CN115990160A - Application of demethyleneberberine hydrochloride in preparation of medicines for preventive treatment of pseudomonas aeruginosa pneumonia - Google Patents

Abstract

The invention relates to the field of biological medicine, in particular to application of demethyleneberberine hydrochloride in preparation of medicines for preventing or treating pseudomonas aeruginosa pneumonia. The invention discovers that the hydrochloric acid demethyleneberberine can prevent or treat the pseudomonas aeruginosa pneumonia for the first time, not only increases the new indication of the compound, but also provides a new thought for developing and researching medicines for treating bacterial pneumonia, and also provides a new thought for solving the problem of multiple drug resistance caused by unreasonable utilization of clinical broad-spectrum antibiotics to a certain extent.

Description

Application of demethyleneberberine hydrochloride in preparation of medicines for preventive treatment of pseudomonas aeruginosa pneumonia

Technical field:

the invention relates to the field of biological medicine, in particular to application of demethyleneberberine hydrochloride in preparation of medicines for preventing or treating pseudomonas aeruginosa pneumonia.

Technical background:

pneumonia refers to acute inflammation of lung parenchyma including terminal airways and alveoli, and most of pathogenic factors are pathogenic microorganisms, immune injury, physical and chemical factors, medicines, allergy and the like, wherein bacterial pneumonia is the most common. Pseudomonas aeruginosa (P.aerosa), also known as Pseudomonas aeruginosa, is a gram negative bacterium. Pseudomonas aeruginosa pneumonia is a type of pneumonia caused by pulmonary infection of Pseudomonas aeruginosa, and is the first cause of nosocomial acquired pneumonia (HAP), and is a great focus of attention in the medical community today.

On one hand, the pseudomonas aeruginosa becomes a first consistent pathogen for nosocomial infection, and on the other hand, the pseudomonas aeruginosa has stronger tolerance and adaptability and can survive in humid environment, ultraviolet irradiation, even cleaning agent and disinfectant; on the other hand, most hospitalized patients have varying degrees of immunodeficiency, making them susceptible to infection by pseudomonas aeruginosa. Along with the gradual enhancement of the infectivity of the pseudomonas aeruginosa, the patient is infected once, the disease progress is rapid, and the death rate is high. Thus, a wide spectrum of antibiotic treatments have emerged, however, pseudomonas aeruginosa is resistant to a variety of antibacterial agents, including antibiotic inactivation by enzymatic action, alterations in efflux pump mechanisms, target mutations, and reduced antibiotic uptake. In addition, the mechanism of Pseudomonas aeruginosa infection is complex. Thus, there is a clinical need to find new drugs and to explore new mechanisms to treat infections caused by pseudomonas aeruginosa.

The body's recognition of pathogens and self-changes must be efficient and highly specific to coordinate the appropriate response while limiting excessive inflammatory and autoimmune responses to normal self. Melanoma deficiency factor 2 (AIM 2) is one of the members of the innate immunosensor that can detect the presence of DNA. In one aspect, AIM2 is a key receptor for pathogens that can detect exogenous DNA accumulated in the cytoplasm during the life cycle of intracellular pathogens such as viruses, bacteria, and parasites; on the other hand, AIM2 can also detect damaged DNA, as well as abnormal presence of DNA within the cytoplasm, such as genomic DNA released into the cytoplasm after loss of nuclear membrane integrity. By recognizing the DNA present in these abnormalities in the cytoplasm, AIM2 is activated to initiate assembly of the inflammatory corpuscles, an innate immune complex, which can lead to activation of apoptosis-related speckle-like proteins (ASCs) and inflammatory cysteine aspartic protease 1 (caspase 1). This triggers maturation and secretion of cytokines IL-1. Beta. And IL-18. It can also initiate cell death, a pro-inflammatory form of cell death. Francisella tularensis (Francisella tularensis) and Listeria monocytogenes (Lysteria monocytogenes) were the first living bacteria identified as being involved in AIM2 inflammation. To date, many bacterial species have been found to activate AIM2, including streptococcus pneumoniae (Streptococcus pneumoniae), mould (Mycobaterium), legionella pneumophila (Legionella pneumophila), staphylococcus aureus (Staphylococcus aureus), etc., which have attracted a great deal of attention. The patent proves for the first time that the pseudomonas aeruginosa infects the lung, AIM2 is activated by identifying the pseudomonas aeruginosa and the DNA damaged by the lung tissue, and causes the release of inflammatory factors, so that AIM2 can be used as a key target for treating the pseudomonas aeruginosa pneumonia.

The hydrochloric acid demethyleneberberine is shown in the formula (I).

The English name of the hydrochloric acid demethylene berberine (formula I) is Demethyleneberberine Hydrochloride or Demethyleneberberine Chloride. The present patent refers to this simply as DMB. The skeleton structure in the formula (I) is demethyleneberberine, which is an active ingredient of the demethyleneberberine hydrochloride. The organic moiety in formula (I) is designated 9, 10-dimthoxy-5, 6-dihydrooisoquino [2,1-b ] isoquino-7-ium-2, 3-diol by International Union of Pure and Applied Chemistry (IUPAC), wherein the designation is 9,10-dimethoxy-5,6-dihydroisoquinolin [2,1-b ] isoquinolin-7-ium-2, 3-dihydroxy. Its molecular formula: c19H18no4+, molecular weight is: 324.35. the chemical abstract number (CAS) is: 25459-91-0. The demethyleneberberine can form various salts with inorganic or organic acids, such as chloride, sulfate, phosphate, bromide, iodide, citrate, fumarate, maleate, malate, succinate, etc.

The demethyleneberberine is present in the natural medicinal plant phellodendron amurense (Cortex Phellodendri Chinensis) of Rutaceae, and related activity is rarely studied due to the low content. It is reported to have antibacterial effect on various gram-positive and gram-negative bacteria in vitro, and has anti-inflammatory, antioxidant and antitumor pharmacological activities. There are studies showing that berberine can treat pneumonia or acute lung injury caused by viruses, lipopolysaccharide, ischemia reperfusion, alcohol, etc., but to date, there has been no study on in vivo treatment of bacterial pneumonia and its mechanism by berberine or demethylene berberine. Therefore, the study adopts the hydrochloric acid demethylene berberine for the first time to investigate whether the hydrochloric acid demethylene berberine can relieve the symptoms of the acute pneumonia of the pseudomonas aeruginosa by inhibiting the activation of AIM2 inflammatory bodies.

The invention comprises the following steps:

in order to overcome the defects of the prior art, the invention aims to provide a compound for preventing or treating pseudomonas aeruginosa pneumonia, in particular to an application of demethyleneberberine hydrochloride (DMB) shown in a formula (I) as a medicine for preventing or treating pseudomonas aeruginosa pneumonia.

According to the invention, by establishing a pseudomonas aeruginosa pneumonia model, whether the demethyleneberberine hydrochloride (DMB) has a preventive or therapeutic effect on pseudomonas aeruginosa pneumonia or not is observed. Research results show that the demethyleneberberine hydrochloride (DMB) has preventive protection and treatment effects on pseudomonas aeruginosa pneumonia.

The hydrochloric acid demethylenetetrahydroberberine product used in the invention is prepared by conventional chemistry, separation and purification. The laboratory adopts High Performance Liquid Chromatography (HPLC) analysis and detection, the purity of the product reaches more than 99 percent, and the chemical structure of the demethylenetetrahydroberberine hydrochloride product used in the laboratory is correct through analysis and identification by a chemical method, a mass spectrometry method and a nuclear magnetic resonance method. The research shows that the purity and chemical structure of the demethylenetetrahydroberberine hydrochloride meet the research requirements of in vivo and in vitro biological activity and pharmacological action.

The invention also relates to pharmaceutical compositions containing as active ingredient demethyleneberberine hydrochloride and conventional pharmaceutical excipients or auxiliaries. Typically, the pharmaceutical compositions of the present invention contain 0.1 to 95% by weight of demethyleneberberine hydrochloride. The compounds of the invention are generally present in the unit dosage form in amounts of from 0.1 to 100mg.

Pharmaceutical compositions of the compounds of the present invention may be prepared according to methods well known in the art. For this purpose, the compounds of the invention may, if desired, be combined with one or more solid or liquid pharmaceutical excipients and/or auxiliaries, in suitable administration forms or dosage forms which can be used as human or veterinary medicine.

The compounds of the present invention or pharmaceutical compositions containing them may be administered in unit dosage form by the enteral or parenteral route, such as oral, intramuscular, subcutaneous, nasal, oral, dermal, peritoneal or rectal, etc.

The route of administration of the compounds of the invention or pharmaceutical compositions containing them may be by injection. The injection includes intravenous injection, intramuscular injection, subcutaneous injection, intradermal injection, acupoint injection, etc.

The administration dosage form may be liquid dosage form or solid dosage form. For example, the liquid dosage form may be true solution, colloid, microparticle, emulsion, or suspension. Other dosage forms such as tablet, capsule, dripping pill, aerosol, pill, powder, solution, suspension, emulsion, granule, suppository, lyophilized powder for injection, etc.

The compound of the invention can be prepared into common preparations, sustained release preparations, controlled release preparations, targeted preparations and various microparticle administration systems.

For example, in order to prepare a unit dosage form into a tablet, various carriers known in the art can be widely used. Examples of carriers are, for example, diluents and absorbents such as starch, dextrin, calcium sulfate, lactose, mannitol, sucrose, sodium chloride, glucose, urea, calcium carbonate, kaolin, microcrystalline cellulose, aluminum silicate, etc.; humectants and binders such as water, glycerin, polyethylene glycol, ethanol, propanol, starch slurry, dextrin, syrup, honey, dextrose solution, acacia slurry, gelatin slurry, sodium carboxymethyl cellulose, shellac, methyl cellulose, potassium phosphate, polyvinylpyrrolidone, and the like; disintegrants such as dry starch, alginate, agar powder, brown algae starch, sodium bicarbonate and citric acid, calcium carbonate, polyoxyethylene sorbitol fatty acid ester, sodium dodecyl sulfate, methylcellulose, ethylcellulose, etc.; disintegration inhibitors such as sucrose, glyceryl tristearate, cocoa butter, hydrogenated oils and the like; absorption promoters such as quaternary ammonium salts, sodium lauryl sulfate, and the like; lubricants such as talc, silica, corn starch, stearate, boric acid, liquid paraffin, polyethylene glycol, and the like. The tablets may be further formulated into coated tablets, such as sugar coated tablets, film coated tablets, enteric coated tablets, or bilayer and multilayer tablets.

For example, in order to make the administration unit into a capsule, the compound of the present invention, desmethylidene berberine hydrochloride, is mixed with the above-mentioned various carriers, and the thus-obtained mixture is placed in a hard gelatin capsule or a soft capsule. The active ingredient of the compound can be prepared into microcapsules, and the microcapsules can be suspended in an aqueous medium to form a suspension, or can be filled into hard capsules or prepared into injection for application.

For example, the compound of the invention, the demethyleneberberine hydrochloride, is prepared into injection preparations, such as solutions, suspension solutions, emulsions, freeze-dried powder injection, which can be aqueous or nonaqueous, and can contain one or more pharmaceutically acceptable carriers, diluents, binders, lubricants, preservatives, surfactants, dispersants, osmotic pressure regulators, solubilizers and pH regulators. Such as water, ethanol, polyethylene glycol, 1, 3-propanediol, ethoxylated isostearyl alcohol, polyoxy isostearyl alcohol, polyoxyethylene sorbitol ester, fatty acid esters, etc. The osmotic pressure regulator can be sodium chloride, mannitol, glycerol, glucose, phosphate, acetate, etc.; the solubilizer or cosolvent can be poloxamer, lecithin, hydroxypropyl beta-cyclodextrin, etc.; the pH regulator may be phosphate, acetate, hydrochloric acid, sodium hydroxide, etc. Mannitol, glucose and the like can be added as propping agents for preparing freeze-dried powder injection.

In addition, coloring agents, preservatives, fragrances, flavoring agents, sweeteners, fragrances, or the like may also be added to the pharmaceutical formulation, if desired. These adjuvants are commonly used in the art.

The sterile media used in the present invention can be prepared by standard techniques well known to those skilled in the art. They may be sterilized, for example, by filtration through a bacterial filter, by adding a sterilizing agent to the composition, by radiation treatment of the composition, or by heat sterilization of the composition. They may also be formulated into sterile injectable media immediately prior to use.

For the purpose of administration, the drug or the pharmaceutical composition of the present invention can be administered by any known administration method to increase the therapeutic effect. The route of administration for practicing the compounds of the present invention will of course depend on the disease and the site where treatment is desired. Because the pharmacokinetic and pharmacodynamic profiles of the compounds of the present invention may vary somewhat, the most preferred method of achieving therapeutic concentrations in tissues is to gradually increase the dose and monitor the clinical effect. For such increasing therapeutic doses, the initial dose will depend on the route of administration.

The dosage of the pharmaceutical composition of the compounds of the present invention administered to any particular patient depends on a number of factors, such as the nature and severity of the disease to be prevented or treated, the sex, age, sex and individual response of the patient or animal, the route of administration, the number of times of administration, the purpose of treatment, and thus the therapeutic dosage of the present invention may vary widely. Depending on the condition of the patient being treated, some change in dosage may be necessary and in any event the physician decides the appropriate dosage for the individual patient.

The dose to be administered refers to the weight of the compound excluding the weight of the carrier (when a carrier is used). Generally, the dosages of pharmaceutical ingredients used in the present invention are well known to those skilled in the art. The prevention or treatment of the present invention may be accomplished by appropriate adjustments to achieve its therapeutically effective amount required based on the actual drug dosage contained in the final formulation of the compound composition of the present invention. May be administered in a single dosage form or divided into several, e.g., two, three or four dosage forms; this is limited by the clinical experience of the administering physician and includes administration regimens that employ other therapeutic means. The compounds or compositions of the present invention may be administered alone or in combination with other therapeutic or symptomatic agents and adjusted in dosage.

Advantageous effects

1. The invention discovers that the hydrochloric acid demethyleneberberine can prevent or treat the pseudomonas aeruginosa pneumonia for the first time, not only increases the new indication of the compound, but also provides a new thought for developing and researching medicines for treating bacterial pneumonia, and also provides a new thought for solving the problem of multiple drug resistance caused by unreasonable utilization of clinical broad-spectrum antibiotics to a certain extent.

The invention takes a C57BL/6 mouse as an animal model, and researches that the hydrochloric acid demethyleneberberine has the effects of preventing and treating the pneumonia model caused by pseudomonas aeruginosa. 100mg/kg and 200mg/kg of the administration component of the hydrochloric acid demethyleneberberine are respectively administered for 7 days, the model group is administered with the same dose of excipient for 7 days, and after the 7 th day of administration component of the hydrochloric acid demethyleneberberine and the model group are respectively administered and excipient for 1 hour, 50ul of pseudomonas aeruginosa suspension is injected through an air pipe, and the injection amount of each mouse is 4x10 ⁶ CFU/CFU. Animals mice were sacrificed after 6 h. According to the lung tissue physical map and lung coefficient, DMB has obvious improvement effect on the pneumonia caused by pseudomonas aeruginosa, and pulmonary edema and congestion condition are relieved. In addition, compared with the model group, from the lung tissue H&E staining and Myeloperoxidase (MPO) content in lung tissue can show that DMB low dose group can effectively reduce infiltration of neutrophil in lung tissue, and has anti-inflammatory effect. Tests of lung tissue Malondialdehyde (MDA), glutathione (GSH) and inflammatory factors TNF-alpha, IL-1 beta and IL-6 thereof show that significant lung tissue lipid peroxidation damage and inflammatory response exist in a pseudomonas aeruginosa pneumonia model, but DMB administration groups have significant lipid peroxidation inhibition and anti-inflammatory biological activity. In addition, protein expression detection of lung tissues AIM2, ASC, clear caspase1 and material IL-beta shows that DMB can inhibit AIM2 activation, thereby inhibiting AIM2 inflammatory body assembly and slowing down inflammatory reaction of mice induced by pseudomonas aeruginosa. The experimental results fully demonstrate that the hydrochloric acid Demethyleneberberine (DMB) can play the roles of anti-inflammatory and antioxidation and inhibiting the activation of AIM2 inflammatory corpuscles, thereby effectively preventing or treatingThe medicine has the potential of treating multiple drug resistance caused by antibiotics due to the special action mechanism of the medicine for treating the pneumonia caused by pseudomonas aeruginosa. .

2. According to the invention, the PA-LPS is used for inducing MH-S cells to simulate a pneumonia inflammation model, and the PA-LPS-induced MH-S cell inflammation is treated through DMB. TNF-alpha, IL-1 beta and IL-6 expression levels and NO content were detected by qRT-PCR technique.

3. The invention establishes a cell level oxidative damage model through the BEAS-2B cells induced by the PA-LPS, treats the BEAS-2B cell damage induced by the PA-LPS through DMB, detects the cell ROS level through a DCFH-DA probe, and detects the cell ROS level through a flow cytometry.

Terminology

DMB: hydrochloric acid demethyleneberberine

MPO myeloperoxidase

MDA: malondialdehyde

GSH: glutathione

NO: nitric oxide

TNF- α: tumor necrosis factor

IL-1. Beta: interleukin-1 beta

IL-6: interleukin-6

COX-2 cyclooxygenase

iNOS nitric oxide synthase

AIM2: melanoma deficiency factor 2

ASC: apoptosis-related spot sample application protein

Caspase1

IG, stomach lavage

Drawings

FIG. 1, DMB inhibits P.aeruginosa growth;

FIG. 2, DMB inhibits the production of ROS by PA-LPS-induced BEAS-2B cells, wherein A is the flow cytometry result and B is the histogram;

FIG. 3, DMB, reduces the inflammatory response (cytokine changes) of PA-LPS induced MH-S cells; wherein A is TNF-alpha, B is IL-1 beta, C is IL-6, D is NO;

FIG. 4 construction of a Pseudomonas aeruginosa pneumonia model;

FIG. 5DMB reduces the extent of Pseudomonas aeruginosa-induced lung swelling in mice;

FIG. 6 P.aeruginosa induced morphological changes in mouse lung tissue;

wherein A represents the lung tissue morphology of a normal mouse, B represents the lung tissue morphology of a lung model mouse induced by pseudomonas aeruginosa, C represents the lung tissue morphology of a DMB 100mg/kg administration group mouse, and D represents the lung tissue morphology of a DMB 200mg/kg administration group mouse

FIG. 7 P.aeruginosa induced pathological changes in lung tissue of mice

Wherein A, A represents a normal mouse lung tissue HE staining pattern, B, B represents a Pseudomonas aeruginosa-induced lung tissue HE staining pattern of a model mouse lung tissue HE, C, C represents a DMB 100mg/kg administration group mouse lung tissue HE staining pattern, D, D1 represents a DMB 200mg/kg administration group mouse lung tissue HE staining pattern

FIG. 8DMB improving Pseudomonas aeruginosa-induced expression of TNF-alpha (A), IL-1 beta (B) and IL-6 (C) in lung tissue of mice

FIG. 9 is a graph showing the MPO results of lung tissue of Pseudomonas aeruginosa-induced mice model for pneumonia in DMB prophylaxis and treatment

FIG. 10 is a graph showing the results of DMB prevention and treatment of lung tissue MDA of Pseudomonas aeruginosa-induced mice model pneumonia

FIG. 11 is a graph showing the results of DMB prevention and treatment of Pseudomonas aeruginosa-induced lung tissue GSH in a model of mouse pneumonia

FIG. 12 is a graph showing Western blotting results of lung tissue inflammatory factors of a Pseudomonas aeruginosa-induced mice model for preventing and treating lung inflammation

Wherein A, A represents TNF-alpha, IL-1 beta and IL-6 inflammatory factor protein expression and quantification patterns, B, B represents COX-2 and iNOS protein expression and quantification patterns

FIG. 13DMB inhibition of AIM2 inflammation minibody activation in Pseudomonas aeruginosa-induced murine pneumonia model

Wherein A represents protein expression of AIM2, ASC, clean caspase1 and material IL-beta, B represents AIM2 quantization map, C represents ASC quantization map, D represents clean caspase1 quantization map, E represents material IL-beta quantization map

In the figure, model and control are compared #, and the DMB administration and Model are compared, wherein #, or p <0.05, # or p <0.01, and the data are obtained through graphpad statistics.

Detailed Description

The following examples will assist those skilled in the art in a more complete understanding of the invention, but are not intended to limit the invention in any way.

EXAMPLE 1 anti-bacterial effect of Demethylene berberine hydrochloride (DMB) on Pseudomonas aeruginosa

The method comprises the following steps: the frozen pseudomonas aeruginosa (ATCC 27853) strain was removed and inoculated onto solid LB medium using a three-plot method and incubated overnight at 37 ℃. After colonies are formed in the solid LB culture dish the next day, single colonies are picked up into 20ml of liquid LB culture medium and placed in a constant temperature shaking incubator (37 ℃,220 rpm) for shaking incubation for 14-16 hours. 200ul of the bacterial suspension is extracted into 20ml of new LB liquid medium, and the bacterial suspension is placed in a constant temperature shaking incubator (37 ℃ C., 220 rpm) for 2.5 to 4 hours until the bacterial suspension grows to the logarithmic phase, and the bacterial suspension grows to 1X10 ⁶ CFU/ml was inoculated into 96-well plates and the Minimum Inhibitory Concentration (MIC) of desmethylidene berberine hydrochloride (DMB) was detected by micro broth dilution (double dilution). DMB concentration interval is 256 mug/ml-4096 mug/ml, corresponding DMSO concentration interval is 0.75% -12%, and the medicine and pseudomonas aeruginosa are incubated at 37 ℃ for 18-20h, and OD value is detected at 600 nm.

Results: as shown in FIG. 1, DMB has an inhibitory effect on the growth of Pseudomonas aeruginosa, and the minimum inhibitory concentration is 2048 μg/ml.

Example 2. Demethyleneberberine hydrochloride (DMB) has protective effect on oxidative damage of BEAS-2B cells induced by PA-LPS.

The method comprises the following steps: BEAS-2B is taken as a study object, lipopolysaccharide (PA-LPS) from pseudomonas aeruginosa is taken as an induction reagent, and the scavenging effect of DMB on ROS is detected at the cellular level by adopting an ROS probe method. Human lung bronchial epithelial cells (BEAS-2B) are inoculated into a culture plate, taken out after the culture plate is attached, pretreated by DMB (10 uM and 20 uM) for 2 hours, taken out, and subjected to PA-LPS (4 ug/ml) for 24 hours to generate ROS to cause oxidative damage to the cells. The fluorescent probe DCFH-DA (50 uM) was used to bind to ROS to generate fluorescence, and the scavenging effect of ROS was detected by flow cytometry.

Results: treatment of the streaming data with flowjo software showed that 10uM and 20uM DMB significantly inhibited ROS produced by PA-LPS in BEAS-2B cells, with the results shown in fig. 2.

EXAMPLE 3DMB reduction of PA-LPS-induced inflammatory response in MH-S cells

The method comprises the following steps: taking MH-S as a study object and PA-LPS as an induction reagent, adopting an RT-PCR method and a kit to detect DMB from a cell level and reducing inflammatory response induced by lipopolysaccharide PA-LPS (1 ug/ml) from pseudomonas aeruginosa. Mouse alveolar macrophages (MH-S) are inoculated into a culture plate and taken out after the mouse alveolar macrophages are attached to the wall, cell culture fluid containing DMB (5 uM, 10uM and 20 uM) is pretreated for 2 hours and taken out, cells are treated by PA-LPS (1 ug/ml) for 24 hours and taken out, and TNF-alpha, IL-1 beta and IL-6 inflammatory factors are detected by a qRT-PCR method. And detecting the content of Nitric Oxide (NO) secreted by cells in the culture solution by adopting the kit.

Results: qRT-PCR shows that DMB can relieve inflammatory response of PA-LPS to MH-S cells. The NO kit results show that DMB can inhibit NO secreted by cells in the culture solution. The results are shown in FIG. 3.

Example 4: preparation of Pseudomonas aeruginosa suspension

The method comprises the following steps: the frozen pseudomonas aeruginosa (ATCC 27853) strain was removed and inoculated onto solid LB medium using a three-plot method and incubated overnight at 37 ℃. After colonies are formed in the solid LB culture dish the next day, single colonies are picked up into 20ml of liquid LB culture medium and placed in a constant temperature shaking incubator (37 ℃,220 rpm) for shaking incubation for 14-16 hours. 200ul of the bacterial suspension was extracted from the bacterial suspension in 20ml of fresh LB liquid medium, placed in a constant temperature shaking incubator (37 ℃ C., 220 rpm) for 2.5-4 hours, the heavy suspension was diluted with liquid LB medium, and the OD was measured at 600nm to 0.5. The titrated heavy suspensions are respectively diluted in a gradient way by taking liquid LB culture medium, 100ul of bacterial suspensions with different concentrations are inoculated into solid culture medium, and incubated overnight at 37 ℃. After the colony formation on the next day, the number of counted colonies was 1.7x10 ⁷ CFU/ml. Centrifuging 7.5ml of the bacterial suspension (at 8000rpm, 4deg.C) for 5min, removing supernatant, and re-suspending with 1.5ml of sterile PBS to obtain Pseudomonas aeruginosaAnd (3) suspension.

Example 5: construction of Pseudomonas aeruginosa mouse pneumonia model

The method comprises the following steps: adult C57BL/6 male mice, 6-8 weeks old, weighing 20-22g, were randomly divided into 4 groups of 6 animals each. The 4 groups are normal control group, model group, DMB low dose group (100 mg/kg) and DMB high dose group (200 mg/kg). The dosing groups were given continuous prophylactic dosing for 7 days, once a day, with the remaining groups given the same volume of vehicle. After 1 hour of prophylactic administration on day 7, animals of each group except the normal control group were anesthetized with 2% isoflurane gas inhalation, and the toe-clamped test was non-responsive. Mice were fixed, the neck dissected, and subcutaneous tissue and muscle were blunt separated, exposing the trachea. 50ul of bacterial suspension was withdrawn, punctured into the trachea, infused into the lungs with inhalation, kept in place for 2 minutes, and the muscle layers and skin were sutured layer by layer, with antibiotic powder applied as shown in figure 4.

Example 6: DMB has protective effect on lung pathological changes of pseudomonas aeruginosa pneumonia model

The method comprises the following steps: after the pseudomonas aeruginosa pneumonia model is finished for 6 hours, cervical dislocation is adopted for killing the mice under isoflurane anesthesia, the mice are weighed, lung tissues are picked up, the mice are rinsed clean in sterile physiological saline, the mice are weighed, left lung lobes of different mice are taken, the mice are fixed for 24 hours by 10% neutral formalin solution, and pathological examination is carried out by HE staining.

Results: the lung coefficient ratio, the lung tissue morphology graph and the HE staining result show that compared with a normal control group, the lung tissue of a model group is obvious in swelling, the color is dark red, inflammatory cell infiltration is obviously increased, compared with the model group, the lung swelling of a low-dose group (100 mg/kg) and a high-dose group (200 mg/kg) of the demethyleneberberine hydrochloride are obviously reduced, the inflammatory cell infiltration is slightly bleeding, and compared with a low-dose group, the lung swelling, the bleeding and the inflammatory cell infiltration are obviously reduced in the high-dose group, as shown in figures 5,6 and 7.

Example 7: influence of DMB on expression of lung tissue inflammatory factor mRNA in Pseudomonas aeruginosa pneumonia model

The method comprises the following steps: after the pseudomonas aeruginosa pneumonia model is ended for 6 hours, cervical dislocation is adopted for killing the mice under isoflurane anesthesia, lung tissues are extracted, 15mg of lung tissues at the same position of different mice are accurately weighed to extract RNA, and mRNA expression changes of the lung tissues TNF-alpha, IL-1 beta and IL-6 are detected.

Results: as shown in FIG. 8, the normal control group had up-regulated mRNA expression of TNF- α, IL-1β and IL-6 in lung tissue compared to the model group, whereas the DMB low dose group (100 mg/kg) and the high dose group (200 mg/kg) had down-regulated mRNA expression of TNF- α, IL-1β and IL-6 in lung tissue compared to the model group, and the high dose group had more pronounced down-regulated mRNA expression of TNF- α, IL-1β and IL-6 in lung tissue compared to the low dose group.

Example 8: DMB can reduce MPO level in lung tissue of mice with Pseudomonas aeruginosa pneumonia.

The method comprises the steps of after a pseudomonas aeruginosa pneumonia model is ended for 6 hours, killing a mouse under isoflurane anesthesia, picking up lung tissues, taking lung tissues of different mice at the same position, grinding the lung tissues on an ice bath with normal saline to prepare 10% lung tissue homogenate, centrifuging, taking supernatant, and determining the MPO level in the lung tissues according to a Nanjing build kit specification.

Results: MPO is generally used as an important index for evaluating tissue inflammation, and its activity unit is defined as 1umol H decomposed per 10% tissue homogenate in a reaction system at 37deg.C ₂ O ₂ Is a combination of the amounts of (a) and (b). In a model of pseudomonas aeruginosa-induced pneumonia, its activity is responsive to neutrophil infiltration. The model group of mice had a significant increase in myeloperoxidase MPO compared to the normal control group. While both DMB low dose (100 mg/kg) and high dose (200 mg/kg) groups significantly reduced MPO compared to the model group. The research result shows that DMB can reduce MPO activity and has anti-inflammatory effect. As shown in fig. 9.

Example 9: DMB can reduce lung tissue MDA levels in pseudomonas aeruginosa pneumonitis mice.

The method comprises the following steps: after the pseudomonas aeruginosa pneumonia model is finished for 6 hours, cervical dislocation is adopted for the mice under isoflurane anesthesia, lung tissues are taken out, lung tissues at the same position of different mice are taken out, physiological saline is used for grinding the mice into 10% lung tissue homogenate on an ice bath, the supernatant is taken after centrifugation, and malondialdehyde MDA level in the lung tissue is measured according to Nanjing build kit instructions.

Results: MDA is lipid peroxide formed by attack of polyunsaturated fatty acids in a biological membrane by oxygen free radicals in a body, and the measurement of the amount of MDA can reflect the degree of lipid peroxidation in the body and indirectly reflect the degree of cell damage. The MDA level in the lung tissue of the mice in the model group was elevated compared to the control group, and both the DMB low dose (100 mg/kg) group and the high dose (200 mg/kg) group were able to significantly reduce MDA compared to the model group. The results of the study show that DMB has MDA-reducing effect and can improve oxidative damage of lung tissue cells, as shown in FIG. 10.

Example 10: DMB can reduce GSH levels in lung tissue of pseudomonas aeruginosa pneumonitis mice.

The method comprises the following steps: after the pseudomonas aeruginosa pneumonia model is finished for 6 hours, cervical dislocation is adopted for the mice under isoflurane anesthesia, lung tissues are taken out, lung tissues at the same position of different mice are taken out, physiological saline is used for grinding the mice into 10% lung tissue homogenate on an ice bath, supernatant is taken after centrifugation, and GSH level in the lung tissues is measured according to Nanjing build kit description.

Results: GSH has antioxidant effects and is used primarily to measure oxidative damage. Pseudomonas aeruginosa pneumonia is often accompanied by oxidative damage along with an inflammatory response. Compared with the control group, the GSH level in the lung tissue of the mice in the model group is reduced, which proves that the lung tissue of the mice with pneumonia induced by pseudomonas aeruginosa has serious oxidative damage, and the high-dose DMB (200 mg/kg) group can obviously increase the GSH content of the lung tissue. The results of the study show that DMB can raise GSH level of Pseudomonas aeruginosa pneumonia mice, and has the function of resisting oxidative damage, as shown in figure 11.

Example 11: influence of DMB on expression of protein related to lung tissue inflammation of mice with Pseudomonas aeruginosa pneumonia model

The method comprises the following steps: after the model of the pseudomonas aeruginosa pneumonia is finished for 6 hours, cervical dislocation is adopted for the mice under the condition of isoflurane anesthesia, lung tissues are extracted, 25mg of extracted proteins of the lung tissues at the same position of different mice are extracted, and the effect of DMB on the expression of TNF-alpha, IL-1 beta, IL-6 and COX-2 proteins of the lung tissues of the mice of the model of the pseudomonas aeruginosa pneumonia is detected by adopting a western immunoblotting method.

Results: the body is stimulated by the outside to cause the unbalance of immunity, thereby releasing a great deal of inflammatory factors such as TNF-alpha, IL-1 beta, IL-6 and the like, and further increasing the degree of inflammatory reaction. COX-2, also known as cyclooxygenase, is the rate-limiting enzyme for arachidonic acid synthesis, and its expression level is low under normal physiological conditions, and under pathological conditions, it participates in inflammatory reactions, and its expression level increases several times. iNOS can induce its massive expression after tissue injury. The protein immunoblotting method shows that TNF-alpha, IL-1 beta, IL-6, COX-2 and iNOS proteins are highly expressed in the model group, and that both the DMB low dose (100 mg/kg) group and the high dose (200 mg/kg) group significantly reduced TNF-alpha, IL-1 beta, IL-6, COX-2 and iNOS expression, thereby reducing inflammatory responses, compared to the normal control group. The results are shown in FIG. 12.

Example 12: effects of DMB on expression of AIM2 inflammatory small associated proteins in lung tissue of Pseudomonas aeruginosa model mice

The method comprises the following steps: after the model of the pseudomonas aeruginosa pneumonia is finished for 6 hours, cervical dislocation is adopted for the mice under the condition of isoflurane anesthesia, lung tissues are extracted, 25mg of extracted proteins of the lung tissues at the same position of different mice are taken, and the effect of DMB on the expression of AIM2, ASC, clear caspase1 and information IL-beta proteins of the lung tissues of the mice model of the pseudomonas aeruginosa pneumonia is detected by adopting a western immunoblotting method.

Results: AIM2 promotes assembly of AIM2 inflammatory bodies by recognizing that pseudomonas aeruginosa DNA and self-damaged DNA are activated, thereby further exacerbating the extent of inflammatory response. The protein immunoblotting method shows that compared with the normal group, the AIM2, ASC, clear caspase1 and information IL-beta protein expression of the model group is increased, and the DMB low dose (100 mg/kg) group and the high dose (200 mg/kg) group can obviously reduce the AIM2, ASC, clear caspase1 and information IL-beta protein expression, thereby inhibiting the assembly of AIM2 inflammatory corpuscles and further relieving the excessive inflammatory reaction and the cell death in a pro-inflammatory form. The results are shown in FIG. 13.

Claims (3)

1. The application of the demethyleneberberine hydrochloride and the pharmaceutical composition thereof in preparing medicaments for preventing or treating pseudomonas aeruginosa pneumonia is provided, wherein the structural formula of the demethyleneberberine hydrochloride is shown as (I).

2. The use according to claim 1, characterized in that the pharmaceutical composition comprises desmethylidene berberine hydrochloride and pharmaceutically acceptable excipients.

3. Use according to claim 1 or 2, characterized in that: the demethyleneberberine includes hydrochloride, phosphate, sulfate and other demethyleneberberine salt forms.

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