CN114748514A - Application of three kinds of bifidobacteria in preventing or treating acute pancreatitis - Google Patents
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Abstract
The invention discloses an application of Bifidobacterium pseudotympanum, Bifidobacterium adolescentis or Bifidobacterium animalis in preventing or treating acute pancreatitis. The invention provides application of Bifidobacterium pseudoatellum, Bifidobacterium adolescentis or Bifidobacterium animalis in products for preventing or treating acute pancreatitis. The strain can relieve acinar cell injury and inflammatory infiltration, reduce the concentration of amylase in serum and reduce the death rate of acute pancreatitis, is used for preparing probiotics medicines, pharmaceutical compositions, health foods, functional foods and feeds for treating acute pancreatitis, and has wide application prospects.
Description
Technical Field
The invention relates to the technical field of acute pancreatitis, in particular to application of bifidobacterium in preventing or treating acute pancreatitis.
Background
Acute pancreatitis is an inflammatory response that results in the activation of pancreatic enzymes in the pancreas, causing the spontaneous digestion, edema, hemorrhage and even necrosis of pancreatic tissue. Clinically, the medicine is characterized by acute epigastric pain, nausea, vomiting, fever, pancreatin increase and the like. The pathological changes are mild and severe, and mild cases mainly include pancreatic edema, which is common in clinic, the disease condition is self-limiting, and the prognosis is good, also called mild acute pancreatitis. The severe patients with hemorrhage and necrosis of pancreas, often secondary infection, peritonitis and shock, have high mortality rate and are called severe acute pancreatitis. According to the atlanta classification of 2012, acute pancreatitis is classified as MAP, MSAP and SAP. The death rate of SAP is 20-30%, and no effective clinical therapeutic medicine exists at present.
There are a large number of microflora in the gut, which adhere to the surface of the intestinal mucosa, maintaining immune homeostasis. Bacteria of the genus bifidobacterium are one of the important members of the human and animal intestinal flora. Bifidobacteria are gram-positive, rod-shaped, sometimes bifurcated, strictly anaerobic bacteria found in the digestive tract and oral cavity of humans and animals.
A large number of studies on bifidobacteria are mostly Bifidobacterium longum, Bifidobacterium breve and Bifidobacterium bifidum. In the aspect of treating acute pancreatitis, the applied bifidobacterium is bifidobacterium longum, and the bifidobacterium preparation used by a patient is a multi-bacterium combined preparation comprising bifidobacterium longum, lactobacillus acidophilus and enterococcus faecalis, so that no scheme for treating the acute pancreatitis by using bifidobacterium is provided at present, and the effect of other bifidobacterium is less understood.
Disclosure of Invention
The invention aims to solve the defects in the prior art, and provides application of three bifidobacteria in preventing or treating acute pancreatitis.
The invention aims to provide application of three bifidobacteria in a product for preventing or treating acute pancreatitis.
Preferably, preventing or treating acute pancreatitis comprises at least one of:
(1) relieving acinar cell injury and inflammatory infiltration;
(2) reducing the concentration of amylase in serum;
(3) reducing mortality of acute pancreatitis.
Preferably, the three bifidobacteria are Bifidobacterium pseudostellatum, Bifidobacterium adolescentis and Bifidobacterium animalis, respectively.
The specific information of the strains is as follows:
bifidobacterium animalis, ATCC25527 strain was purchased from ATCC company, USA.
The Bifidobacterium pseudostellatus ATCC27919 strain was purchased from ATCC company of the USA.
Bifidobacterium adolescentis, ATCC15703 strain was purchased from ATCC company, USA.
Preferably, the product is a health food, a functional food, a feed and a medicament.
A medicine for preventing or treating acute pancreatitis, pharmaceutically acceptable carrier, and at least one of Bifidobacterium pseudoatellum, Bifidobacterium adolescentis, and Bifidobacterium animalis.
Preferably, the pharmaceutically acceptable carrier comprises a pharmaceutical carrier or pharmaceutical excipient.
Preferably, the pharmaceutical excipient comprises an excipient and/or an additive.
Preferably, the dosage form of the medicament comprises milk, mushroom powder, granules, capsules, tablets, pills or oral liquid.
After the applicant carries out pancreatitis modeling on a mouse, metagenome sequencing is carried out on the excrement of the mouse with relatively low inflammation level, and the result shows that the mouse with low inflammation level has more bifidobacteria in comparison with the excrement of the mouse with high inflammation level, and the strain composition of the bifidobacteria in the excrement is shown in figure 1 after deep sequencing.
Fig. 1 shows: the ratio of the Bifidobacterium pseudostellatum to the Bifidobacterium adolescentis in the intestinal tract of the mouse is higher; the proportion of Bifidobacterium animalis is low and only accounts for 0.0004% of the intestinal flora.
However, it has never been reported in the literature whether Bifidobacterium pseudoatellum, Bifidobacterium adolescentis and Bifidobacterium animalis have a potential regulating effect on acute pancreatitis, and we want to further verify the inflammation-inhibiting effect of Bifidobacterium mono-bacterium anaplerosis in acute pancreatitis.
The applicant found and confirmed for the first time through experiments: the effects of Bifidobacterium animalis ATCC25527, Bifidobacterium pseudostellatus ATCC27919 and Bifidobacterium adolescentis ATCC15703 in preventing acute pancreatitis. The Bifidobacterium animalis, B.pseudoatellum and B.adolescentis strains provided by the invention can be used as a substitute of the existing medicament, can be used for preparing probiotic medicaments, pharmaceutical compositions, health-care foods, functional foods and feeds for treating acute pancreatitis, and broadens the application field of the Bifidobacterium animalis, the Bifidobacterium pseudostellatellum and the Bifidobacterium adolescentis strains.
Drawings
FIG. 1 shows the composition of strains of Bifidobacterium in mouse feces.
FIG. 2 is the serum amylase concentration of MAP mice 12 hours after acute pancreatitis modeled using ranulin in example 2.
Fig. 3 is a pathological section of pancreas of MAP mice (comparison between Abx + b. pseudoceratenulatum group, Abx + b. adolescentis group, Abx + b. animalis group and Abx gavage group) 12 hours after modeling acute pancreatitis with frogeurin in example 2.
Fig. 4 is a pathological section of pancreas of MAP mice 12 hours after acute pancreatitis was modeled using ranolanin in example 2 (comparison between PBS group, Abx gavage group, Abx + b. animalis group, and Abx + e. faecalis group).
Fig. 5 is the serum amylase concentration of SAP mice 12 hours after modeling acute pancreatitis with ranophanin in example 3.2.
Fig. 6 is a pathological section of the pancreas of SAP mice 12 hours after modeling acute pancreatitis with ranophanin in example 3.2.
FIG. 7 is the serum amylase concentration of SAP sterile mice 12 hours after modeling acute pancreatitis with ranulin from example 3.3.
Fig. 8 is a pathological section of the pancreas of SAP-free mice 12 hours after modeling acute pancreatitis with ranophangenin in example 3.3.
Figure 9 is the serum amylase concentration of SAP mice 12 hours after modeling acute pancreatitis with ranulin from example 3.4.
Fig. 10 is a pathological section of the pancreas of SAP mice 12 hours after modeling acute pancreatitis with ranophanin in example 3.4.
FIG. 11 is a graph of the survival of the mouse pancreaticobiliary duct of example 3.5 after retrograde injection of sodium taurocholate for 72 hours.
In each of the above figures, P < 0.05, P < 0.01, P < 0.001, and ns > 0.05.
Detailed Description
The present invention will be further illustrated with reference to the following specific examples.
Hereinafter, Bifidobacterium animalis is abbreviated as b.animalis, Bifidobacterium pseudostellatum is abbreviated as b.pseudostellatum, and Bifidobacterium adolescents is abbreviated as b.adolescentum.
Example 1 preparation of B.animallis ATCC25527, B.pseudothecanulus ATCC27919 and B.adolescentis ATCC15703 bacterial agents
Preparing an active microbial inoculum:
bifidobacterium animalis and Bifidobacterium adolescentis were strictly anaerobically cultured in BBL medium. Centrifuging the bacteria liquid of logarithmic phase at 4 deg.C 8000g for 10min, removing supernatant, washing the bacterial mud with sterile normal saline for 2 times, resuspending the bacterial mud in 30% (v/v) glycerol, and storing in-80 deg.C refrigerator.
Bifidobacterium pseudostellatum is strictly anaerobically cultured in MRS medium. Centrifuging the bacteria liquid of logarithmic phase at 4 deg.C 8000g for 10min, removing supernatant, washing the bacterial mud with sterile normal saline for 2 times, resuspending the bacterial mud in 30% (v/v) glycerol, and storing in-80 deg.C refrigerator.
Before use, the active microbial inoculum is applied to a sterile container containing 3% (v/v) glycerolDiluting with normal saline to 1 × 1010CFU/mL, spare.
Example 2 Bifidobacterium animalis, b. pseudoategulatum and b. adolescentis for prevention of Mild Acute Pancreatitis (MAP):
2.1 establishing an acute pancreatitis MAP model:
an acute pancreatitis model is induced by a mode of injecting ranulin into the abdominal cavity. The ranolonin was dissolved in physiological saline, and one injection of ranolonin (25. mu.g/kg body weight) was administered every hour for 7 consecutive injections.
2.2 prevention of Mild Acute Pancreatitis (MAP):
PBS group (representing no clearing of flora): PBS was continuously replenished.
Abx gavage group (representing total clearance of flora): mice were gavaged daily with a mixture of antibiotics consisting of ampicillin (33.2mg), neomycin (33.2mg), metronidazole (33.2mg) and vancomycin (16.7mg) for 5 days. On the fifth day after gavage, antibiotics (ampicillin, neomycin, and metronidazole concentrations were 1g/L, vancomycin concentration was 0.5g/L) were added to the drinking water for the modeling treatment.
Abx + FMT group (representing fecal transplantation after antibiotic-scavenging flora): 200mg of mixed fecal pellets were homogenized with sterile silica beads in 1.5mL PBS at 45Hz for 1 minute, then filtered through a 70 μm filter. After 5 days of Abx gavage, the Abx treated mice were gavaged with 150 μ L of filtered fecal homogenate on day 6.
Abimalis group (representing the antibiotic-cleared flora after replenishing b. animalis): after 5 days of Abx intragastric administration, 1X 10 is supplemented on day 610CFU of Bifidobacterium animalis.
Pseudothecatula group (representing the replacement of antibiotic-scavenging flora by b. pseudothecatula): after 5 days of Abx intragastric administration, 1X 10 is supplemented on day 610CFU of Bifidobacterium pseudostellatum.
Adolescents group (representing the anaplerotic b. adolescents after antibiotic-clearing flora): after 5 days of Abx intragastric administration, 1X 10 is supplemented on day 610Bifidobacterium adolescentis from CFU.
Abex + e. faecalis group (standing for anti-antibody)Replenishing E.faecalis after eliminating flora): after 5 days of Abx lavage, 1X 10 is supplemented on day 610E.faecalis of CFU.
Modeling was performed 48 hours after single-bacterium colonization. The results are shown in FIGS. 2-4. FIG. 2 is a graph showing the serum amylase concentration of MAP mice after 12 hours of modeling of acute pancreatitis with ranolanin; fig. 3 and 4 are both pathological sections of the pancreas of MAP mice 12 hours after modeling acute pancreatitis with ranolanin.
As can be seen from fig. 2-4: the clearance of flora can cause the increase of serum amylase level and the aggravation of pathological damage of pancreas; and the coproparation of the coprophilous fungi and the replenisher of B.animalis, B.pseudotheculatum and B.adolescentis can reduce the level of serum amylase to relieve the pathological damage of pancreas, but the replenisher of E.faecalis has no protective effect.
Example 3 use of Bifidobacterium animalis for prevention of Severe Acute Pancreatitis (SAP)
3.1 establishing acute pancreatitis SAP model:
an acute pancreatitis model is induced by a mode of injecting ranulin into the abdominal cavity. The ranolanin was dissolved in physiological saline, and one needle of ranolanin (25. mu.g/Kg body weight) was injected every hour, 10 needles were continuously injected, and one needle of LPS (7.5mg/Kg) was intraperitoneally injected.
3.2 normal mice are fed back with Bifidobacterium animalis to prevent severe acute pancreatitis:
about 7 weeks old 20-23g male C57BL/6J mice were randomly divided into control group (PBS) and viable Bifidobacterium animalis treated group (PBS + b. animalis), 5 per group, providing controlled feeding conditions.
And (5) performing ranophagnin modeling 48 hours after single bacterium colonization. The results are shown in fig. 5-6, fig. 5 is the serum amylase concentration of SAP mice 12 hours after modeling acute pancreatitis with ranolanin; fig. 6 is a pathological section of the pancreas of SAP mice 12 hours after modeling acute pancreatitis with ranophanin.
As can be seen from FIGS. 5-6: the serum amylase concentration was found to be lower in the PBS + b. animalis group and less pathological lesions of the pancreas were found compared to PBS.
3.3 sterile mice are fed back with Bifidobacterium animalis to prevent severe acute pancreatitis:
about 7 weeks old 20-23g male C57BL/6J sterile (GF) mice were divided into control (Uncolonized) and live Bifidobacterium animalis treated (B.animalis) groups of 5 mice each, providing controlled feeding conditions.
And (5) performing ranophagnin modeling 48 hours after single bacterium colonization. The results are shown in fig. 7-8, fig. 7 is the serum amylase concentration of SAP sterile mice 12 hours after modeling acute pancreatitis with ranolanin; fig. 8 is a pathological section of the pancreas of SAP-free mice 12 hours after modeling acute pancreatitis with ranolanin.
As can be seen from FIGS. 7-8: the serum amylase concentration was found to be lower in the b. animalis group and the pathological damage of the pancreas was less compared to Uncolonized.
3.4Abx mice replete with Bifidobacterium animalis prevents severe acute pancreatitis:
PBS group: PBS was continuously supplemented.
Abx gavage group: mice were gavaged daily with a mixture of antibiotics consisting of ampicillin (33.2mg), neomycin (33.2mg), metronidazole (33.2mg) and vancomycin (16.7mg) for 5 days. On the fifth day after gavage, antibiotics (ampicillin, neomycin, and metronidazole concentrations were 1g/L, vancomycin concentration was 0.5g/L) were added to the drinking water for the duration of the modeling treatment.
Abimalis group: after 5 days of Abx lavage, 1X 10 is supplemented on day 610CFU of Bifidobacterium animalis.
And (3) performing ranophanin modeling 48 hours after single bacterium colonization. The results are shown in fig. 9, fig. 9 is the serum amylase concentration of SAP mice 12 hours after modeling acute pancreatitis with ranolanin; fig. 10 is a pathological section of the pancreas of SAP mice 12 hours after modeling acute pancreatitis with ranophanin.
As can be seen from fig. 9-10: flora clearance leads to elevated serum amylase concentrations and more severe pathological damage to the pancreas, and anaplasia can reduce serum amylase concentrations and alleviate pathological damage to the pancreas.
3.5 supplementation of bifobacterium animalis in Abx mice reduced mortality in severe acute pancreatitis:
PBS group: PBS was continuously supplemented.
Abx gavage group: mice were gavaged daily with a mixture of antibiotics consisting of ampicillin (33.2mg), neomycin (33.2mg), metronidazole (33.2mg) and vancomycin (16.7mg) for 5 days. On the fifth day after gavage, antibiotics (ampicillin, neomycin, and metronidazole concentrations were 1g/L, vancomycin concentration was 0.5g/L) were added to the drinking water for the duration of the modeling treatment.
Abimalis group: after 5 days of Abx intragastric administration, 1X 10 is supplemented on day 610CFU of Bifidobacterium animalis.
Abex + e. faecalis group: after 5 days of Abx lavage, 1X 10 is supplemented on day 610E.faecalis of CFU.
Surgery modeling is carried out 48 hours after single bacterium colonization (each group adopts 5% sodium taurocholate hydrate for bile duct reverse injection to induce an acute pancreatitis model), and the result is shown in figure 11. FIG. 11 is a graph showing survival curves of mice within 72 hours after pancreatic bile duct retrograde injection of sodium taurocholate.
The results show that flora clearance leads to increased mortality, and that anaplerosis can reduce mortality, but anaplerosis has no protective effect.
The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art should be considered to be within the technical scope of the present invention, and the technical solutions and the inventive concepts thereof according to the present invention should be equivalent or changed within the scope of the present invention.
Claims (10)
1. Use of bifidobacteria in a product for the prevention or treatment of acute pancreatitis, wherein the bifidobacteria are Bifidobacterium pseudostellatum, Bifidobacterium adolescentis, or Bifidobacterium animalis, respectively.
2. Use according to claim 1, wherein the Bifidobacterium pseudostellatum is ATCC 27919.
3. Use according to claim 1, wherein the Bifidobacterium adolescentis is specifically ATCC 15703.
4. Use according to claim 1, wherein the Bifidobacterium animalis is specifically ATCC 25527.
5. The use according to claim 1, wherein the product is a health food, a functional food, a feed, and a pharmaceutical.
6. Use according to claim 1, wherein the prevention or treatment of acute pancreatitis comprises at least one of:
(1) relieving acinar cell injury and inflammatory infiltration;
(2) reducing the concentration of amylase in serum;
(3) reducing mortality of acute pancreatitis.
7. A medicament for preventing or treating acute pancreatitis, comprising a pharmaceutically acceptable carrier and at least one of Bifidobacterium pseudoatellum, Bifidobacterium adolescentis, and Bifidobacterium animalis.
8. The medicament of claim 7, wherein the pharmaceutically acceptable carrier comprises a pharmaceutical carrier or pharmaceutical excipient.
9. Pharmaceutical according to claim 8, characterized in that the pharmaceutical excipients comprise excipients and/or additives.
10. The medicament according to any one of claims 7 to 9, wherein the medicament is in the form of a milk preparation, a fungal powder, a granule, a capsule, a tablet, a pill or an oral liquid.
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