CN118121603A - Application of kynurenic acid in medicines for treating sepsis capillary vessel leakage - Google Patents

Abstract

The invention discloses application of kynurenic acid in medicines for treating sepsis capillary vessel leakage. The invention provides application of kynurenic acid in a medicine for treating sepsis capillary vessel leakage, and the medicine can be used as a brand new medicine for treating sepsis capillary vessel leakage.

Description

Application of kynurenic acid in medicines for treating sepsis capillary vessel leakage

Technical Field

The invention relates to the field of biological medicine, in particular to application of kynurenic acid in medicines for treating sepsis capillary vessel leakage.

Background

Sepsis is a life threatening organ dysfunction caused by a host's dysresponsiveness to infection. Despite continual guideline updates, the mortality rate of sepsis is still up to 30%. The difficult-to-control capillary leakage is not only the core pathophysiological mechanism of sepsis, but also the bottleneck of current sepsis treatment. The main clinical treatment methods at present comprise that glucocorticoid controls inflammatory mediators to damage vascular endothelium, liquid resuscitates and restores effective blood volume, albumin increases colloid osmotic pressure to prevent liquid extravasation, and the like, but the methods still cannot well control capillary leakage and prevent the progress of diseases, so that searching for a more effective management strategy has very important significance in the treatment of sepsis capillary leakage.

Disclosure of Invention

In view of the above-mentioned drawbacks of the prior art, the present invention aims to solve the technical problem that the existing medicines for treating sepsis capillary leak cannot well control capillary leak and cannot prevent the progress of the disease. The invention provides application of kynurenic acid in a medicine for treating sepsis capillary vessel leakage, and the medicine can be used as a brand new medicine for treating sepsis capillary vessel leakage.

To achieve the above object, the present invention provides the use of kynurenic acid (C ₁₀H₇NO₃) in a medicament for the treatment of sepsis capillary leak.

Further, kynurenic acid reduces expression of the pro-inflammatory factor IL-1 beta and TNF-alpha of HUVECs.

Further, the effective dose of kynurenic acid at HUVECs was set at 10. Mu.M.

Further, the administration of the drug in HUVECs is direct.

Further, the drug improved the inhibition of HUVECs migration by LPS for 24 hours and 36 hours.

Further, the drug improves the inhibition of proliferation of HUVECs by LPS.

Further, the drug enhances the expression of Claudin-2, claudin-5 at the protein level in HUVECs.

Further, the drug may increase survival of CLP mice.

Further, the effective dose of the drug in the mice was set to 5mg/kg.

Further, the drug was administered in mice by intragastric lavage for three consecutive days.

Further, the medicament can reduce the lung to body weight ratio of the CLP mice and improve pulmonary edema of the CLP mice.

Further, the drug may reduce vascular leakage of lung tissue of CLP mice.

Further, the medicament improves inflammatory cell infiltration and capillary congestion of lung tissues of CLP mice.

Technical effects

In the early stage of the experiment, through collecting peripheral blood samples at different stages of sepsis treatment, metabonomics detection is carried out, and correlation analysis is carried out on all metabolites on the enriched relevant metabolic pathways and clinical indexes of patients, so that kynurenic acid is prompted to play a role in protecting the sepsis patient in the treatment process. Next, the inventor explains that the medicine has remarkable protection effect through in vitro experiments and in vivo experiments of mice, and can improve capillary leakage of sepsis lung tissues, thereby achieving the purpose of improving survival rate of sepsis mice.

The medicine of the invention has no report on the treatment of sepsis capillary vessel leakage, so the innovation is strong. Secondly, the medicine is a human metabolite, so the medicine has high relative safety.

The medicine provided by the invention is expected to improve the survival rate of patients with sepsis in the future, improve the prognosis of patients with sepsis, reduce the medical cost, save medical resources and bring good news to patients.

Drawings

FIG. 1 is a schematic diagram showing the molecular structure of kynurenic acid according to a preferred embodiment of the present invention;

FIG. 2 is a graph showing the effect of kynurenic acid on LPS on HUVECs in accordance with a preferred embodiment of the present invention; among them, FIG. 2A shows that the expression of pro-inflammatory factors IL-1. Beta. And TNF-alpha. Can be significantly improved by detecting cell supernatant cytokines by ELISA, and the results suggest that kynurenic acid. Fig. 2B and 2D (statistical diagram): it was found that kynurenic acid significantly improved inhibition of HUVECs migration by LPS at 24 hours and 36 hours by scratch experiments. Fig. 2C: the inhibition of HUVECs proliferation by LPS was significantly improved by kynurenic acid found by a cell count experiment (Cell Counting Kit-8, CCK-8) experiment. Fig. 2E: through detection of tight junction proteins, channel proteins, adhesion junction proteins and the like, kynurenic acid can obviously improve the expression of Claudin-2 and Claudin-5, wherein the influence of Claudin-5 is most obvious.

FIG. 3 is a graph showing the effect of kynurenic acid on CLP mice in accordance with a preferred embodiment of the present invention. Wherein, fig. 3A: the survival curve of mice shows that kynurenic acid can significantly improve the survival rate of CLP mice when acting on CLP sepsis mice. Fig. 3B: the lung to body weight ratio results suggest that kynurenic acid can reduce the lung to body weight ratio. Fig. 3C and 3D (statistical diagrams): an Evans leakage experiment (Evans) leakage experiment can find that kynurenine can significantly improve the leakage of CLP mouse Evans dye, which suggests that kynurenine can improve the vascular leakage of lung tissues. Fig. 3E: hematoxylin and eosin (H & E) staining showed that kynurenic acid improved inflammatory cell infiltration and capillary congestion in lung tissue of CLP mice.

Detailed Description

In order to make the technical problems, technical schemes and beneficial effects to be solved more clear, the invention is further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the scope of the invention.

The medicine of the invention belongs to the final metabolite of the tryptophan metabolic pathway kynurenine pathway, is a small molecular compound, and the specific structure is shown in the following figure 1.

The invention discovers that tryptophan metabolic pathway is obviously enriched in the liquid resuscitation process of patients with sepsis in the early stage, and the metabolite kynurenine of the tryptophan metabolic pathway is obviously inversely related to interleukin-1 beta and blood lactic acid level of the patients, so that the kynurenine plays a protective role in the liquid resuscitation process of the sepsis. The invention proves that kynurenic acid can relieve sepsis endothelial injury and improve capillary vessel leakage of sepsis mice through the following scheme.

1. In vitro experiments prove that kynurenic acid (C10H 7NO 3) can relieve the damage of the vascular endothelium of sepsis.

(1) FIG. 2A shows that the results of ELISA detection of cytokines from HUVECs cell supernatants suggest that kynurenic acid can significantly reduce the expression of pro-inflammatory factors IL-1. Beta. And TNF-alpha.

The scheme is as follows: kynurenic acid at a final concentration of 10. Mu.M and/or Lipopolysaccharide (LPS) at a final concentration of 100ng/ml were added to the HUVECs cell culture medium, respectively. To quantify the concentration of IL-1 beta, IL-6 and TNF-alpha in plasma, ELISA was performed according to the manufacturer's instructions (MultiSciences, hangzhou, china). All measurements were repeated three times. The minimum detectable concentrations of human IL-1β, IL-6 and TNF- α were 0.15, 0.37 and 0.42pg/mL, respectively, according to the standard curve.

(2) Figures 2B and 2D (statistical graphs) found that kynurenic acid significantly improved inhibition of HUVECs migration by LPS at 24 hours and 36 hours by scratch experiments.

The scheme is as follows: a line was drawn on the back of the six-well plate approximately every 0.5-1 cm. Each hole should pass through at least 5 lines. 5X 10 ⁵ cells were added to the plates and used in culture. The next day was scratched by a 20. Mu.L gun head. Subsequently, the cells were washed with PBS and kynurenic acid at a final concentration of 10. Mu.M and/or LPS at a final concentration of 100ng/ml were added, respectively. Then cultured in a serum-free medium incubator at 37℃with 5% CO ₂. Cells were incubated to 0, 6, 12 and 24 hours, sampled and photographed (scale = 250 μm), respectively.

(3) FIG. 2C shows that kynurenic acid significantly improves the inhibition of HUVECs proliferation by LPS by CCK-8 experiments.

The scheme is as follows: cell viability was detected by cell counting kit-8 (bi yun tian, china). 2000 cells were inoculated into 96-well plates in 100. Mu.L of serum-free medium, and then 10. Mu.L of CCK-8 mixed solution was added. Cell viability of HUVECs was observed 1 hour, 2 hours and 4 hours after addition of kynurenic acid at a final concentration of 10 μm and/or LPS at a final concentration of 100 ng/ml. We used a microplate reader to detect absorbance at the 450nm test wavelength.

(4) In FIG. 2E, the detection of the connexin through western blot shows that kynurenic acid can significantly improve the expression of Claudin-2 and Claudin-5, wherein the influence of Claudin-5 is most significant.

The scheme is as follows: cell lysates isolated from cells were subjected to 10% SDS-PAGE, and the isolated proteins were transferred to polyvinylidene fluoride membranes and blocked with skimmed milk powder. The membrane was incubated with primary antibody overnight at 4 ℃ and then washed three times with PBST. The primary dilution ratio of claudin-1 (Abcam, cambridge, UK) is 1:1000, claudin-2 (Abcam, cambridge, UK) is 1:1000, claudin-5 (Abcam, cambridge, UK) is 1:1000, ZO-1 (Abcam, cambridge, UK) is 1:500, VE-cadherein (Abcam, cambridge, UK) is 1:1000, beta-actin (CST, denver, U.S.A.) is 1:1000. The membrane was incubated with the secondary antibody for 1 hour and the membrane was washed three times. Development photographs were taken with ECL developer.

2. In vivo experiments prove that kynurenic acid can improve capillary leakage of sepsis mice.

(1) Figure 3A shows that kynurenine acting on CLP sepsis mice significantly improves CLP mouse survival by mouse survival curves.

The scheme is as follows: 40C 57BL/6 non-specific pathogen grade male mice, 6-8 weeks old, weighing 18-20g (Shanghai first people hospital animal experiment center), were randomly divided into four groups: control group, kynurenic acid group (kynurenic acid), CLP group (CLP) and clp+kynurenic acid treatment group (clp+kynurenic acid), 10 each. The CLP-induced sepsis model is as follows: mice were fasted for 6 hours and water was stopped for 3 hours prior to surgery. A longitudinal incision of about 1cm was made along the midline of the abdomen, exposing the abdominal cavity, the cecum was found, ligated, and pierced with a clean needle (21G). A small amount of intestinal contents was expressed and the abdominal cavity was closed after returning to the cecum. A warm solution of 0.9% sodium chloride was subcutaneously injected. The mice were then placed on a thermostated blanket until resuscitated. Kynurenic acid (5 mg/kg, intragastric for 3 consecutive days) was applied to a CLP sepsis mouse model.

(2) Fig. 3B lung to body weight ratio results suggest that kynurenic acid can reduce lung to body weight ratio.

The scheme is as follows: animals in the embodiment of fig. 3A were weighed on days 1, 2, and 3 to calculate the lung and body weight ratio.

(3) Figures 3C and 3D (statistical graphs) show that kynurenine can significantly improve the leakage of CLP mouse Evans blue dye through Evans leakage experiments, suggesting that kynurenine can improve pulmonary tissue vascular leakage.

The scheme is as follows: kynurenic acid (5 mg/kg, intragastric for 3 consecutive days) was applied to a CLP sepsis mouse model. Evans blue staining solution (0.5%, 30 mg/mL) was injected into the tail vein of the mice on the third day, giving blue color to their eyes and skin. After 1 hour, mice were sacrificed to determine the amount of Evans dye leakage. The extracted tissue was placed in a 1.5ml centrifuge tube containing 1ml PBS, homogenized and centrifuged. The supernatant was taken, an equal amount of trichloroacetic acid was added, incubated at 4℃and then centrifuged for 15 minutes. Subsequently, absorbance values at 620nm were measured using a spectrophotometer, and then the Evans dye content was calculated. The results show that the lung tissue Evans dye leakage is significantly lower than that of CLP mice in kynurenic acid intragastric CLP mice, suggesting that kynurenic acid can improve the capillary leakage of the lung tissue.

(4) FIG. 3E shows pathological changes of organs and tissues (blood vessels, heart, liver, lung, kidney and intestinal tracts) of mice through H & E staining, especially inflammatory cell infiltration, blood stasis and the like of blood vessels and lung tissues. The results show that kynurenic acid can improve inflammatory cell infiltration and pulmonary congestion of CLP mice.

The scheme is as follows: immediately after mice were sacrificed, their lung tissues were removed, fixed with 4% paraformaldehyde, and then embedded in paraffin. Sections were prepared by dewaxing lung tissue with fractionated xylenes and ethanol and staining with H & E solution. They were then dehydrated and fixed on microscope slides with neutral resin. Subsequently, photographing was performed using an inverted microscope (scale=50 μm).

The foregoing describes in detail preferred embodiments of the present invention. It should be understood that numerous modifications and variations can be made in accordance with the concepts of the invention by one of ordinary skill in the art without undue burden. Therefore, all technical solutions which can be obtained by logic analysis, reasoning or limited experiments based on the prior art by the person skilled in the art according to the inventive concept shall be within the scope of protection defined by the claims.

Claims (13)

1. Use of kynurenic acid in the treatment of sepsis capillary leak in a medicament.

2. The use according to claim 1, wherein kynurenic acid reduces the expression of human umbilical vein endothelial cells HUVECs pro-inflammatory factor IL-1 β and Tumor Necrosis Factor (TNF) - α.

3. The use according to claim 1, wherein the effective dose of kynurenic acid to HUVECs is set at 10 μm.

4. The use of claim 1, wherein the medicament is administered in vitro by direct administration.

5. The use according to claim 1, wherein the medicament improves the inhibition of HUVECs migration by lipopolysaccharide LPS in human umbilical vein endothelial cells for 24 hours and 36 hours.

6. The use according to claim 1, wherein the medicament improves the inhibition of HUVECs proliferation by LPS.

7. The use according to claim 1, wherein the medicament increases the expression of the connexins Claudin-2, claudin-5 of HUVECs at the protein level.

8. The use of claim 1, wherein the medicament significantly improves survival rate of Cecal Ligation Perforated (CLP) sepsis mice.

9. The use according to claim 1, wherein the effective dose of kynurenic acid in mice is set at 5mg/kg.

10. The use of claim 1, wherein the medicament is administered in vivo to a mouse by intragastric lavage for three consecutive days.

11. The use of claim 1, wherein the medicament improves pulmonary edema in CLP mice.

12. The use of claim 1, wherein the medicament reduces vascular leakage from lung tissue of CLP mice.

13. The use of claim 1, wherein the medicament improves inflammatory cell infiltration of lung tissue and capillary congestion in CLP mice.