CN116099002A

CN116099002A - Use of inhibitors of JUN N-terminal kinase in the preparation of a medicament

Info

Publication number: CN116099002A
Application number: CN202210921426.5A
Authority: CN
Inventors: 华子春; 宋丽君; 余传信; 殷旭仁
Original assignee: Changzhou High-Tech Research Institute Of Nanjing University; Jiangsu Institute of Parasitic Diseases
Current assignee: Changzhou High-Tech Research Institute Of Nanjing University; Jiangsu Institute of Parasitic Diseases
Priority date: 2021-10-12
Filing date: 2022-08-02
Publication date: 2023-05-12
Also published as: CN114617966A

Abstract

The invention discloses an application of a JUN N-terminal kinase inhibitor in preparing a medicament for preventing and/or treating schistosomiasis. The JUN N-terminal kinase inhibitor comprises SP600125, can be contained in various medicinal compositions, can be prepared into solution or suspension for intraperitoneal injection or into tablets, capsules or solution for oral administration or intravenous administration, and is used for treating schistosomiasis liver egg granuloma and fibrosis, reducing the degree of schistosomiasis liver fibrosis of mice, reducing the expression level of related fibrosis factors and inflammatory factor mRNA in the liver and the proportion of mononuclear cells, macrophages and T cells, reducing the inflammatory microenvironment in the liver of the mice, so that the JUN N-terminal kinase inhibitor is a novel application of the JUN N-terminal kinase inhibitor for treating schistosomiasis.

Description

Use of inhibitors of JUN N-terminal kinase in the preparation of a medicament

Technical Field

The invention belongs to the technical field of pharmacy, and particularly relates to application of a JUN N-terminal kinase inhibitor in preparation of medicines.

Background

Schistosomiasis is a zoonotic parasitic disease seriously harming human health, the epidemic situation of the schistosome in the whole country is reduced year by year at present, but the distribution area of oncomelania in the epidemic area in the whole country is increased, a certain number of infection sources of schistosomiasis such as cultivated cows, sheep, a large number of wild animal insect-protecting hosts and the like still exist in the epidemic area, objective factors of the epidemic situation are present, and risk factors of the epidemic situation repetition and the rise still exist.

The pathological damage of schistosome infection to a host is mainly granuloma formed by ova deposited in the liver and liver fibrosis formed by excessive tissue repair caused by continuous chronic infection, the liver fibrosis is advanced to a late stage, irreversible liver cirrhosis can be formed, portal blood flow is blocked to cause venous and gastric fundus varices at the lower end of esophagus, massive hemorrhage is caused by rupture, spleen blood stasis and spleen hyperfunction are caused, and hepatic coma and hepatic ascites can occur in serious patients, so that great harm is caused to human health. After the insect body is cleared through treatment, antigen molecules released by eggs remained in the body can continuously induce the development of liver fibrosis, and are root causes of severe symptoms of schistosomiasis in the period. Therefore, after the pathogen treatment is carried out, the liver fibrosis is continuously treated, the sustainable development of liver lesions is prevented, and the method is a non-negligible outstanding problem in the prevention and treatment of schistosomiasis. However, no effective medicine for treating liver fibrosis exists clinically at present, so that the formation mechanism of liver fibrosis is clarified, and the method has important significance in searching for medicine action targets for resisting schistosomiasis liver fibrosis and developing new measures for resisting schistosomiasis liver fibrosis.

There are various causes of fibrosis caused by liver injury, roughly classified into 5, including compound, diet, surgery, transgene or infection, and the mechanisms of liver injury caused by different causes are different, and thus the types of liver fibrosis caused are also different. The type of fibrosis caused by schistosome is liver linear, and fibrous tissue stretches around the lobules to form a stem linear structure, and the liver cells shrink, but the lobules are intact. However, alcoholic liver fibrosis, nonalcoholic fatty liver fibrosis and hepatic fibrosis hepatocytes injury caused by hepatitis B virus, and hepatic lobule are destroyed. Thus, different drugs need to be tested separately for different types of liver fibrosis to verify their effectiveness.

The factors that form liver fibrosis are intricate, and among many factors of liver fibrosis, the inflammatory microenvironment is a key factor that promotes its formation. Previous studies by the applicant have found that necrotic apoptosis mediated by receptor-interacting protein kinase 3 (RIP 3) is a key factor in the formation of inflammatory microenvironments. The expression level of JNK protein in the liver of a mouse with the RIP3 gene knocked out is reduced, and the result shows that the RIP3-JNK axis plays an important role in the generation and development of schistosome liver fibrosis inflammation microenvironment.

JUN N-terminal kinase (JNK) is a stress activated protein kinase family, and can phosphorylate serine residues at 63 rd and 73 rd amino terminal areas of c-Jun genes, wherein three genes of JNK1, JNK2 and JNK3 exist in the JNK family, the JNK1 and the JNK2 are widely expressed in organism tissues, the JNK3 genes are specifically expressed in brain central nervous system, cardiac muscle and testis, and a JNK signal channel can be activated by cytokines such as tumor necrosis factor, epidermal growth factor, interleukin and the like to participate in various biological reactions such as apoptosis, proliferation and differentiation, skeleton construction, inflammation and the like of cells.

Inhibitors of JUN N-terminal kinase can be used to treat brain degenerative diseases such as alzheimer's disease, parkinson's disease, stroke and ischemia-induced brain dysfunction, and also can be used in the treatment of immune diseases such as asthma, rheumatoid arthritis, inflammatory bowel disease, chronic transplant rejection and multiple sclerosis, and also can be used as therapeutic agents for type 2 diabetes, obesity and cancer. The JNK signal channel plays a key role in the occurrence and development of diseases such as ischemia reperfusion liver injury, drug liver injury, alcoholic and nonalcoholic liver injury, liver cancer and the like, and provides a basis for the development of drug targets of the diseases, but the treatment of JUN N-terminal kinase in liver injury caused by schistosome has not been reported so far.

Disclosure of Invention

In order to solve the technical problems in the prior art, the invention aims to provide the application of the JUN N-terminal kinase inhibitor in preparing medicines.

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

use of a JUN N-terminal kinase inhibitor for the preparation of a medicament for the prevention and/or treatment of schistosomiasis.

Further, the medicine is used for inhibiting or improving schistosomiasis liver egg granuloma and fibrosis and inhibiting inflammation of schistosomiasis infection.

Further, the medicament comprises an effective amount of one or several inhibitors of the JUN N-terminal kinase and/or derivatives and/or pharmaceutically acceptable compounds thereof.

Further, the JUN N-terminal kinase inhibitor comprises SP600125.

Further, the medicament is a solution or suspension for intraperitoneal injection, or an oral or intravenous tablet, capsule or solution.

Furthermore, the medicine is a solution or suspension for intraperitoneal injection prepared by dissolving the JUN N-terminal kinase inhibitor in dimethyl sulfoxide, the injection amount is 50-300mg/kg, and the treatment time is 3-4 weeks.

Further, the medicine is used for reducing the granuloma and fibrosis area of schistosomiasis liver insect eggs and reducing the expression level of related fibrosis factors and inflammatory factor mRNA in the liver.

Further, the medicament is used for down-regulating the expression of alpha-smooth muscle agonism protein (alpha-SMA), down-regulating the expression of glutamic-pyruvic transaminase (ALT) and glutamic-oxaloacetic transaminase (AST) in blood, down-regulating the expression of hydroxyproline, down-regulating the expression of a metalloprotease tissue inhibitor and up-regulating the level of matrix metalloprotease degrading extracellular matrix.

Further, the medicament is used for reducing inflammation-related cells and relieving inflammatory responses of macrophages, CD3+ T cells and CD45+ monocytes in non-parenchymal cells.

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

the invention discloses an application of a JUN N-terminal kinase inhibitor in preparing a medicament for preventing and/or treating schistosomiasis. According to the invention, the JUN N-terminal kinase inhibitor is used for treating schistosomiasis liver egg granuloma and fibrosis, so that the degree of schistosomiasis liver fibrosis of mice can be reduced, the inflammatory microenvironment in the livers of the mice can be reduced, the precedent of schistosomiasis treatment is initiated, the market blank is made up, and the JUN N-terminal kinase inhibitor is a new application; inhibitors of JUN N-terminal kinase include, but are not limited to, SP600125, administration of one or more JUN N-terminal kinase inhibitors to human patients for the treatment of liver fibrosis, involving only schistosomiasis liver fibrosis, excluding nonalcoholic liver fibrosis, alcoholic liver fibrosis, CCl ₄ Liver fibrosis; the JUN N-terminal kinase inhibitor can be contained in various medicinal compositions, can be prepared into solution or suspension for intraperitoneal injection or prepared into oral or intravenous tablets, capsules or solution, and the invention fully verifies that when SP100625 in the JUN N-terminal kinase inhibitor is dissolved in dimethyl sulfoxide and is used for intraperitoneal injection in early stage of schistosome liver fibrosis formation, continuous injection treatment is carried out for 3-4 weeks, once a week, the single granuloma area of schistosome infected mouse liver can be reduced, the single ovum fibrosis area is 39.1%, and the liver is reducedThe expression level of related fibrosis factor, inflammatory factor and T cell secretion factor mRNA has the curative effect of inhibiting or improving schistosomiasis liver granuloma and fibrosis within an effective amount, the JUN N-terminal kinase inhibitor can be used for preparing medicines for treating schistosomiasis, the JNK phosphorylation level is reduced by the JUN N-terminal kinase inhibitor, the downstream transcription factor c-JUN phosphorylation level is reduced, the expression level of the regulated inflammatory factor is reduced, and the liver worm granuloma and fibrosis degree is reduced.

Drawings

FIG. 1 shows the results of changes in granuloma of liver eggs of schistosome infected mice treated with JUN N-terminal kinase inhibitor according to example 1 of the present invention, wherein, in FIG. A: HE staining pattern of granuloma of eggs of liver, panel B: a histogram of granuloma area of individual ova of the liver; * P <0.001;

FIG. 2 shows the modified results of liver worm egg fibrosis after treatment of schistosome infected mice with JUN N-terminal kinase inhibitor in example 2 of the present invention, wherein, in FIG. A: MASSON staining pattern of granuloma of eggs of liver, panel B: a histogram of individual worm egg fibrosis areas of the liver; * P <0.001;

FIG. 3 shows the liver modification of the JUN N-terminal kinase inhibitor treated mice infected with schistosome according to example 3 of the present invention, wherein, in FIG. A: expression profile of α -SMA in liver following DMSO control and treatment with JUN N-terminal kinase inhibitor, panel B: a histogram of expression data for α -SMA; * P <0.001;

FIG. 4 is a bar graph showing the expression level of glutamic-pyruvic acid and glutamic-oxaloacetic transaminase in serum of mice infected with schistosome treated with JUN N-terminal kinase inhibitor according to example 4 of the present invention; * P <0.05;

FIG. 5 is a bar graph showing the expression level of hydroxyproline in liver after treatment of schistosome-infected mice with JUN N-terminal kinase inhibitor according to example 5 of the present invention; * P <0.05;

FIG. 6 is a bar graph showing the expression levels of mRNA of type I Collagen Collagen I, type III Collagen Collagen III, α -SMA, metalloproteinase 9MMP9, and metalloproteinase inhibitor-1 TIMP-1 in the liver of a schistosome-infected mouse treated with a JUN N-terminal kinase inhibitor according to example 6 of the present invention; * P <0.05, < P <0.001;

FIG. 7 is a bar graph showing the expression levels of mRNA of tumor necrosis factor TNF- α, growth factor-like model myxoid hormone-like receptor F4/80, interleukin-1 beta IL-1 beta, interleukin-6 IL-6, monocyte chemoattractant factor MCP-1 in the liver of a mouse treated with a JUN N-terminal kinase inhibitor according to example 7 of the invention; * P <0.05, < P <0.001;

FIG. 8 is a graph showing the results of a flow assay for the treatment of macrophages, CD3+ T cells, CD45+ monocytes in non-parenchymal cells in the liver of schistosome-infected mice with JUN N-terminal kinase inhibitor according to example 8 of the present invention, wherein, graph A: macrophages, panel B: cd3+ T cells, panel C: cd45+ monocytes, panel D: histogram of expression levels of macrophages, cd3+ T cells, cd45+ monocytes, P <0.01.

Detailed Description

The present invention is described in detail below with reference to the drawings so that advantages and features of the present invention can be more easily understood by those skilled in the art, thereby making clear and defining the scope of the present invention.

The following presents a simplified summary of one or more aspects in order to provide a basic understanding of such aspects. This summary is not an extensive overview of all contemplated aspects, and is intended to neither identify key or critical elements of all aspects nor delineate the scope of any or all aspects. Its sole purpose is to present some concepts of one or more aspects in a simplified form as a prelude to the more detailed description that is presented later.

The applicant of the present invention has found through great amount of research experiment that JUN N-terminal kinase inhibitor has excellent curative effect on schistosomiasis.

The invention discloses an application of a JUN N-terminal kinase inhibitor in preparing a medicament for preventing and/or treating schistosomiasis.

In particular, the medicament can be used for inhibiting or improving granuloma and fibrosis of schistosomiasis liver insect eggs and inhibiting inflammation of schistosomiasis infection.

The medicament comprises an effective amount of one or several inhibitors of JUN N-terminal kinase and/or derivatives and/or pharmaceutically acceptable compounds thereof, including but not limited to SP600125.

The medicine is solution or suspension for intraperitoneal injection, or tablet, capsule or solution for oral administration or intravenous injection.

More specifically, the medicine is a solution or suspension for intraperitoneal injection prepared by dissolving a JUN N-terminal kinase inhibitor in dimethyl sulfoxide, the injection amount is 50-300mg/kg, and the treatment time is 3-4 weeks.

The medicine is used for reducing the granuloma and fibrosis area of schistosomiasis liver eggs, reducing the expression level of related fibrosis factors and inflammatory factors mRNA in the liver, reducing the expression of alpha-smooth muscle agonism protein (alpha-SMA), reducing the expression of glutamic-pyruvic transaminase (ALT) and glutamic-oxaloacetic transaminase (AST) in blood, reducing the expression of hydroxyproline, reducing the expression of metalloproteinase tissue inhibitor and reducing the level of matrix metalloproteinase degrading extracellular matrix, reducing inflammatory related cells and reducing inflammatory reactions of macrophages, CD3+ T cells and CD45+ monocytes in non-parenchymal cells.

Example 1

Proved by the JUN N-terminal kinase inhibitor to have protective effect on schistosome infected mouse liver granuloma

Detection of area of granuloma of single ova of mouse liver

The positive oncomelania is placed in dechlorinated water and allowed to spontaneously escape cercaria under the illumination condition of 25 ℃. The loop was dipped in dechlorinated water containing cercarias, and the solution was dropped onto a slide and counted under a dissecting microscope. Normal females C57BL/6J for 6-8 weeks, 5 mice per group were fixed on a wood board, abdominal hair was removed, infected skin was moistened with cotton balls dipped in dechlorinated water, and slides (containing 12.+ -. 1 cercaria) were covered on bare skin and left for 20min.

Dissolving SP600125 in dimethyl sulfoxide (DMSO) to prepare a solution or suspension for the next intraperitoneal injection, injecting SP600125 into the abdominal cavity 4 weeks after infection, continuously injecting 50mg/kg for 4 weeks, dissecting a mouse in the eighth week, taking the middle lobe of the liver, immersing the middle lobe into 4% paraformaldehyde for fixation, immersing the tissues in 70%, 80%, 95% and 100% ethanol for dehydration in sequence, and then placing the dehydrated liver tissues into ethanol/xylene for transparency; after tissue waxing and embedding, continuously slicing the wax block on a slicing machine, wherein the slicing thickness is 4 mu m; the slide glass is stuck on the slide glass after the slide glass is spread; HE staining was performed using Beijing Soxhlet staining kit, neutral resin sealing, randomly acquiring 6-8 fields per slice plane under a microscope, taking individual worm's egg granulomas for photographing, and analyzing HE staining positive areas with Image-Pro.

Experimental results show that the granuloma area of single ovum of liver of mice infected with schistosome cercaria is 20881.38 +/-11178.33 mu m ² Single ova granuloma area 9967.421 + -5690.208 μm after treatment with SP600125 ² The differences were statistically significant (P<0.05). The granuloma of ova is inflammation granuloma with ova as center, and aggregated cells such as macrophage, lymphocyte, neutrophil, eosinophil, MFB, etc. The reduction in granuloma area of individual eggs following SP600125 treatment suggests that JUN N-terminal kinase inhibitor SP600125 can reduce inflammatory lesions of the liver by eggs.

Example 2

Proved by the JUN N-terminal kinase inhibitor to have protective effect on liver fibrosis of mice infected by schistosome

Detection of single worm egg fiber area of mouse liver

Dissolving SP600125 in dimethyl sulfoxide (DMSO) to prepare a solution or suspension for the next intraperitoneal injection, injecting SP600125 into the abdominal cavity 4 weeks after infection, continuously injecting 50mg/kg for 4 weeks, dissecting the mice, taking the middle lobe of the liver, immersing the liver into 4% paraformaldehyde for fixation, immersing the tissues in 70%, 80%, 95% and 100% ethanol for dehydration in sequence, and then placing the dehydrated liver tissues into ethanol/xylene for transparency; after tissue waxing and embedding, continuously slicing the wax block on a slicing machine, wherein the slicing thickness is 4 mu m; the slide glass is stuck on the slide glass after the slide glass is spread; masson staining was performed using a Beijing Soxhlet Masson staining kit, neutral resin slides were sealed, 6-8 fields per slice plane were randomly acquired under a microscope, individual ova granulomas were taken for photography, and Image-Pro was used to analyze the Masson staining positive areas.

Experimental results show that the single egg fibrosis area of the liver of the mice infected with schistosome cercaria is 9398.583 +/-3981.51 mu m ² Single worm egg fibrosis area was 5722.46 ± 1548.711 μm after treatment with SP600125 ² The differences were statistically significant (P<0.05). The fibrosis with ova as the center is the repair of granulomatous inflammatory injury, the fibrosis area is reduced after SP600125 treatment, and the JUN N-terminal kinase inhibitor SP600125 is suggested to have a protective effect on schistosome infected mouse liver fibrosis.

Example 1 was followed.

Example 3

Immunohistochemical detection of expression level of alpha-smooth muscle actin (alpha-SMA) in mouse liver

Injecting SP600125 into abdominal cavity 4 weeks after infection, injecting 50mg/kg, continuously injecting for 4 weeks, dissecting mice in eighth week, taking middle lobe of liver, soaking into 4% paraformaldehyde for fixation, soaking tissue in 70%, 80%, 95% and 100% ethanol sequentially for dehydration, and then placing the dehydrated liver tissue into ethanol/xylene for transparency; after tissue waxing and embedding, continuously slicing the wax block on a slicing machine, wherein the slicing thickness is 4 mu m; the slide glass is attached after the slide glass is spread. Heating sodium citrate repairing liquid to boiling with a microwave oven, placing slices on a heat-resistant slice rack, placing into boiling buffer liquid, performing medium-low grade microwave treatment for 15min, taking out running water, naturally cooling, taking out from the buffer liquid, washing with distilled water for 2 times, and washing with PBS for 2 times each for 3min. The antigen-repaired sections were immersed in tap water for 2min. Soaking the slices in endogenous peroxidase blocker 3%H at room temperature ₂ O ₂ For 5min. Washing with distilled water for 2min, and washing with PBS buffer solution for 2min; taking out the slices, wiping the liquid around the tissues, sealing the slices by using 10% goat serum, and incubating the slices at 37 ℃ for 1h; a grouping of pen strokes is used to draw a circle,60 mu L of primary antibody (alpha-SMA 1:200) is added dropwise, and incubated overnight at 4 ℃; the slices are put into PBS buffer solution for full washing for 5min multiplied by 3 times; after wiping the liquid around the tissues, dripping a drop of horseradish enzyme-labeled goat anti-mouse/rabbit IgG secondary antibody, and incubating for 40min at room temperature; washing with PBS buffer for 3min×3 times; 0.05% DAB developer color development. Developing at room temperature for 5-20min, and stopping developing with tap water; hematoxylin dye liquor is used for dying the nucleus, 1% hydrochloric acid alcohol solution is differentiated for 3s, and running water is used for rinsing; sequentially placing the slices into 85% ethanol and 95% ethanol for 2min respectively, and dehydrating the slices in absolute ethanol I and absolute ethanol II for 5min respectively; transparent with xylene, and slicing sequentially into xylene I and II for 5min; a neutral resin is used for sealing. 6-8 fields per slice plane were randomly acquired under a microscope, photographed, and the antibody positive area calculated using Image Proplus.

Experimental results show that SP600125 can reduce expression of alpha-SMA in liver of schistosome infected mice, and the difference has statistical significance (P < 0.001). alpha-SMA is a protein secreted during activation of hepatic stellate cells forming liver fibrosis, and the level of the alpha-SMA is reduced, which suggests that the JUN N-terminal kinase inhibitor SP600125 can reduce the activation degree of hepatic stellate cells in liver of mice infected with schistosome and reduce liver fibrosis.

Example 1 was followed.

Example 4

Proved by the JUN N-terminal kinase inhibitor to have protective effect on liver injury of mice infected by schistosome

Detection of ALT in mouse blood

At 4 weeks post infection, SP600125, 50mg/kg was intraperitoneally injected, and after 4 weeks of continuous injection, mice were dissected at the eighth week, blood was collected by taking the eyeball, collecting the blood in a 1.5mL EP tube, standing overnight at 4℃and centrifuging at 3000rpm for 5min, sucking the supernatant serum, and preserving at-80 ℃. The level of glutamic pyruvic transaminase ALT and glutamic oxalacetic transaminase AST in serum is detected by using a kit of Nanjing built biological technology limited company.

The experimental results show that the ALT and AST levels in blood of mice infected with schistosome cercaria are 44.89+/-10.97U/L and 31.08+/-7.0U/L. ALT and AST levels in the blood of mice after treatment with SP600125 were 23.49 + -48.07U/L and 24.21+ -3.67U/L. The level of ALT in the blood indicates the acute degree of liver inflammation; AST levels indicate the extent of reactive liver injury. The ALT difference before and after treatment with SP600125 is statistically significant, suggesting that SP600125 can reduce liver inflammation and injury.

Example 1 was followed.

Example 5

Detection of hydroxyproline in mouse liver

4 weeks after infection, SP600125 was injected intraperitoneally, 50mg/kg was injected, and after 4 weeks of continuous injection, mice were dissected at the eighth week, blood was collected by taking the eyeball, collecting the blood in a 1.5mL EP tube, standing overnight at 4℃and centrifuging at 3000rpm for 5min, sucking the supernatant serum, and preserving at-80 ℃. 30-100mg of mouse liver left leaf is taken, and the hydroxyproline level in the liver is detected by using a hydroxyproline detection kit of Nanjing built Biotechnology Co.

The level of hydroxyproline in blood of mice infected with schistosome cercaria is 336.77 +/-48.07 mug/g wet liver weight, the level of hydroxyproline in liver of mice treated with SP600125 is 293.78 +/-15.92 mug/g wet liver weight, and the difference is statistically significant. Hydroxyproline is an amino acid, the main component of collagen tissue, and the catabolism of collagen in vivo can be known by measuring the hydroxyproline in blood and urine by utilizing the characteristic that the content of the hydroxyproline in collagen is the highest. A decrease in the level of hydroxyproline in the liver following SP600125 treatment suggests a decrease in the level of collagen in the liver.

Example 1 was followed.

Example 6

mRNA expression levels of related collagen molecules in mouse liver

About 100. Mu.g of mouse liver was taken, total RNA was extracted using Trizol, 500. Mu.g of total RNA was reverse transcribed into cDNA using a Roche reverse transcription kit, and each cytokine qPCR reaction was performed using cDNA as a template (Roche SYBR Green qPCR kit). Primer sequences are shown in Table 1 below:

TABLE 1

Experimental results show that the mRNA levels of Collagen I, collagen III and alpha-SMA in the livers of mice treated with SP600125 were reduced by 1.75-fold, 1.27-fold and 1.45-fold, respectively, compared with mice in the treatment group, suggesting that the Collagen levels in the livers of mice were reduced and the degree of fibrosis was reduced after SP600125 treatment.

Matrix Metalloproteinases (MMPs) and metalloproteinase Tissue Inhibitor (TIMP) are two important factors in the regulation of extracellular matrix (ECM) synthesis and degradation. Wherein MMP-1 mainly degrades type I and III collagen which are main components of extracellular matrix, and TIMP-1 can inhibit the activity of MMP-1. The mRNA level of matrix metalloproteinase (MMP-1) in the liver of mice after SP600125 treatment is not different, but the tissue inhibitor TIMP1 of the matrix metalloproteinase is reduced by 1.52 times compared with the mRNA level of the liver of untreated mice, the inhibition effect on the activity of MMP-1 is reduced, and the extracellular matrix degradation effect of MMP-1 is increased.

Example 1 is followed

Example 7

Demonstrating that inhibitors of JUN N-terminal kinase reduce the level of inflammation in the liver of schistosome infected mice

Detection of inflammatory factors in mouse liver

About 100ug of mouse liver was taken, total RNA was extracted using Trizol, 500 ug of total RNA was reverse transcribed into cDNA using a Roche reverse transcription kit, and each cytokine qPCR reaction (Roche SYBR Green qPCR kit) was performed using cDNA as a template. Primer sequences are shown in Table 2 below:

TABLE 2

/>

The experimental results show that the mRNA levels of inflammatory factors TNFα, F4/80, IL1 beta, IL6 and MCP-1 in the livers of mice after treatment with SP600125 are respectively reduced by 1.34 times, 1.26 times, 2.39 times, 1.61 times and 2.33 times, which suggests that the SP600125 has the effect of reducing the inflammatory levels in the livers.

Example 1 is followed

Example 8

Flow assay demonstrating that the JUN N-terminal kinase inhibitor SP600125 reduces the proportion of inflammatory cells in the liver of schistosome infected mice at levels of inflammation in the liver

Taking mouse liver, cutting the liver into small pieces of tissue, adding collagenase and DNase, digesting for 30min at 37 ℃, sequentially sieving with 200 mesh and 400 mesh sieve, centrifuging for 5min at 300g, and adding PBS to stop digestion; centrifuging for 4min at 50g, and removing liver parenchymal cells; centrifuging for 4min at 25g, and further removing hepatic parenchymal cells; transferring the supernatant into a new centrifuge tube, and centrifuging 300g for 5min; adding 3mL of erythrocyte lysate, incubating for 5min at 37 ℃, and counting after erythrocyte lysis; adding streaming antibody CD3-PE, CD45-FITC, F4/80-PE and CD11b-FITC (BD, USA) into 2X 106 cells, and staining at 4deg.C for 30 min; after washing with PBS, the suspension was resuspended and detected on-machine using a flow detector.

The results show that the ratios of macrophages, CD3+ T cells and CD45+ monocytes in the liver non-parenchymal cells of the DMSO group are 13.37+ -0.56%, 26.48+ -1.59% and 57.80+ -2.00%, respectively; the proportion of macrophages, CD3+ T cells and CD45+ monocytes in the liver non-parenchymal cells of the SP600125 treated mice was 10.13.+ -. 0.69%, 19.00.+ -. 0.61% and 51.20.+ -. 1.32%, respectively. The proportions of macrophages, cd3+ T cells, cd45+ monocytes were all reduced in the SP600125 treated group of non-parenchymal cells compared to DMSO, the differences being statistically significant (P < 0.01), suggesting that SP600125 treatment reduced the proportion of inflammation-related cells in the liver (fig. 8A-D).

The foregoing description is only illustrative of the present invention and is not intended to limit the scope of the invention, and all equivalent structures or equivalent processes or direct or indirect application in other related arts are included in the scope of the present invention.

Claims

Use of a jun N-terminal kinase inhibitor for the preparation of a medicament, characterized in that the medicament is for the prevention and/or treatment of schistosomiasis.
2. Use of a JUN N-terminal kinase inhibitor according to claim 1 for the preparation of a medicament for inhibiting or ameliorating schistosomiasis liver worm granuloma and fibrosis, inhibiting inflammation of schistosomiasis infection.
3. Use of a JUN N-terminal kinase inhibitor according to claim 1 for the preparation of a medicament comprising an effective amount of one or several JUN N-terminal kinase inhibitors and/or derivatives and/or pharmaceutically acceptable compounds thereof.
4. Use of a JUN N-terminal kinase inhibitor according to claim 3, in the manufacture of a medicament, wherein the JUN N-terminal kinase inhibitor comprises SP600125.
5. Use of a JUN N-terminal kinase inhibitor according to claim 1 for the preparation of a medicament, wherein the medicament is a solution or suspension for intraperitoneal injection, or an oral or intravenous tablet, capsule or solution.
6. The use of a JUN N-terminal kinase inhibitor according to claim 5 for the preparation of a medicament, wherein the medicament is a solution or suspension for intraperitoneal injection prepared by dissolving a JUN N-terminal kinase inhibitor in dimethyl sulfoxide, the injection amount is 50-300mg/kg, and the treatment time is 3-4 weeks.
7. Use of a JUN N-terminal kinase inhibitor according to claim 1 for the preparation of a medicament for reducing the granuloma and fibrosis area of schistosomiasis liver worm eggs and for down regulating the expression level of related fibrosis and inflammatory factor mRNA in the liver.
8. Use of a JUN N-terminal kinase inhibitor according to claim 1 for the preparation of a medicament for down-regulating the expression of alpha-smooth muscle agonism, down-regulating the expression of glutamate pyruvate transaminase and glutamate oxaloacetate in blood, down-regulating the expression of hydroxyproline, down-regulating the expression of tissue inhibitor of metalloproteases, up-regulating the level of matrix metalloproteases degrading the extracellular matrix.
9. Use of a JUN N-terminal kinase inhibitor according to claim 1 for the preparation of a medicament for reducing inflammation-related cells, alleviating inflammatory responses of macrophages, cd3+ T cells, cd45+ monocytes in non-parenchymal cells.