Application of traditional Chinese medicine composition in humanized flora transplantation model

Abstract

The invention relates to the technical field of medicines, and discloses an application of a traditional Chinese medicine composition in a humanized flora transplantation model.The application comprises the following steps that an experimental body is pretreated through sterile feeding or broad-spectrum antibiotics, then coprophilous fungus suspension of a functional dyspepsia patient is transplanted, and the humanized flora transplantation model is constructed; acquiring physiological data of the humanized flora transplantation model, and performing intervention on the traditional Chinese medicine composition after the humanized flora transplantation model is completed; acquiring physiological data of the traditional Chinese medicine composition intervened humanized flora transplantation model, and analyzing an evaluation result of the traditional Chinese medicine composition intervened humanized flora transplantation model by combining the evaluation parameters of the humanized flora transplantation model. The bottleneck that the traditional animal model is greatly different from the human microflora and the curative effect and the mechanism are difficult to transform is overcome, so that the research result is closer to the clinical practice, and the molecular mechanism of the traditional Chinese medicine composition is reliably revealed.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, specifically to the application of a traditional Chinese medicine composition in a humanized microbial transplantation model. Background Technology

[0002] Animal models play a crucial role in the research and application of traditional Chinese medicine (TCM) compositions. They allow for the objective and quantitative evaluation of the efficacy of TCM compositions. Through multi-omics testing and histological observation of model animals, specific targets and pathways in the closed-loop regulation of drugs can be revealed. After intervention with TCM compositions, monitoring liver and kidney function indicators, histopathological sections, and behavioral changes in animals can help detect potential toxic side effects or adverse reactions early, ensuring the safety of patients in subsequent clinical trials. Furthermore, animal models can be used for pharmacokinetic studies, providing a scientific basis for optimizing the dosage and developing dosing regimens of TCM compositions by detecting blood drug concentrations, tissue distribution, and metabolites.

[0003] However, the intestinal anatomy, immune system, and metabolic enzyme profile of rodents such as mice and rats used in related technologies differ significantly from those of humans. The absorption, distribution, metabolism, and excretion of drugs in animals cannot fully mimic the human process, leading to insufficient reliability of experimental results in clinical translation. Simple models that use specific pathogens or antibiotics for pretreatment to eliminate endogenous flora, followed by transplantation of only a single strain or a few species, cannot reproduce the complex, diverse, and dynamically balanced microbial community in the human gut, and thus fail to reflect the true network effects of drug-microbe-host interactions. Summary of the Invention

[0004] To address the shortcomings of existing technologies, this invention provides an application of a traditional Chinese medicine composition in a humanized microbial transplantation model, aiming to solve the problem that the difference between animal models and human microbial ecology makes it impossible to reliably evaluate the mechanism of action of the traditional Chinese medicine composition.

[0005] To address the above problems, this invention proposes the application of a traditional Chinese medicine composition in a humanized microbiota transplantation model, comprising the following steps: S1. The experimental subjects were pretreated with sterile feed or broad-spectrum antibiotics, and then fecal microbiota suspension from patients with functional dyspepsia was transplanted to construct a humanized microbiota transplantation model. S2. Obtain physiological data of the humanized microbiota transplantation model, and after the humanized microbiota transplantation model is completed, intervene with a traditional Chinese medicine composition, wherein the traditional Chinese medicine composition includes Shenqu (medicated leaven), charred hawthorn, charred malt, chicken gizzard lining, white peony root, codonopsis root, poria cocos, stir-fried atractylodes macrocephala, yam, costus root, amomum villosum, wine-processed corydalis rhizome, immature bitter orange, prepared licorice root, and fructooligosaccharides. S3, obtaining physiological data of the humanized flora transplantation model after intervention of the traditional Chinese medicine composition, and analyzing the evaluation results of the traditional Chinese medicine composition on the humanized flora transplantation model in combination with the evaluation parameters of the humanized flora transplantation model.

[0006] In some embodiments, step S1 comprises: S1.1, selecting 6-8-week-old female mice, and adapting to the feeding environment for 1 week; S1.2, orally gavaging the mice with the medicament for 10 consecutive days, wherein the gavaging medicament comprises at least one of vancomycin, neomycin, and amoxicillin; S1.3, preparing a functional dyspepsia patient fecal bacteria suspension with a concentration of 109CFU / mL, and gavaging the mice with 0.2 mL of the functional dyspepsia patient fecal bacteria suspension daily under sterile conditions for a total of 4 consecutive weeks, which is marked as an experimental group, and gavaging mice with sterile normal saline, which is marked as a control group, and the humanized flora transplantation model is constructed.

[0007] In some embodiments, step S2 comprises: S2.1, weighing each mouse, and recording the basic food intake and water intake.

[0008] S2.2, gavaging 0.2 mL of 0.5% phenol red starch paste, and sacrificing the mice after 20 minutes, taking out the stomach, and measuring the residual phenol red content at a wavelength of 104nm to calculate the gastric emptying rate; S2.3, gavaging 0.2 mL of 5% activated carbon suspension, and sacrificing the mice after 20 minutes, and measuring the ratio of the activated carbon front distance to the total small intestine length; S2.4, randomly selecting 3-5 mice in each group to collect fresh feces, and immediately placing the feces in a refrigerator at -80°C for 16S rRNA sequencing and LC-MS metabolite analysis; S2.5, taking the orbital venous blood, centrifuging to separate the serum for later use, and homogenizing the ileum tissue for ELISA / LC-MS / MS neurotransmitter and histopathology detection; S2.6, gavaging the mice in the experimental group with the traditional Chinese medicine composition daily, and gavaging the mice in the control group with the same volume of sterile normal saline daily.

[0009] In some embodiments, step S3 comprises: S3.1, weighing the mice every 2 days, and calculating the cumulative weight gain and loss trend, recording the total daily food intake and water intake of the mice within 24 hours after each administration, and using an automatic feeding monitoring system to calculate the single feeding time, food intake per meal, and meal interval to obtain the appetite and feeding evaluation coefficient; S3.2, repeating steps S2.2 and S2.3 to obtain the gastrointestinal function coefficient; S3.3, retrieve the intestinal tissue sections for HE staining, measure the villus height, crypt depth and villus / crypt ratio, detect the inflammatory cell infiltration markers and pro-inflammatory factor IL-1β by immunohistochemical or immunofluorescent staining, semi-quantitatively analyze the number of positively stained cells, and obtain the intestinal stem cell renewal effect coefficient; S3.4, conduct a scale evaluation of the functional improvement score and symptom relief score of the mice, and obtain a comprehensive symptomatology coefficient in combination with the physiological data; S3.5, perform a correlation analysis on the appetite and eating evaluation coefficient, the gastrointestinal function coefficient, the intestinal stem cell renewal effect coefficient, and the comprehensive symptomatology coefficient, and obtain the evaluation result of the traditional Chinese medicine composition on the humanized microbiota transplantation model.

[0010] In some embodiments, step S3.5 includes: S3.5.1, centralizing the appetite and eating evaluation coefficient, the gastrointestinal function coefficient, the intestinal stem cell renewal effect coefficient, and the comprehensive symptomatology coefficient using a standardization method to obtain normalized data; S3.5.2, importing the normalized data into statistical software to calculate the correlation scores between the appetite and eating evaluation coefficient, the gastrointestinal function coefficient, the intestinal stem cell renewal effect coefficient, and the comprehensive symptomatology coefficient using a correlation coefficient, and screening out significant correlation pairs with a correlation score ≥ 0.6 and a P value < 0.05; S3.5.3, based on the significant correlation pairs screened out in S3.5.2, regarding the appetite and eating evaluation coefficient, the gastrointestinal function coefficient, the intestinal stem cell renewal effect coefficient, and the comprehensive symptomatology coefficient as network nodes, identifying core influence parameters by analyzing the centrality of the network nodes; S3.5.4, obtaining the physiological data of the control group, comparing the physiological data of the experimental group and the control group according to the core influence parameters, and obtaining the evaluation result of the traditional Chinese medicine composition on the humanized microbiota transplantation model, which includes the efficacy evaluation result, the safety evaluation result, and the biomarker evaluation result.

[0011] In some embodiments, in step S3.5.4, the efficacy evaluation result includes complete effectiveness, partial effectiveness, and ineffectiveness, the safety evaluation result includes no adverse reactions, mild adverse reactions, and significant adverse reactions, and the biomarker evaluation result includes significant biomarker changes, partial biomarker changes, and no significant changes.

[0012] In some embodiments, the physiological data of the control group and the physiological data of the experimental group both include the appetite and eating evaluation coefficient, the gastrointestinal function coefficient, the intestinal stem cell renewal effect coefficient, and the comprehensive symptomatology coefficient; In step S3.5.4, when the standard deviation values of the appetite and eating evaluation coefficient, the gastrointestinal function coefficient, and the comprehensive symptomatology coefficient in the physiological data of the control group and the physiological data of the experimental group are all greater than or equal to 1.0, a completely effective curative effect evaluation result is output; When any two of the standard deviation values of the appetite and eating evaluation coefficient, the gastrointestinal function coefficient, and the comprehensive symptomatology coefficient in the physiological data of the control group and the physiological data of the experimental group are greater than or equal to 1.0, a partially effective curative effect evaluation result is output; When only one of the standard deviation values of the appetite and eating evaluation coefficient, the gastrointestinal function coefficient, and the comprehensive symptomatology coefficient in the physiological data of the control group and the physiological data of the experimental group is greater than or equal to 1.0 or all of them are less than 1.0, an ineffective curative effect evaluation result is output.

[0013] In some embodiments, in step S3.5.4, When any one of the standard deviation values of the appetite and eating evaluation coefficient, the gastrointestinal function coefficient, and the comprehensive symptomatology coefficient in the physiological data of the control group and the physiological data of the experimental group is less than or equal to -1.0, a safety evaluation result of significant adverse reactions is output; When any one of the standard deviation values of the appetite and eating evaluation coefficient, the gastrointestinal function coefficient, and the comprehensive symptomatology coefficient in the physiological data of the control group and the physiological data of the experimental group is greater than or equal to -1.0 and less than or equal to -0.5, a safety evaluation result of slight adverse reactions is output; When all of the standard deviation values of the appetite and eating evaluation coefficient, the gastrointestinal function coefficient, and the comprehensive symptomatology coefficient in the physiological data of the control group and the physiological data of the experimental group are greater than or equal to -0.5, a safety evaluation result of no adverse reactions is output.

[0014] In some embodiments, in step S3.5.4, When the standard deviation value of the intestinal stem cell renewal effect coefficient in the physiological data of the control group and the physiological data of the experimental group is greater than or equal to 1.0, a biomarker evaluation result of significant biomarker changes is output; When the standard deviation value of the intestinal stem cell renewal effect coefficient in the physiological data of the control group and the physiological data of the experimental group is greater than or equal to 0.5 and less than 1.0, a biomarker evaluation result of partial biomarker changes is output; When the standard deviation value of the intestinal stem cell renewal effect coefficient in the physiological data of the control group and the physiological data of the experimental group is less than 0.5, a biomarker evaluation result of no significant changes is output.

[0015] The application of the traditional Chinese medicine composition in the humanized flora transplantation model in the present application has the beneficial effects compared with the prior art: The step S1 establishes a humanized flora transplantation model, the step S2 gives the intervention of the traditional Chinese medicine composition after the completion of the model, and the step S3 completely acquires and compares various physiological and multi-omics data before and after the intervention, which can truly reproduce the interaction process of human intestinal flora and the host in vivo, and quantitatively evaluate the influence of the traditional Chinese medicine composition on gastrointestinal motility, flora structure and metabolic products. The scheme overcomes the bottleneck that the difference between the traditional animal model and the human microbial community is too large, and the therapeutic effect and mechanism are difficult to transform, so that the research results are closer to the clinical practice. BRIEF DESCRIPTION OF DRAWINGS

[0016] Figure 1 is a flowchart of the application of a kind of traditional Chinese medicine composition in humanized flora transplantation model according to an embodiment of the present application. DETAILED DESCRIPTION

[0017] The technical solutions in the embodiments of the present application will be described clearly and completely below with reference to the drawings in the embodiments of the present application, and all other embodiments obtained by those skilled in the art without creative labor based on the embodiments in the present application belong to the scope of protection of the present application.

[0018] It should be understood that the terms described in the present application are only for describing the specific embodiments, and are not used to limit the present application. In addition, for the numerical range in the present application, it should be understood that each intermediate value between the upper limit and the lower limit of the range is also specifically disclosed. Each smaller range between any stated value or intermediate value in the range and any other stated value or intermediate value in the range is also included in the present application. The upper limit and the lower limit of these smaller ranges can be independently included or excluded from the range.

[0019] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the present application pertains. Although preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present application. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials in connection with which the documents are concerned. In the event of any conflict between the content of this specification and any document incorporated by reference, the content of this specification will control.

[0020] Many modifications and variations of the present application specification can be made without departing from the scope or spirit of the present application, which will be apparent to those skilled in the art. Other embodiments obtained from the specification of the present application will be apparent to the skilled person. The specification and examples of the present application are only exemplary.

[0021] As for "include", "comprise", "have", "contain" and the like used herein, they are all open terms, that is, they mean to include but not limited to.

[0022] Referring to Figure 1 The application provides application of a traditional Chinese medicine composition in a humanized flora transplantation model, which comprises the following steps: S1, the experimental animals are pretreated by sterile feeding or broad-spectrum antibiotics, and then a functional dyspepsia patient's fecal bacteria suspension is transplanted to construct a humanized flora transplantation model.

[0023] Step S1 comprises: S1.1, 6-8-week-old female mice are selected, and the mice are adapted to the feeding environment for one week.

[0024] In this step, 6-8-week-old female mice with stable physiological state and mature immune function are screened, and the mice are given one week of adaptation to feeding, so that the animals can fully eliminate the stress caused by transportation and environmental changes before the experiment starts, thereby ensuring the consistency of the mice in the basic physiological parameters (such as body weight, appetite, activity, etc.), providing a stable biological background for subsequent model construction, and avoiding data bias caused by individual differences.

[0025] S1.2, the mice are orally administered with a medicament for 10 consecutive days, and the medicament comprises at least one of vancomycin, neomycin and amoxicillin.

[0026] In this step, at least one broad-spectrum antibiotic is orally administered for 10 consecutive days, which can effectively eliminate the original intestinal flora in the mice, achieving the effect of shedding the flora. Through the targeted bactericidal effect of the specific antibiotic, the diversity and quantity of the endogenous flora can be significantly reduced, creating a niche vacancy for subsequent transplantation of exogenous humanized flora, and ensuring that the transplanted colonies can successfully colonize and establish a stable humanized flora model.

[0027] S1.3, a functional dyspepsia patient's fecal bacteria suspension with a concentration of 10 9 CFU / mL is prepared, and the mice are orally administered with 0.2 mL of the functional dyspepsia patient's fecal bacteria suspension under sterile conditions every day for 4 consecutive weeks, which is marked as the experimental group, and the mice orally administered with sterile normal saline are marked as the control group, and the humanized flora transplantation model is constructed.

[0028] In this step, the functional dyspepsia patient's fecal bacteria are transplanted in a high-concentration suspension of 10 9 CFU / mL for 4 consecutive days, which can effectively transplant the human flora into the intestinal tract of the antibiotic-treated mice, so that the target bacteria can successfully colonize in the mice and form a stable flora ecology. In comparison, the control group is only administered with sterile normal saline, maintaining the sterile state after the original flora is shed. This step not only realizes the construction of a highly humanized flora transplantation model, but also simulates the microecological characteristics of the intestinal flora of functional dyspepsia patients, ensuring that the subsequent efficacy evaluation can truly reflect the effect of the traditional Chinese medicine composition on the humanized flora transplantation model.

[0029] S2, obtaining physiological data of the humanized flora transplantation model, and intervening with the traditional Chinese medicine composition after the humanized flora transplantation model is completed, wherein the traditional Chinese medicine composition comprises Liu Shenqu, Jiao Shancha, Jiao Maidai, Ji Neijin, Bai Shao, Dangshen, Fuling, Futo Baizhu, Shanyao, Muxiang, Sharen, Jiuyanhuosuo, Zhishi, Zhigancao, and fructooligosaccharide.

[0030] Step S2 comprises: S2.1, weighing the body weight of each mouse and recording the basal food intake and water intake.

[0031] By accurately weighing the body weight of the mouse and continuously recording the basal food intake and water intake, the metabolic and energy balance state of each mouse after the transplantation model is established but before the intervention can be obtained. These basic physiological parameters can reflect the overall health level and gastrointestinal function baseline difference of the mouse, provide reliable control for the quantitative evaluation of the intervention effect of the traditional Chinese medicine composition, and can be used for normalizing the data between groups to exclude the influence of individual differences.

[0032] S2.2, intragastrically administering 0.2 mL of 0.5% phenol red starch paste, and sacrificing the mouse after 20 minutes, taking out the stomach, and measuring the residual phenol red content at 104 nm wavelength to calculate the gastric emptying rate.

[0033] Using phenol red as a tracer dye, by intragastrically administering phenol red starch paste and measuring the residual amount of phenol red in the stomach contents at different times, the gastric emptying rate of the mouse can be accurately quantified. This method is based on the principle that phenol red moves together with chyme in the stomach, and when the residual amount of phenol red decreases, it indicates that the gastric emptying is accelerated, thereby reflecting the regulation effect of the traditional Chinese medicine composition on gastric motility.

[0034] S2.3, intragastrically administering 0.2 mL of 5% activated carbon suspension, and sacrificing the mouse after 20 minutes, measuring the ratio of the leading distance of activated carbon in the small intestine to the total small intestine length.

[0035] Using activated carbon as a non-absorbed tracer, by measuring the ratio of the moving distance of activated carbon in the small intestine to the total length after intragastrical administration, the small intestine propulsion rate can be quantitatively evaluated. This method is based on the principle that activated carbon particles are pushed by intestinal peristalsis, and can sensitively reflect the promotion or inhibition effect of the traditional Chinese medicine composition on small intestine peristalsis function.

[0036] S2.4, randomly selecting 3-5 mice from each group to collect fresh feces, and immediately placing them in a refrigerator at -80°C for 16S rRNA sequencing and LC-MS metabolite analysis.

[0037] By rapidly preserving the fecal samples under low temperature conditions, the original state of the intestinal microbial community structure and metabolites can be maximally preserved, providing reliable samples for subsequent high-throughput 16S rRNA sequencing and non-targeted LC-MS metabolomics analysis. At the same time, the unified processing of samples in different groups can ensure data comparability and reveal the systematic effects of the traditional Chinese medicine composition on intestinal flora and its metabolites.

[0038] S2.5, take the orbital venous blood, centrifugal separation of serum for standby, homogenate ileum tissue, for ELISA / LC-MS / MS neurotransmitter and histopathology detection.

[0039] The collection of mouse serum can be used for quantitative determination of the levels of key neurotransmitters (such as 5-hydroxytryptamine, substance P, VIP) in blood, and the ileum tissue homogenate and section can be used for histopathology analysis and detection of neurotransmitters and inflammatory factors in tissues. This method can evaluate the comprehensive effects of the traditional Chinese medicine composition on the regulation of neurotransmitters and the repair of tissue structure in the "gut-brain axis" at multiple levels by combining liquid chromatography mass spectrometry and enzyme-linked immunoassay technology.

[0040] S2.6, the mice in the experimental group are given the traditional Chinese medicine composition by gavage every day, and the mice in the control group are given the same volume of sterile normal saline by gavage every day.

[0041] By continuously administering the traditional Chinese medicine composition to the mice in the experimental group and treating the control group with the same volume of sterile saline, a strict negative control system can be constructed to exclude the interference of gavage operation itself or gavage solvent on the experimental results. This control design is based on the double-blind principle and can clearly distinguish between drug effects and human operation effects, ensuring the accuracy of evaluating the efficacy and safety of the traditional Chinese medicine composition.

[0042] In an embodiment, to precisely control the input amount of administration, the following equation set is proposed: ; ; ; ; wherein, is a composite efficacy index, for example, a linear combination of the appetite and eating evaluation coefficient, the gastrointestinal function coefficient, the intestinal stem cell renewal effect coefficient, and the comprehensive symptomatology coefficient synthesized according to the weight (dimensionless, normalized). At each sampling time point, it is calculated according to the formula Y(t)=w1A(t)+w2B(t)+w3C(t)+w4D(t), wherein A, B, C, and D are the values of the four coefficients after normalization, and the weights w i The sum of w1, w2, w3, and w4 is equal to 1. The data acquisition frequency is consistent with S3.1-S3.4 (for example, every two days or after each dose), and the real-time calculation is performed by the experimental database. The baseline disturbance term or natural recovery level, estimated as the average of Y over several time points before intervention (e.g., the average of Y over the three time points before intervention) or estimated as the mean of the control group over the same time period. The system recovery coefficient or dissipation rate, representing the rate of regression of the index to baseline, with units of inverse days. u(t) is the controllable input, representing the intensity of administration of the traditional Chinese medicine composition (e.g., the daily dose intensity or dose proportion, which needs to be defined in specific units in the experiment), with a value range of 0 to 1. η is the administration effect coefficient, representing the rate of improvement of Y per unit of administration intensity, with units of inverse days, which can be 0.03 to 0.4. θ is the colonization of the flora to the efficacy index coupling coefficient, representing the current colonization level, with units of inverse days, which can be 0.01 to 0.15. C(t) is dimensionless, which can be 0 to 1, representing the colonization level of the flora or the humanization similarity index, reflecting the degree of colonization of the key human flora in the host intestine. Taking zero means no colonization or very low similarity, and taking one means that the colonization reaches the target level or is highly close to the donor. The quantitative index is calculated using metagenomic or 16S rRNA data, such as the relative abundance of target probiotics, or the normalized similarity of Bray-Curtis similarity to the donor sample is measured and linearly mapped to the 0-1 range. The sampling frequency is consistent with S1 and S2 (recommended daily or every two days). ε(t) has units of inverse days, representing unmodeled disturbances and measurement errors, including environmental fluctuations, individual biological differences, and measurement noise. It is obtained in the form of residuals during model fitting, and is usually assumed to be white noise with mean zero or weakly correlated error, and its variance and distribution characteristics can be estimated by residual analysis. K p is dimensionless, representing the proportional control gain, indicating the multiple of immediate adjustment of the administration intensity when the error increases by one unit. It is determined in the pilot test through controller tuning methods, and needs to ensure that the closed-loop response is stable and not overshooting, which can be 0.2 to 0.6. K i is the integral control gain with units of inverse days, representing the rate of influence of the accumulated amount of error over time on the administration intensity, which can eliminate steady-state error. Together with K p , it is determined through simulation or experimental tuning, and is usually adjusted in the pilot test by gradually increasing the integral action and observing the steady-state error and oscillation, which can be 0.01 to 0.08. u base is dimensionless, representing the initial / baseline administration intensity, representing the value of the conventional or recommended dose on the normalized scale, serving as the starting point of the control law. It is determined by pilot toxicology / efficacy studies, or is normalized after conversion from previous literature and empirical doses. u min and u maxThe unit is dimensionless, indicating the upper and lower boundaries of the administration intensity, which is used to ensure the safety of the administration and the minimum effective dose protection. According to the toxicology and safety research (maximum tolerated dose, no observed adverse effect dose) and the ethical / regulatory constraints, it is normalized and mapped to the corresponding dimensionless upper and lower limits. min = 0.10,u base = 0.50,u max = 1.00. Y target The unit is dimensionless, indicating the expected composite efficacy target value, and the control loop strives to drive Y(t) to reach or exceed the target, which is determined by the clinical transformation target or experimental design, and can be set based on the normal level of the control group or the effective treatment level of the historical group, which can be 0.6-0.8. The unit of e(t) is dimensionless, indicating the target error, which is calculated by the controller to calculate the administration adjustment amount, which is calculated in real time after each sampling of the experiment.

[0043] S3, obtain the physiological data of the humanized flora transplantation model after the intervention of the traditional Chinese medicine composition, and analyze the evaluation results of the humanized flora transplantation model intervened by the traditional Chinese medicine composition combined with the evaluation parameters of the humanized flora transplantation model.

[0044] Step S3 includes: S3.1, weigh the mice every 2 days, and statistically analyze the cumulative body weight gain and loss trend, record the total food intake and water intake of the mice within 24 hours after each administration, calculate the single feeding time, food intake per meal and meal interval by using the automatic feeding monitoring system, and obtain the appetite and feeding evaluation coefficient.

[0045] By accurately weighing the mice every two days and recording the food intake and water intake within 24 hours after administration, combined with the calculation of single feeding time, food intake per meal and meal interval by the automatic feeding monitoring system, the energy intake and appetite changes of the mice can be comprehensively reflected. This method is based on the dynamic correlation principle of food intake and body weight gain and loss, and can sensitively monitor the influence of the traditional Chinese medicine composition on the appetite and metabolism of the mice, so as to quantify the appetite and feeding evaluation coefficient and provide accurate behavioral indicators for subsequent efficacy comparison.

[0046] S3.2, repeat steps S2.2 and S2.3 to obtain the gastrointestinal function coefficient.

[0047] By repeating the phenol red starch paste gastric emptying determination in step S2.2 and the active carbon suspension small intestine propulsion determination in step S2.3, the dual regulation effect of the traditional Chinese medicine composition on the gastric emptying speed and small intestine peristalsis efficiency can be evaluated. This method relies on the movement track of the tracer in the digestive tract, and can quantitatively analyze the gastrointestinal dynamics in real time, and obtain the gastrointestinal function coefficient to objectively reflect the comprehensive influence of the drug on the smooth muscle contraction and neural control function of the gastrointestinal tract.

[0048] S3.3, retrieve the intestinal tissue sections for HE staining, measure the villus height, crypt depth and villus / crypt ratio, detect the inflammatory cell infiltration markers and pro-inflammatory factor IL-1β by immunohistochemical or immunofluorescent staining, and semi-quantitatively analyze the number of positive cells to obtain the intestinal stem cell renewal effect coefficient.

[0049] By measuring the ileal villus height, crypt depth and villus / crypt ratio through HE staining, detecting the inflammatory cell infiltration markers and pro-inflammatory factor IL-1β by immunohistochemical or immunofluorescent staining, and semi-quantitatively analyzing the positive cells, the promoting effect of the traditional Chinese medicine composition on the intestinal epithelial regeneration and stem cell activity can be evaluated from the morphological and molecular levels. This method combines the integrity of the tissue structure and the indicators of the inflammatory state, and can accurately calculate the intestinal stem cell renewal effect coefficient, revealing the efficacy of the drug in repairing the intestinal barrier and inhibiting inflammation.

[0050] S3.4, scale the functional improvement score and symptom relief score of the mice, and obtain the comprehensive symptomatology coefficient by combining the physiological data.

[0051] By scaling the functional improvement score and symptom relief score of the mice, and combining the physiological data such as body weight, eating behavior and gastrointestinal motility, the overall relief degree of the traditional Chinese medicine composition on the symptoms of functional dyspepsia can be reflected in multiple dimensions. This method relies on the fusion of quantitative scores and objective physiological parameters to comprehensively evaluate the effect of the drug in reducing uncomfortable symptoms, restoring gastrointestinal function and improving the quality of life, forming a comprehensive symptomatology coefficient with strong comparability and comprehensiveness.

[0052] S3.5, perform correlation analysis on the appetite and eating evaluation coefficient, gastrointestinal function coefficient, intestinal stem cell renewal effect coefficient and comprehensive symptomatology coefficient to obtain the evaluation results of the traditional Chinese medicine composition on the humanized flora transplantation model.

[0053] By standardizing and centralizing the appetite and eating evaluation coefficient, gastrointestinal function coefficient, intestinal stem cell renewal effect coefficient and comprehensive symptomatology coefficient, the correlation scores between the coefficients are calculated and a weighted network is constructed based on significant correlations. Then, the core influencing parameters are identified through network node centrality analysis, and finally the data of the experimental group and the control group are compared to reveal the key nodes of the traditional Chinese medicine composition in regulating the flora-metabolism-neuro-function closed loop. This method integrates multiple physiological indicators through network science methods, effectively analyzes the drug action mechanism and obtains comprehensive evaluation results in terms of efficacy, safety and biomarkers, providing scientific and systematic evidence support for the application of traditional Chinese medicine composition in humanized flora transplantation model.

[0054] Step S3.5 includes: S3.5.1, the appetite eating evaluation coefficient, the gastrointestinal function coefficient, the intestinal stem cell renewal effect coefficient, and the comprehensive symptomatology coefficient are subjected to centralization processing by a standardization method to obtain normalized data.

[0055] The original physiological indicators of different dimensions and distributions are unified into the same numerical interval with a mean of zero and a standard deviation of one by Z-score standardization centralization processing of the appetite eating evaluation coefficient, the gastrointestinal function coefficient, the intestinal stem cell renewal effect coefficient, and the comprehensive symptomatology coefficient. This not only eliminates the bias caused by different dimensions of each coefficient, but also improves the comparability of subsequent multi-index joint analysis, enabling data from different sources to be effectively cross-compared under the same statistical framework, thereby laying a stable data foundation for correlation calculation.

[0056] S3.5.2, the normalized data is imported into statistical software to calculate the correlation scores between the appetite eating evaluation coefficient, the gastrointestinal function coefficient, the intestinal stem cell renewal effect coefficient, and the comprehensive symptomatology coefficient using a correlation coefficient, and significant correlation pairs with a correlation score ≥ 0.6 and a P value < 0.05 are screened out.

[0057] The normalized data is imported into statistical software, and the correlation strength between the four coefficients is calculated using Pearson or Spearman correlation coefficients, and significant correlation pairs are screened out with a correlation score ≥ 0.6 and a P value < 0.05 as the threshold. This step can objectively identify the most closely related physiological indicator combination after intervention with the traditional Chinese medicine composition, and filter out random noise through statistical significance, thereby ensuring that the subsequent network construction focuses on real and biologically meaningful parameter relationships.

[0058] S3.5.3, based on the significant correlation pairs screened out in S3.5.2, the appetite eating evaluation coefficient, the gastrointestinal function coefficient, the intestinal stem cell renewal effect coefficient, and the comprehensive symptomatology coefficient are regarded as network nodes, and the core influence parameters are identified by analyzing the centrality of the network nodes.

[0059] The significant correlation coefficients screened out in step S3.5.2 are used as network nodes, a weighted undirected network is constructed using edge weights to reflect the correlation strength, and core influence parameters are identified by degree centrality, betweenness centrality, and other network topology indicators. This method, based on complex network theory, visualizes and quantifies the interaction of multi-dimensional physiological coefficients, enabling rapid positioning of key parameters that play a pivotal role in the appetite-gastrointestinal-stem cell-comprehensive symptom closed-loop regulation, providing clear targets for subsequent intervention mechanism analysis and experimental verification.

[0060] S3.5.4, obtaining the physiological data of the control group, comparing the physiological data of the experimental group and the control group according to the core influence parameters, and obtaining the evaluation results of the traditional Chinese medicine composition intervention humanized flora transplantation model, wherein the evaluation results of the traditional Chinese medicine composition intervention humanized flora transplantation model include the efficacy evaluation results, the safety evaluation results and the biomarker evaluation results.

[0061] By statistically comparing the group difference values of the core influence parameters between the experimental group and the control group, the intervention effect of the traditional Chinese medicine composition can be directly quantified, and the efficacy evaluation, safety evaluation and biomarker evaluation results can be formed accordingly. In this step, the key parameters identified by network knowledge are combined with the control data, which not only improves the sensitivity of the intervention effect determination, but also reveals the potential safety risks and biomarker changes, providing comprehensive, systematic and operable evaluation conclusions for the application of the traditional Chinese medicine composition in the humanized flora transplantation model.

[0062] In step S3.5.4, the efficacy evaluation results include complete effectiveness, partial effectiveness and ineffectiveness, the safety evaluation results include no adverse reactions, mild adverse reactions and significant adverse reactions, and the biomarker evaluation results include significant biomarker changes, partial biomarker changes and no significant changes.

[0063] The physiological data of the control group and the physiological data of the experimental group both include appetite and eating evaluation coefficients, gastrointestinal function coefficients, intestinal stem cell renewal effect coefficients and comprehensive symptomatology coefficients. When the standard deviation values of the appetite and eating evaluation coefficients, the gastrointestinal function coefficients and the comprehensive symptomatology coefficients in the physiological data of the control group and the physiological data of the experimental group are all greater than or equal to 1.0, the complete effective efficacy evaluation results are outputted; When any two of the standard deviation values of the appetite and eating evaluation coefficients, the gastrointestinal function coefficients and the comprehensive symptomatology coefficients in the physiological data of the control group and the physiological data of the experimental group are greater than or equal to 1.0, the partial effective efficacy evaluation results are outputted; When only one of the standard deviation values of the appetite and eating evaluation coefficients, the gastrointestinal function coefficients and the comprehensive symptomatology coefficients in the physiological data of the control group and the physiological data of the experimental group is greater than or equal to 1.0 or all of them are less than 1.0, the ineffective efficacy evaluation results are outputted.

[0064] When the standard deviation values of the appetite eating evaluation coefficient, the gastrointestinal function coefficient, and the comprehensive symptomatology coefficient are all ≥1.0, it indicates that the key physiological indicators of the experimental group and the control group have been significantly improved by at least one standard deviation, meaning that the traditional Chinese medicine composition has a comprehensive and strong promoting effect on the appetite, gastrointestinal motility, and overall symptoms of mice, and therefore is determined to be “completely effective”. This one-to-one threshold setting ensures the strictness of the efficacy evaluation. Only when multiple core indicators are simultaneously improved at a high intensity can the drug be confirmed to have a real overall effect; when any two of them are ≥1.0, it is considered that the drug has a strong effect on most physiological links, and therefore is determined to be “partially effective”; when only one or three of them are <1.0, it indicates that the drug is not comprehensive or strong enough, and therefore is determined to be “ineffective”.

[0065] When any one of the standard deviation values of the appetite eating evaluation coefficient, the gastrointestinal function coefficient, and the comprehensive symptomatology coefficient in the physiological data of the control group and the physiological data of the experimental group is ≤-1.0, a safety evaluation result of significant adverse reactions is output; When any one of the standard deviation values of the appetite eating evaluation coefficient, the gastrointestinal function coefficient, and the comprehensive symptomatology coefficient in the physiological data of the control group and the physiological data of the experimental group is ≥-1.0 and ≤-0.5, a safety evaluation result of mild adverse reactions is output; When all of the standard deviation values of the appetite eating evaluation coefficient, the gastrointestinal function coefficient, and the comprehensive symptomatology coefficient in the physiological data of the control group and the physiological data of the experimental group are ≥-0.5, a safety evaluation result of no adverse reactions is output.

[0066] The safety evaluation focuses on whether the traditional Chinese medicine composition induces reverse physiological changes. If the standard deviation value of any core indicator (appetite, gastrointestinal function, comprehensive symptomatology coefficient) is ≤-1.0, it indicates that the corresponding physiological link has deteriorated by one standard deviation or more, reflecting a serious adverse reaction; if -1.0<standard deviation value≤-0.5, it is considered to be a mild decline, only within half a standard deviation of reversible or mild discomfort; if all three are >-0.5, it indicates that the core physiological function remains stable and has no obvious side effects. Such a graded threshold setting can accurately distinguish between strong drug toxicity, mild discomfort, and different risk levels of safe compatibility, providing clear safety definitions for clinical and subsequent research.

[0067] When the standard deviation value of the intestinal stem cell renewal effect coefficient in the physiological data of the control group and the physiological data of the experimental group is ≥1.0, a biomarker evaluation result of significant biomarker changes is output; When the standard deviation value of the intestinal stem cell renewal effect coefficient in the physiological data of the control group and the physiological data of the experimental group is ≥0.5 and <1.0, a biomarker evaluation result of partial biomarker changes is output; When the standard deviation of the intestinal stem cell renewal effect coefficient in the physiological data of the control group and the physiological data of the experimental group is less than 0.5, a biomarker evaluation result of no significant change is output.

[0068] The intestinal stem cell renewal effect coefficient is used alone for biomarker evaluation, reflecting the molecular level effect of the traditional Chinese medicine composition on intestinal epithelial regeneration and barrier repair. When Δ≥1.0, it means that the stem cell marker (such as LGR5, β-catenin) expression is increased by at least one standard deviation, which reflects a significant biological effect; when 0.5≤Δ<1.0, it is a moderate increase of half to one standard deviation, corresponding to "partial change"; when Δ<0.5, the change is less than half a standard deviation, which can be considered as no significant biological regulation. Such threshold distinction ensures that the evaluation of molecular markers is neither too lenient nor ignores the real effect of moderate intensity, making the biomarker evaluation both sensitive and operable.

Claims

1. The application of a traditional Chinese medicine composition in a humanized microbiota transplantation model, characterized in that, Includes the following steps: S1. The experimental subjects were pretreated with sterile feed or broad-spectrum antibiotics, and then fecal microbiota suspension from patients with functional dyspepsia was transplanted to construct a humanized microbiota transplantation model. S2. Obtain physiological data of the humanized microbiota transplantation model, and after the humanized microbiota transplantation model is completed, intervene with a traditional Chinese medicine composition, wherein the traditional Chinese medicine composition includes Shenqu (medicated leaven), charred hawthorn, charred malt, chicken gizzard lining, white peony root, codonopsis root, poria cocos, stir-fried atractylodes macrocephala, yam, costus root, amomum villosum, wine-processed corydalis rhizome, immature bitter orange, prepared licorice root, and fructooligosaccharides. S3. Obtain physiological data of the humanized microbiota transplantation model after intervention with traditional Chinese medicine composition, and analyze the evaluation results of the intervention with traditional Chinese medicine composition in the humanized microbiota transplantation model in combination with the evaluation parameters of the humanized microbiota transplantation model.

2. The application of the traditional Chinese medicine composition according to claim 1 in a humanized microbiota transplantation model, characterized in that, Step S1 includes: S1.1 Select female mice aged 6-8 weeks and acclimate them to the rearing environment for 1 week; S1.

2. Mice were orally administered a drug via gavage for 10 consecutive days. The drug included at least one of vancomycin, neomycin, and azithromycin. S1.3, Preparation concentration is 10 9 A CFU / mL fecal microbiota suspension from patients with functional dyspepsia was administered to mice by gavage at a rate of 0.2 mL daily for 4 consecutive weeks under aseptic conditions. Mice that were gavaged with sterile saline were designated as the control group. The humanized microbiota transplantation model was thus established.

3. The application of the traditional Chinese medicine composition according to claim 2 in a humanized microbiota transplantation model, characterized in that, Step S2 includes: S2.1 Weigh each mouse and record its basal food intake and water intake. S2.

2. 0.2 mL of 0.5% phenol red starch paste was administered by gavage. The mice were sacrificed 20 min later, and the stomachs were removed. The residual phenol red content was measured at a wavelength of 104 nm to calculate the gastric emptying rate. S2.

3. 0.2 mL of 5% activated charcoal suspension was administered by gavage. The mice were sacrificed 20 min later, and the ratio of the distance of the activated charcoal front edge in the small intestine to the total length of the small intestine was measured. S2.

4. Fresh feces were collected from 3 to 5 mice randomly selected from each group and immediately placed in a freezer at -80°C for 16S rRNA sequencing and LC-MS metabolite analysis. S2.

5. Collect orbital venous blood, centrifuge to separate serum for later use, homogenize ileal tissue for ELISA / LC-MS / MS neurotransmitter and histopathological detection; S2.

6. Mice in the experimental group were administered the traditional Chinese medicine composition by gavage daily, while mice in the control group were administered an equal volume of sterile saline by gavage daily.

4. The application of the traditional Chinese medicine composition according to claim 2 in a humanized microbiota transplantation model, characterized in that, Step S3 includes: S3.1 Weigh the mice every 2 days and record the cumulative weight gain and loss trend. Record the total daily food intake and water intake of the mice within 24 hours after each administration. Use an automatic feeding monitoring system to calculate the single feeding time, food intake per meal and feeding interval to obtain the appetite and feeding evaluation coefficient. S3.2 Repeat steps S2.2 and S2.3 to obtain the gastrointestinal function coefficient; S3.

3. Take ileal tissue sections for HE staining, measure villus height, crypt depth and villus / crypt ratio, use immunohistochemistry or immunofluorescence staining to detect inflammatory cell infiltration markers and pro-inflammatory factor IL-1β, semi-quantitatively analyze the number of staining positive cells, and obtain the intestinal stem cell renewal effect coefficient. S3.

4. Scale-based assessment of functional improvement and symptom relief scores in mice, combined with physiological data, to obtain a comprehensive symptomatology coefficient. S3.5 Correlation analysis was performed on the appetite and food intake evaluation coefficient, gastrointestinal function coefficient, intestinal stem cell renewal effect coefficient, and comprehensive symptom coefficient to obtain the evaluation results of the intervention of traditional Chinese medicine composition in the humanized microbiota transplantation model.

5. The application of the traditional Chinese medicine composition according to claim 4 in a humanized microbiota transplantation model, characterized in that, Step S3.5 includes: S3.5.1 The appetite and food intake evaluation coefficient, gastrointestinal function coefficient, intestinal stem cell renewal effect coefficient, and comprehensive symptom coefficient are processed by standardization method to obtain normalized data; S3.5.

2. Import the normalized data into statistical software and use correlation coefficients to calculate the correlation scores between the appetite and food intake evaluation coefficient, gastrointestinal function coefficient, intestinal stem cell renewal effect coefficient, and comprehensive symptom coefficient. Select significant association pairs with a correlation score ≥ 0.6 and a P value < 0.

05. S3.5.3 Based on the significant association pairs selected in S3.5.2, the appetite and food intake evaluation coefficient, gastrointestinal function coefficient, intestinal stem cell renewal effect coefficient, and comprehensive symptom coefficient are regarded as network nodes. By analyzing the centrality of the network nodes, the core influencing parameters are identified. S3.5.4 Obtain physiological data from the control group, compare the physiological data of the experimental group and the control group based on the core influencing parameters, and obtain the evaluation results of the intervention of the traditional Chinese medicine composition in the humanized microbiota transplantation model. The evaluation results of the intervention of the traditional Chinese medicine composition in the humanized microbiota transplantation model include efficacy evaluation results, safety evaluation results, and biomarker evaluation results.

6. The application of the traditional Chinese medicine composition according to claim 5 in a humanized microbiota transplantation model, characterized in that, In step S3.5.4, the efficacy evaluation results include completely effective, partially effective, and ineffective; the safety evaluation results include no adverse reactions, mild adverse reactions, and significant adverse reactions; and the biomarker evaluation results include significant changes in biomarkers, partial changes in biomarkers, and no significant changes.

7. The application of the traditional Chinese medicine composition according to claim 6 in a humanized microbiota transplantation model, characterized in that, The physiological data of both the control group and the experimental group included appetite and food intake evaluation coefficient, gastrointestinal function coefficient, intestinal stem cell renewal effect coefficient, and comprehensive symptom coefficient. In step S3.5.4, when the standard deviation of the appetite and food intake evaluation coefficient, gastrointestinal function coefficient, and comprehensive symptom coefficient in the physiological data of the control group and the experimental group are all greater than or equal to 1.0, the fully effective efficacy evaluation result is output. When any two of the standard deviations of the appetite and food intake evaluation coefficient, gastrointestinal function coefficient, and comprehensive symptom coefficient in the physiological data of the control group and the experimental group are greater than or equal to 1.0, a partially effective efficacy evaluation result is output. If the standard deviation of any one of the following in the physiological data of the control group and the experimental group—the appetite and food intake evaluation coefficient, the gastrointestinal function coefficient, and the comprehensive symptom coefficient—is greater than or equal to 1.0, or if all three are less than 1.0, an invalid efficacy evaluation result will be output.

8. The application of the traditional Chinese medicine composition according to claim 6 in a humanized microbiota transplantation model, characterized in that, In step S3.5.4, When any one of the standard deviations of the appetite-feeding evaluation coefficient, gastrointestinal function coefficient, and comprehensive symptom coefficient in the physiological data of the control group and the experimental group is less than or equal to -1.0, the safety evaluation result of significant adverse reaction is output. When any one of the standard deviations of the appetite-feeding evaluation coefficient, gastrointestinal function coefficient, and comprehensive symptom coefficient in the physiological data of the control group and the experimental group is greater than or equal to -1.0 and less than or equal to -0.5, the safety evaluation result of mild adverse reaction is output. When the standard deviation of the appetite and food intake evaluation coefficient, gastrointestinal function coefficient, and comprehensive symptom coefficient in the physiological data of both the control group and the experimental group is greater than or equal to -0.5, a safety evaluation result of no adverse reactions is output.

9. The application of the traditional Chinese medicine composition according to claim 6 in a humanized microbiota transplantation model, characterized in that, In step S3.5.4, When the standard deviation of the intestinal stem cell renewal effect coefficient in the physiological data of the control group and the physiological data of the experimental group is greater than or equal to 1.0, the biomarker evaluation results of significant biomarker changes are output. When the standard deviation of the intestinal stem cell renewal effect coefficient in the physiological data of the control group and the physiological data of the experimental group is greater than or equal to 0.5 and less than 1.0, the biomarker evaluation results of partial biomarker changes are output. When the standard deviation of the intestinal stem cell renewal effect coefficient in the physiological data of the control group and the physiological data of the experimental group is less than 0.5, the evaluation result of the biomarker with no significant change is output.

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