A strain of Enterococcus faecalis, a detection agent, a detection method and application thereof

Abstract

The present invention belongs to the field of medicine and microbial technology, and specifically relates to a strain of Enterococcus faecalis, a detection agent, a detection method and an application. The present invention provides Enterococcus faecalis xn‑en, with a deposit number of CGMCC No.31850. According to whole genome sequencing analysis, there are many drug-resistant genes and virulence genes in the Enterococcus faecalis xn‑en. The present invention reveals that the Enterococcus faecalis xn‑en extracted from the liver of MCD mice is a drug-resistant Enterococcus faecalis, and has the ability to aggravate the course of NASH. It can be seen that liver flora can also promote the development of liver disease. Enterococcus faecalis xn‑en can be used as a drug target to screen drugs. Drugs with antibacterial activity and the ability to regulate flora may also have anti-NASH and liver fibrosis activity, thereby screening more drugs for the treatment of liver disease.

Description

Technical Field

[0001] The invention belongs to the fields of medicine and microbial technology, and specifically relates to a strain of Enterococcus faecalis, a detection agent, a detection method and an application. Background Art

[0002] Non-alcoholic fatty liver disease (NAFLD) is a metabolic disease characterized by liver fat deposition. It is often closely related to metabolic syndromes such as obesity, dyslipidemia, type 2 diabetes and hypertension, and has become a global health problem. The development stages of NAFLD are divided into early and mid-stage non-alcoholic fatty liver, non-alcoholic steatohepatitis (NASH), liver fibrosis, and late-stage cirrhosis and liver cancer. The etiology of NAFLD is not yet fully understood, but it is generally recognized that the development of the disease is driven by multiple injuries, such as lipid metabolism disorders causing a large amount of lipotoxicity, which in turn produces oxidative stress, insulin resistance, persistent inflammatory response, Kupffer cell immunity, autophagy and apoptosis, etc. Therefore, most of the current clinical treatment drugs also target the above mechanisms or targets, such as statins, anti-inflammatory and liver-protecting drugs, metformin or pioglitazone, which improve insulin resistance, and improve lifestyle and increase exercise.

[0003] In recent years, studies have pointed out a new pathogenesis of NAFLD, namely: the intestinal flora affects the development of NAFLD through the gut-liver axis. The gut-liver axis refers to the close bidirectional connection between the intestine and the liver through the portal vein and bile duct. The intestinal flora can obtain substrates from endogenous (bile acids) and exogenous (high-fat and high-sugar foods or environment) pathways to produce various beneficial or harmful metabolites, which can be transported to the liver through the portal vein, thereby affecting the development of liver disease, such as stimulating inflammatory responses, oxidative stress, or directly causing liver cell damage. In patients with NAFLD, intestinal bacterial overgrowth and intestinal microecological imbalance can be observed, which inspired researchers to use probiotic preparations to regulate disordered intestinal flora, thereby achieving the effect of treating NAFLD. However, the existing technology lacks strains of Enterococcus faecalis for the development of NAFLD treatment drugs. Summary of the invention

[0004] The present invention provides a strain of Enterococcus faecalis, a detection agent, a detection method and an application. The Enterococcus faecalis xn-en can be used as a target to develop new NAFLD therapeutic drugs.

[0005] In order to solve the above technical problems, the following technical solutions are proposed:

[0006] The present invention provides a strain of Enterococcus faecalis ( Enterococcus faecalis )xn-en, the deposit number is CGMCC No. 31850.

[0007] The present invention provides a detection agent, characterized in that it comprises the Enterococcus faecalis xn-en described in the above technical solution.

[0008] Preferably, the application form of the Enterococcus faecalis xn-en includes a bacterial suspension.

[0009] Preferably, the method for preparing the bacterial suspension comprises the following steps: inoculating the Enterococcus faecalis xn-en into a culture medium for culturing to obtain a bacterial suspension.

[0010] The present invention provides the use of the Enterococcus faecalis xn-en described in the above technical solution or the detection agent described in the above technical solution in the preparation of one or both of the following:

[0011] (1) Products that predict or evaluate the efficacy of drugs in treating liver diseases;

[0012] (2) Products used as targets for screening drug types or drug concentrations for the treatment of liver diseases.

[0013] Preferably, the liver disease includes one or more of non-alcoholic fatty liver disease, non-alcoholic steatohepatitis and liver fibrosis.

[0014] The present invention provides a detection method, comprising the following steps: mixing the Enterococcus faecalis XN-EN described in the above technical solution or the detection agent described in the above technical solution with a drug to be screened, and screening the type or concentration of the drug for treating liver disease;

[0015] Alternatively, the Enterococcus faecalis xn-en described in the above technical solution or the detection agent described in the above technical solution is used to feed a liver disease animal model to predict or evaluate the effect of drug treatment of liver disease.

[0016] Preferably, the mixing method includes: mixing the drug to be screened with a culture medium to prepare a drug-containing culture medium, and inoculating the Enterococcus faecalis xn-en into the drug-containing culture medium.

[0017] Preferably, the predictive or evaluated indicators include: reducing one or more of the following: reducing the number of fat vacuoles in the animal liver, reducing inflammatory infiltration, reducing fat deposition, and reducing fibrotic collagen deposition.

[0018] Preferably, when predicting or evaluating the effect of a drug on liver disease, the number of viable Enterococcus faecalis xn-en fed to the animal is ≥10 9 CFU / piece.

[0019] Beneficial effects of the present invention: The present invention provides a strain of Enterococcus faecalis xn-en, with a deposit number of CGMCC No. 31850. The present invention isolates Enterococcus faecalis xn-en from the liver of mice suffering from NASH. After oral administration of Enterococcus faecalis xn-en to mice, it is found that Enterococcus faecalis xn-en can aggravate the fatty vacuolar accumulation and fibrosis of NASH in mice, and increase inflammatory infiltration in mice. The present invention reveals the important role of Enterococcus faecalis xn-en in the liver flora in the development of NAFLD, which will be conducive to the development of new NAFLD therapeutic drugs or targets. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 This is the colony picture of Enterococcus faecalis xn-en on Columbia blood agar plate;

[0021] Figure 2 This is a pie chart showing the distribution of homologous species in the NR database for Enterococcus faecalis xn-en;

[0022] Figure 3 This is the phylogenetic tree of Enterococcus faecalis xn-en based on 16S rRNA. Figure 3 mcd-en in represents xn-en;

[0023] Figure 4 This is the whole genome circle map of Enterococcus faecalis xn-en;

[0024] Figure 5 This is the circle map of the whole genome of Enterococcus faecalis ATCC 29212; Figure 4 and Figure 5 Key information notes:

[0025] The circle diagram displays from the outside to the inside: GC content, sequencing depth, gene element display (CDS: coding sequence; rRNA; pseudo: pseudogene; tRNA), advanced annotation (VFDB: virulence gene; CARD: drug resistance gene) and COG function display on the left (A~Z represent different COG classifications, such as A represents genes related to "RNA processing and modification");

[0026] Figure 6 This is a statistical chart of the types of antibiotics that Enterococcus faecalis xn-en is resistant to;

[0027] Figure 7 This is a statistical chart of the types of antibiotics that Enterococcus faecalis ATCC 29212 is resistant to;

[0028] Figure 8 This is a diagram of the top ten virulence gene types of Enterococcus faecalis xn-en;

[0029] Fig. 9This is a map of the top ten virulence gene types of Enterococcus faecalis ATCC 29212;

[0030] Fig.10 The hematoxylin-eosin staining, Sirius Red staining and Oil Red O staining images of the liver sections of mice in each group, where H&E represents hematoxylin-eosin staining, Sirius Red represents Sirius Red staining, and Oil Red O represents Oil Red staining.

[0031] Biological Deposit Description

[0032] Enterococcus faecalis ( Enterococcus faecalis )xn-en, classified as Enterococcus faecalis ( Enterococcus faecalis ), was deposited in the General Microbiology Center of China Microorganism Culture Collection Committee (CGMCC) on September 4, 2024. The deposit address is: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC No. 31850. DETAILED DESCRIPTION

[0033] The present invention provides a strain of Enterococcus faecalis ( Enterococcus faecalis ) xn-en, the deposit number is CGMCC No. 31850. The 16S rRNA gene sequence of the Enterococcus faecalis xn-en of the present invention is shown in SEQ ID No.1.

[0034] The present invention uses the liver of mice that have already suffered from non-alcoholic fatty hepatitis (NASH) and liver fibrosis as the original material, and isolates a strain of Enterococcus faecalis xn-en. Enterococcus faecalis xn-en can aggravate the liver damage, inflammatory infiltration, fat deposition and fibrotic collagen deposition of mice, and has the ability to aggravate the course of NASH. It can be seen that the liver flora will also promote the development of liver disease, and the liver flora is also a potential pathogenesis of liver disease. According to whole genome sequencing analysis, there are many drug resistance genes and virulence genes in the Enterococcus faecalis xn-en of the present invention, among which the drug resistance genes related to sulfonamide antibiotics, diaminopyrimidine antibiotics, and phosphate antibiotics are unique to Enterococcus faecalis xn-en. It can be seen that Enterococcus faecalis xn-en is a drug-resistant Enterococcus faecalis. In subsequent drug design, Enterococcus faecalis xn-en can be used as a drug target, or it can be inspired to think that drugs with antibacterial activity and the ability to regulate flora may also have anti-NASH and liver fibrosis activity, thereby screening drugs for the treatment of liver disease.

[0035] The colony characteristics of the Enterococcus faecalis xn-en of the present invention are: the colonies formed on the Columbia blood plate are small, round, convex, grayish white, with a diameter between 1 and 2 mm, relatively smooth, and belong to Gram-positive bacteria and facultative anaerobic bacteria. The 16S rRNA sequence of the Enterococcus faecalis xn-en is used to perform a BLAST homologous sequence search in NCBI, and a phylogenetic tree is constructed using the obtained sequences with high homology, and the strain xn-en is determined to be Enterococcus faecalis ( Enterococcus faecalis ).

[0036] The present invention provides a detection agent, comprising the Enterococcus faecalis xn-en described in the above technical solution.

[0037] As an optional embodiment, the application form of the Enterococcus faecalis xn-en of the present invention includes a bacterial suspension.

[0038] As an optional embodiment, the method for preparing the bacterial suspension of the present invention comprises the following steps: inoculating the Enterococcus faecalis xn-en into a culture medium for culturing to obtain a bacterial suspension.

[0039] As another optional embodiment, the Enterococcus faecalis xn-en is streaked and inoculated into Columbia blood culture medium for activation culture to obtain a single colony. As an optional embodiment, the present invention does not specifically limit the method of streaking inoculation, and a conventional method can be used. As an optional embodiment, the temperature of the activation culture of the present invention is 30°C~37°C, or 35~37°C; in a specific embodiment of the present invention, the temperature of the activation culture is 30, 33, 34, 35, 36 or 37°C. The time of the activation culture is 22~26h, or 23~25h. In a specific embodiment of the present invention, the time of the activation culture is 22, 23, 24, 25 or 26h. The activation culture is an aerobic culture.

[0040] As another optional embodiment, a single colony is obtained, and the present invention inoculates the single colony into a brain heart infusion medium for culture to obtain a bacterial suspension. As an optional embodiment, the present invention does not specifically limit the method of inoculation, and a conventional method can be used. As an optional embodiment, the culture temperature is 30°C to 37°C, or 35 to 37°C; in a specific embodiment of the present invention, the culture temperature is 30, 32, 34, 35, 36 or 37°C. The culture time of the present invention is 16 to 18 hours, and in a specific embodiment of the present invention, the culture time is 16, 17 or 18 hours. The culture speed of the present invention is 190 to 210 rpm, and in a specific embodiment of the present invention, the culture speed is 190, 200 or 210 rpm. As an optional embodiment, the composition of the brain heart infusion culture medium includes 5.0 g / L peptone, 4 g / L dehydrated calf brain infusion powder, 4 g / L dehydrated buffalo heart infusion powder, 5 g / L sodium chloride, 2 g / L glucose and 2.5 g / L disodium hydrogen phosphate.

[0041] The effective viable count of Enterococcus faecalis xn-en in the bacterial suspension of the present invention is ≥1×10 9 CFU / mL.

[0042] The present invention provides the use of the Enterococcus faecalis xn-en described in the above technical solution or the detection agent described in the above technical solution in the preparation of one or both of the following:

[0043] (1) Products that predict or evaluate the effects of drugs in treating liver diseases;

[0044] (2) Products used as targets for screening drug types or drug concentrations for the treatment of liver diseases.

[0045] As an optional embodiment, the liver disease described in the present invention includes one or more of non-alcoholic fatty liver disease, non-alcoholic steatohepatitis and liver fibrosis.

[0046] As an optional embodiment, the product of the present invention includes a reagent, a test paper or a test kit.

[0047] The present invention provides a detection method, comprising the following steps: mixing the Enterococcus faecalis XN-EN described in the above technical solution or the detection agent described in the above technical solution with a drug to be screened, and screening the type or concentration of the drug for treating liver disease.

[0048] As an optional embodiment, the present invention does not specifically limit the mixing method, and a conventional method can be used. In the present invention, the mixing method includes: mixing the drug to be screened with a culture medium to prepare a drug-containing culture medium, and inoculating the Enterococcus faecalis xn-en into the drug-containing culture medium. The present invention does not specifically limit the type and type of the culture medium, and a culture medium in which Enterococcus faecalis xn-en can grow normally can be used.

[0049] When the type of drug to be screened is determined, the present invention inoculates the Enterococcus faecalis xn-en into a drug-containing culture medium. If the Enterococcus faecalis xn-en does not grow or its growth state is inhibited, the drug to be screened can be used as a potential drug for treating liver disease.

[0050] When determining the drug concentration of the drug to be screened, the present invention dilutes the drug to be screened into different concentrations, and the Enterococcus faecalis xn-en of the present invention is inoculated in a culture medium containing different drug concentrations. The MIC value of the drug is the lowest drug concentration that can inhibit the growth and reproduction of Enterococcus faecalis xn-en. The MIC value of the drug to be screened is the potential drug concentration for screening and treating liver disease. The present invention does not specifically limit the method for determining the MIC value of the drug to be screened, and conventional methods can be used.

[0051] The present invention also provides a detection method, comprising the following steps: feeding the Enterococcus faecalis XN-EN described in the above technical solution or the detection agent described in the above technical solution to a liver disease animal model to predict or evaluate the effect of drug treatment of liver disease.

[0052] As an optional embodiment, the predicted or evaluated index includes: reducing the number of fat vacuoles in the animal liver, reducing inflammatory infiltration, reducing fat deposition and reducing fibrotic collagen deposition, or more than two of them. When the number of fat vacuoles in the animal liver, reducing inflammatory infiltration, reducing fat deposition and reducing fibrotic collagen deposition are reduced, it indicates that Enterococcus faecalis xn-en is inhibited or killed, the liver disease is improved, and the therapeutic effect of the drug is good.

[0053] As an optional embodiment, when predicting or evaluating the effect of a drug on liver disease in the present invention, the number of viable Enterococcus faecalis xn-en fed to the animal is ≥10 9 CFU / unit; As an optional embodiment, the feeding method includes gavage. The present invention has no special limitation on the preparation method of the animal model, and a conventional method can be used. As an optional embodiment, the animal model includes a mouse model.

[0054] In order to further illustrate the present invention, the technical solution provided by the present invention is described in detail below in conjunction with the accompanying drawings and embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0055] Example 1 Cultivation and preservation of strains

[0056] 1. Cultivation and purification of strains

[0057] (1) Strain source: A mouse model with non-alcoholic fatty hepatitis was constructed. The specific construction method is as follows: C57BL / 6 male mice were fed MCD feed for seven weeks, the liver was removed under sterile operation, and serum tests and pathological sections of the liver were performed. Serum tests and pathological sections showed that the mice had non-alcoholic fatty hepatitis and mild liver fibrosis, indicating that the model was successfully established.

[0058] (2) Preparation of tissue samples: Under a sterile environment, place the liver of a mouse that has been successfully modeled into a tissue grinder, add sterile PBS in a certain proportion, and grind until there is no obvious lumpy tissue to obtain a tissue suspension.

[0059] (3) Preparation of Columbia blood agar plates: Prepare the culture medium according to the dosage specified in the instructions for use of Columbia agar medium powder. Specifically, take a certain amount of Columbia agar medium powder (purchased from Qingdao Haibo Biotechnology Co., Ltd.), add 200 mL of purified water, heat and stir until the powder dissolves, sterilize with high-pressure steam at 120°C for 20 min, and when the culture medium temperature cools to 40-50°C, add sterile defibrinated sheep blood (purchased from Nanjing Xavier Biotechnology Co., Ltd.) to make the mass concentration of sterile defibrinated sheep blood in the culture medium 10%. Mix well and pour into a plate, cool and set aside.

[0060] (4) Strain culture: Take 200 μL of the tissue suspension from step (2) and spread it evenly on the Columbia blood plate from step (3), and culture it in a 37°C constant temperature incubator under aerobic conditions for 24 h.

[0061] (5) Isolation and purification: Pick single colonies of different shapes, sizes, and colors on the plate, inoculate them into brain heart infusion liquid culture medium, and culture them on a constant temperature shaker at 37°C and 200 rpm for 16-18 hours to obtain a bacterial suspension. The bacterial suspension is then frozen in 20% glycerol and stored at -80°C for a long time.

[0062] 2. Strain identification

[0063] The above-mentioned isolated strains were subjected to 16S rRNA bacterial species identification, and the identification unit was Beijing Ruiboxingke Biotechnology Co., Ltd.

[0064] The primer sequences used in the identification process are as follows:

[0065] 27F: 5'-AGAGTTTGATCATGGCTC-3' (SEQ ID No. 2);

[0066] 1492R: 5'-TAGGGTTACCTTGTTACGACTT-3' (SEQ ID No. 3);

[0067] The obtained sequences were compared with the NCBI database by blast, and one of the strains was identified as Enterococcus faecalis ( Enterococcus faecalis ), named xn-en, that is, Enterococcus faecalis xn-en.

[0068] The 16S rRNA gene sequence of Enterococcus faecalis is shown in SEQ ID No. 1:

[0069]

[0070] 3. Strain characteristics

[0071] The colony characteristics of Enterococcus faecalis xn-en are: the colonies formed on Columbia blood agar are small, round, raised, grayish white, with a diameter of 1 to 2 mm, relatively smooth, and are Gram-positive bacteria with facultative anaerobic properties. Figure 1 .

[0072] 4. Whole genome sequencing

[0073] DNA of Enterococcus faecalis xn-en was extracted using a bacterial DNA extraction kit, and whole genome sequencing analysis was completed by Sangon Biotech (Shanghai) Co., Ltd. The original sequencing data was quality controlled and genome spliced, and NCBI-PGAP / Prokka, CRT and other software were used to predict gene elements, such as CDS, tRNA, rRNA, genomic islands, prophages, etc. The gene protein sequence was compared with multiple databases such as NR, KOG, COG, NR, GO, KEGG, VFDB, CARD, etc. to obtain its functional annotation information. The results are as follows:

[0074] (1) Basic information of the whole genome

[0075] The GC content of Enterococcus faecalis xn-en is 37.61%, the chromosome is circular, the length is 2822643bp, the Gap N (bp) is 0, and there is only 1 spliced ​​sequence / contig. Other specific information is shown in Table 1.

[0076] Table 1 Complete genome information of Enterococcus faecalis xn-en

[0077]

[0078] (2) Analysis of homologous genes and closely related strains

[0079] Ten strains of Enterococcus faecalis and Enterococcus faecalis xn-en were selected for comparison with the NR library, and the similarity of the transcript sequences of the species with those of similar species, as well as the functional information of the homologous sequences, were obtained. The results are shown in Figure 2 , 10 strains covered 85.6% of the E. faecalis xn-en sequences, including E. faecalis ( Enterococcus faecalis ) 1083 had the largest number of homologous sequences, accounting for 41.5%; no strains with homologous sequence alignment data of >99% with Enterococcus faecalis xn-en were found in the alignment, indicating that Enterococcus faecalis xn-en isolated from the liver of mice with NASH is a new strain.

[0080] The NCBI Blast+ whole genome was used to compare the homologous strain information with the NR database. The 16S rRNA sequence predicted by the gene was compared with the NCBI 16S database using Blast, and the parameter identify>95 was set. The top 30 16S rRNA sequences with the highest identify were selected to construct the phylogenetic tree. The results are shown in Figure 3 , Enterococcus faecalis xn-en has obvious homology with other strains, Enterococcus faecalis The evolutionary relationship of K137-la is relatively close.

[0081] (3) Analysis of drug resistance genes and virulence genes

[0082] The whole genome sequence of the reference strain Enterococcus faecalis ATCC 29212 was downloaded from NCBI, a whole genome circle map was drawn, and it was compared with the VFDB and CARD databases to obtain the annotation information of virulence genes and drug resistance genes. The results of drug resistance gene and virulence gene analysis are as follows:

[0083] Based on the whole genome sequence information of Enterococcus faecalis xn-en and Enterococcus faecalis ATCC 29212 and the annotation information of VFDB and CARD databases, the whole genome circle map was drawn, see Figure 4 and Figure 5 , where the inner blue circle represents drug resistance genes and the red circle represents virulence genes. According to statistics, there are 135 drug resistance genes, 210 core virulence genes, and 313 virulence factor-related genes annotated to Enterococcus faecalis xn-en. There are 93 drug resistance genes, 136 core virulence genes, and 217 virulence factor-related genes annotated to Enterococcus faecalis ATCC 29212.

[0084] The results of drug resistance gene prediction are shown in Figure 6 and Figure 7 ,according to Figure 6 and Figure 7 It can be seen that both Enterococcus faecalis xn-en and Enterococcus faecalis ATCC 29212 contain genes that are resistant to common antibiotics such as tetracycline antibiotics, macrolide antibiotics, and fluoroquinolone antibiotics, but Enterococcus faecalis xn-en has more and richer types of resistance genes. Among them, resistance genes related to sulfonamide antibiotics, diaminopyrimidine antibiotics, and phosphate antibiotics are unique to Enterococcus faecalis xn-en.

[0085] In the prediction of virulence genes, the top ten virulence gene types were counted, and the results are shown in Figure 8 and Fig. 9 It can be seen that the virulence genes related to adhesion, immune escape, and iron transport system in Enterococcus faecalis xn-en are richer than those in Enterococcus faecalis ATCC29212, and the virulence genes related to the quorum sensing system and ferrous iron uptake only exist in Enterococcus faecalis xn-en.

[0086] Example 2 Preparation of Chinese medicine self-microemulsion with antibacterial effect and investigation of its in vitro antibacterial ability

[0087] 1. Preparation of Chinese medicine self-microemulsion with antibacterial effect

[0088] A Chinese medicine self-microemulsion that can "inhibit the growth of Enterococcus faecalis xn-en" was designed to investigate whether it can offset the effect of Enterococcus faecalis xn-en on NASH. Natural Chinese medicine ingredients with antibacterial activity such as cinnamaldehyde, dihydrotanshinone, berberine, berberine, tea tree oil, etc. were selected, and an oil phase and an emulsifier were added to prepare a Chinese medicine self-microemulsion. The Chinese medicine ingredients selected by the present invention are cinnamaldehyde and dihydrotanshinone, and the prepared Chinese medicine self-microemulsion with antibacterial effect is referred to as CDR. At the same time, Enterococcus faecalis ATCC29212 was selected as a reference strain.

[0089] 2. Investigation of in vitro antibacterial ability

[0090] (1) Resuscitation and expansion of strains

[0091] The frozen Enterococcus faecalis xn-en in Example 1 was streaked and revived in Columbia blood agar plates and cultured aerobically at 37°C for 24 hours. A single colony was then picked and inoculated into brain heart infusion medium and cultured on a constant temperature shaker at 37°C and 200 rpm for 16-18 hours to obtain xn-en bacterial suspension, which was diluted into bacterial suspension 1. The number of viable bacteria in bacterial suspension 1 was 5×10 5 CFU / mL.

[0092] The preparation method of Enterococcus faecalis ATCC 29212 bacterial suspension was the same as that of Enterococcus faecalis xn-en bacterial suspension to obtain 29212 bacterial suspension, which was diluted to form bacterial suspension 2. The number of viable bacteria in bacterial suspension 2 was 5×10 5 CFU / mL.

[0093] (2) MIC determination

[0094] The first group of drugs (cinnamaldehyde API): Cinnamaldehyde API (purchased from Shanghai MacLean Biochemical Technology Co., Ltd.) was weighed and dissolved in dimethyl sulfoxide (DMSO) to prepare a 256 mg / mL stock solution, which was then added to the culture medium as needed to prepare a drug-containing agar plate with a final concentration of 0.1-1280 μg / mL.

[0095] The second group of drugs (dihydrotanshinone API): Weigh dihydrotanshinone API (purchased from Nanjing Yuanzhi Biotechnology Co., Ltd.), dissolve it in DMSO, prepare a 6.4 mg / mL stock solution, and then add it to the culture medium as needed to prepare a drug-containing agar plate with a final concentration of 0.1~1280 μg / mL.

[0096] The third group of drugs (physical mixture): Weigh the cinnamaldehyde and dihydrotanshinone raw materials, dissolve them in DMSO at a concentration ratio of cinnamaldehyde: dihydrotanshinone of 28:1, and then add them to the culture medium as needed to prepare a drug-containing agar plate with a final concentration of 0.1~1280 μg / mL.

[0097] The fourth group of drugs (CDR): Weigh 1g of the raw material cinnamaldehyde and 35mg of dihydrotanshinone, add 1g of oil phase (medium chain triglyceride) and 1g of emulsifier (polyoxyethylene castor oil EL), and mix well under magnetic stirring. The concentration ratio of cinnamaldehyde to dihydrotanshinone is 28:1, and then add it to the culture medium as needed to prepare a drug-containing agar plate with a final concentration of 0.1~1280 μg / mL.

[0098] The above-mentioned groups of drugs were added to uncoagulated Columbia blood culture medium to prepare drug-containing agar plates with a final concentration of 0.1-1280 μg / mL. Bacterial suspension 1 and bacterial suspension 2 were spotted ten times on the drug-containing plates, respectively, and cultured in a 37 ℃ constant temperature incubator under an aerobic environment for 24 h. The lowest CDR concentration at which no bacteria grew on the plate was recorded as the MIC value.

[0099] The results are shown in Table 2. It can be seen that CDR showed significant antibacterial effects on both Enterococcus faecalis xn-en and Enterococcus faecalis ATCC 29212. From the MIC value, the MIC value of CDR against Enterococcus faecalis xn-en was 4 times higher than that of Enterococcus faecalis ATCC 29212, which indicates that Enterococcus faecalis xn-en has higher drug resistance than ATCC 29212.

[0100] Table 2 MIC values ​​of drugs in each group

[0101]

[0102] In summary, the Chinese medicine self-microemulsion (CDR) with antibacterial effect prepared by the present invention has good antibacterial ability against Enterococcus faecalis xn-en, and can significantly inhibit the growth of Enterococcus faecalis at the MIC concentration, but the drug concentration required to inhibit Enterococcus faecalis xn-en is 4 times that of Enterococcus faecalis ATCC 29212, indicating that Enterococcus faecalis xn-en has higher drug resistance than ATCC 29212.

[0103] Example 3 Investigating the effect of Enterococcus faecalis xn-en on non-alcoholic steatohepatitis (NASH) in mice

[0104] 1. Experimental Animals

[0105] Male SPF-grade C57BL / 6 mice, weighing 20 g, were purchased from Beijing Huafukang Biotechnology Co., Ltd. The mice were kept at a constant temperature (22°C) and humidity (50%±10%) and had free access to drinking water. During the adaptation period, all mice were fed with experimental mouse growth and reproduction feed.

[0106] 2. Experimental methods

[0107] (1) Establishment of non-alcoholic steatohepatitis (NASH) model

[0108] The NASH model was established using MCD (methionine and choline deficient) feed. 25 experimental animals were randomly divided into 5 groups, 5 in each group. The specific conditions of experimental grouping, diet and drug administration are as follows:

[0109] Control group: The mice were given a normal diet for 7 weeks and gavaged with normal saline twice a week at a volume of 0.2 mL each time.

[0110] MCD group: The mice were given MCD diet for 7 weeks and gavaged with normal saline at a volume of 0.2 mL each time, twice a week.

[0111] The xn-en group was given an MCD diet for 7 weeks and gavaged with Enterococcus faecalis xn-en twice a week, with each gavage dose of 10 9 CFU / piece.

[0112] MCD+CDR group: The mice were given MCD diet for 7 weeks. Starting from the 5th week, they were gavaged daily with a Chinese medicine self-microemulsion with antibacterial effect (abbreviated as CDR, the preparation method of CDR was the same as that of Example 2). The dosage of CDR was cinnamaldehyde: 82 mg / kg / d, and dihydrotanshinone: 2.9 mg / kg / d.

[0113] The xn-en+CDR group was given an MCD diet for 7 weeks and gavaged with Enterococcus faecalis xn-en twice a week, with each gavage dose of 10 9 CFU / mouse. Starting from the 5th week, the rats were gavaged daily with a Chinese medicine self-microemulsion (abbreviated as CDR, the preparation method of CDR was the same as that of Example 2), and the dosage of CDR was cinnamaldehyde: 82 mg / kg / d, dihydrotanshinone: 2.9 mg / kg / d.

[0114] (2) Sampling and testing

[0115] After 7 weeks, the mice were fasted but not watered for 12 h, and blood and liver tissues of each group were collected for subsequent testing.

[0116] Liver section staining: The liver tissues of the animals in each group were fixed and then stained with hematoxylin-eosin (H&E), oil red (Oil Red O) and Sirius Red. The staining results are shown in Table 1. Fig.10As shown. The results showed that compared with the Control group, the livers of the mice in the MCD group showed a large amount of fat vacuoles, lipid degeneration and inflammatory infiltration, and showed a certain amount of collagen deposition. In the xn-en group, which was gavaged with Enterococcus faecalis xn-en on the basis of the MCD diet, the fat vacuoles in the livers of mice increased, and the inflammatory infiltration and fibrotic collagen deposition were aggravated, indicating that Enterococcus faecalis xn-en can aggravate the development of NASH and liver fibrosis. In the MCD+CDR group and the xn-en+CDR group, which were given the Chinese medicine self-microemulsion (CDR) with antibacterial effect, there was no obvious fat vacuoles and collagen deposition in the slice field of view, indicating that drugs with antibacterial effects on Enterococcus faecalis xn-en have potential anti-NASH effects.

[0117] In summary, the present invention reveals that the Enterococcus faecalis xn-en extracted from the liver of MCD mice is a drug-resistant Enterococcus faecalis and has the ability to aggravate the course of NASH. It can be seen that liver flora can also promote the development of liver disease. Enterococcus faecalis xn-en can be used as a drug target to screen drugs. Drugs with antibacterial activity and the ability to regulate flora may also have anti-NASH and liver fibrosis activity, thereby screening more drugs for the treatment of liver disease.

[0118] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A strain of Enterococcus faecalis ( Enterococcus faecalis )xn-en, the deposit number is CGMCC No. 31850.

2. A detection agent, characterized in that Comprising the Enterococcus faecalis xn-en described in claim 1.

3. The detection agent according to claim 2, characterized in that The application form of the Enterococcus faecalis xn-en includes a bacterial suspension.

4. The detection agent according to claim 3, characterized in that The preparation method of the bacterial suspension comprises the following steps: inoculating the Enterococcus faecalis xn-en into a culture medium for culturing to obtain a bacterial suspension.

5. Use of the Enterococcus faecalis xn-en according to claim 1 or the detection agent according to any one of claims 2 to 4 in the preparation of one or both of the following: (1) Products that predict or evaluate the efficacy of drugs in treating liver diseases; (2) Products used as targets for screening drug types or drug concentrations for the treatment of liver diseases.

6. The use according to claim 5, characterized in that: The liver disease is selected from one or more of non-alcoholic fatty liver disease, non-alcoholic steatohepatitis and liver fibrosis.

7. A detection method, characterized in that: The method comprises the following steps: mixing the Enterococcus faecalis xn-en described in claim 1 or the detection agent described in any one of claims 2 to 4 with a drug to be screened, and screening the type or concentration of the drug for treating liver disease; Alternatively, the Enterococcus faecalis XN-EN described in claim 1 or the detection agent described in any one of claims 2 to 4 is used to feed an animal model of liver disease to predict or evaluate the effect of drug treatment of liver disease; the liver disease is selected from one or more of non-alcoholic fatty liver disease, non-alcoholic steatohepatitis and liver fibrosis.

8. The method according to claim 7, characterized in that: The mixing method includes: mixing the drug to be screened with a culture medium to prepare a drug-containing culture medium, and inoculating the Enterococcus faecalis xn-en into the drug-containing culture medium.

9. The method according to claim 7, characterized in that: The prediction or evaluation indexes include: reducing one or more of the following: reducing the number of fat vacuoles in the animal liver, reducing inflammatory infiltration, reducing fat deposition, and reducing fibrotic collagen deposition.

10. The method according to claim 7 or 9, characterized in that: When predicting or evaluating the effect of a drug on liver disease, the viable count of Enterococcus faecalis xn-en fed to the animal is ≥10 9 CFU / piece.

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