Application of isodan leaf emodin in preparation of Icam1 inhibitor

Abstract

The invention belongs to the technical field of medicines, and particularly relates to application of isodan leaf emodin in preparation of an Icam1 inhibitor, related experiments of establishment of a sepsis liver injury model induced by cecum ligation puncture (CLP) show that by applying the isodan leaf emodin, the Icam1 inhibitor can be used for preparing Icam1. According to the present invention, the level of liver tissue proinflammatory factors TNF-alpha, IL-1beta and IL-6 in the liver injury caused by sepsis is reduced, the level of the anti-inflammatory factor IL-10 is increased, and the HE dyeing of the liver tissue proves that the emodin can alleviate the CLP induced inflammatory cell infiltration in the liver tissue, can protect the structural integrity of the liver tissue, and can provide the anti-inflammatory effect on the liver tissue, such that the liver injury caused by sepsis can be inhibited, and the liver injury caused by sepsis can be inhibited. The data shows that the isodan leaf emodin can relieve sepsis liver injury and reduce liver function indexes. In research, it is found for the first time that the action mechanism of the isodan-leaf emodin in relieving the sepsis liver injury is that liver function indexes and inflammatory response are relieved by inhibiting expression of a transmembrane protein intercellular adhesion molecule-1 (Icam1), and it is indicated that the isodan-leaf emodin has a good application prospect in treating sepsis-related tissue injury.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, specifically relating to the use of isorhapontigenin (ISO) in the preparation of Icam1 inhibitors. Background Technology

[0002] Sepsis is a systemic inflammatory response syndrome (SIRS) caused by infection. The development of sepsis can cause damage to multiple tissues, including lung damage, kidney damage, and liver damage. It is estimated that about 30%-50% of sepsis patients will develop acute liver injury (ALI), which is characterized by elevated transaminase and bilirubin levels. In patients with severe sepsis or septic shock, the incidence of liver injury can be as high as 60%-80%, especially for patients with pre-existing chronic liver diseases (such as cirrhosis and fatty liver). Summary of the Invention

[0003] The present invention aims to provide the use of isodanthroline in the preparation of Icam1 inhibitors, with the aim of alleviating acute liver injury caused by sepsis.

[0004] Emodin is a dietary polyphenol, a resveratrol derivative found in grapes and traditional Chinese medicine. It can be obtained through extraction or chemical synthesis. Previous studies have found that emodin has an ameliorative effect on lung injury caused by sepsis (see the use of emodin in the preparation of CXCL10 inhibitors as described in Chinese patent document CN120549898A).

[0005] Since sepsis can cause damage to other tissues besides the lungs, including liver damage, and sometimes even both lung and liver damage simultaneously, if isopantheline bromide can improve both lung and liver damage caused by sepsis, then its application in antiseptic drugs would have a broader prospect. Therefore, further research was conducted on isopantheline bromide preparations for treating sepsis-induced liver damage, and a proposed use of isopantheline bromide in the preparation of Icam1 inhibitors was proposed, as follows: The first aspect of this invention is to provide the use of isodane emodin in the preparation of a drug having one or more of the following effects: (1) Icam1 inhibitors; (2) Reduce the secretion of pro-inflammatory factors in the liver; (3) Increase the secretion of anti-inflammatory factors in the liver; (4) Treatment of liver damage caused by sepsis.

[0006] Preferably, in use (2), the liver pro-inflammatory factors include TNF-α, IL-6, and IL-1β; Preferably, in use (3), the liver anti-inflammatory factor includes IL-10.

[0007] Preferably, the drug is a tablet, capsule, pill, oral liquid, traditional Chinese medicine, or injection.

[0008] Preferably, the drug further comprises pharmaceutically acceptable excipients.

[0009] For example, in preparing tablets and pills containing isopantherin, the isopantherin powder can be mixed with excipients such as starch and dextrin for weight adjustment, or the taste can be improved by mixing it with excipients such as sucrose and lactose. Preferably, the drug is administered once daily, with a single dose of 15–35 mg / kg. For example, the single dose can be 15, 20, 25, 30, or 35 mg / kg.

[0010] Preferably, the single dose of the drug is 25 mg / kg.

[0011] A second aspect of the present invention is to provide a medicament for treating liver damage caused by sepsis, with isopantherin as the active ingredient, as shown in Formula I. (I).

[0012] Compared with existing technologies, the beneficial effects of this invention are as follows: This invention relates to the application of isodanthroline in the treatment of septic liver injury. Through experiments using a cecal ligation-puncture (CLP)-induced septic liver injury model, it was confirmed that the polyphenol monomer isodanthroline can significantly improve pathological liver tissue damage and reduce the level of inflammatory factors in liver tissue. Experiments showed that the application of isodanthroline reduced the levels of pro-inflammatory factors TNF-α, IL-1β, and IL-6 in liver tissue and increased the level of the anti-inflammatory factor IL-10. HE staining of liver tissue confirmed that isodanthroline can alleviate CLP-induced inflammatory cell infiltration in liver tissue and protect the structural integrity of liver tissue. These data indicate that isodanthroline can alleviate septic liver injury and reduce liver function indicators. This study is the first to discover that the mechanism of isodanthroline in alleviating septic liver injury is through inhibiting the expression of the transmembrane protein intercellular adhesion molecule-1 (Icam1), thereby reducing liver function indicators and inflammatory responses, providing a new treatment option for sepsis-related liver injury. Attached Figure Description

[0013] The present invention will be explained in detail below with reference to the accompanying drawings. It should be noted that the drawings are used to provide a further understanding of the present invention and form part of the specification. They are used together with the embodiments of the present invention to explain the present invention, but should not impose any limitation on the implementability of the present invention.

[0014] In the attached figures: Sham group: sham surgery group, used as blank control group; ISO group: drug administration group, used as control group; CLP group: CLP sepsis liver injury model group; ISO+CLP group: CLP model drug administration group, 25mg / kg treatment group.

[0015] Figure 1 The results of qPCR show the effects of isopanthelenol on the expression of inflammatory factor genes in the liver tissue of CLP sepsis model mice. Figure 1 In this table, “Relative TNF-α mRNA” represents the expression level of the TNF-α gene, “Relative IL-6 mRNA” represents the measured expression level of the IL-6 gene, “Relative IL-1β mRNA” represents the measured expression level of the IL-1β gene, and “Relative IL-10 mRNA” represents the measured expression level of the IL-10 gene.

[0016] Figure 2 HE pathological section of mouse liver tissue.

[0017] Figure 3 To Figure 2 Damage scores of liver tissue pathological sections in each experimental group; Figure 3 The “Liver histology score” refers to the liver histology score (Smith score).

[0018] Figure 4 The results of liver function tests for AST (aspartate aminotransferase) and ALT (alanine aminotransferase) were obtained for each experimental group. Figure 4 In this table, "AST U / L" represents the concentration of aspartate aminotransferase, and "ALT U / L" represents the concentration of alanine aminotransferase.

[0019] Figure 5 Western blot results showing the inhibition of Icam1 protein expression in liver tissue of CLP sepsis model mice by isotannins ethinyl arvense.

[0020] Figure 6 The results of qPCR show that isopanol inhibits the expression of the Icam1 gene in the liver tissue of a CLP sepsis model mouse. Figure 6 In this context, "Relative mRNA expression" refers to the relative expression level of the Icam1 gene. Detailed Implementation

[0021] The technical solutions of the embodiments of this disclosure will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of this disclosure. All other embodiments obtained by those skilled in the art based on the embodiments of this disclosure without creative effort are within the scope of protection of this disclosure.

[0022] Experimental subjects To evaluate whether isodane can improve acute liver injury caused by sepsis, the study used cecal ligation and puncture (CLP) to induce sepsis as an experimental model, and set up a sham operation group and a drug administration group as controls. That is, the entire experimental subjects were divided into four groups: sham operation group (Sham), drug administration group (ISO, 25 mg / kg), model group (CLP), and model administration group (CLP+ISO, 25 mg / kg).

[0023] Constructing CLP model mice CLP model mice are created by ligating and puncturing the cecum of mice to simulate the pathophysiological process of sepsis. They are used to study treatment methods and evaluate drugs for sepsis. The specific CLP modeling method is as follows: 1. Preparation before the experiment (1) Animal selection: Commonly used strain: C57BL / 6 (adult males 7-8 weeks old, weighing 20-25g).

[0024] Ethical approval: It must be reviewed by the animal experiment ethics committee and follow the "3R principle".

[0025] (2) Instruments and reagents: Surgical instruments: sterile scalpel, forceps, sutures (5-0 or 6-0 absorbable sutures), 21G needles (for puncture).

[0026] Anesthesia: Intraperitoneal injection of sodium pentobarbital (50 mg / kg).

[0027] Postoperative care: Preheat normal saline (37℃).

[0028] 2. Surgical procedure steps (1) Anesthesia and skin preparation: Anesthetize the mice until they are painless. Fix them in a prone position, shave their abdomens, and disinfect them with iodine.

[0029] (2) Opening the abdomen to expose the cecum: Make a midline abdominal incision (1-1.5 cm), separate the muscle layers one by one to expose the cecum; gently pull out the cecum to avoid damaging the mesenteric vessels.

[0030] (3) Cecal ligation: Ligation location: approximately 75% from the end of the cecum.

[0031] (4) Cecal perforation: Use a 21G needle to puncture the distal end of the ligation 1-2 times; gently squeeze the cecum to allow feces to leak into the abdominal cavity, simulating bacterial dissemination.

[0032] (5) Abdominal closure and resuscitation: The cecum was repositioned, and the abdominal wall muscles and skin were sutured layer by layer. 1 mL of preheated saline solution was injected subcutaneously to replenish body fluids and prevent shock.

[0033] Constructing a sham surgery group (Sham) The sham group serves as the control group for sepsis animal experiments. It uses the same animal strain and simulates the cecal ligation and puncture (CLP) procedure without causing substantial damage. This is used to eliminate the interference of surgical trauma on the experimental results. The difference between the sham group and the CLP model mouse model described above is that the cecal ligation and puncture are not performed. Only other surgical steps such as skin disinfection, anesthesia, incision, and abdominal closure are performed.

[0034] Preparation of Emodin Solution The isodanthroline powder used in this embodiment was purchased from MedChemExpress (MCE) in the United States, catalog number HY-N2593. The parameters of isodanthroline are as follows: molecular weight: 258.27 g / mol, molecular formula: C15H14O4, molecular structure formula as follows: .

[0035] Preparation method of isodanthroline solution (taking 25 mg per kg body weight as an example, other dosages are prepared similarly): Dissolve 100 mg of pure isodanthroline powder in 2 mL of DMSO (dimethyl sulfoxide) and mix thoroughly. Take 100 μL of the mixed liquid and add it to 2 mL of physiological saline and mix thoroughly to obtain a working solution with a concentration of 2.5 mg per mL.

[0036] Application of isopanol in inhibiting inflammatory factors in a CLP-induced sepsis-induced liver injury model Experimental methods for detecting inflammatory factors in liver tissue: (1) Grouping and treatment of experimental animals: Grouping: Mice were divided into four groups: Sham, ISO, CLP, and ISO+CLP. Sham was the sham-operated group; ISO was the drug-only group, which was injected with the previously prepared working solution; CLP was the model group, which was a CLP model mouse that was constructed but did not receive the prepared working solution; ISO+CLP was a CLP model mouse that was injected with the working solution.

[0037] In this group, no drugs were administered to the Sham and CLP groups, and samples were collected from the Sham and CLP groups 8 hours after modeling. Samples were collected from the ISO and ISO+CLP groups after being administered drugs at a dose of 12.5 mg / kg for 5 consecutive days.

[0038] Sampling and preservation: Mouse liver tissue was taken, immediately immersed in liquid nitrogen, and then stored at -80℃.

[0039] (2) RNA extraction from liver tissue (FastPure Cell / Tissue Total RNA Isolation Kit RC112): Take about 50 mg of liver tissue, lyse it using the pestle method or a kit, add 500 μl of Buffer PL (lysis buffer) to homogenize it, transfer the homogenized sample to FastPure gDNA Filter Column II, centrifuge at 12000 rpm (13400×g) for 30 s, discard the supernatant, and collect the filtrate.

[0040] Add 0.5 times the volume of anhydrous ethanol to the filtrate and mix well. Transfer the mixture to FastPure RNA Column III, centrifuge at 12000 rpm (13400×g) for 30 s, and discard the filtrate. Add 700 μl of Buffer RW1 to the RNA column, centrifuge at 12000 rpm for 30 s, and discard the filtrate. Add 700 μl of Buffer RW2 (containing anhydrous ethanol), centrifuge at 12000 rpm for 30 s, and then centrifuge for another 1 min. Carefully remove the adsorption column to avoid contamination. Transfer to a new centrifuge tube (DNase-free Collection Tubes), add 50-200 μl (50 μl recommended) of RNase-free ddH2O to the center of the adsorption column, and incubate at room temperature for 10 min. Elute the RNA by centrifuging at 12000 rpm for 1 min. To increase yield, the ddH2O can be preheated to 65°C, or a second elution can be performed after centrifugation. Store the RNA at -80°C.

[0041] (4) Reverse transcription: Detect RNA concentration and adjust RNA loading volume to 1000ng-1500ng; Reverse transcription system: Enzyme Mix: 0.5 μl; Primer Mix: 0.5 μl; 5×RT Buffer: 2 μl; RNA + H2O: 7 μl; Mix the eight tubes by vortexing, centrifuge, and then reverse transcribe using a cytometer. After reverse transcription, dilute the cDNA with 40-50 μl of ddH2O.

[0042] (5) qPCR detection (detection indicators: TNF-α, IL-6, IL-1β, IL-10): qPCR system: SYBR Green: 5 μl; forward and reverse primers (F+R): 0.4 μl each; H2O: 3.6 μl; cDNA: 1 μl; Procedure: Use the Roche qPCR instrument and set up 40-50 cycles.

[0043] Combination Figure 1 As shown, Figure 1 From left to right: Real-time quantitative qPCR analysis of TNF-α expression, real-time quantitative qPCR analysis of IL-6 expression, real-time quantitative qPCR analysis of IL-1β expression, and real-time quantitative qPCR analysis of IL-10 expression. The experimental results show that isopanthelin significantly reduced the expression of tumor necrosis factor α, IL-1β, and IL-6 in the liver, while increasing the expression of IL-10. The detection results are shown below. Figure 1 ( * p、 # p <0.05, ** p , ## p <0.01, *** p , ### p <0.001, **** p , #### p <0.0001, the statistical result is statistically significant.

[0044] Figure 1 In this context, tumor necrosis factor α (TNF-α) is a pro-inflammatory cytokine mainly produced by macrophages and monocytes; IL-1β is interleukin-1β, a pro-inflammatory cytokine; IL-6 is interleukin-6, a pleiotropic cytokine with broad functions; and IL-10 is interleukin-10, which mainly maintains the balance of the immune system by inhibiting the production of pro-inflammatory factors and regulating the activity of immune cells.

[0045] Application of isopanol in the inhibition of liver injury in a CLP model (1) Experimental grouping: The grouping was based on CLP modeling. The Sham group and CLP group were not given any medication. The Sham group and CLP group were sampled 8 hours after modeling. The ISO group and ISO+CLP group were given 25mg / kg for 5 consecutive days before sample collection.

[0046] (2) Post-molding processing: Anesthesia and fixation: After anesthesia with 2% sodium pentobarbital, the mouse was fixed to a plate; Dissection and blood collection: Disinfect the skin with 75% alcohol, open the chest and collect blood from the heart; Liver tissue processing: Mouse liver tissue was pre-cooled and washed with PBS, dried, embedded in OCT, and then quick-frozen at -80℃.

[0047] (3) Slicing: Embedding process: OCT is coated on the back of the cryopreserved embedding blocks and fixed to a microtome; Sectioning parameters: 30μm trimming thickness, 4μm sectioning thickness, glass slides were labeled and stored at -20℃.

[0048] (4) Staining: Fixation: Fix tissues with 4% paraformaldehyde for 20 minutes; Washing: Wash 3 times with 1×PBS (shake 30 rpm, 3 minutes each time); Mark: Use the Super PAP Pen to draw circles on the slide (avoiding tissue areas).

[0049] (5) Permeation treatment: Prepare a 0.1% Triton X-100 solution and add it dropwise into the tissue circle; Processing time: 15 minutes; Operating environment: Humidity box (to maintain humidity); Wash the slides with 1×PBS buffer; repeat 3 times, 3 minutes each time (shake gently).

[0050] (6) Closed: Blocking solution: 5% BSA (prepared with PBS); Sealing conditions: Keep the humidified box sealed and protected from light for 1 hour.

[0051] (7) Antibody incubation: Primary antibody incubation: Follow the dilution ratio in the antibody instructions; conditions: 4°C, protected from light, in a humidified chamber overnight; remove the antibody the next day and allow it to warm to room temperature for 30 minutes.

[0052] Secondary antibody incubation: In a light-protected, humidified box, follow the instructions.

[0053] (8) Nucleus staining and mounting: DAPI staining of nuclei: Preparation ratio: 1:200 (diluted with PBS); Staining conditions: stain at room temperature in the dark for 20 minutes; Washing after staining: wash once with 1×PBS.

[0054] Mounting: Use 50% glycerol to mount the slide, add 15-20 μL; avoid moving the slide after adding the coverslip.

[0055] (9) Observe and photograph under a microscope.

[0056] Experimental results showed that HE staining of liver tissue in the isopanol-treated group revealed a significant reduction in inflammatory cell infiltration and improved liver structural integrity. Microscopic observation revealed... Figure 2 The results are statistically scored digitally and are then visible. Figure 3 ( **** p <0.0001).

[0057] Effects of emodin on liver function indicators in a CLP sepsis liver injury model In each experimental group, AST (aspartate aminotransferase) and ALT (alanine aminotransferase) levels were measured in mouse blood samples. The results are as follows: Figure 4 As shown ( * p、 # p <0.05, ### p <0.01), indicating that isopantheroside can effectively reduce AST and ALT levels in a CLP-induced sepsis-induced liver injury model.

[0058] Inhibitory effect of isopantheroside on Icam1 expression in liver tissue of CLP model mice Methods for detecting Icam1 expression in liver tissue: (1) Detection of Icam1 protein expression level: Grouping and treatment of experimental animals: The grouping and modeling process of experimental animals is the same as above.

[0059] Liver tissue protein extraction: Modeling was performed according to the above procedure, and mouse liver tissue was collected. Prepare lysis buffer (per sample): 500 uL RIPA lysis buffer; PMSF (1:100 v / v), Cocktail protease inhibitor (1:25 v / v), Phosphatase inhibitor (1:100 v / v); first mechanically homogenize the tissue, then add 500 uL lysis buffer.

[0060] Ultrasonic fragmentation: 3-second pulse / 6-second interval, 75W power; number of cycles: 3-5 (complete tissue fragmentation is the criterion).

[0061] Centrifuge at 4℃: 12000 rpm for 20 minutes; after centrifugation, transfer the supernatant to a new EP tube; Adjust the protein concentration to 3 μg / μL, then denature 400 μL of protein with 100 μL of 5×SDS in a metal bath at 98°C for 10 min.

[0062] Western blot analysis: Western blot analysis was performed on the Icam1 gene. The results showed that isopantherin significantly reduced Icam1 protein expression in the liver, as indicated by the detection results. Figure 5 , Figure 5 The groups are arranged in order of Sham group, ISO group, CLP group, and ISO+CLP group. The rightmost group is ISO+CLP group, whose Icam1 protein expression level is significantly lower than that of CLP group.

[0063] (2) Detection of Icam1 mRNA levels: The modeling process, animal grouping, and sample processing were the same as above. qPCR was used to detect the Icam1 gene. Experimental results showed that isopantherin significantly reduced Icam1 expression in the liver, as indicated by the detection results. Figure 6 ( **** p <0.0001), the expression results of Icam1 by real-time quantitative qPCR analysis in the figure show that the expression of Icam1 in the ISO+CLP group was significantly lower than that in the CLP group at the mRNA level, indicating that isopanthelenol can significantly reduce the expression of Icam1 in the liver.

[0064] Experimental conclusion: The above examples demonstrate that isodanthroline provides a new candidate drug for the clinical treatment of septic liver injury by regulating the expression of Icam1. Isodanthroline inhibits the expression of Icam1 in the liver, thereby inhibiting the infiltration of inflammatory cells, reducing the expression of TNF-α, IL-1β, and IL-6, and increasing the expression of IL-10, ultimately effectively inhibiting the death of the CLP-induced septic liver injury model.

[0065] Based on the above experimental examples, it is known that the application of isodanthroline reduced the levels of pro-inflammatory factors TNF-α, IL-1β, and IL-6 in liver tissue and increased the level of the anti-inflammatory factor IL-10. HE staining of liver tissue confirmed that isodanthroline could alleviate CLP-induced inflammatory cell infiltration in liver tissue and protect the structural integrity of liver tissue. These data indicate that isodanthroline can alleviate septic liver injury and reduce liver function indicators. This study is the first to discover that the mechanism by which isodanthroline alleviates septic liver injury is through inhibiting the expression of the transmembrane protein intercellular adhesion molecule-1 (Icam1), thereby reducing acute liver injury caused by sepsis.

[0066] It should be noted that, unless otherwise defined, all terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains, and terms such as those defined in a common dictionary should be interpreted as having a meaning consistent with their meaning in the context of the relevant art. It should also be understood that the above is a description of the disclosure and should not be considered as a limitation thereof. Although several exemplary embodiments of the disclosure have been described, those skilled in the art will readily understand that various changes, modifications, substitutions, and variations can be made to these embodiments without departing from the principles and spirit of the invention. Therefore, all such modifications are intended to be included within the scope of the disclosure as defined in the claims, and will not be detailed here.

Claims

1. Use of isorhaponticin in the preparation of a medicament having one or more of the following effects: (1) Icam1 inhibitor; (2) reducing secretion of proinflammatory factors in liver; (3) increasing secretion of anti-inflammatory factors in liver; (4) treating liver injury caused by sepsis.

2. The use according to claim 1, wherein in the use (2), the proinflammatory factors in liver include TNF-α, IL-6, IL-1β.

3. The use according to claim 1, wherein in the use (3), the anti-inflammatory factors in liver include IL-10. The medicament is a tablet, a capsule, a pill, an oral solution, a traditional Chinese medicine or an injection solution. The medicament further comprises a pharmaceutically acceptable excipient.

4. Use according to claim 1, characterized in that: The medicament is administered once a day, and the single administration dose is 15-35 mg / kg.

5. Use according to claim 4, characterized in that: The single administration dose of the medicament is 25 mg / kg.

6. Use according to claim 1, characterized in that:

8. A medicament for treating liver injury caused by sepsis, wherein isorhaponticin shown in formula I is a pharmacologically active ingredient, 7. Use according to claim 5, characterized in that: ​ ​ 。

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