JP2013056851A - Type ii alveolar epithelial cell activator - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for activating proliferation of type II alveolar epithelial cells by oral ingestion thereby allowing influenza or infectious lung disorder to be cured or alleviated, and to provide a type II alveolar epithelial cell activator drug.SOLUTION: The method for activating proliferation of type II alveolar epithelial cells thereby allowing lung disorder to be cured or alleviated, includes oral ingestion of lactobacillus. A type II alveolar epithelial cell activator is also provided. The lactobacillus is preferably genus Enterococcus, particularly, faecalis species.

Description

  The present invention relates to a food or a pharmaceutical that activates the proliferation of type II alveolar epithelial cells by oral ingestion to improve lung injury.

  Many lactic acid bacteria of the genus Enterococcus have so far been reported to activate immunity by ingestion (Patent Documents 1 to 5, Non-Patent Documents 1 to 3). For example, in Patent No. 2969017, we have reported that ingestion of lactic acid bacteria belonging to the genus Enterococcus to mice orally prevents infection by Candida and leads to reduction of symptoms. Japanese Patent No. 3272023 reports that leukopenia caused by drinking ethanol or administering an anticancer agent is improved by oral administration of lactic acid bacteria belonging to the genus Enterococcus.

  The mechanism by which Enterococcus lactic acid bacteria exert an immunostimulatory effect is considered to be the same as the mechanism by which general lactic acid bacteria exert immunity. That is, when orally ingested lactic acid bacteria or lactic acid bacteria components reach the small intestine, they are taken into Peyer's patches from M cells present in the intestinal epithelium, and antigens are presented by dendritic cells to activate T cells. It leads to strengthening systemic immunity. (Non-Patent Document 4)

  In addition to the immunostimulatory action, the lactic acid bacteria belonging to the genus Enterococcus have an effect of improving liver function (Patent Document 6, Non-Patent Document 5), an obesity-suppressing action (Non-Patent Document 6), an allergy-suppressing action (Patent Documents 7-8, Patent Document 7) and the improvement effect of skin diseases (Patent Documents 9 to 11) have been reported. There are also reports on safety when ingested in large quantities. (Non-patent document 8)

The lungs are the place where carbon dioxide produced in the body exchanges oxygen in the atmosphere. Specifically, gas exchange is performed in a pouch-like organ called an alveoli, which is further branched from the trachea divided into left and right in the bronchi. Capillaries surround the alveoli. Type I alveolar epithelial cells form basement membranes with capillary vascular endothelial cells surrounding the alveoli and perform gas exchange. Type II alveolar epithelial cells produce lung surfactant, reduce alveolar surface tension, and prevent fibrosis. In addition, type II alveolar epithelial cells
It also functions as a cell. When type I alveolar epithelial cells are damaged, they proliferate and differentiate into type I alveolar epithelial cells. It has been reported that hepatocyte growth factor is effective in activating type II alveolar epithelial cells. (Patent Document 12) Normally, since the lungs of adults are organs that do not regenerate naturally, it is considered that the best way to prevent lung injury is to increase the capacity of reserve cells and suppress deterioration.

  When pneumonia is caused by an infection such as influenza, it is currently mainly to eliminate pathogens with antiviral agents and antibiotics, but this depends on the person's ability to recover and the emergence of resistant viruses and resistant bacteria. There is a risk of doing. A method for treating pulmonary dysfunction using probiotic lactic acid bacteria has also been reported, but this is due to the anti-inflammatory action of probiotics and does not promote recovery of the lung itself. (Patent Document 13)

Japanese Patent No. 2969017 Japanese Patent No. 3272023 JP2008-194001 JP-A-9-12496 JP-A-8-99887 JP-A-8-176000 JP-A-11-124336 JP2003-137795 JP 2000-95698 A JP 2004-26670 A JP 2005-126342 A Special table 2007-528366 Special table 2007-514738

Pharmaceutical Journal 1993 Journal of Japanese Agricultural Chemistry 1995 International Journal of Immunopharmacology 1996 Probiotics / Prebiotics / Biogenics Research and Significance with Special Reference to the Relationship with Enterobacteria Bifidobacteria Center Japan Foundation Japan Red Wakayama Magazine 2004 Journal of Japan Society for Food Science and Technology 2009 Clin Exp Allergy 2004 Applied pharmacology 2009

  An object of the present invention is to provide a food or drug capable of activating proliferation of type II alveolar epithelial cells by oral ingestion and reducing influenza and infectious pulmonary inflammation.

  In researching lactic acid bacteria belonging to the genus Enterococcus, we found that mice treated with lactic acid bacteria belonging to the genus Enterococcus had mild pulmonary inflammation and low mortality even when infected with influenza. The present invention was completed by ascertaining that this was due to activation of type II alveolar epithelial cell proliferation.

  The present invention treats lung injury by activating the growth of type II alveolar epithelial cells by orally ingesting Enterococcus lactic acid bacteria. What is already sold as a health food can be used for this invention, and since a prescription is not required, it can always be used and can be drunk easily when needed. The lactic acid bacteria belonging to the genus Enterococcus are not limited to viable bacteria but may be killed bacteria, and if killed bacteria, they are safe without causing secondary infections even to sick people with reduced resistance.

It is the immuno-staining image by the anti-prosurfactant protein C antibody of the lung section. An example of prosurfactant protein C-positive cells is indicated by arrows. It is a graph which shows the ratio of the type II alveolar epithelial cell number in a lung.

  The microorganism belonging to the genus Enterococcus used in the present invention may be any microorganism belonging to the genus Enterococcus, and examples thereof include bacteria such as Enterococcus faecalis and Enterococcus faecium. A microorganism belonging to the genus Enterococcus is sometimes called enterococci. The microorganisms can be ordered from domestic and international distribution agencies such as ATCC, IFO and JCM. Since these microorganisms are bacteria generally present in foods, bacteria used for food production, or bacteria isolated from the stool of healthy persons, there is no risk of side effects. Moreover, since it can be easily obtained in a large amount by culturing, the use of microbial cells obtained by culturing is inexpensive and economical. As a useful microorganism, Enterococcus faecalis (Enterococcus faecalis) is preferable because of its cell wall structure, and a particularly useful microorganism is Enterococcus faecalis NF-1011 strain, which is a strain isolated from the intestines of healthy individuals. It is. This strain is deposited under the accession number FERM BP-10902 of the National Institute of Advanced Industrial Science and Technology (AIST). The bacteriological (morphological, biochemical and serological) properties of the strain are described in Patent Document 1.

  The microbial cells belonging to the genus Enterococcus used in the present invention may be those obtained from the above microorganisms, and one or two or more microbial cells may be used in combination. Ideally, it should contain at least one faecalis species, regardless of whether the fungus is alive or dead. Although an example of the method of obtaining the microbial cell used for this invention is described, it does not specifically limit to this. The above microorganisms are ingested into an appropriate amount of a sterilized Rogoza liquid medium and statically cultured at 35 to 37 ° C. for 10 to 16 hours to obtain a preculture solution. This is added to 10 liters of sterilized Rogoza liquid medium, and statically cultured in the same manner. The obtained culture solution is centrifuged to obtain viable cells as a precipitate. Viable cells can be washed several times with an isotonic solution such as physiological saline and centrifuged in the same manner to obtain cells. The cells may be further suspended in an isotonic solution such as physiological saline and used as a cell solution, or may be used after being dried by a freeze-drying method or the like.

  Furthermore, in the present invention, a treated product of a microbial cell in which the cell wall of the microbial cell is completely or partially destroyed to release at least one intracellular component can be used. Examples of the treated product of the microbial cells used in the present invention include those obtained by treating the microbial cells with heat, physical force, or lytic enzyme. In the case of destruction by heat, the cell suspension may be heated at 100 ° C. or more for several minutes or more, but considering the destruction efficiency, a temperature (110 ° C. to 125 ° C.) at which autoclaving can be performed is desirable. For example, a processed bacterial cell can be obtained by heat treatment at 110 ° C. for 10 minutes. Moreover, in the case of the destruction process by a physical force, it is also possible to obtain a processed product by destroying the cell wall using a physical method such as ultrasonic treatment or French press. Furthermore, in the case of the lysis treatment with a lytic enzyme, it can be obtained by treating the bacterial cells with an effective amount of the lysis enzyme. In particular, enzyme treatment is preferable because high destruction efficiency can be obtained. In addition, the above processing methods can be used in combination.

  The dosage form of the oral composition of this invention is not specifically limited, It can prepare in a well-known dosage form suitably. Even if a large amount of microbial cells or a processed product thereof can be prepared, it can be prepared into an administrable dosage form by concentrating the microbial cells or processed product thereof as an active ingredient if necessary. Depending on the use of the composition, it can take a dosage form commonly used for foods and pharmaceuticals, and is usually a solid, semi-solid or liquid, preferably a solid or liquid. Specifically, tablets (including orally disintegrating tablets, chewable tablets, effervescent tablets, troches, jelly drops, etc.), pills, granules, fine granules, powders, hard capsules, soft capsules , Dry syrups, liquids (including drinks, suspensions, syrups), jellys, etc., preferably tablets, granules, fine granules, powders, hard capsules, soft capsules Capsules and liquids. It is desirable to contain 100 to 10 trillion lactic acid bacteria of the genus Enterococcus, and at least 500 billion to 5 trillion lactic acid bacteria of the genus Enterococcus are included for the amount consumed at one time.

  The use of the oral composition of the present invention is not particularly limited as long as the effects of the present invention are achieved. For example, pharmaceuticals, foods [health foods, nutritional supplements (balanced nutritional foods, supplements, etc.), functional nutritional foods, and special health uses Including food]. Specifically, vitamins (including tablets, granules, and drinks) for pharmaceuticals, drugs for women, etc., foods such as gums, confectionery such as tablet confectionery, and balanced nutritional foods such as beverages such as nutritional drinks, powders And supplements having forms such as capsules and tablets, health foods, nutritional functional foods, foods for specified health use, and the like.

  In the oral composition of the present invention, in addition to the above components, components usually used in foods and pharmaceuticals may be appropriately blended depending on the use or dosage form of the composition. The ingredients that can be blended are not particularly limited, but include, for example, amino acids, alcohols, polyhydric alcohols, saccharides, gums, polysaccharides and other high molecular compounds, surfactants, antiseptic / antibacterial / bactericides, and pH adjustments. Agents, chelating agents, antioxidants, enzyme components, binders, disintegrants, lubricants, fluidizing agents, cooling agents, minerals, cell activators, nutrient tonics, excipients, thickeners , Stabilizers, preservatives, tonicity agents, dispersants, adsorbents, disintegration aids, wetting agents or wetting regulators, moisture-proofing agents, coloring agents, flavoring agents or fragrances, fragrances, reducing agents, solubilizing agents An agent, a solubilizing agent, a foaming agent, a thickening agent or a thickening agent, a solvent, a base, an emulsifier, a plasticizer, a buffering agent, a brightening agent and the like can be mentioned.

  The dose of the oral composition of the present invention can be appropriately increased or decreased according to symptoms, patient age, body weight, dosage form and the like. A daily dose of 0.001 to 0.5 g / Kg body weight, preferably 0.002 to 0.1 g / Kg body weight, can be administered per day per adult, Furthermore, it can be administered once a day or divided into several times.

Preparation of bacterial cell sample Enterococcus faecalis NF-1011 (patent biological deposit center accession number FERM BP-10902) was inoculated into 10 ml of Rogoza liquid medium having the composition shown below at 37 ° C. for 15 hours. Aerobic static culture (pre-culture) was performed to obtain about 10 ⁹ cells / ml of a cell solution (seed). This was inoculated into 10 L of Rogosa liquid medium (the number of bacteria: 10 ⁶ cells / ml) and statically cultured at 37 ° C. for 16 hours to obtain a cell solution of about 10 ⁹ cells / ml of viable cells. The obtained bacterial liquid is collected by centrifugation (12,000 × g, 20 minutes), washed twice with physiological saline (0.85% aqueous sodium chloride solution), and suspended in 100 ml of distilled water. It became cloudy and a cell suspension was obtained. Lysozyme was added to this bacterial cell suspension to a final concentration of 0.1 mg / ml, treated at 37 ° C. for 4 hours, and then heated at 110 ° C. for 10 minutes to obtain a treated bacterial cell product (LFK). It was.

Preparation method of Rogoza liquid medium The components in Table 1 below were dissolved in 1000 ml of distilled water, adjusted to pH 7.0, and then prepared by autoclaving at 121 ° C. for 15 minutes.

C57BL / 6 (male, 5 weeks old) was orally administered once a day for 7 days with 0.2 ml of LFK suspension (75 mg / ml) adjusted with physiological saline. In the control group, physiological saline was administered. On day 7 after the start of administration, the mice were euthanized under isoflurane anesthesia, and the lungs were removed. The removed lung was washed with PBS, fixed with 10% neutral buffered formalin for 2 days, and embedded in paraffin. A 4 mm section was prepared with a microtome (Sakura Seiki), attached to a glass slide, stretched and dried overnight at 42 ° C. The sample was deparaffinized using xylene (5 minutes repeated 3 times), and the endogenous peroxidase inhibition treatment was performed using 0.3% hydrogen peroxide solution diluted with methanol (5 minutes). The activation treatment was performed by boiling for 1 minute in a microwave using a 10 mM citrate buffer (pH 6). After cooling by standing at room temperature, washing was performed with PBS (5 minutes was repeated three times). Blocking was performed with 2% normal goat serum / 0.2% Triton X-100 / PBS for 2 hours at room temperature. 2% normal goat serum / 0.2% Triton after PBS washing (5 minutes repeated 3 times)
The reaction was performed overnight at 4 ° C. using a primary antibody solution (anti-prosurfactant protein C, Millipore) diluted 4000 times with X-100 / PBS. Wash with 0.2% Triton X-100 / PBS (repeat 5 minutes 6 times), then react with HRP labeled polymer (DAKO, Envision + system HRP labeled polymer anti rabbit) to develop color (DAKO, Envision + kit) / HRP (DAB)). After washing with PBS, hematoxylin staining, tap water washing (10 minutes), dehydration (95% ethanol for 3 minutes, 100% ethanol for 5 minutes), xylene penetration (5 minutes repeated twice), enteran Enclosed with Merck. Prosurfactant is a marker of type II alveolar epithelial cells
Stained images of protein C positive cells were observed under a microscope. By counting hematoxylin-stained cells and prosurfactant protein C-positive cells, the ratio of the number of type II alveolar epithelial cells in the lung cells in the specimen was calculated.

Results FIG. 1 shows an immunostaining image with anti-prosurfactant protein C antibody. Compared to the control group given saline, the alveolar wall was thickened and the number of type II alveolar epithelial cells increased in the LFK administration group. In addition, Figure 2 shows the prosurfactant in lung cells
The percentage of protein C positive cells is shown. Significantly prosurfactant in LFK administration group
The percentage of protein C positive cells increased. These show that the number of type II alveolar epithelial cells increases when the treated bacterial cell product of the present invention is orally administered.

  The present invention activates the proliferation of type II alveolar epithelial cells by orally ingesting lactic acid bacteria, and is expected to reduce or recover pneumonia resulting from infections such as influenza and lung injury caused by drugs and smoking it can. Although it can be used as a food, it can also be used as a medical ingredient.

Claims (5)

  A method of activating the growth of type II alveolar epithelial cells by ingesting lactic acid bacteria and healing or reducing lung injury   The method of claim 1, wherein the lactic acid bacteria is of the genus Enterococcus.   The method of claim 1, wherein the lactic acid bacterium is Enterococcus sp. Faecalis species   Type II alveolar epithelial cell activator containing Enterococcus lactic acid bacteria or cell components as active ingredients   The type II alveolar epithelial cell activator according to claim 3, wherein the lactic acid bacterium of the genus Enterococcus is a faecalis species