Application of meloxicam in preparation of pyroptosis inhibitor and treatment of sepsis lung injury

Abstract

The invention discloses application of meloxicam in preparation of a pyroptosis inhibitor and treatment of sepsis. It is found that meloxicam can inhibit NLRP3 inflammasome assembly and the pyroptosis level of cells mediated by meloxicam. In addition, in-vivo experiments in mice find that meloxicam can relieve lung injury caused by sepsis by inhibiting pyroptosis. The meloxicam is expected to serve as an NLRP3 inflammasome mediated pyroptosis inhibitor to be developed into a medicine for clinically treating the sepsis lung injury.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and specifically refers to the application of meloxicam in preparing cell pyroptosis inhibitors and treating septic lung injury. Background Art

[0002] Sepsis refers to a life-threatening systemic inflammatory response syndrome caused by host immune response disorders caused by infectious factors (bacteria, fungi, parasites, viruses, etc.). It is a common complication and one of the main causes of death in clinical critically ill patients such as trauma, burns, shock, infection, and major surgical operations. There are more than 18 million sepsis patients worldwide each year, and the incidence rate increases by 1.5% year by year. In my country, a multicenter epidemiological survey showed that the incidence of severe sepsis in the ICU was 8.68%, the mortality rate was as high as 44.7%, and the per capita medical expenditure was US$11,390 / year. In recent years, experimental and clinical research on sepsis has received attention, which has improved people's understanding of it, but the incidence and mortality of sepsis are still high, which seriously threatens human health and poses a great challenge to the world's public health system. The conquest of sepsis is a global problem. In my country, the development of sepsis drugs is a clinically urgently needed drug for the treatment of critical illnesses that the country encourages to develop, and it is also a national major new drug creation support variety. However, to date, there is still no approved specific treatment for sepsis.

[0003] In recent years, the relationship between host cell death and sepsis has attracted much attention. Researchers have found that macrophages induce cell pyroptosis by activating inflammasomes, and over-activated cell pyroptosis can induce an inflammatory storm. Due to the structural characteristics of the lung, it is the primary organ affected by the systemic inflammatory response. Sepsis lung injury then further aggravates the sepsis process through multiple pathways, forming a vicious cycle, which seriously threatens the life and health of patients. Therefore, timely treatment of sepsis lung injury is crucial to improving the prognosis of sepsis patients. NLRP3 inflammasome-mediated pyroptosis is considered to be a programmed cell lytic death that plays a key role in clearing intracellular pathogens. However, when NLRP3 inflammasomes are abnormally activated, inflammatory diseases can result. Studies have shown that the activation of NLRP3 inflammasomes plays a promoting role in the formation of the body's inflammatory storm, which affects the severity and prognosis of sepsis lung injury.

[0004] It can be seen that abnormal activation of NLRP3 inflammasome and its mediated cell pyroptosis signaling pathway play an important role in the occurrence of sepsis, and inhibiting excessive cell pyroptosis has become a new idea for the treatment of septic lung injury.

[0005] Meloxicam is a nonsteroidal anti-inflammatory drug used to treat rheumatoid arthritis, ankylosing spondylitis and osteoarthritis. It was developed by Boehringer Heinz and first launched in Germany in 1996. Two years after its launch, its sales exceeded 300 million. It was subsequently launched in the United States in 2000 and then approved for marketing in China in 2002. Studies have shown that most nonsteroidal anti-inflammatory drugs inhibit the activity of cyclooxygenase (COX) and thus block the synthesis of prostaglandins (PG) involved in inflammation, cancer, and various cardiovascular diseases. Research data show that the inhibitory activity of meloxicam on inducible COX2 is much greater than its inhibitory activity on structural COX1 (the IC50 of COX2:COX1 in LPS-stimulated human peripheral blood mononuclear cells is 0.09), so meloxicam is a selective COX2 inhibitor. Its weakened inhibitory effect on COX1 means that gastrointestinal and kidney adverse reactions during medication can be reduced.

[0006] In recent years, researchers have found that the mechanism of action of nonsteroidal anti-inflammatory drugs such as meloxicam can be either COX2-dependent or COX2-independent. Currently, no studies have been found on the effects of meloxicam on NLRP3 inflammasome assembly and its mediated pyroptosis of macrophages, nor have there been any reports on the treatment of septic lung injury with meloxicam. Summary of the invention

[0007] The technical problem to be solved by the present invention is to provide a new use of meloxicam in the preparation of NLRP3 inflammasome assembly and its mediated cell pyroptosis inhibitor.

[0008] Preferably, the meloxicam is used to reduce the degree of macrophage pyroptosis.

[0009] Preferably, the meloxicam inhibits the assembly of NLRP3 inflammasome induced by LPS+Nigericin.

[0010] Preferably, the meloxicam inhibits the binding of the adaptor protein ASC, the assembly element NLRP3 and the Caspase-1 precursor in the NLRP3 inflammasome.

[0011] Preferably, the NLRP3 inflammasome-related disease is an inflammatory disease.

[0012] Preferably, the inflammatory disease is sepsis.

[0013] Preferably, the cell pyroptosis inhibitor further comprises a pharmaceutically acceptable carrier or excipient.

[0014] Preferably, the medicine includes powder, tablet, granule, capsule, pill or other preparations corresponding to the administration route.

[0015] Another object of the present invention is to provide the use of meloxicam in treating septic lung injury mediated by pyroptosis.

[0016] Preferably, the meloxicam reduces pyroptosis in septic lung tissue.

[0017] The advantages of the present invention compared with the prior art are: it has been successfully discovered that meloxicam can effectively inhibit the assembly of NLRP3 inflammasomes, which play a key role in many inflammatory diseases. Accurate inhibition of NLRP3 inflammasomes is a key breakthrough in the treatment of sepsis and other related diseases. Meloxicam blocks the key link of the inflammatory response from the source by acting on this key target, providing strong theoretical support and practical basis for treatment. Moreover, meloxicam can also significantly reduce the level of macrophage pyroptosis mediated by meloxicam, which is one of the important factors leading to the deterioration of sepsis. By inhibiting this process, the damage to the body caused by the inflammatory response can be greatly reduced, providing a direct and effective way to improve the condition of patients with sepsis. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 Used to illustrate the effect of meloxicam on cell pyroptosis;

[0019] Among them, 1a is the effect on cell activity; 1b is the effect on cell membrane integrity; 1c is the effect on cell morphology.

[0020] Figure 2 Used to illustrate the effect of meloxicam on NLRP3 inflammasome assembly in macrophages.

[0021] Figure 3 Used to illustrate the effect of meloxicam on mortality and lung tissue damage in septic mice;

[0022] Among them, 3a shows the effect on the mortality rate of septic mice; 3b shows the effect on the amount of IL-1β, a key factor of cell pyroptosis, in the serum of septic mice; and 3c shows the effect on lung tissue damage in septic mice.

[0023] Figure 4 Used to illustrate the effect of meloxicam on pyroptosis of lung tissue cells in septic mice;

[0024] 4a is the effect on the pyroptosis rate of lung tissue cells; 4b is the effect on pyroptosis proteins in lung tissue. DETAILED DESCRIPTION

[0025] The present invention is further described in detail below in conjunction with the accompanying drawings.

[0026] Example 1 Effect of Meloxicam on Macrophage Pyroptosis

[0027] Experimental materials: RAW264.7 cells Cell culture: RAW264.7 cell line was purchased from the cell bank of the Institute of Biochemistry and Cell Biology, Shanghai Branch of the Chinese Academy of Sciences. The cells were cultured at 37°C, 5% CO2 using DMEM complete culture medium (Wisent) containing 10% fetal bovine serum (Wisent) and antibiotics (100 U / mL penicillin and 100 μg / mL streptomycin, Wisent).

[0028] Implementation steps: Take RAW264.7 cells in the logarithmic growth phase and add 4×10 3 The cells were inoculated into 96-well plates. After culturing in a cell culture incubator for 24 hours, they were pretreated with meloxicam (50, 75, 100 μM, purchased from Taoshu) for 2 hours, and then stimulated with LPS (1 μg / mL, 19 hours, purchased from Sigma) combined with ATP (5 mM, 1 hour, purchased from Selleck) or Nigericin (10 μM, 1 hour, purchased from Taoshu). After that, 10 μL of CCK8 reagent (purchased from Abbkine) was added to each well and incubated with the cells in the incubator for 2 hours. The absorbance was measured at 450 nm on an ELISA reader (1a); or 60 μL of LDH detection working solution (purchased from Biyuntian) was added to each well, mixed, incubated at room temperature (about 25°C) in the dark for 30 minutes, and then the absorbance was measured at 490 nm (1b). RAW264.7 cells were cultured at 5×10 5 / well were inoculated in a 12-well plate. When the cell density reached 70%-80%, the cells were pretreated with meloxicam (100 μM, purchased from Taoshu Company) for 2 hours, and then stimulated with LPS (1 μg / mL, 19 hours, purchased from Sigma Company) combined with Nigericin (10 μM, 1 hour, purchased from Taoshu Company). The culture medium was discarded from the cells, and 2.5% room temperature glutaraldehyde fixative was added. The cells were fixed at room temperature in the dark for about 5 minutes. The cells were gently scraped off in one direction with a cell scraper, and the cell fluid was aspirated into a centrifuge tube. The tube was centrifuged at 2000 rpm for 2 minutes. The fixative was discarded and new electron microscopy fixative was added. The cell clusters were gently picked up and suspended in the fixative. The cells were fixed at room temperature in the dark for 30 minutes, and then transferred to 4°C for storage. The cells were photographed using a transmission electron microscope (1c).

[0029] Experimental results: Figure 1 As shown in a, meloxicam can reverse the decrease in cell viability caused by LPS+ATP or LPS+Nigericin; Figure 1 As shown in b, after stimulation with LPS+ATP or LPS+Nigericin, the release of LDH increased, while meloxicam could significantly reduce the release of LDH, indicating that meloxicam could reverse the cell membrane rupture caused by cell pyroptosis; Figure 1As shown in Figure c, meloxicam can significantly improve the morphological changes of cell pyroptosis induced by LPS+Nigericin, such as cell swelling, cell membrane protrusion and rupture, organelle cavitation, and mitochondrial shrinkage. This shows that meloxicam can weaken the degree of macrophage pyroptosis.

[0030] Example 2 Effect of Meloxicam on NLRP3 Inflammasome Assembly

[0031] Experimental materials: RAW264.7 cells

[0032] Implementation steps: RAW264.7 cells were cultured at 5×10 5 / well were inoculated in a 12-well plate. When the cell density reached 70%-80%, they were pretreated with meloxicam (100 μM, purchased from Taoshu Company) for 2 hours, and then treated with LPS (1 μg / mL, 19 hours, purchased from Sigma Company) and Nigericin (10 μM, 1 hour, purchased from Taoshu Company). Immunoprecipitation was performed with corresponding antibodies to detect the binding of the adapter protein ASC with the assembly element NLRP3 and Caspase-1 precursor.

[0033] Experimental results: Figure 2 The results shown in the figure indicate that meloxicam can inhibit the assembly of NLRP3 inflammasome induced by LPS+Nigericin. It can be seen that meloxicam reduces the degree of cell pyroptosis by affecting the assembly of NLRP3.

[0034] Example 3 Effect of Meloxicam on Mortality and Lung Tissue Damage in Sepsis Mice

[0035] Experimental materials: Male 6-week-old BALB / c mice (20-22 g) were purchased from Jiangsu Jicui Pharmaceutical Biotechnology Co., Ltd. (certificate number: SCXK (Su) 2023-0009).

[0036] Cecal ligation and puncture (CLP) modeling: fasting but not water for 12 hours before modeling. After weighing the mice, 3% sodium pentobarbital (30 mg / kg) was injected intraperitoneally for anesthesia according to body weight. After fixing the mice, the skin was prepared and disinfected with iodine, and an incision of about 1 cm was made along the midline of the abdomen. Forceps were inserted into the abdomen to find the cecum. Then, a No. 1 silk thread was used to make a ligature at 2 / 3 of the cecum (from the free end), and a 21G needle was used to penetrate the distal end of the ligature 3 times. After the cecal leakage was formed, the intestinal contents were squeezed out of the needle by about 1 mm. Finally, the cecum was returned to the abdominal cavity, and the abdominal wall incision was sutured layer by layer. After the operation, it was disinfected again with iodine. After the operation, 1 mL of 37°C normal saline was injected subcutaneously on the back immediately to replenish the body fluids lost during the operation. After the sham operation group (Sham), only the cecum was taken out after laparotomy, and no ligation or puncture was performed.

[0037] Implementation steps: Mice were randomly divided into 5 groups: sham operation group (Sham), CLP model group, CLP + 5 mg / kg meloxicam (MEL) treatment group, CLP + 10 mg / kg meloxicam (MEL) treatment group, CLP + 20 mg / kg meloxicam (MEL) treatment group, with 10 mice in each group. According to the group, the corresponding dose of meloxicam or blank solvent was injected intraperitoneally 1 hour before surgery and 8 hours after surgery. The mice were monitored continuously for 72 hours and the death time of the experimental mice was recorded (3a); the mice were randomly divided into 3 groups: sham operation group (Sham), CLP model group, CLP + 20 mg / kg meloxicam (MEL) treatment group, 5 mice in each group, and the eyeballs were removed and blood was collected 18 hours after CLP modeling. The whole blood was allowed to stand at room temperature for 1 hour, and then centrifuged at 3000 rpm, 4°C for 10 minutes to collect serum. After dilution as required, the IL-1β ELISA kit (purchased from Proteintech) was used for detection. 100 μL of standard or test sample was added to each well, the ELISA plate was thoroughly mixed and placed at 37°C for 40 minutes, and the ELISA plate was thoroughly washed with washing solution. After that, 50 μL of distilled water and the first antibody working solution were added to each well (except the blank). After the ELISA plate was thoroughly mixed The plate was placed at 37°C for 20 minutes. After washing the plate, 100 μL of the working solution of the enzyme-labeled antibody was added to each well. The plate was fully mixed and placed at 37°C for 10 minutes. After washing the plate, 100 μL of the substrate working solution was added to each well. The plate was fully mixed and placed in the dark at 37°C. After 15 minutes, 100 μL of the stop solution was added to each well and mixed. The absorbance was measured at 450 nm using an enzyme reader within 30 minutes (3b). The mice were randomly divided into 3 groups: sham operation group (Sham), CLP model group, and CLP+20 mg / kg meloxicam (MEL) treatment group, with 3 mice in each group. The mice were anesthetized 18 hours after CLP modeling, and the residual blood was removed by cardiac perfusion. The mouse lung tissue was obtained, the tissue was rinsed with 1×PBS and fixed in 4% paraformaldehyde, then paraffin sectioned and H&E stained, and lung histology and image acquisition were performed under 200× and 400× optical microscopes (3c).

[0038] Experimental results: Figure 3 As shown in a, medium (10 mg / kg) and high doses (20 mg / kg) of meloxicam can effectively prolong the survival time of model mice, and the survival rate of mice reaches 30% and 60%; Figure 3 As shown in b, high-dose (20 mg / kg) meloxicam can significantly reduce the level of IL-1β in the serum of mice with CLP-induced sepsis; Figure 3As shown in Figure c, the main histological changes in the lungs of mice in the CLP model group were lung collapse, some areas were solid masses, the adjacent alveoli were over-expanded, the alveolar septa were broken to form large alveolar cavities, and inflammatory cells were infiltrated in large numbers, while the lung histological damage and inflammatory cell accumulation in the high-dose (20 mg / kg) meloxicam treatment group were weakened. It can be seen that meloxicam can improve the survival rate of sepsis model mice and reduce sepsis-induced lung damage, and has a good therapeutic effect on sepsis mice.

[0039] Example 4 Effect of Meloxicam on Pyroptosis in Lung Tissue of Sepsis Mice

[0040] Experimental materials: Male 6-week-old BALB / c mice (20-22 g) were purchased from Jiangsu Jicui Pharmaceutical Biotechnology Co., Ltd. (certificate number: SCXK (Su) 2023-0009).

[0041] Implementation steps: Mice were randomly divided into 3 groups: sham group, CLP model group, CLP + 20 mg / kg meloxicam (MEL) treatment group, 3 mice in each group. After 18 hours of CLP modeling, the mice were anesthetized and the residual blood was removed by cardiac perfusion. The lung tissues of the mice were taken, rinsed with 1× PBS and fixed in 4% paraformaldehyde, then paraffin sectioned and TUNEL stained, and the degree of cell apoptosis was observed under a 400× fluorescence microscope (4a); Mice were randomly divided into 3 groups: sham group The mice were anesthetized 18 hours after CLP modeling and the residual blood was removed by cardiac perfusion. The lung tissues were obtained and washed with 1×PBS. The lung tissues were crushed with a tissue crusher and lysed with RIPA lysis buffer for 30 minutes. The lung tissues were centrifuged at 12000 rpm and 4°C for 5 minutes to extract the proteins. The execution of pyroptosis-related proteins was detected by Western Blot using Caspase-1, IL-1β and GSDMD antibodies.

[0042] Experimental results: Figure 4 As shown in a, high-dose (20 mg / kg) meloxicam treatment can inhibit CLP-induced cell pyroptosis in the lung tissue of mice; Figure 4 As shown in b, high-dose (20 mg / kg) meloxicam treatment can inhibit the shear activation of pyroptosis-related proteins Caspase-1, IL-1β, and GSDMD. It can be seen that meloxicam treatment can reduce the degree of pyroptosis in the lung tissue of septic mice.

[0043] The present invention has achieved remarkable results in in vivo experiments on mice, clearly proving that meloxicam can effectively reduce sepsis-induced lung injury by inhibiting cell pyroptosis. Lung injury is one of the common and serious complications of sepsis. This experimental result directly proves the therapeutic potential of meloxicam in practical applications and provides solid animal experimental evidence for its further application in clinical treatment.

[0044] As an "old drug", meloxicam has gone through the preclinical and clinical trial stages, which means that it has accumulated certain clinical data and experience in terms of safety and effectiveness. Compared with new drugs, it can save a lot of time and resources in the research and development and approval process. Today, when the cost of drug research and development is rising, this advantage of meloxicam can greatly reduce research and development costs and improve the success rate and efficiency of research and development.

[0045] Based on the above advantages, meloxicam is expected to be rapidly developed as a drug for the clinical treatment of sepsis-induced lung injury as an inhibitor of NLRP3 inflammasome-mediated macrophage pyroptosis. This will bring new treatment hope to the majority of sepsis patients and provide strong support for improving patients' prognosis and quality of life.

[0046] The present invention and its embodiments are described above, and such description is not restrictive. The drawings show only one embodiment of the present invention, and the actual structure is not limited thereto. In short, if ordinary technicians in the field are inspired by it, without departing from the purpose of the invention, they can design a structure and embodiment similar to the technical solution without creativity, which should belong to the protection scope of the present invention.

Claims

1. Application of meloxicam in the preparation of cell pyroptosis inhibitors.

2. The use of meloxicam in the preparation of a cell pyroptosis inhibitor according to claim 1, characterized in that: The meloxicam is used to weaken the degree of macrophage pyroptosis.

3. The use of meloxicam in the preparation of a cell pyroptosis inhibitor according to claim 1, characterized in that: The meloxicam inhibits the assembly of NLRP3 inflammasome induced by LPS+Nigericin.

4. The use of meloxicam in the preparation of a cell pyroptosis inhibitor according to claim 3, characterized in that: The meloxicam inhibits the binding of the adaptor protein ASC, the assembly element NLRP3 and the Caspase-1 precursor in the NLRP3 inflammasome.

5. The use of meloxicam in the preparation of a cell pyroptosis inhibitor according to claim 4, characterized in that: The NLRP3 inflammasome-related disease is an inflammatory disease.

6. The use of meloxicam in the preparation of a cell pyroptosis inhibitor according to claim 5, characterized in that: The inflammatory disease is sepsis.

7. The use of meloxicam in the preparation of a cell pyroptosis inhibitor according to claim 1, characterized in that: The cell pyroptosis inhibitor also includes a pharmaceutically acceptable carrier or excipient.

8. The use of meloxicam in the preparation of a cell pyroptosis inhibitor according to claim 1, characterized in that: The medicine includes powder, tablet, granule, capsule, pill or other preparations corresponding to the administration route.

9. The use according to any one of claims 1 to 8, characterized in that: Used in the treatment of septic lung injury.

10. The treatment of septic lung injury according to claim 9, characterized in that: Meloxicam reduces pyroptosis in septic lung tissue.

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