CN116098923A - Application of enterobacteria in preparing medicine for treating liver injury diseases caused by apoptosis and composition - Google Patents
Application of enterobacteria in preparing medicine for treating liver injury diseases caused by apoptosis and composition Download PDFInfo
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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- A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
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- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
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Abstract
The invention belongs to the technical field of biology, and particularly relates to application of intestinal bacteria in preparing a medicament for treating liver injury diseases caused by apoptosis and a composition. The intestinal bacteria are Bacteroides acidifaciens; the liver injury diseases caused by apoptosis are liver cell apoptosis-related liver diseases mediated by CD95/CD95L death signals, including but not limited to autoimmune hepatitis, alcoholic hepatitis, viral hepatitis, drug/toxic hepatitis, liver fibrosis, liver cirrhosis or liver cancer. Experiments show that when the abundance of Bacteroides acidifaciens in intestinal tracts is increased, the strain can inhibit the apoptosis of liver cells by inhibiting the expression of liver death receptor CD95 and/or apoptosis protease caspase-3, thereby achieving the effect of protecting the liver, and therefore, the strain has the treatment effect on liver injury diseases caused by apoptosis.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to application of intestinal bacteria in preparing a medicament for treating liver injury diseases caused by apoptosis and a composition.
Background
The liver is one of the most important immune organs of the human body, is responsible for eliminating toxic products in the body, and is rich in a large number of immune cells. Liver injury is a fatal syndrome that endangers humans and reflects the serious consequences of autoimmune hepatitis, viral hepatitis, alcoholic hepatitis, drug or toxic hepatitis, etc., and liver injury is closely related to abnormal death of hepatocytes.
CD95 is a transmembrane protein belonging to a member of the tumor necrosis factor receptor family and plays an important role in transmitting apoptosis signals. CD95/CD95L mediated apoptosis of liver cells is widely involved in various liver diseases such as alcoholic hepatitis, viral hepatitis, autoimmune hepatitis, drug or poison hepatitis, liver fibrosis, cirrhosis, and hepatocellular carcinoma.
Mice of different intestinal microbiota exhibit varying degrees of susceptibility to canavalin a-induced liver injury, possibly due to modulation of the CD95/CD95L pathway by the intestinal microbiota.
The bacteria in human intestinal tracts are hundreds of millions and have 150 times more genes than host genes, which are called microbiome (microbiome), and are a complex, dynamic and diverse microbial population under normal conditions, so as to form microecological balance, play an important role in body health and diseases, participate in energy acquisition and storage, metabolism and interaction with intestinal immune systems, and promote the maturation of immune cells and the occurrence of normal immune responses. Once the flora structure is deregulated, it can lead to the development of various diseases such as obesity, diabetes, nonalcoholic fatty liver, inflammatory bowel disease, depression, multiple sclerosis, etc.
CN107184613a discloses a probiotic composition for promoting hepatitis b virus clearance and its use, preferably selected from the group consisting of: one or more of bifidobacteria, lactobacilli, escherichia coli and enterococcus faecalis. The invention determines the influence of the probiotic combination intervention on the HBV capability of HBV mice through in vitro animal experiments. The invention finally determines that two different probiotic compositions have obvious effect of promoting HBV clearance in serum HbsAg level, serum HBV DNA level, liver tissue pgRNA and cccDNA level, and the effect of promoting clearance is realized by changing intestinal flora through probiotics, thus being hopeful to develop a new situation in a strategy for clinically treating hepatitis B. However, the invention requires a combination of probiotics to achieve a certain effect, and is relatively complex.
Studies have shown that there is a close interaction between the intestinal flora and the liver. There is a high correlation between the abundance of some bacteria in the gut and the extent of liver damage, and the flora can affect liver disease processes by modulating various cells in the liver. For example, the abundance of Veilonella and Ruminococcea in the gut is highly correlated with the extent of liver injury and serum transaminase levels, whereas Bactoides are reversed; akkermansia muciniphila is effective in protecting mice from autoimmune liver injury. Many studies have shown that intervention of the intestinal flora is helpful for treating liver diseases, and thus, it is found that a specific strain of intestinal bacteria capable of exerting a therapeutic effect on hepatitis is of great importance for the therapeutic strategy of intestinal flora intervention of liver diseases. However, most of the existing findings only show the correlation between certain intestinal bacteria and liver injury, and lack of definite experimental evidence proves that a single strain can inhibit liver injury at the cellular and animal level and the action mechanism thereof.
In view of this, the present invention has been made.
Disclosure of Invention
The invention aims at the technical problems, and a first object of the invention is to provide an application of intestinal bacteria in preparing a medicament for treating liver injury diseases caused by apoptosis. The intestinal bacteria is Bacteroides acidifaciens, and the strain is a normal inhabited flora in intestinal tracts of human beings and animals, and experiments show that when the abundance of the strain in the intestinal tracts is increased, the strain can inhibit the expression of death receptor CD95 in liver cells, so that the apoptosis of the liver cells is inhibited, and the effect of protecting the liver is achieved, so that the strain has a therapeutic effect on liver injury diseases caused by the apoptosis.
A second object of the present invention is to provide a composition containing enterobacteria Bacteroides acidifaciens as an ingredient, which is useful for treating liver injury diseases caused by apoptosis.
In order to achieve the first object, the present invention adopts the following technical scheme:
the invention provides application of intestinal bacteria in preparing a medicament for treating liver injury diseases caused by apoptosis, wherein the intestinal bacteria is Bacteroides acidifaciens.
The human intestinal tract has more than 1000 and trillions of bacteria, most of which belong to the phylum bacteroides (bacterioides), firmicutes (Firmicutes) and actinomycetes (actinomycetes). The bacteroides mainly comprises bacteroides, proteus and Mucor; the phylum Thick-wall bacteria mainly includes Clostridium, lactobacillus and ruminococcus, and butyrate-producing eubacterium, clostridium and Roche. In addition, there are also some fungal species in the gut, such as candida, saccharomyces, etc.
According to the invention, firstly, two intestinal flora structures are found to have different sensitivities to the autoimmune hepatitis of mice induced by canavalin A from mice with different intestinal flora structures, and through the analysis of intestinal flora metagenomics of the two mice, the inventor locks the eyes of bacteria with the abundance ranking 5 top in the intestinal flora of the mice with low hepatitis degree, namely bacteroides acidophilus (Bacteroides acidifaciens), bacteroides dirachta (Parabacteroides distasonis), bacteroides thetaiotaomicron (Bacteroides thetaiotaomicron), bacteroides mulberkoviceae (Bacteroides dorei) and bacteroides simplex (Bacteroides uniformis) respectively. Through repeated culture, separation and identification, the inventor separates the 5 strains from the mouse feces with low hepatitis degree, and then carries out pure culture, mouse gastric lavage and hepatitis model detection on the 5 strains respectively, and finally determines that only Bacteroides acidifaciens gastric lavage can reduce the autoimmune hepatitis level of the mouse. The inventor further detects the treatment effect of Bacteroides acidifaciens on alcoholic hepatitis, and discovers that the strain can reduce the alcoholic hepatitis level of mice by lavage. Through intensive mechanism studies, the present inventors found that Bacteroides acidifaciens exerts therapeutic effects on autoimmune liver and alcoholic liver by inhibiting the expression of the death signal receptor CD95 in hepatocytes, thereby inhibiting apoptosis of hepatocytes. While CD 95-mediated apoptosis is widely involved in the mechanism of liver injury occurrence in a variety of liver diseases.
Further, the liver injury disease caused by apoptosis is liver cell apoptosis-related liver disease mediated by CD95/CD95L death signals.
In particular, the liver apoptosis-related liver disease includes, but is not limited to, autoimmune hepatitis, alcoholic liver, viral hepatitis, drug/poison hepatitis, liver fibrosis, cirrhosis or liver cancer.
Further, the Bacteroides acidifaciens can inhibit hepatic cell apoptosis and liver injury by inhibiting the expression of the liver death receptor CD95 and/or apoptosis protease caspase-3, so as to treat liver injury diseases caused by apoptosis.
Further, the cell concentration of Bacteroides acidifaciens is not lower than 3×10 9 CFU/ml。
Further, the Bacteroides acidifaciens is prepared by the following steps:
anaerobic culturing Bacteroides acidifaciens strain with BLHK medium, centrifuging to collect thallus, washing with sterile PBS, and re-suspending to adjust thallus concentration to 3×10 or more 9 CFU/ml。
Specifically, the BLHK medium comprises the following components: BHI38.5g/L, L-cysteine 0.25g/L, heme 5mg/L, vitamin K 1 10mg/l。
Further, the anaerobic culture is anaerobic culture at 37 ℃ for 36 hours.
Further, the centrifugation was 9200 rpm/min at 4 ℃.
Further, the washing is 2 times.
Further, the Bacteroides acidifaciens strain is obtained by the following separation method:
mashing fresh feces of mice or people in PBS, centrifuging, removing sediment, diluting the supernatant to a proper proportion, coating on a blood agar plate, performing anaerobic culture, transferring a single colony to a BLHK culture plate for anaerobic culture, performing colony PCR identification on the single colony by adopting an identification primer designed according to a 16S rDNA sequence of Bacteroides acidifaciens, selecting positive colonies of a PCR result for streaking separation, performing PCR identification again after anaerobic culture, performing complete sequence sequencing confirmation of the 16S rDNA, and storing in a refrigerator for standby after confirming.
In order to achieve the second object of the present invention, the present invention adopts the following technical scheme:
a composition for treating liver injury diseases caused by apoptosis, wherein the composition comprises intestinal bacteria Bacteroides acidifaciens.
Experiments show that the intestinal bacteria Bacteroides acidifaciens can inhibit apoptosis of liver cells by inhibiting the expression of death signal receptor CD95 in the liver cells, thereby exerting the therapeutic effects on the autoimmune liver and the alcoholic liver. While CD 95-mediated apoptosis is widely involved in the mechanism of liver injury occurrence in a variety of liver diseases. Therefore, the invention further provides a composition taking the enterobacteria Bacteroides acidifaciens as one of the components, the composition can be used for treating liver injury diseases caused by apoptosis, and the composition can also be used together with other medicines for treating the liver injury diseases caused by apoptosis.
Compared with the prior art, the invention has the following advantages:
the invention adopts intestinal flora intervention mode to treat liver injury diseases caused by apoptosis, the intervention mode is to orally treat liver injury diseases caused by apoptosis by bacterial Bacteroides acidifaciens isolated from intestinal tracts, can effectively reduce clinical indexes such as serum transaminase level and liver pathological damage, has better safety and smaller side effect compared with the traditional liver injury disease treatment medicine caused by apoptosis, can be used simultaneously with the liver injury disease medicine caused by apoptosis, and Bacteroides acidifaciens has better protection effect on intestinal barrier, and can protect liver and digestive tract.
Drawings
FIG. 1-a is a graph showing the effect of Bacteroides acidifaciens on serum transaminase levels of autoimmune hepatitis mice in example 1;
FIG. 1-b is a graph showing the results of immunohistochemistry of Bacteroides acidifaciens on liver pathological injury (HE), liver apoptosis (TUNEL) and liver CD95 IHC in the autoimmune hepatitis mice of example 1;
FIG. 2-a is a graph showing the effect of Bacteroides acidifaciens on serum transaminase levels in alcoholic hepatitis mice in example 1;
FIG. 2-b is a graph showing the results of Bacteroides acidifaciens in example 1 on liver pathological damage (HE) and fat staining (Oil red) of alcoholic hepatitis mice;
FIG. 3-a is a comparison of serum transaminase levels of Bacteroides acidifaciens pair Jo2 injected mice in example 2;
FIG. 3-b is a comparison of the pathological damage (HE) and apoptosis (TUNEL) of the liver of Jo2 injected mice by Bacteroides acidifaciens in example 2;
FIG. 4-a is the immunoblot detection results of liver CD95 and caspase-3 from mice in example 38 hours after injection of Canavalia ectropis A;
FIG. 4-b shows the result of immunohistochemistry of liver CD95 IHC from the liver of the mice of example 3;
FIG. 4-c is the immunoblot detection results of liver CD95 and caspase-3 from mice with alcoholic liver in example 38 hours after gavage with 50% ethanol;
FIG. 4-d shows the immunohistochemical results of liver CD95 IHC in mice with alcoholic liver disease in example 3;
FIG. 5-a is a graph showing the effect of Bacteroides acidifaciens and other 4 strains isolated from the intestinal tract on serum transaminase levels in autoimmune hepatitis mice in example 4;
FIG. 5-b is a graph showing the results of immunohistochemistry of Bacteroides acidifaciens and other 4 strains isolated from intestinal tract on liver pathological damage (HE), apoptosis (TUNEL) and CD95 of autoimmune hepatitis mice in example 4.
Detailed Description
The following are specific embodiments of the present invention, which are described in order to further illustrate the invention, not to limit the invention.
Examples 1, bacteroides acidifaciens use of the Strain in autoimmune hepatitis and alcoholic hepatitis
Five mixed antibiotics (neomycin sulfate 10mg/ml, ampicillin 10mg/ml, metronidazole 10mg/ml, vancomycin 5mg/ml, amphotericin B0.1 mg/ml) were used for the intestinal flora removal of mice, and each mouse was subjected to gastric lavage for 200 μl, 2 times per day, 2 weeks after continuous gastric lavage, and then sterilized water was exchanged for two days of rest. The Bacteroides acidifaciens strain was treated with BLHK medium (BHI 38.5g/L, L-cysteine 0.25g/L, heme 5mg/L, vitamin K) 1 10 mg/l) was anaerobically cultured at 37℃for 36 hours, the culture was subjected to 9200 rpm/isolation at 4℃for 5 minutes to collect the cells, washed 2 times with sterilized PBS and resuspended to adjust the cell concentration to 3X 10 9 CFU/ml. This bacterial suspension was gavaged at a dose of 200. Mu.l/mouse to the above mixed antibiotic treated mice, 2 times per day for 1 week, and the mice that were gavaged Bacteroides acidifaciens were labeled BA, and the control group (control) was gavaged with wild-type mouse fecal bacteria, and Parabacteroides distasonis isolated from the intestinal tract of the mice was used as a control single bacteria, and the group labeled PD, parabacteroides distasonis was cultured and gavaged in the same manner as Bacteroides acidifaciens.
Treatment of autoimmune hepatitis in mice:
canavalia gladiata was injected at a dose of 10mg/kg through the tail vein to induce autoimmune hepatitis in mice. After injection for 12 hours, the eye vein was sampled, and after leaving the blood for 2 hours, serum was obtained at 3700 rpm/min from the heart, and the serum transaminase level was measured using the serum transaminase measurement kit. After mouse livers are fixed by paraformaldehyde, HE staining is carried out to observe pathological damage conditions of the livers, a TUNEL apoptosis detection kit is used for detecting apoptosis conditions in liver slices, and CD95 immunohistochemical staining is carried out.
The results of the effect on serum transaminase levels in autoimmune hepatitis mice are shown in FIG. 1-a.
The results of immunohistochemistry for hepatocase injury (HE), hepatocyte apoptosis (TUNEL) and hepatic CD95 IHC in autoimmune hepatitis mice are shown in fig. 1-b, where HE plot shows that Bacteroides acidifaciens treated mice (BA) had significantly fewer areas of liver plaque lesions than Control and Control single Parabacteroides distasonis treated groups (PD); TUNEL plot shows that the area and brightness of light fluorescent region (representing apoptotic region) of liver in BA group mice is significantly smaller than that of Control group (Control group) and PD group; the dark areas in the CD95 IHC plot represent the death receptor CD95 staining positive areas, which shows that the area and shade of the liver CD95 positive areas of the BA group mice are significantly smaller than in the Control and PD groups.
The results show that Bacteroides acidifaciens can significantly reduce serum transaminase levels, pathological liver injury and apoptosis of liver cells of mice induced by canavalin A.
Treatment of alcoholic hepatitis in mice:
the analytically pure absolute ethanol was prepared as 50% ethanol, and the mice were subjected to gastric lavage at a dose of 7.5ml/kg body weight to induce alcoholic hepatitis in the mice, after 12 hours of ocular venous blood collection, after 2 hours of blood resting, serum was obtained at 3700 rpm/15 minutes from heart separation, and then the serum transaminase level was detected using a serum transaminase detection kit. After mouse livers are fixed by paraformaldehyde, HE staining is carried out to observe pathological damage conditions of the livers, and TUNEL apoptosis detection kit is used for detecting apoptosis conditions in liver slices.
The effect on serum transaminase levels in alcoholic hepatitis mice is shown in FIG. 2-a.
The results of liver case damage (HE) and fat staining (Oil red) of alcoholic hepatitis mice are shown in fig. 2-b, wherein white dots or blocks in the HE diagram represent pathological damage areas of alcoholic liver, and the areas of liver and alcoholic liver damage areas of mice in the BA group are shown to be obviously smaller than those of the Control group and the PD group; the light and dark color in the Oil red plot represents fat content, and the plot shows that the liver fat content of mice in the BA group is significantly less than that in the Control group and the PD group.
The results show that Bacteroides acidifaciens can obviously reduce serum transaminase level, liver pathological damage and liver fat condition of mice with alcoholic hepatitis.
Example 2, application of Bacteroides acidifaciens Strain to liver injury caused by apoptosis of hepatic cells mediated by CD95/CD95L death Signal pathway
Five mixed antibiotics (neomycin sulfate 10mg/ml, ampicillin 10mg/ml, metronidazole 10mg/ml, vancomycin 5mg/ml, amphotericin B0.1 mg/ml) were used for the intestinal flora removal of mice, and each mouse was subjected to gastric lavage for 200 μl, 2 times per day, 2 weeks after continuous gastric lavage, and then sterilized water was exchanged for two days of rest. The Bacteroides acidifaciens strain was treated with BLHK medium (BHI 38.5g/L, L-cysteine 0.25g/L, heme 5mg/L, vitamin K) 1 10 mg/l) was anaerobically cultured at 37℃for 36 hours, the culture was subjected to 9200 rpm/isolation at 4℃for 5 minutes to collect the cells, washed 2 times with sterilized PBS and resuspended to adjust the cell concentration to 3X 10 9 CFU/ml. This bacterial suspension was gavaged at a dose of 200. Mu.l/mouse to the above mixed antibiotic treated mice, 2 times per day for 1 week, and the mice that were gavaged Bacteroides acidifaciens were labeled BA, and the control group (control) was gavaged with wild-type mouse fecal bacteria, and Parabacteroides distasonis isolated from the intestinal tract of the mice was used as a control single bacteria, and the group labeled PD, parabacteroides distasonis was cultured and gavaged in the same manner as Bacteroides acidifaciens.
The 3 groups of mice were each given intraperitoneal injections (dose 0.2mg/kg body weight) with CD95 agonistic antibody Jo2 to activate the hepatic cell CD95/CD95L death signal pathway to cause liver injury, and after 12 hours the eyes were collected by intravenous blood sampling, and after 2 hours of blood standing, serum was obtained at 3700 rpm/15 minutes from heart separation, and the serum transaminase level was detected using the serum transaminase detection kit. After mouse livers are fixed by paraformaldehyde, HE staining is carried out to observe pathological damage conditions of the livers, and TUNEL apoptosis detection kit is used for detecting apoptosis conditions in liver slices.
The results of the serum transaminase level comparison of Jo 2-injected mice are shown in FIG. 3-a.
The comparison result of liver pathological injury (HE) and hepatocyte apoptosis (TUNEL) of Jo 2-injected mice is shown in fig. 3-b, wherein the HE graph shows that the area of the liver dark shadow-like injury area of the mice in the BA group is obviously smaller than that of the mice in the Control group and the PD group; TUNEL plot shows that the area and brightness of light fluorescent region (representing apoptotic region) of liver in BA group mice is significantly smaller than in Control and PD groups.
The results show that Bacteroides acidifaciens can obviously reduce the serum transaminase level, liver pathological damage and hepatic cell apoptosis of mice induced by Jo2 antibodies relative to the control group and the PD group, and show that Bacteroides acidifaciens has an inhibitory effect on CD95 agonist Jo2 induced liver damage of mice, so that Bacteroides acidifaciens can inhibit CD95 mediated hepatic cell apoptosis and hepatic damage on animal level by inhibiting the expression of death receptor CD 95.
Example 3, bacteroides acidifaciens use of Strain for inhibiting expression of hepatocyte apoptosis Signal protein in autoimmune hepatitis and alcoholic hepatitis
Five mixed antibiotics (neomycin sulfate 10mg/ml, ampicillin 10mg/ml, metronidazole 10mg/ml, vancomycin 5mg/ml, amphotericin B0.1 mg/ml) were used for the intestinal flora removal of mice, each mice was filled with 200 μl of stomach, 2 times per day, 2 weeks after continuous gastric filling, sterile water was changed, and the mice were allowed to rest for two days. The Bacteroides acidifaciens strain was treated with BLHK medium (BHI 38.5g/L, L-cysteine 0.25g/L, heme 5mg/L, vitamin K) 1 10 mg/l) was anaerobically cultured at 37℃for 36 hours, the culture was subjected to 9200 rpm/isolation at 4℃for 5 minutes to collect the cells, washed 2 times with sterilized PBS and resuspended to adjust the cell concentration to 3X 10 9 CFU/ml. The method comprisesThe bacterial suspension was gavaged at a dose of 200. Mu.l/mouse to the above mixed antibiotic treated mice, 2 times per day for 1 week, and the mice were labeled BA by gavage Bacteroides acidifaciens, and the control group (control) was gavaged with wild-type mouse fecal bacteria, and Parabacteroides distasonis isolated from the intestinal tract of the mice was used as a control single bacteria, and the group was labeled PD, and the culture and gavage manners of Parabacteroides distasonis were the same as Bacteroides acidifaciens.
Canavalia gladiata was injected at a dose of 10mg/kg through the tail vein to induce autoimmune hepatitis in mice. After 8 hours of injection, mouse livers were taken, total liver proteins were extracted, western antibodies to CD95 and caspase-3 were used for immunoblotting experiments, and immunohistochemical detection was performed using immunohistochemical antibodies to CD 95.
The immunoblotting detection results of liver CD95 and caspase-3 of the self-liver-free mice 8 hours after the injection of the canavalin A are shown in a figure 4-a, wherein the black stripe depth represents the expression level of CD95 or caspase-3 protein, and the figure shows that the expression level of CD95 and caspase-3 protein in the liver of the mice in the BA group is obviously lower than that of the mice in the control group (Con) and the PD group.
The immunohistochemical results of liver CD95 of the self-liver-free mice are shown in FIG. 4-b, the dark areas in the CD95 IHC graph represent the death receptor CD95 staining positive areas, and the graph shows that the area and the color depth of the liver CD95 positive areas of the mice in the BA group are obviously smaller than those of the Control group and the PD group.
The results show that Bacteroides acidifaciens can significantly reduce the expression of mouse liver death receptor CD95 and apoptosis protease caspase-3 relative to the control group and PD group, thereby inhibiting hepatocyte apoptosis.
Preparing 50% ethanol from analytically pure absolute ethanol, and performing gastric lavage on a mouse at a dosage of 7.5ml/kg body weight to induce alcoholic hepatitis of the mouse, taking the liver of the mouse after 8 hours, extracting total protein of the liver, performing an immunoblotting experiment by using a Western antibody of CD95 and caspase-3, and performing immunohistochemical detection by using an immunohistochemical antibody of CD 95.
The immunoblotting detection results of liver CD95 and caspase-3 of the mice with alcoholic liver are shown in figure 4-c 8 hours after 50% ethanol is infused into the stomach, wherein the black stripe depth represents the level of the expression level of CD95 or caspase-3 protein, and the figure shows that the expression level of CD95 and caspase-3 protein in the liver of the mice in the BA group is obviously lower than that in the control group (Con) and the PD group;
the immunohistochemical results of liver CD95 of mice with alcoholic liver are shown in FIG. 4-d, the dark area in the CD95 IHC graph represents the death receptor CD95 staining positive area, and the graph shows that the area and the color depth of the liver CD95 positive area of mice in the BA group are obviously smaller than those in the Control group and the PD group.
The results show that Bacteroides acidifaciens can significantly reduce the expression of mouse liver death receptor CD95 and apoptosis protease caspase-3 relative to the control group and PD group, thereby inhibiting hepatocyte apoptosis.
Example 4 comparison of the effects of the Bacteroides acidifaciens Strain and other isolated strains on inhibition of autoimmune hepatitis
The strain isolation method comprises the following steps: mashing fresh feces of mice in PBS at 4 ℃, centrifuging for 5 minutes at 200 rpm, removing precipitates, diluting the supernatant to a proper proportion, coating on a blood agar plate, performing anaerobic culture, transferring single colonies to a BLHK culture plate for anaerobic culture, simultaneously performing colony PCR identification on the single colonies by adopting identification primers designed according to corresponding 16S rDNA sequences, selecting positive colonies of PCR results, performing streak separation, performing PCR identification again after anaerobic culture, performing complete sequence sequencing confirmation of 16S rDNA, storing in a glycerol tube form in a refrigerator at-80 ℃ after confirmation, and separating 5 bacterial strains according to the method, wherein the bacterial strains are respectively: bacteroides Acidifaciens (BA), parabacteroides Distasonis (PD), bacteroides Thetaiotaomicron (BT), bacteroides Dorei (BD) and Bacteroides Uniformis (BU).
Five mixed antibiotics (neomycin sulfate 10mg/ml, ampicillin 10mg/ml, metronidazole 10mg/ml, vancomycin 5mg/ml, amphotericin B0.1 mg/ml) were used for the intestinal flora removal of mice, each mice was filled with 200 μl of stomach, 2 times per day, 2 weeks after continuous gastric filling, sterile water was changed, and the mice were allowed to rest for two days. The 5 strains were treated with BLHK medium (BHI 38.5g/L, L-cysteine 0.25g/L, heme 5mg/L, vitamin K) 1 10 mg/l) was anaerobically incubated at 37℃for 36 hours, and the cultures were isolated at 9200 rpm at 4℃for 5 minutesThe cells were collected, washed 2 times with sterile PBS and resuspended to adjust the cell concentration to 3X 10 9 CFU/ml. The bacterial suspension was inoculated into the above-mentioned mixed antibiotic-treated mice at a dose of 200. Mu.l/mouse, 2 times per day for 1 week, the mice in which the stomach was inoculated Bacteroides acidifaciens were labeled BA, the mice in which the stomach was Parabacteroides distasonis were labeled PD, the mice in which the stomach was Bacteroides thetaiotaomicron were labeled BT, the mice in which the stomach was Bacteroides dorei were labeled BD, the mice in which the stomach was Bacteroides uniformis were labeled BU, and the Control group (Control) was inoculated with wild-type mouse fecal bacteria in the same manner as the 5 strains were cultured and inoculated.
Canavalia gladiata was injected at a dose of 10mg/kg through the tail vein to induce autoimmune hepatitis in mice. After injection for 12 hours, the eye vein was sampled, and after leaving the blood for 2 hours, serum was obtained at 3700 rpm/min from the heart, and the serum transaminase level was measured using the serum transaminase measurement kit. After mouse livers are fixed by paraformaldehyde, HE staining is carried out to observe pathological damage conditions of the livers, a TUNEL apoptosis detection kit is used for detecting apoptosis conditions in liver slices, and CD95 immunohistochemical staining is carried out.
The results of the effect on serum transaminase levels in autoimmune hepatitis mice are shown in FIG. 5-a.
The results of immunohistochemistry for hepatocase injury (HE), hepatocyte apoptosis (TUNEL) and hepatic CD95 in autoimmune hepatitis mice are shown in fig. 5-b, where HE plot shows that the area of injury in the liver plaques of Bacteroides acidifaciens treated mice (BA) is significantly less than that of Control and Control single bacteria Parabacteroides distasonis treated groups (PD), control single bacteria Bacteroides thetaiotaomicro treated group (BT), control single bacteria Bacteroides dorei treated group (BD), control single bacteria Bacteroides uniformis treated group (BU); TUNEL plot shows that the area and brightness of light fluorescent region (representing apoptotic region) of liver in BA group mice is significantly smaller than that of Control and PD, BD, BT, BU groups; the dark areas in the CD95 IHC plot represent the death receptor CD95 positive areas, which shows that the area and shade of the liver CD95 positive areas of the BA group mice are significantly smaller than those of the Control and PD, BD, BT, BU groups.
The results show that only Bacteroides acidifaciens can significantly reduce the serum transaminase level, liver pathological damage and hepatocyte apoptosis of the mice induced by canavalin a relative to other 4 strains of single bacteria isolated from the intestinal tract.
Claims (10)
1. The application of the intestinal bacteria in preparing the medicines for treating the liver injury diseases caused by apoptosis is characterized in that the intestinal bacteria is Bacteroides acidifaciens.
2. The use according to claim 1, wherein the apoptosis-induced liver injury disease is a liver apoptosis-related disease mediated by CD95/CD95L death signal.
3. The use according to claim 2, wherein said liver apoptosis-related disease includes, but is not limited to, autoimmune hepatitis, alcoholic liver disease, viral hepatitis, drug/poison hepatitis, liver fibrosis, cirrhosis or liver cancer.
4. The use according to any one of claims 1 to 3 wherein Bacteroides acidifaciens inhibits liver cell apoptosis and liver damage by inhibiting expression of the liver death receptor CD95 and/or the apoptosis protease caspase-3, to treat liver disease.
5. The use according to claim 4, wherein the cell concentration of Bacteroides acidifaciens is not less than 3X 10 9 CFU/ml。
6. The use according to claim 5, wherein Bacteroides acidifaciens is prepared by the following steps:
anaerobic culturing Bacteroides acidifaciens strain with BLHK medium, centrifuging to collect thallus, washing with sterile PBS, and re-suspending to adjust thallus concentration to 3×10 or more 9 CFU/ml。
7. The use according to claim 6, wherein the BLHK medium comprises the following components: BHI38.5g/L, L-cysteine 0.25g/L, heme 5mg/L, vitamin K 1 10mg/l。
8. The use according to claim 6, wherein said anaerobic cultivation is anaerobic cultivation for 36 hours at 37 ℃.
9. The use according to any one of claims 6 to 8, wherein the Bacteroides acidifaciens strain is obtained by the following isolation method:
mashing fresh feces of mice or people in PBS, centrifuging, removing sediment, diluting the supernatant to a proper proportion, coating on a blood agar plate, performing anaerobic culture, transferring a single colony to a BLHK culture plate for anaerobic culture, performing colony PCR identification on the single colony by adopting an identification primer designed according to a 16S rDNA sequence of Bacteroides acidifaciens, selecting positive colonies of a PCR result for streaking separation, performing PCR identification again after anaerobic culture, performing complete sequence sequencing confirmation of the 16S rDNA, and storing in a refrigerator for standby after confirming.
10. A composition useful for treating liver injury caused by apoptosis, wherein the composition comprises enterobacteria Bacteroides acidifaciens.
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| US20190083599A1 (en) * | 2014-08-28 | 2019-03-21 | Yale University | Compositions and Methods for Treating an Inflammatory Disease or Disorder |
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| US20190083599A1 (en) * | 2014-08-28 | 2019-03-21 | Yale University | Compositions and Methods for Treating an Inflammatory Disease or Disorder |
| CN107206030A (en) * | 2014-12-08 | 2017-09-26 | 财团法人峨山社会福祉财团 | Include the pharmaceutical compositions that are used to prevent or treat metabolic disease of the raw sour bacteroid as active ingredient |
| US20170333492A1 (en) * | 2014-12-08 | 2017-11-23 | The Asan Foundation | Pharmaceutical composition for preventing or treating metabolic diseases, comprising bacteroides acidifaciens as active ingredient |
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