Application of Erysipelastostridioeae as non-alcoholic steatohepatitis marker and kit

Abstract

The invention relates to an application of Erysipelastostridioeae as a marker of non-alcoholic steatohepatitis and a kit of the Erysipelastostridioeae. The disease degree of the non-alcoholic steatohepatitis is judged according to the abundance level of the Erysipelastostridioeae. The invention further relates to an application of the Erysipelastostridioeae as the marker of the non-alcoholic steatohepatitis and a kit of the Erysipelastostridioeae. According to the invention, a novel association between nicotine and NASH is found, through 16S rRNA sequencing, the phenomenon that Erysipelastostridioeae can be obviously increased in an NASH model due to nicotine exposure is revealed, and a potential action mechanism of the Erysipelastostridioeae in a disease progress is clarified.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to an application of Erysipelatoclostridiaceae as a marker of non-alcoholic fatty hepatitis and a kit. Background Art

[0002] Smoking is one of the most common unhealthy behaviors of human beings. The World Health Organization lists smoking as the leading cause of preventable death in the world. Quitting smoking is the most effective way to extend life expectancy, but it is not easy to quit smoking because nicotine is highly addictive and has withdrawal symptoms. Harmful substances in tobacco include nicotine, coal tar, carbon monoxide, etc., among which nicotine is the most important harmful substance.

[0003] Non-alcoholic fatty liver disease (NAFLD) includes a series of pathological changes, usually progressing from simple steatosis to non-alcoholic steatohepatitis (NASH), and then to cirrhosis and hepatocellular carcinoma in some cases. The diagnosis of NASH usually requires liver tissue biopsy or medical imaging examinations, which are risky, invasive and costly. Since metabolites produced in the intestine play a key role in the progression of NASH through the gut-liver axis, the intestinal flora refers to the symbiotic microbial community in the human intestine, and its composition and abundance are closely related to the health status of the host. In recent years, some studies have shown that dysbiosis of the intestinal flora is associated with the occurrence and progression of NASH. Therefore, the development of a non-invasive and effective diagnosis based on biomarkers is of great clinical significance for studying the role of smoking-related NASH. Summary of the invention

[0004] Purpose of the present invention: In order to overcome the defects of the prior art, the present invention provides an application of Erysipelatoclostridiaceae as a marker for non-alcoholic fatty hepatitis.

[0005] The technical solution of the present invention is to use Erysipelatoclostridiaceae as a marker for non-alcoholic fatty liver disease, and to judge the degree of pathology of non-alcoholic fatty liver disease by the abundance level of Erysipelatoclostridiaceae.

[0006] Preferably, the abundance of Erysipelatoclostridiaceae is obtained by sequencing 16SrRNA of microbial DNA in feces.

[0007] Another object of the present invention: In order to overcome the defects of the prior art, the present invention provides an Erysipelatoclostridiaceae kit.

[0008] The technical solution of the present invention is: an Erysipelatoclostridiaceae kit, comprising specific primers and probes suitable for detecting Erysipelatoclostridiaceae.

[0009] This study discovered a new association between nicotine and NASH. Through 16S rRNA sequencing, it was revealed that nicotine exposure can lead to a significant increase in Erysipelatoclostridiaceae in the NASH model, clarifying its potential mechanism of action in the disease process. A non-invasive diagnostic solution for specific intestinal flora markers has been developed for smokers, providing new possibilities for early prevention and intervention of NASH and avoiding the invasive operation of traditional liver biopsy. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] Figure 1 α diversity graph of each group of mice in a specific embodiment of the present invention; Figure 2 β diversity diagram of each group of mice in a specific embodiment of the present invention; Figure 3 It is a genus-level bacterial flora distribution diagram of a specific embodiment of the present invention; Figure 4 This is a diagram showing the effect of nicotine exposure on the structure of the NASH intestinal microbial community according to a specific embodiment of the present invention. DETAILED DESCRIPTION

[0011] The technical scheme in this embodiment will be described clearly and completely below in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0012] The relevant experimental steps are as follows: S1. Construction of nicotine exposure mouse model The following experiments all used male 8-week-old C57BL / 6J mice weighing 20-22 g purchased from Gaofei Biotechnology Co., Ltd. The mice (6 per cage) were housed in a pathogen-free environment with free access to water and food at a controlled temperature of 22°C and a photoperiod of 12 h light and 12 h dark.

[0013] The mice were divided into four groups and fed with different diets, namely, normal diet + saline group (Ctrl), normal diet + nicotine group (Ctrl+NIC), high-fat diet + saline group (CDHFD), and high-fat diet + nicotine group (CDHFD+NIC). Normal diet feed (1010086) was from Jiangsu Xietong Pharmaceutical Bioengineering Co., Ltd., and high-fat diet feed (A06071302) was from Research Diets (New Brunswick, NJ, USA). The mice were fed for 10 weeks.

[0014] The nicotine dosage was 1.5 mg / kg (N3876, Sigma-Aldrich) and was administered by intraperitoneal injection once a day for 10 weeks; at the end of the 10th week, the mice were fasted overnight before euthanasia. This project was conducted in the Experimental Animal Center of the First Affiliated Hospital of Wenzhou Medical University and was supervised and approved by the Institutional Animal Care and Use Committee of the First Affiliated Hospital of Wenzhou Medical University (WYYY-AEC-2023-154).

[0015] S2. Fecal samples collection of mice in each group Place the mouse in a sterile cage (such as a sterilized empty mouse cage or feces collector) and wait for it to defecate naturally; use a sterile toothpick or tweezers to collect the excrement in time to avoid contamination by the mouse. After collecting enough samples, put the mouse back into the cage.

[0016] S3. Detection and analysis of fecal flora abundance 1. Extract genomic DNA from stool samples using a DNA kit and assess the purity and concentration of the DNA.

[0017] 2. Amplify the V3-V4 variable region of the 16S rRNA gene by PCR using region-specific primers 338F 5'-barcode+ATCCTACGGGAGGCAGCA-3' and 806R 5'-GGACTACHVGGGTWTCTAAT-3' and high-fidelity DNA polymerase. The barcode in the front primer is a 7-10 base oligonucleotide sequence used to distinguish different samples in the same library.

[0018] 3. After the components required for the PCR reaction are configured, pre-denature at 98°C for 5 minutes on the PCR instrument to fully denature the template DNA, and then enter the amplification cycle. In each cycle, first keep it at 98°C for 30 seconds to denature the template, then lower the temperature to 52°C and keep it for 30 seconds to fully anneal the primers to the template; keep it at 72°C for 45 seconds to extend the primers on the template and synthesize DNA to complete a cycle, and repeat the cycle 25 times to enrich the amplified DNA fragments. Finally, keep it at 72°C for 5 minutes to extend the product completely and store it at 12°C. The amplification results were subjected to 2% agarose gel electrophoresis, and the target fragment was cut and the target fragment was recovered by the sorting magnetic bead recovery method.

[0019] 3. Library construction: Illumina's TruSeq Nano DNA LT Library Prep Kit was used to construct the library. Step 1: Perform end repair: Use End Repair Mix2 in the kit to remove the protruding bases at the 5' end of the DNA, fill in the missing bases at the 3' end, and add a phosphate group to the 5' end. Step 2: Add A to the 3' end. During this process, an A base will be added to the 3' end of the DNA to prevent the self-ligation of the DNA fragment, while ensuring that the DNA is connected to the sequencing adapter with a protruding T base at the 3' end. Step 3: Add an adapter with a specific label. This process is to allow the DNA to hybridize to the Flow Cell in the end. Step 4: Amplify the DNA fragments with the adapter added by PCR, and then use BECKMAN AMPureXP beads to purify the PCR system. Step 5: Perform final fragment selection and purification of the library by 2% agarose gel electrophoresis.

[0020] 4. Library quality inspection and quantification: Take 1 μL of the library and perform 2100 quality inspection on the library using the Agilent HighSensitivity DNA Kit on the Agilent Bioanalyzer machine. The qualified library has a single peak and no connector. Use the Quant-iTPicoGreen dsDNA Assay Kit to quantify the library on the Promega QuantiFluor. The concentration of the qualified library should be above 2 nM after calculation.

[0021] 5. Sequencing: For qualified libraries, we used the NovaSeq 6000 SPReagent Kit (500 cycles) on the Illumina NovaSeq machine to perform 2×250bp double-end sequencing. First, the library to be loaded on the machine (Index cannot be repeated) was gradient diluted to 2nM, and then mixed according to the required data volume ratio. The mixed library was denatured into a single strand with 0.1N NaOH for sequencing. The amount of library loaded can be controlled between 15-18pM according to the actual situation.

[0022] 6. Diversity analysis, using QIIME2 software to calculate alpha and beta diversity indices. Alpha diversity refers to the average species diversity in an area of ​​the environment, and beta diversity refers to the differences between different areas of the environment. The Shannon index is the most commonly used diversity index in ecological research. It not only considers species richness, but also species uniformity, so it is a comprehensive indicator of community structure. The principal coordinate analysis (PCoA) analysis method was used to find the differences between different samples (groups).

[0023] Experimental Results Compared with the CDHFD group, the alpha diversity of the CDHFD+NIC group was significantly reduced; PCoA analysis (beta diversity) showed that there were significant differences in the clustering of the intestinal microbiota of the four groups of mice due to different diets; at the genus level, Erysipelatocostridium (Erysipelothrix) was significantly increased in the CDHFD+NIC group. In addition, compared with the Ctrl group, the serum alanine aminotransferase (ALT) and serum aspartate aminotransferase (AST) levels of mice in the CDHFD+NIC group were significantly increased. The results showed that the relative abundance of Erysipelatocostridium was significantly increased in the CDHFD+NIC group, and nicotine exposure may affect the intestinal microbial composition of these specific bacterial families, leading to intestinal microecological balance. The increase in Erysipelatocostridium is a key bacterium for the aggravation of NASH. The severity of NASH can be judged according to the abundance of Erysipelatoclostridiaceae, which has the characteristics of accurate diagnosis, convenient operation and strong clinical practicality, and provides new possibilities for the early prevention and intervention of NASH.

Claims

1. A use of Erysipelatoclostridiaceae as a marker for non-alcoholic fatty hepatitis, characterized in that: The severity of nonalcoholic steatohepatitis was determined by the abundance level of Erysipelatoclostridiaceae.

2. The use of Erysipelatoclostridiaceae as a marker for non-alcoholic fatty hepatitis according to claim 1, characterized in that: The abundance of Erysipelatoclostridiaceae was obtained by 16S rRNA sequencing of microbial DNA in feces.

3. A kit for Erysipelatoclostridiaceae, characterized in that: Includes specific primers and probes suitable for detecting Erysipelatoclostridiaceae.

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