CN110898225A

CN110898225A - Application of mTOR/S6K1 and MAPK signal pathway inhibition in improvement of insulin resistance

Info

Publication number: CN110898225A
Application number: CN201911324320.1A
Authority: CN
Inventors: 韩宇博; 田苗; 刘莉; 邹国良; 隋艳波; 金娟; 彭鹏; 刘紫君; 娄宏君
Original assignee: Heilongjiang University of Chinese Medicine
Current assignee: Heilongjiang University of Chinese Medicine
Priority date: 2019-12-23
Filing date: 2019-12-23
Publication date: 2020-03-24

Abstract

The invention discloses application of mTOR/S6K1 and MAPK signal pathway inhibition in improvement of insulin resistance, and belongs to the technical field of medical biology. Specifically, the invention discloses application of an mTOR/S6K1 signaling pathway and/or MAPK signaling pathway inhibitor in preparation of a medicament for improving insulin resistance, and application of an mTOR/S6K1 signaling pathway labeling agent and/or an MAPK signaling pathway labeling agent in preparation of a kit for evaluating the effect of the medicament on improving insulin resistance. The invention provides a new drug target for insulin resistance, has important medical and pharmaceutical values for clinically improving insulin resistance, and can conveniently and quickly evaluate the effect of improving insulin resistance by drugs by monitoring related signal paths.

Description

Application of mTOR/S6K1 and MAPK signal pathway inhibition in improvement of insulin resistance

Technical Field

The invention belongs to the technical field of medical biology, and particularly relates to application of mTOR/S6K1 and MAPK signal pathway inhibition in improvement of insulin resistance.

Background

Insulin Resistance (IR) due to various causes is a major cause of Metabolic Syndrome (MS), including genetic mutations in insulin receptors, glucose transporters or signaling proteins, as well as some genetic mutations that have not yet been identified, overweight, lack of exercise, excessive eating, increased free fatty acids in the blood, aging, etc. The consequences of IR will certainly lead to the development of elevated blood glucose, hyperlipidemia and hyperinsulinemia, which are the fundamental factors for the development of MS. Therefore, IR participates in each link of MS, plays a key role in the link, and mutually promotes with other links to be causal, so that the MS rapidly progresses.

In addition, IR can occur in a plurality of tissues and organs sensitive to insulin, so that the balance of the body on blood sugar and blood fat and the control of body weight are damaged, and the progress of the MS is promoted jointly, wherein the IR generated in the central nervous system causes obesity, the IR generated in fat tissues is related to hyperlipidemia and obesity-related inflammatory reactions, the IR generated in liver tissues is closely related to the development of hyperglycemia, the IR generated in heart tissues accelerates the progress of heart failure, the IR generated in pancreatic tissues damages the regeneration of islet β cells, so that the utilization disorder of blood sugar is aggravated, the generation of IR generated in skeletal muscles is also found to shorten the life span, the IR generated in vascular endothelial cells causes hypertension, the bone tissues are also related to hypertension if the IR generated, and the progress of the IR is related to various pathological states, so that the improvement of the IR state is a problem to be solved at present.

Researches find that the coptis chinensis gallbladder-warming decoction has a remarkable curative effect on treating MS, and a plurality of indexes can prove that the relieving effect of the coptis chinensis gallbladder-warming decoction on IR is an important reason for effectively treating MS. However, the action mechanism of Huanglian Wen Dan Tang has not been studied yet.

Disclosure of Invention

In order to solve the technical problems, the inventor observes the improvement effect of the golden thread gallbladder warm decoction medicated serum on an insulin resistance 3T3-L1 adipocyte model and researches the participation condition of an mTOR/S6K1 signal pathway and an MAPK signal pathway in the process, and unexpectedly finds that the golden thread gallbladder warm decoction medicated serum can inhibit the mRNA and protein expression levels of the mTOR and S6K1 of IR-3T3-L1 adipocyte and can down-regulate the expression of p-ERK1/2, thereby achieving IR improvement.

In one aspect, the invention provides the use of an inhibitor for the manufacture of a medicament for use in improving insulin resistance, wherein the inhibitor is capable of inhibiting the mTOR/S6K1 signalling pathway and/or the MAPK signalling pathway.

In some embodiments of the invention, the inhibition of mTOR/S6K1 signaling pathway refers to inhibition of mTOR expression and/or inhibition of S6K1 expression. mTOR is a member of the phosphatidylinositol-related protein kinase (PIKK) family, exerts effects downstream of the PI3K/Akt signaling pathway, and has silk/threonine kinase activity. Kinases such as PI3K/Akt can activate mTOR serine 2448 site, and phosphorylated mTOR has biological activity and further phosphorylates downstream effector protein ribosome 6 kinase 1(S6K 1).

In some embodiments of the invention, the inhibition of mTOR expression refers to a reduction in mTOR mrna levels and/or a reduction in mTOR protein levels; the inhibition of S6K1 expression refers to a reduction in S6K1mRNA levels and/or a reduction in S6K1 protein levels.

In some preferred embodiments of the invention, the inhibitor is capable of simultaneously reducing mtorr mrna levels and protein levels. In other preferred embodiments of the invention, the inhibitor is capable of simultaneously reducing S6K1mRNA levels and protein levels. In some more preferred embodiments of the invention, the inhibitor is capable of simultaneously reducing the levels of mRNA and protein of mTOR and S6K 1.

In other embodiments of the invention, the inhibition of the MAPK signaling pathway refers to inhibition of the ERK1/2 pathway.

In some embodiments of the invention, the inhibition of the ERK1/2 pathway is a reduction in the level of phosphorylation of the ERK1/2 protein, even if the level of p-ERK1/2 protein is reduced.

In some more preferred embodiments of the invention, the inhibitor is capable of simultaneously reducing the levels of mTOR and S6K1mRNA and protein, and reducing the levels of p-ERK1/2 protein.

In some embodiments of the present invention, the inhibitor is selected from at least one of coptidis decoction and metformin.

In some embodiments of the present invention, the coptis chinensis gallbladder-warming decoction comprises the following components: 10 parts of coptis chinensis, 15 parts of rhizoma pinelliae preparata, 15 parts of immature bitter orange, 15 parts of bamboo shavings, 15 parts of dried orange peel, 20 parts of poria cocos, 10 parts of liquorice and 5 parts of ginger.

In a second aspect, the invention provides the use of an mTOR/S6K1 signalling pathway labeling agent and/or an MAPK signalling pathway labeling agent in the manufacture of a kit for assessing the effect of a drug on improving insulin resistance.

In some embodiments of the invention, the mTOR/S6K1 signaling pathway labeling agent is an mTOR labeling agent and/or an S6K1 labeling agent.

In some embodiments of the invention, the mTOR labeling agent is selected from at least one of an mTOR mRNA labeling agent and an mTOR protein labeling agent; the S6K1 labeling reagent is at least one selected from S6K1mRNA labeling reagent and S6K1 protein labeling reagent.

In other embodiments of the invention, the MAPK signaling pathway labeling reagent is a p-ERK1/2 protein labeling reagent.

In some preferred embodiments of the present invention, the mRNA labeling reagent refers to a primer combination capable of specifically amplifying the mRNA. In some more preferred embodiments of the invention, the mRNA labeling reagent further comprises a reverse transcription reagent.

In some more preferred embodiments of the present invention, the primer combination capable of specifically amplifying mTOR mRNA comprises an upstream primer having a nucleotide sequence shown in SEQ ID No.1 and an upstream primer having a nucleotide sequence shown in SEQ ID No. 2; the primer combination capable of specifically amplifying the S6K1mRNA comprises an upstream primer with a nucleotide sequence shown in SEQ ID No.3 and an upstream primer with a nucleotide sequence shown in SEQ ID No. 4.

Further, the mRNA labeling reagent further comprises a reverse transcription reagent.

In other preferred embodiments of the present invention, the protein labeling reagent is an antibody capable of specifically binding to the protein.

In some more preferred embodiments of the invention, the antibody capable of specifically binding to an mTOR protein is a rabbit Anti-mTOR antibody (abcam, cat # ab 32028); the antibody capable of specifically binding to the S6KI protein is a rabbit Anti-S6KI antibody (abcam, cat # ab 32529); the antibody capable of specifically binding to the p-ERK1/2 protein is rabbit Anti-p-ERK1/2(Thr202/Tyr204) antibody (abcam, cat # ab 235890).

In some embodiments of the present invention, the medicament includes at least one selected from the group consisting of coptidis decoction and metformin.

The invention has the advantages of

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

1. provides a new drug target for insulin resistance and has important medical and pharmaceutical values for clinically improving insulin resistance.

2. By utilizing the invention, the effect of improving insulin resistance by the medicament can be conveniently and quickly evaluated by monitoring the relevant signal path.

Drawings

FIG. 1 shows a comparison of glucose consumption for each group. Comparison with model groups: represents P <0.05, represents P < 0.01.

Figure 2 shows the respective group of GOD concentration levels. Comparison with model groups: represents P < 0.05.

FIG. 3 shows the melting peak and amplification curve of each gene. A: mTOR dissolution peak, B: an mTOR amplification curve; c: S6K1 peak, D: S6K1 amplification curve; e: ERK1 peak dissolution, F: ERK1 amplification curve; g: ERK2 peak dissolution, H: ERK2 amplification curve; i: JNK1 peak lysis, J: JNK1 amplification curve; k: GAPDH dissolution peak, L: GAPDH amplification curve.

FIG. 4 shows a comparison of mTOR mRNA expression for groups of 3T3-L1 adipocytes. Compared to the normal group, # P < 0.01; p <0.05 compared to model group.

FIG. 5 shows a comparison of the expression of S6K1mRNA by groups of 3T3-L1 adipocytes. P <0.01 compared to normal; p <0.01 compared to model group.

FIG. 6 shows a comparison of the expression of ERK1mRNA by the various groups of 3T3-L1 adipocytes. Compare # P >0.05 to normal group; compare P >0.05 to model group.

FIG. 7 shows a comparison of the expression of ERK2 mRNA by groups of 3T3-L1 adipocytes. Compare # P >0.05 to normal group; compare P >0.05 to model group.

FIG. 8 shows a comparison of the expression of JNK 1mRNA by groups of 3T3-L1 adipocytes. Comparison with normal group # P < 0.05; compare P >0.05 to model group.

FIG. 9 shows bands comparing the inner control of mTOR protein and GAPDH for groups of 3T3-L1 adipocytes. A: a normal group; b: a model group; c: coptidis decoction for warming gallbladder; d: the metformin group.

FIG. 10 shows a comparison of mTOR protein expression for groups of 3T3-L1 adipocytes. Comparison with normal group # P < 0.05; p <0.05, P <0.01 compared to model groups.

FIG. 11 shows the band diagram of the internal control of the S6K1 protein and GAPDH of the 3T3-L1 adipocytes in each group. A: a normal group; b: a model group; c: coptidis decoction for warming gallbladder; d: the metformin group.

FIG. 12 shows a comparison of the expression of S6K1 protein by groups of 3T3-L1 adipocytes. P <0.01 compared to normal; p <0.01 compared to model group.

FIG. 13 shows the comparison of the expression of proteins in groups 3T3-L1 adipocytes ERK1/2 and p-ERK1/2 in band A: a normal group; b: a model group; c: coptidis decoction for warming gallbladder; d: the metformin group.

FIG. 14 shows a comparison of the expression of ERK1/2, p-ERK1/2 proteins in groups of 3T3-L1 adipocytes. Comparison with normal group # P < 0.05; p <0.01 compared to model group.

FIG. 15 shows the band diagram of the internal control of 3T3-L1 adipocyte JNK1 protein and GAPDH in each group. A: a normal group; b: a model group; c: coptidis decoction for warming gallbladder; d: the metformin group.

FIG. 16 shows a comparison of the expression of JNK1 protein by groups of 3T3-L1 adipocytes.

Detailed Description

In order to make the technical problems, technical solutions and advantageous effects solved by the present invention more apparent, the present invention is further described in detail below with reference to the following embodiments.

Examples

The following examples are used herein to demonstrate preferred embodiments of the invention. It will be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function in the invention, and thus can be considered to constitute preferred modes for its practice. Those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit or scope of the invention.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs and the disclosures and references cited herein and the materials to which they refer are incorporated by reference.

Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

The experimental procedures in the following examples are conventional unless otherwise specified. The instruments used in the following examples are, unless otherwise specified, laboratory-standard instruments; the test materials used in the following examples were purchased from a conventional biochemical reagent store unless otherwise specified.

Examples

1 materials of the experiment

1.1 cell lines

3T3-L1 mouse embryonic fibroblasts were purchased from the cell bank of Chinese academy of sciences.

1.2 Experimental animals

30 male SD rats of SPF grade were purchased from the university of Chinese medicine, Heilongjiang, laboratory animal center, weighing 200 + -20 g. Animal license number: SCXK (black) 2015006. After adaptive feeding for 1 week, the feed is used for experimental research, and the temperature and humidity of the feeding environment are 22-24 ℃ and 40-50% respectively; the feeding density is 5 per cage, the food water is freely drunk, and the padding is replaced every other day.

1.3 Experimental drugs

Coptis chinensis gallbladder-warming decoction: 10g of coptis chinensis, 15g of rhizoma pinelliae preparata, 15g of immature bitter orange, 15g of bamboo shavings, 15g of dried orange peel, 20g of poria cocos, 10g of liquorice and 5g of ginger, wherein all decoction pieces are purchased from the department of herbaceous medicine affiliated to the first hospital in Heilongjiang traditional Chinese medicine.

Metformin hydrochloride: specification: 200mg, cargo number: AL4147, batch number: 20171022, Tianjin alpha Biotech limited.

1.4 Experimental reagents

2 method of experiment

2.1 preparation of medicated serum from Coptidis decoction for warming gallbladder

Adding 14 times of water into the decoction pieces of the traditional Chinese medicines, and decocting for 3 times, 1 hour each time. Decocting the decoction according to the method: weighing all Chinese medicinal decoction pieces (crude drug amount is 105g), adding 14 times of distilled water, and decocting for 3 times (wrapping with caulis Bambusae in Taenia). Decocting with slow fire for 1 hr after each boiling, filtering with gauze, mixing the obtained 3 times medicinal liquids, concentrating under reduced pressure to obtain soft extract, and making into lyophilized powder.

The final concentration of 200g rat drug, converted to the body surface equivalent dose, is 24 times the dose administered to an adult. The dose of the drug for gastric lavage of SD rat is 2mL of drug solution per 100g of body weight, so that the concentration of the drug for gastric lavage is 2.1g crude drug/mL. The administration mode of the decoction before breakfast and after supper in clinic is simulated by performing intragastric administration 2 times every day, the intragastric administration is performed for 1 time respectively at 8 am and 20 am, a proper amount of rat food is given after intragastric administration at 8 am, and the fasting is not forbidden after intragastric administration at 20 am; performing intragastric administration for 5 times to stabilize the blood concentration of the rat; blood was collected 1h after the last gavage.

30 SD rats are divided into 15 in the coptis chinensis gallbladder-warming decoction group and the blank group respectively by a random digital method. Diluting Coptidis rhizoma gallbladder warming decoction lyophilized powder with distilled water to obtain medicinal liquid with crude drug concentration of 2.1g/mL before intragastric administration every day, and administrating the above diluted solution in Coptidis rhizoma gallbladder warming decoction group, and substituting distilled water with equal volume in blank group for administrating medicinal liquid. Continuously taking the medicine, and collecting blood from abdominal aorta under aseptic condition after 1h of last intragastric administration. Standing the blood collecting tube for 1h, centrifuging at 3000rpm for 15min to obtain serum, filtering with 0.22 μm microporous membrane, inactivating at 56 deg.C for 30min, subpackaging and labeling blank serum and medicated serum, and storing at-80 deg.C for use.

2.2 preparation of Primary reagents

Adipogenic induction differentiation medium A (induction medium A)

Induction medium a composition: using OriCell^TMDifferentiation medium induced by adipogenesis of SD rat bone marrow mesenchymal stem cellsThe kit specifically comprises: 175mL of basal medium, 20mL of FBS, 400 μ L of insulin, 200 μ L of IBMX, 200 μ L of dexamethasone, 2mL of glutamine, 200 μ L of rosiglitazone and 2mL of double antibody.

Adipogenic differentiation medium B (Induction medium B)

Induction medium B composition: OriCell^TMThe SD rat bone marrow mesenchymal stem cell adipogenesis induction differentiation medium kit comprises: 175mL of basal medium, 20mL of FBS, 400 mu L of insulin, 2mL of glutamine and 2mL of double antibody.

Dexamethasone solution

25mM of mother liquor: dissolving 100mg of dexamethasone powder in 10.1912mL of DMSO, and mixing uniformly;

100 μ M working solution: adding 996. mu.L of culture medium into 4. mu.L of mother liquor to obtain working solution; when in use, 1. mu.L of the working solution is added to 100. mu.L of the culture medium.

Metformin hydrochloride solution

4mg/mL of mother liquor: dissolving 10mg of metformin hydrochloride in 2.5mL of DMSO, and filtering to obtain the metformin hydrochloride;

100. mu.g/mL of working solution: adding 975. mu.L DMSO into 25. mu.L of the mother solution, and adding 1. mu.L of the working solution into 100. mu.L of the culture medium.

DEPC water

100 μ L of diethyl Dicarbonate (DEPC) was added to 100mL of deionized water, stirred with a glass rod, left overnight, autoclaved for 30min, and dispensed into 1mL centrifuge tubes in a clean bench.

Tris-Glycine electrophoresis buffer

5 × Tris-Glycine electrophoresis buffer (mother liquor): stirring and dissolving 15.1g of Tris alkali, 5g of sodium dodecyl sulfate and 94g of glycine by using 800mL of deionized water, adjusting the pH to 8.3, then using the deionized water to fix the volume to 1000mL, and uniformly mixing;

1 × Tris-Glycine electrophoresis buffer (working solution): 200mL of 5 XTTris-Glycine electrophoresis buffer (mother solution) was added to 800mL of deionized water and mixed well.

Transfer buffer

Adding 5.8g of Tris alkali, 0.37g of sodium dodecyl sulfate and 2.9g of glycine into 700mL of deionized water, stirring for dissolving, adding 200mL of methanol, diluting to 1000mL with deionized water, and mixing uniformly.

TBST buffer

10 × TBST buffer (stock): weighing 24.2g of Tris alkali and 80g of NaCl, dissolving in 800mL of deionized water, adjusting the pH value to 7.6, metering the volume to 1000mL, and mixing uniformly;

1 × TBST buffer (working solution): 100mL of 10 XTSST buffer (mother solution) was added with 1mL of Tween-20 to a volume of 1000mL and mixed well.

5% confining liquid

1g of skim milk powder was weighed and dissolved in 20mL of 1 XTSST buffer and stirred well.

Anti-dilution liquid

1g of bovine serum albumin was dissolved in 20mL of 1 XTSST buffer, stirred well, filtered through a 0.22. mu.M microporous membrane, and stored at 4 ℃.

2.33 induced differentiation and characterization of T3-L1 fibroblasts

Using OriCell^TMThe SD rat bone marrow mesenchymal stem cell adipogenesis induced differentiation medium kit is recommended to be induced and differentiated:

(1) 5% CO in a 60mm petri dish₂Culturing 3T3-L1 cells at 37 deg.C, changing proliferation culture medium 1 time every 3 days, and inducing when cell fusion degree reaches 100% or over fusion state under microscope observation;

(2) carefully removing the cell proliferation medium by aspiration, and adding 3mL of induction medium A liquid which is placed at room temperature in advance;

(3) after 3 days of culture, removing the solution A, and adding 2mL of induction culture medium solution B;

(4) after 24h, the solution B is sucked and discarded, and the solution A is replaced for continuous induction;

(5) repeating steps (3) and (4), proceeding with step (3), and maintaining culture with solution B for 4-7 days until lipid droplets become large enough and round. During the period, the fresh induction culture medium B liquid is required to be replaced every 2 to 3 days;

(6) the fat cells are stained by an oil red staining method and are placed under a microscope to observe the staining effect of the fat cells.

2.4 construction of insulin resistance model and detection of glucose consumption and glucose oxidase content

2.4.1 measurement of glucose consumption

Culturing 3T3-L1 fibroblasts on a 96-well plate, inducing differentiation according to the method, replacing a proliferation culture medium containing 1 mu M/L dexamethasone after differentiation, completing model construction of cultured 96hIR-3T3-L1 fat cells, and stabilizing the model within 36 hours. The IR-3T3-L1 fat cells are randomly divided into a model group, a coptis chinensis wendan soup group and a metformin group, and then differentiated and mature 3T3-L1 fat cells are used as a normal group, and the normal group comprises 4 groups, wherein each group is provided with 3 compound holes.

The optimal concentration of the medicated serum of the golden thread gallbladder-warming decoction for regulating IR-3T3-L1 fat cells is that the medicated serum accounts for 4 percent of the culture system, and the action time is 36 hours.

Normal group: 3T3-L1 adipocytes + DMEM high glucose + 8% serum blanks + 5% FBS;

model group: IR-3T3-L1 adipocytes + DMEM high glucose + 8% serum blanks + 5% FBS;

coptis chinensis gallbladder-warming decoction group: IR-3T3-L1 adipocytes + DMEM high glucose + 4% drug-containing serum + 4% serum blank + 5% FBS;

metformin group: IR-3T3-L1 adipocytes + DMEM high sugar + 8% blank serum + 5% FBS + metformin hydrochloride solution (1 mM/L);

after 36h, the remaining culture medium from each well of the plate was collected and used for the measurement of glucose consumption.

2.4.2 measurement of glucose consumption

The glucose content in the culture solution is measured by using a glucose oxidase method glucose measurement kit, the specific method is carried out according to the specification, and the blank tube, the calibration tube and the sample tube are set as follows:

blank tube: 10 mu L of distilled water and 1000 mu L of working solution;

sample tube: sample solution 10 μ L + working solution 1000 μ L;

calibrating the tube: 10 mu L of calibration solution and 1000 mu L of working solution;

placing each tube in water bath at 37 deg.C for 10min, adjusting blank tube to zero, and measuring absorbance value of each tube at 505nm wavelength with semi-automatic biochemical analyzer; glucose content (mmol/L) ═ sample absorbance/calibration absorbance × calibrator concentration (5.55 mmol/L).

Glucose consumption was calculated for each group: glucose consumption (mmol/L) — glucose content of non-inoculated cells in culture broth (mmol/L) -glucose content of sample culture broth (mmol/L).

2.4.3ELISA method for detecting glucose oxidase content in culture solution

(1) Balancing the required lath at room temperature for 20min in advance, and diluting 20 times of concentrated washing liquid with distilled water for later use;

(2) setting standard holes and sample holes, wherein 50 mu L of standard substance of 80, 40, 20, 10, 5, 0U/L is added in each standard hole; 40 mu L of sample diluent is added into the sample hole, and 10 mu L of sample to be detected is added. Adding a sample to the bottom of the hole of the enzyme label plate during sample adding, slightly shaking and uniformly mixing the sample and the hole wall to the greatest extent;

(3) adding an enzyme: adding 100 mu L of enzyme-labeled reagent into each hole, and not adding blank holes;

(4) and (3) incubation: sealing the reaction hole with a sealing plate membrane, and incubating for 60min at 37 ℃;

(5) washing: carefully uncovering the unsealing plate film, discarding liquid, spin-drying, filling each hole with cleaning solution, and standing

Standing for 30s, discarding, repeating the above steps for 5 times, and patting to dry;

(6) color development: sequentially adding 50 μ L of color-developing agent A and color-developing agent B into each well, shaking gently, mixing, and developing at 37 deg.C in dark for 15 min;

(7) and (4) terminating: adding 50 mu L of stop solution into each hole, and stopping the reaction;

(8) and opening the microplate reader, detecting the OD value of each hole at the wavelength of 450nm, and recording and deriving.

2.5 extraction of RNA and cellular proteins

The grouping and treatment method was the same as 2.4, and 3T3-L1 adipocytes in a 60mm dish were randomly divided into 4 groups, a normal group, a model group, a Coptis chinensis Wendan decoction group and a metformin group, and the treatment was performed accordingly.

2.5.1 extraction of Total RNA from cells

2.5.1.1 Pre-extraction preparation

(1) DEPC with the final concentration of 0.1% is prepared, is a virulent substance with strong activity, and is prepared and used in a fume hood;

(2) putting the used plastic product into a container which can be sterilized at high temperature, and soaking the plastic product in DEPC aqueous solution overnight;

(3) carefully discarding the DEPC aqueous solution, sealing the container of the soaked plastic product with aluminum foil, and sterilizing at high temperature and high pressure for at least 30 min;

(4) baking to dry, and placing in a clean place for later use.

2.5.1.2 extraction of RNA

(1) Discarding cell supernatant, adding 1ml of Trizol, slightly shaking to uniformly distribute Trizol on the cell surface, standing at room temperature for 2min, blowing with a gun head, collecting, and transferring to a 1.5ml LEP tube;

(2) adding chloroform 200 μ L, reversing, mixing, standing at room temperature for 15 min;

(3)13000rpm, centrifuging at 4 ℃ for 15min, and the operations are all carried out by taking care to avoid DNA pollution;

(4) after centrifugation, the macroscopic liquid was divided into three layers: the upper layer is a colorless water phase and contains RNA; the middle layer is a white interface phase and contains protein and DNA; the lower layer is a colored organic phase containing DNA; carefully sucking the upper aqueous phase about 200-;

(5) adding isopropanol according to the volume ratio of the isopropanol to the supernatant of 1:1, repeatedly reversing and uniformly mixing, and standing at room temperature for 10 min;

(6) centrifuging at 4 deg.C for 10min at 10000rpm, and discarding the supernatant as much as possible without contacting with precipitate;

(7) resuspending and precipitating: adding 1mL of 75% ethanol, and gently oscillating the centrifugal tube;

(8) centrifuging at 8000rpm and 4 deg.C for 5min, discarding supernatant as much as possible, naturally air drying, dissolving with 150 μ L of DEPC water 100-;

(9) determination of RNA purity: dissolving 1 μ L sample in 20 μ L RNase-free deionized water, and dissolving

The OD260nm and OD280nm of the RNA were measured by ultraviolet spectrophotometry, and the RNA purity was calculated from the ratio OD260nm/OD280 nm. The RNA purity of the sample extracted by the method is good, and the ratio of OD260nm to OD280nm is 1.8-2.0.

(10) Calculating the concentration and yield of total RNA of the cells: the RNA solution with OD260nm value of 1 contained about 40. mu.g/mLRNA, and the yield of cellular RNA was calculated. The concentration of extracted RNA was: RNA sample concentration (μ g/μ L) ═ OD260nm × dilution factor × 40/L000; total amount of RNA (μ g) RNA concentration × total volume (μ L).

2.5.2 extraction of Total cellular protein

2.5.2.1 protein extraction

(1) Scraping the cells with a cell scraper, transferring into an EP tube, centrifuging at 1200r/min for 3min, and discarding the supernatant;

(2) adding 5 times volume of completeripapbuffer containing protease inhibitor, repeatedly blowing, and vortex vibrating for 20 s; standing on ice for 20min, and vortexing for 20 s;

(3) performing centrifugation at 13000r/min and 4 ℃ for 10min, transferring the supernatant into a precooled 1.5mL centrifuge tube, and preserving at-80 ℃;

2.5.2.2 determination of protein concentration

(1) Preparing a working solution: according to the following weight ratio of 50: 1, preparing a BCA working solution from a bovine serum albumin (BCA) reagent and a Cu reagent, and fully and uniformly mixing;

(2) diluting the standard substance: adding 90 μ L PBS 10 μ LBSA standard substance to obtain final concentration of 0.5mg/mL, sequentially adding 0, 2, 4, 6, 8, 12, 16, and 20 μ L standard substances into protein standard substance well of 96-well plate, and filling each well to 20 μ L with PBS;

(3) 20 μ L of the sample diluted 21-fold with PBS was added to the sample wells of a 96-well plate; and adding each well together with the standard substance into 200 mu LBCA working solution, standing at 37 ℃ for 20min, and then measuring the OD value by using a microplate reader. According to the OD value of the standard substance and the calculated concentration, a standard curve, a formula and an R value can be obtained; the concentration of the sample to be measured can be obtained by calculation, and the original concentration of the sample can be obtained by multiplying the concentration by the dilution times.

2.6Real Time qPCR method for detecting mRNA expression level

2.6.1RNA reverse transcription reaction

(1) RNA was removed from-80 ℃ on ice, 2000ng was removed and added to a 200. mu.L PCR tube (no RNase);

(2) reverse transcription system:

reagent	Adding amount of
		5×RT Buffer	4μL
RT Enzyme Mix	1μL
		Primer Mix	1μL

Preparing (n +1) mixed systems in 1 1.5mL centrifuge tubes according to the system;

(3) mixing the above systems, centrifuging instantly, and packaging each system with 6 μ L;

(4) adding nucleic-free Water to each system to 20 mu L;

(5) the following program was set up on a PCR instrument:

37℃，15min

98℃，5min

4℃，+∞

(6) the resulting product was stored at-20 ℃.

2.6.2RT-qPCR reaction

Primer sequences primers were designed using primer5.0 software based on the whole gene sequence of mTOR, S6K1, ERK1, ERK2, JNK1 and GAPDH mRNA. All primers were identified as specific primers by homology comparison. The designed primer sequences are as follows:

gene name primer name sequence (5 '→ 3')

(1) Quantifying the cDNA to 100 ng/. mu.L based on the cDNA concentration;

(2) n +1 mixed systems were formulated according to the following system:

reagent	Volume (20 μ L)
		SYBR qPCR Mix	10μL
Upstream primer	1μL
		Downstream primer	1μL
DEPC water	7μL

(3) Subpackaging into 0.2ml PCR tubes according to 19 μ L each tube;

(4) adding 1 mu L of cDNA template;

(5) flicking and mixing evenly after instantaneous centrifugation, and then instantaneous centrifugation again;

(6) the PCR machine started the PCR amplification with the following procedure:

10s at 95 ℃; 5s at 95 ℃, 10s at 55 ℃ and 15s at 72 ℃ for 40 cycles; 4 ℃ and + ∞.

(7) And (5) sorting and exporting data for analysis.

2.6.3Real-time qPCR data analysis and processing

Relative quantitative method was used to determine the relative amount of change in the target gene by comparing the difference in expression of the target gene between the treated and untreated samples using mouse GAPDH as an internal control, 2^-ΔΔCtThe method is a simple algorithm for analyzing the relative change of gene expression in real-time quantitative PCR experiments. First, a threshold cycle value (Ct value) is obtainedThe cycle number of the fluorescence signal of each detection product reaching a set value is calculated to obtain the Ct value average value. The delta Ct is the average difference of Ct values of the gene to be detected and the GAPDH gene of the mouse, and the delta Ct value is obtained by the difference of the Ct values of the corresponding genes of the control sample. Finally by comparison 2^-ΔΔCtAnd analyzing the change of the expression of the gene to be detected.

2.7Western Blot method for detecting protein expression level

(1) Adding RIPALysis Buffer to 20 μ g of protein to make up to uniform volume, adding 5 XProteinLoading Buffer, and placing in 100 deg.C metal bath for 5min to inactivate protein denaturation;

(2) loading: the solution containing 20 μ g of protein was taken as the loading volume for each group, and 5 μ L of Page rudained proteinladder was used as reference;

(3) electrophoresis: and (4) putting the mould filled with the glue into an electrophoresis tank, filling the electrophoresis buffer solution, and connecting an electrophoresis apparatus. The initial voltage is 80V, when the bromophenol blue reaches the separation gel, the voltage is increased to 120V, the power supply is turned off when the bromophenol blue runs to the bottom of the separation gel, and the electrophoresis is finished;

(4) film transfer: firstly, soaking the sponge and the PVDF membrane by using a transfer buffer solution, cutting off the concentrated gel, making a sandwich in the sequence of the sponge → the PVDF membrane → the gel → the sponge, carefully exhausting bubbles, and putting the sandwich into a semi-dry transfer electrophoresis tank together. Connecting an electrophoresis apparatus at 110V for 60-100 min;

(5) and (3) sealing: taking out the PVDF membrane, soaking the PVDF membrane in 5% sealing solution, placing the PVDF membrane in a shaking table, and shaking and sealing the PVDF membrane for 2 hours at room temperature;

(6) in combination with an antibody: diluting the corresponding primary antibody diluent according to a certain proportion, immersing the PVDF membrane in the diluted primary antibody liquid, and incubating overnight at 4 ℃;

(7) washing the membrane: washing PVDF membrane with pre-prepared 1 × TBST buffer solution for 4 times, each time for 15 min;

(8) binding of secondary antibody: immersing the PVDF membrane in a corresponding secondary antibody diluted by a confining liquid, and incubating for 1h at room temperature;

(9) washing the membrane: washing PVDF membrane with 1 × TBST buffer solution for 4 times, each time for 15 min;

(10) ECL color development: and (3) soaking the PVDF membrane in ECL luminescent solution, developing in a dark state for 3min, sucking the membrane by using filter paper, and scanning by using a full-automatic chemiluminescence image analysis system, wherein the protein expression amount is the target protein gray value/GAPDH gray value.

2.8 statistical methods

The IBM SPSS Statistics 24.0 software is used for data Statistics, and the measured data is expressed by mean plus or minus standard deviation. If the samples conform to normal distribution through the normality test, the comparison of the samples among the groups can adopt single-factor analysis of variance: when the variance is uniform, the multiple comparison of the samples adopts LSD-t test, and when the variance is not uniform, GamesHowell test is adopted; if the samples do not conform to normal distribution after the normality test, the non-parameter test is adopted for the comparison of the samples among the groups. The above test methods all consider that the difference in time when P is less than 0.05 has statistical significance.

3 results

3.1 glucose consumption levels in each group

As shown in table 1 and fig. 1, glucose consumption was measured for 36h of each group administered intervention, and the model group glucose consumption was lower than that of the normal group (P < 0.01); compared with the model group, the glucose consumption of the coptis gallbladder-warming decoction group and the metformin group is increased, and the difference has statistical significance (P <0.05), while the difference between the coptis gallbladder-warming decoction group and the metformin group has no statistical significance (P > 0.05).

TABLE 1 comparison of glucose consumption levels among groups (x. + -.s, n. RTM. 3)

3.2 glucose oxidase levels in each group

Drawing a standard curve: and (3) taking the concentration value of the measured standard substance as an abscissa and the OD value of the standard hole as an ordinate to obtain a standard curve regression equation: y0.03502 +0.01992X, R2 0.99971. And (3) removing the OD value of the pore plate per se from the OD value obtained by each group of detection, and inputting the obtained OD value into a regression equation to obtain the concentration of each group of Glucose Oxidase (GOD) (shown in table 2). As can be seen from FIG. 2, the GOD concentration in the culture medium of the Huanglian Wendantang group and the metformin group was higher than that in the model group, and the difference was statistically significant (P < 0.05).

Table 2 comparison of glucose oxidase levels between groups (x ± s, n ═ 3)

3.3 mRNA expression results of mTOR/S6K1 signaling pathway and MAPK signaling pathway-related genes of each group of 3T3-L1 adipocytes

Real-time qPCR was used to analyze the mRNA expression of the mTOR/S6K1 signaling pathway and MAPK pathway-associated genes of 3T3-L1 adipocytes, including the expression changes of mTOR, S6K1, ERK1, ERK2 and JNK1 relative to the reference GAPDH gene, and 3 samples were taken in each group. As shown in FIG. 3, the lysis peak and amplification curve of each gene show that no primer dimer and no non-specific amplification occur, indicating that each primer has good specificity, the amplification curve has good repeatability, and the fluorescence intensity is moderate. The amplification efficiency is consistent, the result can be relatively quantitatively analyzed, the mRNA expression quantity of the normal group is set as '1', the Ct value of each group is measured, and 2 is calculated^-ΔΔCtThe relative expression change of mRNA of each gene can be obtained by collating the internal reference GAPDH gene.

3.3.1 expression results of 3T3-L1 adipocyte mTORmRNA in each group

As shown in Table 3 and FIG. 4, compared with the normal group, the expression of mTOR mRNA in the model group is increased, the difference is statistically significant (P <0.01), and the expression level of mTOR mRNA in the coptis chinensis gallbladder warming decoction group and the metformin group is not obviously changed (P > 0.05); compared with the model group, the mTORmRNA expression of the coptis gallbladder-warming decoction group and the metformin group is reduced, and the difference has statistical significance (P is less than 0.05). Compared with the metformin group, the expression level of mTOR mRNA in the coptis chinensis gallbladder-warming decoction group 3T3-L1 fat cells is not obviously different (P > 0.05). Therefore, mTOR participates in the occurrence and development of IR, and the coptis chinensis gallbladder-warming decoction can regulate the expression of mTOR from the transcription level.

Table 3 comparison of mTOR mRNA expression by groups of 3T3-L1 adipocytes (x ± s, n ═ 3)

3.3.2 expression results of S6K1mRNA from groups of 3T3-L1 adipocytes

As shown in Table 4 and FIG. 5, compared with the normal group, the mRNA expression of the S6K1 in the model group was increased, the difference was statistically significant (P <0.01), and the mRNA expression levels of the S6K1mRNA in the Huanglian Wendantang group and the metformin group were not significantly changed (P > 0.05); compared with the model group, the mRNA expression of the S6K1mRNA of the coptis chinensis gallbladder-warming decoction group and the metformin group is reduced, and the difference has statistical significance (P is less than 0.01). Compared with the metformin group, the Coptis chinensis Wendan decoction group has no obvious difference in the expression level of the mRNA of the S6K1 of the 3T3-L1 fat cell (P > 0.05). Therefore, S6K1 is also involved in the generation and development of IR as an effector protein of mTOR, and the Huanglian Wendan decoction can regulate the expression of S6K1 from the transcriptional level.

Table 4 comparison of 3T3-L1 adipocytes S6K1mRNA expression (x ± S, n ═ 3) for each group

3.3.3 expression results of 3T3-L1 adipocyte ERK1mRNA in each group

As shown in Table 5 and FIG. 6, the expression level of ERK1mRNA in 3T3-L1 fat cells has no obvious change and the difference has no statistical significance (P >0.05) when compared with each other in4 groups.

Table 5 comparison of 3T3-L1 adipocyte ERK1mRNA expression in each group (x ± s, n ═ 3)

3.3.4 expression results of 3T3-L1 adipocyte ERK2 mRNA in each group

As shown in Table 6 and FIG. 7, the expression level of ERK2 mRNA in 3T3-L1 adipocytes has no obvious change and the difference has no statistical significance (P >0.05) when compared with each other in4 groups.

Table 6 comparison of 3T3-L1 adipocytes ERK2 mRNA expression (x ± s, n ═ 3)

3.3.5 expression results of 3T3-L1 adipocytes JNK 1mRNA

As shown in table 7 and fig. 8, the expression level of JNK 1mRNA of the adipocytes 3T3-L1 of the model group was up-regulated, the difference was statistically significant (P <0.05), and the expression levels of JNK 1mRNA of the coptis wendan decoction group and the metformin group did not significantly change (P > 0.05); compared with the model group, the expression of JNK 1mRNA of the fat cells of the coptis chinensis gallbladder-warming decoction group and the metformin group 3T3-L1 is not obviously different (P is more than 0.05); compared with the metformin group, the expression level of JNK 1mRNA of the fat cells of the coptis chinensis wendan decoction group 3T3-L1 is not obviously different (P > 0.05). As shown by the results, JNK1 may be involved in the formation of IR, but the Huanglian Wendan decoction has no influence on the expression.

Table 7 comparison of expression of JNK 1mRNA by 3T3-L1 adipocytes (x ± s, n ═ 3)

3.4 expression results of mTOR/S6K1 signal pathway and MAPK signal pathway-related protein of each group of 3T3-L1 adipocytes

Detecting the expression conditions of mTOR/S6K1 signal channels and MAPK signal channel related proteins of 3T3-L1 fat cells by using a Western Blot method, wherein the expression conditions comprise mTOR, S6K1, ERK1/2, p-ERK1/2 and JNK1 proteins, scanning by using a gel image processing system to obtain the gray value result of an X-ray film protein band, taking the ratio of the gray value of each histone to the gray value of an internal reference GAPDH as the relative expression of the corresponding protein, and comparing the change of the expression level of each histone.

3.4.1 expression of mTOR protein in groups of 3T3-L1 adipocytes

As shown in fig. 9 and fig. 10, compared with the normal group, the expression level of mTOR protein in the model group 3T3-L1 adipocytes was up-regulated, and the difference was statistically significant (P <0.05), and the expression levels of mTOR protein in the coptis chinensis wendan decoction group and the metformin group 3T3-L1 adipocytes were not significantly changed (P > 0.05); compared with the model group, the expression level of mTOR protein is reduced, and the difference has statistical significance (P is less than 0.05, and P is less than 0.01) in the coptis chinensis gallbladder-warming decoction group and the metformin group 3T3-L1 fat cells; compared with the metformin group, the expression level of mTOR protein of the fat cells of the coptis chinensis wendan decoction group 3T3-L1 is not obviously different (P is more than 0.05). Consistent with the mRNA results, mTOR was involved in the formation of IR, and Coptidis rhizoma Wendan decoction decreased its expression level.

3.4.2 expression of S6K1 protein of group 3T3-L1 fat cells

As shown in fig. 11 and fig. 12, compared with the normal group, the protein expression level of the model group 3T3-L1 fat cell S6K1 was significantly up-regulated, the difference was statistically significant (P <0.01), and the protein expression level of the coptis chinensis wendan decoction group and the metformin group 3T3-L1 fat cell S6K1 was not significantly changed (P > 0.05); compared with the model group, the protein expression levels of the 3T3-L1 fat cells S6K1 of the coptis chinensis gallbladder-warming decoction group and the metformin group are reduced, and the difference has statistical significance (P is less than 0.01); compared with the metformin group, the protein expression level of the golden thread Wendan decoction group 3T3-L1 fat cell S6K1 is not obviously different (P > 0.05). Consistent with the mRNA results, S6K1 was involved in the formation of IR, and Huanglian Wendan Tang decreased the expression level.

3.4.3 expression of groups 3T3-L1 adipocytes ERK1/2, p-ERK1/2 protein

The expression level of ERK1/2 and its active form P-ERK1/2 protein is shown in figure 13 and figure 14, the expression level of ERK1/2 protein of each group of 3T3-L1 fat cells has no obvious change, and the difference has no statistical significance (P > 0.05); compared with the normal group, the expression level of the P-ERK1/2 protein of the adipocyte of the model group 3T3-L1 is up-regulated, and the difference has statistical significance (P is less than 0.05); compared with the model group, the expression level of P-ERK1/2 protein of the fat cells of the coptis gallbladder-warming decoction group and the metformin group is reduced, and the difference has statistical significance (P is less than 0.01). As can be seen, although the mRNA level and the protein level of ERK1/2 are not changed among the groups, the phosphorylation form is obviously increased in the IR model group, and the phosphorylation level of the Huanglian Wendan decoction can be reduced.

3.4.4 expression of 3T3-L1 adipocyte JNK1 proteins in groups

As shown in fig. 15 and 16, the expression level of JNK1 protein was not significantly changed in each group of adipocytes, and the difference was not statistically significant (P > 0.05).

All documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the present invention as defined by the appended claims.

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Claims

1. Use of an inhibitor for the manufacture of a medicament for use in improving insulin resistance, wherein the inhibitor is capable of inhibiting the mTOR/S6K1 signalling pathway and/or the MAPK signalling pathway.

2. The use of claim 1, wherein said inhibition of the mTOR/S6K1 signaling pathway is inhibition of mTOR expression and/or inhibition of S6K1 expression.

3. The use of claim 2, wherein the inhibition of mTOR expression is a reduction in mTOR mRNA levels and/or a reduction in mTOR protein levels; the inhibition of S6K1 expression refers to a reduction in S6K1mRNA levels and/or a reduction in S6K1 protein levels.

4. The use of claim 1, wherein said inhibition of the MAPK signaling pathway is inhibition of the ERK1/2 pathway.

5. The use of claim 4, wherein the inhibition of the ERK1/2 pathway is a decrease in the phosphorylation level of ERK1/2 protein.

6. The use according to any one of claims 1 to 5, wherein the inhibitor is selected from at least one of coptidis decoction and metformin.

Use of a mTOR/S6K1 signaling pathway labeling agent and/or a MAPK signaling pathway labeling agent in the manufacture of a kit for evaluating an effect of a drug on improving insulin resistance.

8. The use of claim 7, wherein said mTOR/S6K1 signaling pathway labeling agent is an mTOR labeling agent and/or an S6K1 labeling agent.

9. The use of claim 8, wherein the mTOR labeling agent is selected from at least one of an mTOR mRNA labeling agent and an mTOR protein labeling agent; the S6K1 labeling reagent is at least one selected from S6K1mRNA labeling reagent and S6K1 protein labeling reagent.

10. The use of claim 7, wherein said MAPK signaling pathway labeling agent is a p-ERK1/2 protein labeling agent.