CN112076223A - Use of intestinal probiotics - Google Patents
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- KVWDHTXUZHCGIO-UHFFFAOYSA-N olanzapine Chemical compound C1CN(C)CCN1C1=NC2=CC=CC=C2NC2=C1C=C(C)S2 KVWDHTXUZHCGIO-UHFFFAOYSA-N 0.000 claims abstract description 26
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/66—Microorganisms or materials therefrom
- A61K35/74—Bacteria
- A61K35/741—Probiotics
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P3/00—Drugs for disorders of the metabolism
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- Public Health (AREA)
- Mycology (AREA)
- Chemical & Material Sciences (AREA)
- Medicinal Chemistry (AREA)
- Pharmacology & Pharmacy (AREA)
- Veterinary Medicine (AREA)
- Animal Behavior & Ethology (AREA)
- Microbiology (AREA)
- Engineering & Computer Science (AREA)
- Epidemiology (AREA)
- Molecular Biology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Diabetes (AREA)
- Hematology (AREA)
- Obesity (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Organic Chemistry (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
Abstract
The invention discloses application of intestinal probiotics. The use is for improving metabolic side effects caused by olanzapine administration. The application of the intestinal probiotics can provide a safer and more effective new strategy and theoretical basis for clinically preventing and treating OLZ-related metabolic side effects.
Description
Technical Field
The invention belongs to the technical field of probiotics, and particularly relates to application of intestinal probiotics.
Background
Probiotics are beneficial active microorganisms and are closely related to human health. The probiotics can synthesize digestive enzymes, and the digestive enzymes synthesized by animals participate in the digestion of nutrient substances in intestinal tracts, stimulate the animals to secrete the digestive enzymes, reduce the depth of crypts of small intestines, increase the height of villi, increase the surface area of the small intestines, promote the absorption of nutrient substances in the intestinal tracts, improve the immunity and the like.
Statistically, about one third of the psychiatric patients are accompanied by metabolic syndrome, while the prevalence of metabolic syndrome in chronic psychiatric patients is as high as 69%, and the prevalence of obesity, type 2 diabetes mellitus and hypercholesterolemia in psychiatric patients is even 3-5 times that of the general population. These factors ultimately lead to an increased risk of cardiovascular disease, which is also one of the leading causes of death in schizophrenic patients. Olanzapine is one of the most widely used atypical antipsychotics for the treatment of schizophrenia and other psychiatric disorders. However, patients are prone to severe metabolic side effects including elevated blood glucose, weight gain, elevated blood lipids, and increased food intake after olanzapine administration. Currently, metformin is used for improving metabolic disorder caused by olanzapine, but the metformin can only partially relieve impaired glucose tolerance of rats caused by long-term use of olanzapine; and only improve olanzapine-induced insulin resistance of the liver but not peripheral tissues. The side effect research of metformin and other patients who relieve mental diseases and take olanzapine has found that metformin has certain effect at present, but further research is needed. It is of great practical significance to find a new strategy for more effective and safe prevention and treatment of olanzapine-related metabolic disorders.
Disclosure of Invention
In order to overcome the defects in the prior art, the invention provides application of intestinal probiotics.
The invention realizes the purpose through the following technical scheme:
use of intestinal probiotic bacteria for ameliorating metabolic side effects caused by the administration of olanzapine.
Experiments prove that the intestinal probiotics have a better improvement effect on metabolic side effects caused by olanzapine administration such as glucose-induced metabolic disorder and the like, and provide a safer and more effective new strategy and theoretical basis for clinically preventing and treating OLZ-related metabolic side effects.
In one embodiment, the metabolic side effects include hyperglycemia, insulin resistance, serum and tissue inflammatory factors, overexpression of liver tissue gluconeogenic key enzymes, and/or intestinal mucosal barriers.
In one embodiment, the key hepatic tissue gluconeogenesis enzymes include glucose-6-phosphatase and phosphoenolpyruvate carboxykinase.
In one embodiment, the intestinal probiotics are prepared by the following method:
preparing a mucin liquid culture medium and a mucin solid culture medium;
collecting a stool sample and inoculating the stool sample into the mucin liquid culture medium for the first anaerobic culture to obtain a first bacterial liquid;
taking the first bacterial liquid as a template to carry out a PCR experiment to obtain a second bacterial liquid;
selecting a bacterial liquid in a PCR reaction positive tube, inoculating the bacterial liquid on the mucin solid culture medium, and performing anaerobic culture for the second time to obtain a first bacterial colony;
inoculating the first bacterial colony on a chocolate plate, and performing anaerobic culture for the third time to obtain a second bacterial colony;
inoculating the second colony on a Columbia blood plate, and purifying and culturing to obtain a third colony;
and taking the third colony, and carrying out PCR sequencing verification through a 16sRNA universal primer to separate the intestinal probiotics.
In one embodiment, the mucin liquid medium and the mucin solid medium both use mucin as raw materials, and the mucin is purified mucin.
In one embodiment, the method for purifying mucin comprises:
anhydrous NaH is added2PO4、Na2HPO4·12H2Dissolving O and NaCl in anhydrous ethanol, adjusting pH to 7-8, diluting to constant volume with anhydrous ethanol, sterilizing, and cooling to room temperature to obtain solution A;
dissolving NaCl in anhydrous ethanol, adjusting pH to 7-8, diluting to constant volume with anhydrous ethanol, sterilizing, and cooling to room temperature to obtain solution B;
adding mucin powder into the solution A, uniformly stirring, adjusting the pH value to 7-8, adding toluene, uniformly stirring, centrifuging, and collecting supernatant to obtain a first protein solution;
adding absolute ethyl alcohol into the first egg protein liquid to enable the concentration of the first egg protein liquid to be 55% -65%, cooling, centrifuging, and collecting precipitates to obtain a first precipitate;
dissolving the first precipitate in the solution B, stirring and centrifuging, and collecting supernatant to obtain a second protein solution;
adding absolute ethyl alcohol into the second protein solution to enable the concentration of the second protein solution to be 55% -65%, cooling, centrifuging, and collecting precipitates to obtain a second precipitate;
dissolving the second precipitate in water to obtain purified mucin;
in one embodiment, the mucin liquid medium is prepared by the method comprising:
FeCl is added2·4H2O、H3BO4、ZnCL2、CuCL2·7H2O、MnCL2、CoCL2·6H2O and NiCl2·6H2Adding O into HCl solution, and adding water to constant volume to obtain acidic trace elements;
mixing Na2SeO3、Na2MoO4·2H2Mixing O and NaOH0.4g, and diluting to constant volume with water to obtain alkaline microelement;
mixing biotin and VitB3、VitB6、VitB2、VitB1、VitB12Mixing with pantothenic acid, and diluting with water to desired volume to obtain biotin solution;
adding CaCl2、MgCl2Mixing the acidic trace elements and the alkaline trace elements, adding water to a constant volume, sterilizing, and cooling to room temperature to obtain solution C;
mixing Na2HPO4NaCl and KH2PO mixing, adding water to constant volume, sterilizing, and cooling to room temperature to obtain solution D;
NaHCO is added3Adding into vitamin solution, adding water to desired volume, filtering, and collecting filtrate to obtain solution E;
mixing Na2S, adding water, fixing the volume, filtering, and taking filtrate to obtain solution F;
and respectively adding the solution C, the solution D, the solution E and the solution F into the purified mucin, and uniformly obtaining the mucin liquid culture medium.
In one embodiment, the mucin solid medium is prepared by the following method:
FeCl is added2·4H2O、H3BO4、ZnCL2、CuCL2·7H2O、MnCL2、CoCL2·6H2O and NiCl2·6H2Adding O into HCl solution, and adding water to constant volume to obtain acidic trace elements;
mixing Na2SeO3、Na2MoO4·2H2Mixing O and NaOH0.4g, and diluting to constant volume with water to obtain alkaline microelement;
mixing biotin and VitB3、VitB6、VitB2、VitB1、VitB12Mixing with pantothenic acid, and diluting with water to desired volume to obtain biotin solution;
adding CaCl2、MgCl2Mixing the acidic trace elements and the alkaline trace elements, adding water to a constant volume, sterilizing, and cooling to room temperature to obtain solution C;
mixing Na2HPO4NaCl and KH2PO mixing, adding water to constant volume, sterilizing, and cooling to room temperature to obtain solution D;
NaHCO is added3Adding into vitamin solution, adding water to desired volume, filtering, and collecting filtrate to obtain solution E;
mixing Na2S, adding water, fixing the volume, filtering, and taking filtrate to obtain solution F;
adding agar into water, sterilizing, adding the solution C, solution D, solution E, solution F and purified mucin, homogenizing, pouring into a sterile culture container, cooling and solidifying to obtain mucin solid culture medium.
In one embodiment, the PCR experiment uses a PCR reaction solution as a raw material, the PCR reaction solution includes a first primer and a second primer, the sequence of the first primer is CAGCACGTGAAGGTGGGGAC, and the sequence of the second primer is CCTTGCGGTTGGCTTCAGAT.
In one embodiment, the temperature of the first anaerobic culture is 35-40 ℃ and the time is 2-6 days; the temperature of the second anaerobic culture is 35-40 ℃, and the time is 2-6 days; the temperature of the third anaerobic culture is 35-40 ℃, and the time is 1-5 days; the purification culture is repeated for 2-5 times for culture at 35-40 deg.C for 3-8 days.
Drawings
Fig. 1 is a technical experimental roadmap for a probiotic in the gut versus mouse experiment as described in an embodiment of the present invention;
FIG. 2 is a graph showing fasting plasma glucose in mice after administration of the probiotic bacteria;
FIG. 3 is a graph illustrating glucose tolerance and fasting plasma glucose in mice administered the probiotic bacteria according to one embodiment of the present invention;
FIG. 4 is a graph showing the serum insulin levels of mice administered the probiotic gut in accordance with one embodiment of the present invention;
FIG. 5 is a graph showing the insulin resistance coefficient of mice administered the probiotic bacteria;
FIG. 6 is a graph showing the level of ATGL transcription in mice administered the gut probiotic of one embodiment of the invention;
FIG. 7 is a graphical representation of the transcript levels of PTP1B following administration of said gut probiotic to a mouse, in accordance with one embodiment of the present invention;
FIG. 8 is a graph showing the IL-6 levels in serum of mice after administration of the probiotic to the gut in accordance with one embodiment of the present invention;
FIG. 9 is a graph showing the serum TNF- α levels in mice administered the probiotic bacteria of the gut according to one embodiment of the present invention;
FIG. 10 is a graph showing the serum IL-1 β levels of mice administered the probiotic in the gut according to one embodiment of the invention;
FIG. 11 is a schematic representation of the transcription of the gluconeogenic key enzyme G6Pase in mice after administration of said gut probiotic in accordance with one embodiment of the present invention;
FIG. 12 is a schematic representation of the transcription of the key gluconeogenic enzyme PEPCK in mice administered with the probiotic bacteria of the gut according to one embodiment of the present invention;
FIG. 13 is a graph showing the expression of gut claudin following administration of the gut probiotic to mice in accordance with one embodiment of the present invention.
Detailed Description
In order to make the aforementioned objects, features and advantages of the present invention comprehensible, specific embodiments thereof will be described in detail below. In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present invention. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein.
The embodiment of the invention provides application of intestinal probiotics in improving metabolic side effects caused by olanzapine taking.
Experiments prove that the intestinal probiotics have a better improvement effect on metabolic side effects caused by olanzapine administration such as glucose-induced metabolic disorder and the like, and provide a safer and more effective new strategy and theoretical basis for clinically preventing and treating OLZ-related metabolic side effects.
In one embodiment, the metabolic side effects include hyperglycemia, insulin resistance, serum and tissue inflammatory factors, overexpression of hepatic tissue gluconeogenic key enzymes, and/or intestinal mucosal barriers.
In one embodiment, the key hepatic tissue gluconeogenesis enzymes include glucose-6-phosphatase and phosphoenolpyruvate carboxykinase.
The intestinal probiotic is preferably the intestinal probiotic disclosed in publication CN 105176822A.
Namely, the intestinal probiotics are prepared by the following method:
preparing a mucin liquid culture medium and a mucin solid culture medium;
collecting a fecal sample and inoculating the fecal sample into a mucin liquid culture medium for first anaerobic culture to obtain a first bacterial liquid;
taking the first bacterial liquid as a template to carry out a PCR experiment to obtain a second bacterial liquid;
selecting bacterial liquid in the PCR positive tube, inoculating the bacterial liquid on a mucin solid culture medium, and performing anaerobic culture for the second time to obtain a first bacterial colony;
inoculating the first bacterial colony on a chocolate plate, and performing anaerobic culture for the third time to obtain a second bacterial colony;
inoculating the second colony on a Columbia blood plate, and purifying to culture a third colony;
taking the third colony, and performing PCR sequencing verification through a 16sRNA universal primer to separate the intestinal probiotics.
In one embodiment, the mucin liquid medium and the mucin solid medium are both prepared from mucin, which is purified mucin.
In one embodiment, the method for purifying mucin comprises:
anhydrous NaH is added2PO4、Na2HPO4·12H2Dissolving O and NaCl in anhydrous ethanol, adjusting pH to 7-8, diluting to constant volume with anhydrous ethanol, sterilizing, and cooling to room temperature to obtain solution A;
dissolving NaCl in anhydrous ethanol, adjusting pH to 7-8, diluting to constant volume with anhydrous ethanol, sterilizing, and cooling to room temperature to obtain solution B;
adding mucin powder into the solution A, uniformly stirring, adjusting the pH value to 7-8, adding toluene, uniformly stirring, centrifuging, and collecting supernatant to obtain a first protein solution;
adding absolute ethyl alcohol into the first egg protein liquid to enable the concentration of the first egg protein liquid to be 55% -65%, cooling, centrifuging, and collecting precipitates to obtain a first precipitate;
dissolving the first precipitate in the solution B, stirring and centrifuging, and collecting supernatant to obtain a second protein solution;
adding absolute ethyl alcohol into the second protein solution to enable the concentration of the second protein solution to be 55% -65%, cooling, centrifuging, and collecting precipitates to obtain a second precipitate;
dissolving the second precipitate in water to obtain purified mucin;
in one embodiment, the mucin liquid medium is prepared by the method comprising:
FeCl is added2·4H2O、H3BO4、ZnCL2、CuCL2·7H2O、MnCL2、CoCL2·6H2O and NiCl2·6H2Adding O into HCl solution, adding water to constant volume to obtain acidic trace elements；
Mixing Na2SeO3、Na2MoO4·2H2Mixing O and NaOH0.4g, and diluting to constant volume with water to obtain alkaline microelement;
mixing biotin and VitB3、VitB6、VitB2、VitB1、VitB12Mixing with pantothenic acid, and diluting with water to desired volume to obtain biotin solution;
adding CaCl2、MgCl2Mixing the acidic trace elements and the alkaline trace elements, adding water to a constant volume, sterilizing, and cooling to room temperature to obtain solution C;
mixing Na2HPO4NaCl and KH2PO mixing, adding water to constant volume, sterilizing, and cooling to room temperature to obtain solution D;
NaHCO is added3Adding into vitamin solution, adding water to desired volume, filtering, and collecting filtrate to obtain solution E;
mixing Na2S, adding water, fixing the volume, filtering, and taking filtrate to obtain solution F;
and respectively adding the solution C, the solution D, the solution E and the solution F into the purified mucin, and uniformly obtaining the mucin liquid culture medium.
In one embodiment, the mucin solid medium is prepared by the following method:
FeCl is added2·4H2O、H3BO4、ZnCL2、CuCL2·7H2O、MnCL2、CoCL2·6H2O and NiCl2·6H2Adding O into HCl solution, and adding water to constant volume to obtain acidic trace elements;
mixing Na2SeO3、Na2MoO4·2H2Mixing O and NaOH0.4g, and diluting to constant volume with water to obtain alkaline microelement;
mixing biotin and VitB3、VitB6、VitB2、VitB1、VitB12Mixing with pantothenic acid, and diluting with water to desired volume to obtain biotin solution;
adding CaCl2、MgCl2Mixing the acidic trace elements and the alkaline trace elements, adding water to a constant volume, sterilizing, and cooling to room temperature to obtain solution C;
mixing Na2HPO4NaCl and KH2PO mixing, adding water to constant volume, sterilizing, and cooling to room temperature to obtain solution D;
NaHCO is added3Adding into vitamin solution, adding water to desired volume, filtering, and collecting filtrate to obtain solution E;
mixing Na2S, adding water, fixing the volume, filtering, and taking filtrate to obtain solution F;
adding agar into water, sterilizing, adding the solution C, solution D, solution E, solution F and purified mucin, homogenizing, pouring into a sterile culture container, cooling and solidifying to obtain mucin solid culture medium.
The amount of each substance used may be selected according to the purpose of culture.
In one embodiment, the PCR reaction solution is used as a raw material for the PCR experiment, and the PCR reaction solution includes a first primer and a second primer, wherein the sequence of the first primer is CAGCACGTGAAGGTGGGGAC, and the sequence of the second primer is CCTTGCGGTTGGCTTCAGAT.
In one embodiment, the temperature of the first anaerobic culture is 35-40 ℃ for 2-6 days; the temperature of the second anaerobic culture is 35-40 ℃, and the time is 2-6 days; the temperature of the third anaerobic culture is 35-40 ℃, and the time is 1-5 days; the purification culture is repeated for 2-5 times for culture at 35-40 deg.C for 3-8 days. The culture time is the preferable culture time, and each culture time can be flexibly selected according to the culture effect.
For example, the intestinal probiotic isolation method is as follows:
step one, preparing a basic culture medium: preparing a mucin liquid culture medium and a mucin solid culture medium, wherein mucin in the mucin liquid culture medium and the mucin solid culture medium is purified mucin, and the purification method of the mucin comprises the following steps:
preparation of reagents:
the absolute ethyl alcohol is pre-cooled at 4 ℃.
Solution A: anhydrous NaH2PO40.2g；Na2HPO4·12H2O7 g; mixing NaCl6g, dissolving with above anhydrous ethanol, adjusting pH to 7.8, and metering to 1000mL, storing at 125 deg.C under high pressure for 30min, sterilizing, and storing at room temperature.
And B, liquid B: dissolving NaCl5.85g in the above anhydrous ethanol, adjusting pH to 7.0, diluting to 1000mL, storing at 125 deg.C under high pressure for 30min, sterilizing, and storing at room temperature.
Weighing 10g-15g of mucin powder, adding into a 500mL flask of the solution A, uniformly stirring for 2h on a magnetic stirrer, adjusting the pH value of the solution to 7.2 +/-0.2 by using 1M sodium hydroxide, dropwise adding 500uL-1000uL of toluene, continuously uniformly stirring for 18h on the magnetic stirrer, centrifuging at 3000rpm for 10min, collecting the supernatant into another autoclaved flask, removing the precipitate, and adding the absolute ethyl alcohol into the solution to enable the concentration to be about 60%. Placing in a refrigerator at 4 deg.C for 30min, centrifuging at 3000rpm for 10min, collecting precipitate, discarding supernatant, dissolving the precipitate in 200mLB liquid, stirring for 6h, centrifuging at 3000rpm for 10min, collecting supernatant to another autoclave-sterilized flask, discarding precipitate, adding the above anhydrous ethanol into the solution to make its concentration about 60%, placing in a refrigerator at 4 deg.C for 30min, centrifuging at 3000rpm for 10min, collecting precipitate, discarding supernatant, dissolving the precipitate in 100mL distilled water, and storing at 4 deg.C under sealed condition. The purified mucin has insoluble impurities removed and has obviously raised concentration. After purification mucin showed a salt peak at A230.
Preparation of mucin liquid medium:
preparation of reagents:
acid trace elements: FeCl2·4H2O1.491g；H3BO40.06g；ZnCl20.068g；CuCl2·7H2O0.1725g；MnCl20.0635g；CoCl2·6H2O0.0119；NiCl2·6H2Adding O0.0235g into HCL4.18mL, and making the volume of distilled water to be 100 mL;
alkaline trace elements: na (Na)2SeO30.01729g；Na2MoO4·2H2O0.0242g; adding 0.4g of NaOH0 into distilled water, shaking up, and then adding distilled water to a constant volume of 100 mL;
vitamin solution: 0.02g of biotin; VitB30.21；VitB60.5g；VitB20.1g；VitB10.2g；VitB120.25 g; 0.1g pantothenic acid, addShaking up in distilled water, and then adding distilled water to a constant volume of 100 mL;
and C, liquid C: CaCL21.1g；MgCL21.0 g; 1mL of acidic trace elements; 1mL of alkaline trace element, diluting distilled water to 100mL, sterilizing at 125 ℃ under high pressure for 30min, and storing at normal temperature in a sealing manner;
and (3) liquid D: na (Na)2HPO45.3g；NaCL3g；KH2PO44g, adding distilled water, shaking up, then adding distilled water to a constant volume of 100mL, sterilizing at a high pressure of 125 ℃ for 30min, and sealing and storing at normal temperature;
e, liquid E: NaHCO 23Adding 2g of the extract into 2mL of vitamin solution, diluting to 100mL with water, filtering with a 0.22-micron filter membrane, and storing at 4 ℃ in a sealed manner;
and F, liquid: na (Na)2S2.5g of the active carbon is added into distilled water, then the distilled water is used for fixing the volume to 100mL, a filter membrane with the diameter of 0.22 mu m is used for filtering, and the active carbon is sealed and stored at the temperature of 4 ℃;
taking 200mL of sterile distilled water, putting the sterile distilled water into an autoclaved wide-mouth bottle, adding 2mL of solution C, 2mL of solution D, 1mL of solution E, 2mL of solution F and 30-40 mL of purified mucin solution into a super clean workbench, shaking uniformly after adding each solution, sucking 5mL of solution by an electric pipettor, subpackaging the solution in a 15mL centrifuge tube, tightly covering a tube cover, and sealing and storing at 4 ℃.
Preparation of mucin solid medium:
weighing 2g of agar (OXIOD, UK) in 200mL of distilled water, sterilizing at 125 deg.C under high pressure for 30min, immediately adding 2mL of solution C, 2mL of solution D, 1mL of solution E, 2mL of solution F and 30-40 mL of purified mucin solution in a clean bench, and shaking up after adding each solution; pouring into sterile plates, wherein each plate is 20 mL; after cooling and solidifying, placing in a sterile plastic bag, and sealing and storing at 4 ℃. The mucin solid medium should be flat, semi-permeable and cloudy, and have a thickness of about 5 mm.
Collecting a fecal specimen:
collecting 1g-2g of normal human fecal specimen, immediately inoculating in mucin liquid culture medium, diluting by 10 times, and separating by 10 times-1Diluting to 10-6Marking, and immediately placing in an anaerobic glove box at 37 ℃ for culturing for 4 days. Note that the caps are unscrewed in the anaerobic tank for gasThe bodies are exchanged.
Selection and identification of mucin liquid culture medium:
1. the liquid in the medium was shaken up, and 200. mu.L of each mucin liquid medium was aspirated under anaerobic conditions into a sterile 1.5mL EP tube and labeled.
2. The EP tube was placed in a metal bath at 100 ℃ for 15 min. The subsequent PCR experiment is carried out by using the template.
3. The PCR reaction solution was prepared at the following concentrations:
specific primer 1: CAGCACGTGAAGGTGGGGAC
Specific primers 2: CCTTGCGGTTGGCTTCAGAT
4. The PCR reaction was performed according to the following procedure:
Plating on mucin plates:
1. and selecting the PCR reaction positive tube, and discarding the negative tube. The highest dilution positive tube was selected for each specimen for the next experiment.
2. The broth was shaken up in an anaerobic environment and inoculated with a disposable inoculating loop (2. mu.l) onto mucin plates, each plate divided into 6-8 zones by continuous streaking.
3. Anaerobic culture at 37 ℃ for 3 days.
Inoculation on chocolate plates:
1. colonies were picked directly from the mucin plates in a loop and inoculated onto chocolate plates, each of which was continuously streaked in zone 4.
2. After anaerobic culture at 37 ℃ for 2 days, observation was carried out, and relatively fine colonies scattered among colonies were selected and inoculated on a Columbia blood plate. The specific colony morphology is shown in FIG. 5. When colonies are picked, small white colonies which are included among large wet colonies or exist independently and have bulges, roundness, humidity and smooth edges are picked.
Purification was performed on columbia blood plates:
individual colonies were picked for purification culture on Columbia blood plates, and after about 5 days, individual colonies were picked for culture, and so on 3 times. Finally obtaining the purified convex, round, white and semitransparent microcolonies.
PCR verification was again performed by specific primers:
the confirmation was performed using colony PCR, the template was replaced with a single loop of colonies as described above.
Sequencing validation by 16sRNA universal primers:
primer 1: 1492R: GGTTACCTTGTTACGACTT
Primer 2: 27F: AGAGTTTGATCCTGGCTCA
The PCR reaction was performed according to the following procedure:
gel imaging of PCR products:
The subsequent inventor successfully separates 38 intestinal C-IFH strains from 200 fecal specimens, and the separation success rate of the intestinal C-IFH strains in Chinese population is about 20%.
The intestinal probiotics are used for carrying out experiments on mice.
60 female C57BL/6 mice of 6 weeks size were housed under a barrier system and randomly divided into 2 cohorts including 30 in cohort 1 and 30 in cohort 2, and after 2 weeks acclimatized 1 cohort 30 mice were on a high fat diet for 8 weeks and 30 mice in cohort two cohorts were on a regular diet for 8 weeks. After 8 weeks the 1 st cohort of 30 mice was randomized into 3 groups of 10 mice each, including: continuing the high fat diet + water + PBS (phosphate buffered saline) intragastric group, continuing the high fat diet + olanzapine (5mg/kg) + PBS intragastric group, and continuing the high fat diet + olanzapine (5mg/kg) + Akk (intestinal probiotics of the invention) intragastric group; the 2 nd large group of 30 mice, randomly divided into 3 groups of 10, included: continuing the normal diet + water + PBS intragastric group, continuing the normal diet + olanzapine (5mg/kg) + Akk gavage group. Akk therein, Akkermansia muciniphila, the intestinal probiotic of the present invention. PBS was gavaged at 10ml/kg, Akkermansia muciniphila at 109Gavage is carried out at the dose of cfu/kg, and each mouse is gavage twice a day, wherein the gavage period is 2 months. Mice were sacrificed after the experiment for subsequent experimental analysis. The experimental technical route is shown in figure 1.
The intestinal probiotics (Akk) are tested by using several subtypes, and the test results of the several subtypes are the same. The following high fat diet group was HFD and the normal diet group was NCD.
The results of the above intestinal probiotics in improving olanzapine-induced hyperglycemia in mice:
after 8 weeks of treatment with different diets, mice on the high-fat diet had significantly higher fasting plasma glucose than mice on the normal diet, as shown in fig. 2. Olanzapine impaired glucose tolerance and fasting glucose in the general diet mice, and a comparison of fig. 3, Akk improved glucose tolerance and fasting glucose. Olanzapine increased serum insulin levels in mice, see figure 4, and increased insulin resistance coefficient, see figure 5, indicating that Akk improved insulin resistance in mice.
The result of the intestinal probiotics reduces the expression of part of insulin resistance related proteins is that:
akk decreased olanzapine-induced increased transcription level of ATGL (fatty triglyceride lipase), as shown in FIG. 6. Akk decreased the transcript level of PTP1B (protein tyrosine phosphatase 1B) in mice in the group of high fat diets, as shown in FIG. 7, demonstrating that Akk decreased olanzapine-induced increase in the transcript level of PTP 1B.
The above intestinal probiotics improve intestinal barrier and reduce olanzapine-induced inflammatory factors as a result:
akk reduced olanzapine induced increases in serum IL-6 and TNF- α levels, as shown in FIG. 8 and FIG. 9, Akk also reduced serum IL-1 β levels in mice on a high fat diet, as shown in FIG. 10.
The results of the above intestinal probiotics reducing the overexpression of gluconeogenesis key enzymes:
akk reduces the olanzapine-induced over-transcription of the gluconeogenic key enzymes G6Pase (glucose 6 phosphatase) and PEPCK (phosphoenolpyruvate carboxykinase), as shown in FIGS. 11 and 12, respectively.
The result of the intestinal probiotics repairing the intestinal mucosa barrier of the mouse is as follows:
akk increase olanzapine-induced loss of intestinal claudin expression, see FIG. 13.
The experimental results prove that the intestinal probiotics can improve the metabolic side effect caused by olanzapine taking.
The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.
Claims (10)
1. Use of intestinal probiotic bacteria for ameliorating metabolic side effects caused by the administration of olanzapine.
2. Use of intestinal probiotics according to claim 1, characterized in that: the metabolic side effects include hyperglycemia, insulin resistance, serum and tissue inflammatory factors, overexpression of liver tissue gluconeogenic key enzymes, and/or intestinal mucosal barriers.
3. Use of intestinal probiotics according to claim 2, characterized in that: the liver tissue gluconeogenesis key enzyme comprises glucose-6-phosphatase and phosphoenolpyruvate carboxykinase.
4. Use of intestinal probiotics according to claim 1, characterized in that: the intestinal probiotics are prepared by the following method:
preparing a mucin liquid culture medium and a mucin solid culture medium;
collecting a stool sample and inoculating the stool sample into the mucin liquid culture medium for the first anaerobic culture to obtain a first bacterial liquid;
taking the first bacterial liquid as a template to carry out a PCR experiment to obtain a second bacterial liquid;
selecting a bacterial liquid in a PCR reaction positive tube, inoculating the bacterial liquid on the mucin solid culture medium, and performing anaerobic culture for the second time to obtain a first bacterial colony;
inoculating the first bacterial colony on a chocolate plate, and performing anaerobic culture for the third time to obtain a second bacterial colony;
inoculating the second colony on a Columbia blood plate, and purifying and culturing to obtain a third colony;
and taking the third colony, and carrying out PCR sequencing verification through a 16sRNA universal primer to separate the intestinal probiotics.
5. Use of intestinal probiotics according to claim 4, characterized in that: the mucin liquid culture medium and the mucin solid culture medium both adopt mucin as raw materials, and the mucin is purified mucin.
6. Use of intestinal probiotics according to claim 5, characterized in that: the method for purifying the mucin comprises the following steps:
anhydrous NaH is added2PO4、Na2HPO4·12H2Dissolving O and NaCl in anhydrous ethanol, adjusting pH to 7-8, diluting to constant volume with anhydrous ethanol, sterilizing, and cooling to room temperature to obtain solution A;
dissolving NaCl in anhydrous ethanol, adjusting pH to 7-8, diluting to constant volume with anhydrous ethanol, sterilizing, and cooling to room temperature to obtain solution B;
adding mucin powder into the solution A, uniformly stirring, adjusting the pH value to 7-8, adding toluene, uniformly stirring, centrifuging, and collecting supernatant to obtain a first protein solution;
adding absolute ethyl alcohol into the first egg protein liquid to enable the concentration of the first egg protein liquid to be 55% -65%, cooling, centrifuging, and collecting precipitates to obtain a first precipitate;
dissolving the first precipitate in the solution B, stirring and centrifuging, and collecting supernatant to obtain a second protein solution;
adding absolute ethyl alcohol into the second protein solution to enable the concentration of the second protein solution to be 55% -65%, cooling, centrifuging, and collecting precipitates to obtain a second precipitate;
and dissolving the second precipitate in water to obtain the purified mucin.
7. Use of intestinal probiotics according to claim 4, characterized in that: the preparation method of the mucin liquid culture medium comprises the following steps:
FeCl is added2·4H2O、H3BO4、ZnCL2、CuCL2·7H2O、MnCL2、CoCL2·6H2O and NiCl2·6H2Adding O into HCl solution, and adding water to constant volume to obtain acidic trace elements;
mixing Na2SeO3、Na2MoO4·2H2Mixing O and NaOH0.4g, and diluting to constant volume with water to obtain alkaline microelement;
mixing biotin and VitB3、VitB6、VitB2、VitB1、VitB12Mixing with pantothenic acid, and diluting with water to desired volume to obtain biotin solution;
adding CaCl2、MgCl2Mixing the acidic trace elements and the alkaline trace elements, adding water to a constant volume, sterilizing, and cooling to room temperature to obtain solution C;
mixing Na2HPO4NaCl and KH2PO mixing, adding water to constant volume, sterilizing, and cooling to room temperature to obtain solution D;
NaHCO is added3Adding into vitamin solution, adding water to desired volume, filtering, and collecting filtrate to obtain solution E;
mixing Na2S, adding water, fixing the volume, filtering, and taking filtrate to obtain solution F;
and respectively adding the solution C, the solution D, the solution E and the solution F into the purified mucin, and uniformly obtaining the mucin liquid culture medium.
8. Use of intestinal probiotics according to claim 4, characterized in that: the preparation method of the mucin solid medium comprises the following steps:
FeCl is added2·4H2O、H3BO4、ZnCL2、CuCL2·7H2O、MnCL2、CoCL2·6H2O and NiCl2·6H2Adding O into HCl solution, and adding water to constant volume to obtain acidic trace elements;
mixing Na2SeO3、Na2MoO4·2H2Mixing O and NaOH0.4g, and diluting to constant volume with water to obtain alkaline microelement;
mixing biotin and VitB3、VitB6、VitB2、VitB1、VitB12Mixing with pantothenic acid, and diluting with water to desired volume to obtain biotin solution;
adding CaCl2、MgCl2Mixing the acidic trace elements and the alkaline trace elements, adding water to a constant volume, sterilizing, and cooling to room temperature to obtain solution C;
mixing Na2HPO4NaCl and KH2PO mixing, adding water to constant volume, sterilizing, and cooling to room temperature to obtain solution D;
NaHCO is added3Adding into vitamin solution, adding water to desired volume, filtering, and collecting filtrate to obtain solution E;
mixing Na2S, adding water, fixing the volume, filtering, and taking filtrate to obtain solution F;
adding agar into water, sterilizing, adding the solution C, solution D, solution E, solution F and purified mucin, homogenizing, pouring into a sterile culture container, cooling and solidifying to obtain mucin solid culture medium.
9. Use of intestinal probiotics according to claim 4, characterized in that: the PCR experiment adopts PCR reaction liquid as a raw material, the PCR reaction liquid comprises a first primer and a second primer, the sequence of the first primer is CAGCACGTGAAGGTGGGGAC, and the sequence of the second primer is CCTTGCGGTTGGCTTCAGAT.
10. Use of intestinal probiotics according to claim 4, characterized in that: the temperature of the first anaerobic culture is 35-40 ℃, and the time is 2-6 days; the temperature of the second anaerobic culture is 35-40 ℃, and the time is 2-6 days; the temperature of the third anaerobic culture is 35-40 ℃, and the time is 1-5 days; the purification culture is repeated for 2-5 times for culture at 35-40 deg.C for 3-8 days.
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Application publication date: 20201215 |