacccaagccgccaccacaaaaaccgaagtaaaggacaccgggccagcaggctcacagtcctgcgagagtatgcctatgacgtcccaacaagtgtggaaggaagcgtgcagaatggcttacctaaaagccggccaggcagcaatgaaggacattcaaggagtaggagagcttatttagcctacagagagagacagtacaacccaccccaacaagacagcagtgatgcttgtagcacacttcccaaaagtagccgaaatgttgaaaagcctgtttcaactagtagtaagaaagatgcaccaaggaagccagctgcagccgaccttgaaagtcagcagaaatcttacggcctgaacttggctgttcagaatggaccagttaaaagcaatgggcaggaaggacccttgctagctaccgacgtcactggtaatgttaggactgggttatggaaacacgaaacaactgtg

(2) Detection of mRNA levels of adipose tissue/mature adipocytes:

RNA extraction, extracting RNA of the treated adipose tissue/mature adipocytes using a total RNA extraction reagent (BS 258A), the extraction method being referred to the instructions of the extraction reagent;

mRNA level detection, namely, carrying out related detection on cDNA obtained by reverse transcription by using a universal fluorescent quantitative PCR kit (BL 697A), wherein a specific experimental method is shown in a kit instruction, and finally calculating the mRNA level of adipose tissue/mature adipocytes under different treatment conditions.

(3) Protein level detection of adipose tissue/mature adipocytes:

Cell samples the treated 6-well plate was placed on ice, washed 3 times with PBS, and then added with the appropriate amount of 1 XRIPA lysis buffer (CST). Placing at room temperature for 2min, scraping the wall of a dish with a cell scraper, collecting lysis buffer in a 1.5ml centrifuge tube, boiling at 95 ℃ for 10min to thoroughly denature the Protein, quantifying the collected Protein concentration with a Kevlar BCA Protein quantification kit, preparing a Protein upper sample according to the proportion of adding 1 mu L of Protein buffer (beta-mercaptoethanol, 0.4% bromophenol blue) into each 9 mu L of denatured Protein sample, boiling at 95 ℃ for 10min, taking a prepared SDS-PAGE gel (prepared by 10% separating gel and 5% concentrating gel), adding 20 mu g/hole of the upper sample, adding Protein LADDER MARKER, carrying out 80V electrophoresis on the concentrated gel part, changing the voltage to 120V when the bromophenol blue is run to the separating gel, and stopping electrophoresis when the bromophenol blue is run to a proper position. Placing PVDF film on the surface of PAGE gel, transferring protein on the PAGE gel to PVDF film by 300mA constant flow transfer film for 80min, closing PVDF film by 7% milk for 2h after the end of the electrotransfer, reducing non-specific background on PVDF film, adding target protein antibody and placing on 4 deg.C shaking table for shaking overnight, adding corresponding secondary antibody and placing on 4 deg.C shaking table for shaking 4h. Washing the membrane with TBST for 3 times and 10 min/time, and adding ECL hypersensitive chemiluminescence liquid onto PVDF membrane for development.

Tissue sample 50mg of tissue sample was taken and added to 1ml of RIPA lysis buffer, the tissue was minced with scissors, homogenized on ice and sonicated. After centrifugation at 10,000rpm for 10min, the supernatant was transferred to a new EP tube and the subsequent steps were identical to the cell treatment.

(4) Body temperature measurement the body temperature of the animal was measured at room temperature using a non-contact infrared thermometer (DT-8806S, CEM) at 9:00 am. The body temperature of the abdomen of each animal was measured three times as its average temperature.

(5) Thermal imaging of small animals thermal imaging pictures were taken with a thermal imager (flirone PRO) at 9:00 am at room temperature.

(6) HE staining:

1) The tissue sections were first placed in a 65 ℃ incubator for 30 minutes;

2) Xylene dewaxing (10 min/time; 3 times);

3) Sequentially hydrating, namely sequentially placing 100%, 95%, 90%, 80% and 70% alcohol for 5min each time;

4) Staining with hematoxylin for 3min;

5) Washing with water, differentiating with 1% ethanol hydrochloride, and controlling differentiation time;

6) Washing with flowing clear water for 20min;

7) Dyeing with eosin for 1min, and washing with flowing clear water for 20min;

8) Sequentially placing in 70%, 80%, 90%, 95% and 100% alcohol for 5min;

9) Soaking in xylene for 10min twice;

10 Air-drying in a special fume hood, sealing with neutral resin, and photographing with a microscope.

(6) Immunohistochemical staining:

1) The tissue sections were placed in a 65 ℃ incubator and incubated for 20 minutes;

2) Dewaxing with xylene (20 min/time, 3 times);

3) Hydrating, and standing in 100%, 95%, 90%, 80% and 70% alcohol for 5min;

4) Antigen retrieval, namely placing the slice in an autoclave, adding 0.01M citric acid buffer solution for boiling, timing for 2min after the complete air outlet, standing the slice to room temperature, and washing with PBST for 3 times each for 5 min;

5) Blocking, namely incubating for 30min in a 37 ℃ incubator with 3% hydrogen peroxide solution, and flushing with PBST three times for 5 min each;

6) Blocking, namely blocking for 1 hour by goat serum at normal temperature, and washing for 5 minutes by PBST for 3 times;

7) The sections were dried with absorbent paper, and UCP1 primary antibody (1:200 dilution) was added, washed 3 times with PBST for 5 minutes at 4 ℃ overnight;

8) Adding a secondary antibody A solution in an immunohistochemical staining kit, incubating for 1 hour at normal temperature, and washing with PBST for 3 times each for 5 minutes;

9) DAB color development, namely adding DAB color development liquid (B liquid: C liquid=50:1), and immediately flushing with running water for 20min after UCP1 protein turns brown;

10 Counterstaining, namely staining with hematoxylin for 2 minutes, and then washing with clear water for 20 minutes;

11 Dewatering, namely respectively standing in alcohol with 100 percent concentration, 95 percent concentration, 90 percent concentration, 80 percent concentration and 70 percent concentration for 5 minutes;

12 Standing twice in xylene for 10 minutes each;

13 After the sections are dried, the sections are sealed by a cover glass after neutral resin is added, and finally, the photographed sections are observed by a microscope.

Analysis of results:

As shown in FIG. 2, the mRNA levels of Adgra and Ucp1 and the protein levels of ADGRA3 and UCP1 in mature adipocytes induced by 3T3-L1 under different treatment conditions were found to be ADGRA to up-regulate the UCP1 expression level in adipocytes (FIGS. 2A-B). At the same time ADGRA3 increased the body temperature of the mice and promoted brown fat thermogenesis (FIGS. 2C-D), combined with ADGRA3 over-expression of protein levels of ADGRA3-3×FLAG and UCP1 (FIG. 2F) on the inguinal subcutaneous white adipose tissue (FIG. 2E) and brown adipose tissue of the mice, ADGRA3 increased UCP1 levels on white adipose tissue and brown adipose tissue. From the haematoxylin eosin HE staining of inguinal subcutaneous white adipose tissue and brown adipose tissue of ADGRA over-expressed mice (fig. 2G) and UCP1 immunohistochemical staining (fig. 2H), ADGRA3 induced multicavigation of white fat and brown fat. The above results demonstrate ADGRA that can induce white fat browning.

The mechanism by which hesperetin Hes and ADGRA induce fat browning is shown in fig. 3, which is drawn using Figdraw in fig. 3, with an authorized derivation ID of IWAIA0d9f9.

Although embodiments of the present application have been described above in connection with the above, the present application is not limited to the specific embodiments and fields of use described above, which are intended to be illustrative, instructive, and not limiting. Those skilled in the art, having the benefit of this disclosure, may effect numerous forms of the application without departing from the scope of the application as claimed.

Claims

1. The application of a preparation for promoting G protein coupled receptor ADGRA to the preparation of a medicament for treating obesity is disclosed, wherein the preparation for promoting ADGRA3 expression is a Adgra gene expression promoter, and the Adgra gene expression promoter is a Adgra3 over-expression vector.

2. The use according to claim 1, wherein the vector is a plasmid vector or a viral vector.