Application of epigallocatechin gallate in preparation of medicine for preventing mouse cecum ligation perforation sepsis
Prophylactic administration of EGCG to mice with cecal ligation and perforation sepsis inhibited the inflammatory response, improved survival rate, and reduced bacterial infection and liver tissue damage. This approach addresses the lack of effective preventative measures for sepsis in existing technologies, achieving good preventative efficacy and safety.
Patent Information
- Application Number
- CN202511000381.8
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-07-21
- Publication Date
- 2025-10-28
AI Technical Summary
There is a lack of effective preventive measures to reduce the incidence of sepsis in the current technology. EGCG may enhance the antioxidant and anti-inflammatory properties of the human body when treating sepsis, and its synergistic effect with other drugs has not been fully explored.
Epimorphyll catechin gallate (EGCG) was used as the active ingredient to prepare a drug for the prevention of cecal ligation perforation sepsis in mice via intraperitoneal or intravenous injection at a dose of 0-20 mg/kg and a concentration of 0-200 μM. The drug was directly added to the culture medium and co-cultured with cells to inhibit inflammatory signaling pathways such as TLR4/NF-κB, block GSDMD-mediated pyroptosis, reduce the release of pro-inflammatory factors, and enhance antioxidant defense capabilities.
EGCG significantly improves the survival rate of mice, reduces bacterial infection in various organs, improves liver tissue damage and inflammation, alleviates tissue damage, reduces mortality, and has high safety and good tolerability, making it suitable for a wide range of applications.
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Figure CN120837489A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of medicine, specifically to the application of EGCG in the prevention of CLP sepsis in mice. Background Technology
[0002] Sepsis is a systemic inflammatory response syndrome caused by infection. It can be caused by bacteria, fungi, viruses, or parasites, and is commonly seen in diseases such as pneumonia, abdominal infections, urinary tract infections, and bacteremia. In severe cases, it can lead to multiple organ failure and endanger life. Due to the high cost of treatment and the heavy consumption of medical resources, sepsis has become a major global health threat, and there is an urgent need to develop new treatments.
[0003] Cecal ligation and perforation (CLP) model-induced multimicrobial sepsis is the most widely used model. CLP-induced rodent sepsis models exhibit disease patterns with typical symptoms of sepsis or septic shock, such as hypothermia, tachycardia, and tachypnea. CLP models more accurately reflect the clinical features of sepsis than endotoxin (LPS)-induced sepsis models.
[0004] Epigallocatechin gallate (EGCG) is the most potent active ingredient in tea polyphenols. Belonging to the catechin family, it possesses antibacterial, antioxidant, anti-inflammatory, and antitumor effects, making it highly valuable for drug development. Studies have shown that EGCG can reduce the risk of excessive inflammatory responses induced by infection or endotoxemia by inhibiting inflammatory signaling pathways such as TLR4 / NF-κB, blocking GSDMD-mediated pyroptosis, reducing the release of pro-inflammatory factors, and enhancing antioxidant defense capabilities. Furthermore, EGCG's protective effect on the intestinal barrier may reduce bacterial translocation and lower the likelihood of sepsis.
[0005] However, it is important to clarify that therapeutic effects (intervention for existing symptoms) are not the same as preventive effects (reducing the likelihood of developing the disease). Existing evidence shows that daily cumulative intake of EGCG may enhance the body's antioxidant and anti-inflammatory properties, suggesting that EGCG may have preventive potential for inflammatory diseases. Future research should continue to explore the application of EGCG in drug preparation and its synergistic effects with other drugs to fully realize its application value in the prevention and treatment of sepsis. Summary of the Invention
[0006] In view of the above, this invention has conducted an in-depth study on the application of EGCG in the prevention of CLP sepsis in mice. To achieve the above-mentioned technical effects, this invention is implemented through the following technical means:
[0007] This invention first discloses the application of epimorphyll catechin gallate in the preparation of a drug for preventing cecal ligation perforation sepsis in mice, wherein:
[0008] The molecular formula of the table for catechin gallate is C. 12 H 18 O 11 Its molecular weight is 458.37, and its chemical structure is as follows:
[0009]
[0010] Furthermore, the drug also includes a pharmaceutically acceptable carrier.
[0011] Furthermore, the drug administration routes include: intraperitoneal injection or intravenous injection, as well as direct addition to culture medium for co-culture with cells.
[0012] Furthermore, the intravenous injection is a tail vein injection.
[0013] Furthermore, during administration, epimercurate catechin gallate is a solution preparation made from dry powder (purchased from Sigma-Aldrich, CAS No.: 989-51-5).
[0014] Furthermore, the dosage for intraperitoneal or intravenous injection is 0-20 mg / kg.
[0015] Furthermore, the concentration used for direct addition of culture medium and co-culture with cells is 0-200 μM.
[0016] The beneficial effects of this invention are as follows:
[0017] (1) EGCG can intervene in the pathophysiological process of sepsis in mice by inhibiting the inflammatory response. In the mouse CLP sepsis model, EGCG can significantly improve the survival rate of mice, reduce bacterial infection in various organs, and improve liver tissue damage and inflammation. EGCG pretreatment can also effectively inhibit the increase of macrophage inflammatory factors under LPS stimulation. Applying EGCG to prevent sepsis can effectively control the inflammatory response, reduce tissue damage, and reduce mortality in CLP sepsis mice, showing good efficacy in preventing sepsis.
[0018] (2) EGCG, as a natural compound, has high safety and good tolerability. Compared with traditional drugs, it has fewer side effects and a wider range of applications. Attached Figure Description
[0019] Figure 1 A is a schematic diagram of the experimental procedure for establishing a mouse cecal perforation sepsis model and EGCG treatment; Figure 1 B shows the 7-day survival curves of mice in the CLP group and the sham-operated group (n=10 for each group); Figure 1C represents the 7-day survival curves of mice in the intraperitoneal injection EGCG treatment group and the CLP group (n=10 for each group); Figure 1 D is the 7-day survival curve of mice in the EGCG treatment group and the CLP group treated by tail vein injection (n=8-9 for each group).
[0020] Figure 2 A is a schematic diagram of the experimental process for establishing a mouse CLP sepsis model, treating and extracting mouse organs and primary bone marrow-derived macrophages using EGCG; Figure 2 BF shows the colony count results of heart, liver, spleen, lung, and kidney tissues from each group of mice after grinding, dilution, and agar dispensing.
[0021] Figure 3 The figures below show the results of qPCR detection of the expression of inflammatory factors IL-1β, TNF-α, CXCL10, CCL5, CCL2 and CXCL11 in primary bone marrow-derived macrophages of mice in each group.
[0022] Figure 4 AB was used to detect the serum levels of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) in mice in each group using a fully automated biochemical analyzer. Figure 4 The figures in Figure CF show the results of qPCR detection of the expression of inflammatory factors IL-1β, TNF-α, CCL5, and CCL2 in the liver tissues of mice in each group.
[0023] Figure 5 The images above show the results of qPCR detection of the expression of inflammatory factors IL-1β, TNF-α, CCL5, CCL2, CXCL10, and CXCL11 in immortalized bone marrow-derived macrophages (iBMDM) after pretreatment with EGCG (200 μM) and stimulation with LPS (1 μg / mL). Detailed Implementation
[0024] The preferred embodiments of the present invention will now be described in detail so that the advantages and features of the present invention can be more easily understood by those skilled in the art, thereby providing a clearer and more explicit definition of the scope of protection of the present invention.
[0025] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.
[0026] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. The following embodiments are only for illustrating the present invention and should not be regarded as limiting the scope of the present invention. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall be followed. Where the manufacturers of reagents or instruments are not specified, they are all conventional products that can be purchased commercially.
[0027] Example 1
[0028] This embodiment uses CLP to establish a mouse model of sepsis and verifies the effect of EGCG on the survival rate of CLP-induced sepsis mice. The specific operation method is as follows:
[0029] (1) Experimental materials:
[0030] SPF-grade female C57BL / 6j mice (6-8 weeks old, purchased from the animal facility of Chongqing Medical University, acclimatized for one week, fasted for 12 hours before surgery), electric razor, 1mL syringe (0.45mm needle), sterile 4.0 sutures with needle, scissors, forceps, needle holder, povidone-iodine solution, 75% alcohol, cotton balls, heating pad (in the following examples, the above experimental materials are collectively referred to as CLP experimental equipment and animals), EGCG.
[0031] (2) Experimental methods and specific steps:
[0032] Twelve hours prior to CLP, mice were pretreated with either EGCG (20 mg / kg) or sterile water. They were then deeply anesthetized and placed on the surgical platform. The lower left quadrant of the abdomen was shaved using an electric razor. The area was first sterilized with a cotton ball soaked in 75% alcohol, followed by wiping with an iodine-soaked cotton ball for further sterilization. An incision was made in the skin of the sterilized area, and then the peritoneum was incised 1 cm to expose the cecum. Approximately one-third of the cecum was tightly ligated near the tail using sterile 4.0 sutures. A puncture was then performed in this area using a clean 0.45 mm syringe needle. A small amount of feces was expelled with forceps. After ligation and puncture, the cecum was gently returned to the abdominal cavity, and the incisions in the muscle and skin were sutured with sterile 4.0 sutures. The incisions were then wiped with an iodine-soaked cotton ball for further sterilization. Postoperatively, the mice were placed on a heating pad to ensure complete recovery from anesthesia. In the CLP model, sepsis is concentrated in the peritoneal cavity, particularly the cecum, which is the primary site of infection. This site allows for the controlled release of fecal matter into the peritoneal space, mimicking the multimicrobial nature of intraperitoneal sepsis in humans (flowchart shown). Figure 1 (As shown in A). The sham surgery control group underwent only laparotomy, without cecal ligation or perforation.
[0033] EGCG (20 mg / kg) or sterile water was injected again 24 h and 48 h after CLP surgery. The weight and survival of the mice were monitored daily for 7 consecutive days.
[0034] (3) Experimental results:
[0035] like Figure 1 As shown, compared with the sham-operated group, the 7-day survival rate of mice in the CLP group was significantly reduced ( Figure 1 B, **P<0.01); whether intraperitoneal injection ( Figure 1 C, **P<0.01) or tail vein injection ( Figure 1 D, *P<0.05), the 7-day survival rate of mice in the EGCG treatment group was significantly higher than that in the CLP group.
[0036] Example 2
[0037] This embodiment verifies the effect of EGCG pretreatment on bacterial load in various organs of CLP-induced septic mice through animal experiments. The specific operation method is as follows:
[0038] (1) Experimental materials:
[0039] CLP experimental equipment and animals, EGCG, PBS solution, homogenization tubes, magnetic beads, and blood agar plates.
[0040] (2) Experimental methods and specific steps:
[0041] Twelve hours prior to CLP, mice were pretreated with either intraperitoneal injection of EGCG (20 mg / kg) or sterile water. Mice were euthanized at specific time points post-CLP. Hearts, livers, spleens, lungs, and kidneys were isolated under sterile conditions, weighed, and placed in homogenization tubes. 700 μL of PBS and two magnetic beads were added, and the tissues were homogenized using a homogenizer. After homogenization, the tissues were diluted to different concentration gradients, and 10 μL of each gradient dilution was inoculated onto blood agar plates. The homogenized volume of each tissue was measured. After incubation at 37°C for 24 hours, the colony-forming units (CFU) were counted. CFU = number of colonies per milliliter * homogenate volume / organ weight.
[0042] (3) Experimental results:
[0043] like Figure 2 As shown, compared with the CLP group mice, the bacterial load in all organs of the EGCG-treated group mice was significantly reduced. Figure 2 BF (**P<0.01; ***P<0.001) indicates that EGCG has a preventive effect on bacterial infection of various organs in mice caused by sepsis.
[0044] Example 3
[0045] This embodiment uses animal experiments to verify the effects of EGCG pretreatment on serum AST and ALT levels and the expression of inflammatory factors in liver tissue of CLP-septic mice. The specific operation method is as follows:
[0046] (1) Experimental materials:
[0047] CLP experimental equipment and animals, EGCG, biochemical blood collection tubes, homogenization tubes, magnetic beads, sterile enzyme-free 1.5mL centrifuge tubes, Trizol.
[0048] (2) Experimental methods and specific steps:
[0049] Twelve hours before CLP, mice were pretreated with EGCG (20 mg / kg) or sterile water via intraperitoneal injection. At a specific time point after CLP, the mice were deeply anesthetized, placed in a lateral position on a flat surface, and the skin around the eyes was pressed with the index finger and thumb to cause the eyeballs to protrude and become congested. The eyeballs were then quickly removed with ophthalmic forceps, and the mice were inverted with their heads down. The mice's bodies could be massaged from the back of the body towards the eyes to allow blood in the orbit to flow into the prepared biochemical blood collection tubes. After collecting the blood, the mice were euthanized, and the livers were separated under sterile conditions. Blood was centrifuged at 3000g for 10 minutes to separate serum. Serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels were detected using an automated biochemical analyzer. Liver tissue was placed in a homogenization tube, 1 mL of Trizol was added for tissue lysis, and two magnetic beads were added for homogenization. After centrifugation, total RNA was extracted from the liver tissue by adding chloroform and isopropanol. cDNA was synthesized by reverse transcription and used as a template to detect the expression levels of IL-1β, TNF-α, CCL5, and CCL2 in the liver of mice in each group by qPCR.
[0050] (3) Experimental results:
[0051] like Figure 3 As shown, compared with the CLP group mice, the EGCG treatment group mice showed a significant improvement in liver function. Figure 3 AB, **P<0.01; ***P<0.001), and the expression of inflammatory factors in liver tissue was also significantly inhibited ( Figure 3 CF, ***P<0.001).
[0052] Example 4
[0053] This embodiment uses animal experiments to verify the effect of EGCG pretreatment on the expression of inflammatory factors in primary bone marrow macrophages of CLP-secreting mice. The specific operation method is as follows:
[0054] (1) Experimental materials:
[0055] CLP experimental equipment and animals, EGCG, PBS solution, cell sieve, red blood cell lysis buffer, sterile enzyme-free 1.5mL centrifuge tubes, Trizol.
[0056] (2) Experimental methods and specific steps:
[0057] Twelve hours prior to CLP, mice were pretreated with EGCG (20 mg / kg) or sterile water via intraperitoneal injection. At specific time points post-CLP, the mice were euthanized and immersed in 75% alcohol for 5 minutes for disinfection. Under aseptic conditions, the femur and tibia were separated, muscle tissue was removed, and the bones were rinsed thoroughly with PBS. Subsequently, both ends of the bones were cut, and the bone marrow cavity was repeatedly flushed with PBS using a syringe until the bone turned white. Bone marrow cell suspension was collected into centrifuge tubes, and the cell suspension was pipetted 10-15 times. The suspension was then passed through a 70 μm cell sieve to remove bone fragments and impurities, and centrifuged (400 g, 5 minutes) before discarding the supernatant. Red blood cells were lysed with 3-5 mL of red blood cell lysis buffer for 5 minutes. The lysis was terminated by adding 10 mL of PBS. After centrifugation, the cells were washed once with PBS and collected into sterile 1.5 mL centrifuge tubes without enzymes. 500 μL of Trizol was added for lysis and total RNA was extracted from macrophages. The expression levels of IL-1β, TNF-α, CXCL10, CCL5, CCL2 and CXCL11 in primary bone marrow-derived macrophages of mice in each group were detected by qPCR.
[0058] (3) Experimental results:
[0059] like Figure 4 As shown, compared with the CLP group mice, the expression of inflammatory factors in primary bone marrow-derived macrophage tissue of the EGCG-treated group mice was significantly inhibited. Figure 4 The results (*P<0.05; **P<0.01; ***P<0.001) indicate that EGCG plays a preventive role in the dysregulation of inflammatory factors caused by sepsis.
[0060] Example 5
[0061] This embodiment verifies the effect of EGCG pretreatment on the expression of inflammatory factors in immortalized bone marrow macrophages through cell experiments. The specific operation method is as follows:
[0062] (1) Experimental materials:
[0063] Immortalized bone marrow-derived macrophages (iBMDM), DMEM complete medium (10% fetal bovine serum, 1% penicillin-dextrose antibody), EGCG, lipopolysaccharides (LPS), cell culture plates, sterile enzyme-free 1.5 mL centrifuge tubes, Trizol.
[0064] (2) Experimental methods and specific steps:
[0065] iBMDM cells were seeded at 5 × 10⁶ cells per well in 6-well cell culture plates. 5 Cells were cultured in a 37℃, 5% CO2 incubator. The incubation settings were: A: control, B: LPS, and C: EGCG + LPS. After the initial incubation period, EGCG was added to group C at a final concentration of 200 μM. Sterile water was added to groups A and B as controls. All cells were co-cultured in the incubator for 2 hours. After 2 hours, 1 μg / mL LPS was added to groups B and C to stimulate the cells for 6 hours. After stimulation, the culture medium was discarded, and 500 μL of Trizol was added for lysis. Total RNA was extracted from the cells. The expression levels of IL-1β, TNF-α, CCL5, CCL2, CXCL10, and CXCL11 in each group of iBMDM cells were detected by qPCR.
[0066] (3) Experimental results:
[0067] like Figure 5 As shown, compared with group B, EGCG pretreatment can effectively inhibit the expression of inflammatory factors in iBMDM cells (***P<0.001), which is consistent with the results of primary macrophages.
[0068] The above description and specific embodiments are intended to demonstrate the practical application of the technical solution provided by this invention and should not be construed as limiting the scope of protection of this invention. Those skilled in the art can make various modifications, equivalent substitutions, or improvements within the spirit and principle of this invention. The scope of protection of this invention is defined by the appended claims.
Claims
1. Application of epimorphyll catechin gallate in the preparation of a drug for preventing cecal ligation perforation sepsis in mice, wherein: The molecular formula of the table for catechin gallate is C. 12 H 18 O 11 The chemical structural formula is as follows:
2. The application according to claim 1, wherein: The drug also includes a pharmaceutically acceptable carrier.
3. The application according to claim 1, wherein: The drug administration routes include: intraperitoneal injection or intravenous injection, as well as direct addition to culture medium for co-culture with cells.
4. The application according to claim 3, wherein: When administered, epimercurate catechin gallate is a solution preparation made from dry powder.
5. The application according to claim 3, wherein: The dosage for intraperitoneal or intravenous injection is 0-20 mg / kg.
6. The application according to claim 3, wherein: The concentration used for direct addition to the culture medium and co-culture with cells is 0-200 μM.