A characteristic metabolite profile of tortoise shell glue and its application in evaluating myocardial protection

Abstract

The application discloses a characteristic metabolite spectrum of tortoise shell glue and application thereof in evaluation of myocardial protection, relates to the technical field of tortoise shell glue, and the characteristic metabolite spectrum of tortoise shell glue is a compound spectrum composed of a tortoise shell glue-derived metabolite spectrum and a myocardial protection response spectrum, comprising the following steps: acquiring tortoise shell glue-derived markers and myocardial protection response markers meeting screening conditions from a tortoise shell glue-derived marker set and a myocardial protection response marker set as characteristic metabolites, and generating a tortoise shell glue characteristic metabolite spectrum. The application forms the tortoise shell glue characteristic metabolite spectrum by establishing the compound spectrum composed of the tortoise shell glue-derived metabolite spectrum and the myocardial protection response spectrum, performs synergistic analysis on the tortoise shell glue-derived substances and the myocardial protection response substances, is favorable for identifying specific action objects of the tortoise shell glue in myocardial protection, and provides a basis for researching a molecular mechanism of the tortoise shell glue in myocardial protection.

Description

Technical Field

[0001] This invention relates to the field of tortoise shell glue technology, specifically to a characteristic metabolite profile of tortoise shell glue and its application in evaluating myocardial protection. Background Technology

[0002] Tortoise shell glue, a traditional Chinese medicine derived from animal glue, is prepared through processes such as boiling and concentration. It is believed to have effects such as nourishing yin and suppressing yang, tonifying the kidneys and strengthening bones. Recent studies have found that tortoise shell glue has potential applications in cardiovascular protection, particularly in improving myocardial injury, inhibiting myocardial fibrosis, reducing oxidative stress, and improving chronic heart failure. However, the specific mechanisms of action of tortoise shell glue in cardioprotection remain unclear, making it difficult to establish the correlation and potential molecular mechanisms between the effective components of tortoise shell glue and cardioprotection. Summary of the Invention

[0003] The purpose of this invention is to provide a characteristic metabolite profile of tortoise shell glue and its application in evaluating myocardial protection, so as to solve the problems mentioned in the background art.

[0004] To achieve the above objectives, the present invention provides the following technical solution: a characteristic metabolite spectrum of tortoise shell glue, wherein the characteristic metabolite spectrum of tortoise shell glue is a complex spectrum composed of a tortoise shell glue-derived metabolite spectrum and a myocardial protective response spectrum, and the method for establishing the characteristic metabolite spectrum of tortoise shell glue includes the following steps: Step 1: Establish a mouse model of chronic heart failure and set up a normal control group, a model control group, and a tortoise shell glue intervention group; Step 2: Collect samples, obtain at least one of serum, heart tissue and liver tissue from each group of mice, and use liquid chromatography-mass spectrometry to detect the samples and obtain the candidate metabolic feature set and characterization data of the candidate metabolic features. Step 3: Annotate the candidate feature set and characterization data, perform multivariate statistical analysis or differential analysis to obtain the set of tortoise shell collagen-derived biomarkers and the set of myocardial protective response biomarkers; Step 4: Set screening criteria, select tortoise shell colloid-derived biomarkers and myocardial protective response biomarkers that meet the screening criteria from the set of tortoise shell colloid-derived biomarkers and the set of myocardial protective response biomarkers as characteristic metabolites, and generate a tortoise shell colloid characteristic metabolite profile.

[0005] Preferably, the tortoise shell collagen-derived metabolite profile includes characterization data of tortoise shell collagen-derived biomarkers. Tortoise shell collagen-derived markers include at least two of the following: hydroxyproline, proline, Pro-Hyp, Hyp-Gly, and Ala-Hyp. The myocardial protective response profile includes characterization data of myocardial protective response biomarkers in the tortoise shell glue intervention group samples; Myocardial protective response markers include B-type natriuretic peptide, N-terminal B-type natriuretic peptide precursor, malondialdehyde, antioxidant enzyme SOD, lactate dehydrogenase, and calcium. 2+ -At least three of the following: ATPase, Pink1 protein expression level, and Drp1 protein expression level.

[0006] Preferably, the tortoise shell glue intervention group includes a low-concentration tortoise shell glue intervention group, a medium-concentration tortoise shell glue intervention group, and a high-concentration tortoise shell glue intervention group.

[0007] Preferably, the metabolite characterization data includes identification data and abundance data, wherein the identification data includes one or more of retention time, mass number, mass deviation, and ion mass-to-charge ratio, and the abundance data includes one or more of peak height, peak area, and absolute concentration.

[0008] Preferably, the screening criteria include: The abundance of metabolic markers belonging to tortoise shell glue was higher in the tortoise shell glue intervention group than in the normal control group and the model control group; Metabolic markers that are biomarkers of myocardial protective response showed a regression towards the normal control group in the tortoise shell glue intervention group compared to the model control group, and the regression rate was not less than 10%. Metabolic markers that are biomarkers of myocardial protective response showed a greater rate of regression to the normal control group in the high-concentration or medium-concentration tortoise shell glue intervention group than in the low-concentration tortoise shell glue intervention group.

[0009] An application of a characteristic metabolite profile of tortoise shell glue in evaluating myocardial protection, wherein the characteristic metabolite profile of tortoise shell glue is used to establish a myocardial protection response evaluation model, and the myocardial protection response evaluation model is used to evaluate the degree of myocardial protection response induced by tortoise shell glue in a chronic heart failure model.

[0010] Preferably, the establishment of the myocardial protection response model includes the following steps: A characteristic metabolite data matrix was constructed based on characterization data of tortoise shell collagen biomarkers and characterization data of myocardial protective response biomarkers; A myocardial protection response model was established using one or more of the following methods: weighted scoring method, partial least squares discriminant analysis method, logistic regression method, random forest method, or XGBoost method. The characteristic metabolite data matrix was used as an input variable, and an evaluation score was obtained based on the myocardial protection response model.

[0011] Preferably, the evaluation score based on the myocardial protection response model is obtained according to the following formula: ; Where G is the assessment score, For the level score of tortoise shell collagen-derived markers, The response marker callback score is represented by α and β, which are the weighting coefficients of the corresponding scores.

[0012] Preferably, the calculation method for the level score of the tortoise shell collagen-derived marker is as follows: ; Where n is the number of tortoise shell collagen-derived markers, The weighting coefficient of the j-th tortoise shell collagen-derived biomarker, denoted as the standardized abundance value of the j-th tortoise shell collagen-derived biomarker.

[0013] Preferably, the callback score of the response marker is obtained by the following method: ; ; in For the callback rate of the i-th myocardial protection response biomarker, Let be the weighting coefficient of the i-th myocardial protective response marker. This represents the average expression value of the i-th myocardial protective response biomarker in the model control group. represents the average expression value of the i-th biomarker in the tortoise shell glue intervention group. This represents the average expression value of the i-th biomarker in the normal control group.

[0014] Compared with the prior art, the beneficial effects of the present invention are: 1. This invention establishes a complex spectrum of tortoise shell glue-derived metabolites and myocardial protective response substances to form a characteristic metabolite spectrum of tortoise shell glue. This allows for synergistic analysis of tortoise shell glue-derived substances and myocardial protective response substances, which is beneficial for identifying the specific targets of tortoise shell glue in myocardial protection and provides a basis for studying the molecular mechanism of tortoise shell glue in myocardial protection.

[0015] 2. Based on the characteristic metabolite spectrum of tortoise shell glue, which includes the metabolite spectrum of tortoise shell glue and the myocardial protective response spectrum, this invention constructs a myocardial protective response evaluation model, which is beneficial for obtaining the relationship between tortoise shell glue and myocardial protective response and can provide guidance for the use of tortoise shell glue in myocardial protection. Detailed Implementation

[0016] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0017] One embodiment of the present invention provides: a characteristic metabolite spectrum of tortoise shell glue, wherein the characteristic metabolite spectrum of tortoise shell glue is a complex spectrum composed of a tortoise shell glue-derived metabolite spectrum and a myocardial protective response spectrum, and the method for establishing the characteristic metabolite spectrum of tortoise shell glue includes the following steps: Step 1: Establish a mouse model of chronic heart failure and set up a normal control group, a model control group, and a tortoise shell glue intervention group; In this embodiment, the mouse chronic heart failure model was induced by injection of doxorubicin (DOX) solution at a concentration of 1 mg / ml (specifically, by intraperitoneal injection at a dose of 5 mg / kg / time, once a week for four consecutive weeks). Mice injected with DOX were divided into a model control group and a tortoise shell glue intervention group. The tortoise shell glue intervention group included a low-concentration tortoise shell glue intervention group (administration method: 1.3 g / kg, once a day, for 8 weeks), a medium-concentration tortoise shell glue intervention group (administration method: 2.6 g / kg, once a day, for 8 weeks), and a high-concentration tortoise shell glue intervention group (administration method: 5.2 g / kg, once a day, for 8 weeks). The normal control group was injected with an equal volume of physiological saline during the establishment of the heart failure model. The number of mice in each group was 8. By setting up a normal control group, a baseline of normal metabolic state in healthy mice can be established, and the unhealthy metabolic state of mice in a chronic heart failure model and the metabolic regression state of mice after intervention with tortoise shell glue can be obtained. Step 2: Collect samples, obtain at least one of serum, heart tissue and liver tissue from each group of mice, and use liquid chromatography-mass spectrometry to detect the samples and obtain the candidate metabolic feature set and characterization data of the candidate metabolic features. This embodiment also includes using tortoise shell glue extract as a reference detection object to obtain a basic set of tortoise shell glue-derived biomarkers. Serum, heart tissue and liver tissue are all used as test samples and are detected by liquid chromatography-mass spectrometry. The detection data are processed by peak extraction, peak alignment and noise reduction to obtain candidate metabolic feature characterization data. Metabolite characterization data includes identification data and abundance data. Identification data includes one or more of retention time, mass number, mass deviation, and ion mass-to-charge ratio. Identification data is used to determine whether candidate metabolic features in different samples are the same metabolic feature. Abundance data includes one or more of peak height, peak area, and absolute concentration. Abundance data is used to compare the changes in metabolic features in the normal control group, the model control group, and the tortoise shell glue intervention group. Step 3: Annotate the candidate feature set and characterization data, perform multivariate statistical analysis or differential analysis to obtain the set of tortoise shell collagen-derived biomarkers and the set of myocardial protective response biomarkers; Data for identifying candidate metabolic features were analyzed. When a metabolic feature in the tortoise shell glue-derived biomarker control set coexists with the same metabolic feature in the tortoise shell glue intervention group, and this metabolic feature is not present in the model control group and normal control group, the metabolic feature is recorded as a tortoise shell glue-derived biomarker, and a tortoise shell glue-derived biomarker set is generated. When a metabolite is present in the normal control group, model control group, and tortoise shell glue intervention group, and the change state of this metabolite in the tortoise shell glue intervention group tends to be closer to the change state of the normal control group compared to the model control group, the metabolite is recorded as a myocardial protective response biomarker, and a myocardial protective response biomarker set is generated. Furthermore, the tortoise shell collagen-derived biomarkers include at least two of hydroxyproline, proline, Pro-Hyp, Hyp-Gly, and Ala-Hyp, while the myocardial protective response biomarkers include B-type natriuretic peptide, N-terminal B-type natriuretic peptide precursor, malondialdehyde, the antioxidant enzyme SOD, lactate dehydrogenase, and Ca2+. 2+ -At least three of the following: ATPase, Pink1 protein expression level, and Drp1 protein expression level; Step 4: Set screening criteria, obtain tortoise shell collagen-derived biomarkers and myocardial protective response biomarkers that meet the screening criteria from the set of tortoise shell collagen-derived biomarkers and the set of myocardial protective response biomarkers as characteristic metabolites, and generate a tortoise shell collagen characteristic metabolite profile. The filtering criteria include: The abundance of metabolic markers belonging to tortoise shell glue in the tortoise shell glue intervention group was higher than that in the normal control group and the model control group, and was positively correlated with the tortoise shell glue intervention concentration; Metabolic markers that are biomarkers of myocardial protective response showed a regression towards the normal control group in the tortoise shell glue intervention group compared to the model control group, and the regression rate was not less than 10%. Metabolic markers that are biomarkers of myocardial protective response showed a greater rate of regression to the normal control group in the high-concentration or medium-concentration tortoise shell glue intervention group than in the low-concentration tortoise shell glue intervention group.

[0018] Example 2: An embodiment of the present invention: the application of a characteristic metabolite profile of tortoise shell glue in evaluating myocardial protection. The characteristic metabolite profile of tortoise shell glue is used to establish a myocardial protection response evaluation model, which is used to evaluate the degree of myocardial protection response induced by tortoise shell glue in a chronic heart failure model.

[0019] The establishment of a myocardial protection response model includes the following steps: A characteristic metabolite data matrix was constructed based on the characterization data of tortoise shell collagen biomarkers and the characterization data of myocardial protective response biomarkers. The rows of the matrix represent the sample groups collected, and the columns are the characteristic metabolites and their characterization data. A myocardial protection response model was established using one or more of the following methods: weighted scoring method, partial least squares discriminant analysis method, logistic regression method, random forest method, or XGBoost method. The characteristic metabolite data matrix was used as an input variable, and the evaluation score was obtained based on the myocardial protection response model. The assessment score is obtained based on the myocardial protection response model, using the following formula: ; Where G is the assessment score, For the level score of tortoise shell collagen-derived markers, In response to the marker callback score, α and β are the weighting coefficients of the corresponding scores, respectively; The level score of the tortoise shell glue source marker is used to reflect the expression level of the source marker in the chronic heart failure model, and the response marker callback score is used to assess the degree of recovery of the chronic heart failure model from the damaged state to the normal state. When the myocardial protection response model is established using partial least squares discriminant analysis, the weight coefficients are determined based on the standardized regression coefficients of each variable. When the myocardial protection response model is established using random forest or XGBoost, the weight coefficients are determined based on the normalization of the importance weights of the variables in the model. The calculation method for the level score of tortoise shell collagen-derived markers is as follows: ; Where n is the number of tortoise shell collagen-derived markers, The weighting coefficient of the j-th tortoise shell collagen-derived biomarker, Let be the standardized abundance value of the j-th tortoise shell collagen-derived biomarker, and It is obtained by Z-score standardization of abundance data.

[0020] The callback score for the response marker is obtained using the following method: ; ; in For the callback rate of the i-th myocardial protection response biomarker, Let be the weighting coefficient of the i-th myocardial protective response marker. This represents the average expression value of the i-th myocardial protective response biomarker in the model control group. represents the average expression value of the i-th biomarker in the tortoise shell glue intervention group. This represents the average expression value of the i-th biomarker in the normal control group; When the callback score of the response marker is greater than 0, it indicates that tortoise shell glue can induce myocardial protective response in the chronic heart failure model. The degree of myocardial protective response induced by tortoise shell glue in the chronic heart failure model is evaluated according to the value of the evaluation score. As the evaluation score increases, it indicates that the expression level of tortoise shell glue-derived components in the chronic heart failure model gradually increases and / or the myocardial protective effect induced by tortoise shell glue gradually strengthens. Furthermore, by establishing a complex spectrum composed of the metabolite spectrum of tortoise shell glue-derived substances and the myocardial protective response spectrum, the relationship between tortoise shell glue-derived substances and myocardial protective response can be established, providing a basis for the treatment of chronic heart failure with tortoise shell glue.

[0021] Working principle: A characteristic metabolite spectrum of tortoise shell glue, which is a complex spectrum composed of tortoise shell glue-derived metabolites and myocardial protective response substances. The method for establishing the characteristic metabolite spectrum of tortoise shell glue includes the following steps: Step 1: Establish a mouse model of chronic heart failure and set up a normal control group, a model control group, and a tortoise shell glue intervention group; Step 2: Collect samples, obtain at least one of serum, heart tissue and liver tissue from each group of mice, and use liquid chromatography-mass spectrometry to detect the samples and obtain the candidate metabolic feature set and characterization data of the candidate metabolic features. Step 3: Annotate the candidate feature set and characterization data, perform multivariate statistical analysis and differential analysis to obtain the set of tortoise shell collagen-derived biomarkers and the set of myocardial protective response biomarkers; Step 4: Set screening conditions, obtain tortoise shell collagen-derived biomarkers and myocardial protective response biomarkers that meet the screening conditions from the set of tortoise shell collagen-derived biomarkers and the set of myocardial protective response biomarkers as characteristic metabolites, verify the characteristic metabolites by targeted liquid chromatography-mass spectrometry, and generate a tortoise shell collagen characteristic metabolite spectrum. An application of the characteristic metabolite profile of tortoise shell glue in evaluating cardioprotection: The characteristic metabolite profile of tortoise shell glue is used to establish a cardioprotective response evaluation model, which is then used to assess the degree of cardioprotective response induced by tortoise shell glue in a chronic heart failure model. A set of biomarkers for tortoise shell glue and a set of biomarkers for myocardial protective response were used in a kit to analyze the therapeutic effect of the tortoise shell glue on chronic heart failure.

[0022] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above, and that the invention can be implemented in other specific forms without departing from the spirit or essential characteristics of the invention. Therefore, the embodiments should be considered in all respects as exemplary and non-limiting, and the scope of the invention is defined by the appended claims rather than the foregoing description. Thus, it is intended that all variations falling within the meaning and scope of equivalents of the claims be included within the present invention.

Claims

1. A characteristic metabolite profile of tortoise shell glue, characterized in that, The characteristic metabolite spectrum of tortoise shell glue is a complex spectrum composed of tortoise shell glue-derived metabolites and myocardial protective response substances. The method for establishing the characteristic metabolite spectrum of tortoise shell glue includes the following steps: Step 1: Establish a mouse model of chronic heart failure and set up a normal control group, a model control group, and a tortoise shell glue intervention group; Step 2: Collect samples, obtain at least one of serum, heart tissue and liver tissue from each group of mice, and use liquid chromatography-mass spectrometry to detect the samples and obtain the candidate metabolic feature set and characterization data of the candidate metabolic features. Step 3: Annotate the candidate feature set and characterization data, perform multivariate statistical analysis or differential analysis to obtain the set of tortoise shell collagen-derived biomarkers and the set of myocardial protective response biomarkers; Step 4: Set screening criteria, select tortoise shell colloid-derived biomarkers and myocardial protective response biomarkers that meet the screening criteria from the set of tortoise shell colloid-derived biomarkers and the set of myocardial protective response biomarkers as characteristic metabolites, and generate a tortoise shell colloid characteristic metabolite profile.

2. The characteristic metabolite profile of tortoise shell glue according to claim 1, characterized in that: The tortoise shell collagen-derived metabolite profile includes characterization data of tortoise shell collagen-derived biomarkers. Tortoise shell collagen-derived markers include at least two of the following: hydroxyproline, proline, Pro-Hyp, Hyp-Gly, and Ala-Hyp. The myocardial protective response profile includes characterization data of myocardial protective response biomarkers in the tortoise shell glue intervention group samples; Myocardial protective response markers include B-type natriuretic peptide, N-terminal B-type natriuretic peptide precursor, malondialdehyde, antioxidant enzyme SOD, lactate dehydrogenase, and calcium. 2+ -At least three of the following: ATPase, Pink1 protein expression level, and Drp1 protein expression level.

3. The characteristic metabolite profile of tortoise shell glue according to claim 2, characterized in that: The tortoise shell glue intervention groups include a low-concentration tortoise shell glue intervention group, a medium-concentration tortoise shell glue intervention group, and a high-concentration tortoise shell glue intervention group.

4. The characteristic metabolite profile of tortoise shell glue according to claim 3, characterized in that: The metabolite characterization data includes identification data and abundance data, wherein the identification data includes one or more of retention time, mass number, mass deviation, and ion mass-to-charge ratio, and the abundance data includes one or more of peak height, peak area, and absolute concentration.

5. The characteristic metabolite profile of tortoise shell glue according to claim 4, characterized in that: The filtering criteria include: The abundance of metabolic markers belonging to tortoise shell glue was higher in the tortoise shell glue intervention group than in the normal control group and the model control group; Metabolic markers that are biomarkers of myocardial protective response showed a regression towards the normal control group in the tortoise shell glue intervention group compared to the model control group, and the regression rate was not less than 10%. Metabolic markers that are biomarkers of myocardial protective response showed a greater rate of regression to the normal control group in the high-concentration or medium-concentration tortoise shell glue intervention group than in the low-concentration tortoise shell glue intervention group.

6. The application of a characteristic metabolite profile of tortoise shell glue in evaluating myocardial protection, characterized in that: The characteristic metabolite profile of tortoise shell glue is used to establish a myocardial protective response evaluation model, which is used to assess the degree of myocardial protective response induced by tortoise shell glue in a chronic heart failure model.

7. The application of the characteristic metabolite profile of tortoise shell glue according to claim 6 in evaluating myocardial protection, characterized in that: The establishment of the myocardial protection response model includes the following steps: A characteristic metabolite data matrix was constructed based on characterization data of tortoise shell collagen biomarkers and characterization data of myocardial protective response biomarkers; A myocardial protection response model was established using one or more of the following methods: weighted scoring method, partial least squares discriminant analysis method, logistic regression method, random forest method, or XGBoost method. The characteristic metabolite data matrix was used as an input variable, and an evaluation score was obtained based on the myocardial protection response model.

8. The application of the characteristic metabolite profile of tortoise shell glue according to claim 7 in evaluating myocardial protection, characterized in that: The evaluation score based on the myocardial protection response model is obtained according to the following formula: ; Where G is the assessment score, For the level score of tortoise shell collagen-derived markers, The response marker callback score is represented by α and β, which are the weighting coefficients of the corresponding scores.

9. The application of the characteristic metabolite profile of tortoise shell glue according to claim 8 in evaluating myocardial protection, characterized in that: The calculation method for the level score of the tortoise shell collagenous marker is as follows: ; Where n is the number of tortoise shell collagen-derived markers, The weighting coefficient of the j-th tortoise shell collagen-derived biomarker, denoted as the standardized abundance value of the j-th tortoise shell collagen-derived biomarker.

10. The application of the characteristic metabolite profile of tortoise shell glue according to claim 8 in evaluating myocardial protection, characterized in that: The callback score of the response marker is obtained through the following method: ； ; in For the callback rate of the i-th myocardial protection response biomarker, Let be the weighting coefficient of the i-th myocardial protective response marker. This represents the average expression value of the i-th myocardial protective response biomarker in the model control group. represents the average expression value of the i-th biomarker in the tortoise shell glue intervention group. This represents the average expression value of the i-th biomarker in the normal control group.

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