CN111205299B

CN111205299B - STAT3 inhibitor and application thereof

Info

Publication number: CN111205299B
Application number: CN202010054496.6A
Authority: CN
Inventors: 郭澄; 杨全军; 陈林林
Original assignee: Shanghai Sixth Peoples Hospital
Current assignee: Shanghai Sixth Peoples Hospital
Priority date: 2020-01-17
Filing date: 2020-01-17
Publication date: 2021-12-10
Anticipated expiration: 2040-01-17
Also published as: CN111205299A

Abstract

The invention relates to the technical field of biology, and discloses a STAT3 inhibitor and application thereof, wherein the STAT3 inhibitor has a structure shown as S-I. The invention provides an application of a STAT3 inhibitor in preparing a medicine for treating tumor cachexia. The STAT3 inhibitor provided by the invention has a simple separation and purification method, can inhibit the activation of STAT3, further improve skeletal muscle atrophy, and provides a new strategy for preparing a medicament for treating or improving tumor cachexia.

Description

STAT3 inhibitor and application thereof

Technical Field

The invention relates to the technical field of biology, and particularly relates to a STAT3 inhibitor and application thereof.

Background

Cachexia is a wasting syndrome caused by multiple factors including injury, organ failure, sepsis, rheumatoid arthritis, and HIV/AIDS, with loss of body weight and imbalances in energy and protein metabolism by tumors or other chronic diseases. Tumor cachexia, a leading factor in cachexia, is the interaction of tumors and the body to produce excessive amounts of inflammatory cytokines, leading to extensive oxidative stress and activation of the ubiquitin protein degradation system, increased protein catabolism, insufficient anabolism, resulting in severe weight loss and muscle loss. The occurrence of cachexia not only reduces the quality of life of patients and shortens the life cycle of patients, but also seriously affects the implementation of basic disease treatment schemes of patients, such as reducing the sensitivity of drug treatment and increasing the occurrence of complications. Current conventional treatments for tumor cachexia include trophic support and appetite-increasing drugs (megestrol and medroxyprogesterone), but they do not prevent weight loss and skeletal muscle atrophy.

The sustained weight loss of tumor cachexia is mainly caused by skeletal muscle atrophy, the mechanism of which is very complex and is manifested by excessive production of inflammatory cytokines and endogenous hormone level disorder, activation of catabolic signaling pathways mainly based on JAK-STAT3, SMAD-FOXO and IKK-nfkb, leading to activation of the ubiquitin-protease system (UPS), mediating protein degradation and skeletal muscle atrophy. There is increasing evidence that activation of signal transducer and activator of transcription 3(STAT3) is associated with a variety of disease-associated cachexia, and studies have shown that STAT3 activates myostatin (myostatin) and E3 ubiquitin ligase MuRF1 and Atrogin-1 by modulating CAAT/enhancer-binding protein δ (C/EBP δ), ultimately activating protein degradation pathways, leading to muscle atrophy.

The inhibition of skeletal muscle atrophy is an important way for preventing and treating tumor cachexia, and the existing drugs for treating skeletal muscle atrophy caused by tumor cachexia mainly comprise natural products, beta-adrenoceptor agonists, enzyme inhibitors, cytokine antibodies and the like. STAT3 is an important target for preventing and treating tumor cachexia, and the current research finds that the small-molecule inhibitor C188-9 of STAT3 can reverse the muscle consumption of C26 and LLC tumor mice by increasing muscle protein synthesis and inhibiting protein degradation. In addition, it has been reported that C2C12 myotubes treated with cell permeability STAT3 SH2 domain mimetic peptide (SIP) were found to cause moderate myofiber hypertrophy and to inhibit IL-6-induced myofiber atrophy. In phase II clinical trials, the STAT3 upstream kinase Jak1/2 inhibitor Ruxolitinib was also found to significantly increase the body weight of pancreatic cancer patients. The current study of STAT3 small molecule inhibitors in the treatment of tumor cachexia is in preclinical or clinical trials. In conclusion, the findings indicate that STAT3 is an effective target for treating cachexia, and the development of a novel STAT3 inhibitor has potential application value in treating tumor cachexia.

Disclosure of Invention

The invention provides a STAT3 inhibitor and application thereof, which are used for solving the problems in the prior art.

In order to achieve the purpose, the invention adopts the following technical scheme:

in a first aspect of the invention there is provided a STAT3 inhibitor, the STAT3 inhibitor having the structure shown in S-I,

further, the method for separating and purifying the STAT3 inhibitor comprises the following steps:

step one, crushing and sieving angelica dahurica, then adding ethanol for soaking, and percolating to obtain an extracting solution;

step two, carrying out reduced pressure concentration on the extracting solution obtained in the step one to obtain an extract containing the STAT3 inhibitor;

and step three, separating and purifying the extract obtained in the step two by adopting a high performance liquid chromatography to obtain the STAT3 inhibitor.

Further, the radix angelicae dahuricae adopted in the step one is a 20-mesh sieve, and the concentration of ethanol is 60-75%.

Further, the weight ratio of the angelica dahurica to the ethanol solution in the first step is 1: 8-10.

Further, the speed of percolation in the first step is 3-6m L/(min × kg).

Further, the temperature used for the concentration under reduced pressure in the second step is 70 to 90 ℃.

Further preferably, the temperature used in the concentration under reduced pressure is 80 ℃ and the concentration is carried out until the relative density is 1.25 to 1.30.

Further, the conditions of the high performance liquid chromatography in the third step are as follows: acetonitrile: water 65: 35; the wavelength is 300 nm; the flow rate was 1.2 mg/mL.

The second aspect of the invention provides an application of the STAT3 inhibitor in preparing a medicine for treating tumor cachexia, wherein the medicine comprises one or more of a STAT3 inhibitor, an isomer of the STAT3 inhibitor, a hydrate pharmaceutically acceptable for the STAT3 inhibitor, a solvate pharmaceutically acceptable for the STAT3 inhibitor, a salt pharmaceutically acceptable for the STAT3 inhibitor, a hydrate pharmaceutically acceptable for the isomer of the STAT3 inhibitor, a solvate pharmaceutically acceptable for the isomer of the STAT3 inhibitor, and a salt pharmaceutically acceptable for the isomer of the STAT3 inhibitor.

Further, the drug is a drug that specifically binds to SH2 domain of STAT3 protein.

Further, the drug is a drug that inhibits activation and transcriptional activity of STAT3 protein.

Furthermore, the drug is a drug for inhibiting the mRNA and protein expression level of E3 ubiquitination ligase MuRF1 and MAFbx/Atrogin-1.

Further, the medicine also comprises a pharmaceutically acceptable carrier.

Further, the carrier is selected from any one or more of the following: fillers, binders, solubilizers, disintegrants, and glidants.

Further, the dosage form of the medicament is selected from any one of the following: capsule, tablet, granule, powder, pill or injection.

Furthermore, the dosage form of the medicine is capsule, tablet or injection.

By adopting the technical scheme, compared with the prior art, the invention has the following technical effects:

the STAT3 inhibitor provided by the invention can inhibit activation of STAT3, so that skeletal muscle atrophy is improved, and a new strategy is provided for preparing a medicine for treating or improving tumor cachexia.

Drawings

FIG. 1 is a chromatogram of a STAT3 inhibitor according to an embodiment of the present invention;

FIG. 2 is a 1H-based NMR spectrum of a STAT3 inhibitor in accordance with an embodiment of the present invention;

FIG. 3 is a 13C-based NMR spectrum of a STAT3 inhibitor in accordance with an embodiment of the present invention;

FIG. 4 is a graph of the binding profile of STAT3 inhibitor to STAT3 using surface plasmon resonance analysis in accordance with a validated embodiment of the invention; wherein, panel a is a response sensorgram of STAT3 inhibitor binding to STAT3, and panel B is a steady-state affinity model fitting schematic diagram for calculating the affinity constant of a compound to STAT 3;

FIG. 5 is a schematic diagram of molecular modelling docking to analyse binding of STAT3 inhibitor to STAT3 SH2 domain in a validated embodiment of the invention; wherein panel a is a graph of the results of molecular docking assays for the interaction between STAT3 inhibitor and STAT3 SH2 domain; panel B is a schematic model of the binding of STAT3 inhibitor to STAT3 SH2 domain;

FIG. 6 is a graph of the results of an in vitro study of the inhibition of STAT3 inhibitors on STAT3 protein activation in a validated example of the invention; wherein, the graph A is a result graph of the effect of the STAT3 inhibitor on the vitality of the C2C12 myotube, the graph B is an electrophoresis graph of the expression quantity of p-STAT3 in different treatment time after the TCM stimulation, and the graph C is a result graph of the inhibition effect of the STAT3 inhibitor on the phosphorylation of STAT3 protein; (#) indicates a statistical difference (P <0.05) compared to the untreated control group, and (#) indicates a statistical difference (P <0.05) compared to the TCM control group;

FIG. 7 is a graph showing the results of an in vitro study of the effect of STAT3 inhibitors on myotube atrophy and myoprotein degradation in a validated embodiment of the invention; wherein, the graph A is a statistical graph of an influence image and a diameter of the STAT3 inhibitor on myotube morphology, the graph B is a result graph of the inhibition effect of the STAT3 inhibitor on MuRF1 and Atrogin-1 protein expression, and the graph C and the graph D are a result graph of the inhibition effect of the STAT3 inhibitor on MuRF1 and Atrogin-1mRNA expression; (#) indicates a statistical difference (P <0.05) compared to the untreated control group, and (#) indicates a statistical difference (P <0.05) compared to the TCM control group;

FIG. 8 is a graph showing the results of an in vitro study of the effect of STAT3 inhibitors on the signal pathway associated with muscle protein degradation in a validated embodiment of the invention; wherein, the graph A is a graph of the result of the effect of the STAT3 inhibitor on the myoprotein degradation related signal pathway protein, the graph B is an immunofluorescence image of the effect of the STAT3 inhibitor on p-STAT3 nuclear expression, and the graph C is a graph of the result of the inhibition of the STAT3 inhibitor on STAT3 transcriptional activity; (#) indicates a statistical difference (P <0.05) compared to the untreated control group, and (#) indicates a statistical difference (P <0.05) compared to the TCM control group;

FIG. 9 is a graph showing the results of an in vivo study of the reversal of skeletal muscle atrophy and muscle degradation of tumor cachexia by an inhibitor of STAT3 in a validated example of the invention; wherein, the graph A is a flow chart of experimental design, the graph B is a graph of the influence of the STAT3 inhibitor on the weight of the mouse, the graph C is a graph of the influence result of the STAT3 inhibitor on the muscle mass of the mouse, the graph D is a graph of the influence result of the STAT3 inhibitor on the fat mass of the mouse, the graph E is a graph of the influence result of the STAT3 inhibitor on the organ mass of the mouse, and the graph F is a graph of the influence result of the STAT3 inhibitor on the tumor load; (#) indicates a statistical difference (P <0.05) compared to the healthy control group, and (#) indicates a statistical difference (P <0.05) compared to the CT26 tumor group;

FIG. 10 is a graph of the results of an in vivo study of the effect of STAT3 inhibitors on gastrocnemius pathological changes in a validated example of the invention; wherein, the STAT3 inhibitor has the effects of improving the size and distribution of mouse muscle in a CT26 tumor-induced cachexia skeletal muscle atrophy model (A), protecting MyHC protein expression in muscle (B) and inhibiting MuRF1 and Atrogin-1 (C); (#) indicates a statistical difference (P <0.05) compared to the healthy control group, and (#) indicates a statistical difference (P <0.05) compared to the CT26 tumor group;

FIG. 11 is a graph showing the effect of STAT3 inhibitors on the myoprotein degradation-associated signaling pathway in a validated embodiment of the invention; the effect of STAT3 inhibitors on muscular atrophy-related signaling pathways in CT26 tumor-induced cachexia skeletal muscle atrophy model muscles (a), and inhibition of p-STAT3 nuclear expression (B); (#) indicates a statistical difference (P <0.05) compared to the healthy control group, and (#) indicates a statistical difference (P <0.05) compared to the CT26 tumor group.

Detailed Description

The invention provides a STAT3 inhibitor and application thereof, the STAT3 inhibitor has a structure shown as S-I,

the present invention will be described in detail and specifically with reference to the following examples to facilitate better understanding of the present invention, but the following examples do not limit the scope of the present invention. It is noteworthy that in the examples and figures below, the STAT3 inhibitor is abbreviated as IMP.

Example 1

This example provides a method for separating and purifying IMP, comprising the following steps:

step one, 10.0 kg of angelica dahurica is crushed and sieved by a 20-mesh sieve, then 60-75% ethanol with the amount of 6-10 times of the angelica dahurica is added for soaking, and the mixture is percolated at the speed of 4-6m L/(min kg) to obtain an extracting solution;

step two, concentrating the extracting solution obtained in the step one under the condition of 80 ℃ under reduced pressure until the relative density is 1.25-1.30, and obtaining the extract containing the STAT3 inhibitor;

and step three, separating the extract obtained in the step two by adopting a high performance liquid chromatography, wherein the separation conditions are acetonitrile: water 65: 35, wavelength 300nm, flow rate 1.2 mg/mL. The results obtained are shown in FIG. 1, with an IMP content of 1.5 mg/g.

And step four, obtaining an NMR spectrum of the IMP obtained by separation and purification by using an NMR method based on 1H and 13C (shown in figure 2 and figure 3).

Example 2

The embodiment provides an application of IMP in preparing a medicine for treating tumor cachexia, wherein the medicine comprises the IMP, isomers of the IMP and one or more of pharmaceutically acceptable hydrates, solvates and salts of the IMP.

In addition, the medicament also comprises a pharmaceutically acceptable carrier selected from any one or more of the following: fillers, binders, solubilizers, disintegrants, and glidants.

The dosage form of the medicine is selected from any one of the following: capsule, tablet, granule, powder, pill or injection.

Verification example 1

In the present verification embodiment, a Surface Plasmon Resonance (SPR) technique is used to analyze an interaction mode between IMP and STAT3, and the specific method is as follows:

1. the isoelectric point of STAT3 protein is calculated to be pH 6.34, acetate buffer solutions with pH of 4.0, 4.5, 5.0 and 5.5 are selected at 10mmol/L, the coupling protein STAT3 is diluted to the final concentration of 20 mu g/ml, the coupling protein STAT3 is respectively flowed on the surface of the chip, and the pH 4.0 buffer solution capable of obtaining the maximum reaction value (RU) is used as the coating buffer solution with the optimal pH value.

2. STAT3 was coupled to the FC2 channel of CM5 chip using an amino coupling method: the conjugated protein STAT3 was diluted to a final concentration of 10. mu.g/ml with 10mM acetate buffer, pH 4.0. The chip surface was treated with 0.2M EDC and 50mM NHS mixed at a ratio of 1:1 at a flow rate of 10. mu.l/min for 7min, followed by introduction of a coupled protein STAT3 solution, 10min later, introduction of a pH 8.5, 1M ethanolamine solution to block the chip surface active groups, and blocking overnight.

3. Configured IMP sample solution concentrations of 0, 0.125, 0.25, 0.5, 1, 2, 4, 8, 16, 32 μ M were injected at a flow rate of 30 μ l/min for 60s, respectively, and dissociation was performed for 120s for kinetic experiments, with data obtained from 1:1 kinetic model or stable binding model for fitting analysis, the results are shown in figure 4.

As can be seen from fig. 4A and 4B, IMP specifically binds STAT3 concentration-dependently with a maximal response at 9.59RU with a binding dissociation constant Kd of 9.98 μ M.

Verification example 2

The embodiment adopts molecular simulation docking to analyze the binding effect of IMP and STAT3 SH2 structural domain, and the specific method is as follows:

1. molecular docking was performed using the CDOCKER module of Discovery studio 3.0, the protein structure was downloaded from the PDB database, numbered 1BG1, and the DNA strands and water molecules were removed. The docking site is defined in the SH2 domain with coordinates (103.59, 70.59, 68.19) and a radius of 15 angstroms.

2. After the docked molecules were given the CHARMM force field, the sites were placed for docking, Top hits set to 10, Random consistency set to 10, organic to clearance set to 10, and the rest as per default.

3. The CDOCKER ENERGY of the molecule was evaluated for optimal conformation and the docking results were treated with PYMOL. The results are shown in FIG. 5.

As can be seen from FIG. 5, IMP binds to the SH2 domain of STAT3 protein with a binding energy of-4.7689 kJ/mol. IMP is capable of forming hydrogen bond interactions with Arg-609 and Sigma-Pi interactions with Lys-591. In addition, the IMP can also form intermolecular hydrophobic interaction with amino acid residues such as Glu-594, Arg-595, Ile-634, Gln-635, Ser-636, Val-637, Glu-638, Pro-639, Thr-620, Ser-611, Ser-612, Ser-613 and the like.

Verification example 3

The verification embodiment is used for in vitro research on the inhibition effect of IMP on STAT3 protein activation, and the specific method is as follows:

at present, a plurality of cachexia skeletal muscle atrophy in vitro models mainly comprise tumor cell culture supernatant, dexamethasone, TNF-alpha, IFN-gamma, IL-6, chemotherapeutic drugs and the like which can be used as in vitro induction factors to induce C2C12 myotube atrophy. The tumor cell culture supernatant can simulate the in vivo tumor cachexia environment, so that in subsequent experiments, the tumor cell culture supernatant (TCM) is used as an inducer of the skeletal muscle atrophy in vitro model. CT26 is a colon adenocarcinoma cell derived from mouse, cultured in 100 μ l dmm complete medium containing 10% FBS, replaced with DMEM medium containing 2% horse serum until the cell confluence reaches about 90%, cultured for 36h, the supernatant of the cultured CT26 cells was collected and centrifuged at 4500rpm for 5min at 4 ℃, and the obtained TCM was mixed in a 1: 4 diluted in a differentiation medium as a myotube atrophy stimulating agent of the present invention.

(1) The cytotoxicity of IMP on C2C12 cells was determined by the method of CCK-8. Mouse-derived C2C12 myoblasts at 1X 10 per well⁴The density of individual cells was seeded in a 96-well plate, cultured in 100. mu. LDMEM complete medium containing 10% FBS, and when the cells reached 80-90% confluency, the medium was changed to DMEM differentiation medium containing 2% horse serum for 3 days to form myotubes, and three duplicate wells were prepared for each concentration of IMP while a blank control containing an equal amount of DMSO was set. After 48 hours of incubation, the original medium was discarded, 100. mu.L of CCK-8DMEM medium containing 10. mu.g/mL was replaced, and after 1 hour of incubation at 37 ℃, the Optical Density (OD) was measured at 450 nm. The results are shown in figure 6A, where IMP above 30 μm is toxic to C2C12 myotube cells, and therefore doses below this concentration were selected for subsequent treatments.

(2) Mouse myoblast C2C12 at 2X 10⁵The density of individual cells was seeded in 6-well plates, stimulated with TCM solution for 0, 15, 30, 60, 120, 240min, respectively, the cells were collected at different time points, lysed using RIPA buffer on ice for 30min, and the lysate was centrifuged at 12000rpm for 10min at 4 ℃. Protein quantification was performed using the BCA kit, and the protein was denatured in 4 × loading buffer with boiling water for 5 min. The loading volume was calculated from the loading amount of 30. mu.g of protein and separated by 10% SDS-PAGE electrophoresis, and the protein was transferred to a PVDF membrane. Blocking in PBS buffer containing 5% skimmed milk powder at room temperature for 2h, and incubating at 4 ℃ overnight. PBST membrane washing three times, each time for 5 min. Incubating the secondary antibody for 1h at room temperature in a dark place, washing the membrane, and performing Odessey scanning imaging. The result is shown in fig. 6B, the expression of p-STAT3 is most significant around 15min of TCM stimulation, so that 15min is adopted as TCM stimulation time for researching the inhibition effect of IMP on STAT3 protein phosphorylation.

(3) Westernblot detects the inhibitory effect of IMP on phosphorylation of STAT3 protein in C2C12 cells. The mouse myoblast C2C12 is induced by 2% horse serum for 3 days, after being differentiated into myotube cells, IMP with different concentrations is added for pre-incubation for 2h, then TCM stimulation is carried out for 15min, the cells are cracked, and the protein expression conditions of p-STAT3 and STAT3 are detected by using western blot, and the result is shown in figure 6C.

As can be seen from fig. 6C, 10 μ M and 20 μ MIMP can significantly inhibit the phosphorylation level of STAT3 protein, but have no inhibitory effect on the activation of STAT1 and JAK2, and thus, it is confirmed that IMP is a specific inhibitor of STAT 3.

Verification example 4

The verification is implemented by researching the influence of IMP on myotube atrophy in vitro, and the specific method is as follows:

under the condition of verifying the in vitro muscle atrophy model established in the embodiment 3, the method for detecting the influence of IMP on the morphology of TCM-induced myotube atrophy cells by the immunofluorescence technology comprises the following steps:

mouse myoblast C2C12 at 4X 10⁴The density of individual cells was seeded in 24-well plates and 2% horse serum induced differentiation of C2C12 myoblasts into multinucleated myotubes.

(1) 5, 10, 20 μ MIMP were added to the well plates for pre-incubation for 2h followed by induction with TCM for 72 h. The medium was aspirated and washed 3 times with PBS.

(2) Fixed with 4% paraformaldehyde for 15min, and washed with PBS 3 times for 3min each.

(3) 0.5% TritonX-100 (in PBS) was incubated for 20min at room temperature.

(4) PBS was washed 3 times for 3min each, the PBS was blotted dry with absorbent paper, 5% BSA was added to cover the bottom of the dish, and the dish was blocked at room temperature for 30 min.

(5) The blocking solution was aspirated off the absorbent paper, washed free, and MyHC (1:250) antibody was added and incubated overnight at 4 ℃.

(6) And (3) PBST washing for 3 times in the next day, wherein each time is 3min, the water absorption paper absorbs the excess liquid, then a fluorescent secondary antibody is added, and the mixture is incubated for 1h in a dark room temperature, and PBST washing is performed for 3 times, and each time is 3 min.

(7) Adding DAPI dropwise, incubating for 5min in dark, staining the sample with PBS for 3 times (3 min each time), and washing off excessive DAPI.

(8) And (4) observing under a fluorescence microscope, collecting images, and drawing a Diameter distribution curve, wherein the result is shown in a figure 7A.

As can be seen from fig. 7A, after TCM induced for 72h, myotube morphology changed significantly, myotube diameter became smaller, myotube atrophy was significantly protected after 10 μ M and 20 μ MIMP treatment, and diameter statistics show that IMP significantly improved TCM induced C2C12 myotube atrophy.

Verification example 5

This example is an in vitro study of the effect of IMP on myoprotein degradation, as follows:

the ubiquitin protein degradation pathway is the most important way for protein degradation in the skeletal muscle atrophy process caused by tumor cachexia. Various signaling pathways are involved in myocyte protein degradation, but both ultimately exert proteolytic degradation via two muscle-specific E3 ubiquitin protein ligases MuRF-1 and Atrogin-1. Both MuRF-1 and Atrogin-1 were significantly upregulated in a variety of models of skeletal muscle atrophy caused by tumor cachexia, and are highly specific markers of skeletal muscle atrophy.

(1) Mouse myoblast C2C12 at 2X 10⁵The density of each cell is inoculated in a 6-well plate, 2% horse serum is induced and differentiated into a multinuclear muscle tubule, 5 mu MIMP, 10 mu MIMP are added to pre-incubate for 2h respectively, then TCM is used for inducing for 48h, the cells are lysed, and western blot is used for detecting the influence of IMP on the protein expression levels of E3 ubiquitin protein ligase MuRF1 and Atrogin-1 in C2C12 cells induced by the TCM, and the result is shown in figure 7B.

(2) Under the same model, collecting each group of cells, extracting cell mRNA by a Trizol method, and performing reverse transcription on 1 mu g of mRNA under the conditions of 37 ℃ and 15 min; cDNA was obtained at 85 ℃ for 15 seconds and diluted 10-fold. Preparing a qPCR reaction system of a SYBR Green detection method: mu.l 2 × Master Mix, 0.5. mu.l primer (specific sequence shown in Table 1), 1. mu.l cDNA, ddH₂Make up to 10. mu.l of O. PCR was performed in 2 steps with pre-denaturation at 95 ℃ for 30s, followed by 95 ℃ for 10s and extension at 60 ℃ for 30s for 40 cycles. For each pair of primers, 3 replicate wells were made in each template, dataTreatment adopted 2^-ΔΔCtThe results are shown in FIG. 7C.

As can be seen from FIG. 7B, 10. mu.M and 20. mu.MIMP were able to significantly suppress the expression levels of MuRF1 and Atrogin-1 proteins.

As can be seen from FIG. 7C, in this model, the mRNA expression levels of MuRF-1 and Atrogin-1 were up-regulated in C2C12 myotubes induced by TCM, while IMP significantly inhibited the expression of MuRF-1 and Atrogin-1mRNA and was dose-dependent.

MuRF-1, Atrogin-1 and GAPDH primers were designed as shown in Table 1:

TABLE 1 basic information on primer sequences

Verification example 6

The verification example is to study the influence of IMP on a myoprotein degradation related signal channel in vitro, and the specific method is as follows:

(1) study of the effect of IMP on myoprotein degradation-associated signaling pathways: mouse myoblast C2C12 at 2X 10⁵The density of each cell is inoculated in a 6-well plate, 2% horse serum is induced and differentiated into a multinuclear muscle tubule, 5 mu MIMP, 10 mu MIMP are added to pre-incubate for 2h respectively, then TCM is used for inducing for 48h, the cells are cracked, and western blot is used for detecting the influence of IMP on the expression levels of muscle degradation related signal channels STAT3 and downstream C/EBP delta, NF-Kb, SMAD3 and FOXO3 proteins in C2C12 cells induced by TCM, and the result is shown in figure 8A.

(2) Study of the effect of IMP on STAT3 nuclear entry: mouse myoblast C2C12 at 4X 10⁴The density of individual cells was seeded in 24-well plates and 2% horse serum induced differentiation of C2C12 myoblasts into multinucleated myotubes. After pre-incubation for 2h by adding 20 mu MIMP, stimulating for 30min by TCM, and detecting the influence of IMP on the nuclear invasion condition of p-STAT3 by immunofluorescence technique, the result is shown in FIG. 8B.

(3) Study of the effect of IMP on TCM activation STAT3 transcriptional activity: REPO for Hela cell^TMAfter the STAT3 reporter gene lentivirus is infected for 96 hours, the concentration (8ng/ml) of blasticidin capable of completely killing normal Hela cells is selected for infecting the STAT3 reporter geneAnd (4) screening the cells of the lentivirus to obtain a STAT3 reporter gene stable transgenic cell line. The cell line was treated at 1X 10⁴The density of individual cells was seeded in 96-well plates, and the next day 25ng of TK-Renilla plasmid was transfected with fugene HD (Promega) as an internal control. After 24h of cell transfection, IMP with different concentrations was added for pretreatment for 4h, then TCM was added for 24h of stimulation, and fluorescence values were detected using the Dual-Luciferase Reporter Assay System (Promega), the results are shown in FIG. 8C. IMP is able to dose-dependently inhibit STAT3 transcriptional activity at concentrations of 5-20. mu.M.

As can be seen from FIG. 8A, 10 μ M and 20 μ MIMP can remarkably inhibit STAT3 protein phosphorylation and C/EBP delta expression levels while inhibiting MuRF1 and Atrogin-1 protein expression, but have no obvious inhibition effect on the activation of NF-Kb, SMAD3 and FOXO3, and the in vitro confirmation that IMP specifically inhibits STAT3 signal channel.

As can be seen from FIG. 8B, 20 μ MIMP was able to significantly inhibit TCM-induced p-STAT3 nuclear expression.

As can be seen in FIG. 8C, IMP was able to dose-dependently inhibit STAT3 transcriptional activity at concentrations of 5-20. mu.M.

Verification example 7

The verification example is to research the reversal effect of IMP on tumor cachexia skeletal muscle atrophy and muscle degradation in vivo, and the specific method is as follows:

(1) establishing an animal model of tumor cachexia: taking out mouse CT26 colon adenocarcinoma cells from ultra-low temperature refrigerator, thawing, transferring to 4 th generation, collecting cells, centrifuging, washing with PBS buffer solution for 2 times, adding small amount of PBS buffer solution to suspend CT26 cells to density of 2 × 10⁶Cells/ml, 0.2ml of cell suspension was subcutaneously inoculated on the dorsal side of the right forelimb of each Balb/c mouse. As shown in FIG. 9A, the mice were administered 25mg/kg and 50mg/kg IMP on day 15 after inoculation with CT26 cells, and were each gavaged at the above doses, and the control group was gavaged with an equal volume of 0.5% CMCNA solution once a day for 15 days. The weight and food intake of the mice were measured every other day after the inoculation, and the tumor mass length and width were measured to calculate the tumor mass. The mice were sacrificed on day 30, and hearts, liver, spleen, kidney, epididymal fat, gastrocnemius muscle, and tibialis anterior muscle were taken and weighed, and the results are shown in fig. 9B-E. Weighing the above organs, quickly freezing in liquid nitrogen, transferring to-80 deg.C refrigeratorAnd (5) freezing and storing.

(2) Pathological changes of gastrocnemius muscle were observed by Hematoxylin and Eosin (HE) staining, and the results are shown in fig. 10A to 10C.

As can be seen from fig. 9B-E, the CT26 tumor caused significant tumor cachexia symptoms, mainly including significant reduction in body weight, muscle, and fat mass, and the data show that IMP treatment effectively ameliorated these symptoms caused by the CT26 tumor; IMP also improves cardiac and renal quality, but does not alleviate splenomegaly in cachectic mice.

As can be seen in fig. 10A, IMP treatment significantly increased the reduced muscle cross-sectional area in cachectic mice, improving skeletal muscle atrophy. Immunofluorescence of gastrocnemius sections showed that IMP treatment significantly reduced the expression of MuRF1 in muscle and increased MyHC expression (fig. 10B); in addition, the Real-time qPCR method detected MuRF-1 and Atrogin-1mRNA expression in gastrocnemius, and the results showed that IMP was able to dose-dependently inhibit the expression levels of MuRF1 and Atrogin-1mRNA (FIG. 10C). These data indicate that IMP has a therapeutic effect on tumor cachexia and improves muscle wasting in tumor cachexia mice.

Verification example 8

The verification example is to study the influence of IMP on a myoprotein degradation related signal channel in vivo, and the specific method is as follows:

(1) study of the effect of IMP on myoprotein degradation-associated signaling pathways: the mouse gastrocnemius muscle was lysed on ice using RIPA lysate for 30min and the lysate was centrifuged at 12000rpm for 10min at 4 ℃. Protein quantification was performed using the BCA kit, and the protein was denatured in 4 × loading buffer with boiling water for 5 min. The western blot is used for detecting the influence of IMP on the expression levels of the signal pathway STAT3 related to the degradation of tumor cachexia muscle and the downstream C/EBP delta, NF-Kb, SMAD3 and FOXO3 proteins, and the result is shown in FIG. 11A.

(2) Study of the effect of IMP on nuclear expression of p-STAT3 in muscle: immunohistochemical staining was performed on gastrocnemius muscle of each group of mice, and the results are shown in fig. 11B.

As can be seen from FIG. 11A, 25mg/kg and 50mg/kg IMP can remarkably inhibit phosphorylation of STAT3 protein and expression level of downstream C/EBP delta while inhibiting expression of MuRF1 and Atrogin-1 protein, but have no obvious inhibition effect on activation of NF-Kb, SMAD3 and FOXO3, and in vivo results also confirm that IMP can specifically inhibit STAT3 signal channel.

As can be seen in figure 11B, IMP treatment was able to dose-dependently inhibit the nuclear expression of p-STAT3 in the muscle of cachectic mice, which further confirms that IMP improvement in muscle atrophy is closely related to inhibition of STAT3 activation in the muscle.

From the verification examples, the IMP provided by the invention improves skeletal muscle atrophy by inhibiting the activation of STAT3, and further provides a new strategy for preparing a medicament for treating or improving tumor cachexia.

The embodiments of the present invention have been described in detail, but the embodiments are merely examples, and the present invention is not limited to the embodiments described above. Any equivalent modifications and substitutions to those skilled in the art are also within the scope of the present invention. Accordingly, equivalent changes and modifications made without departing from the spirit and scope of the present invention should be covered by the present invention.

Claims

1. Application of compound with structure shown as S-I in preparation of STAT3 inhibitor for treating tumor cachexia skeletal muscle atrophy

2. The use according to claim 1, wherein the compound is isolated and purified by:

step one, crushing and sieving angelica dahurica, then adding an ethanol solution for soaking, and percolating to obtain an extracting solution;

step two, carrying out reduced pressure concentration on the extracting solution obtained in the step one to obtain an extract containing the compound;

and step three, separating and purifying the extract obtained in the step two by adopting a high performance liquid chromatography to obtain the compound.

3. The use of claim 2, wherein the sieve used in the radix angelicae in the first step is a 20-mesh sieve; the concentration of the ethanol solution is 60-75%.

4. The use of claim 2, wherein the weight ratio of the radix angelicae to the ethanol solution in the first step is 1: 8-10.

5. The use of claim 2, wherein the percolation rate in step one is 3-6 mL-min^-1·kg^-1。

6. The use according to claim 2, wherein the temperature used for the concentration under reduced pressure in step two is 70-90 ℃.

7. The application of the compound with the structure shown as S-I as STAT3 inhibitor in preparing the medicine for treating tumor cachexia skeletal muscle atrophy is characterized in that the active ingredient of the medicine is the compound and/or the pharmaceutically acceptable salt thereof;

8. the use of claim 7, wherein the medicament further comprises a pharmaceutically acceptable carrier.

9. The use according to claim 8, wherein the carrier is selected from any one or more of: fillers, binders, solubilizers, disintegrants, and glidants.

10. The use according to claim 7, wherein the medicament is in a dosage form selected from any one of: capsule, tablet, granule, powder, pill or injection.