TWI688394B - Use of parabacteroides goldsteinii to prevent or treat chronic obstructive pulmonary disease - Google Patents

Abstract

The present invention describes a method to improve the weight loss, the emphysema, the infiltration of inflammatory cells in lung tissues, the thickening of the tracheal wall, the pulmonary fibrosis, and abnormal lung function, which caused by chronic obstructive pulmonary disease, by administering the probiotic bacterium Parabacteroides goldsteiniitoa subject. The bacterium Parabacteroides goldsteinii can also modulate the expression of genes involved in lung fibrosis and mitochondrial activity in lung tissue. The bacterium Parabacteroides goldsteiniican therefore be used to treat chronic obstructive pulmonary disease.

Description

Use of Parabacterium gordonii for preventing or treating chronic obstructive pulmonary disease

The present invention relates to the use of Parabacteroides goldsteinii , in particular to the application of using Parabacterium gordonii to prevent or treat chronic obstructive pulmonary disease; wherein the Parabacterium gordonii can improve chronic obstructive pulmonary disease The patient's lung function, proinflammatory cytokine accumulation, airway smooth muscle hyperplasia, emphysema, pulmonary fibrosis, and mitochondrial dysfunction.

Chronic obstructive pulmonary disease (COPD) is a compound term used to describe emphysema and chronic bronchitis. It is estimated that by 2020, chronic obstructive pulmonary disease will become the third highest human mortality disease in the world, among which chronic obstructive pulmonary disease is significantly associated with chronic cigarette smoke (CS) exposure. The lung tissue of patients with chronic obstructive pulmonary disease has the characteristics of chronic inflammation, mucus metaplasia, damaged alveoli, and structural apoptosis. At the same time, COPD patients often suffer from weight loss, mainly due to increased basal energy consumption, reduced nutrient intake and poor metabolic efficiency. Malnutrition in COPD patients can cause respiratory muscle weakness, and further prone to complications such as hypoxia or carbon dioxide retention. In addition, malnutrition can also cause a decline in immune function, increasing the frequency and severity of lung infections.

Recent research has also pointed out that the airways in the tissues of patients with chronic obstructive pulmonary disease are often remodeled and fibrotic, and the nearby alveoli will show the characteristics of tissue thinning and interval rupture. Inflammatory symptoms in the lung tissue of chronic obstructive pulmonary disease are considered to be causally related to emphysema and other pathological changes in the patient's lungs, and will worsen as the disease progresses. However, the relationship and mechanism between these inflammatory symptoms and the remodeling response of lung tissue in chronic obstructive pulmonary disease are unknown. Onset of COPD The rate and mortality rate remain high due to the onset of acute symptoms such as coughing, shortness of breath, sputum production, or decreased expiratory airflow. The overall health status of these patients and the rate of progression and frequency of deterioration of these diseases , And the loss of lung function are closely related. Therefore, these acute symptoms are regarded as the main targets for the treatment of chronic obstructive pulmonary disease. However, the regulatory mechanisms of these acute symptoms and their effects on the inflammation and remodeling of lung tissue have not been fully studied.

Recent studies have shown that airway microbiota is associated with persistent chronic respiratory tract inflammation in patients with chronic obstructive pulmonary disease, and it plays an important role in the aggravation of the condition and can cause the disease to worsen. In addition, previous studies have also shown that dysbiosis of dynamic respiratory microbiota and changes in microbial composition may affect the development and course of chronic obstructive pulmonary disease. Current research indicates that in "healthy" (ie, not suffering from chronic obstructive pulmonary disease) smokers, the abundance of fecal microbiota ( Prevotella ) will increase, and it is thicker than non-smokers. The ratio of Firmicutes/Bacteroidetes will decrease; and the number of Bifidobacterium spp. in smokers will also decrease, so individuals may lose the anti-inflammatory effects associated with the genus. However, to date there have been no relevant studies on the changes in the gut microbiota of patients with chronic obstructive pulmonary disease.

One object of the present invention is to provide a use of Parabacteroides goldsteinii , its metabolites, or its bacterial components. By the role of Parabacteroides gordonii , the physiological function of the individual of the recipient is adjusted to achieve inhibition Or the purpose of treating chronic obstructive pulmonary disease.

To achieve the aforementioned object, the present invention provides a use of Parabacteroides goldsteinii , the metabolite, or the bacterial component for the preparation of a pharmaceutical composition for preventing and/or treating chronic pulmonary obstructive diseases, wherein the The deposit number of B. gordonii is DSM 32939. By administering the pharmaceutical composition to individuals with chronic obstructive pulmonary disease, improve weight loss, proinflammatory cytokine accumulation, airway smooth muscle hyperplasia, improve emphysema, improve pulmonary fibrosis, and improve deterioration due to chronic obstructive pulmonary disease Lung function, and/or mitochondrial dysfunction, to achieve the purpose of inhibiting or treating chronic obstructive pulmonary disease; and the B. gordonii by reducing IL-1 β gene, TNF -α gene in lung cells, COL3A1 gene, and/or PGC-1 α gene expression, to reduce lung cell inflammation, pulmonary fibrosis and/or excessive oxidative stress, and by simultaneously enhancing Cytb gene and NRF-1 gene in lung cells , RNR-1 gene, RNR-2 gene, SIRT1 gene, and/or TFAM gene expression to enhance the activity of mitochondria in lung cells.

In an embodiment of the present invention, the B. gordonii is a live bacterium; and the pharmaceutical composition may further include other bacteria than the B. gordonii.

In an embodiment of the present invention, the effective dose of the B. gordonii is that each individual is given at least 5×10 ⁹ CFUs/kg per day, and the administration period may be 12 weeks.

In the prevention or treatment of chronic obstructive pulmonary disease by using the B. gordonii according to the embodiment of the present invention, it can be orally administered. When it is administered, it can be eaten together with general foods. Therefore, when preparing a pharmaceutical composition containing B. gordonii, the pharmaceutical composition may further include protein, monosaccharide, disaccharide, oligosaccharide, oligosaccharide, polysaccharide, carbohydrate , Amino acids, lipids, vitamins or any combination thereof.

In addition, in the preparation of the pharmaceutical composition containing Parabacterium gordonii according to the embodiment of the present invention, excipients, carriers, adjuvants and/or food additives well known in the technical field may be further added. The dosage form may be, but not limited to, a spray gas, a solution, a semi-solid, a solid, a gelatin capsule, a soft capsule, a lozenge, a lozenge, a chewing gum, and/or a freeze-dried powder formulation. At the same time, the B. gordonii or the pharmaceutical composition containing other components in the embodiment of the present invention can be further used for preparing food, health food, dietary supplement or vaccine composition.

By the use of the B. gordonii of the present invention for preparing a pharmaceutical composition for preventing or treating chronic obstructive pulmonary disease, the B. gordonii can improve the weight loss and emphysema caused by chronic obstructive pulmonary disease in animals or humans Phenomenon, infiltration of inflammatory cells in lung tissue, thickening and hyperplasia of tracheal wall, pulmonary fibrosis, and abnormal lung function to reduce the accumulation of proinflammatory cytokines, airway smooth muscle hyperplasia, emphysema, etc. Inflammatory reaction and reduce the worsening lung function; the B. gordonii can also effectively reduce the expression of IL-1 β gene, TNF -α gene, COL3A1 gene, and PGC-1 α gene in lung tissue to reduce Inflammation of lung cells, pulmonary fibrosis and/or excessive oxidative stress, and at the same time can effectively improve the Cytb gene, NRF-1 gene, RNR-1 gene, RNR-2 gene, SIRT1 gene, and TFAM gene in lung tissue Performance level to enhance the activity of mitochondria in lung cells and repair mitochondrial dysfunction caused by chronic obstructive pulmonary disease. Therefore, the use of the characteristics of the B. gordonii of the present invention provides an innovative strategy for preventing or treating chronic obstructive pulmonary disease.

The embodiments of the present invention will be further described below. The examples listed below are used to clarify the present invention, and are not intended to limit the scope of the present invention. Anyone who is familiar with this art, without departing from the spirit and scope of the present invention, Some changes and retouching can be done, so the scope of protection of the present invention shall be deemed as defined by the scope of the attached patent application.

FIG. 1 is a bar graph of P. goldsteinii improving the weight loss of chronic obstructive pulmonary disease mice according to an embodiment of the present invention.

2 is a bar graph of P. goldsteinii ( P. goldsteinii ) improving the percentage of weight loss in mice with chronic obstructive pulmonary disease according to an embodiment of the present invention.

FIG. 3 is a histological image of P. goldsteinii improving emphysema in mice with chronic obstructive pulmonary disease according to an embodiment of the present invention.

4 is a histological image of P. goldsieinii of one embodiment of the present invention to improve the infiltration of inflammatory cells, thickening of the tracheal wall, and pulmonary fibrosis in the lung tissue of mice with chronic obstructive pulmonary disease. .

FIG. 5 is a bar graph of P. goldsteinii ( P. goldsteinii ) improving abnormal inspiratory volume in mice with chronic obstructive pulmonary disease according to an embodiment of the present invention.

6 is a bar graph of P. goldsteinii improving lung compliance in mice with chronic obstructive pulmonary disease according to an embodiment of the present invention.

7 is a bar graph of P. goldsteinii ( P. goldsteinii ) improving functional abnormality of residual lung volume in mice with chronic obstructive pulmonary disease according to an embodiment of the present invention.

8 is a bar graph of P. goldsteinii ( P. goldsteinii ) improving lung abnormalities in mice with chronic obstructive pulmonary disease according to an embodiment of the present invention.

FIG. 9 is a bar graph showing that P. goldsteinii improves the abnormal forced expiratory volume/forced vital capacity ratio at 100 milliseconds in mice with chronic obstructive pulmonary disease according to an embodiment of the present invention.

10 is a graph showing the results of reducing the expression of IL-1 β gene in the lung tissue of mice with chronic obstructive pulmonary disease by P. goldsteinii according to an embodiment of the present invention. * p value <0.05.

11 is a graph showing the results of reducing the expression level of TNF -α gene in the lung tissue of mice with chronic obstructive pulmonary disease by P. goldsteinii according to an embodiment of the present invention. * p value <0.05.

12 is a graph showing the results of reducing the expression of COL3A1 gene in the lung tissue of mice with chronic obstructive pulmonary disease by P. goldsteinii according to an embodiment of the present invention. ***p value <0.001.

FIG. 13 is a graph showing the results of increasing the expression level of Cytb gene in the lung tissue of mice with chronic obstructive pulmonary disease by P. goldsteinii according to an embodiment of the present invention. ***p value <0.001.

FIG. 14 is a graph showing the results of the enhancement of NRF-1 gene expression in lung tissue of mice with chronic obstructive pulmonary disease by P. goldsteinii according to an embodiment of the present invention. * p-value <0.05; ** p-value <0.01.

15 is a graph showing the results of reducing the expression level of PGC-1 α gene in the lung tissue of mice with chronic obstructive pulmonary disease by P. goldsteinii according to an embodiment of the present invention. * p-value <0.05; ** p-value <0.01.

FIG. 16 is a graph showing the results of increasing the expression level of RNR-1 gene in the lung tissue of mice with chronic obstructive pulmonary disease by P. goldsteinii according to an embodiment of the present invention. * p-value <0.05; ** p-value <0.01.

FIG. 17 is a graph showing the results of increasing the expression level of RNR-2 gene in the lung tissue of mice with chronic obstructive pulmonary disease by P. goldsteinii according to an embodiment of the present invention. * p-value <0.05; ** p-value <0.01.

18 is a graph showing the results of increasing the expression of SIRT1 gene in lung tissue of mice with chronic obstructive pulmonary disease by P. goldsteinii according to an embodiment of the present invention. * p-value <0.05; ** p-value <0.01.

FIG. 19 is a graph showing the results of enhancing the expression of TFAM gene in the lung tissue of mice with chronic obstructive pulmonary disease by P. goldsteinii according to an embodiment of the present invention. * p-value <0.05; ** p-value <0.01.

definition

"Effective dose" as used herein means that it can improve the weight loss and emphysema caused by chronic obstructive pulmonary disease in mammals or humans, the infiltration of inflammatory cells in the lung tissue, the thickening and hyperplasia of the tracheal wall, and pulmonary fibrosis Phenomenon, abnormal lung function, or can effectively reduce the expression of IL-1 β gene, TNF -α gene, COL3A1 gene, and PGC-1 α gene in lung tissue, as well as effectively enhance Cytb gene, NRF-1 gene, The number of RNR-1 gene, RNR-2 gene, SIRT1 gene, and TFAM gene required for the performance of the amount of Paracobacterium gordonii . The effective dose may vary depending on the biological species or individual being treated, but the effective dose can be experimentally determined by, for example, dose escalation.

The numerical values used in this article are approximate values, and all experimental data are expressed within a range of 20%, preferably within a range of 10%, and most preferably within a range of 5%.

According to the present invention, the operation procedures and parameter conditions related to bacterial culture fall within the professional literacy and routine technical scope of those skilled in the art.

As used herein, the term "metabolite" means the substance secreted into the bacterial culture fluid after the bacteria are metabolized when the bacteria is cultivated, including the culture fluid in which the bacteria is cultivated.

As used herein, the term "bacterial component" means a derivative substance directly or indirectly related to the bacterium when cultivating the bacterium, including but not limited to the metabolite of the bacterium, the structure of the bacterium, the bacterium-related active and inactive ingredients Wait.

The present invention provides a use of Paracobacterium gordonii for preparing a pharmaceutical composition for preventing or treating chronic obstructive pulmonary disease. The following experiments show that the B. gordonii of the present invention can improve the weight loss and emphysema, the infiltration of inflammatory cells in the lung tissue, the thickening and hyperplasia of the tracheal wall, and the lung fibers in mammals or humans due to chronic obstructive pulmonary disease. Phenomenon, abnormal lung function, or can effectively reduce the expression of IL-1 β gene, TNF -α gene, COL3A1 gene, and PGC-1 α gene in lung tissue, and effectively enhance Cytb gene, NRF-1 gene , RNR-1 gene, RNR-2 gene, SIRT1 gene, and TFAM gene performance. In general, the effective dose of Paramycobacterium gordonii according to the present invention is at least 5×10 ⁹ CFUs/kg administered daily to a mammal or a human, as detailed below.

Parabacterium gordonii

Ge's sub-embodiment of the present invention used in the embodiment of Bacteroides (Parabacteroides goldsteinii, P.goldsteinii) MTS01 capable of preventing or treating a chronic obstructive pulmonary disease probiotic strains (Probiotic bacteria). The Bacteroides gordonii is deposited at the German Microbial Strains Preservation Center (Deutsche Sammlung von Mikroorganismen und Zellkulturen, DSMZ); October 29, 107; No. DSM 32939. And the B. gordonii was also deposited in the Food Industry Development Institute; February 15, 108; No. BCRC 910869. B. gordonii is an absolute anaerobic bacterium, which needs to be cultured in an anaerobic incubator at 37℃ for about 48 hours. The liquid culture medium of the bacteria is NIH thioglycollate broth (TGC II) (purchased from BD, United States, number 225710), and the solid culture medium is Anaerobic blood agar plate (Ana.BAP) (purchased from Qixin Biotechnology Company, Taiwan) . The bacteria are stored in a refrigerator at -80 degrees for a long time. The protective solution is 25% glycerin. No special cooling treatment is required, and it can be stored by freeze-drying to stabilize its activity. In the examples of the present invention, it has been confirmed by animal experiments that feeding the live strain of Parabacterium gordonii of the present invention can improve the weight loss and emphysema caused by chronic obstructive pulmonary disease, and improve the inflammatory cells in the lung tissue caused by chronic obstructive pulmonary disease The phenomenon of infiltration, thickening and hyperplasia of the tracheal wall, and pulmonary fibrosis, and the improvement of abnormal lung function caused by chronic obstructive pulmonary disease can also effectively reduce the IL-1β gene, TNF-α gene, COL3A1 gene in lung tissue, And PGC-1α gene expression to reduce lung cell inflammation, pulmonary fibrosis and/or excessive oxidative stress, and can effectively enhance Cytb gene, NRF-1 gene, RNR-1 gene, RNR in lung tissue -2 gene, SIRT1 gene, and TFAM gene expression levels to enhance the activity of mitochondria in lung cells and repair mitochondrial dysfunction caused by chronic obstructive pulmonary disease. It shows the use of the B. gordonii of the present invention for preparing a pharmaceutical composition for preventing or treating chronic obstructive pulmonary disease.

Probiotic strains (Probiotic or Probiotic bacteria) are microorganisms whose bacteria, mixed strains, extracts or metabolites have a positive effect on the host's own system, usually derived from living bacteria in the human body that are beneficial to intestinal health. It can refer to some microorganisms that are supplemented from outside and may be beneficial to the body.

According to the present invention, the operation procedures and parameter conditions related to bacterial culture fall within the professional literacy and routine technical scope of those skilled in the art.

Experimental mice

In the examples of the present invention, C57BL/6 female mice 8 to 10 weeks old and free of specific pathogens were used. The mice were purchased from Jackson Laboratory (Bar Harbor, USA), and all the mice were fed food and water at will. The mice are housed in a facility free of specific pathogens and are treated according to the guidelines of the National Institutes of Health.

Example 1 The effect of B. gordonii to improve the weight loss of mice with chronic obstructive pulmonary disease

One embodiment of the present invention is to test the efficacy of the B. gordonii of the present invention to improve the weight loss of mice with chronic obstructive pulmonary disease. First, C57BL/6Narl female mice of 8-10 weeks old are divided into the following three groups (each Group n=10): (1) control group exposed to indoor air and given 100 μL of PBS once a day for 12 weeks by intragastric infusion; (2) exposed to 12 3R4F cigarettes twice a day (Kentucky) University) in the smoke and once a day by intragastric infusion of PBS for a total of 12 weeks, and (3) twice daily exposure to the smoke of 12 3R4F cigarettes (University of Kentucky) and once a day Intragastric administration of 1x10 ⁸ CFU/20g of P. goldsteinii (CS+MTS01) for a total of 12 weeks in the experimental group; of which, twice a day was exposed to the smoke of 12 3R4F cigarettes (University of Kentucky) for 12 weeks It is a model of chronic obstructive pulmonary disease induced by cigarette smoke (CS) in mice.

During the 12 weeks of feeding, the weight of each group of mice was monitored weekly, and the final weight of week 12 minus the initial weight of week 0 was used as the value of weight gain. The results are shown in Figure 1; The value of weight gain is divided by the initial body weight and expressed as a percentage to calculate the rate of change in weight of each mouse in each group. The results are shown in FIG. 2. The data of the experimental results are displayed as the mean ± standard deviation, and statistical analysis is performed by One-way ANOVA (*p value<0.05; **p value<0.01; ***p value<0.001) .

The experimental results of the present invention to improve the weight loss of CS-induced chronic obstructive pulmonary disease mice are shown in FIG. 1 and FIG. 2. It can be seen from the figure that compared with the control group mice exposed to indoor air, the weight gain value of the mice in the chronic obstructive pulmonary disease induced by CS will be significantly reduced, and the weight change rate will also be significantly reduced Reduced; and when CS-induced mice are chronic obstructive pulmonary disease, the paramycobacterium gordonii of the present invention is fed at the same time, compared with the mice in the comparison group, the value of weight gain can be significantly increased, and the rate of weight change It will also improve significantly. These results show that the B. gordonii of the present invention can effectively improve the problem of individual weight loss caused by chronic obstructive pulmonary disease.

Example 2 Paracobacterium gordonii improves pulmonary emphysema, tracheal wall remodeling hyperplasia, and pulmonary fibrosis in chronic obstructive pulmonary disease mice

One embodiment of the present invention is to observe the efficacy of the B. gordonii of the present invention in improving emphysema, tracheal wall remodeling hyperplasia and pulmonary fibrosis in mice with chronic obstructive pulmonary disease. First, the mice were divided into groups for experimentation as in the previous Example 1, and after 12 weeks of feeding, the lung tissues of the mice in each group were taken out, fixed with formalin solution, and embedded in paraffin, followed by a thickness of 4 mm Tissue sections were prepared and stained with hematoxylin (Hematoxylin) and eosin (Eosin, H&E), and then observed and recorded with a light microscope (Olympus, Japan). Stained sections of lung tissues of each group of mice, and using Image J software ( National Institutes of Health (USA) analyzes the histological images of each group, where the images are selected from 2 random regions of 10-15 slices in each group.

The experimental results of the present invention to improve the emphysema of chronic obstructive pulmonary disease mice are shown in FIG. 3. It can be seen from the figure that the alveolar wall of the mice in the control group induced by CS to be chronic obstructive pulmonary disease is thinner and damaged more than the mice in the positive control group exposed to indoor air; When the rat is chronic obstructive pulmonary disease, the B. gordonii of the present invention is fed at the same time, and the emphysema phenomenon is significantly reduced compared with the mice in the comparative group. This result shows that the B. gordonii of the present invention can effectively improve emphysema caused by chronic obstructive pulmonary disease.

The experimental results of the present invention to improve the airway wall remodeling hyperplasia and pulmonary fibrosis of Paracetobacter gordonii in mice with chronic obstructive pulmonary disease are shown in FIG. 4. It can be seen from the figure that compared with the positive control group mice exposed to indoor air, the infiltration of inflammatory cells, thickening and hyperplasia of the tracheal wall in the lung tissue of the comparative group mice induced by CS to chronic obstructive pulmonary disease, And the situation of pulmonary fibrosis is more serious; and if the CS-induced mice are chronic obstructive pulmonary disease, and the paramycobacterium gordonii of the present invention is fed at the same time, the inflammation and the tracheal wall become thicker than the mice in the comparative group Hyperplasia and pulmonary fibrosis were significantly reduced. This result shows that the B. gordonii of the present invention can effectively improve the infiltration of inflammatory cells, thickening and hyperplasia of the tracheal wall, and pulmonary fibrosis in the lung tissue caused by chronic obstructive pulmonary disease.

Example 3 Efficacy of B. gordonii to improve lung function in mice with chronic obstructive pulmonary disease

One embodiment of the present invention is to observe the efficacy of the B. gordonii of the present invention in improving lung function in mice with chronic obstructive pulmonary disease. First, the mice were grouped for experiments as in the previous Example 1, and after 12 weeks of feeding, all mice were anesthetized to perform tracheostomy, and the mice were placed in a forced lung action system (Buxco Research Systems, USA , Hereinafter referred to as Buxco system). First, apply an average breathing rate of 100 breaths/min to the anesthetized mice, and use the Buxco system to perform three semi-automatic maneuvers, including wave-ear law to measure functional residual capacity (FRC), quasi-static PV (Quasistatic PV), and Fast flow volume maneuver; where the FRC is determined by Boyle’s law.

The operation of quasi-static PV is to measure total lung capacity (TLC), residual volume (Residual volume, RV), inspiratory capacity (IC), vital capacity (Vital capacity, VC), and expiratory reserve Expiratory reserve volume (ERV), and lung compliance (Chord compliance, Cchord).

The fast flow-driven operation is to record forced expiratory flows (FEFs), average expiratory time (Times of expiration, Te) and average inspiratory time (Times of inspiration, Ti), and forced expiratory volume (Forced expiratory volume, FEV); wherein, the FEFs include forced expiratory flow rate (FEF) and peak expiratory flow rate (PEF); and the FEVs include forced vital capacity (Forced vital capacity) , FVC), forced expiratory volume at 100 milliseconds (FEV100), and forced expiratory volume at 200 milliseconds (FEV200).

All the above operations and driving are continued until the three correct measurements are completed, and the three averages of the above various parameters measured from each mouse in each group are used as the result value of the parameter of the mouse . Next, the result values are displayed as mean±standard deviation, and statistical analysis is performed by one-way ANOVA (One-way ANOVA) and Dunnett multiple comparison test (GraphPad prism 6.01; GraphPad Software Inc., United States) (* p value<0.05; **p value<0.01; ***p value<0.001).

The experimental results of the present invention to improve the abnormal phenomenon of inspiratory volume in mice with chronic obstructive pulmonary disease are shown in Fig. 5; the improvement of abnormal lung compliance in chronic obstructive pulmonary disease mice The experimental results are shown in Figure 6; the experimental results for improving the abnormal phenomenon of functional residual lung volume in mice with chronic obstructive pulmonary disease are shown in Figure 7; the experimental results for improving the abnormal phenomenon of total lung volume in mice with chronic obstructive pulmonary disease are Fig. 8; the experimental result of improving the abnormal phenomenon of forced expiratory volume/forced vital capacity ratio at 100 milliseconds in the lungs of mice with chronic obstructive pulmonary disease is shown in FIG. 9.

As can be seen from FIGS. 7 to 9, compared to the control group mice exposed to indoor air, the inspiratory volume, lung compliance, and functional lung residual of the control group mice induced by CS into chronic obstructive pulmonary disease The volume and total lung volume will increase significantly, and the ratio of forced expiratory volume/forced vital capacity at 100 milliseconds of the lung will decrease significantly; and if the CS-induced mice are chronic obstructive pulmonary disease, the Compared with the mice in the control group, the B. gordonii has a significant reduction in inspiratory volume, lung compliance, functional residual lung volume, and total lung volume, and its lungs are forced to exhale at 100 ms The volume/forced vital capacity ratio will increase significantly. This result shows that the B. gordonii of the present invention can effectively improve the inspiratory volume, lung compliance, functional residual lung volume, total lung volume, and forced expiratory volume of the lung at 100 milliseconds caused by chronic obstructive pulmonary disease /The abnormal ratio of forced vital capacity can effectively improve the lung function of chronic obstructive pulmonary disease.

Example 4 Efficacy of B. gordonii in regulating the expression of genes related to lung fibrosis and mitochondrial activity in the lungs of mice with chronic obstructive pulmonary disease

One embodiment of the present invention is to observe the efficacy of the B. gordonii of the present invention in regulating the gene expression level of lung fibrosis and mitochondrial activity in the lungs of mice with chronic obstructive pulmonary disease. First, the mice were divided into groups for experimentation as in the previous Example 1, and after 12 weeks of feeding, RNeasy ^® MiniKit (Qiagen, Valencia, CA, USA) was used to extract total ribonucleic acid (Total) from the lung tissues of each group of mice. RNA), then use the Quant II rapid reverse transcriptase reagent kit (Tools, Taiwan), using the extracted Total RNA as a template and the primers of Table 1 for reverse transcription to generate cDNA products corresponding to the mRNA of these specific genes, Then mix 1μL of the obtained cDNA with 1μL of the specific gene primer, 5μL of 2xqPCRBIO SyGreen Blue Mix Lo-ROX (PCR Biosystems, UK), and 3μL of double-distilled water. real-time polymerase chain reaction (qPCR), the PCR conditions are after the initial step of pre-incubation at 95°C for 3 minutes, reaction at 95°C for 10 seconds, 60°C for 20 seconds, and 72°C for 5 seconds, a total of 50 cycles are repeated After the circle, another melting curve cycle was performed to detect the expression level of genes related to pulmonary fibrosis and mitochondrial activity. Among them, 18S ribosome (18S ribosome) was used as an internal control for qPCR measurement.

The present invention's Ge sub Bacteroides decreased results of IL-1 β expression levels of the gene of COPD mice shown in Figure 10; reducing TNF results chronic obstructive pulmonary disease manifestations of mice amount -α genes such as Figure 11; the experimental results of reducing the COL3A1 gene expression of mice with chronic obstructive pulmonary disease are shown in Figure 12; the experimental results of increasing the Cytb gene expression of mice with chronic obstructive pulmonary disease are shown in Figure 13; improving chronic obstruction The experimental results of NRF-1 gene expression in mice with sexual lung disease are shown in Figure 14; the experimental results of reducing the PGC-1 α gene expression in mice with chronic obstructive pulmonary disease are shown in Figure 15; The experimental results of RNR-1 gene expression in mice are shown in Figure 16; the experimental results of increasing RNR-2 gene expression in mice with chronic obstructive pulmonary disease are shown in Figure 17; the SIRT1 gene is increased in mice with chronic obstructive pulmonary disease The experimental results of performance are shown in Figure 18; the experimental results of improving the expression of TFAM gene in mice with chronic obstructive pulmonary disease are shown in Figure 19.

Among them, IL-1 β gene and TNF -α gene are important genes that promote inflammation; COL3A1 gene is an important gene that causes lung fibrosis; Cytb gene is an important gene that affects mitochondrial function; NRF-1 gene, PGC- 1 α gene, RNR1 gene, RNR2 gene, SIRT1 gene, and TFAM gene are important genes that affect mitochondrial function and anti-oxidative stress, in which the performance of PGC-1 α gene in mice with chronic obstructive pulmonary disease is known The volume will rise.

As can be seen from FIGS. 10 to 19, compared to the non-smoking control group mice exposed to indoor air, the IL-1 β gene in the lung tissue cells of the comparative group mice induced by CS to chronic obstructive pulmonary disease, The expression levels of TNF -α gene, COL3A1 gene, and PGC-1 α gene will be significantly improved; while the expression levels of Cytb gene, NRF-1 gene, RNR-1 gene, RNR-2 gene, SIRT1 gene, and TFAM gene Will be significantly reduced. If the CS-induced mice are chronic obstructive pulmonary disease, the paramycobacterium gordonii of the present invention is fed at the same time. Compared with the mice in the comparative group, the IL-1 β gene, TNF -α gene in the lung tissue cells, The expression levels of COL3A1 gene and PGC-1 α gene will be significantly reduced; while the expression levels of Cytb gene, NRF-1 gene, RNR-1 gene, RNR-2 gene, SIRT1 gene, and TFAM gene will be significantly increased . This result shows that the B. gordonii of the present invention can effectively reduce the expression levels of IL-1 β gene, TNF -α gene, COL3A1 gene, and PGC-1 α gene related to pulmonary fibrosis, as well as effectively improve The expression levels of Cytb gene, NRF-1 gene, RNR-1 gene, RNR-2 gene, SIRT1 gene, and TFAM gene related to the body activity.

In the prevention or treatment of chronic obstructive pulmonary disease, the B. gordonii according to the embodiment of the present invention can be administered orally. When it is administered, it can be eaten together with general food. Therefore, when preparing a pharmaceutical composition containing Parabacterium gordonii, the pharmaceutical composition may further include protein, monosaccharide, disaccharide, oligosaccharide, polysaccharide, carbohydrate, amino acid , Lipid, vitamin, or any combination thereof.

In addition, in the preparation of the pharmaceutical composition containing the B. gordonii according to the embodiment of the present invention, a carrier or other adjuvants well known in the art may be further added. The dosage form may be, but not limited to, a solution, a gelatin capsule, a soft capsule, or a lozenge, so as to facilitate the transmission of the B. gordonii of the present invention to the intestinal tract and/or to colonize the intestinal tract partially or completely. At the same time, the B. gordonii or the pharmaceutical composition containing other ingredients in the embodiments of the present invention can also be added to foods, health foods or dietary supplements.

It can be known from the above test that the embodiment of the present invention provides a use of Paracobacterium gordonii for the preparation of a pharmaceutical composition for the prevention or treatment of chronic obstructive pulmonary disease. The effect of weight loss and emphysema caused by lung disease; it also has the effect of improving the infiltration of inflammatory cells caused by chronic obstructive pulmonary disease, thickening of the tracheal wall, and pulmonary fibrosis in animal or human lung tissues , Can reduce proinflammatory cytokine accumulation, airway smooth muscle hyperplasia, emphysema and other lung inflammation; and can improve the inspiratory volume, lung compliance, and functional residual lung volume of animals or humans caused by chronic obstructive pulmonary disease , The total lung volume, and the abnormal ratio of forced expiratory volume/forced vital capacity in the 100 milliseconds of the lung to reduce the deterioration of lung function; the B. gordonii can also effectively reduce the IL-1 β gene and TNF in the lung tissue -Alpha gene, COL3A1 gene, and PGC-1 alpha gene expression, to reduce lung cell inflammation, pulmonary fibrosis and/or excessive oxidative stress, and also effectively enhance Cytb gene, NRF-1 in lung tissue Gene, RNR-1 gene, RNR-2 gene, SIRT1 gene, and TFAM gene expression levels to enhance the activity of mitochondria in lung cells and repair mitochondrial dysfunction caused by chronic obstructive pulmonary disease. Therefore, the B. gordonii of the present invention can be applied to the prevention or treatment of chronic obstructive pulmonary disease and the preparation of related pharmaceutical compositions.

【Biological Material Storage】

German Microbial Culture Preservation Center (Deutsche Sammlung von Mikroorganismen und Zellkulturen, DSMZ); October 29, 107; No. DSM 32939. Food Industry Development Research Institute; February 15, 108; No. BCRC 910869.

<110> Xingjufan Biotechnology Co., Ltd.

<120> The use of B. gordonii for the prevention or treatment of chronic obstructive pulmonary disease

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<212> DNA

<213> Artificial sequence

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<223> Synthetic primer

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<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic primer

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<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic primer

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<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic primer

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<210> 11

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<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic primer

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<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic primer

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<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic primer

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<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic primer

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<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic primer

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<212> DNA

<213> Artificial sequence

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<223> Synthetic primer

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<212> DNA

<213> Artificial sequence

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<223> Synthetic primer

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<212> DNA

<213> Artificial sequence

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<223> Synthetic primer

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<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic primer

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<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic primer

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<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic primer

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<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic primer

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<212> DNA

<213> Artificial sequence

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<223> Synthetic primer

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<400> 24

Claims (11)

A use of Parabacteroides goldsteinii and its metabolites, which is used for the preparation of a pharmaceutical composition for the prevention and/or treatment of chronic pulmonary obstructive diseases, wherein the deposit number of Parabacteroides gordonii is DSM 32939. The use as described in item 1 of the patent application scope, wherein the Bacteroides gordonii is a live bacterium. The use as described in item 1 of the patent application scope, wherein the effective dose of the B. gordonii is that each individual is given at least 5×10 ⁹ CFUs/kg per day. The use as described in item 1 of the patent application scope, wherein the B. gordonii and its metabolites improve weight loss, proinflammatory cytokine accumulation, airway smooth muscle hyperplasia, emphysema, caused by chronic pulmonary obstructive disease Pulmonary fibrosis, worsening lung function, and/or mitochondrial dysfunction. The use as described in item 1 of the scope of the patent application, wherein the pharmaceutical composition may further include other bacteria than the B. gordonii. The use as described in item 1 of the patent application scope, wherein the B. gordonii and its metabolites reduce inflammation of lung cells, pulmonary fibrosis and/or excessively high oxidative stress. The use as described in item 1 of the patent application scope, wherein the B. gordonii and its metabolites reduce the expression of IL-1β gene, TNF-α gene, COL3A1 gene, and/or PGC-1α gene in lung cells the amount. The use as described in item 1 of the patent application scope, wherein the B. gordonii and its metabolites increase the activity of mitochondria in lung cells. The use as described in item 1 of the patent application scope, in which the B. gordonii and its metabolites enhance the Cytb gene, NRF-1 gene, RNR-1 gene, RNR-2 gene, SIRT1 gene in lung cells, and /Or TFAM gene expression. The use as described in item 1 of the patent application scope, wherein the pharmaceutical composition further includes a pharmaceutically acceptable excipient, carrier, adjuvant and/or food additive. The use as described in item 1 of the patent application scope, wherein the dosage form of the pharmaceutical composition is a spray gas, a solution, a semi-solid, a solid, a gelatin capsule, a soft capsule, a lozenge, a lozenge, Chewing gum and/or a freeze-dried powder preparation.

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