CN112691098A

CN112691098A - Application of ferulic acid in preparation of medicine for treating sepsis intestinal mucosa barrier function damage

Info

Publication number: CN112691098A
Application number: CN202110193134.XA
Authority: CN
Inventors: 何莎莎; 刘清泉; 徐霄龙; 郭玉红; 赵京霞
Original assignee: Beijing Hospital of Traditional Chinese Medicine Affiliated to Capital University of Medicine Sciences
Current assignee: Beijing Hospital of Traditional Chinese Medicine Affiliated to Capital University of Medicine Sciences
Priority date: 2021-02-20
Filing date: 2021-02-20
Publication date: 2021-04-23

Abstract

The invention discloses an application of ferulic acid in preparation of a medicament for treating sepsis intestinal mucosa barrier function damage, and belongs to the technical field of medicines. According to the invention, a mouse sepsis model is established by adopting cecal ligation and perforation operation, ferulic acid can reduce the death rate, improve the intestinal mucosa morphological structure, inhibit intestinal bacteria displacement and protect the intestinal mucosa barrier function. The CAS number of ferulic acid used in the present invention is 1135-24-6, which can be used alone or in the form of a pharmaceutical composition comprising ferulic acid as an active ingredient and a pharmaceutically acceptable carrier. Ferulic acid, whether used alone or in a composition, can repair the barrier function of intestinal mucosa excellently.

Description

Application of ferulic acid in preparation of medicine for treating sepsis intestinal mucosa barrier function damage

Technical Field

The invention belongs to the technical field of medicines, and particularly relates to application of ferulic acid in preparation of a medicine for treating sepsis intestinal mucosa barrier function injury.

Background

Sepsis has a high incidence and mortality, which is a major problem and challenge in the medical field at present. A cross-sectional survey of ICU patients at 44 hospitals across the country in 2020 showed that sepsis occurred at 20.6% and mortality at 35.5%. Sepsis (sepsis3.0) is defined as uncontrolled response of the body to infection that leads to life-threatening organ dysfunction, new definitions emphasize organ dysfunction caused by body infection, and protection of organ function is the key to prevention and treatment of sepsis. The intestinal tract is particularly special in a plurality of organs affected by sepsis, and has important barrier functions besides normal digestion, absorption and excretion functions. The intestinal mucosa barrier is the first line of defense to prevent bacteria and toxins in the intestinal tract from passing through the intestinal mucosa into the lymphatic system or blood circulation of the body. Pathological stress caused by severe trauma, infection, shock and the like can cause intestinal mucosa barrier dysfunction of critically ill patients, so that the permeability of intestinal mucosa is changed, the intestinal flora is shifted, and endotoxin is released into blood. Thereby causing enterogenous infection or systemic inflammatory response syndrome and even causing the dysfunction of multiple organs of the body. Clinical studies show that the occurrence and development of sepsis are often accompanied by intestinal mucosal barrier dysfunction, and the evaluation of the intestinal mucosal barrier function is an important link for judging the outcome and prognosis of the sepsis patient.

The current treatment regimen for sepsis intestinal barrier dysfunction is mainly to control infection, enteral nutrition, improve intestinal tissue perfusion, and the like. No sepsis intestinal barrier dysfunction treatment medicine with definite curative effect and high safety exists. Therefore, the research on molecular mechanisms of the sepsis intestinal mucosa barrier dysfunction, the research and development of treatment drugs for the intestinal mucosa barrier dysfunction and the discovery of key action targets of the treatment drugs have very important clinical significance.

Ferulic Acid (FA) is one of cinnamic Acid derivatives, is a phenolic Acid widely existing in plants, has pharmacological effects of resisting oxidation, resisting inflammation, resisting thrombi, etc., and has low toxicity. Has wide application in the aspects of medicines, health products, cosmetic raw materials, food additives and the like. At present, the traditional Chinese medicine composition is mainly used for treating diseases such as coronary heart disease, nephropathy, cerebral infarction, Alzheimer disease and the like in clinic. At present, no report of the application of ferulic acid in the treatment of intestinal mucosa barrier of sepsis exists.

Disclosure of Invention

In order to solve the technical problems, one of the purposes of the invention is to provide an application of ferulic acid in preparing a medicament for treating sepsis intestinal mucosa barrier function injury, wherein the ferulic acid is purchased from China institute for testing and drug administration, CAS number is 1135-24-6, and structural formula is as follows:

further, the sepsis is caused by a microbial infection.

Further, the sepsis includes early sepsis, severe sepsis, and septic shock.

Further, the sepsis gut mucosal barrier function impairment is accompanied by increased permeability and bacterial translocation.

The invention also aims to provide a medicament for treating sepsis intestinal mucosa barrier function damage, which contains ferulic acid.

Further, the medicine also comprises medically acceptable auxiliary materials or carriers.

The ferulic acid used in the invention can be used alone or in the form of a pharmaceutical composition comprising ferulic acid as an active ingredient and a pharmaceutically acceptable carrier. The composition can be administered by oral, sublingual, transdermal, intramuscular or subcutaneous, mucocutaneous, intravenous routes. The pharmaceutical composition can be in the form of tablets, capsules, granules, powders, pills or sustained release formulations. The various pharmaceutical dosage forms provided by the invention can be prepared by a conventional pharmaceutical method.

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

the invention provides a new application of ferulic acid in preparing a medicament for treating sepsis intestinal mucosa barrier function damage, wherein the ferulic acid can be used independently or in the form of a medicinal composition, and the ferulic acid can excellently repair intestinal mucosa regardless of being used independently or in the form of the composition.

Drawings

FIG. 1 is a graph showing the survival of mice in example 1.

FIG. 2 is a graph of HE staining of mouse ileum tissue in example 1.

FIG. 3 is a transmission electron micrograph of ileum tissue of the mouse in example 1.

FIG. 4 is a graph showing the results of measuring the serum diamine oxidase (DAO) content of the mouse in example 1.

FIG. 5 is a graph showing the results of measurement of the endotoxin content in serum of mouse in example 1.

FIG. 6 is a plate diagram of the bacterial culture of Escherichia coli and Staphylococcus aureus in the mouse of example 1.

FIG. 7 is a graph showing the statistical results of bacterial translocation of Escherichia coli and Staphylococcus aureus in mice in example 1.

FIG. 8 is HuProt in example 2^TMHuman proteome chip scan.

Fig. 9 is a graph of 5 ferulic acid vs biological cadaverine significant binding protein information in example 2.

FIGS. 10 to 12 are graphs showing the results of Western Blot experiments in example 2.

FIG. 13 is a graph showing the results of analysis of the interaction pattern of ferulic acid with AKT1 protein in example 2.

FIG. 14 is the ferulic acid versus sepsis AKT1 of example 2^-/-Graph of HE staining results of mouse ileum tissue.

FIG. 15 is a transmission electron micrograph of mouse ileum tissue in example 2.

FIG. 16 shows sepsis AKT1 in example 2^-/-Mouse serum DAO test result chart.

FIG. 17 is the sepsis AKT1 in example 2^-/-Test result chart of mouse serum endotoxin.

FIG. 18 is the sepsis AKT1 in example 2^-/-And (3) a statistical result graph of bacterial translocation of escherichia coli and staphylococcus aureus of the mice.

Detailed Description

Example 1 protective Effect of Ferulic acid on intestinal mucosal barrier function of septic mice

1. The experimental steps are as follows: establishing a Caecum Ligation Perforation (CLP) sepsis mouse model, and counting the survival rate of the mouse for 7 days to evaluate the curative effect of ferulic acid on sepsis mice. And taking intestinal tissues of each group of mice 24 hours after CLP operation to perform HE staining and electron microscope observation, detecting the bacteria numbers of Escherichia coli and staphylococcus aureus in serum DAO, endotoxin and mesenteric lymph nodes, and determining the effect of ferulic acid on the intestinal mucosa barrier function of the sepsis mice.

The specific experimental operations were as follows:

1) pyemia modeling: c57BL/6J male mice are adaptively fed for 1 week at the age of 8 weeks (20-22g), fasted without water supply for 12h before molding, and then are subjected to Cecal Ligation and Perforation (CLP) surgery to establish a sepsis model.

2) And (3) counting the survival rate of the mice: mice were randomly divided into 3 groups: (1) sham group (Sham); (2) sepsis model group (CLP); (3) the ferulic acid group (FA) is administered by gavage 0.5mL per day at a dose of 100mg/kg, and then subjected to CLP surgery for molding. Mice in sham and CLP groups were given the same volume of saline. Survival rates were recorded for 7 days and survival curves were plotted, with the results shown in fig. 1, which is seen from the survival plots of fig. 1: compared with a model group, the survival rate of the sepsis mouse is improved by 19.2% by ferulic acid in 7 days.

3) HE staining: at 24 hours after mouse CLP modeling, ileum tissues of each group were fixed in 4% paraformaldehyde, dehydrated, cleared, waxed, embedded, sliced, deparaffinized, stained, and sealed with neutral gum, and observed under a microscope and images were collected, and the results are shown in fig. 2, and the results of ileum tissue HE in fig. 2 show: the ileum villus of the mice in the sham operation group has complete structure, the epithelium is arranged closely and orderly, and the mucous membrane and the submucosa have no edema and inflammatory cell infiltration. The ileum villi of mice in the CLP group is atrophic compared with that in the sham operation group, and the mucous membrane and the submucosa layer have obvious swelling and congestion and inflammatory cell proliferation and infiltration phenomena. Compared with the CLP group, the ferulic acid treatment group has the advantages that the villus structure and inflammatory cell infiltration of the ileum of the mice are obviously improved.

4) Transmission electron microscopy: after 24 hours from the mouse CLP molding, 1mm of each group of mice was taken out with a sharp blade³Of ileumTissues were fixed in 2.5% glutaraldehyde, fixed again with 1% osmic acid and washed with 0.1M phosphate buffer, dehydrated, semi-soaked, fully soaked, ultrathin sections were made, stained with lead and observed for ileal microvilli and tight junction structure by transmission electron microscopy, as shown in FIG. 3, from the results of FIG. 3 for ileal tissue transmission electron microscopy: the micro villi of the ileum of the mice in the sham operation group are arranged in a comb shape, and the tight connection structure is complete; the CLP group mice ileum microvilli structure becomes short and sparse, the falling-off occurs, the tight connection structure is obviously damaged, and the gap between adjacent cells is obviously increased. The ferulic acid treatment group obviously improves microvilli and a tight junction structure, so that the number of the microvilli is increased, and the tight junction structure is basically complete.

5) Detecting the content of diamine oxidase (DAO) in mouse serum: collecting serum of each group of mice 24 hours after the mice CLP are molded; setting an ultraviolet spectrophotometer at 340nm and a 0.5cm light path quartz cuvette, and adjusting to zero by using double distilled water; adding 80 mul of sample to be detected into the test tube with the corresponding number, taking 800 mul of reagent to flush into the test tube, immediately mixing uniformly and timing; the absorbance value (A1 value) was read at 20 seconds after pouring into a quartz cuvette; pouring the colorimetric solution into a primary test tube, placing the primary test tube in a 37 ℃ water bath for 10 minutes, then quickly pouring the colorimetric solution into a quartz cuvette, and measuring the absorbance value (A2 value) again at 10 minutes and 20 seconds; DAO activity (U/L) ═ A1-A2 x 10³X total volume of reaction solution (880. mu.l)/6.3X colorimetric optical path X reaction time (10 minutes). times.sampling amount (80. mu.l). As a result, as shown in fig. 4, it was found from fig. 4 that the DAO concentration in the serum of the CLP group was significantly increased. The ferulic acid pretreated group reduced DAO concentration compared to the CLP group.

6) Detecting the content of endotoxin in the serum of the mouse: 24 hours after the mouse CLP model is made, the endotoxin content in each group of serum samples is detected by using an endotoxin detection limulus kit (microplate quantitative chromogenic matrix method), and the result is shown in FIG. 5, and as can be seen from FIG. 5, the endotoxin concentration in the CLP group serum is obviously increased. The ferulic acid pretreated group reduced endotoxin concentrations compared to the CLP group.

7) Coli, staphylococcus aureus bacterial translocation assay: 24 hours after mouse CLP modeling, 0.1g of mesenteric lymph node is respectively weighed in each group, tissue homogenate is carried out to prepare tissue bacterium liquid, and the stock solution is made into 1: 100-fold dilution, 100. mu.l each, was inoculated into the medium on E.coli plates and Staphylococcus aureus plates. When the culture medium in the culture dish is stable in shape, the culture medium is put into an incubator at 37 ℃ for aerobic culture for 24 hours, photographing and counting are carried out under a colony counter, the result is shown in figure 6, and the statistical result is shown in figure 7. As can be seen from FIGS. 6 and 7, the numbers of Escherichia coli and Staphylococcus aureus in mesenteric lymph nodes of mice in the CLP group were significantly increased, and the numbers of Escherichia coli and Staphylococcus aureus in the ferulic acid-treated group were significantly decreased compared to the CLP group, as compared to the sham-operated group.

Example 2 Effect of Ferulic acid on improving intestinal mucosal barrier function of septic mice by targeting AKT1

1. The experimental steps are as follows: by Huprot^TMHuman protein chip, Pulldown combined WB, and computer simulation small molecule and protein interaction mode analysis, to determine ferulic acid and AKT1 target binding and predict amino acid binding site, then to construct AKT1 gene knockout mice sepsis model, through HE staining, electron microscope, serum DAO, endotoxin and mesenteric lymph node bacteria displacement count, to discuss the role of ferulic acid in knockout mice intestinal mucosa barrier function of sepsis AKT1 gene.

The following experiments were all performed 24 hours after CLP molding of mice.

The specific experimental operations were as follows:

1) and (3) AKT1 knockout mouse sepsis modeling and treatment: after 1 week of adaptive feeding of C57BL/6J male wild type and AKT1 knockout mice, they were randomized: sham group (n-10), CLP group (n-20), CLP + ferulic acid group (n-20). Fasting is carried out 12h before model building, water is not forbidden, and CLP operation is carried out to establish a sepsis model. The ferulic acid treatment group (FA) was gavaged at a dose of 100mg/kg of 0.5mL per day, and subjected to CLP surgery for molding. Mice in sham and CLP groups were given the same volume of saline.

2)HuProt^TMHuman proteome chips: dissolving biotin cadaverine-labeled ferulic acid and biotin cadaverine samples into 10mM mother liquor, and then carrying out chip hybridization, washing and detection according to a chip detection standard flow provided by CDI chip company, wherein GenePiThe x 4000B scanner scans the chip at 635nm, and the result is shown in fig. 8, and it can be seen from fig. 8 that the biotin cadaverine-labeled ferulic acid group has many red fluorescence enhancement points, which represent possibly bound proteins, compared with the biotin cadaverine control group.

Through analysis of human proteome chips and bioinformatics results and review of literature of binding differential proteins, 5 ferulic acid _ vs _ biological cadaverine significant binding proteins (NT5C3, APOC3, PDGFRA, AKT1, RAC3) were screened, with fold differences of 16.10, 15.68, 8.94, 5.44, 5.33, respectively, and detailed information is shown in fig. 9.

3) Pulldown verified ferulic acid binding protein: a Pierce Spin Column tube was prepared and the streptavidin gel was equilibrated immobilized. 300. mu.l (10 mM mother liquor dissolved in DMSO was used after 2-fold dilution) of biotin-labeled small molecules was added to the Spin Column tube, biotin cadaverine-labeled ferulic acid and biotin cadaverine immobilization was performed, 250. mu.l of a biotin blocking solution was added to the Spin Column tube to perform biotin blocking, and then 300. mu.l of a capture protein sample solution was added to the Spin Column tube to perform capture of the biotin-labeled small molecules.

4) Western Blot experiment: mu.l of each of the Biotin beads and the Oridnin-Biotin beads were added to 70. mu.l of 2 Xloading Buffer, mixed by shaking and then centrifuged in a boiling water bath for 5min, allowed to stand on ice until cooled, and centrifuged at 8000rpm for 2min, and 20. mu.l of each of the supernatants (from step 3)) was applied. According to the molecular weight of the target band, carrying out glue preparation, electrophoresis, film transfer and sealing. Putting the membrane into a hybridization box, adding PDGFRA, AKT1/2/3, AKT1, APOC3, RAC3 and NT5C3 primary antibody, and putting the membrane in a decoloring shaker at 4 ℃ overnight; using VeriBlot for IP Detection reagent (HRP), putting the secondary antibody and the membrane into a hybridization box again, placing the hybridization box on a shaking table to shake slowly, adding ECL luminous liquid after incubating for 1h at room temperature, and using a digital imaging system to photograph the membrane, the results are shown in fig. 10-12, and as can be seen from fig. 10-12, AKT1, APOC3 and PDGFRA can be captured by streptavidin agarose and detected by a Westernblot method, wherein the AKT1 protein changes most obviously, so AKT1 is selected subsequently to perform ferulic acid action target research.

5) Analysis of interaction pattern of ferulic acid with AKT 1: the schrodinger software was used for molecular dynamics simulation. The whole protein system adopts gaff and ff14SB force fields, takes protein as a center, adds a 10A cubic water box, adds Na + to make the system be electrically neutral, preserves a topological and coordinate structure, and then carries out simulation. And carrying out flexible docking on the protein and the small molecules by utilizing Glide software to obtain a primary docking phase structure. In the butt joint, the phase with the best butt joint energy is selected for structure extraction, and is used for subsequent molecular dynamics research.

The results of the interaction pattern analysis of ferulic acid with AKT1 protein are shown in fig. 13, and it can be seen that: ferulic acid can form better hydrogen bond system polarity effect with hydrophilic amino acid segments Ser205 and Thr 211. Hydrophobic interactions are formed with the hydrophobic amino acids Trp80, Gln79, Leu210 in the protein binding pocket.

6) HE staining: each group of ileum tissues was fixed in 4% paraformaldehyde 24 hours after mouse CLP modeling, and observed and images were collected under a microscope after dehydration, clearing, waxing, embedding, slicing, spreading, dewaxing, staining, and sealing with neutral gum. Ferulic acid to sepsis AKT1^-/-The HE staining results of mouse ileum tissue are shown in fig. 14, and it can be seen that: AKT1^-/-The group mice had intact ileal tissue morphological structures, consistent with the Sham group. The CLP + AKT 1-/-group has obviously desquamated intestinal villus apical epithelial cells, naked lamina propria, obvious swelling and congestion of mucous membrane and submucosa, and inflammatory cell proliferation and infiltration, and more obvious pathological changes than the CLP group. FA + CLP + AKT1^-/-Group and CLP + AKT1^-/-Compared with the group, the villus structure and inflammatory cell infiltration phenomenon of the ileum of the mouse are obviously improved, but the enterocyte is still obviously shed.

7) Transmission electron microscopy: after 24 hours from the mouse CLP molding, 1mm of each group of mice was taken out with a sharp blade³The ileum tissues were fixed in 2.5% glutaraldehyde fixing solution, 1% osmic acid was fixed again and washed with 0.1M phosphate buffer, and then ileal microvilli and tight junction structures were observed by transmission electron microscopy after dehydration, semi-permeation, full-permeation, ultrathin section making, lead staining, and the results are shown in FIG. 15.

As can be seen from fig. 15: and AKT1^-/-Group comparison, CLP + AKT1^-/-The microvilli structure and the tight connection structure of the ileum tissue are obviously damaged, so that the microvilli are disorderly arranged and basically fall off, tight connection compact substances disappear, and the gaps between adjacent cells are obviously increased; and CLP + AKT1^-/-Group comparison, FA + CLP + AKT1^-/-The micro villus and the tight connection structure are improved, but the micro villus can still be seen to be disorderly arranged, and the tight connection compact substance structure is incomplete.

8) Detecting the content of diamine oxidase (DAO) in mouse serum: collecting serum of each group of mice 24 hours after the mice CLP are molded; setting an ultraviolet spectrophotometer at 340nm and a 0.5cm light path quartz cuvette, and adjusting to zero by using double distilled water; adding 80 mul of sample to be detected into the test tube with the corresponding number, taking 800 mul of reagent to flush into the test tube, immediately mixing uniformly and timing; the absorbance value (A1 value) was read at 20 seconds after pouring into a quartz cuvette; pouring the colorimetric solution into a primary test tube, placing the primary test tube in a 37 ℃ water bath for 10 minutes, then quickly pouring the colorimetric solution into a quartz cuvette, and measuring the absorbance value (A2 value) again at 10 minutes and 20 seconds; DAO activity (U/L) ═ A1-A2 x 10³X total volume of reaction solution (880. mu.l)/6.3X colorimetric optical path X reaction time (10 minutes). times.sampling amount (80. mu.l).

9) Detecting the content of endotoxin in the serum of the mouse: 24 hours after mouse CLP modeling, endotoxin content in each group of serum samples was detected by using a limulus kit for endotoxin detection (microplate quantitative chromogenic substrate method).

Ferulic acid to sepsis AKT1^-/-The results of mouse serum DAO and endotoxin assays are shown in fig. 16 and 17, respectively, and it is known that: and AKT1^-/-Group comparison, CLP + AKT1^-/-Group DAO and endotoxin concentrations rose significantly; and CLP + AKT1^-/-Group comparison, FA + CLP + AKT1^-/-Group DAO levels were reduced but the differences were not significant, while endotoxin concentrations were significantly reduced. CLP + AKT1 compared to CLP group^-/-Both group DAO and endotoxin concentrations increased significantly. Compared with FA group, FA + CLP + AKT1^-/-Both group DAO and endotoxin concentrations increased significantly.

9) Coli, staphylococcus aureus bacterial translocation assay: 24 hours after mouse CLP modeling, 0.1g of mesenteric lymph node is respectively weighed in each group, tissue homogenate is carried out to prepare tissue bacterium liquid, and the stock solution is made into 1: 1Diluting at 00 times, respectively taking 100ul of each concentration, and inoculating the culture medium to an escherichia coli culture plate and a staphylococcus aureus culture plate. When the culture medium in the culture dish is stable in shape, the culture medium is sent into an incubator at 37 ℃ for aerobic culture for 24 hours, photographing and counting are carried out under a colony counter, and the statistical result is shown in figure 18, and the following results can be known: and AKT1^-/-Group comparison, CLP + AKT1^-/-The number of colibacillus and staphylococcus aureus in the group is obviously increased; and CLP + AKT1^-/-Group comparison, FA + CLP + AKT1^-/-The groups escherichia coli and staphylococcus aureus were reduced in number, but the differences were not significant. CLP + AKT1 compared to CLP group^-/-Both the group escherichia coli and the staphylococcus aureus numbers increased significantly. Compared with FA group, FA + CLP + AKT1^-/-Both the group escherichia coli and the staphylococcus aureus numbers increased significantly.

The above-described embodiments are merely illustrative of the preferred embodiments of the present invention, and do not limit the scope of the present invention, and various modifications and improvements of the technical solutions of the present invention can be made by those skilled in the art without departing from the spirit of the present invention, and the technical solutions of the present invention are within the scope of the present invention defined by the claims.

Claims

1. The application of ferulic acid in the preparation of a medicament for treating sepsis intestinal mucosa barrier function damage.

2. Use according to claim 1, wherein the sepsis is caused by a microbial infection.

3. Use according to claim 1, wherein the sepsis comprises early sepsis, severe sepsis and septic shock.

4. The use according to claim 1, wherein the impairment of gut mucosal barrier function in sepsis is accompanied by increased permeability and bacterial translocation.

5. The use according to claim 1, wherein the medicament is a tablet, capsule, granule, powder, pill or sustained release formulation.

6. Use according to claim 1, wherein the ferulic acid acts as an agonist of AKT 1.

7. A medicine for treating sepsis intestinal mucosa barrier function injury is characterized in that the medicine contains ferulic acid.

8. The medicament of claim 7, further comprising a pharmaceutically acceptable adjuvant or carrier.