Application of P.distassonis ATCC: 8503 in treatment of intestinal ischemia-reperfusion injury

Abstract

The invention provides application of P.distassonis ATCC: 8503 in treatment of intestinal ischemia reperfusion injury, and belongs to the technical field of microorganisms. According to the application disclosed by the invention, the P.distaxis ATCC: 8503 is used for treating the intestinal ischemia-reperfusion injury, the treatment effect of the P.distaxis ATCC: 8503 is detected, and a result proves that the P.distaxis ATCC: 8503 can be used for inhibiting the pathological morphology injury of intestinal tissues induced by the intestinal ischemia-reperfusion injury, reducing the content of FITC (Fluorescein Isothiocyanate) and LPS (Lipopolysaccharide) in serum and reducing the permeability of an intestinal barrier, so that the P.distaxis ATCC: 8503 can be used for treating the intestinal ischemia-reperfusion injury. The intestinal barrier function injury induced by the intestinal ischemia reperfusion injury is relieved, and the endotoxin displacement and the kidney injury induced by the intestinal ischemia reperfusion injury of the mouse are relieved.

Description

Technical Field

[0001] The invention relates to the technical field of microorganisms, and in particular to application of Parabacteroides distasonis ATCC: 8503 in treating intestinal ischemia-reperfusion injury. Background Art

[0002] Intestinal ischemia-reperfusion injury (IIR) refers to the phenomenon in medicine that after a period of intestinal ischemia, intestinal function further deteriorates when blood flow is restored. It is also called intestinal ischemia-reperfusion injury.

[0003] Intestinal ischemia-reperfusion injury involves multiple pathophysiological processes, including: 1. Damage to intestinal barrier function: When ischemia and hypoxia occur, the intestinal mucosa edema, intestinal permeability increases, and intestinal mucosal secretion decreases, which damages the intestinal barrier function, causing intestinal flora displacement and toxins entering the blood. 2. Inflammatory cascade reaction: After endotoxin enters the blood, it can activate the reticuloendothelial system, promote the release of a large number of inflammatory cells and toxins, and cause an inflammatory cascade reaction. Microcirculatory disorders, metabolic acidosis, increased oxygen free radicals, calcium overload, complement system activation, leukocyte adhesion, and the release of proinflammatory factors are all involved in the occurrence of intestinal ischemia-reperfusion injury.

[0004] Parabacteroides distasonis (P. distasonis) is a Gram-negative, non-spore-forming, anaerobic, rod-shaped bacterium belonging to the genus Parabacteroides and one of the core flora of the human body. Correlation analysis showed that its content was significantly negatively correlated with obesity, non-alcoholic fatty liver disease, diabetes and other disease states, suggesting that it may play a positive regulatory role in glucose and lipid metabolism. However, the role and mechanism of P. distasonis in intestinal ischemia-reperfusion injury have not been reported. Summary of the invention

[0005] The purpose of the present invention is to provide an application of Parabacteroides distasonis ATCC: 8503 in treating intestinal ischemia-reperfusion injury, wherein Parabacteroides distasonis can be used to alleviate intestinal barrier function damage, endotoxin translocation and renal damage induced by intestinal ischemia-reperfusion injury.

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0007] The present invention provides an application of Parabacteroides distasonis in preparing a medicine for treating intestinal ischemia-reperfusion injury. The standard strain number of the Parabacteroides distasonis is P. distasonis ATCC: 8503.

[0008] Furthermore, the Parabacteroides distichum can alleviate intestinal barrier function damage, endotoxin translocation, and renal damage induced by intestinal ischemia-reperfusion injury.

[0009] The present invention also provides a medicine for treating intestinal ischemia-reperfusion injury, wherein the medicine comprises Parabacteroides distasonis and / or its metabolites, and the standard strain number of the Parabacteroides distasonis is P. distasonis ATCC: 8503.

[0010] Furthermore, the drug also includes pharmaceutically acceptable excipients.

[0011] Furthermore, the dosage form of the pharmaceutical preparation is any one of a liquid preparation, a solid preparation, and a semisolid preparation.

[0012] The beneficial effects of the present invention compared with the prior art are:

[0013] The present invention uses Parabacteroides distasonis ATCC: 8503 to treat intestinal ischemia-reperfusion injury, and detects the therapeutic effect of Parabacteroides distasonis ATCC: 8503. The results show that administration of Parabacteroides distasonis ATCC: 8503 can inhibit the pathological morphological damage of intestinal tissue induced by intestinal ischemia-reperfusion injury, reduce the content of FITC and LPS in serum, reduce intestinal barrier permeability, and slow down intestinal barrier function damage induced by intestinal ischemia-reperfusion injury. In addition, Parabacteroides distasonis ATCC: 8503 can also slow down endotoxin translocation and renal damage induced by intestinal ischemia-reperfusion injury. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative labor.

[0015] Figure 1 This is a graph showing the pathological results of the improvement of intestinal tissue injury induced by intestinal ischemia-reperfusion injury in mice by Parabacteroides distichous in Example 1 of the present invention, wherein A is a HE staining graph of intestinal tissue morphological changes in each group, and B is a quantitative scoring result of intestinal tissue injury in each group, and * indicates that the difference compared with the I / R group is statistically significant p<0.05;

[0016] Figure 2This is the result diagram of Example 2 of the present invention, showing that Parabacteroides distichous slows down the intestinal barrier function damage induced by intestinal ischemia-reperfusion injury in mice, where AD is the result diagram of the expression of intestinal barrier-related indicators in intestinal tissues of each group. E is the result diagram of FITC content in serum of each group, * indicates that the difference compared with the I / R group is statistically significant p<0.05;

[0017] Figure 3 This is a graph showing the results of Example 3 of the present invention in which Parabacteroides distichous slows down endotoxin translocation and renal injury induced by intestinal ischemia-reperfusion injury in mice, wherein A is a graph showing the results of LPS content in serum of each group, and B is a graph showing the results of BUN content in serum of each group, and * indicates that the difference compared with the I / R group is statistically significant, p<0.05. DETAILED DESCRIPTION

[0018] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0019] It should be understood that the terms described in the present invention are only for describing special embodiments and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. Each smaller range between the intermediate value in any stated value or stated range and any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0020] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0021] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0022] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0023] Example 1

[0024] Example 1 of the present invention detected the effect of Parabacteroides distichous on pathological morphological damage of intestinal tissue induced by intestinal ischemia-reperfusion injury in mice, and the specific steps were as follows:

[0025] (1) Experimental Materials

[0026] Experimental animals

[0027] Twenty-four male C57BL / 6J mice aged 6-8 weeks and weighing 16-22 g were selected for the experiment. They were purchased from the Animal Center of Nanfang Hospital and raised in the SPF-grade Animal Experiment Department of Nanfang Hospital of Southern Medical University. All operations involved in the animal breeding process were approved by the Ethics Committee and met the requirements of animal ethics.

[0028] Reagents and instruments

[0029] Parabacteroides distasonis (ATCC: 8503, Beina Cell Microbial Collection Management Center); CLARIOstar all-function multifunctional microplate reader (BMG, Germany); blood plate (Guangdong Huankai Microbiological Technology Co., Ltd.); round-bottom vertical anaerobic culture bag; anaerobic gas-generating bag (Guangdong Huankai Microbiological Technology Co., Ltd.); isoflurane (Reward Life Science Technology Co., Ltd.); microvascular artery clamp (Chengdu North America Jiarui Biotechnology Co., Ltd.); sterile silk thread (Ningbo Medical Suture Co., Ltd.); phosphate buffered saline (PBS) pH 7.4 buffer (Gibco); hematoxylin-eosin staining (Beijing Regen Biological Company); anhydrous ethanol (Guangdong Guanghua Technology Co., Ltd.); xylene (Guangdong Guanghua Technology Co., Ltd.); paraffin (Leica); 4% paraformaldehyde (Beijing Solarbio Technology Co., Ltd.); neutral gum (Solarbio); fully automatic fluorescence microscope (Olympus).

[0030] (2) Experimental methods and results

[0031] 1) Cultivation of Parabacteroides distiliensis: First, activate the strain in the freeze-dried tube and perform subculture to restore its vitality: draw about 0.5 ml of sterile water into the freeze-dried tube, fully dissolve it, add it to 2 blood agar plates, about 200 μL / plate, and spread it evenly; place the plate upright in a round-bottom vertical culture bag, add an anaerobic gas-producing bag and anaerobic indicator, and place it in a 37°C constant temperature incubator for anaerobic culture for 48 hours, and colonies can be seen. Then, use 4-5 mL of sterile PBS solution to blow down the colonies on the blood plate, then centrifuge the suspension at 8000 rpm for 10 minutes, discard the supernatant, resuspend it with sterile PBS, and measure the concentration on a McFarland turbidimeter after withering correction. The colony concentration is 9×10 9CFU / ml, mix the resuspended bacterial solution and glycerol in a 4:1 ratio, and store in a -80℃ refrigerator for later use.

[0032] 2) Animal experiments

[0033] A. Establishment of the mouse superior mesenteric artery ischemia / reperfusion (I / R) model (the intestinal ischemia / reperfusion injury animal model is a perioperative intestinal injury model constructed by classic superior mesenteric artery clamping): fasting for 12 hours before surgery, free drinking water, isoflurane inhalation anesthesia mice, and the superior mesenteric artery is clamped with a non-invasive microvascular artery clamp to block blood flow. After intestinal ischemia lasts for 60 minutes, the artery clamp is released to restore blood supply, and intestinal reperfusion is performed. After checking that there is no bleeding in the abdominal cavity, the peritoneum, muscle and skin are sutured layer by layer with sterile silk thread. After blocking and during reperfusion, 0.5 ml of warm saline at about 37°C is subcutaneously injected for fluid resuscitation. After 2 hours of intestinal reperfusion, the blood and tissue of the mice are collected for examination.

[0034] B. Experimental Grouping:

[0035] Twenty-four C57BL / 6J mice aged 6-8 weeks were randomly divided into Sham group, I / R group, and intestinal I / R group treated with oral administration of Parabacteroides distasonis (I / R+P.distasonis).

[0036] Sham group: C57BL / 6J mice were subjected to routine abdominal midline incision and suture without superior mesenteric artery I / R treatment;

[0037] I / R group: C57BL / 6J mice were treated with 200 μL PBS for 7 consecutive days by intragastrical gavage, and the superior mesenteric artery was I / R treated by conventional midline abdominal incision and suture;

[0038] I / R+P.distasonis group: C57BL / 6J mice were treated with 200μL B.vulgatus by oral gavage for 7 consecutive days, and the superior mesenteric artery I / R was performed after routine abdominal midline incision and suture.

[0039] 3) Detection of pathological morphological changes in intestinal tissue

[0040] Fresh intestinal tissue was fixed by immersion in 4% paraformaldehyde for 24 hours, then dehydrated, embedded, sliced, stained with hematoxylin-eosin, and sealed with neutral gum. The pathological morphological changes of intestinal tissue were observed under an automatic fluorescence microscope, and the intestinal mucosal damage was graded and scored using the modified Chiu method. The results are shown in Table 1. Figure 1 shown.

[0041] Figure 1A, B The results of HE staining of intestinal tissue and the histomorphological injury score showed that compared with the Sham group, the intestinal villi at the top of the intestinal tissue of mice in the I / R group fell off and bled; compared with the I / R group, P. distasonis pretreatment could significantly reduce intestinal tissue damage. The experimental results show that P. distasonis can inhibit the pathological morphological damage of intestinal tissue of mice induced by intestinal ischemia-reperfusion injury.

[0042] Example 2

[0043] Example 2 of the present invention detected the therapeutic effect of Parabacteroides distichous on intestinal barrier function damage induced by intestinal ischemia-reperfusion injury in mice. The results are as follows: Figure 2 As shown, the specific steps are as follows:

[0044] (1) Experimental Materials

[0045] Experimental animals

[0046] Twenty-four male C57BL / 6J mice aged 6-8 weeks and weighing 18-22 g were selected for the experiment. They were purchased from the Animal Center of Nanfang Hospital of Southern Medical University and raised in the SPF-level Animal Experiment Department of Nanfang Hospital of Southern Medical University. All operations involved in the animal breeding process were approved by the Ethics Committee and met the requirements of animal ethics.

[0047] Reagents and instruments

[0048] Parabacteroides distasonis (ATCC: 8503, Beina Cell Microbial Collection Management Center); CLARIOstar all-function multifunctional microplate reader (BMG, Germany); blood plate (Guangdong Huankai Microbiological Technology Co., Ltd.); round-bottom vertical anaerobic culture bag; anaerobic gas-generating bag (Guangdong Huankai Microbiological Technology Co., Ltd.); isoflurane (Reward Life Science Technology Co., Ltd.); microvascular artery clamp (Chengdu North America Jiarui Biotechnology Co., Ltd.); sterile silk thread (Ningbo Medical Suture Co., Ltd.); physiological saline (Shijiazhuang Siyao Co., Ltd.); phosphate buffered saline (PBS) pH 7.4 buffer (Gibco); TRIZOL lysis buffer (Invitrogen); chloroform (Guangdong Guanghua); isopropanol (Guangdong Guanghua); anhydrous ethanol (Guangdong Guanghua); DEPC water (Sigma); SYBR Green fluorescent dye (Toyobo); ReverTraAce qPCR RT Kit (Toyobo); PCR instrument (Eppendorf, Germany); Fluorescence quantitative PCR instrument (Applied Biosystems AB, USA).

[0049] (2) Experimental methods and results

[0050] 1) Animal experiments:

[0051] A. Establishment of the mouse superior mesenteric artery I / R model (the intestinal ischemia-reperfusion injury animal model is a classical perioperative intestinal injury model constructed by superior mesenteric artery clamping): Same as Example 1.

[0052] B. Experimental Grouping:

[0053] Twenty-four C57BL / 6J mice aged 6-8 weeks were randomly divided into Sham group, I / R group, and intestinal I / R group treated with oral administration of Parabacteroides distasonis (I / R+P.distasonis).

[0054] Sham group: C57BL / 6J mice were subjected to routine abdominal midline incision and suture without superior mesenteric artery I / R treatment;

[0055] I / R group: C57BL / 6J mice were treated with 200 μL PBS for 7 consecutive days by intragastrical gavage, and the superior mesenteric artery was I / R treated by conventional midline abdominal incision and suture;

[0056] I / R+P.distasonis group: C57BL / 6J mice were treated with 200 μL P.distasonis by oral gavage for 7 consecutive days, and the superior mesenteric artery I / R treatment was performed after routine abdominal midline incision and suture.

[0057] 2) Intestinal barrier marker mRNA expression

[0058] A. RNA extraction method:

[0059] Take 20-50mg of intestinal tissue and place it in a 1.5mL RNAse-free EP tube. Add 500μL TRIZOL lysis buffer and homogenize. Add 100μL chloroform to the homogenate, shake 15-20 times, and let stand at room temperature for 1-2min. Centrifuge at 12,000rpm, 4℃ for 15min, and aspirate the upper aqueous phase to a new RNAse-free 1.5mL EP tube. Add an equal volume of isopropanol, shake 15-20 times, and let stand at room temperature for 10min. Centrifuge at 12,000rpm, 4℃ for 10min, discard the supernatant, add 1mL 80% ethanol (prepared with DEPC water), shake and oscillate, and centrifuge at 7500rpm, 4℃ for 5min. Discard the supernatant, centrifuge at 7500rpm, 4℃ for 1min, absorb the residual ethanol, let stand at room temperature for 5-10min, and add 50-100μL DEPC water to resuspend. The RNA product is stored in a -80℃ refrigerator for later use.

[0060] B. Reverse transcription of extracted RNA:

[0061] Take 1μL of extracted RNA and add it to 6μL of Nuclease-free Water. Denature it at 65℃ for 5min on a PCR amplifier. Immediately take it out and cool it on ice after denaturation. Add 2μL of 5×reaction buffer, 0.5μL of RT Enzyme mix, and 0.5μL of Prime mix to the reaction system. Slightly shake it and centrifuge it for 3-5s. Place the reaction system on a PCR amplifier, reverse transcribe it at 37℃ for 15min, and inactivate the enzyme at 98℃ for 5min. After the reaction, add 190μL of sterile water to obtain a cDNA solution.

[0062] C. Real-time PCR reaction:

[0063] 6 μL SYBR Green, 1 μL inflammatory factor primer and 5 μL cDNA solution were added to the reaction system and then placed in a fluorescent quantitative PCR instrument. Real-time PCR reaction conditions: Cycling: stage 95℃15s; 60℃1min, 40 cycles; Melt cure stage: 95℃15s; 60℃1min; 95℃30s; 60℃15s.

[0064] 3) Intestinal barrier permeability test:

[0065] Mice were treated with 0.6 mg / kg fluorescein isothiocyanate-labeled dextran (FITC-dextran) by gavage 4 hours before detection, and the FITC content in peripheral blood was detected by an enzyme marker (485 nm, 530 nm).

[0066] Figure 2 The results of A and B showed that P. distasonis pretreatment could significantly upregulate the mRNA expression levels of intestinal mechanical barrier markers Occludin and Zo-1 compared with the I / R group. Figure 2 Data from C and D show that the expression levels of Ki67, a marker of intestinal tissue cell proliferation, and Lgr5, a marker of intestinal rapid proliferation stem cells, in the P. distasonis group were significantly upregulated compared with those in the I / R group. Figure 2 E The FITC content in serum showed that compared with the I / R group, P. distasonis pretreatment could significantly reduce the FITC content in serum and reduce the intestinal barrier permeability. The above experimental results indicate that P. distasonis pretreatment can alleviate the intestinal barrier function damage induced by intestinal ischemia-reperfusion injury in mice.

[0067] Example 3

[0068] Example 3 of the present invention detected the therapeutic effect of Parabacteroides distichous on endotoxin translocation and renal injury induced by intestinal ischemia-reperfusion injury in mice. The results are as follows: Figure 3 As shown, the specific steps are as follows:

[0069] (1) Experimental Materials

[0070] Experimental animals

[0071] Twenty-four male C57BL / 6J mice aged 6-8 weeks and weighing 18-22 g were selected for the experiment. They were purchased from the Animal Center of Nanfang Hospital of Southern Medical University and raised in the SPF-level Animal Experiment Department of Nanfang Hospital of Southern Medical University. All operations involved in the animal breeding process were approved by the Ethics Committee and met the requirements of animal ethics.

[0072] Reagents and instruments

[0073] Parabacteroides distasonis (ATCC: 8503, Beina Cell Microbiology Collection Management Center); CLARIOstar all-function multifunctional microplate reader (BMG, Germany); blood agar plate (Guangdong Huankai Microbiology Technology Co., Ltd.); round-bottom vertical anaerobic culture bag; anaerobic gas-producing bag (Guangdong Huankai Microbiology Technology Co., Ltd.); isoflurane (Reward Life Science Technology Co., Ltd.); microvascular artery clamp (Chengdu North America Jiarui Biotechnology Co., Ltd.); sterile silk thread (Ningbo Medical Suture Co., Ltd.); physiological saline (Shijiazhuang Siyao Co., Ltd.); phosphate buffered saline (PBS) pH 7.4 buffer (Gibco); blood urea nitrogen (BUN) detection kit (Nanjing Jiancheng Bioengineering Institute).

[0074] (2) Experimental methods and results

[0075] 1) Animal experiments:

[0076] Establishment of the mouse superior mesenteric artery I / R model (the intestinal ischemia-reperfusion injury animal model is a classical perioperative intestinal injury model constructed by superior mesenteric artery clamping): Same as Example 1.

[0077] 2) Experimental groups:

[0078] Twenty-four C57BL / 6J mice aged 6-8 weeks were randomly divided into Sham group, I / R group, and intestinal I / R group treated with oral administration of Parabacteroides distasonis (I / R+P.distasonis).

[0079] Sham group: C57BL / 6J mice were subjected to routine abdominal midline incision and suture without superior mesenteric artery I / R treatment;

[0080] I / R group: C57BL / 6J mice were treated with 200 μL PBS for 7 consecutive days by intragastrical gavage, and the superior mesenteric artery was I / R treated by conventional midline abdominal incision and suture;

[0081] I / R+P.distasonis group: C57BL / 6J mice were treated with 200 μL P.distasonis by oral gavage for 7 consecutive days, and the superior mesenteric artery I / R treatment was performed after routine abdominal midline incision and suture.

[0082] 3) Detection of LPS content in serum

[0083] The serum was obtained after centrifugation of blood collected from the dissected mouse heart, and the procedure was performed according to the instructions of the Mouse Serum LPS Detection Kit.

[0084] 4) Detection of BUN content in serum

[0085] The serum was obtained after centrifugation of blood collected from the dissected mouse heart, and the procedure was performed according to the instructions of the mouse serum BUN detection kit.

[0086] Figure 3 A. The LPS content in serum showed that, compared with the I / R group, P. distasonis pretreatment could significantly reduce the LPS content in serum. Figure 3 B results showed that compared with the Sham group, the serum BUN content of mice in the I / R group increased significantly; compared with the I / R group, the serum BUN content was significantly reduced after pretreatment with P. distasonis bacteria. The above experimental results indicate that P. distasonis bacteria can slow down the endotoxin translocation and renal injury induced by intestinal ischemia-reperfusion injury in mice.

[0087] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A use of Parabacteroides distichous in the preparation of a drug for treating intestinal ischemia-reperfusion injury, characterized in that: The standard strain number of the Parabacteroides distichum is P. distasonis ATCC:8503.

2. The use of Parabacteroides distichous according to claim 1 in the preparation of a medicament for treating intestinal ischemia-reperfusion injury, characterized in that: The Parabacteroides distichum can alleviate intestinal barrier function damage, endotoxin translocation and kidney damage induced by intestinal ischemia-reperfusion injury.

3. A drug for treating intestinal ischemia-reperfusion injury, characterized in that: The drug includes Parabacteroides distichous and / or its metabolites, and the standard strain of Parabacteroides distichous is numbered as P. distasonis ATCC:8503.

4. The drug for treating intestinal ischemia-reperfusion injury according to claim 3, characterized in that: The drug also includes pharmaceutically acceptable excipients.

5. The drug for treating intestinal ischemia-reperfusion injury according to claim 4, characterized in that: The dosage form of the pharmaceutical preparation is any one of a liquid preparation, a solid preparation, and a semisolid preparation.

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