CN115097044A - Construction method for coprophilous bacteria transplantation through intervention of intestinal flora in rheumatoid arthritis model - Google Patents
Construction method for coprophilous bacteria transplantation through intervention of intestinal flora in rheumatoid arthritis model Download PDFInfo
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- CN115097044A CN115097044A CN202210772033.2A CN202210772033A CN115097044A CN 115097044 A CN115097044 A CN 115097044A CN 202210772033 A CN202210772033 A CN 202210772033A CN 115097044 A CN115097044 A CN 115097044A
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Abstract
The invention discloses a construction method for intervening rheumatoid arthritis models through intestinal flora in coprophilous fungi transplantation, and belongs to the technical field of model construction. The invention adopts UHPLC-Q-active Orbitrap-MS technology, analyzes the serum of a rat with rheumatoid arthritis through metabonomics, and screens differential metabolites to reveal the action mechanism of the coprophilic bacterium transplantation for interfering RA process by influencing intestinal flora. The invention screens 13 different metabolites including arachidonic acid, docosahexaenoic acid, 13S-hydroxy octadecadienoic acid and L-phenylalanine from rat serum, screens 3 metabolic pathways through the enrichment of the pathways: phenylalanine, tyrosine and tryptophan biosynthesis, phenylalanine metabolism and arachidonic acid metabolism. And proves that the coprophila rheumatica transplantation can influence the arachidonic acid metabolic pathway by improving the intestinal flora imbalance of the rheumatoid arthritis rats and intervene the progress of the rheumatoid arthritis by regulating the metabolic disturbance.
Description
Technical Field
The invention belongs to the technical field of model construction, and particularly relates to a construction method for intervening a rheumatoid arthritis model through intestinal flora in coprophilous fungi transplantation.
Background
Rheumatoid Arthritis (RA) is an autoimmune disease, mostly occurs in small joints of hands, wrists, feet, etc., has a long course of disease, and is mainly affected by environmental and genetic factors. Epidemiological characteristics show that the domestic morbidity reaches 0.32-0.38 percent, and the number of the diseases is accumulated to exceed 450 ten thousand, so that the Chinese medicinal preparation has high disability rate.
There are a large number of cells formed by the co-evolution of a large number of microorganisms and hosts in the human intestinal tract. The number and type of microflora can vary depending on a variety of factors such as the environment, diet, and the like. The normal growth and development of human body can not leave the balance of intestinal flora. The metabolic disturbance of the intestinal flora can cause the damage of the intestinal immune system, thereby causing immune inflammatory reaction and possibly leading to metabolic diseases, nervous system diseases, cancer and autoimmune system diseases. The intestinal flora is subject to metabolic changes in the affected body, which metabolizes fatty acids before they are absorbed, the changes in the microbiota composition lead to an imbalance in these metabolites, and elevated lipids can drive inflammation, thereby promoting metabolic disorders, inflammation and tumor growth. Clinical studies show that the number and the species of intestinal flora in RA patients are remarkably changed compared with those of healthy people, the proportion of harmful bacteria is increased, and the proportion of beneficial bacteria is decreased. Early RA patients underwent significant changes in microbial composition, mainly manifested by a decrease in bifidobacteria and bacteroides and an increase in prevotella. In the treatment of RA patients, the structure of intestinal flora is changed by intervention modes such as diet regulation, probiotic supplementation, excrement transplantation and the like, so that the effects of controlling inflammation and improving symptoms are achieved.
Metabonomics can quantitatively determine metabolites during the occurrence and development of diseases, reflect the change of metabolites at early metabolic stages in human bodies, and are considered to be an optimal means for revealing the pathogenesis of the diseases. Feces and serum from clinical cases can be used for metabonomics research, and a new way is provided for early diagnosis of diseases by finding metabolic markers in early diseases. The LC-MS technology is the most commonly used analysis technology in metabonomics at present due to its characteristics of high sensitivity, high speed, high resolution and the like.
Disclosure of Invention
Aiming at the problem that metabolites changing the structure of intestinal flora are undefined based on the intervention mode of coprophilous fungus transplantation at present, the invention provides a construction method for the coprophilous fungus transplantation to intervene in a rheumatoid arthritis model through the intestinal flora.
The invention adopts UHPLC-Q-active Orbitrap-MS technology, analyzes the serum of a rat with rheumatoid arthritis through metabonomics, and screens differential metabolites to reveal an action mechanism that coprophilic transplantation interferes RA process by influencing intestinal flora.
In order to achieve the purpose, the invention adopts the following technical scheme:
a construction method for intervening a rheumatoid arthritis model by coprophilous implantation through intestinal flora comprises the following steps:
step 3, using Compound discover to normalize the peak area;
step 4, observing the influence of FMT-F group on rat metabolites by performing PCA analysis and OPLS-DA analysis on SIMCA-P;
step 6, identifying and determining differential metabolites through an HMDB database;
and 7, performing channel enrichment analysis on the differential metabolites through MetabioAnalyst, and screening a key metabolic pathway with a channel Impact value larger than 0.1.
Further, the differential metabolites include Cholic Acid (Cholic Acid), Ethyl myristate (Ethyl myristate), Oleic Acid (Oleic Acid), Arachidonic Acid (Arachidonic Acid), Docosahexaenoic Acid (DHA, Docosahexaenoic Acid), L-Phenylalanine (L-Phenylalanine), D-Tryptophan (D-Tryptophan), D-galactose (Hexose), GINGEROL (10-GINGEROL), 2, 4-Dichlorotoluene (2, 4-Dichlorotoluene), N-undecylenic benzene (N-unsaturated sulfonic Acid), 12-Hydroxylauric Acid (12-hydroxyarylauric Acid), 13S-hydroxyoctadecadienoic Acid (13S-hydroxyoctadecadienoic Acid).
Further, the key metabolic pathway includes three metabolic pathways, which are: phenylalanine, tyrosine and tryptophan biosynthetic metabolic pathways, phenylalanine metabolic pathways and arachidonic acid metabolic pathways.
Further, the step 1 randomly divides the rats into a normal group (NC), a model group (CIA), a coprophila transplantation-Fengshining group (FMT-F group), a Tripterygium wilfordii polyglycoside group (TG group) and a Fengshining group (FSN group), then carries out drug administration treatment on the rats of each group, and the specific method for completing the preparation before detection is as follows:
after 1 week of adaptive feeding of rats, 30 rats were randomly divided into 5 groups of 6 per group, namely, normal group (NC group), model group (CIA group), coprophilic transplantation-Fengshining group (FMT-F group), Tripterygium wilfordii polyglycoside group (TG group) and Fengshining group (FSN group);
normal group (NC group) and model group (CIA group) are perfused with normal saline for 2 mL/d;
after an intestinal sterile model is established for a fecal bacteria transplantation-Fengshining group (FMT-F group), 2mL of fecal bacteria liquid is extracted by a needle tube every day, a needle head is removed, a transparent hose (about 1cm in length) is replaced, the fecal bacteria liquid is slowly injected into the anus of a rat in the FMT-F group, the anus of the rat is pressed by fingers after enema, the rat is inverted by 45 degrees to be in a head-down posture, the state is maintained for 1min, and the enema solution is prevented from flowing out;
tripterygium glycosides tablet (9mg/kg) is prepared into solution with normal saline, and administered at a dose of 2mL/d for intragastric administration;
2mL/d of the rheumatism group (FSN group) rat gavage rheumatism;
each group of rats was administered with the drug continuously for 21 days; all rats in each group were fasted for the first 1 day of the last dose; after administration for 1h in the last day, collecting blood from abdominal artery of all rats, centrifuging for 10min (4 deg.C, 14000rpm), collecting supernatant, subpackaging in EP tube, and storing at-80 deg.C for use; adding 300 mu L of acetonitrile into 100 mu L of serum, vortexing for 1min, centrifuging for 10min (4 ℃, 14000rpm), taking 200 mu L of supernatant, blowing the supernatant by nitrogen, adding 200 mu L of acetonitrile for redissolving, vortexing, centrifuging, and taking the supernatant for sample injection analysis.
Further, the model group (CIA group) needs to inject emulsifying agents at the root, the back and the sole of the tail of the rat respectively; the emulsifier is obtained by fully emulsifying bovine type II collagen and Freund's complete adjuvant on ice in a volume ratio of 1:1, and the collagen concentration of the obtained emulsifier is 1 mg/mL;
further, 3 antibiotics are adopted for processing to construct an intestinal sterile model, and the specific method comprises the following steps: 3 antibiotics of ampicillin (1g/mL), vancomycin (1g/mL) and imipenem (1g/mL) are dissolved in drinking water to prepare an antibiotic solution, the antibiotic solution is replaced every two days for 4 days continuously, and an intestinal sterile model is built.
Further, the preparation method of the fecal bacteria liquid comprises the steps of collecting fresh feces of Fengshining rats within 3 hours every day by using 2mL of EP tubes during an experiment, mixing 6 rat feces, fully and uniformly stirring, weighing 1.0g, adding 10mL of physiological saline, uniformly mixing for 2min in a vortex mode to prepare FMT-F suspension, filtering the FMT-F suspension by using a filter screen with the thickness of 1mm, centrifuging the FMT-F suspension for 15min (4 ℃, 14000rpm) by using a high-speed refrigerated centrifuge, removing supernatant, adding 10mL of physiological saline for redissolving, repeating the steps for 3 times to prepare the fecal bacteria liquid, and storing the fecal bacteria liquid in a refrigerator with the temperature of 4 ℃ for later use.
Further, the specific parameter conditions of the ultra-high performance liquid chromatography-quadrupole-electrostatic field orbital trap high resolution mass spectrometry combined technology (UHPLC-Q-active Orbitrap-MS) in the step 2 are as follows:
chromatographic conditions are as follows: using an ACQUITY BEH C18 chromatographic column; the mobile phase A is acetonitrile, and the mobile phase B is 0.01 percent formic acid aqueous solution;
mass spectrum conditions: the temperature of the ion transmission tube is 320 ℃; the S-Lens RF Level is 50; the full scanning/data depend on secondary scanning, and the scanning range is 100-1000 m/z; the primary mass resolution is 70000FWHM, the secondary resolution is 17500FWHM, and the collision energy is 30 eV;
further, the specific method for observing the effect of FMT-F on rat metabolites by PCA analysis and OPLS-DA analysis in SIMCA-P14.1 in said step 4 is: firstly, analyzing the data subjected to normalization processing, wherein Principal Component Analysis (PCA) in an unsupervised mode indicates the internal difference among groups, and orthogonal partial least squares discriminant analysis (OPLS-DA) in a supervised mode indicates the difference of metabolites in the groups; parameter R 2 Y (explanatory of evaluation model) and Q 2 (to evaluate the predictive nature of the model) to assess the quality of the model, the closer its value is to 1, the more successful the model is built; the reliability of the model was verified by permutation test (n ═ 200 times), preventing overfitting of the OPLS-DA model.
Compared with the prior art, the invention has the following advantages:
the invention screens 13 different metabolites including arachidonic acid, docosahexaenoic acid, 13S-hydroxy octadecadienoic acid and L-phenylalanine from rat serum, and screens 3 metabolic pathways through the enrichment of pathways: phenylalanine, tyrosine and tryptophan biosynthesis, phenylalanine metabolism and arachidonic acid metabolism. And proves that coprophilous bacterium transplantation can influence the arachidonic acid metabolic pathway by improving the intestinal flora imbalance of the rheumatoid arthritis rats and intervene the progress of the rheumatoid arthritis by regulating the metabolic disturbance.
Drawings
FIG. 1 is a total ion flow diagram of the sera of NC group, CIA group and FMT-F group under negative and positive ion modes;
FIG. 2A. PCA diagrams of NC, CIA, FMT-F, TG sets; diagrams of Hotelling' T2 of NC group, CIA group, FMT-F group and TG group;
FIG. 3A is a graph of OPLS-DA for NC and CIA groups; a permation diagram of an NC group and a CIA group; c, S-Plot of NC group and CIA group; OPLS-DA graphs of CIA group and FMT-F group; a permatation chart of a CIA group and an FMT-F group; S-Plot of CIA group and FMT-F group;
FIG. 4A. different metabolite Venn plots of CIA group versus NC group, CIA group versus FMT-F group; b.heat map of 13 differential metabolites (positive color yellow for high expression, positive correlation, and near negative color green for low expression, negative correlation);
FIG. 5 content variation of different differential metabolites in each group: (
N ═ 6), CIA group P < 0.05 compared to NC group; FMT-F group compared to CIA group # P＜0.05；
FIG. 6A.13 differential metabolite pathway analysis B.13 differential metabolite enrichment analysis;
FIG. 7 is a diagram showing the mechanistic effect of arachidonic acid metabolism on rheumatoid arthritis (PLC: phospholipase C; PLD: phospholipase D; PLA2: phospholipase A2; TLRs/NF-kB: Toll-like receptor/nuclear transcription factor-kB; COX: cyclooxygenase; LOX: lipoxygenase; CYP450: cytochrome P450).
Detailed Description
Materials and instruments
Animals: 30 Wistar female rats (SPF grade) with a body weight of about 225g were purchased from Beijing Wintorlington laboratory animal technology Limited, license number: SCXK (Beijing) 2016-. The temperature in the rat feeding room is kept at 15-25 ℃, the relative humidity is 45-55%, the rat is adaptively fed for a week in a natural light environment (12 hours of day and night alternation), the rat is freely fed with food and water, and padding is replaced for 1 time every 3 days.
Medicine preparation: the compound medicinal material for treating rheumatism is purchased from Ling Huan Tang pharmacy linkage company Limited in Jinzhong city.
Preparing the rheumatism medicament: decocting Notopterygii rhizoma, radix Angelicae Pubescentis, caulis Sinomenii, radix Clematidis, rhizoma Wenyujin Concisa, radix Saposhnikoviae, rhizoma Ligustici Chuanxiong, herba Ephedrae, cortex Cinnamomi, rhizoma Sparganii, sanguis Draxonis, rhizoma corydalis, radix Cyathulae, radix rehmanniae Preparata, fructus Amomi, rhizoma Zingiberis recens, and Glycyrrhrizae radix for two times, mixing, concentrating to 2g/mL, and storing in refrigerator at 4 deg.C for use.
The instrument comprises the following steps: a four-stage rod-electrostatic field orbit trap high-resolution mass spectrometer, a high-speed refrigerated centrifuge and a vortex mixer are selected.
TABLE 1 Instrument table
Reagent: selecting bovine type II Collagen (CII), Freund's complete adjuvant (CFA), sodium chloride injection (normal saline), tripterygium glycosides tablet, ampicillin capsule, vancomycin hydrochloride for injection, imipenem cilastatin sodium for injection, methanol (mass spectrum grade), acetonitrile (mass spectrum grade), formic acid (mass spectrum grade) and Wahaha purified water.
TABLE 2 reagent Table
Emulsification of collagen: bovine type II collagen in a ratio of 1:1 and Freund's complete adjuvant were emulsified to obtain collagen with a concentration of 1 mg/mL.
0.1mL of emulsifier is injected into the root part, the back part and the sole of the tail of the rat in the CIA group respectively, and 0.1mL of physiological saline is injected into the same part of the rat in the normal group. After one week, the immunization was boosted and a CIA rat model was established.
Treatment and administration of drugs
Establishing an intestinal tract sterile model
The fecal bacteria transplant group rats were treated with 3 antibiotics to construct an intestinal sterility model. The specific method comprises the following steps: 3 antibiotics of ampicillin (1g/mL), vancomycin (1g/mL) and imipenem (1g/mL) were dissolved in drinking water to prepare antibiotic solutions, which were changed every two days for 4 days. The remaining groups were given normal drinking water.
Preparation of FMT bacterial liquid
Collecting fresh feces of rats in Fengshining group within 3h by using a 2mL EP tube every day during an experimental period, mixing 6 rat feces, fully stirring, weighing 1.0g, adding 10mL of physiological saline, uniformly mixing for 2min in a vortex manner to prepare FMT-F suspension, filtering by using a 1mm filter screen, centrifuging by using a high-speed refrigerated centrifuge for 15min (4 ℃, 14000rpm), discarding supernatant, adding 10mL of physiological saline for redissolution, repeating the steps for 3 times to prepare fecal strain liquid, and storing the fecal strain liquid in a refrigerator at 4 ℃ for later use.
After constructing the intestinal sterile model, 2mL of fecal inoculum solution was taken out of the FMT-F group daily with a needle cannula, the needle was removed, the tube was replaced with a transparent tube, the fecal inoculum solution was slowly injected into the anus of the rat in the FMT-F group, the anus of the rat was pressed with fingers after the enema was performed, and the rat was inverted by 45 ℃ to keep the rat in a head-down posture for 1min, thereby preventing the enema solution from flowing out. The NC group and the CIA group are intragastrically filled with 2mL/d of physiological saline; preparing tripterygium glycosides tablet (9mg/kg) with normal saline to obtain solution, and administering 2mL/d of TG group for intragastric administration; the FSN group of rats was 2mL/d gastric lavage rheumatism. Rats in each group were dosed continuously for 21 days.
Serum collection and processing
All rats in the experimental group were fasted 1 day prior to the last dose. After 1h of administration, all rats were bled from the abdominal artery, centrifuged for 10min (4 ℃, 14000rpm), and the supernatant was taken and aliquoted into EP tubes and stored at-80 ℃ for future use.
Adding 300 mu L of acetonitrile into 100 mu L of serum, vortexing for 1min, centrifuging for 10min (4 ℃, 14000rpm), taking 200 mu L of supernatant, blowing the supernatant by nitrogen, adding 200 mu L of acetonitrile for redissolving, vortexing, centrifuging, and taking the supernatant for sample injection analysis.
UHPLC-Q-active Orbitrap-MS detection condition
Chromatographic conditions
An ACQUITY BEH C18 column (100 mm. times.2.1 mm, 1.7 μm) was used; the column temperature is 40 ℃, the volume flow is 0.3mL/min, and the sample injection amount is 5 mu L; mobile phase a was acetonitrile and mobile phase B was 0.01% aqueous formic acid, with gradient elution as in table 3 below.
TABLE 3 gradient elution conditions of mobile phase
Conditions of Mass Spectrometry
Electrospray ionization (ESI) was used for detection in positive and negative ionization mode (Table 4). The temperature of the ion transmission tube is 320 ℃; the S-Lens RF Level is 50; the full scanning/data depend on two-stage scanning, and the scanning range m/z is 100-1000; the primary mass resolution is 70000FWHM, the secondary resolution is 17500FWHM, and the collision energy is 30 eV.
TABLE 4 Mass Spectrometry conditions
Step 3, importing mass spectrum data into CD software (Compound discovery 3.0, Thermo, US) and exporting three-dimensional data of Retention Time (RT), mass-to-charge ratio (m/z) and peak area (A). After the peak area is normalized by baseline correction, denoising and the like,
step 4, the normalized data is led into SIMCA software (SIMCA14.1, China) to analyze the data, and the main mode of unsupervised modeComponent Analysis (PCA) indicates intrinsic differences between groups, and supervised-mode orthogonal partial least squares discriminant analysis (OPLS-DA) indicates differences in metabolites within groups. Parameter R 2 Y (explanatory of evaluation model) and Q 2 (predictive evaluation of the model) to assess the quality of the model, the closer its value is to 1, the more successful the model is built. The reliability of the model was verified by permutation test (n ═ 200 times) to prevent overfitting of the OPLS-DA model. Points in the score Plot (S-Plot) that are far from the center are considered to contribute more, with the more closely two angles the more strongly related the metabolite.
And 5, carrying out PCA analysis on the data of the rats in the NC group, the CIA group, the FMT-F group and the TG group, respectively carrying out OPLS-DA analysis on the rats in the NC group, the CIA group and the FMT-F group, and screening out the differential metabolites by combining a VIP value more than or equal to 1 and a P value less than 0.05 in an S-Plot.
And step 6, determining differential metabolites by combining an HMDB database, analyzing the content variation trend of each metabolite by using GraphPad Prism 8.0.2 software, and performing multi-group comparison by adopting One-way ANOVA analysis.
And 7, analyzing the metabolic pathways of the differential metabolites through an online analysis platform MetabioAnalyst, and performing visualization processing on involved metabolic pathways by using KEGG.
Results
UHPLC-MS analysis of in vivo metabolites: total ion flow graph (TIC) of metabolites under negative and positive ion modes of an NC group, a CIA group and an FMT-F group (figure 1), retention time is concentrated in 0-14.01 min, and the rat serum metabolites of the NC group, the CIA group and the FMT-F treatment group have obvious difference.
And (3) metabolic mapping analysis: principal Component Analysis (PCA) has visibility into general clusters, trends, or outliers in the observed values. PCA analysis is carried out on the NC group, the CIA model group, the FMT-F treatment group and the TG group, and the four groups of samples are found to have significant separation tendency in a score map (figure 2.A), the difference between the groups is large, and R is 2 Y＝0.872，Q 2 0.739, which proved to explain the metabolic differences between serum samples, was successfully established. The results show that the NC group and the CIA group are clearly distinguished in the horizontal direction, indicating that the metabolic profile of the CIA group is significantly separated from the NC group. FMT-F group andthe TG groups deviate from the CIA group and are close to the NC group, which shows that the 2 treatment methods have an improvement effect on RA, the metabolic contour center of the FMT-F group is closer to the NC group, and the regulation effect of the FMT-F group on the CIA rat serum metabolic disorder is better than that of the TG group. No outliers were shown in the Hotelling' T2 plot (fig. 2.B), therefore all data were included in further multivariate and univariate analyses.
CIA group vs. NC group
Constructing an OPLS-DA model between the CIA group and the NC group, wherein the model parameter R 2 Y＝0.937，Q 2 ＝0.974。 R 2 Y shows that the OPLS-DA model can account for group separation. Q 2 >0.5 indicates that the model has a high predictive power for the group-to-group discrimination.
The OPLS-DA score plot (fig. 3.a) shows that the CIA group had a significant separation trend from the NC cohort, indicating that the body metabolic profile of rheumatoid arthritis rats was altered. At the same time, an alignment test (n 200) was performed to verify the overfitting of the model, with the point in the upper right corner higher than the sample point on the left, Q 2 The negative half of the y-axis is crossed by the regression line of (c), indicating a high predictability of the model, and the results indicate no overfitting (fig. 3. B). Differential metabolites were screened by S-Plot (FIG. 3.C) in combination with VIP values ≥ 1 and P values < 0.05, and the metabolites were identified by HMDB database to obtain 55 differential metabolites.
fmt-F group of CIA group vs
In order to evaluate the effectiveness of the coprophila transplantation treatment group on improving the RA process, OPLS-DA and model verification (figure 3.D.E) are carried out on a CIA group and an FMT-F group, and the results show that the CIA group and the FMT-F treatment group can be separated obviously, and the coprophila transplantation treatment has a callback effect on the metabolic disorder of the rheumatoid arthritis rats. 20 differential metabolites were obtained after screening and identification by S-Plot (FIG. 3.F) in combination with VIP values ≥ 1 and P values < 0.05.
Screening for differential metabolites
The 55 and 20 different metabolites selected from the above CIA group and NC group, and CIA group and FMT-F group were assigned to Venn diagram (FIG. 4.A), and 13 different metabolites were selected (Table 5). The 13 differential metabolite clustering layers were subjected to heat mapping (FIG. 4.B), which indicated the degree of difference between the expression levels of the 13 metabolites among the different groups and the mean value by the shade of color based on the mean value of the expression levels of the metabolites of the same group.
The content of different metabolites in rats among the groups is changed (figure 5), and compared with the NC group, the levels of cholic acid, arachidonic acid, L-phenylalanine and 13S-hydroxyoctadecadienoic acid in the CIA group are obviously increased; the levels of ethyl myristate, oleic acid, docosahexaenoic acid, D-tryptophan, D-galactose, gingerol, 2, 4-dichlorotoluene, N-undecylenic benzene and 12-hydroxylauranic acid are obviously reduced.
Compared with the CIA group, the levels of FMT-F group cholic acid, docosahexaenoic acid, gingerol, 2, 4-dichlorotoluene, N-undecylenic benzene and 12-hydroxylauranic acid are obviously increased; the levels of ethyl myristate, oleic acid, arachidonic acid, L-phenylalanine, D-tryptophan, D-galactose and 13S-hydroxyoctadecadienoic acid are obviously reduced.
Wherein, 8 metabolites, namely arachidonic acid, docosahexaenoic acid, L-phenylalanine, gingerol, 2, 4-dichlorotoluene, N-undecylenic benzene, 12-hydroxy lauric acid and 13S-hydroxy octadecadienoic acid have obvious callback function.
TABLE 5 differential metabolites
Metabolic pathway analysis
MetabioAnalyst analysis was performed using an online analysis platform to find metabolic pathways relevant to affecting RA disease progression. When 13 different metabolites were introduced into MetabioAnalyst and analyzed, the pathway was considered to be closely related if the pathway Impact value > 0.1. Thus, the metabolic pathways involved by the differential metabolites were found by the pathway enrichment analysis (fig. 6.a) to comprise 8 (table 6). Wherein the correlation between three metabolic pathways and the action mechanism of influencing RA by coprophilous fungus transplantation is highest: -Phenylalanine, tyrosine and tryptophan biosynthesis (phenylalkane, tyrosine and tryptophan biosynthesis); ② Phenylalanine metabolism (Phenylalanine metabolism), and L-Phenylalanine participates in two metabolic pathways; ③ Arachidonic acid metabolism (Arachidonic acid metabolism), and Arachidonic acid is involved in the metabolic pathway. Although the value of Galactose metabolism (Galactose metabolism) Impact is less than 0.1, the pathway also contributes to a certain extent. In the enrichment analysis plot (FIG. 6.B), the higher position and the darker color indicate a strong correlation between the enriched metabolic pathways. The three metabolic pathways described above were observed in both pathway analysis and enrichment analysis. Therefore, the three approaches are proved to have important connection with the treatment effect of rheumatoid arthritis and coprophilous fungi transplantation.
TABLE 6 MetaboAnalyst pathway analysis results
Note: total is the number of compounds in the pathway; hit: (match value) number of metabolites matched to the pathway; raw p value-p value obtained by enrichment analysis; holm p: p-values corrected via Bonferroni; FDR p: p-value corrected via False Discovery Rate; impact: value of path impact
The experiments show that the metabolism of the CIA group is obviously changed compared with the NC group. Metabonomics analysis results show that the path with the largest contribution degree of the path influence in the process of the FMT-F group interfering rheumatoid arthritis is as follows: biosynthesis of phenylalanine, tyrosine and tryptophan; ② metabolism of phenylalanine; ③ metabolism of arachidonic acid. The metabolites affecting RA are mainly 13S-hydroxyoctadecadienoic Acid (13S-hydroxyoctadecadienoic Acid, 13S-HODE), Arachidonic Acid (Arachidonic Acid methylalism, AA), Docosahexaenoic Acid (DHA) and L-Phenylalanine (L-phenylalkanine).
13S-HODE is a secondary oxidation product from linoleic acid catalyzed by Lipoxygenase (LOX) and can also be converted from gamma-linolenic acid, which in turn can be converted to AA. HODE has effects of inducing monocyte and macrophage to accelerate apoptosis, promoting inflammation, etc., and during the disease development process, the number of inflammatory cells exceeds the scavenging ability of macrophage, and the disease process is accelerated. The 13S-HODE content level in the CIA model is increased and shows a proinflammatory effect, and after the treatment of the coprophila transplantation group, the content is reduced, so that the coprophila transplantation treatment group has a certain regulation effect on metabolic disorder of the coprophila transplantation treatment group, which suggests that coprophila transplantation can regulate the 13S-HODE level of the proinflammatory substance by improving intestinal flora and inhibit macrophage apoptosis to achieve the treatment effect.
AA is an omega-6 polyunsaturated fatty acid, exogenous arachidonic acid is converted from linoleic acid and arachidonic acid in food, endogenous arachidonic acid is mainly stored in cell membranes, mostly in the form of phospholipids. AA is produced by phospholipase A when the cells are in a stressed state 2 (PLA 2 ) Phospholipase C (PLC) and phospholipase D (PLD) are converted to free arachidonic acid and converted to pro-inflammatory substances such as eicosanoids by three pathways including Cyclooxygenase (COX), Lipoxygenase (LOX) and cytochrome P450(CYP 450). The COX, LOX pathway produces interleukin (IL-6), tumor necrosis factor-alpha (TNF-alpha), and interferon-alpha (INF-alpha), and the CYP450 pathway produces epoxyeicosatrienoic acids (EETs). It has been found that AA metabolite EETs has antagonistic effect on IL-6 and TNF-alpha, and the increase of EETs level in inflammatory body can cause the decrease of IL-6 and TNF-alpha expression. The AA metabolism pathway plays a key role in RA, and the AA metabolism produces inflammatory factors, triggers a defense mechanism of an organism and produces inflammatory reaction. It was found that a large number of T cells were aggregated in synovial tissue and synovium of RA patients. RA pathogenesis is complex, and researches show that T cells and antigen combine to promote macrophages to release inflammatory factors, and inflammatory signals activate synovial fibroblasts and chondrocytes to secrete various substances to damage joint tissues. Regulatory T cells (Treg) play a role in immune regulation and immune suppression, and CD4 in peripheral blood of patients with diseases + The number of Treg cells is small, the function of Treg is reduced, and Th1 cells release proinflammatory factors to drive harmful autoimmune response, so that the Th1 cells generate inflammatory response and induce RA. AA and AA metabolites in the CIA model are proinflammatory substances, the content of the CIA group is increased compared with that of the NC group, and the metabolic quantity of the CIA group is obviously reduced after the CIA group is treated by the fecal strain transplantation group, so that the fecal strain transplantation is supposed to be possible to change the composition of intestinal flora, inhibit the metabolism of arachidonic acid and improve the function reduction of TregThe low body drives the self-generated immune response to achieve the anti-inflammatory effect.
Toll-like receptors (TLRs) recognize inflammatory molecular signals and introduce the signals into cells, so that nuclear factor-kappa B (NF-kappa B) and the like are activated, and inflammatory reaction caused by the release of various inflammatory cell transmitters is also considered to be one of pathogenesis of RA. DHA can be converted from alpha-linolenic acid, can inhibit the release of proinflammatory cytokines such as interleukin IL-1 beta, TNF-alpha, IL-6 and the like, inhibit a nuclear factor-kB pathway, and achieve the effect of inhibiting inflammatory reaction. The experimental result shows that the content of the CIA group is reduced compared with that of the NC group, and the FMT-F can effectively improve the DHA content, which is probably one of the mechanisms of the FMT-F for playing the anti-inflammatory action.
The metabolism of the 13S-HODE, AA and DHA is catalyzed by the same enzyme, and antagonism exists among the three substances. The main pathway for the production of the proinflammatory substance eicosanoids is the arachidonic acid metabolic pathway, and at this time, 13S-HODE and DHA compete with AA for catalytic enzymes, resulting in reduced AA metabolism and decreased eicosanoid production, which is manifested by anti-inflammatory action.
Phenylalanine, tyrosine and tryptophan biosynthetic pathways synthesize precursors required for amino acid metabolism. Serum amino acid levels are positively correlated with the severity of osteoarthritis. Research shows that amino acid metabolism in RA patients is disordered, and the content of phenylalanine is obviously increased. Plasma concentrations of aspartic acid, tryptophan, phenylalanine, etc., may be valuable biomarkers for clinical diagnosis of RA patients. Compared with the NC group, the L-phenylalanine in the CIA model is up-regulated in the experiment, and consistent with the research result, the coprophilous fungus transplantation can regulate the amino acid metabolism of the organism and enable the phenylalanine concentration in blood plasma to tend to be normalized, so that the arthrocele and pain degree of RA patients are improved, and the condition is relieved.
In conclusion, the present invention adopts the LC-MS technology to identify 13 different metabolites related to RA from CIA rat serum, wherein 13S-hydroxy octadecadienoic acid, arachidonic acid, docosahexaenoic acid and L-phenylalanine. The following 3 metabolic pathways are presumed to be the most relevant metabolic pathways for fecal transplantation treatment of RA: phenylalanine, tyrosine and tryptophan biosynthesis, phenylalanine metabolism and arachidonic acid metabolism. Therefore, RA metabolic disorder may be related to amino acid anabolism and arachidonic acid metabolism, the treatment effect of fecal bacteria transplantation is determined based on metabonomics indexes, and the action mechanism (figure 7) of RA metabolic disorder may be that fecal bacteria transplantation influences the progress of rheumatoid arthritis by giving a rheumatoid arthritis rat a new intestinal flora, improving flora disorder, regulating organism arachidonic acid metabolism to be normalized and reducing the synthesis of a proinflammatory substance eicosanoid to relieve inflammatory reaction.
Those matters not described in detail in the present specification are well known in the art to which the skilled person pertains. Although illustrative embodiments of the present invention have been described above to facilitate the understanding of the present invention by those skilled in the art, it should be understood that the present invention is not limited to the scope of the embodiments, and various changes may be made apparent to those skilled in the art as long as they are within the spirit and scope of the present invention as defined and defined by the appended claims, and all matters of the invention which utilize the inventive concepts are protected.
Claims (9)
1. A construction method for intervening rheumatoid arthritis models through intestinal flora in coprophilous fungus transplantation is characterized by comprising the following steps: the method comprises the following steps:
step 1, randomly dividing rats into a normal group, a model group, a coprophila rheumatica transplanting group, a tripterygium glycosides group and a phenpromethazine group, then carrying out administration treatment on the rats in each group, and completing preparation before detection;
step 2, performing metabonomics analysis on each group of rat serum by adopting an ultra-high performance liquid chromatography-quadrupole-electrostatic field orbit trap high-resolution mass spectrometry technology;
step 3, using Compound discover to normalize the peak area;
step 4, observing the change of the metabolites of the rat by PCA analysis and OPLS-DA analysis through SIMCA-P;
step 5, performing visualization processing on data by using GraphPad Prism, and primarily screening out different metabolites;
step 6, identifying and determining differential metabolites through an HMDB database;
and 7, performing channel enrichment analysis on the differential metabolites through MetabioAnalyst, and screening a key metabolic pathway with a channel Impact value larger than 0.1.
2. The method for constructing the rheumatoid arthritis model through the intervention of the intestinal flora in coprophilous fungi transplantation according to the claim 1, which is characterized in that: the differential metabolites include cholic acid, ethyl myristate, oleic acid, arachidonic acid, docosahexaenoic acid, L-phenylalanine, D-tryptophan, D-galactose, gingerol, 2, 4-dichlorotoluene, N-undecylbenzene, 12-hydroxy lauric acid, and 13S-hydroxy octadecadienoic acid.
3. The method for constructing the rheumatoid arthritis model through the intervention of the intestinal flora in coprophilous fungi transplantation according to the claim 1, which is characterized in that: the key metabolic pathway comprises three metabolic pathways, which are respectively: phenylalanine, tyrosine and tryptophan biosynthetic metabolic pathways, phenylalanine metabolic pathways and arachidonic acid metabolic pathways.
4. The method for constructing the rheumatoid arthritis model through the intervention of the intestinal flora in coprophilous fungi transplantation according to the claim 1, which is characterized in that: the step 1 randomly divides the rats into a normal group, a model group, a coprophilous fungi transplantation-fengshining group, a tripterygium glycosides group and a fengshining group, then carries out administration treatment on the rats of each group, and the specific method for completing the preparation before detection is as follows:
after 1 week of adaptive feeding of rats, 30 rats were randomly divided into 5 groups, namely a normal group, a model group, a fecal strain transplantation-Fengshining group, a Tripterygium wilfordii polyglycoside group and a Fengshining group, and 6 rats in each group;
the normal group and the model group are perfused with normal saline for 2 mL/d;
after an intestinal sterile model is established for a coprophilous fungus transplantation-Fengshining group, 2mL of coprophilous fungus liquid is extracted by a needle tube every day, a needle head is removed, a transparent hose is replaced, the coprophilous fungus liquid is slowly injected into the anus of a rat in an FMT-F group, the anus of the rat is pressed by fingers after enema, the rat is turned over by 45 degrees to be in a head-down posture, the state is maintained for 1min, and enema solution is prevented from flowing out;
the tripterygium glycosides tablet is prepared into solution by normal saline and then is administered with 2mL/d for intragastric administration;
2mL/d of gavage rheumatism treatment agent for rats in the rheumatism treatment group;
each group of rats was administered with the drug continuously for 21 days; all rats in each group were fasted for the first 1 day of the last dose; after administration for 1h on the last day, collecting blood from abdominal artery of all rats, centrifuging for 10min at 4 deg.C and 14000rpm, collecting supernatant, subpackaging in EP tube, and storing at-80 deg.C for use; adding 300 mu L of acetonitrile into 100 mu L of serum, vortexing for 1min, centrifuging for 10min at 4 ℃, 14000rpm, taking 200 mu L of supernatant, blowing the supernatant by nitrogen, adding 200 mu L of acetonitrile for redissolving, vortexing, centrifuging, and taking the supernatant for sample injection analysis.
5. The method for constructing the rheumatoid arthritis model through the intervention of the intestinal flora in coprophilous fungi transplantation according to the claim 1, which is characterized in that: the model group is required to inject emulsifying agents at the root part, the back part and the sole part of the tail of the rat respectively; the emulsifier is obtained by fully emulsifying bovine type II collagen and Freund's complete adjuvant on ice in a volume ratio of 1:1, and the collagen concentration of the obtained emulsifier is 1 mg/mL.
6. The method for constructing the rheumatoid arthritis model through the intervention of the intestinal flora in coprophilous fungi transplantation according to the claim 1, which is characterized in that: 3 antibiotics are adopted for processing to construct an intestinal sterile model, and the specific method comprises the following steps: dissolving 3 antibiotics of ampicillin, vancomycin and imipenem in drinking water to prepare an antibiotic solution, replacing the antibiotic solution every two days for 4 days continuously, and building an intestinal sterile model.
7. The method for constructing the rheumatoid arthritis model through the intervention of the intestinal flora in coprophilous fungi transplantation according to the claim 1, which is characterized in that: collecting fresh excrement of a Fengshining rat within 3 hours by using a 2mL EP tube every day during an experiment period, mixing 6 rat excrement, fully stirring uniformly, weighing 1.0g, adding 10mL of physiological saline, carrying out vortex mixing for 2min to prepare FMT-F suspension, filtering by using a filter screen of 1mm, centrifuging by using a high-speed refrigerated centrifuge for 15min, carrying out centrifugation at 4 ℃ and 14000rpm, removing supernatant, adding 10mL of physiological saline for redissolving, repeating the steps for 3 times to prepare the fecal strain liquid, and storing the fecal strain liquid in a refrigerator at 4 ℃ for later use.
8. The method for constructing the rheumatoid arthritis model through the intervention of fecal flora in intestinal flora according to claim 1, which is characterized in that: the specific parameter conditions of the ultra-high performance liquid chromatography-quadrupole-electrostatic field orbitrap high-resolution mass spectrometry technology in the step 2 are as follows:
chromatographic conditions are as follows: using an ACQUITY BEH C18 chromatographic column; the mobile phase A is acetonitrile, and the mobile phase B is 0.01 percent formic acid aqueous solution;
mass spectrum conditions: the temperature of the ion transmission tube is 320 ℃; the S-Lens RF Level is 50; the full scanning/data depend on secondary scanning, and the scanning range is 100-1000 m/z; first order mass resolution 70000FWHM, second order resolution 17500FWHM, collision energy 30 eV.
9. The method for constructing the rheumatoid arthritis model through the intervention of the intestinal flora in coprophilous fungi transplantation according to the claim 1, which is characterized in that: in the step 4, PCA analysis and OPLS-DA analysis are carried out by SIMCA-P, so that the specific method for observing the influence of the coprophila rheumatica transplantation group on the rat metabolites is as follows: firstly, analyzing the data subjected to normalization processing, wherein principal component analysis in an unsupervised mode shows the internal difference among groups, and orthogonal partial least square discriminant analysis in a supervised mode shows the difference of metabolites in the groups; by a parameter R 2 Y and Q 2 To evaluate the quality of the model; the reliability of the model is verified by permutation and permutation test, and overfitting of the OPLS-DA model is prevented.
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