Application of linoleoyl carnitine in screening of medicine with regulating effect on post-PCI pro-inflammatory phase extension of ACS patient, screening method and medicine

Abstract

The invention belongs to the technical field of prevention and treatment of ventricular remodeling after ACS (acute coronary syndrome), and particularly relates to application of linoleoyl carnitine in screening of a medicine with an adjusting effect on post-PCI pro-inflammatory phase extension of an ACS patient, a screening method and the medicine. Research finds that the content of linoleoyl carnitine in plasma of an ACS patient after PCI can reflect the myocardial ischemia injury degree and can aggravate activity inhibition of hypoxia / reoxygenation on myocardial cells for the first time, so that the linoleoyl carnitine can be used for screening drugs with an adjusting effect on prolonging of the proinflammatory period of the ACS patient after PCI. Based on the complex balance relation between the proinflammatory period and the anti-inflammatory repair period, the invention provides a method for reversely screening the medicine with the regulating effect on the extension of the proinflammatory period after the PCI of the ACS patient, and the screened medicine can be used for regulating the polarization of Mo and inhibiting the extension of the proinflammatory period, so that the adverse remodeling of the heart after the ACS is relieved, and the effective recovery of the heart function is promoted.

Description

Technical Field

[0001] The invention belongs to the technical field of preventing and treating ventricular remodeling after ACS, and particularly relates to the application of linoleoylcarnitine in screening drugs having a regulating effect on the extension of the proinflammatory phase after PCI in ACS patients, as well as a screening method and drugs. Background Art

[0002] Acute coronary syndrome (ACS) is a group of clinical syndromes caused by acute myocardial ischemia. Ventricular remodeling after ACS is one of the main causes of heart failure (HF). The regulation of adaptive remodeling and adverse remodeling is the key to prevent and treat ventricular remodeling after ACS. The balance between adaptive remodeling and adverse remodeling is currently a new therapeutic target for preventing and treating ventricular remodeling after ACS.

[0003] The innate immune response is an important regulator between adaptive and maladaptive remodeling and consists of three key phases: pro-inflammatory, anti-inflammatory, reparative, and maturation.

[0004] Monocytes (Mo) play an important role in both the pro-inflammatory and anti-inflammatory repair phases of ventricular remodeling. Studies have shown that high levels of pro-inflammatory Mo in the peripheral blood of ACS patients are associated with prolonged pro-inflammatory phases and impaired left ventricular function recovery. Therefore, it is very important to find substances that reflect the prolonged pro-inflammatory phase after PCI in ACS patients and find methods to inhibit the high levels of pro-inflammatory Mo to inhibit the prolonged pro-inflammatory phase after ACS and prevent and treat ventricular remodeling after ACS. At present, research on balancing the pro-inflammatory phase and anti-inflammatory repair phase in the innate immune response after ACS has not been translated into effective clinical treatment for myocardial ischemic injury. Finding drugs that can balance the pro-inflammatory phase and anti-inflammatory repair phase in the innate immune response after ACS and regulate the prolonged pro-inflammatory phase after PCI in ACS patients will provide new targets for inhibiting the prolonged pro-inflammatory phase after ACS and preventing and treating ventricular remodeling after ACS. Summary of the invention

[0005] In view of the above technical problems, the present invention provides the use of linoleoylcarnitine in screening drugs that have a regulatory effect on the prolongation of the proinflammatory phase after PCI in ACS patients, as well as a screening method and a drug. The present invention proposes for the first time to use linoleoylcarnitine to screen drugs that have a regulatory effect on the prolongation of the proinflammatory phase after PCI in ACS patients. The drugs screened out by the screening method provided by the present invention can be used to regulate the polarization of Mo, inhibit the prolongation of the proinflammatory phase, thereby reducing the adverse remodeling of the heart after ACS and promoting the effective recovery of cardiac function.

[0006] In order to achieve the above-mentioned object of the invention, the present invention adopts the following technical solutions:

[0007] The first aspect of the present invention provides the use of linoleoylcarnitine in screening drugs having a regulatory effect on the prolongation of the pro-inflammatory phase after PCI in ACS patients.

[0008] The present invention first discovered through research that linoleylcarnitine in plasma of ACS patients after PCI is related to the prolongation of the proinflammatory phase and fatty acid metabolism disorder in energy metabolism disorder, and its content can reflect the degree of myocardial ischemic injury and aggravate the inhibition of hypoxia / reoxygenation on the activity of myocardial cells. Therefore, linoleylcarnitine can be used to screen drugs that have a regulating effect on the prolongation of the proinflammatory phase of ACS patients after PCI.

[0009] The second aspect of the present invention is a method for screening a drug having a regulatory effect on the prolongation of the pro-inflammatory phase after PCI in ACS patients, which specifically comprises the following steps:

[0010] S1. Co-culture the cardiomyocytes with the drug to be screened for 8 to 12 hours;

[0011] S2, replacing the culture medium with serum-free and sugar-free DMEM medium containing linoleylcarnitine and the drug to be screened for hypoxic culture for at least 12 hours;

[0012] S3. Replace the culture medium with complete DMEM medium containing linoleoylcarnitine and the drug to be screened. After 4-6 hours of reoxygenation culture, detect the cell activity and screen out the drugs that significantly improve the activity of myocardial cells based on the cell activity, that is, the drugs that have a regulatory effect on the prolonged pro-inflammatory period after PCI in ACS patients.

[0013] Based on the complex balance between the pro-inflammatory phase and the anti-inflammatory repair phase, the present invention provides the above-mentioned method for reverse screening of cells for drugs that have a regulatory effect on the prolongation of the pro-inflammatory phase after PCI in ACS patients. The screened drugs can be used to regulate the polarization of Mo, inhibit the prolongation of the pro-inflammatory phase, thereby alleviating the adverse remodeling of the heart after ACS and promoting the effective recovery of cardiac function.

[0014] Here, "significant improvement" means that the value of cell activity produces a significant difference (such as P<0.05).

[0015] Preferably, the content of linoleylcarnitine in the serum-free and sugar-free DMEM medium is 20 μmol / L.

[0016] Preferably, the cardiomyocytes are H9c2 cardiomyocytes.

[0017] Preferably, the hypoxic culture time is 12 hours.

[0018] Preferably, the reoxygenation culture time is 4 hours.

[0019] Preferably, the method for detecting cell activity in S3 can adopt CCK-8 method. Exemplarily, the cell activity detection can be performed according to the following operation: after the reoxygenation culture in S3 is completed, the original drug-containing serum is aspirated and discarded, and a complete culture medium containing 10% CCK-8 reagent is added again, and the culture medium is incubated at 37°C in the dark for 1 to 2 hours, and the OD value is detected. The drug that improves the activity of cardiomyocytes is screened according to the OD value.

[0020] The third aspect of the present invention provides the use of the above method in screening active ingredients of traditional Chinese medicine that have an improving effect on the prolonged pro-inflammatory phase after PCI in ACS patients.

[0021] By using the above method and the model of linoleoylcarnitine aggravating myocardial cell damage caused by hypoxia / reoxygenation, it is possible to screen out active ingredients of traditional Chinese medicine that can inhibit linoleoylcarnitine from aggravating myocardial cell damage caused by hypoxia / reoxygenation and significantly improve myocardial cell damage caused by hypoxia / reoxygenation.

[0022] The fourth aspect of the present invention provides a drug that has a regulating effect on the prolongation of the pro-inflammatory phase after PCI in ACS patients, wherein the drug contains at least one of salvianolic acid B, cryptotanshinone, tanshinone ⅡA, salvianolic acid C, salvianolic acid F, and asiatic acid. The above 6 active ingredients have the effect of significantly improving the effect of linoleylcarnitine exacerbating hypoxia / reoxygenation myocardial cell injury (P<0.05).

[0023] A fifth aspect of the present invention provides the use of a Salvia miltiorrhiza (SM) extract in the preparation of a medicament for improving the prolonged pro-inflammatory phase after PCI in ACS patients.

[0024] Preferably, the preparation method of the Danshen extract is: extracting Danshen slices with 50% to 70% v / v ethanol aqueous solution by refluxing, and drying the obtained extract. Further preferably, the concentration of the ethanol aqueous solution is 60% v / v.

[0025] The salvia miltiorrhiza extract prepared by the preparation method contains 5 of the above 6 active ingredients, which can significantly improve the inhibitory activity of linoleylcarnitine on hypoxia / reoxygenation cardiomyocytes, increase the activity of superoxide dismutase (SOD), reduce the content of malondialdehyde (MDA), improve the lipid peroxidation damage of linoleylcarnitine to hypoxia / reoxygenation cardiomyocytes, and have therapeutic potential for the damage of hypoxia / reoxygenation to cardiomyocytes aggravated by linoleylcarnitine. After studying the MI / RI mouse model, it was found that the salvia miltiorrhiza extract can significantly increase the left ventricular ejection fraction (LVEF) and left ventricular short axis shortening rate (LVFS) of mice, significantly reduce the left ventricular end-diastolic diameter (LVID,d) and left ventricular end-systolic diameter (LVID,s), and improve the cardiac function of MI / RI mice in a dose-dependent manner. 4g / kg and 2g / kg of salvia miltiorrhiza extract can significantly reduce the inflammatory infiltration of myocardial tissue in MI / RI mice and improve myocardial fibrosis. Through the analysis of metabolic differentials and pathway enrichment, it was found that Danshen extract can regulate the metabolic disorders of MI / RI mice by improving linoleylcarnitine accumulation, promoting fatty acid oxidation, inhibiting glycolysis, etc.; it can increase the SOD activity of MI / RI mice, reduce the MDA content, and improve lipid peroxidation damage. In addition, Danshen extract can also inhibit the expression of proinflammatory factors such as IL-6, IL-1β, TNF-α, IFN-γ and proinflammatory Mo-related chemokines such as GM-CSF, MCP-1 / CCL2, MCP-3 / CCL7 in the myocardium of MI / RI mice, thereby inhibiting the polarization of Mo to the proinflammatory type, inhibiting the extension of the proinflammatory phase after ACS, and regulating the balance between the proinflammatory phase and the anti-inflammatory repair phase in the innate immune response.

[0026] More preferably, the preparation method of the salvia miltiorrhiza extract is as follows: extracting the salvia miltiorrhiza slices twice with 60% v / v ethanol aqueous solution under reflux, combining the obtained extracts, and drying.

[0027] The beneficial effects of the present invention are as follows: the present invention first discovered that linoleylcarnitine in the plasma of ACS patients after PCI is related to fatty acid metabolism disorders in the prolonged pro-inflammatory phase and energy metabolism disorders, and its content can reflect the degree of myocardial ischemic damage, and will aggravate the inhibition of hypoxia / reoxygenation on the activity of myocardial cells, so it can be used to screen drugs that have a regulatory effect on the prolonged pro-inflammatory phase after PCI in ACS patients. The theoretical basis of the method for screening drugs that have an improving effect on the prolonged pro-inflammatory phase after PCI in ACS patients provided by the present invention is the correlation between linoleylcarnitine and fatty acid metabolism disorders in the prolonged pro-inflammatory phase and energy metabolism disorders found by the present invention through research, as well as the complex balance between the pro-inflammatory phase and the anti-inflammatory repair phase. The drugs screened out by this method can be used to regulate the polarization of Mo, inhibit the prolonged pro-inflammatory phase, and then reduce the adverse remodeling of the heart after ACS, and promote the effective recovery of cardiac function. The salvia miltiorrhiza extract contains a variety of active monomers of traditional Chinese medicine screened out by this method. The study found that Salvia miltiorrhiza extract can improve the accumulation of long-chain acylcarnitines such as linoleoylcarnitine in the heart by promoting fatty acid oxidation and inhibiting glycolysis in MI / RI mice, and can also affect the polarization of Mo toward the pro-inflammatory type and regulate the balance between the pro-inflammatory phase and the anti-inflammatory repair phase in the innate immune response after ACS. The results of this study confirmed the feasibility of the above screening method and provided a new therapeutic idea for the clinical prevention and treatment of ventricular remodeling after ACS by combining traditional Chinese and Western medicine. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 The protective effects of the 39 active ingredients of traditional Chinese medicine in Example 1 of the present invention on hypoxia / reoxygenation H9c2 cardiomyocytes; "***" indicates that compared with the Con group, P < 0.001, and "#" indicates that compared with the H12R4 group, P < 0.05;

[0029] Figure 2 The protective effects of the eight active ingredients in Example 1 of the present invention on the hypoxia / reoxygenation H9c2 cardiomyocytes after linoleylcarnitine intervention; "***" indicates that compared with the Con group, P < 0.001, "#" indicates that compared with the H12R4 group, P < 0.05, and "$" indicates that compared with the linoleylcarnitine group, P < 0.05;

[0030] Figure 3 The salvia miltiorrhiza extract in Example 1 of the present invention can improve the inhibitory effect of linoleoylcarnitine on the activity of H9c2 cardiomyocytes after hypoxia / reoxygenation; "***" indicates that compared with the Con group, P < 0.001, "#" indicates that compared with the H12R4 group, P < 0.05, and "￥" indicates that compared with the H12R4 + linoleoylcarnitine group, P < 0.05;

[0031] Figure 4The salvia miltiorrhiza extract in Example 1 of the present invention improves the lipid peroxidation damage of linoleoylcarnitine to H9c2 cardiomyocytes after hypoxia / reoxygenation; A: the effect of salvia miltiorrhiza extract on the SOD activity of H9c2 cardiomyocytes after hypoxia / reoxygenation after linoleoylcarnitine intervention, B: the effect of salvia miltiorrhiza extract on the MDA content of H9c2 cardiomyocytes after hypoxia / reoxygenation after linoleoylcarnitine intervention; "*" indicates that compared with the Con group, P<0.05, "#" indicates that compared with the H12R4 group, P<0.05, "￥" indicates that compared with the H12R4+linoleoylcarnitine group, P<0.05;

[0032] Figure 5 The effect of the Danshen extract in Example 2 of the present invention on the body weight and various organs of MI / RI mice; A: body weight coefficient, B: heart organ index, C: liver organ index, D: spleen organ index, E: lung organ index, F: kidney organ index; "**" indicates P<0.01 compared with the Sham group; "#" indicates P<0.05 compared with the Model group, "##" indicates P<0.01, n=10;

[0033] Figure 6 This is the mouse heart ultrasound during the modeling process in Example 2 of the present invention; A: echocardiographic image, B: left ventricular ejection fraction; "***" indicates that compared with the Sham group, P<0.001, n=10;

[0034] Figure 7 These are ultrasound images of the hearts of mice in each group 3 days after reperfusion in Example 2 of the present invention;

[0035] Figure 8 The effect of the Danshen extract in Example 2 of the present invention on the cardiac function of MI / RI injured mice; A: left ventricular ejection fraction, B: left ventricular minor axis shortening rate, C: left ventricular end-diastolic diameter, D: left ventricular end-systolic diameter; compared with the Sham group, "*" indicates P<0.05, "**" indicates P<0.01; compared with the Model group, "#" indicates P<0.05, "##" indicates P<0.01, n=10;

[0036] Fig. 9 is the HE staining image (40×) of the myocardial tissue of each group of MI / RI mice in Example 2 of the present invention;

[0037] Fig.10 is a Masson staining image (40×) of the myocardial tissue of each group of MI / RI mice in Example 2 of the present invention;

[0038] Fig.11 The principal component analysis results of plasma (A) and heart tissue (B) of each group of MI / RI mice 3 days after PCI in Example 2 of the present invention are shown;

[0039] Fig.12 OPLS-DA, 200 permutation tests and S-Plot of plasma of MI / RI mice 3d post-PCI in Example 2 of the present invention;

[0040] Fig.13 OPLS-DA, 200 permutation tests and S-Plot diagram of heart tissue of MI / RI mice 3d post-PCI in Example 2 of the present invention;

[0041] Fig.14 This is the metabolic pathway analysis of plasma differential metabolites between the SM (4 g / kg) group and the Model group at 3 days after Post-PCI in Example 2 of the present invention;

[0042] Fig.15 It is the differential metabolite of cardiac tissue between SM (4 g / kg) group and Model group at 3 days after Post-PCI in Example 2 of the present invention;

[0043] Fig.16 This is the effect of the Danshen extract on lipid peroxidation injury in MI / RI mice at 3 days after post-PCI in Example 2 of the present invention; compared with the Sham group, "*" indicates P<0.05, "**" indicates P<0.01; compared with the Model group, "#" indicates P<0.05, "##" indicates P<0.01, n=10;

[0044] Fig.17 The expression of pro-inflammatory and anti-inflammatory cytokines in each group of MI / RI mice at 3 days after post-PCI in Example 2 of the present invention; compared with the Sham group, "*" indicates P<0.05, "**" indicates P<0.01; compared with the Model group, "#" indicates P<0.05, "##" indicates P<0.01, n=10;

[0045] Fig.18 The expression of chemokines related to pro-inflammatory Mo in each group of MI / RI mice at 3 days after post-PCI in Example 2 of the present invention; compared with the Sham group, "*" indicates P < 0.05, "**" indicates P < 0.01; "#" indicates P < 0.05 compared with the Model group, n = 10;

[0046] Fig.19 This is the expression of chemokines related to repair Mo in each group of MI / RI mice at 3 days post-PCI in Example 2 of the present invention; "*" indicates that compared with the Sham group, P<0.05, n=10;

[0047] Fig. 20The model of hypoxia / reoxygenation of H9c2 cardiomyocytes in Example 1 of the present invention is established; A: optimal hypoxia / reoxygenation time screening result, B: optimal hypoxia / reoxygenation time screened; "***" indicates that compared with the Con group, P < 0.001;

[0048] Fig.21 The cell morphology of the H9c2 cardiomyocytes Con group and H12R4 group in Example 1 of the present invention;

[0049] Fig. 22 It is the identification spectrum of propionylcarnitine in hypoxia / reoxygenation H9c2 cardiomyocytes in Example 1 of the present invention; A: primary spectrum, the upper part is the metabolite in the cell, and the lower part is the propionylcarnitine standard; B: secondary fragment, the upper part is the metabolite in the cell, and the lower part is the propionylcarnitine standard;

[0050] Fig.23 It is the identification spectrum of butyrylcarnitine in hypoxia / reoxygenation H9c2 cardiomyocytes in Example 1 of the present invention; A: primary spectrum, the upper part is the metabolite in the cell, and the lower part is the butyrylcarnitine standard; B: secondary fragment, the upper part is the metabolite in the cell, and the lower part is the butyrylcarnitine standard;

[0051] Fig.24 This is the identification spectrum of valerylcarnitine in hypoxia / reoxygenation H9c2 cardiomyocytes in Example 1 of the present invention; A: primary spectrum, the upper part is the metabolite in the cell, and the lower part is the valerylcarnitine standard; B: secondary fragment, the upper part is the metabolite in the cell, and the lower part is the valerylcarnitine standard;

[0052] Fig.25 is the effect of the five potential biomarkers in Example 1 of the present invention on the activity of H9c2 cardiomyocytes; A is the effect of the five potential biomarkers on the activity of hypoxia / reoxygenation H9c2 cardiomyocytes, B is the effect of different doses of linoleoylcarnitine on hypoxia / reoxygenation H9c2 cardiomyocytes, and C is the effect of linoleoylcarnitine on healthy H9c2 cardiomyocytes; "***" indicates that compared with the Con group, P < 0.001, compared with the H12R4 group, "#" indicates P < 0.05, and "###" indicates P < 0.001;

[0053] Fig.26 The linoleoylcarnitine in Example 1 of the present invention aggravates the lipid peroxidation damage of H9c2 cardiomyocytes after hypoxia / reoxygenation; A is the effect of linoleoylcarnitine on the SOD activity of H9c2 cardiomyocytes after hypoxia / reoxygenation, and B is the effect of linoleoylcarnitine on the MDA content of H9c2 cardiomyocytes after hypoxia / reoxygenation; "*" indicates that compared with the Con group, P<0.05, and "#" indicates that compared with the H12R4 group, P<0.05. DETAILED DESCRIPTION

[0054] In order to make the purpose, technical scheme and advantages of the present invention clearer, the present invention is further described in detail below in conjunction with specific embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work fall within the scope of protection of the present invention.

[0055] The present invention has found through research that the content of linoleylcarnitine in the plasma of ACS patients is related to the extension of the pro-inflammatory phase after PCI in ACS patients, which can reflect the degree of myocardial ischemic injury after ACS, and reproduced and verified in the model of ischemia-reperfusion in C57BL / 6J mice that linoleylcarnitine does exist and accumulates in the heart tissue, and the accumulation of linoleylcarnitine in the heart tissue is related to myocardial injury. Based on the H9c2 myocardial cell hypoxia / reoxygenation model, the present invention has found that linoleylcarnitine has a dose-dependent inhibitory effect on the activity of hypoxic / reoxygenated H9c2 myocardial cells. The present invention has also found through research that linoleylcarnitine has a significant inhibitory effect on the SOD activity of hypoxic / reoxygenated H9c2 myocardial cells, and can increase the MDA content of hypoxic / reoxygenated H9c2 myocardial cells. MDA is a lipid peroxide produced after the biological membrane is attacked by ROS, and its level indirectly reflects the degree of lipid peroxidation damage in the cell; SOD is a key antioxidant enzyme in the cell and an important indicator for evaluating the antioxidant capacity of the cell. Therefore, linoleylcarnitine can aggravate lipid peroxidation injury in H9c2 cardiomyocytes after hypoxia / reoxygenation.

[0056] With its diversified ingredients, multi-pathway regulation and multi-target effects, traditional Chinese medicines that replenish qi and promote blood circulation have significant advantages and broad application prospects in the prevention and treatment of cardiovascular diseases. Not only are the drugs flexible to use, but they also have fewer side effects. They have the advantages of multi-target, multi-angle and multi-mechanism improvements in inhibiting Mo-related inflammatory reactions and preventing and treating ventricular remodeling after ACS. This part of the study starts from the active ingredients of traditional Chinese medicines that replenish qi and promote blood circulation, and focuses on the effects of potential biomarkers on cardiomyocytes, and reversely searches for traditional Chinese medicines that have protective effects on cardiomyocytes.

[0057] The present invention experimentally studies the H9c2 cardiomyocyte model of hypoxia / reoxygenation based on linoleylcarnitine intervention, and screens 39 active ingredients of qi-invigorating and blood-activating Chinese medicines from qi-invigorating and blood-activating Chinese medicines. The results show that 8 active ingredients, including salvianolic acid B, cryptotanshinone, and ginsenoside Rg3, can significantly improve the activity of H9c2 cardiomyocytes hypoxia / reoxygenation intervened by linoleylcarnitine. The above 8 active ingredients are given on the basis of H9c2 cardiomyocytes hypoxia / reoxygenation intervened by linoleylcarnitine, and it is found that 6 active ingredients, including salvianolic acid B, cryptotanshinone, and tanshinone ⅡA salvia miltiorrhiza, have a protective effect on the activity of H9c2 cardiomyocytes hypoxia / reoxygenation after linoleylcarnitine intervention, and 5 of them are effective active ingredients of salvia miltiorrhiza.

[0058] The present invention found through experimental research that the salvia miltiorrhiza extract can improve the activity inhibition and lipid peroxidation damage of linoleoylcarnitine on hypoxia / reoxygenation H9c2 cardiomyocytes, and may have a certain therapeutic potential for fatty acid metabolism disorders and inflammation such as linoleoylcarnitine accumulation.

[0059] Based on the above research, the embodiments of the present invention provide the use of linoleoylcarnitine in screening drugs that have a regulatory effect on the extension of the pro-inflammatory phase after PCI in ACS patients.

[0060] The embodiment of the present invention also provides a method for screening a drug having a regulating effect on the prolongation of the pro-inflammatory phase after PCI in ACS patients, which specifically comprises the following steps:

[0061] S1. Co-culture the cardiomyocytes with the drug to be screened for 8 to 12 hours;

[0062] S2, replacing the culture medium with serum-free and sugar-free DMEM medium containing linoleylcarnitine and the drug to be screened for hypoxic culture for at least 12 hours;

[0063] S3. Replace the culture medium with complete DMEM medium containing linoleoylcarnitine and the drug to be screened. After 4-6 hours of reoxygenation culture, detect the cell activity and screen out the drugs that significantly improve the activity of myocardial cells based on the cell activity, that is, the drugs that have a regulatory effect on the prolonged pro-inflammatory period after PCI in ACS patients.

[0064] The embodiment of the present invention also provides the use of the above method in screening active ingredients of traditional Chinese medicine that have an improving effect on the prolongation of the proinflammatory phase caused by the prolongation of the proinflammatory phase after PCI in ACS patients.

[0065] The embodiment of the present invention also provides a drug that has a regulating effect on the extension of the pro-inflammatory phase after PCI in ACS patients.

[0066] The embodiment of the present invention also provides the use of the Danshen extract in preparing a drug for improving the prolonged pro-inflammatory phase of ACS patients after PCI.

[0067] The scheme of the present invention is described below through specific embodiments.

[0068] The main analytical instruments used in the following examples are: flow cytometer, BD FACSVerse TM (BD Biosciences, USA), Ultimate 3000 ultra-high performance liquid chromatography system (Thermo Fisher Scientific, USA), high-resolution mass spectrometer Q-Exactive-Orbitrap MS (Thermo Fisher Scientific, Germany).

[0069] The main reagents used in the following examples: high glucose culture medium, fetal bovine serum, trypsin, double antibody, phosphate buffered saline (PBS) were purchased from BI, Israel; RPMI-1640 sugar-free culture medium was purchased from Gibco; dimethyl sulfoxide (DMSO) was purchased from Solarbio, China; CCK-8 reagent was purchased from Dojin Chemical Research Institute, Japan; propionylcarnitine standard and suspending agent sodium carboxymethylcellulose (CMC-Na) were purchased from Shanghai MacLean Biochemical Technology Co., Ltd.; butyrylcarnitine standard, valerylcarnitine standard, hydroxyisovalerylcarnitine standard, linoleylcarnitine standard were purchased from Sigma-Aldrich, Merck, USA; total SOD activity detection kit and lipid oxidation (MDA) detection kit were purchased from Beyotime Biotechnology; Omni-Easy TM Ready-to-use BCA protein quantification kit was purchased from Shanghai Yamei Biopharmaceutical Technology Co., Ltd.; Danshen decoction pieces were purchased from Hebei Chunkai Pharmaceutical Co., Ltd., batch number 230902077; EDTA was purchased from Beijing Solebow Biotechnology Co., Ltd.; xylene, environmentally friendly dewaxing solution, anhydrous ethanol, and neutral gum were purchased from Meiyao Group Chemical Reagent Co., Ltd.; HE staining solution set, Masson staining solution set, differentiation solution, and 4% tissue cell fixative were purchased from Servicebio; Bio-plex Pro TM Mouse Chemokine Assays and PE-streptavidin were purchased from Bio-RAD; total superoxide dismutase (T-SOD) and malondialdehyde (MDA) enzyme-linked immunosorbent assay kits were purchased from Elabscience.

[0070] The rat cardiomyocyte cell line (H9c2) used in the following examples was purchased from the Meijia Biomedical Experimental Cell Resource Bank.

[0071] Unless otherwise specified, other raw materials, reagents, drugs or instruments used in the following examples are conventional commercial products obtained from commercial channels. The methods used in the following examples are conventional methods in the art unless otherwise specified.

[0072] The statistical analysis used in the following examples is as follows: experimental data are expressed as mean ± standard deviation SPSS26.0 was used for statistical analysis. The t test was used for comparison between two groups, and One-way ANOVA was used for comparison between multiple groups. When the variances were equal, the LSD test method was used, and when the variances were unequal, the Dunnett's T3 test method was used. P<0.05 was considered statistically significant.

[0073] Example 1

[0074] This example provides a method for screening drugs that have a regulatory effect on the prolonged pro-inflammatory phase in ACS patients after PCI.

[0075] 1. Experimental methods

[0076] 1.1 Recovery, passaging and culture of H9c2 cardiomyocytes

[0077] H9c2 cardiomyocytes were revived, passaged and cultured according to routine procedures.

[0078] 1.2 Screening of drugs that have protective effects on linoleylcarnitine-treated H9c2 cardiomyocytes subjected to hypoxia / reoxygenation

[0079] Focusing on the Chinese medicines for invigorating qi and promoting blood circulation, based on the H9c2 cardiomyocyte model of hypoxia / reoxygenation intervened by linoleylcarnitine, the protective effects of 39 monomer active ingredients from Chinese medicines for invigorating qi and promoting blood circulation, such as salvia miltiorrhiza, ginseng, astragalus, ginkgo, panax notoginseng, angelica, ligusticum chuanxiong, saffron, etc. (Table 1) on hypoxia / reoxygenation of H9c2 cardiomyocytes were investigated.

[0080] Table 1 39 active ingredients from traditional Chinese medicines that replenish qi and promote blood circulation

[0081]

[0082] The method of pre-administration for 12 hours was selected, 1 μmol / L was selected as the initial administration concentration, and the CCK-8 method was used to detect cell activity to screen the active ingredients of traditional Chinese medicine with obvious protective effects and the subsequent concentrations used. The specific operation steps are as follows:

[0083] 1.2.1Planning

[0084] H9c2 cardiomyocytes of similar generation were cultured at 6×10 4 The cells were inoculated at a density of 100 μL / mL in a 96-well plate, with 100 μL per well, and cultured with complete DMEM medium. After the cells adhered to the wall, the original medium was discarded and replaced with complete DMEM medium containing 1 μmol / L active ingredient. The cells were then returned to a constant temperature incubator at 37°C and 5% CO2 for 12 h.

[0085] 1.2.2 Intervention

[0086] After 12 hours of pre-administration, the original medium was discarded and replaced with serum-free and sugar-free DMEM medium containing 20 μmol / L linoleoylcarnitine and 1 μmol / L active ingredient, and placed in a hypoxic chamber. A mixed gas containing 95% N2 and 5% CO2 (O2≤1%) was filled into the hypoxic chamber for hypoxia intervention. After 12 hours of hypoxic culture, the cells were removed from the hypoxic chamber, the original drug-containing medium was discarded and rinsed with PBS, and the medium was replaced with complete DMEM medium containing 20 μmol / L linoleoylcarnitine and 1 μmol / L active ingredient, and returned to a constant temperature incubator at 37°C, 95% O2 and 5% CO2 for reoxygenation culture for 4 hours. The cells in the control group were operated in the same way, but complete DMEM medium was always used.

[0087] The specific groups are as follows:

[0088] ① Control group (Con): Complete DMEM medium was given for synchronization at each stage and placed in a constant temperature incubator at 37°C and 5% CO2 for 16 h;

[0089] ② Model group (H12R4): According to the intervention method, the corresponding culture medium (without linoleylcarnitine and the drug to be screened) and culture conditions were given at each stage, with hypoxia culture for 12 hours and reoxygenation culture for 4 hours;

[0090] ③ Hypoxia / reoxygenation + active ingredient group (traditional Chinese medicine monomer active ingredient): pre-administered for 12 hours, followed by administration of the corresponding culture medium (containing 20 μmol / L linoleoylcarnitine and 1 μmol / L of the drug to be screened) and culture conditions at each stage, hypoxia culture for 12 hours, and reoxygenation culture for 4 hours.

[0091] 1.2.3 CCK-8 assay to determine cell viability

[0092] The original drug-containing serum was discarded, and complete DMEM medium containing 10% CCK-8 reagent was added again, 100 μL per well, and placed in a 37°C constant temperature incubator in the dark for about 1.5 hours. The OD value was detected at 450nm using an enzyme marker to screen out the active ingredients and concentrations of traditional Chinese medicine that best promoted cell activity.

[0093] 1.2.4 Results

[0094] like Figure 1 As shown, compared with the Con group, the cardiomyocyte activity of the H12R4 group was significantly decreased (P<0.001); salvianolic acid B, angelica lactone A, cryptotanshinone, pachymic acid, ginsenoside Rg3, ginsenoside Rg1, tanshinone ⅡA, salvianolic acid C, salvianolic acid F, and asiatic acid could significantly improve the activity of H9c2 cardiomyocytes subjected to hypoxia / reoxygenation induced by linoleylcarnitine (P<0.05).

[0095] The above 10 active ingredients of traditional Chinese medicine were tested at concentrations of 0.01μmol / L, 0.1μmol / L, 1μmol / L, and 10μmol / L according to the above method. The results showed that 8 of the 10 active ingredients could significantly improve the hypoxia / reoxygenation inhibition of H9c2 cardiomyocytes by linoleylcarnitine intervention (P<0.05), namely, salvianolic acid B, cryptotanshinone, tanshinone ⅡA, salvianolic acid C, salvianolic acid F, ginsenoside Rg3, ginsenoside Rg1, and asiatic acid. Except for ginsenoside Rg3, which had an optimal intervention concentration of 10μmol / L, the optimal intervention concentrations of the other 7 were all 1μmol / L (Table 2).

[0096] Table 2 8 active ingredients of traditional Chinese medicine that improve the activity of H9c2 cardiomyocytes induced by linoleylcarnitine and hypoxia / reoxygenation

[0097]

[0098] Note: Probability refers to the ratio of the number of times P<0.05 to the total number of experiments.

[0099] The above eight traditional Chinese medicine active ingredients, including salvianolic acid B, cryptotanshinone, and ginsenoside Rg3, which can significantly improve the activity of H9c2 cardiomyocytes induced by linoleylcarnitine and hypoxia / reoxygenation, were tested at their respective optimal intervention concentrations according to the above method. The results are as follows Figure 2 As shown. Compared with the Con group, the cardiomyocyte activity of the H12R4 group was significantly decreased (P<0.001); compared with the H12R4 group, 20μmol / L linoleylcarnitine had a significant inhibitory effect on the cell activity of H9c2 cardiomyocytes after hypoxia / reoxygenation (P<0.05); on the basis of linoleylcarnitine intervention on hypoxia / reoxygenation H9c2 cardiomyocytes, the administration of salvianolic acid B, cryptotanshinone, tanshinone ⅡA, salvianolic acid C, salvianolic acid F, and asiatic acid could significantly improve the inhibitory effect of linoleylcarnitine accumulation on the activity of hypoxia / reoxygenation H9c2 cardiomyocytes (P<0.05), and 5 of the above 6 effective active ingredients belong to the effective ingredients of Salvia miltiorrhiza, indicating that Salvia miltiorrhiza may have a certain protective effect on H9c2 cardiomyocytes after linoleylcarnitine intervention.

[0100] 1.3 Protective effect of SM extract on H9c2 cardiomyocytes subjected to hypoxia / reoxygenation

[0101] 1.3.1 Preparation of SM extract

[0102] 1 kg of SM slices were reflux extracted twice with 60% v / v ethanol aqueous solution, each time for 2 hours, with a solid-liquid ratio of 1:8 and 1:6. The combined extracts were filtered and concentrated to the end, and vacuum dried to obtain SM extracts with an extraction rate of 45.05%. The obtained SM extracts were used for the efficacy studies of cells and subsequent animal experiments.

[0103] 1.3.2 Effects of different concentrations of SM extract on the activity of H9c2 cardiomyocytes induced by linoleylcarnitine and hypoxia / reoxygenation

[0104] Three experimental concentrations (low, medium, and high) were selected. The CCK-8 method was used to detect cell activity to investigate whether different concentrations of SM extract had the best protective effect on the linoleylcarnitine-intervention hypoxia / reoxygenation H9c2 cardiomyocyte model. The specific steps were the same as "1.2".

[0105] The specific groups are as follows:

[0106] ① Control group (Con): Complete DMEM medium was given for synchronization at each stage and placed in a constant temperature incubator at 37°C and 5% CO2 for 16 h;

[0107] ② Model group (H23R4): According to the model establishment method, the corresponding culture medium (without linoleylcarnitine and SM extract) and culture conditions were given at each stage, with hypoxia culture for 12 hours and reoxygenation culture for 4 hours;

[0108] ③ Hypoxia / reoxygenation + SM extract low-dose group (100 μg / mL): pre-administered for 12 hours, followed by administration of the corresponding culture medium (containing 20 μmol / L linoleylcarnitine and 100 μg / mL SM extract) and culture conditions at each stage, hypoxia culture for 12 hours, reoxygenation culture for 4 hours;

[0109] ④ Hypoxia / reoxygenation + SM extract medium dose group (200 μg / mL): pre-administered for 12 hours, followed by administration of the corresponding culture medium (containing 20 μmol / L linoleylcarnitine and 200 μg / mL SM extract) and culture conditions at each stage, hypoxia culture for 12 hours, and reoxygenation culture for 4 hours;

[0110] ⑤ Hypoxia / reoxygenation + SM extract high-dose group (300 μg / mL): pre-administered for 12 hours, followed by administration of the corresponding culture medium (containing 20 μmol / L linoleoylcarnitine and 300 μg / mL SM extract) and culture conditions for each stage, hypoxia culture for 12 hours, and reoxygenation culture for 4 hours.

[0111] The results are as follows Figure 3Compared with the Con group, the cardiomyocyte activity of the H12R4 group was significantly decreased (P<0.001); compared with the H12R4 group, 20μmol / L linoleylcarnitine had a significant inhibitory effect on the cell activity of H9c2 cardiomyocytes under hypoxia / reoxygenation (P<0.05); on the basis of linoleylcarnitine intervention on H9c2 cardiomyocytes under hypoxia / reoxygenation, 300μg / mL SM extract could significantly improve the inhibitory effect of linoleylcarnitine on the activity of H9c2 cardiomyocytes under hypoxia / reoxygenation (P<0.05), indicating that Danshen has an improving effect on the activity of H9c2 cardiomyocytes under hypoxia / reoxygenation after linoleylcarnitine intervention.

[0112] 1.3.3 Effects of SM extract on lipid peroxidation injury in H9c2 cardiomyocytes induced by linoleylcarnitine-induced hypoxia / reoxygenation

[0113] (1) Total SOD activity detection

[0114] ① Preparation of cell samples: cells were cultured in anoxic conditions for 12 hours, and reoxygenated for 4 hours before the culture dish was removed from the incubator, the original drug-containing medium was discarded, and the cells were rinsed twice with pre-cooled PBS, digested with 0.25% trypsin, and digestion was stopped when the cells became round, and the cells were transferred to a centrifuge tube and centrifuged at 1000 rpm for 5 minutes at room temperature to collect the cells. SOD sample preparation solution was added to the cell pellet at a ratio of 100 μL of SOD sample preparation solution per 1 million cells, and gently pipetted to promote lysis. Subsequently, the cells were lysed on ice for 10-15 minutes, and then centrifuged (4°C, 14000 rpm, 20 minutes), and the supernatant was taken for testing.

[0115] ② Determination of protein concentration by bicinchoninic acid (BCA) method: Detect total protein concentration according to the instruction manual of BCA protein concentration determination kit (containing non-dilution standard), that is, according to the number of samples, prepare an appropriate volume of BCA working solution with reagent A and reagent B in the BCA kit at a ratio of 50:1. According to the gradient concentration of protein standards (Table 3), add 20 μL of diluted sample, add 200 μL of BCA working solution to each well, and place at 37°C for 20-30 minutes. Use an enzyme reader to measure the OD value at 562nm, and calculate the protein concentration of the sample according to the standard curve.

[0116] Table 3 BCA protein standard concentration gradient

[0117]

[0118] ③Prepare WST-8 / enzyme working solution: Each sample requires 160 μL of WST-8 / enzyme working solution (151 μL SOD detection buffer, 8 μL WST-8, 1 μL enzyme solution). Prepare the corresponding volume of WST-8 / enzyme working solution according to the number of samples.

[0119] ④ Sample measurement: Add the sample to be tested, SOD detection buffer, WST-8 / enzyme working solution and diluted reaction start working solution to each detection well in sequence according to the instructions, mix well and incubate at 37°C for 30 minutes. Use an ELISA reader to measure the OD value at 450nm and calculate the SOD activity.

[0120] (2) MDA content detection

[0121] ① Preparation of cell samples: After 12 hours of hypoxic culture and 4 hours of reoxygenation culture, take out the culture dish from the incubator, discard the original drug-containing medium, rinse twice with pre-cooled PBS, digest with 0.25% trypsin, and collect the cells after centrifugation at 1000rpm for 5 minutes at room temperature. Add 100μL lysis solution to the cell pellet at a ratio of 1000000 cells, add lysis solution to the cell pellet after gentle pipetting and lysis on ice for 10-15 minutes, centrifuge (4℃, 14000rpm, 20min), and take the supernatant for testing.

[0122] ②The BCA method for determining protein concentration is the same as above.

[0123] ③Prepare TBA storage solution: Weigh an appropriate amount of TBA and use TBA preparation solution to prepare TBA storage solution with a final concentration of 0.37%. After fully dissolved, store at room temperature away from light.

[0124] ④ Prepare MDA working solution: Each sample requires 203 μL of MDA working solution (150 μL TBA diluent, 50 μL TBA storage solution, 3 μL antioxidant). Prepare the corresponding volume according to the number of samples.

[0125] ⑤Prepare a standard curve: dilute the standard in the kit to 1μM, 2μM, 5μM, 10μM, 20μM, 50μM, and 100μM with PBS to prepare a standard curve.

[0126] ⑥ Sample measurement: Take 1 1.5mL EP tube and add 100μL PBS as a blank control, take 7 other tubes and add 100μL of different concentrations of standard to make a standard curve, add 100μL sample to the sample tube for measurement, and finally add 200μL MDA detection working solution to each EP tube, mix well, heat at 100℃ for 15min, cool to room temperature, centrifuge (room temperature, 1000rpm, 10min), and take the supernatant. Add 200μL of supernatant from each group to a 96-well plate, measure the OD value at 532nm with an enzyme reader, and calculate the MDA content.

[0127] (3) Results

[0128] like Figure 4Compared with the Con group, the SOD activity of cardiomyocytes in the H12R4 group was significantly decreased (P<0.01), and the MDA content was significantly increased (P<0.05); compared with the H12R4 group, linoleylcarnitine could significantly reduce the SOD activity of H9c2 cardiomyocytes after hypoxia / reoxygenation (P<0.05), and significantly increase the MDA content (P<0.05). On the basis of linoleylcarnitine intervention in hypoxia / reoxygenation H9c2 cardiomyocytes, 300μg / mL SM extract could significantly increase SOD activity (P<0.05) and reduce MDA content (P<0.05), indicating that SM extract can improve the lipid peroxidation damage of linoleylcarnitine to H9c2 cardiomyocytes after hypoxia / reoxygenation.

[0129] Example 2

[0130] This example provides the effects of Danshen extract on energy metabolism and inflammatory response in mice with ischemia-reperfusion injury.

[0131] 1. Experimental methods

[0132] 1. Animal model preparation, grouping and dosing regimen

[0133] SPF-grade healthy C57BL / 6J male mice were used as research subjects, weighing 25±5g, provided by Sibeifu (Beijing) Co., Ltd., animal qualification certificate number: SCXK (Beijing) 2019-0010, and the experiment was approved by the Ethics Committee of Tianjin University of Traditional Chinese Medicine (TCM-LAEC2023125). All the above animals were raised in the SPF-grade animal room of the Experimental Animal Center of Tianjin University of Traditional Chinese Medicine, with a room temperature of 20-25℃ and a relative humidity of 40-60%. The feed was provided by the animal center and was sterilized granular feed. Drinking bottles filled with clean tap water were placed for mice to drink freely. The drinking bottles were changed daily, and the light and dark environments were alternated for 12 hours each. Before the experiment, all mice were adaptively fed, had free drinking water, had no adverse reactions, and were included in the experiment with normal eating, drinking and activities.

[0134] 1.1 Preparation of mouse ischemia-reperfusion model

[0135] The specific steps are as follows:

[0136] (1) The day before modeling, the mice were skinned and depilated from the neck and chest. After intraperitoneal injection of tribromoethanol for anesthesia, the mice were fixed on a heated operating table, intubated and assisted with breathing by a ventilator, with a respiratory rate of 120 times / min and a tidal volume of 0.2 mL.

[0137] (2) Thoracotomy: After the mouse breathes evenly, make a transverse incision on the chest and use curved forceps to bluntly separate the cortex, muscle and other tissues to expose the ribs and intercostal spaces. Clamp the lower edge of the fourth rib with bone forceps, and use curved forceps to enter the chest cavity at an angle of 20 degrees. Expand the curved forceps, place a chest expander, and adjust the field of view.

[0138] (3) Ligation: Before ligation, place a reperfusion thread above the LAD, use a 7-0 suture needle to tie a loose knot 2-3 mm below the lower edge of the left atrial appendage to ligate the LAD. The whitening of the myocardium in the left ventricular area indicates that the ligation is successful. Time for 45 minutes, close the chest and ventilate. Use a 5-0 suture needle to suture the skin, disinfect with iodine, and try to stop the ventilator. After the mouse breathes evenly, remove the ventilator and place the mouse on an electric blanket.

[0139] (4) Reperfusion: 45 minutes after successful ligation, the mouse's cardiac function was tested by ultrasound, and then the slipknot was opened for reperfusion. After reperfusion, the mouse was placed on an electric blanket and waited for awakening.

[0140] 1.2 Experimental groups and drug administration methods

[0141] According to body weight, mice were randomly divided into sham group (Sham), model group (Model), positive drug group (Asipirin), SM extract low-dose group, SM extract medium-dose group, and SM extract high-dose group, with a total of 6 groups, 15 in each group. Each group of mice was given oral administration, starting from 5 days before modeling, once a day, and continuously until the third day after modeling. According to the clinical drug dose (2020 edition of the Pharmacopoeia dose: 10-15g / 70kg / d) and the conversion relationship between the mouse dose and the human dose, the raw drug doses of SM extract in the high, medium and low groups were 4g / kg, 2g / kg and 1g / kg, respectively. Among them, 2g / kg is approximately the clinical equivalent dose. The mice in the positive control group were gavaged with 0.2% CMC-Na before modeling and 13mg / kg of aspirin after modeling. The intervention was performed by gavage of 0.1mL per 10g body weight, and the sham group and the model group were given the same volume of 0.2% CMC-Na. The mice were weighed once a day, and the dosing volume was adjusted in time according to the changes in body weight (Table 4).

[0142] Table 4 Experimental groups and drug administration

[0143]

[0144]

[0145] 2. Collection of plasma and tissue samples

[0146] 2.1 Plasma samples

[0147] Coat the centrifuge tube with EDTA anticoagulant in advance. After the mouse is anesthetized, remove the eyeball to collect blood, mix thoroughly by turning it upside down, and allow the blood to coagulate. After standing at room temperature for 30 minutes, centrifuge at 4°C, 3000 rpm for 10 minutes, collect the supernatant, and store it at -80°C after aliquoting.

[0148] 2.2 Tissue samples

[0149] After the blood collection is completed, the heart is perfused with pre-cooled saline, and the heart, liver, spleen, lung, and kidney are immediately removed, washed with saline, and dried with filter paper, and weighed for calculation of organ index. Three organs are fixed in tissue fixative. The remaining organs are wrapped with tin foil, placed in cryopreservation tubes, marked, and quickly frozen in liquid nitrogen. After the sampling is completed, they are transferred to a -80℃ refrigerator for storage in preparation for other index detection.

[0150] 3. Experimental methods

[0151] 3.1 Echocardiography

[0152] 45 minutes after modeling and before sampling 3 days after reperfusion, each group of mice underwent cardiac ultrasound examination. The specific method was as follows: the mice were anesthetized and fixed in a supine position on a small animal heating operating table, the skin was prepared (hair removal at the neck and chest), ultrasound coupling agent was applied to the left chest, the probe was close to the left chest, and the detection position was the left side of the sternal midline. After the complete heart was presented in the B-mode ultrasound image field, the echocardiogram was collected in the M-mode ultrasound image mode. LVEF, LVFS, LVID,d, LVID,s were measured, and the measurement was repeated for 3 cycles, and the average value was recorded.

[0153] 3.2 Cardiac tissue pathology

[0154] The mouse heart was fixed and sliced ​​according to routine procedures, and then HE staining and Masson staining were performed.

[0155] 3.3 Metabolomics analysis of plasma and cardiac tissue based on UHPLC / Q-Orbitrap-MS

[0156] Metabolite elution was completed on the Ultimate 3000 ultra-high performance liquid chromatography system: the chromatographic column was HSS T3 (Waters, 2.1×100mm, 1.8μm), the mobile phase was 0.1% formic acid in water (A), acetonitrile (B). The elution gradient was as follows: 0-7min, 5%-100% B; 7-10min, 100% B; 10-10.5min, 100%-5% B; 10.5-13min, 5% B. The column temperature was 35°C, the flow rate was 0.3mL / min, and the injection volume was 2μL.

[0157] Mass spectrometry analysis was performed on a Q-ExactiveTM hybrid Q-Orbitrap mass spectrometer system equipped with a heated electrospray ionization source (HESI). The HESI source parameters were set as follows: spray voltage -3.5 kV / +3.5 kV, sheath gas (N2) 35 L / h, auxiliary gas (N2) 10 L / h, sweep gas (N2) 0 L / h, capillary temperature 320 °C, auxiliary gas heating temperature 350 °C.

[0158] Full MS / dd-MS 2 Scanning method, MS 1 The full scan range was m / z 60-900, with a resolution of 70,000; MS 2 The mass spectrometer was scanned over a dynamic mass range with a resolution of 17,500.

[0159] 3.4 Detection of lipid peroxidation damage activity indicators in plasma

[0160] The SOD activity and MDA content of mouse plasma were detected according to routine procedures.

[0161] 3.6 The detection of myocardial tissue inflammatory factors and chemokine-related indicators adopts the cytokine multiple detection method, as follows:

[0162] 3.6.1 Tissue protein extraction

[0163] Weigh 10 mg of mouse heart tissue, put it into a 1.5 mL centrifuge tube, add protein lysis buffer (add 10 μL of protease inhibitor to every 1 mL of protein lysis buffer), add 100 μL of protein lysis buffer to every 10 mg of tissue, cut it into pieces with small scissors, use an ultrasonic disruptor to fully disrupt it, place the disrupted tissue in a pre-cooled centrifuge, centrifuge at 4°C, 3000 rpm for 10 min, collect the supernatant, and store it at -80°C.

[0164] 3.6.2 Protein concentration detection

[0165] The method was the same as that in Example 1, “Bicinchoninic acid (BCA) method for protein concentration determination”, and the sample protein concentration was uniformly diluted to 3.9 mg / mL according to the test results.

[0166] 3.6.3 Detection methods

[0167] According to the instructions of the kit, 50 μL of beads, standards, quality control products, and samples were added in sequence, the plate was sealed in the dark, and incubated at room temperature at 800 rpm for 0.5 h. The microplate was placed on a magnetic stand for 3 min to ensure that the beads were adsorbed, 100-200 μL of cleaning solution was added to rinse the beads 3 times, and then 50 μL of detection antibody was added, the plate was sealed in the dark, and the plate was incubated at room temperature at 800 rpm for 0.5 h. The microplate was placed on a magnetic stand for 3 min, 100-200 μL of cleaning solution was added to rinse the beads 3 times, and then 50 μL of PE-streptavidin was added to incubate at room temperature at 800 rpm for 20 min, and the plate was sealed in the dark. After the end, 100-200 μL of cleaning solution was added to rinse the beads 3 times, and 125 μL of sheath solution was added again to resuspend the beads, incubated at room temperature at 800 rpm for 2 min, and tested on the machine.

[0168] 3.6.4 Data processing

[0169] The original output data of Luminex instrument were analyzed by Milliplex Analyst 5.1 system according to the standard curve measurement results generated by the standard samples.

[0170] 4. Results

[0171] 4.1 Effects of Salvia miltiorrhiza extract on body weight and organs of MI / RI mice

[0172] Effects of Danshen extract on body weight and organs of MI / RI mice Figure 5 There was no significant difference in body weight among the mice in each group ( Figure 5 Figure A, P>0.05), but compared with the Sham group, the heart coefficient of the Model group mice was significantly increased ( Figure 5 Figure B, P<0.05), considering that the increase in the heart coefficient of the Model group mice may be related to the inflammatory infiltration caused by the high level of pro-inflammatory Mo 3d post-PCI; compared with the Model group, the heart mass coefficient of the Aspirin and different doses of SM extract groups was significantly reduced ( Figure 5 B, P<0.05), there was no significant difference in the organ indexes of liver, spleen, lung and kidney among the mice in each group ( Figure 5 C-F, P>0.05).

[0173] 4.2 Effects of Salvia miltiorrhiza extract on cardiac function in MI / RI mice

[0174] like Figure 6 As shown in Figure A, compared with the Sham group, the ST segment of the electrocardiogram of mice with LAD ligation for 45 minutes was significantly elevated, and the anterior wall M wave disappeared. Before reperfusion, compared with the Sham group, the LVEF of the Model, Aspirin and SM extract groups was significantly reduced (P<0.05), and there was no significant difference in the LVEF of the mice in the treatment groups (P>0.05) ( Figure 6 Figure B), indicating that the model was successful.

[0175] 3 days after reperfusion (Post-PCI 3d), the cardiac ultrasound measurement results of mice in each group showed ( Figure 7 and Figure 8), compared with the Sham group, the LVEF and LVFS of the mice in the Model group were significantly decreased (P<0.01), and the LVID,d and LVID,s were significantly increased (P<0.01, P<0.05), indicating that the cardiac function of the mice in the Model group was decreased. Compared with the Model group, after intervention with Aspirin and different doses of SM extract, the LVEF and LVFS of the mice were significantly increased (P<0.01), and the LVID,d and LVID,s were significantly decreased (P<0.01 or P<0.05), and the SM extract treatment groups at different doses were better than the Aspirin group, indicating that SM improved the cardiac function of MI / RI mice in a dose-dependent manner at 3d Post-PCI.

[0176] 4.3 Effect of Salvia miltiorrhiza extract on HE staining of MI / RI mice

[0177] The results show that Fig. 9 ), the myocardial interstitial gaps in the Sham group were dense, there was no interstitial edema, no inflammatory cell infiltration, and the myocardial fibers were arranged neatly and orderly with uniform coloring; in the Model group, the myocardial interstitial gaps were widened, the interstitial edema, the structure was severely damaged, the blood vessels proliferated and expanded, the myocardial fibers were arranged disorderly, the local muscle fiber stripes disappeared and broke, and a large number of inflammatory cells infiltrated were visible; in the Aspirin group, the myocardial cells were obviously edematous, the gaps were larger, the cell structure damage was alleviated compared with the Model group, inflammatory cell infiltration was visible, and some muscle fibers were arranged sparsely and disorderly; in the SM (1g / kg) group, the myocardial cells were slightly edematous, the gaps were widened, inflammatory cell infiltration was visible locally, and the myocardial fibers were slightly disordered, but the myocardial pathological damage was alleviated compared with the Model group; compared with the Model group, the pathological changes of myocardial tissue in the SM (4g / kg) and SM (2g / kg) groups were significantly improved, mainly reflected in the denser myocardial interstitial gaps, the reduced inflammatory cell infiltration to varying degrees, the neater arrangement of myocardial fibers, and the more uniform coloring, and the SM (4g / kg) group had the best improvement effect.

[0178] 4.4 Effect of Salvia miltiorrhiza extract on Masson staining in MI / RI mice

[0179] The results show that Fig.10 ), compared with the Sham group, the Model group had significantly more interstitial blue collagen deposits and aggravated myocardial fibrosis. Compared with the Model group, the interstitial blue collagen deposits in each drug group were significantly reduced, and the myocardial fibrosis was significantly improved. The SM (4g / kg) group and the SM (2g / kg) group were better than the Aspirin group, and the SM (4g / kg) group had the best improvement effect.

[0180] 4.5 Effects of Salvia miltiorrhiza extract on differential metabolites and metabolic pathways in MI / RI mice

[0181] 4.5.1 Multivariate statistical analysis of plasma and cardiac metabolomics based on UHPLC / Q-Orbitrap-MS

[0182] The principal component analysis results are as follows Fig.11 As shown, the QC samples clustered together, indicating that the instrument had good precision during the run.

[0183] Compared with principal component analysis, OPLS-DA can more clearly show the differences between pairs. Fig.12 and Fig.13 As shown in the figure, there is significant separation between each two groups, whether in plasma or heart samples, indicating that the metabolite abundance between each two groups measured by UHPLC / Q-Orbitrap-MS is significantly different. The permutation test analysis results show that the R 2 The intercept is greater than zero, Q 2 The intercept is less than zero, indicating that the model is not overfitted and is reliable, with good predictive ability. The identified metabolites with VIP>1 and P<0.05 are marked in red in the S-Plot, and these are considered to be potential differential metabolites.

[0184] In OPLS-DA analysis, R 2 X represents the explanatory power of the model on the X variable, R 2 Y represents the explanatory power of the model for the Y variable, Q 2 The predictability of the model and the differences between groups are shown in Table 1. 2 In terms of metabolites, compared with the Model group, SM (4 g / kg) > SM (2 g / kg) > SM (1 g / kg), indicating that the metabolite abundance between the high-dose group of SM extract and the Model group was the highest.

[0185] 4.5.2 Identification of differential metabolites

[0186] According to the screening criteria of the OPLS-DA model, compounds with VIP>1 and P<0.05 were selected as differential metabolites, and the differential metabolites were identified by comparing the standard, the internal database of the Compound Discoverer software (mzCloud, Chemspider, etc.), the online database HMDB, Pubchem and Metlin databases, etc. The differential metabolites in the plasma and heart tissue of the mice in the SM (4g / kg) group and the Model group were deeply analyzed. The results showed that 19 metabolites were significantly different in the plasma of the two groups of mice (Table 5): compared with the Model group, 9 differential metabolites such as linoleoylcarnitine, palmitoylcarnitine, and N-acetyl-L-methionine were significantly increased in the SM (4g / kg) group, and 10 differential metabolites such as L-acetylcarnitine, propionylcarnitine, and 2-methylbutyrylcarnitine were significantly reduced in the SM (4g / kg) group. There were significant differences in the cardiac tissues of 18 metabolites between the two groups of mice (Table 6): compared with the Model group, 15 differential metabolites such as linoleoylcarnitine, eicosenoylcarnitine, and 6-phosphoglucose were significantly decreased in the SM (4 g / kg) group, while hydroxyeicosatrienoic acid, N-palmitoyl-L-methionine, and N-oleoylleucine were significantly increased in the SM (4 g / kg) group.

[0187] Table 5 Differential metabolites in plasma between SM (4 g / kg) group and Model group at 3 days post-PCI

[0188]

[0189]

[0190] Table 6 Differential metabolites in cardiac tissue between SM (4 g / kg) group and Model group at 3 days post-PCI

[0191]

[0192]

[0193] 4.5.3 Metabolic pathway enrichment analysis

[0194] MetaboAnalyst 5.6 software was used to enrich the differential metabolites in plasma and heart tissue of the SM (4 g / kg) group and the Model group at 3 days after PCI to explore the effect of SM extract on the metabolic pathways of MI / RI mice at 3 days after PCI. The enrichment analysis results are shown in Fig.14 and Fig.15. The results showed that the metabolic pathways in plasma related to the improvement of MI / RI by SM extract mainly included the oxidation of short-chain fatty acids, metabolism of phenylacetate, β-oxidation of long-chain fatty acids, urea cycle, degradation of lysine, and metabolism of glutamate. Further summarizing the metabolic pathways with significant changes, it was found that the metabolic pathways affected by SM extract in plasma were mainly fatty acid oxidation and amino acid metabolism, glycine and serine metabolism, etc. In cardiac tissue, the main metabolic pathways related to the improvement of MI / RI by SM extract were arginine and spermine biosynthesis, glycolysis, β-oxidation of mitochondria, urea cycle, pentose phosphate pathway, amino sugar metabolism, gluconeogenesis, and glutamate metabolism. Further summarizing the metabolic pathways with significant changes, it was found that the metabolic pathways affected by SM extract in cardiac tissue were mainly mitochondrial fatty acid oxidation, glycolysis, and amino acid metabolism.

[0195] 4.6 Effects of Salvia miltiorrhiza extract on lipid peroxidation in MI / RI mice

[0196] like Fig.16 As shown in the figure, compared with the Sham group, the SOD activity of the Model group was significantly reduced (P<0.01), and the MDA content was significantly increased (P<0.05). Compared with the Model group, all doses of SM extract treatment groups could increase SOD activity and reduce MDA content. The 4g / kg SM extract had the best effect of increasing SOD activity and reducing MDA content (P<0.01, P<0.05), indicating that SM extract can improve lipid peroxidation damage in MI / RI mice, further confirming the metabolomics results that SM extract improves energy metabolism disorders such as insufficient fatty acid metabolism in MI / RI mice.

[0197] 4.7 Effects of Salvia miltiorrhiza extract on the levels of inflammatory factors and chemokines in cardiac tissue of MI / RI mice

[0198] 4.7.1 Effects of Salvia miltiorrhiza extract on pro-inflammatory and anti-inflammatory factors in cardiac tissue

[0199] The results are as follows Fig.17 As shown, at 3d Post-PCI, compared with the Sham group, the levels of IL-6, IL-1β, TNF-α, and IFNγ in the heart tissue of the Model group mice were significantly upregulated (P<0.05), while the levels of IL-4 (P<0.05) and IL-10 were downregulated. Compared with the Model group, 4g / kg SM extract treatment significantly reduced the expression of IL-6, IL-1β, TNF-α, and IFNγ in the myocardial tissue of mice (P<0.05), and promoted the expression of IL-10 (P<0.05), while Aspirin significantly inhibited the expression of IL-6 in the myocardial tissue of mice (P<0.05) and promoted the expression of IL-10 (P<0.05).

[0200] 4.7.2 Effects of Salvia miltiorrhiza extract on chemokines in cardiac tissue

[0201] The results are as follows Fig.18 As shown in the results, compared with the Sham group, the levels of GM-CSF, MCP-1 / CCL2, MCP-3 / CCL7, CX3CL1, CXCL10, and CXCL11 in the heart tissue of the Model group mice were significantly upregulated (P<0.05). Compared with the Model group, SM (4g / kg) treatment significantly reduced the expression of GM-CSF, MCP-1 / CCL2, MCP-3 / CCL7, CX3CL1, CXCL10, and CXCL11 (P<0.05), while Aspirin significantly inhibited the expression of MCP-1 / CCL2, MCP-3 / CCL7, and CX3CL10 (P<0.05).

[0202] CCR5 is one of the receptors for CCL3, CCL4, and CCL5, which can inhibit inflammation and alleviate adverse ventricular remodeling after AMI by recruiting the repair Mo subpopulation. CCL17 is a chemokine produced by M2-type Ma, which is conducive to the recruitment of repair Mo. Studies have shown that repair Mo can produce SDF-1α / CXCL12 and TGF-β to enhance the mobilization and activity of regulatory T cells, thereby inhibiting the extension of the pro-inflammatory phase. By analyzing the above chemokines related to repair Mo ( Fig.19 ), and found that compared with the Model group, the expressions of MCP-1α / CCL3, MCP-1β / CCL4, CCL5, TARC / CCL17, and SDF-1α / CXCL12 showed an upward trend after SM (4 g / kg) treatment.

[0203] By summarizing the changes in inflammatory factors and chemokines related to repair-type Mo in myocardial tissue of MI / RI mice after treatment with SM extract, it can be seen that anti-inflammatory-related inflammatory factors and chemokines only showed an upward trend, but were not significantly upregulated. This shows that at 3 days post-PCI, SM extract improves linoleylcarnitine accumulation, promotes fatty acid metabolism, and inhibits glycolysis, while mainly inhibiting the extension of the proinflammatory phase after myocardial injury, balancing the polarization of Mo subpopulations, and thus preventing and treating ventricular remodeling after ACS.

[0204] Experimental Example 1

[0205] This experimental example provides the effects of different hypoxia and reoxygenation conditions on cardiomyocyte activity.

[0206] 1. Recovery, passaging and culture of H9c2 cardiomyocytes

[0207] H9c2 cardiomyocytes were revived, passaged and cultured according to routine procedures.

[0208] 2. Effects of different hypoxia and reoxygenation conditions on cardiomyocyte activity

[0209] 2.1 Paving

[0210] Same as “1.2.1” in Example 1.

[0211] 2.2 Hypoxia intervention

[0212] The method is basically the same as Example 1 "1.2.2", except that the culture medium does not contain linoleylcarnitine and drugs, and the hypoxia time is set to 4h, 6h, 8h, and 12h, respectively.

[0213] 2.3 Reoxygenation intervention

[0214] The method is basically the same as Example 1 "1.2.3", except that the culture medium does not contain linoleoylcarnitine and drugs, and the reoxygenation time is set to 2h, 4h, and 6h, respectively.

[0215] 2.4 CCK-8 assay to determine cell viability

[0216] After reoxygenation intervention, add 10 μL of CCK-8 reagent to each well, mix well, place in a 37°C constant temperature incubator, and incubate for about 1.5 hours in the dark. Detect OD value at 450 nm using an enzyme reader.

[0217] 2.5 Results

[0218] like Fig. 20 As shown in the figure, the cell viability (X±S) of H4R6, H6R4, H6R2, H6R12, H8R2, H12R4, and H12R6 (the value after H represents the number of hours of hypoxia, and the value after R represents the number of hours of reoxygenation) was 0.86±0.09, 0.81±0.05, 0.86±0.05, 0.94±0.04, 0.86±0.04, 0.70±0.04, and 0.63±0.06, respectively, compared with the Con group. Compared with the Con group, the cells were moderately damaged after 12h of hypoxia, 4h of reoxygenation, and 6h of reoxygenation. The hypoxia / reoxygenation model of H9c2 cardiomyocytes was successfully established, and the cell morphology was shown in Figure 1. Fig.21 , among which the degree of cell damage in H12R4 was moderate.

[0219] Experimental Example 2

[0220] This experimental example provides the effects of five biomarkers on H9c2 cardiomyocytes.

[0221] 1. Recovery, passaging and culture of H9c2 cardiomyocytes

[0222] H9c2 cardiomyocytes were revived, passaged and cultured according to routine procedures.

[0223] 2. H9c2 cardiomyocyte sample preparation

[0224] 2.1 Sample pretreatment

[0225] The cell clumps after digestion and centrifugation were rinsed three times with 4°C pre-cooled PBS, centrifuged at 4°C, 800 rpm for 5 min, the supernatant was discarded, 3 mL of pre-cooled PBS was added, the cell clumps were gently blown off with a pipette, and the cells were counted.

[0226] 2.2 Cell sample preparation

[0227] ① Collect 1×10 6 Place cells into a centrifuge tube, rinse three times with 4°C precooled PBS, centrifuge (4°C, 800 rpm, 5 min), discard the supernatant, add 1 mL of ice methanol / water (2:1, v / v), and gently blow the sample apart with a pipette;

[0228] ②Ice bath ultrasonic 200W crushing, crushing for 3s, stop for 7s, continue crushing for 5min, centrifuge (4℃, 14000rpm, 20min);

[0229] ④ Take the supernatant into a 1.5mL centrifuge tube and blow with nitrogen;

[0230] ⑤ Redissolve with 50 μL pre-cooled methanol / water (2:1, v / v), vortex for 5 min, centrifuge (4°C, 14000 rpm, 20 min), take the supernatant, filter with a 0.22 μm organic phase pinhole filter, transfer to an injection vial, and store at -80°C;

[0231] ⑥QC was prepared by mixing equal volumes (10 μL) of all sample extracts.

[0232] 3. Qualitative analysis conditions of 5 biomarkers

[0233] Same as "3.3" in Example 2.

[0234] 4. Effects of five biomarkers on H9c2 cardiomyocytes

[0235] According to the present invention, in the previous clinical study, the relative contents of linoleoylcarnitine and other five acylcarnitines in the plasma of ACS patients 3 days after PCI were obtained as the dosing concentration for intervention cells (see Table 7).

[0236] Table 7 Dosing concentration references for 5 potential biomarkers

[0237]

[0238] The CCK-8 method was used to detect cell activity to determine the concentration range used subsequently. The specific steps are as follows:

[0239] 4.1 Paving

[0240] Same as “1.2.1” in Example 1.

[0241] 4.2 Intervention

[0242] After the cells reached the desired density, serum-free and sugar-free DMEM medium containing 5 biomarkers at the dosage concentrations shown in Table 7 was given. Ten replicate wells were set up in each group. After 12 hours of hypoxic culture, the original drug-containing medium was discarded and replaced with complete DMEM medium containing 5 biomarkers at the initial dosage concentrations, and reoxygenated culture was performed for 4 hours.

[0243] The specific groups are as follows:

[0244] ① Control group (Con): Complete medium was given for synchronization of each stage, and the cells were placed in a constant temperature incubator at 37°C and 5% CO2 for 16 h;

[0245] ② Model group (H12R4): According to the intervention method, the corresponding culture medium (without biomarkers) and culture conditions were given at each stage, with hypoxia culture for 12 hours and reoxygenation culture for 4 hours;

[0246] ③Hypoxia / reoxygenation + propionylcarnitine group (A): on the basis of hypoxia and reoxygenation, the initial concentration of propionylcarnitine was given at the same time, and the hypoxia culture was carried out for 12 hours and the reoxygenation culture was carried out for 4 hours;

[0247] ④Hypoxia / reoxygenation + butyrylcarnitine group (B): On the basis of hypoxia and reoxygenation, butyrylcarnitine at the initial concentration was given at the same time, and the hypoxia culture was carried out for 12 hours and the reoxygenation culture was carried out for 4 hours;

[0248] ⑤Hypoxia / reoxygenation + valerylcarnitine group (C): On the basis of hypoxia and reoxygenation, valerylcarnitine at the initial concentration was given at the same time, and the hypoxia culture was carried out for 12 hours and the reoxygenation culture was carried out for 4 hours;

[0249] ⑥Hypoxia / reoxygenation + hydroxyisovalerylcarnitine group (D): On the basis of hypoxia and reoxygenation, hydroxyisovalerylcarnitine at the initial concentration was given at the same time, and the hypoxia culture was carried out for 12 hours and the reoxygenation culture was carried out for 4 hours;

[0250] ⑦Hypoxia / reoxygenation + linoleoylcarnitine group (E): On the basis of hypoxia and reoxygenation, the initial concentration of linoleoylcarnitine was given at the same time, and the hypoxia culture was carried out for 12 hours and the reoxygenation culture was carried out for 4 hours.

[0251] 4.3 CCK-8 assay to determine cell viability

[0252] After 12 hours of hypoxic culture and 4 hours of reoxygenation culture, the original drug-containing serum was discarded and complete culture medium containing 10% CCK-8 reagent was added again, 100 μL per well, and placed in a 37°C constant temperature incubator for incubation in the dark for about 1.5 hours, and the OD value was detected at 450 nm using an enzyme marker.

[0253] 4.4 Effects of five acylcarnitines on H9c2 cardiomyocytes

[0254] like Fig. 22 , 23 As shown in Figures 24 and 34, the primary mass spectra and secondary fragmentation patterns of propionylcarnitine, butyrylcarnitine, and valerylcarnitine in the samples were consistent with those of the standard, indicating that propionylcarnitine, butyrylcarnitine, and valerylcarnitine are endogenous metabolites of hypoxia / reoxygenation H9c2 cardiomyocytes, that is, the above three acylcarnitines exist in hypoxia / reoxygenation H9c2 cardiomyocytes. After comparison with the standard, hydroxyisovalerylcarnitine and linoleylcarnitine were not qualitatively detected in hypoxia / reoxygenation H9c2 cardiomyocytes, indicating that hydroxyisovalerylcarnitine and linoleylcarnitine are exogenous metabolites of hypoxia / reoxygenation H9c2 cardiomyocytes, and their effects on H9c2 cardiomyocytes are determined by external factors.

[0255] The results of cell viability assay were Fig.25 As shown in Figure A, compared with the Con group, the cardiomyocyte activity of the H12R4 group was significantly decreased (P<0.05); on the basis of hypoxia / reoxygenation intervention, 5 types of acylcarnitine (AE) were given to intervene in the cells. The results showed that compared with the H12R4 group, the cell activity of group E (linoleoylcarnitine) was significantly decreased (P<0.01), indicating that linoleoylcarnitine can aggravate the inhibitory effect of hypoxia / reoxygenation on the activity of H9c2 cardiomyocytes.

[0256] 4.5 Effects of linoleylcarnitine concentration on hypoxia / reoxygenation-induced H9c2 cardiomyocytes

[0257] The cells were plated according to the procedure of "1.2.1" of Example 1. After the cells reached the desired density, serum-free and sugar-free DMEM culture medium containing 5 μmol / L, 10 μmol / L, 20 μmol / L, 40 μmol / L and 80 μmol / L linoleoylcarnitine was given. Ten replicate wells were set up for each group. After 12 hours of anoxic culture, the original drug-containing culture medium was aspirated and discarded, and replaced with complete DMEM culture medium containing the same concentration of linoleoylcarnitine. After 4 hours of reoxygenation culture, the original drug-containing serum was aspirated and discarded, and complete culture medium containing 10% CCK-8 reagent was re-added, 100 μL per well, and the cells were placed in a 37°C constant temperature incubator and incubated in the dark for about 1.5 hours. The OD value was detected at 450 nm using a microplate reader.

[0258] The results are as follows Fig.25 As shown in Figure B, 5μmol / L, 10μmol / L, 20μmol / L, 40μmol / L and 80μmol / L linoleoylcarnitine all had a significant inhibitory effect on the cell activity of H9c2 cardiomyocytes subjected to hypoxia / reoxygenation (P<0.05), indicating that the accumulation of linoleoylcarnitine has a dose-dependent inhibitory effect on the activity of H9c2 cardiomyocytes subjected to hypoxia / reoxygenation.

[0259] 4.6 Effect of linoleylcarnitine concentration on healthy H9c2 cardiomyocytes

[0260] Different concentrations of linoleylcarnitine were used to intervene in healthy H9c2 cardiomyocytes. The specific method was the same as "1.5.5". Fig.25 As shown in Figure C, there was no significant difference in the activity of cardiomyocytes intervened by 1μmol / L, 5μmol / L, 10μmol / L, 20μmol / L, 40μmol / L and 80μmol / L linoleoylcarnitine and that of the Con group (P>0.05), indicating that the accumulation of linoleoylcarnitine has no damaging effect on healthy H9c2 cardiomyocytes.

[0261] 5. Effects of linoleylcarnitine on lipid peroxidation injury in H9c2 cardiomyocytes after hypoxia / reoxygenation

[0262] The total SOD activity detection and MDA content detection methods are the same as in Example 1.

[0263] The results are as follows Fig.26 As shown. Compared with the Con group, the SOD activity of myocardial cells in the H12R4 group was significantly decreased (P<0.01); compared with the H12R4 group, linoleylcarnitine had a significant inhibitory effect on the SOD activity of H9c2 myocardial cells after hypoxia / reoxygenation (P<0.05). At the same time, the MDA content of myocardial cells in the H12R4 group was significantly increased (P<0.05); compared with the H12R4 group, linoleylcarnitine could further increase the MDA content of H9c2 myocardial cells after hypoxia / reoxygenation (P<0.05), indicating that linoleylcarnitine can aggravate the lipid peroxidation damage of H9c2 myocardial cells after hypoxia / reoxygenation.

[0264] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. Application of linoleylcarnitine in screening drugs that regulate the prolonged proinflammatory phase in ACS patients after PCI.

2. A method for screening a drug that has a regulatory effect on the prolongation of the pro-inflammatory phase after PCI in ACS patients, characterized in that: The specific steps include: S1. Co-culture the cardiomyocytes with the drug to be screened for 8 to 12 hours; S2, replacing the culture medium with serum-free and sugar-free DMEM medium containing linoleylcarnitine and the drug to be screened for hypoxic culture for at least 12 hours; S3. Replace the culture medium with complete DMEM medium containing linoleoylcarnitine and the drug to be screened. After 4-6 hours of reoxygenation culture, detect the cell activity and screen out the drugs that significantly improve the activity of myocardial cells based on the cell activity, that is, the drugs that have a regulatory effect on the prolonged pro-inflammatory period after PCI in ACS patients.

3. The method according to claim 2, characterized in that The content of linoleylcarnitine in the serum-free and sugar-free DMEM medium is 20 μmol / L.

4. The method according to claim 2 or 3, characterized in that: The cardiomyocytes are H9c2 cardiomyocytes.

5. The method according to claim 2 or 3, characterized in that: The hypoxic culture time is 12 hours; and / or The reoxygenation culture time is 4 hours.

6. The method according to claim 2 or 3, characterized in that: The method for detecting cell activity in S3 is the CCK-8 method.

7. Use of the method according to any one of claims 2 to 6 in screening active ingredients of traditional Chinese medicine that have an improving effect on the prolonged pro-inflammatory phase in ACS patients after PCI.

8. A drug having a regulatory effect on the prolongation of the pro-inflammatory phase after PCI in ACS patients, characterized in that: The medicine contains at least one of salvianolic acid B, cryptotanshinone, tanshinone ⅡA, salvianolic acid C, salvianolic acid F and asiatic acid.

9. Application of Salvia miltiorrhiza extract in the preparation of drugs for improving the prolonged pro-inflammatory phase in ACS patients after PCI.

10. The use according to claim 9, characterized in that: The preparation method of the salvia miltiorrhiza extract is as follows: extracting salvia miltiorrhiza slices by refluxing with 50-70% v / v ethanol aqueous solution, and drying the obtained extract.

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