CN102100704B - Application of probiotic strain GM-080 in preparing composition for treating carditis and heart apoptosis - Google Patents
Application of probiotic strain GM-080 in preparing composition for treating carditis and heart apoptosis Download PDFInfo
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Abstract
The invention relates to application of a probiotic strain GM-080 in preparing a compound for treating carditis and heart apoptosis. A composition for treating carditis and heart apoptosis is prepared from lactobacillus paracasei GM-080 in an effective content with the collection number of China Center for Type Culture Collection (CCTCC) M204012.
Description
Technical field
The present invention relates to a kind of purposes of probiotic strain, particularly relate to the application of a kind of probiotic strain GM-080 in the composition of preparation treatment heart inflammation and heart cell apoptosis.
Background technology
Probiotic bacterium (probiotics or probiotic bacteria) refers generally to stem from the viable bacteria that human body is interior, be of value to intestinal health, also can refer to external additional, possible useful to health certain micro-organisms, for example milk-acid bacteria (lacticacid bacteria; LAB) and the part yeast, wherein milk-acid bacteria for lactose or other carbohydrate being converted to the general designation of the microorganism of lactic acid.The genus lactubacillus gram-positive microorganism, the use of Chang Zuowei foodstuffs industry fermentation.
Recent studies have shown that, milk-acid bacteria can be improved irritated relative disease and gastrointestinal upset, for example inflammatory enteropathy (inflammatory bowel disease; IBD) etc.generally speaking, milk-acid bacteria is with lactobacillus genus (Lactobacillus), Leuconostoc (Leuconostoc), Pediococcus (Pediococcus), lactic acid Coccus (Lactococcus) and streptococcus (Streptococcus) etc. are main, Aerococcus (Aerococcus) is separately arranged, meat Bacillaceae (Carnobacterium), enterococcus spp (Enterococcus), wine Coccus (Oenococcus), spore milk-acid bacteria (Sporolactobacillus) is arranged, limbs Coccus (Teragenococcus), hover Coccus (Vagococcus) and Wei Si Bordetella (Weisella) etc., wherein most milk-acid bacteria is under the jurisdiction of lactobacillus genus.Above-mentioned Pseudomonas is under the jurisdiction of lactobacillus order (Lactobacillales), and wherein the part bacterial classification can be used as probiotic bacterium (probiotics).For lactobacillus genus and the genus bifidobacterium (Bifidobacterium) of probiotic bacterium, its research is comparatively thorough at present.
recently studies show that about EPDML, but milk-acid bacteria is immune response stimulating more, with development, the immunotolerance (tolerance) of harmless allergen (innocent allergens) (is consulted Penders, J., Stobberingh, E.E., van den Brandt, P.A., Thijs, C.The role of the intestinalmicrobiota in the development of atopic disorders.European Journal of Allergyand Clinical Immunology 62 (11), 1223-1236. (2007)).
other research also shows, milk-acid bacteria can effectively improve the relevant diarrhoea (antibiotic-associated diarrhea) of people and animals' microbiotic and traveler's diarrhea (travellers ' diarrhea), infantile diarrhea (pediatric diarrhea), inflammatory enteropathy (IBD), the hot-tempered disease of intestines (irritable bowel syndrome) (and more than consult Furrie, E.Probiotics and allergy.Proceedings of the Nutrition Society 64 (4), 465 – 469,2005, Goossens, D., Jonkers, D., Stobberingh, E., van den Bogaard, A., Russel, M., Stockbrugger, R.Probiotics in gastroentrology:indication and futureperspectives.Scandinavian Journal of Gastroenterology 239 (Suppl.), 15 – 23,2003, Kalliomaki, M., Salminen, S., Poussa, T., Arvilommi, H., Isolauri, E.Probiotics andprevention of atopic disease:4-year follow-up of a randomised placebo-controlledtrial.Lancet 361,1869 – 1871,2003, Pfruender, H., Amidjojo, M., Hang, F., Weuster-Botz, D.Production of Lactobacillus kefir cells for asymmetric synthesisof a 3,5-dihydroxycarboxylate.Applied Microbiology and Biotechnology 67,619 – 622,2005, Shanahan, F.Probiotics and inflammatory bowel disease:is there ascientific rationale Inflammatory Bowel Disease 6 (2), 107 – 115, 2000), atopic disease (atopic disease) (more than consult Penders, J., Stobberingh, E.E., van den Brandt, P.A., Thijs, C.The role of the intestinal microbiota in the development of atopicdisorders.European Journal of Allergy Clinical Immunology 62 (11), 1223 – 1236, 2007, Lee, J., Seto, D., Bielory, L.Meta-analysis of clinical trials of probiotics forprevention and treatment of pediatric atopic dermatitis.Journal of Allergy andClinical Immunology 123 (1), 266 – 267,2009) etc.
Utilize the correlative study of lactic acid bacteria for treatment cardiovascular disorder over 50 years.Studies show that of past, milk-acid bacteria can effectively reduce blood pressure with high-cholesterol disease (hypercholesterolemia) (more than consult Pfruenderet al., 2005; Lye, H.S., Kuan, C.Y., Ewe, J.A., Fung, W.Y., Liong, M.T.Theimprovement of hypertension by probiotics:Effects on cholesterol, diabetes, renin, and phytoestrogens.International Journal of Molecular Sciences 10,3755-3775,2009).
Yet above-mentioned research is seldom inquired into probiotic strain and whether be can be applicable to heart inflammation and the heart cell apoptosis that allergy causes, does not also propose the regulatory mechanism that probiotic strain may relate to.
In view of this, needing badly provides a kind of probiotic strain in the new purposes that treatment heart inflammation and heart cell apoptosis are provided, with other application surface of exploitation probiotic strain.
Summary of the invention
One aspect of the present invention is to provide the application of a kind of probiotic strain GM-080 in the composition of preparation treatment heart inflammation and heart cell apoptosis, and wherein the content of this probiotic strain GM-080 in said composition can effectively be treated heart inflammation and heart cell apoptosis.
According to above-mentioned aspect of the present invention, propose the application of a kind of probiotic strain GM-080 in the composition of preparation treatment heart inflammation and heart cell apoptosis, and the content of this probiotic strain GM-080 in this composition can effectively be treated heart inflammation and heart cell apoptosis.In one embodiment, this probiotic strain GM-080 for example can be lactobacillus paraceasi (Lactobacillus paracasei) GM-080 (be preserved in Chinese Typical Representative culture collection center, preserving number is CCTCC M 204012).
According to one embodiment of the invention, above-mentioned probiotic strain GM-080 for live or do not activate.
According to one embodiment of the invention, above-mentioned composition is that medical composition, diet supplement, food or its composition divide.
The application of probiotic strain GM-080 of the present invention in the composition of preparation treatment heart inflammation and heart cell apoptosis, can utilize above-mentioned probiotic strain GM-080, see through the expression that specificity suppresses p-JNK, Bad and Bax, and then effectively treat heart inflammation and heart cell apoptosis, thereby other application surface of exploitation probiotic strain.
Brief Description Of Drawings
For above and other objects of the present invention, feature, advantage and embodiment can be become apparent, being described in detail as follows of accompanying drawing:
Fig. 1 illustrates the schematic diagram of the signal transduction path of probiotic strain GM-080 participation treatment heart inflammation according to an embodiment of the invention and heart cell apoptosis.
Fig. 2 shows the Western blotting analysis chart of mouse heart tissue according to an embodiment of the invention.
Fig. 3 illustrates the histogram of the p-JNK expression amount of the expression amount stdn Fig. 2 that utilizes alpha-tubulin.
Fig. 4 illustrates the histogram of JNK 1/2 expression amount of the expression amount stdn Fig. 2 that utilizes alpha-tubulin.
Fig. 5 shows the Western blotting analysis chart of mouse heart tissue according to an embodiment of the invention.
Fig. 6 illustrates the histogram of the p-NF κ B expression amount of the expression amount stdn Fig. 5 that utilizes alpha-tubulin.
Fig. 7 illustrates the histogram of the p-I κ B expression amount of the expression amount stdn Fig. 5 that utilizes alpha-tubulin.
Fig. 8 shows the Western blotting analysis chart of mouse heart tissue according to an embodiment of the invention.
Fig. 9 illustrates the histogram of the Bad expression amount of the expression amount stdn Fig. 8 that utilizes alpha-tubulin.
Figure 10 illustrates the histogram of the Bax expression amount of the expression amount stdn Fig. 8 that utilizes alpha-tubulin.
Figure 11 shows the Western blotting analysis chart of mouse heart tissue according to an embodiment of the invention.
Figure 12 illustrates the histogram of the cytoplasmic expression of cytochrome C amount of the expression amount stdn Figure 11 that utilizes alpha-tubulin.
Figure 13 illustrates the histogram of apoptotic proteins enzyme 3 expression amounts of the activated state of the expression amount stdn Figure 11 that utilizes alpha-tubulin.
Embodiment
Brought forward is described, the invention provides a kind of probiotic strain GM-080 in the purposes for the treatment of heart inflammation and heart cell apoptosis, it utilizes probiotic strain GM-080 to make a composition, and the content of this probiotic strain GM-080 can effectively be treated heart inflammation and heart cell apoptosis.
The present invention alleged " probiotic strain GM-080 " herein referred to lactobacillus paraceasi (Lactobacillus paracasei) GM-080, and this bacterial strain GM-080 has been preserved in Chinese Typical Representative culture collection center (China Center for Type Culture Collection on February 19th, 2004; CCTCC; Hubei China province wuchang, wuhan Luo Jiashan), preserving number is CCTCC M204012.Aforementioned probiotic strain GM-080 can utilize usual manner, or utilizes the disclosed separation of China Patent Publication No. CN1696281A and cultural method to make, and does not separately give unnecessary details at this.In brief, aforementioned probiotic strain GM-080 can utilize MRS meat soup (broth medium;
Add penbritin 100 μ g/mL, final pH 6.5 ± 0.2), at the temperature of 37 ℃ with anaerobism or aerobic cultivation.In another illustration, can be with the aforementioned bacterium liquid streak inoculation on agar plate (streak plating) that utilizes MRS meat soup.
In one embodiment, probiotic strain GM-080 of the present invention is through (invivo) animal immune experiment confirm in vivo, but specificity suppresses the genetic expression of p-JNK, Bad and Bax and treatment heart inflammation and heart cell apoptosis really.Heart inflammation and heart cell apoptosis may cause myocarditis (myocarditis) and cardiomyopathy (cardiomyopathies), such as allergic myocarditis (hypersensitivity myocarditis), rheumatic heart disease (or claiming valvular heart disease), hypertrophic cardiomyopathy (hypertrophic cardiomyopathy), dilated cardiomyopathy disease (dilated cardiomyopathy), restricted type cardiomyopathy (restrictive cardiomyopathy) etc.Furthermore, the content of this probiotic strain GM-080 can effectively be treated myocardial inflammation and the apoptosis of cardiac muscle that for example brings out because of anaphylaxis.It should be noted that at this, the present invention's alleged " animal immune experiment " herein refers to utilize " anaphylactogen sensitized animal ", that is utilizes anaphylactogen, for example Protalbinic acid (ovalbumin; OVA), the people is the anaphylaxis of bringing out laboratory animal, with the effect of assessment probiotic strain GM-080 for myocardial inflammation and apoptosis of cardiac muscle.
Consult Fig. 1, it illustrates the schematic diagram of the signal transduction path of probiotic strain GM-080 participation treatment heart inflammation according to an embodiment of the invention and heart cell apoptosis.In one embodiment, as shown in the signal transduction path 103 on Fig. 1 right side, probiotic strain GM-080101 can suppress p-c-Jun N terminal kinase (phosphorylated c-Jun N-terminal kinase by specificity; P-JNK), and the expression of inhibition downstream p-NF κ B and TNF-α, thereby cardiac inflammatory (cardiac inflammation) slowed down, the myocardial inflammation that for example brings out because of anaphylaxis (myocardial inflammation).
In another embodiment, above-mentioned probiotic strain GM-080 also can suppress the expression of Bad and Bax by specificity, prevents that effectively the Bcl-2 associated death from promoting the factor (Bcl-2-associated death promoter; Bad) X protein relevant to Bcl-2 (Bcl-2-associated X protein; Bax) be piled up in a surface of line body 111, with the expression with apoptotic proteins enzyme 3 (caspase 3) of the cytochrome C (cytochrome C) that suppresses the downstream, thereby the apoptosis of being controlled by grain line body 111 in the inhibition heart cell (or claiming programmed cell death), the apoptosis of cardiac muscle that for example brings out because of anaphylaxis (myocardial apoptosis) is as shown in the signal transduction path 105 in Fig. 1 left side.
In another embodiment, more alternative other hybrid bacterial strain that comprises of above-mentioned probiotic strain GM-080 is with the composition for the manufacture for the treatment of heart inflammation and heart cell apoptosis.in an illustration, aforesaid other hybrid bacterial strain can include but not limited to lactobacillus acidophilus (Lactobacillus acidophilus), lactobacillus plantarum (Lactobacillus plantarum), bifidus longum bb (Bifidobacterium longum), fermentation lactobacillus (Lactobacillus fermentum), bulgaricus ccm (Lactobacillus bulgaricus), thermophilus streptococcus (Streptococcus thermophilus), cheese Bacterium lacticum (Lactobacillus cremors), the secondary cheese subspecies (Lactobacillus paracasei subsp.paracasei) of secondary lactobacillus johnsonii, rhamnose lactic acid bacteria (Lactobacillus rhamnosus GG) or above-mentioned arbitrary combination.
What this replenished be, in one embodiment, above-mentioned probiotic strain GM-080 (lactobacillus paraceasi GM-080 for example; Preserving number is CCTCC M 204012) during for the manufacture of the composition for the treatment of heart inflammation and heart cell apoptosis, can be (live) alive or (inactive) that does not activate.In an illustration, above-mentioned probiotic strain GM-080 can be medical composition, diet supplement, food or its composition and divides.In another illustration, above-mentioned probiotic strain GM-080 can be cryodesiccated form, and this probiotic strain GM-080 more can comprise other composition, for example glucose, maltodextrin, infant formula, Nutriflora P (fructo-oligosaccharides), Magnesium Stearate, cheese spices (yogurt spices), other composition that is difficult to separate or its above-mentioned arbitrary combination.
Below utilize embodiment so that application of the present invention to be described, so it is not to limit the present invention, and the person skilled in the art of the present invention can do various modifications and modification without departing from the spirit and scope of the present invention.
Embodiment one: the foundation of animal evaluation profile
1. the preparation of probiotic strain GM-080
This embodiment utilizes lactobacillus paraceasi GM-080 (preserving number is CCTCC M 204012), carries out the animal immune experiment, with the effect of the GM-080 treatment heart inflammation of assessment probiotic strain and heart cell apoptosis.The bacterium amount of lactobacillus paraceasi GM-080 (CCTCC M 204012) can be every kilogram approximately 1 * 106 to about 1 * 1011 colony-forming unit (CFU) (CFU/g).This lactobacillus paraceasi GM-080 (CCTCC M204012) can be cryodesiccated form, and can comprise other composition, for example glucose, maltodextrin, infant formula, Nutriflora P, Magnesium Stearate, cheese spices, other composition that is difficult to separate or its above-mentioned arbitrary combination.
this embodiment also can select other hybrid bacterial strain collocation lactobacillus paraceasi, aforesaid other hybrid bacterial strain can include but not limited to lactobacillus acidophilus (Lactobacillus acidophilus), lactobacillus plantarum (Lactobacillus plantarum), bifidus longum bb (Bifidobacterium longum), fermentation lactobacillus (Lactobacillus fermentum), bulgaricus ccm (Lactobacillus bulgaricus), thermophilus streptococcus (Streptococcus thermophilus), cheese Bacterium lacticum (Lactobacillus cremors), the secondary cheese subspecies (Lactobacillus paracasei subsp.paracasei) of secondary lactobacillus johnsonii, rhamnose lactic acid bacteria (Lactobacillus rhamnosus GG) or above-mentioned arbitrary combination.Also can use the commercially available product that contains above-mentioned hybrid bacterial strain to carry out.
in an illustration, aforesaid other hybrid bacterial strain can comprise the first hybrid bacterial strain, wherein the first hybrid bacterial strain can include but not limited to for example lactobacillus acidophilus (Lactobacillus acidophilus), lactobacillus plantarum (Lactobacillus plantarum), bifidus longum bb (Bifidobacterium longum), fermentation lactobacillus (Lactobacillus fermentum), bulgaricus ccm (Lactobacillus bulgaricus), thermophilus streptococcus (Streptococcus thermophilus), cheese Bacterium lacticum (Lactobacillus cremors) or above-mentioned arbitrary combination.The bacterium amount of the first hybrid bacterial strain can be more than or equal to 1 * 107CFU/g.
In another illustration, aforesaid other hybrid bacterial strain also can comprise the second hybrid bacterial strain, and wherein the second hybrid bacterial strain can include but not limited to that for example aforesaid the first hybrid bacterial strain is added the secondary cheese subspecies (Lactobacillus paracasei subsp.paracasei) of secondary lactobacillus johnsonii, rhamnose lactic acid bacteria (Lactobacillusrhamnosus GG) or above-mentioned arbitrary combination.The bacterium amount of the second hybrid bacterial strain can be more than or equal to 1 * 107 CFU/g.
2. the foundation of anaphylactogen sensitized animal test model
This embodiment sets up anaphylactogen sensitized animal test model with BALB/c mouse (Taipei Le Sike biotech inc).At first, experiment mice is divided into 3 groups of test group (hypersensitive test group) and 2 groups of control groups (normal healthy controls group, irritated control group), and wherein every group is 5 all 7 of male mices in age (normal healthy controls group) or 8 (irritated control group, hypersensitive test group).The mouse of normal healthy controls group and irritated control group utilizes mouthful stomach tube to fill with the mode of (orogastric intubation), throws 0.2mL distilled water once every day.The mouse oral different probiotic strains (being respectively the first hybrid bacterial strain, the second hybrid bacterial strain, lactobacillus paraceasi GM-080 (CCTCC M 204012)) once every day of each hypersensitive test group, every feeding are approximately 1 * 106 to 1 * 1011CFU/g.The mouse of irritated control group and hypersensitive test group is in the 0th day and the 14th day, respectively at the Protalbinic acid (ovalbumin of intraperitoneal injection 2 micrograms (μ g) with 6 micrograms (μ g); OVA) with complete Freund's adjuvant (complete Freund ' s adjuvant; CFA), to bring out the anaphylaxis of mouse.All mouse after weighing, sacrifice, are taken out heart and also clean with distilled water after processing 28 days, and separately left atrium and left and right ventricles carry out weighing again.
The raising temperature of mouse is for being 65 ± 5% in 25 ± 1 ℃, relative humidity and being maintained at the light that switched in 12 hours and secretly circulating, with the feed (MF-18 of standard laboratory grade; East yeast industry Co., Ltd., Oriental Yeast Co.Ltd, Japan) feeding, the not supply of limiting feed and water between feeding period.The raising condition of mouse all follows the related experiment the care of animal guide of NIH's bulletin to carry out.
3. the extraction of heart tissue
Aforementioned mouse left ventricle is placed in cracking buffered soln, with the ratio of 100mg tissue/1mL cracking buffered soln, carried out approximately 1 minute, with (homogenize) left ventricular tissues that homogenizes, and then cracking goes out the protein in the myocardial cell.Aforesaid cracking buffered soln can comprise Tutofusin tris (Tris) solution of 20mM, the ethylenediamine tetraacetic acid (EDTA) (EDTA) of 2mM, the 2 mercapto ethanol of 50mM, 10% glycerine, proteinase inhibitor (Roche), inhibitors of phosphatases mixed solution (phosphatase inhibitorcocktail; Sigma), pH 7.4.Afterwards, the equal chylema (homogenate) of gained was placed on ice approximately 10 minutes, then with centrifugal 40 minutes of the centrifugal force of about 12000 * g, centrifugal twice altogether.Then, get supernatant liquor and deposit in-80 ℃, with in the follow-up dependent evaluation of carrying out.
Embodiment two: assessment probiotic strain GM-080 is for the effect for the treatment of heart inflammation
This embodiment utilizes electrophoretic analysis and Western blotting assessment probiotic strain GM-080 for the effect for the treatment of heart inflammation and heart cell apoptosis.Following division they.
With the equal chylema of above-mentioned gained, utilize 10% SDS-PAGE electrophoretic analysis, carried out 3.5 hours with 85 volts.Afterwards, in Tris-HCl solution (pH 8.3) balance of the 25mM that contains 192mM glycine and 20% (v/v) methyl alcohol 15 minutes.The preparation of above-mentioned SDS-PAGE and relevant device thereof should be in the technical field of the invention any those of ordinary skill to be known, and does not separately give unnecessary details at this.
The running gel of aforementioned SDS-PAGE can then carry out the Western blotting and analyze (Westernblotting assay).In this embodiment, utilize the test kit of Western blotting, Bio-RadScientific Instruments Transfer Unit for example), with 85 volts, 2.5 hours, the protein transduction of aforementioned running gel is printed to transfer film, wherein transfer film for example can be poly(vinylidene fluoride) transfer film (polyvinylidenedifluoride membrane, pvdf membrane; Aperture 0.45m; Millipore, Bedford, MA, U.S.A.).Afterwards, add 5% skim-milk to be dissolved in TBS damping fluid (Tris-Base, NaCl, Tween-20, pH 7.4) in as lock solution, in room temperature approximately after 1 hour, add one-level antibody (with 500 times of antibodies solution dilutions, the formula aftermentioned of antibodies solution), in 4 ℃ of reactions to every other day.Then, with TBS buffer solution for cleaning three times, each 10 minutes.Subsequently, add the secondary antibody with 500 times of TBS damping fluid dilutions, in 37 ℃ of reactions after 1 hour, with TBS buffer solution for cleaning three times, each 10 minutes.Then, add the cold light photoghraphic coupler, for example enhanced chemical cold light (Enhanced ChemiLuminescence; ECL) Western trace photosensitized reaction reagent (Western blotting luminal reagent; Santa Cruz Biotechnology, CA), and utilize cold light fluorometric analysis instrument system (for example Fujifilm LAS-3000 chemiluminescence detection system, Tokyo, Japan) to analyze sentence read result.
The one-level antibody of aforementioned use for example can be for tumor necrosis factor-alpha (TNF-α; Cell Signaling.Technology, Beverly, MA, USA), Toll sample acceptor 4 (Toll-like receptor 4; TLR4), p-JNK, c-Jun N terminal kinase 1/2 (c-Jun N-terminal kinase; JNK 1/2), phosphorylation nuclear Factor-Kappa B (phosphorylated nuclear factor-κ B; P-NF κ B), phosphorylation NF-κ B supressor (phospholate-I κ B; P-I κ B), the monoclonal antibody of p-p38, Bad, Bax, cytochrome C, apoptotic proteins enzyme 3 and alpha-tubulin (above product comes from Santa Cruz Biotechnology, Santa Cruz, CA, USA).The secondary antibody of aforementioned use for example can be goat anti-mouse IgG (goat anti-mouse IgG-HRP) in conjunction with horseradish peroxidase, in conjunction with the goat anti-rabbit igg (goat anti-rabbitIgG-HRP) of horseradish peroxidase or (above product comes from Santa Cruz Biotechnology in conjunction with the anti-goat IgG of the donkey of horseradish peroxidase (donkey anti-goat IgG-HRP), Santa Cruz, CA, USA).
The antibodies solution of aforementioned use can comprise for example Tween-20 of the NaCl and 0.1% (v/v) of the Tris-HCl solution of 100mM (pH 7.5), 0.9% (v/v).
1. assessment probiotic strain GM-080 is for the impact of anaphylactogen sensitized mice heart p-JNK and JNK 1/2 expression amount
Consult Fig. 2, it shows the Western blotting analysis chart of mouse heart tissue according to an embodiment of the invention, wherein the 1-2 road represents the normal healthy controls group, the 3-4 road represents irritated control group, the 5-6 road represents hypersensitive test group (hybrid bacterial strain A, i.e. the first hybrid bacterial strain), and the 7-8 road represents hypersensitive test group (hybrid bacterial strain B, i.e. the second hybrid bacterial strain), the 9-10 road represents hypersensitive test group (lactobacillus paraceasi GM-080 (CCTCC M 204012)).The in-house TLR4 of mouse heart (approximately 89kDa), the p-JNK (approximately 54kDa and 46kDa) of the former sensitization of Fig. 2 diagnosis allergy and the expression of JNK 1/2 (approximately 54kDa and 46kDa).As for alpha-tubulin (approximately 57kDa) as internal contrast group (internal control), with the above-mentioned protein expression amount of stdn.
Consult Fig. 3, it illustrates the histogram of the p-JNK expression amount of the expression amount stdn Fig. 2 that utilizes alpha-tubulin, wherein longitudinal axis representative utilizes the relative expression quantity (being the relative expression quantity of p-JNK/ alpha-tubulin) of the expression amount stdn p-JNK expression amount of alpha-tubulin, and take the relative expression quantity of the p-JNK/ alpha-tubulin of normal healthy controls group as 1.0.
Consult Fig. 4, it illustrates the histogram of JNK 1/2 expression amount of the expression amount stdn Fig. 2 that utilizes alpha-tubulin, wherein longitudinal axis representative utilizes the relative expression quantity (being the relative expression quantity of JNK 1/2/ alpha-tubulin) of the expression amount stdn p-JNK expression amount of alpha-tubulin, and take the relative expression quantity of JNK 1/2/ alpha-tubulin of normal healthy controls group as 1.0.
By the result of Fig. 2 to Fig. 4 as can be known, compared to normal healthy controls group (Fig. 2 1-2 road), mouse is after anaphylactogen sensitization, and the p-JNK of the mouse heart tissue of irritated control group (Fig. 2 3-4 road) and the expression amount of JNK 1/2 present remarkable increase.Secondly, compared to irritated control group (Fig. 2 3-4 road), the expression amount of the p-JNK of the heart tissue of the mouse of hypersensitive test group and JNK 1/2 (Fig. 2 9-10 road) is significantly lower than hypersensitive test group (hybrid bacterial strain A and hybrid bacterial strain B, Fig. 2 5-8 road) with irritated control group (Fig. 2 3-4 road), as Fig. 3 to shown in Figure 4, but the probiotic strain GM-080 that represents oral embodiment one really specificity suppress the expression amount of p-JNK and JNK 1/2.
2. assessment probiotic strain GM-080 is for the impact of anaphylactogen sensitized mice heart p-NF κ B and p-I κ B expression amount
Consult Fig. 5, it shows the Western blotting analysis chart of mouse heart tissue according to an embodiment of the invention, wherein the 1-2 road represents the normal healthy controls group, the 3-4 road represents irritated control group, the 5-6 road represents hypersensitive test group (hybrid bacterial strain A, i.e. the first hybrid bacterial strain), and the 7-8 road represents hypersensitive test group (hybrid bacterial strain B, i.e. the first hybrid bacterial strain), the 9-10 road represents hypersensitive test group (lactobacillus paraceasi GM-080 (CCTCC M 204012)).The in-house p-NF κ of the mouse heart B (approximately 65kDa) of the former sensitization of Fig. 5 diagnosis allergy and the expression of p-I κ B (approximately 40kDa) are used for the above-mentioned protein expression amount of stdn as for alpha-tubulin.
Consult Fig. 6, it illustrates the histogram of the p-NF κ B expression amount of the expression amount stdn Fig. 5 that utilizes alpha-tubulin, wherein longitudinal axis representative utilizes the relative expression quantity (being the relative expression quantity of p-NF κ B/ alpha-tubulin) of the expression amount stdn p-NF κ B expression amount of alpha-tubulin, and take the relative expression quantity of the p-NF κ B/ alpha-tubulin of normal healthy controls group as 1.0.
Consult Fig. 7, it illustrates the histogram of the p-I κ B expression amount of the expression amount stdn Fig. 5 that utilizes alpha-tubulin, wherein longitudinal axis representative utilizes the relative expression quantity (being the relative expression quantity of p-I κ B/ alpha-tubulin) of the expression amount stdn p-I κ B expression amount of alpha-tubulin, and take the relative expression quantity of the p-I κ B/ alpha-tubulin of normal healthy controls group as 1.0.
By the result of Fig. 5 to Fig. 7 as can be known, compared to normal healthy controls group (Fig. 5 1-2 road), mouse is after anaphylactogen sensitization, and the p-NF κ B of the mouse heart tissue of irritated control group (Fig. 5 3-4 road) and the expression amount of p-I κ B present a little increase.secondly, compared to irritated control group (Fig. 5 3-4 road), the expression amount (Fig. 5 9-10 road) of the p-NF κ B of the heart tissue of the mouse of hypersensitive test group is significantly lower than irritated control group (Fig. 5 3-4 road), and the expression amount (Fig. 5 9-10 road) of the p-I κ B of the heart tissue of the mouse of hypersensitive test group is more remarkable in hypersensitive test group (hybrid bacterial strain A and hybrid bacterial strain B, Fig. 5 5-8 road) with irritated control group (Fig. 5 3-4 road), as extremely shown in Figure 7 in Fig. 5, but the probiotic strain GM-080 that represents oral embodiment one specificity really suppresses the expression amount of p-NF κ B and p-I κ B.
3. assessment probiotic strain GM-080 is for the impact of anaphylactogen sensitized mice heart Bad and Bax expression amount
Consult Fig. 8, it shows the Western blotting analysis chart of mouse heart tissue according to an embodiment of the invention, wherein the 1-2 road represents the normal healthy controls group, the 3-4 road represents irritated control group, the 5-6 road represents hypersensitive test group (hybrid bacterial strain A, i.e. the first hybrid bacterial strain), and the 7-8 road represents hypersensitive test group (hybrid bacterial strain B, i.e. the first hybrid bacterial strain), the 9-10 road represents hypersensitive test group (lactobacillus paraceasi GM-080 (CCTCC M 204012)).The in-house Bad of mouse heart (approximately 25kDa) of the former sensitization of Fig. 8 diagnosis allergy and the expression of Bax (approximately 23kDa) are used for the above-mentioned protein expression amount of stdn as for alpha-tubulin.
Consult Fig. 9, it illustrates the histogram of the Bad expression amount of the expression amount stdn Fig. 8 that utilizes alpha-tubulin, wherein longitudinal axis representative utilizes the relative expression quantity (being the relative expression quantity of Bad/ alpha-tubulin) of the expression amount stdn Bad expression amount of alpha-tubulin, and take the relative expression quantity of the Bad/ alpha-tubulin of normal healthy controls group as 1.0.
Consult Figure 10, it illustrates the histogram of the Bax expression amount of the expression amount stdn Fig. 8 that utilizes alpha-tubulin, wherein longitudinal axis representative utilizes the relative expression quantity (being the relative expression quantity of Bax/ alpha-tubulin) of the expression amount stdn Bax expression amount of alpha-tubulin, and take the relative expression quantity of the Bax/ alpha-tubulin of normal healthy controls group as 1.0.
By the result of Fig. 8 to Figure 10 as can be known, compared to normal healthy controls group (Fig. 8 1-2 road), mouse is after anaphylactogen sensitization, and the Bad of the mouse heart tissue of irritated control group (Fig. 8 3-4 road) and the expression amount of Bax present remarkable increase.Secondly, compared to irritated control group (Fig. 8 3-4 road), the Bad of the heart tissue of the mouse of hypersensitive test group and the expression amount of Bax (Fig. 8 9-10 road) are significantly lower than hypersensitive test group (hybrid bacterial strain A and hybrid bacterial strain B, the 82nd figure 5-8 road) with irritated control group (Fig. 8 3-4 road), as Fig. 8 to shown in Figure 10, but the probiotic strain GM-080 that represents oral embodiment one really specificity suppress the expression amount of Bad and Bax.
4. assessment probiotic strain GM-080 is for the impact of cytochrome C and apoptotic proteins enzyme 3 expression amounts of anaphylactogen sensitized mice heart
Consult Figure 11, it shows the Western blotting analysis chart of mouse heart tissue according to an embodiment of the invention, wherein the 1-2 road represents the normal healthy controls group, the 3-4 road represents irritated control group, the 5-6 road represents hypersensitive test group (hybrid bacterial strain A, i.e. the first hybrid bacterial strain), and the 7-8 road represents hypersensitive test group (hybrid bacterial strain B, i.e. the second hybrid bacterial strain), the 9-10 road represents hypersensitive test group (lactobacillus paraceasi GM-080 (CCTCC M 204012)).The expression of the apoptotic proteins enzyme 3 (approximately 17kDa) of the in-house cytoplasmic cytochrome C of the mouse heart of the former sensitization of Figure 11 diagnosis allergy (approximately 11kDa) and activated state is used for the above-mentioned protein expression amount of stdn as for alpha-tubulin.
Consult Figure 12, it illustrates the histogram of the cytoplasmic expression of cytochrome C amount of the expression amount stdn Figure 11 that utilizes alpha-tubulin, wherein longitudinal axis representative utilizes the relative expression quantity (being the relative expression quantity of cytochrome C/alpha-tubulin) of the expression amount stdn expression of cytochrome C amount of alpha-tubulin, and take the relative expression quantity of the cytochrome C/alpha-tubulin of normal healthy controls group as 1.0.
Consult Figure 13, it illustrates the histogram of apoptotic proteins enzyme 3 expression amounts of the activated state of the expression amount stdn Figure 11 that utilizes alpha-tubulin, wherein longitudinal axis representative utilizes the relative expression quantity (being the relative expression quantity of apoptotic proteins enzyme 3/ alpha-tubulin of activated state) of expression amount stdn apoptotic proteins enzyme 3 expression amounts of alpha-tubulin, and take the relative expression quantity of apoptotic proteins enzyme 3/ alpha-tubulin of the activated state of normal healthy controls group as 1.0.
By the result of Figure 11 to Figure 13 as can be known, compared to normal healthy controls group (Figure 11 1-2 road), mouse is after anaphylactogen sensitization, and the cytochrome C of the mouse heart tissue of irritated control group (Figure 11 3-4 road) and the expression amount of apoptotic proteins enzyme 3 present a little increase.Secondly, compared to irritated control group (Figure 11 3-4 road), the expression amount of the cytochrome C of the heart tissue of the mouse of hypersensitive test group and apoptotic proteins enzyme 3 (Figure 11 9-10 road) is significantly lower than hypersensitive test group (hybrid bacterial strain A and hybrid bacterial strain B, Figure 11 5-8 road) with irritated control group (Figure 11 3-4 road), as Figure 12 to shown in Figure 13, but the probiotic strain GM-080 that represents oral embodiment one really specificity suppress the expression amount of the apoptotic proteins enzyme 3 of cytoplasmic cytochrome C and activated state.
In sum, probiotic strain GM-080 of the present invention (secondary cheese Bacterium lacticum GM-080) (preserving number is CCTCC M 204012) confirms to can be applicable to heart inflammation that allergy causes and the treatment of heart cell apoptosis, and the regulatory mechanism that milk-acid bacteria may relate to proposed, namely can see through the expression that specificity suppresses p-JNK, Bad and Bax, and then effectively treat heart inflammation and heart cell apoptosis, thereby other application surface of exploitation probiotic strain.what this need replenish be, though the present invention is with specific bacterial strain, the particular analysis mode, the particular animal pattern, special reaction condition, specific immunization ways, certain material or particular device etc. are as illustration, illustrate that probiotic strain GM-080 of the present invention is in the purposes for the treatment of heart inflammation and heart cell apoptosis, but any those of ordinary skill as can be known in the technical field of the invention, the present invention is not limited to this, without departing from the spirit and scope of the present invention, probiotic strain GM-080 of the present invention also can use other probiotic strain, other analysis mode, other zootype, other reaction conditions, other immunization ways, the material that other grade is suitable or miscellaneous equipment etc. carry out.In addition, probiotic strain GM-080 of the present invention is during for the manufacture of the composition for the treatment of heart inflammation and heart cell apoptosis, for example medical composition, diet supplement, food or its form minute, and probiotic strain GM-080 can be alive or do not activate, or is cryodesiccated form.Moreover probiotic strain GM-080 of the present invention also can comprise other composition, for example glucose, maltodextrin, infant formula, Nutriflora P, Magnesium Stearate, cheese spices, other composition that is difficult to separate or its above-mentioned arbitrary combination.
By the above embodiment of the present invention as can be known, the application of probiotic strain GM-080 of the present invention in the composition of preparation treatment heart inflammation and heart cell apoptosis, its advantage is to utilize probiotic strain GM-080 to see through the expression that specificity suppresses p-JNK, Bad and Bax, and then effectively treat heart inflammation and heart cell apoptosis, thereby other application surface of exploitation probiotic strain GM-080.
Although the present invention discloses as above with a plurality of embodiment; yet they are not to limit the present invention; at any those of ordinary skill in the technical field of the invention without departing from the spirit and scope of the present invention; can do various modifications and modification, so protection scope of the present invention is as the criterion with claim.
Claims (4)
1. the application of probiotic strain GM-080 in the composition of preparation treatment heart inflammation and heart cell apoptosis, wherein this probiotic strain GM-080 is lactobacillus paraceasi (Lactobacillusparacasei) GM-080, it is preserved in Chinese Typical Representative culture collection center, preserving number is CCTCC M204012, and the content of this probiotic strain GM-080 in said composition can effectively be treated heart inflammation and heart cell apoptosis.
2. the application of probiotic strain GM-080 according to claim 1 in the composition of preparation treatment heart inflammation and heart cell apoptosis, wherein this probiotic strain GM-080 for work or through not activating.
3. the application of probiotic strain GM-080 according to claim 1 in the composition of preparation treatment heart inflammation and heart cell apoptosis, wherein the content of this probiotic strain GM-080 can effectively be treated myocardial inflammation and the apoptosis of cardiac muscle that brings out because of anaphylaxis.
4. the application of probiotic strain GM-080 according to claim 1 in the composition of preparation treatment heart inflammation and heart cell apoptosis, wherein said composition is that medical composition, diet supplement, food or its composition divide.
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