Application of N-acetylglutamic acid in preparation of anti-myocardial fibrosis drugs

Abstract

The invention relates to the field of biological medicines, and discloses application of N-acetylglutamic acid in preparation of anti-myocardial fibrosis medicines. According to the application disclosed by the invention, the N-acetylglutamic acid and the carglutamic acid are found to have the effects of obviously improving the systolic function of the heart, reducing the energy metabolism deposition of the fibroblasts and reducing the collagen deposition so as to inhibit excessive fibrosis of the myocardial fibroblasts after myocardial infarction for the first time. The discovery provides a'switch 'for fibroblast repair of myocardial infarction and excessive fibrosis, and has important value for avoiding the excessive fibrosis of the heart after myocardial infarction.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and particularly to the application of N-acetylglutamic acid in the preparation of anti-myocardial fibrosis drugs. Background Art

[0003] Myocardial infarction is one of the refractory diseases seriously endangering human health. Recent studies have shown that fibrosis mediated by cardiac fibroblasts is a key factor leading to heart failure after myocardial infarction, but there is still a lack of effective treatment means for fibrosis at present. Therefore, deeply understanding and intervening in cardiac fibrosis after myocardial infarction is one of the key scientific issues in the field of cardiovascular research.

[0004] After myocardial cells are damaged during the acute myocardial infarction period, fibroblasts need to secrete collagen to fill the absence of myocardial cells. After the myocardial infarction repair period, due to the continuous secretion of collagen by myocardial fibroblasts, excessive fibrosis occurs, causing collagen cross-linking into hard scars, which greatly affects the cardiac compliance and pumping function after myocardial infarction, and even produces paradoxical wall motion, leading to the occurrence of heart failure in the long term after myocardial infarction.

[0005] In previous experiments, we accidentally found that the SGLT2 inhibitor dapagliflozin can significantly reduce fibrosis after myocardial infarction ( Figure 1 ), and increase the content of N-acetylglutamic acid (N-AG), the key product at the beginning of the urea cycle ( Figure 2 ). Further research on the differential metabolites of metabolomics ( Figure 3 ) shows that fumarate (Fum), the product generated by the arginine synthesis pathway of the urea cycle that generates N-acetylglutamic acid, can enter the mitochondria through the mitochondrial membrane, reduce the production of ATP in the mitochondria, and thus cut off the energy required for fibroblast transdifferentiation and collagen secretion, but its specific mechanism is still unclear. If the mechanism can be further understood and the research gap in the energy metabolism of fibroblasts after myocardial infarction can be filled, it can provide a new theoretical basis and potential intervention strategies for the treatment of heart failure after myocardial infarction, which has positive significance.

[0006] In addition, the present invention also confirms through previous research that both the transdifferentiation and collagen secretion of fibroblasts require a large amount of ATP as energy support, and the energy metabolism process has an important regulatory role in the generation of ATP by fibroblasts. However, the specific energy metabolism regulation method during the transdifferentiation and collagen secretion of cardiac fibroblasts is currently unclear. Therefore, clarifying the specific mechanism of regulating fibroblast energy generation is of great significance for exploring the treatment targets of cardiac fibrosis after myocardial infarction.

[0007] After myocardial cells are damaged during the acute myocardial infarction (AMI) period, fibroblasts secrete collagen to fill the gaps left by the damaged myocardial cells. After the AMI repair period, continuous secretion of collagen by myocardial fibroblasts leads to excessive fibrosis, causing collagen cross-linking into a hard scar, which greatly affects the cardiac compliance and pumping function after AMI, and even causes paradoxical wall motion, leading to the occurrence of heart failure in the long term after AMI. Our work provides a "switch" between fibroblast repair of AMI and excessive fibrosis, and identifies N-acetylglutamate (N-AG) and its substitute, carnitine glutamate (Carg), as potential therapeutic drugs for myocardial fibrosis after AMI.

[0008] The present invention provides the use of N-acetylglutamate (N-AG) and its substitute, carnitine glutamate (Carg), in improving the effect of myocardial fibrosis after AMI, and the protective effect improves the function of cardiac excessive fibrosis. Summary of the Invention

[0009] The specific technical solution of the present invention is as follows: In the first aspect, the present invention provides the use of N-acetylglutamate (N-AG) in the preparation of anti-myocardial fibrosis drugs.

[0010] N-acetylglutamate is catalyzed by N-acetylglutamate synthase, the key enzyme at the start of the urea cycle, and is the primary product of the arginine synthesis pathway. Fumarate (Fum) is the most important intermediate in the exchange between the arginine synthesis pathway and intracellular mitochondria; in in vitro studies, it was found that the energy metabolism of fibroblast transdifferentiation can be inhibited by exogenous addition of N-acetylglutamate (N-AG) or fumarate (Fum), achieving the effect of inhibiting collagen secretion. There are many intermediate products in the urea cycle, most of which maintain dynamic balance within the cycle and do not participate in other metabolic pathways, but only fumarate is released without participating in the cycle, and this metabolite is also an important intermediate in mitochondrial energy metabolism. The present invention reveals the amino acid synthesis pathway and discovers that the intermediate products of arginine synthesis and the urea cycle, N-acetylglutamate (N-AG) and fumarate (Fum), can improve cardiac contractile function in rats and neonatal rat cardiac fibroblasts, and inhibit collagen secretion, thereby inhibiting myocardial fibrosis caused by cardiac remodeling. There is no relevant report on the above information internationally at present. Therefore, this new discovery of the present invention is expected to provide a new theoretical basis and new drug treatment targets for the treatment of myocardial fibrosis after AMI.

[0011] In the second aspect, the present invention provides the use of carnitine glutamate (Carg) in the preparation of anti-myocardial fibrosis drugs. The present invention discovers through experiments that carnitine glutamate has an effect similar to that of N-acetylglutamate.

[0012] Thirdly, the present invention provides the use of an SGLT2 inhibitor in the preparation of a drug having the effect of increasing the content of N-acetylglutamate. Further, the SGLT2 inhibitor is dapagliflozin.

[0013] The present invention discovers through experiments that SGLT2 inhibitors, especially dapagliflozin, have the effect of increasing the content of N-acetylglutamate.

[0014] Further, the myocardial fibrosis is the myocardial fibrosis caused after myocardial infarction.

[0015] Preferably, the drug further comprises one or more of pharmaceutically acceptable excipients, carriers, and excipients.

[0016] More preferably, the dosage form of the drug is oral or injection.

[0017] Fourthly, the present invention provides the use of N-acetylglutamate as a target in the screening of drugs having an anti-myocardial fibrosis effect.

[0018] Based on the above new discovery of the present invention, N-acetylglutamate can be used as a target for screening drugs having anti-myocardial fibrosis efficacy.

[0019] Fifthly, the present invention provides a method for inhibiting the fibrosis of cardiac fibroblasts in vitro: adding one or more of N-acetylglutamate (N-AG) and carglutamate (Carg) to the culture medium of cardiac fibroblasts in vitro, thereby inhibiting the fibrosis of cardiac fibroblasts.

[0020] Sixthly, the present invention provides a method for inhibiting the transdifferentiation or collagen secretion of cardiac fibroblasts in vitro, which is characterized in that: adding one or more of N-acetylglutamate (N-AG) and carglutamate (Carg) to the culture medium of cardiac fibroblasts in vitro, entering the mitochondria through the mitochondrial membrane to reduce the production of ATP in the mitochondria, thereby cutting off the energy required for fibroblast transdifferentiation and collagen secretion.

[0021] Compared with the prior art, the beneficial effect of the present invention is that: the present invention discovers for the first time that N-acetylglutamate and carglutamate have obvious effects of improving cardiac systolic function, reducing the energy metabolism deposition of fibroblasts, reducing collagen deposition, and thus inhibiting the excessive fibrosis of cardiac fibroblasts after myocardial infarction. This discovery provides a "switch" between fibroblast repair of myocardial infarction and excessive fibrosis, and has important value for avoiding excessive fibrosis of the heart after myocardial infarction. Description of the Drawings

[0022] Figure 1M-wave graph of cardiac systolic function detected by echocardiography before and after ligation of the coronary artery in a 6-week-old rat acute myocardial infarction model; the groups were: normal control group (Sham), model group (MI+DMSO), and dapagliflozin group (MI+Dapa).

[0023] Figure 2 Metabolomics detection results of neonatal rat cardiac fibroblasts cultured in vitro after TGF-β1-induced in vitro fibroblast transdifferentiation and treatment with dapagliflozin.

[0024] Figure 3 Detection results of all metabolites in cardiac fibroblasts.

[0025] Figure 4 M-wave graph of cardiac systolic function detected by echocardiography before and after ligation of the coronary artery in a 6-week-old rat acute myocardial infarction model; the model group was MI, the fumaric acid group was MI+Fum, and the N-acetylglutamic acid group was MI+N-AG.

[0026] Figure 5 Results of cardiac fibrosis staining and immunofluorescence staining in rats after myocardial infarction; the model group was MI, and the N-acetylglutamic acid group was MI+N-AG.

[0027] Figure 6 Collagen expression levels in neonatal rat cardiac fibroblasts cultured in vitro after TGF-β1-induced in vitro fibroblast transdifferentiation and treatment with the drug N-acetylglutamic acid (N-AG) or its substitute carnosine (Carg).

[0028] Figure 7 Phalloidin staining results of neonatal rat cardiac fibroblasts cultured in vitro after TGF-β1-induced in vitro fibroblast transdifferentiation and treatment with the N-acetylglutamic acid (N-AG) administration group and its substitute carnosine (Carg) drug; red indicates the cytoskeleton, blue indicates the nucleus, and cell size is mainly depicted by the cytoskeleton.

[0029] Figure 8 Results of related energy metabolism of neonatal rat cardiac fibroblasts cultured in vitro inoculated in a special plate for real-time ATP rate detection and oxygen consumption detection after TGF-β1-induced modeling and treatment with the drug N-acetylglutamic acid (N-AG) or its substitute carnosine (Carg). Detailed implementation methods

[0031] N-Acetylglutamic acid (N-AG) and fumaric acid (Fum): Two drugs were purchased from MCE. In vivo feeding: N-Acetylglutamic acid (N-AG) was directly dissolved in sterile water to a 25 mg / ml solution, and the pH was adjusted to 7.0 - 7.4 with NaOH. After filtration and sterilization, it was directly fed to rats weighing about 200 g and about 6 weeks old; Fumaric acid (Fum) was directly dissolved in sterile water to a 10 mg / ml solution, and the pH was adjusted to 7.0 - 7.4 with NaOH. After filtration and sterilization, it was directly fed to rats weighing about 200 g and about 6 weeks old; In vitro experiment, N-Acetylglutamic acid (N-AG) and fumaric acid (Fum) were dissolved in sterile ddH2O to 100 mM, and after suction filtration, they were administered at a ratio of 1:20, that is, 5 mM.

[0032] Example 1: Detection of cardiac systolic function EF (ejection fraction) and FS (fractional shortening) percentage by small animal ultrasound before and after myocardial infarction in rats Experimental animals: Male rats were housed in the SPF environment of the Experimental Animal Center of the Second Affiliated Hospital of Zhejiang University School of Medicine.

[0033] Experimental instruments and reagents: Small animal ultrasound imaging system (Vevo 3100), dapagliflozin (MCE BMS-512148), medical coupling agent (Hangzhou Kaiple Medical Devices Co., Ltd.).

[0034] 1) Rat acute myocardial infarction model (left ventricular coronary artery) Rats weighing 200 g were depilated and prepared for skin. Anesthesia was induced by intraperitoneal injection of 4% chloral hydrate. After the rats completely lost consciousness, they were fixed in the supine position on the operating table. Subsequently, tracheal intubation was performed and connected to a small animal ventilator for assisted ventilation. The skin of the surgical field was disinfected with iodophor, and then the chest was opened layer by layer. The ribs were cut at the 3rd - 4th ribs to enter the chest. After removing the pericardium, the heart was exposed. At 2 - 3 mm below the pulmonary artery conus, the coronary artery was permanently ligated with 6-0 nylon thread. It could be seen that the area below the ligation line immediately turned pale and the movement amplitude weakened. At this time, it could be regarded as the successful construction of the rat myocardial infarction model.

[0035] 2) Grouping and drug administration The above rats were divided into a normal control Sham group, a myocardial infarction model MI + DMSO group, and a dapagliflozin-administered MI + Dapa group. The drug concentration in the MI + Dapa group was 10 mg / kg (10 mg drug / 1000 g body weight) for dose treatment.

[0036] 3) Index detection On the day before surgery (before modeling), 3 days after modeling, and 28 days after modeling, the rats were anesthetized with isoflurane gas and then underwent small animal ultrasonic imaging to observe the left ventricular cardiac systolic function of each group. The chest hair of the rats was removed to expose the chest cavity, and the rats were placed supine on a mouse board connected to an electrophysiological detection system. The ultrasonic data of the left ventricular long axis B-mode and M-mode of the rats were collected using a 25 Hz probe, and finally, the Auto LV automatic analysis software of the VevoLAB 5.7.1 version analysis software was used to analyze the cardiac systolic function of the rats in all M-modes.

[0037] 4) Statistical analysis GraphPad 9.0 software was used for data processing and analysis. The results were expressed as mean ± variance. Two-way ANOVA was used for inter-group comparison. *P < 0.05, ***p < 0.001, ****p < 0.0001 indicated that the differences were statistically significant.

[0038] 5) Experimental results Figure 1 An M-waveform graph was collected for the cardiac systolic function of a 6-week-old rat acute myocardial infarction model 28 days after ligating the coronary artery by small animal ultrasonic detection; among them, EF represents the left ventricular ejection fraction of the heart, and FS represents the fractional shortening of the short axis. Figure 1 The results showed that there were no significant differences in the preoperative baseline EF and FS data among the normal control (Sham), model group (MI + DMSO), and drug administration group (MI + Dapa). ****p < 0.0001 indicated that the EF of the model group was significantly lower than that of the normal control 3 days after myocardial infarction, and ***p < 0.001 indicated that the FS of the model group was significantly lower than that of the normal control 3 days after myocardial infarction; ns indicated that there were no significant changes in EF and FS in the drug administration group compared with the model group 3 days after myocardial infarction, indicating that both groups had reached the level of myocardial infarction. ****p < 0.0001 indicated that both EF and FS in the model group were significantly decreased compared with the normal group 28 days after myocardial infarction, and *P < 0.05 indicated that both EF and FS in the drug administration group were significantly increased compared with the model group 28 days after myocardial infarction, and the differences were statistically significant, indicating that dapagliflozin has the function of improving cardiac contraction after myocardial infarction.

[0039] Example 2: Results of metabolomic differences in neonatal rat cardiac fibroblasts Experimental animals: Male neonatal rats were purchased from Shanghai Slack Experimental Animal Co., Ltd.

[0040] Experimental instruments and reagents: cell incubator (Thermo 240i), primary cardiomyocyte isolation kit (Miltenyi Biotec 130-098-373), TGF-β1 (R&D 100-21C), dapagliflozin (MCE BMS-512148), PBS (Zhejiang Senrui Biotechnology Co., Ltd.), high-glucose DMEM medium (Gibco 11965092), newborn calf serum (Biological Industries), metabolomics (Beijing Novogene Bioinformatics Technology Co., Ltd. metabolome).

[0041] 1) Isolation of primary fibroblasts and TGF-β1 modeling The primary cardiomyocyte isolation kit was used to isolate the hearts of neonatal rats into single cells, and the cells were cultured in low-glucose (1 g / L) DMEM containing 10% newborn calf serum. When the cell confluence reached 80%, the cells were digested with EDTA-trypsin and seeded into 6-well plates. When the cell confluence reached 80% the next day, the cells were treated with serum-free medium overnight. On the third day, the cells were induced to transdifferentiate in vitro with 10 ng / ml TGF-β1.

[0042] 2) Grouping and drug administration The above cells were divided into a WT group, a TGF-β1 group, and a TGF-β1 + Dapa group, with the Dapa concentration being 10 μM.

[0043] 3) Index detection Cell counting: 5×10 6 Cells were seeded in 6-cm culture dishes and treated with serum-free medium overnight to synchronize the cells, and then divided into a WT group, a TGF-β1 group, and a TGF-β1 + Dapa group. The cells in each group were observed under a microscope (n = 6 replicates for each group), and another 2 quality control groups were set up. After 24 hours of drug administration in each group, the cells were rinsed twice with cold PBS, collected with a cell scraper, frozen in fresh liquid nitrogen for 15 seconds, quickly thawed at room temperature, and lysed by repeated freezing. The collected cells were sent to the company on dry ice and waited for the metabolomics results.

[0044] 4) Statistical analysis Figure 3 A list of all metabolites with significant changes (p < 0.05) was listed, and data processing and analysis were performed using GraphPad software. The results were expressed as mean ± variance. One-way ANOVA was used for comparison between groups. *P < 0.05, **p < 0.01, ***p < 0.001 indicated statistical significance.

[0045] 5) Experimental results Figure 2The differences in the abundances of cell metabolites after treatment with TGF-β1 and then dapagliflozin in vitro-cultured neonatal rat cardiac fibroblasts. Among them: ***p < 0.001 indicates that the N-A-DL-G metabolite in the TGF-β1 model group of cells was significantly reduced compared with that in WT cells, **p < 0.01 indicates that the N-A-DL-G metabolite in the TGF-β1 + Dapa group of cells was significantly increased compared with that in the TGF-β1 group, and the difference was statistically significant, indicating that Dapa has the ability to increase the production of N-A-DL-G; **p < 0.01 indicates that the N-A-L-G metabolite in the TGF-β1 model group of cells was significantly reduced compared with that in WT cells, *p < 0.05 indicates that the N-A-L-G metabolite in the TGF-β1 + Dapa group of cells was significantly increased compared with that in the TGF-β1 group, and the difference was statistically significant, indicating that Dapa has the ability to increase the production of N-A-L-G. This result indicates that Dapa has the ability to increase the N-acetylglutamate products of two isomers of N-AG. Figure 3 Represents the list of all statistically different metabolites in metabolomics. all_wt1-6 represents 6 replicates of normal controls, all_tgfb1-6 represents 6 replicates of TGF-β1, and all_t_dapa1-6 represents 6 replicates of TGF-β1 + Dapa.

[0046] Example 3: Detection of cardiac systolic function EF (ejection fraction) and FS (fractional shortening) percentage by small animal ultrasound before and after myocardial infarction in rats Experimental animals: Male rats were housed in the SPF environment of the Experimental Animal Center of the Second Affiliated Hospital of Zhejiang University School of Medicine.

[0047] Experimental instruments and reagents: Small animal ultrasound imaging system (Vevo 3100), N-acetylglutamic acid (MCE HY-W015240), fumaric acid (MCE HY-W015883), medical coupling agent (Hangzhou Kaiple Medical Device Co., Ltd.).

[0048] 1) Rat acute myocardial infarction model (left ventricular coronary artery) Rats weighing 200 g were depilated and prepared for skin. Anesthesia was induced by intraperitoneal injection of 4% chloral hydrate. After the rats were completely unconscious, they were fixed in the supine position on the operating table. Subsequently, tracheal intubation was performed, and a small animal ventilator was connected for assisted ventilation. After the skin of the surgical field was disinfected with iodophor, the chest was opened layer by layer, and the ribs were cut at the 3rd - 4th ribs to enter the chest. After removing the pericardium, the heart was exposed. At 2 - 3 mm below the pulmonary artery conus, the coronary artery was permanently ligated with 6-0 nylon thread. It could be seen that the area below the ligation line immediately turned pale and the movement amplitude decreased. At this time, it could be considered that the construction of the rat myocardial infarction model was successful.

[0049] 2) Grouping and administration The above rats were divided into an MI group, an MI + N-acetylglutamic acid (N-AG) drug group, and an MI + fumaric acid (Fum) administration group. The MI + N-acetylglutamic acid (N-AG) drug group was treated with a concentration of 5 g / kg (5 g of drug / 1000 g of body weight), and the MI + fumaric acid (Fum) drug group was treated with a concentration of 2 g / kg (2 g of drug / 1000 g of body weight).

[0050] 3) Index detection The rats were subjected to small animal ultrasonic imaging detection under isoflurane gas anesthesia one day before surgery (before modeling), 3 days after modeling, and 28 days after modeling to observe the left ventricular cardiac systolic function of each group. The chest hair of the rats was removed to expose the chest cavity, and they were placed supine on a mouse board connected to an electrophysiological detection system. The ultrasonic data of the left ventricular long axis B-mode and M-mode of the rats were collected using a 25 Hz probe, and finally, the Auto LV automatic analysis software of the VevoLAB 5.7.1 version analysis software was used to analyze the cardiac systolic function of the rats in all M-modes.

[0051] 4) Statistical processing GraphPad 9.0 software was used for data processing and analysis. The results were expressed as mean ± variance. Two-way ANOVA was used for inter-group comparison. *P < 0.05, **p < 0.01, ***p < 0.001 indicated that the differences were statistically significant.

[0052] 5) Experimental results Figure 4 The M-waveform diagram was collected for the cardiac systolic function detected by small animal ultrasound 28 days after ligating the coronary artery in a 6-week-old rat acute myocardial infarction model; among them, EF represents the left ventricular ejection fraction of the heart, and FS represents the shortening rate of the short axis. Figure 4 The results showed that there were no significant differences in the EF and FS data of the model group (MI), the drug administration group (MI + N-AG), and the drug administration group (MI + Fum). The EF and FS of the drug administration group (MI + Fum) 28 days after myocardial infarction were significantly increased compared with the MI group, with *P < 0.05, and the differences were statistically significant. The EF of the (MI + N-AG) group 28 days after myocardial infarction was *P < 0.05 compared with the MI group, and the FS was **p < 0.01, both of which were significantly increased and the differences were statistically significant, indicating that N-acetylglutamic acid (N-AG) and fumaric acid (Fum) have the function of improving cardiac contraction after myocardial infarction.

[0053] Example 4: Specific staining and immunofluorescence staining of cardiac tissue fibrosis after myocardial infarction in rats to clarify the inhibition of cardiac fibrosis by N-acetylglutamic acid Experimental animals: Male rats were raised in the SPF environment of the Experimental Animal Center of the Second Affiliated Hospital of Zhejiang University School of Medicine.

[0054] Experimental instruments and reagents: upright white light microscope (Leica DM3000), upright fluorescence microscope (Leica DM6b), paraffin slicer (Leica), N-acetylglutamic acid (MCE HY-W015240), Sirius red (Solarbio G1472), WGA (Thermo Fisher W11262).

[0055] 1) Rat acute myocardial infarction model (left ventricular coronary artery) Same as Example 3.

[0056] 2) Grouping and administration Same as Example 3.

[0057] 3) Index detection After ultrasound, the rats were anesthetized with an overdose of anesthetic. Then, the chest cavity was opened to expose the heart, and the aorta was cut. Subsequently, the heart was perfused with 50 ml of cold PBS to remove excess blood, rinsed twice in PBS to ensure no residual blood, and the heart was placed in 4% formaldehyde solution for fixation. After one day of fixation, dehydration and paraffin embedding were performed. The heart was cut into 4-mm-thick sections on a paraffin slicer. After dewaxing, fibrosis-specific staining was performed and photographed with a 5× lens under an upright microscope. After dewaxing and antigen repair, immunofluorescence staining was performed and photographed with a 20× lens under an upright fluorescence microscope.

[0058] 4) Statistical analysis GraphPad 9.0 software was used for data processing and analysis. The results were expressed as mean ± variance. Two-way ANOVA was used for inter-group comparison. **p < 0.01 indicated that the difference was statistically significant.

[0059] 5) Experimental results Figure 5 Results of fibrosis-specific staining and immunofluorescence staining of cardiac tissue after myocardial infarction in 6-week-old rats; among them, Scar circumference% represents the ratio of the scar circumference of the inner and outer diameters of the heart, and WGA+Area Per Cell in Border Zoom represents the proportion of WGA-positive cells in the infarct border zone. Figure 5 The results showed that: **p < 0.01, the ratio of the scar circumference of the inner and outer diameters of the heart in the administration group (MI+N-AG) was significantly lower than that in the model group (MI), and **p < 0.01, the proportion of WGA-positive cells in the infarct border zone in the administration group (MI+N-AG) was significantly lower than that in the model group (MI), and the difference was statistically significant, indicating that N-acetylglutamic acid (N-AG) has the function of improving cardiac fibrosis after myocardial infarction.

[0060] Example 5: Collagen expression in neonatal rat cardiac fibroblasts Experimental animals: Male neonatal rats, purchased from Shanghai Slac Laboratory Animal Co., Ltd.

[0061] Experimental instruments and reagents: Cell incubator (Thermo 240i), primary cardiomyocyte isolation kit (Miltenyi Biotec 130 - 098 - 373), TGF-β1 (R&D 100 - 21C), N-acetylglutamate (MCE HY-W015240), carnosine (MCE HY-B0711), PBS (Zhejiang Senrui Biotechnology Co., Ltd.), high-glucose DMEM medium (Gibco 11965092), fetal bovine serum (Biological Industries), EDA-Fibronectin (Abcam 2413), Collagen I (Abcam 270993), Periostin (R&D AF2955), Vimentin (Abcam 92547), α-SMA (Abcam 5694).

[0062] 1) Isolation of primary fibroblasts and TGF-β1-induced modeling The primary cardiomyocyte isolation kit was used to isolate neonatal rat hearts into single cells, and the cells were cultured in low-glucose (1 g / L) DMEM containing 10% fetal bovine serum. When the cell confluence reached 80%, the cells were digested with EDTA-trypsin and seeded into 6-well plates. On the second day, when the confluence reached 80%, the cells were treated with serum-free medium overnight. On the third day, the cells were induced to undergo in vitro transdifferentiation with 10 ng / ml TGF-β1.

[0063] 2) Grouping and drug administration The above cells were divided into a WT group, a TGF-β1 group, an N-AG group, and a Carg group. The concentration of the N-AG group was 5 mM, and the Carg group was treated with 5 mM.

[0064] 3) Index detection Cell counting: 1×10 6 After seeding in 6-well plates and treating with serum-free medium overnight to synchronize the cells, a WT group, a TGF-β1 group, an N-AG group, and a Carg group were set up. The cells in each group were observed under a microscope (n = 3 replicates per group). After 24 hours of drug administration in each group, the cells were collected and lysed to obtain the corresponding protein solutions. Finally, the proteins were separated by SDS-PAGE, and the collagen secretion of the cells in each group was detected with the corresponding antibodies.

[0065] 4) Statistical analysis GraphPad software was used for data processing and analysis. The results were expressed as mean ± variance. One-way ANOVA was used for inter-group comparison. *P < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 indicated statistically significant differences.

[0066] 5) Experimental results Figure 6Expression of collagen synthesis-related proteins EDA-Fibronectin, Collagen I, Periostin, Vimentin, and α-SMA on neonatal rat cardiac fibroblasts cultured in vitro after treatment with TGF-β1 and then with N-AG and Carg drugs. Among them: In the N-AG administration group, **p < 0.01 indicates that EDA-Fibronectin in the model group was significantly increased compared with WT cells, **p < 0.01 indicates that EDA-Fibronectin in the N-AG administration group was significantly decreased compared with the model group, ****p < 0.0001 indicates that Collagen I in the model group was significantly increased compared with WT cells, **p < 0.01 indicates that Collagen I in the N-AG administration group was significantly decreased compared with the model group, *P < 0.05 indicates that Periostin in the model group was significantly increased compared with WT cells, *P < 0.05 indicates that Periostin in the N-AG administration group was significantly decreased compared with the model group, *P < 0.05 indicates that Vimentin in the model group was significantly increased compared with WT cells, *P < 0.05 indicates that Vimentin in the N-AG administration group was significantly decreased compared with the model group, ns indicates that there was an increasing trend but no significant difference in α-SMA in the model group compared with WT cells, *P < 0.05 indicates that α-SMA in the N-AG administration group was significantly decreased compared with the model group. In the Carg administration group, **p < 0.01 indicates that EDA-Fibronectin in the model group was significantly increased compared with WT cells, *p < 0.05 indicates that EDA-Fibronectin in the Carg administration group was significantly decreased compared with the model group, *p < 0.05 indicates that Collagen I in the model group was significantly increased compared with WT cells, *p < 0.05 indicates that Collagen I in the Carg administration group was significantly decreased compared with the model group, ***p < 0.001 indicates that Periostin in the model group was significantly increased compared with WT cells, **p < 0.01 indicates that Periostin in the Carg administration group was significantly decreased compared with the model group, ***p < 0.001 indicates that Vimentin in the model group was significantly increased compared with WT cells, **p < 0.01 indicates that Vimentin in the Carg administration group was significantly decreased compared with the model group, **p < 0.01 indicates that α-SMA in the model group was significantly increased compared with WT cells, *P < 0.05 indicates that α-SMA in the Carg administration group was significantly decreased compared with the model group, and the differences were statistically significant, indicating that N-AG (5 mM) and Carg (5 mM) have the ability to inhibit the transdifferentiation of cardiac fibroblasts stimulated by TGF-β1.

[0067] Example 6: Cytoskeleton staining of neonatal rat cardiac fibroblasts Experimental animals: Male neonatal rats were purchased from Shanghai SLAC Laboratory Animal Co., Ltd.

[0068] Experimental instruments and reagents: cell incubator (Thermo 240i), inverted fluorescence microscope (Leica DMi8), primary cardiomyocyte isolation kit (Miltenyi Biotec 130-098-373), TGF-β1 (R&D 100-21C), N-acetylglutamate (MCE HY-W015240), carbamyl glutamate (MCE HY-B0711), PBS (Zhejiang Senrui Biotechnology Co., Ltd.), high-glucose DMEM medium (Gibco 11965092), fetal bovine serum (Biological Industries), phalloidin (Invitrogen P1951), Hoechst 33342 (Thermo Fisher R37165).

[0069] 1) Isolation of primary fibroblasts and TGF-β1 modeling Same as Example 2.

[0070] 2) Grouping and drug administration The above cells were divided into WT group, TGF-β1 group, N-AG group and Carg group. The concentration of N-AG group was 5 mM, and Carg was treated at 5 mM.

[0071] 3) Index detection Cell counting 2×10 5 Inoculated into 24-well plates and treated without serum overnight to synchronize the cells. Then set up WT group, TGF-β1 group, N-AG group and Carg group. Observe the cells in each group under the microscope (n = 3 replicates for each group). After 24 hours of drug administration in each group, fix the cells with 4% formaldehyde solution, rinse with PBS 3 times, disrupt the cell membrane with 0.2% Triton X-100, fix the cells with 5% BSA-PBS, stain with 1 μM phalloidin at room temperature for 30 minutes, stain with Hoechst (1:10000) at room temperature for 10 minutes, and record the cell fluorescence signal with an inverted fluorescence microscope to obtain the cytoskeleton condition.

[0072] 4) Statistical processing Use Image Pro Plus software to analyze the area of cell red fluorescence signal and the number of blue cell nuclei, and use GraphPad software for data processing and analysis. The results are expressed as mean ± variance. One-way ANOVA is used for comparison between groups. *P < 0.05, **p < 0.01, ***p < 0.001 indicate that the differences are statistically significant.

[0073] 5) Experimental results Figure 7The cytoskeleton sizes of neonatal rat cardiac fibroblasts cultured in vitro after treatment with TGF-β1 and then with N-AG and Fum drugs. Among them: ***p < 0.001 indicates that the cell area of the TGF-β1 model group is significantly increased compared with that of the WT cells, ***p < 0.001 for N-AG (5 mM) and **p < 0.01 for Carg (5 mM), and the cell area is significantly decreased compared with the TGF-β1 group, and the difference is statistically significant, indicating that N-AG (5 mM) and Carg (5 mM) have the ability to inhibit the increase in cell area of cardiac fibroblasts caused by TGF-β1 stimulation. This result shows that the administration groups of N-acetylglutamate (N-AG) and the substitute carnosine (Carg) significantly improve the cell size, thus indicating the result of inhibiting cell transdifferentiation. ***p < 0.001 indicates that the cell volume of the TGF-β1 model group is significantly larger than that of the WT, **p < 0.01 indicates that the N-acetylglutamate (N-AG) drug is significantly smaller than TGF-β1, and ***p < 0.001 indicates that the carnosine (Carg) drug, the substitute for N-acetylglutamate, is significantly smaller than TGF-β1.

[0074] Example 7: Real-time ATP production rate and mitochondrial oxygen consumption rate of neonatal rat cardiac fibroblasts Experimental animals: Male neonatal rats, purchased from Shanghai Slack Experimental Animal Co., Ltd.

[0075] Experimental instruments and reagents: Cell incubator (Thermo 240i), cell energy metabolism detector (Agilent Seahorse XF96), primary myocardial cell isolation kit (Miltenyi Biotec 130-098-373), TGF-β1 (R&D 100-21C), N-acetylglutamate (MCE HY-W015240), carnosine (MCE HY-B0711), PBS (Zhejiang Senrui Biotechnology Co., Ltd.), high-glucose DMEM medium (Gibco 11965092), newborn fetal bovine serum (Biological Industries), Seahorse XF real-time ATP production rate assay kit (Agilent 103592-100), Seahorse XF cell mitochondrial stress test kit (Agilent 103015-100).

[0076] 1) Isolation of primary fibroblasts and establishment of the TGF-β1 model Same as Example 2.

[0077] 2) Grouping and drug administration The above cells were divided into the WT group, TGF-β1 group, N-AG group and Carg group. The concentration of the N-AG group was 5 mM, and Carg was treated at 5 mM.

[0078] 3) Index detection Cell counting: 2×10 4 Cells were seeded in a Seahorse XF 96-well plate dedicated for the experiment. After overnight serum-free treatment to synchronize the cells, the WT group, TGF-β1 group, N-AG group and Carg group were set up. Cells in each group were observed under a microscope (n = 4 replicates per group). After 24 hours of drug administration in each group, the cells were processed according to the instructions of the Seahorse XF Real-Time ATP Production Rate Assay Kit and the Seahorse XF Cell Mitochondrial Stress Test Kit, and then the ATP production rate and mitochondrial oxygen consumption rate of the cells were detected by the instrument to evaluate the energy metabolism conditions.

[0079] 4) Statistical analysis GraphPad software was used for data processing and analysis. The results were expressed as mean ± variance. One-way ANOVA was used for inter-group comparison. *P < 0.05, **P < 0.01, ***P < 0.001 indicated statistically significant differences.

[0080] 5) Experimental results Figure 8The real-time ATP production rate and mitochondrial oxygen consumption rate of neonatal rat cardiac fibroblasts cultured in vitro after treatment with TGF-β1 and then with N-AG and Fum drugs. Among them: A, B represent the proportions of ATP produced by glycolysis (Glyco ATP), oxidative phosphorylation (Mito ATP), and the total cell (Total ATP) in cells of different groups. **p < 0.01 indicates that the ATP produced by glycolysis in the TGF-β1 model group is significantly higher than that in WT cells. ***p < 0.001 indicates that the ATP produced by oxidative phosphorylation in the TGF-β1 model group is significantly higher than that in WT cells. ***p < 0.001 indicates that the total ATP produced by cells in the TGF-β1 model group is significantly higher than that in WT cells. ns indicates that there is no significant change in the ATP produced by glycolysis and the total ATP produced by cells in the N-AG (5 mM) treatment group compared with the TGF-β1 model group. *P < 0.05 indicates that the ATP produced by oxidative phosphorylation in the N-AG (5 mM) treatment group is significantly less than that in the TGF-β1 model group, and the difference is statistically significant. C, D and E, F represent the mitochondrial-related respiration conditions such as basal respiration and maximal respiration detected by adding different cell respiration inhibitors to the mitochondrial oxygen consumption rate in cells of different groups. ****p < 0.0001, ***p < 0.001 indicate that the oxygen consumption of basal respiration and maximal respiration in the TGF-β1 model group is significantly higher than that in WT cells. **p < 0.01, *P < 0.05 indicate that the oxygen consumption of basal respiration and maximal respiration in the N-AG (5 mM) treatment group is significantly less than that in the TGF-β1 model group respectively. **p < 0.01, ***p < 0.001 indicate that the oxygen consumption of basal respiration and maximal respiration in the Carg (5 mM) treatment group is significantly less than that in the TGF-β1 model group respectively, and the difference is statistically significant. This shows that N-AG (5 mM) and Carg (5 mM) can reduce the energy metabolism of cell oxidative phosphorylation and thus inhibit the cell energy metabolism caused by TGF-β1 stimulation of cardiac fibroblasts on the premise of not affecting the energy production of glycolysis in improving cardiac fibroblasts.

Claims

1. Use of N-acetylglutamic acid in the preparation of an anti-myocardial fibrosis drug.

2. Use of carnitine glutamate in the preparation of an anti-myocardial fibrosis drug.

3. The application according to claim 1 or 2, characterized in that: The myocardial fibrosis is the myocardial fibrosis caused by myocardial infarction.

4. The application according to claim 1 or 2, characterized in that: The drug further comprises one or more of pharmaceutically acceptable excipients, carriers and excipients.

5. The application according to claim 4, wherein: The preparation form of the drug is oral or injection.

6. Use of N-acetylglutamic acid as a target in screening for drugs with anti-myocardial fibrosis effects.

7. A method for inhibiting fibrosis of myocardial fibroblasts in vitro, characterized in that: Adding one or more of N-acetylglutamic acid and carnitine glutamate to the culture medium of in vitro cardiac fibroblasts, thereby inhibiting the fibrosis of cardiac fibroblasts.

8. A method for inhibiting the transdifferentiation or collagen secretion of cardiac fibroblasts in vitro, characterized in that: Adding one or more of N-acetylglutamic acid and carnitine glutamate to the culture medium of in vitro cardiac fibroblasts, entering the mitochondria through the mitochondrial membrane to reduce the production of ATP in the mitochondria, thereby cutting off the energy required for fibroblast transdifferentiation and collagen secretion.

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