阅读说明：本技术 非戈替尼在细胞毒性损伤中的应用 (Application of non-golitinib in cytotoxic injury ) 是由 文先杰 李逸群 陈美欣 毋朝霞 杨淑璇 梁肖霞 陈伟健 于 2021-04-21 设计创作，主要内容包括：本发明公开了非戈替尼在细胞毒性损伤中的应用,涉及医药配制品技术领域。本发明公开的非戈替尼在制备修复或改善或预防由酰胺类局部麻醉类药物引起的细胞损伤的产品等方面的新用途,以及在制备用于修复或改善或预防与炎症反应相关的疾病的药物中的新应用,为修复或改善或预防酰胺类局部麻醉类药物引起的中枢神经系统疾病提供了理论依据和新的思路。(The invention discloses an application of non-gautinib in cytotoxic injury, and relates to the technical field of pharmaceutical preparations. The invention discloses a new application of non-gautinib in preparation of products for repairing or improving or preventing cell damage caused by amide local anesthesia drugs, and a new application in preparation of drugs for repairing or improving or preventing diseases related to inflammatory reaction, and provides a theoretical basis and a new thought for repairing or improving or preventing central nervous system diseases caused by amide local anesthesia drugs.)

1. Use of non-golitinib for the preparation of a product for repairing or ameliorating or preventing cytotoxic damages caused by amide local anesthetics.

2. The use of claim 1, wherein the type of cellular injury comprises one or more of apoptosis, decreased cell viability, and cellular inflammatory response.

3. The use of claim 1, wherein the reduction in cell viability comprises cell contraction rounding, membrane rupture, and content spillage.

4. The use according to claim 1, wherein the cellular inflammatory response is one or more of a decrease in the expression level of IL-10, an increase in the expression level of IL-6, and an increase in the expression level of TNF-a.

5. The use of claim 1, wherein the amide local anesthetic comprises one of ropivacaine hydrochloride, bupivacaine hydrochloride, and lidocaine hydrochloride.

6. Use of non-golitinib for the preparation of a medicament for the treatment or prevention of a peripheral nervous system disease and/or a central nervous system disease associated with an inflammatory response.

7. Use according to claim 6, wherein the central nervous system disorder comprises nerve damage caused by exposure to toxic compounds selected from the group consisting of industrial solvents, heavy metals, drugs or chemotherapeutic agents, drug-induced central neurotoxic damage, convulsions.

Technical Field

The invention relates to the field of research of pharmaceutical preparations, in particular to application of non-golitinib in cytotoxic injury.

Background

Filgotinib (chinese name non-golitinib or felonitinib) is a drug used for the treatment of Rheumatoid Arthritis (RA), Ulcerative Colitis (UC), psoriatic arthritis (PsA) and crohn's disease. Agotinib is a Janus kinase inhibitor, selective for the enzyme subtype JAK 1. It is considered a promising drug because it selectively inhibits JAK 1.

Filtinib has undergone a three-phase FINCH test. FINCH 1 was a randomized, placebo and adalimumab control trial administered in combination with MTX for a period of 52 weeks, and 1, 759 adult moderate severe active RA patients who were not responding adequately to MTX (methotrexate) treatment were enrolled. The primary endpoint for FINCH 1 was ACR20 at week 12. The trial included radiologic assessments at weeks 24 and 52. FINCH2 is a 24-week global randomized, double-blind, placebo-controlled phase 3 study in which non-gonitinib was evaluated on 449 adult moderately active RA patients who had an inadequate response to bdamds (biological alteration disease-type antirheumatic) against a conventional synthetic disease-modifying antirheumatic (csDMARD) background. The primary endpoint for FINCH2 was ACR20 at week 12. FINCH 3 is a randomized trial over a 52-week period and 1, 252 MTX-naive patients were evaluated for the efficacy of 200mg of non-golitinib administered alone and 100mg or 200mg of non-golitinib administered in combination with MTX administered alone. The primary endpoint for FINCH 3 was ACR20 at week 24. The trial included radiologic assessments at weeks 24 and 52.

Disclosure of Invention

The invention aims to provide the application of the non-golitinib in a cytotoxic injury scene, provide a new idea for treating cytotoxic injury and widen the application range of the non-golitinib.

The invention provides an application of non-gautinib in preparation of a product for repairing or improving or preventing cell damage caused by ropivacaine hydrochloride.

In some embodiments of the invention, the type of cell injury comprises one or more of apoptosis, decreased cell viability, and cellular inflammatory response.

In some embodiments of the invention, the reduction in cell viability comprises cell contraction rounding, membrane rupture, and content spillage.

In some embodiments of the invention, the cellular inflammatory response is one or more of increased IL-10 expression, decreased IL-6 expression, and decreased TNF-a expression.

In some application embodiments of the present invention, the amide local anesthetic includes one of ropivacaine hydrochloride, bupivacaine hydrochloride, and lidocaine hydrochloride.

Through tests, the situation that cell apoptosis is relieved after the non-golitinib is applied to a sample can be observed, the activity of the sample cell is improved, the expression quantity of p-STAT1 and p-STAT2 proteins of the sample cell is reduced, the expression quantity of an anti-inflammatory factor IL-10 of the sample is increased, and the expression quantity of inflammatory factors IL-6 and TNF-a factors of the sample is reduced.

In another aspect, the invention provides the use of non-golitinib for the preparation of a medicament for the treatment or prevention of a peripheral nervous system disease and/or a central nervous system disease associated with an inflammatory response.

Neurotoxic injury caused by local anesthetics is often manifested as numbness, paresthesia, spasmodic and radioactive burning-like pain in the affected area, and in severe cases, sensory loss and even paralysis are caused, which can cause discomfort to the patient. The nerve damage mechanism of local anesthetics is not clear at present, and may be related to intracellular calcium overload, low-voltage dependent calcium channels (T-type calcium channels), calcium dependent calmodulin kinase (camkii), p38MAPK, apoptosis and the like. After I-IFN is combined with its receptor IFNAR (divided into IFNAR1 and IFNAR 2), IFNAR phosphorylates, JAK1 and non-receptor tyrosine kinase TYK2 are simultaneously activated, transcription factors STAT1 and STAT2 are induced to phosphorylate and activate, STAT1 and STAT2 polymerize to form a dimer, and a STAT1-STAT2-IRF9 trimer complex is formed with IFN regulatory factor 9(IRF9), and the complex is transferred into a cell nucleus, is combined with an IFN Stimulation Response Element (ISRE) in the cell nucleus to activate the transcription of IFN Stimulation Genes (ISGs), and inflammatory response is induced to occur through different action mechanisms by ISGs encoded proteins. The inventor unexpectedly finds that the JAK1-STAT signal pathway blockage of the non-golitinib can reduce the occurrence of inflammatory reaction, and further relieve the nerve cell toxicity damage caused by local anesthesia drugs.

In some embodiments of the invention, the central nervous system disorder comprises nerve damage caused by exposure to a toxic compound selected from the group consisting of industrial solvents, heavy metals, drugs, or chemotherapeutic agents, drug-induced central neurotoxic damage, convulsions.

Drawings

FIG. 1 is a bar graph comparing the viability of groups of cells;

FIG. 2 is a graph comparing the apoptosis rates of various groups;

FIG. 3 is a graph showing the expression of STAT1, STAT2, p-STAT1, and p-STAT2 proteins in each group of cells;

FIG. 4 is a graph showing the IL-6, IL-10, and TNF-. alpha.content in each group of cells.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to specific embodiments, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

1) Experimental Material

Filgotinib (non-gonitinib) is a product of Selleck Chemicals USA (batch No. S760501), Ropivacaine (Ropivacaine hydrochloride) is a product of Aslicon England (batch No. NAZL), Annexin-APC/7-AAD kit is a product of Kaiki Biotech in Nanjing (batch No. 20200103), p-STAT1 and p-STAT2 antibodies are from Abclonal Biotech Limited, Wuhan, China (batch No. p-STAT 1: 1000540, batch No. p-STAT 2: LOT 21155940101); IL-6ELISA (batch: BWWE6KXLEX), IL-10ELISA (batch: 76ZTQQPFTN), TNF-a (batch: BNTAU5UJAP) kits were from Elabscience biotech, Inc., Wuhan, China.

2) Experimental methods

2-1) Experimental grouping and treatment protocol

The experiment was divided into four groups, and the culture criteria were: injecting the cellsAdding into DMEM/F12 medium containing 15% fetal calf serum, and culturing at 37 deg.C with 5% CO₂Culturing in an incubator under saturated humidity condition, and changing the culture medium every 2 days. Standard group: SH-SY5Y cells were cultured according to the standard. Non-golitinib group: SH-SY5Y cells were cultured for 4h with a final concentration of 5. mu.M of nonglutinous cell culture medium and then cultured as standard. Ropivacaine hydrochloride group: SH-SY5Y cells were treated with 3mM ropivacaine hydrochloride for 4h and cultured as standard. Test groups: SH-SY5Y cells were previously treated with ropivacaine hydrochloride at a final concentration of 3mM and a non-golitinib cell culture solution at a final concentration of 5. mu.M for 4h, and then cultured according to a standard culture.

2-2) detection of cell viability by MTT method

After each group was treated according to the treatment protocol, 10. mu.l MTT was added to each well and incubated at 37 ℃ for 4 h; removing the culture medium, adding 150 μ l DMSO, and shaking for 10 min; the microplate reader measures the light absorption value OD568 of each well. By OD568_{Control group}Value and OD568_{Blank control}Difference in value, and OD568_{Control group}The value ratio is 100%. OD568 value and OD568 of experimental group cells_{Blank control}The ratio of the difference to the control group is the cell viability of the experimental group.

The results are shown in FIG. 1. Compared with the standard group, the cell viability of the non-golitinib group is not statistically different, the cell viability of the ropivacaine hydrochloride group and the cell viability of the experimental group are remarkably reduced, and the difference has statistical significance. Compared with the ropivacaine hydrochloride group, the cell viability of the test group is obviously increased, and the difference is statistically significant (Mean + -SD, n-3),^aP<the 0.05vs standard set was used,^bP<0.05vs test group.

2-3) detecting apoptosis rate by flow cytometry

4 groups of SH-SY5Y cells were cultured at a cell density of 1X 10⁶Perml into 6-well plates, 1ml cell suspension per well, 5% CO₂Incubated overnight at 37 ℃. Collecting cells, centrifuging at 1200rpm for 5min, removing supernatant, and adding PBS for resuspension; the cells were rinsed 2 times with PBS, 1200rpm, 5 min; the following steps are carried out according to the AnnexinV-APC/7-AAD apoptosis detection kit operation instructions: adding 500 μ l Binding Buffer, and resuspending the cells; 5 mul of annexin V-FITC is evenly mixed, 5 mul of PI is added and evenly mixed; reacting at room temperature in dark place for 5-15min, and detecting fine particles on loss-of-flow cytometerThe rate of apoptosis.

The results are shown in FIG. 2. The apoptosis rate of the standard group was (4. + -. 0.05)%. Compared with the standard group, the apoptosis rate of the non-gautinib group has no statistical difference, the apoptosis rate of the ropivacaine hydrochloride group and the test group is obviously increased, and the difference has statistical significance. Compared with ropivacaine hydrochloride group cells, the apoptosis rate of the test group cells is obviously reduced, and the difference has statistical significance, (Mean + -SD, n ═ 3),^aP<the 0.05vs standard set was used,^bP<0.05vs test group.

2-4) WB detection of expression of p-STAT1 and p-STAT2 proteins

After 4 groups of SH-SY5Y cells were seeded in six-well cell plates and treated according to the protocol, the supernatant was removed, 1ml of 4 ℃ pre-cooled PBS was added to each well to co-wash the cells three times, the cell plates were placed on ice, 120. mu.l of lysate containing PMSF (0.1mM) was added to each well and lysed on ice for 30min, and the cell debris and lysate were transferred to a 1.5ml centrifuge tube. Centrifuge at 12000rpm for 5min at 4 ℃. The centrifuged supernatant was transferred to a 0.5ml centrifuge tube and stored at-20 ℃. And measuring OD568 by taking 2 mu l of the protein sample by using a BCA kit and a DG-3022A enzyme-linked immunosorbent assay, and calculating the concentration of the protein in the sample. And (3) carrying out boiling water bath on each protein sample for 10min for denaturation, cooling to room temperature, and then placing at-20 ℃ for storage. Respectively preparing 5% concentrated gel and 12% separation gel, sampling 40 μ g total protein, loading, performing electrophoresis at 80V until the bromophenol blue indicator forms a linear shape at the junction of the concentrated gel and the separation gel, and changing to constant pressure of 120V until bromophenol blue reaches the bottom of the gel. And taking out the gel, cutting a target strip according to a Marker, sequentially putting the gel in a black plate-fiber pad-filter paper-gel-PVDF membrane-filter paper-fiber pad-white plate, putting the gel into a membrane transferring instrument, and filling an electro-transfer liquid into a membrane transferring groove to start membrane transferring. The film transferring condition is that the film transferring time is 70min-120min under 200mA, after the film transferring is finished, the PVDF film is soaked in TBST (sealing liquid) of 5% skimmed milk powder, and the film is sealed for 2h by a shaking table at room temperature. The corresponding primary antibody was diluted with blocking solution, and the PVDF membrane was immersed in the primary antibody incubation solution and incubated overnight at 4 ℃. The PVDF membrane was washed well by TBST. Adding HRP labeled secondary antibody, diluting at a ratio of 1:50000, and incubating for 2h at room temperature in a shaking table. Uniformly mixing an enhancement solution and a stable peroxidase solution in an ECL reagent according to a ratio of 1:1, dropwise adding a working solution on a PVDF membrane, reacting for several minutes until a fluorescence band is obvious, absorbing excess substrate solution by using filter paper, covering a preservative film, tabletting an X-ray film, sequentially putting into a developing solution for developing, fixing by using a fixing solution, and washing the film. The film grey values were analyzed using BandScan. Antibody concentration: GAPDH is 1:1000, JAK1, STAT1, STAT2, p-JAK1, p-STAT1, p-STAT2 is 1: 500.

The results are shown in FIG. 3. The expression of JAK1, STAT1 and STAT2 in each group of cells was not statistically different. Compared with the cells in the standard group, the cells in the nonglutinib group have reduced p-JAK1, p-STAT1 and p-STAT2 proteins, the cells in the ropivacaine hydrochloride group have up-regulated p-JAK1, p-STAT1 and p-STAT2 proteins, the cells in the experimental group have no statistical difference between p-JAK1 and p-STAT1, and the expression of p-STAT2 is increased. Compared with the ropivacaine hydrochloride group, the expression of the cells p-JAK1, p-STAT1 and p-STAT2 in the test group is increased, and the difference is statistically significant (Mean + -SD, n is 3), aP<The 0.05vs standard set was used,^bP<0.05 vs. non-gautinib group,^cP<the 0.05vs ropivacaine hydrochloride group.

2-5) detecting the concentration of TNF-alpha, IL-6 and IL-10 by ELSA method

After 4 groups of SH-SY5Y cells are treated according to the experimental scheme, the cells are centrifuged at 3000rpm for 10min, and the supernatant is collected for detection. And (3) detecting according to the experimental steps of the ELISA detection kit of IL-6/IL-10/TNF-alpha, and respectively setting a blank hole, a standard hole and a sample hole to be detected. And adding 100 mul of sample diluent into the blank hole, and adding 100 mul of standard sample or sample to be detected into the rest holes respectively. Incubate at 37 ℃ for 90 minutes. The well was discarded, 100. mu.l of biotinylated antibody working solution was added to each well, and incubated at 37 ℃ for 1 hour. The liquid was discarded and 100. mu.l of the enzyme conjugate working solution was added to each well and incubated at 37 ℃ for 30 minutes. The liquid in the wells is discarded, 90. mu.l of color developing agent (TMB) is added in each well, and the ELISA plate is incubated for 15 minutes at 37 ℃ in the dark. The reaction was terminated by adding 50. mu.l of a terminator. The optical density (OD value) of each well was immediately measured at a wavelength of 450nm with a microplate reader. TNF-alpha, IL-6, IL-10 concentrations were calculated from the standards and OD values.

The results are shown in FIG. 4.

The content of IL-6 is measured: compared with the standard group, the IL-6 content of the non-gautinib group has no statistical significance, and the IL-6 content of the ropivacaine hydrochloride group and the test group is increased; the IL-6 content in the test group was reduced compared to the ropivacaine hydrochloride group.

The content of IL-10 is measured: compared with the standard group, the difference of the IL-10 content of the non-golitinib group and the test group has no statistical significance; the IL-10 content in the test group was increased compared to the ropivacaine hydrochloride group.

The content of TNF-alpha is measured: compared with the standard group, the content of the TNF-alpha in the non-gautinib group is not statistically significant, and the content of the TNF-alpha in the ropivacaine hydrochloride group and the test group is increased; the test group showed a decrease in TNF-alpha content compared to the ropivacaine hydrochloride group, (Mean ± SD, n ═ 3),^aP<the 0.05vs standard set was used,^bP<the 0.05vs ropivacaine hydrochloride group.

It will be evident to those skilled in the art that the invention is not limited to the details of the foregoing illustrative embodiments, and that the present invention may be embodied in other specific forms without departing from the spirit or essential attributes thereof. The present embodiments are therefore to be considered in all respects as illustrative and not restrictive, the scope of the invention being indicated by the appended claims rather than by the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein.