Application of high-purity natural borneol in preparation of medicine for relieving heart failure and improving myocardial injury

Abstract

The invention relates to novel medical application of high-purity natural borneol, in particular to application of the high-purity natural borneol in preparation of drugs for relieving heart failure and improving myocardial injury. According to the high-purity natural borneol disclosed by the invention, the expression levels of TGF-beta and Smad3 are reduced and the expression level of Smad7 is increased by adjusting a TGF-beta / Smad signal channel, so that the left ventricular ejection fraction of heart failure is improved, the myocardial fibrosis proportion is reduced and the myocardial cell apoptosis rate is reduced. The pharmaceutical composition takes high-purity natural borneol as a main component and can be administered in a subcutaneous injection or oral administration way, the dosage range of the pharmaceutical composition is 0.2 g / kg to 0.8 g / kg, and the pharmaceutical composition can be prepared into tablets, capsules and injections. According to the invention, the heart function is obviously improved, the level of serum myocardial injury markers is reduced, the biocompatibility and the safety are good, an efficient and safe new scheme is provided for the treatment of heart failure, and a theoretical basis and a practical support are provided for the development of natural medicines.

Description

Technical Field

[0001] The present invention relates to a new medical application of high-purity natural borneol, and in particular to an application of high-purity natural borneol in the preparation of a medicine for alleviating heart failure and improving myocardial damage. Background Art

[0002] Heart failure (HF) is a common and complex clinical syndrome characterized by impaired cardiac systolic or diastolic function, which prevents the body from meeting its normal blood circulation needs. With the accelerated aging of the population and the increasing incidence of common cardiovascular diseases such as hypertension and coronary heart disease, the prevalence of HF is steadily increasing, making it one of the most important chronic cardiovascular conditions of the 21st century. Western medicine primarily treats HF with medication, mechanical ventilation, cardiac resynchronization therapy (CRT), and heart transplantation. However, these treatment strategies all have varying degrees of drawbacks, such as side effects, poor patient compliance, and high surgical costs. Therefore, seeking new treatments and approaches to address these shortcomings has become a crucial task in the field of HF treatment.

[0003] In recent years, natural medicines have gradually become a research hotspot in the field of heart failure treatment due to their advantages such as wide sources, low toxicity and side effects, and diverse mechanisms of action. Natural borneol is a crystal obtained by distilling and cooling the trunk of the Lauraceae plant borneol, also known as "borneol borneol" and "plum slices", and is a precious and fine medicinal material. It has the effects of opening all the orifices, dispersing stagnant heat, improving eyesight and removing cataracts, reducing swelling and relieving pain, and is widely used in various clinical internal and external departments. In particular, its application in critical cardiovascular and cerebrovascular diseases has further demonstrated its clinical value. For example, borneol is contained in emergency Chinese patent medicines such as Suxiao Jiuxin Pills, Suhexiang Pills, Angong Niuhuang Pills, Compound Danshen Drops, and Liushen Pills. In clinical use, borneol is used to formulate prescriptions to treat various heart failure diseases, which can quickly relieve patients' symptoms such as chest tightness, dyspnea, and palpitations.

[0004] The literature "Wang Liying, Wang Jian, Fan Yamei, et al. Anti-inflammatory and HIF-1α / VEGF regulation of three borneols in the prevention and treatment of AMI model rats [J]. Chinese Journal of Experimental Traditional Chinese Medicine, 2022, 28(19): 61-72." shows that borneol has anti-inflammatory and antibacterial effects, improves coronary blood flow, and resists hypoxia. It has a protective effect on rats with acute myocardial ischemia, can alleviate myocardial necrosis in rats with myocardial ischemia, and alleviates myocardial damage. Myocardial ischemia can lead to a decrease in myocardial contraction and relaxation function, further affecting the overall pumping function of the heart, thereby increasing the burden on the heart.

[0005] At the same time, myocardial ischemia can also lead to myocardial cell apoptosis and hypertrophy, and may trigger the transformation of fibroblasts into myofibroblasts, promoting myocardial remodeling. Myocardial remodeling is the most important pathogenesis of heart failure. Ventricular remodeling leads to increased ventricular muscle weight, ventricular volume and changes in ventricular shape, such as myocardial hypertrophy. Myocardial hypertrophy can cause left ventricular diastolic dysfunction, decreased cardiac pumping function, abnormal cardiac function, and heart failure. Modern pharmacological studies have shown that natural borneol has multiple biological activities such as anti-inflammatory, antioxidant, improvement of coronary blood flow, and anti-myocardial ischemia. However, the potential role of natural borneol in the treatment of heart failure and its specific mechanism have not been systematically studied.

[0006] The pathophysiology of heart failure is closely related to myocardial fibrosis and cardiomyocyte apoptosis, and the TGF-β / Smad signaling pathway is a key pathway regulating myocardial fibrosis. High expression of TGF-β can promote the transformation of fibroblasts into myofibroblasts, leading to the progression of myocardial fibrosis. Smad3 is a key downstream effector of this pathway, directly involved in the transcriptional regulation of fibrosis-related genes. Conversely, Smad7, as an inhibitory Smad protein, can inhibit the activation of the TGF-β / Smad signaling pathway through negative feedback. Therefore, targeted regulation of the TGF-β / Smad signaling pathway is considered an important strategy for the treatment of heart failure. Summary of the Invention

[0007] To address the above technical issues, the present invention aims to provide a method for alleviating heart failure and improving myocardial damage using high-purity natural borneol. This method is based on its unique mechanism of improving left ventricular ejection fraction, reducing myocardial fibrosis area, and reducing myocardial cell apoptosis rate in patients with heart failure by regulating the TGF-β / Smad signaling pathway, reducing the expression levels of TGF-β and Smad3, and increasing the expression level of Smad7. This method not only provides a new drug option for the treatment of heart failure, but also provides a scientific basis for the development and utilization of natural medicines.

[0008] In order to achieve the above-mentioned purpose, the present invention adopts the following technical solutions: Application of high-purity natural borneol in the preparation of medicines for alleviating heart failure and improving myocardial damage.

[0009] Preferably, the high-purity natural borneol reduces the expression levels of TGF-β and Smad3 and increases the expression level of Smad7 by regulating the TGF-β / Smad signaling pathway, so as to improve the left ventricular ejection fraction, reduce the proportion of myocardial fibrosis and reduce myocardial cell apoptosis.

[0010] Preferably, the amount of the high-purity natural borneol is 0.2 g / kg to 0.8 g / kg.

[0011] Furthermore, the present invention also provides a pharmaceutical composition for alleviating heart failure and improving myocardial damage, wherein the pharmaceutical composition comprises high-purity natural borneol, which regulates the TGF-β / Smad signaling pathway, reduces the expression levels of TGF-β and Smad3, and increases the expression level of Smad7, thereby achieving the effects of improving left ventricular ejection fraction, reducing the proportion of myocardial fibrosis, and reducing myocardial cell apoptosis.

[0012] Preferably, the amount of the high-purity natural borneol is 0.2 g / kg to 0.8 g / kg.

[0013] Preferably, the pharmaceutical composition is administered by subcutaneous injection or oral administration.

[0014] Preferably, the pharmaceutical composition further comprises auxiliary ingredients, including cardiotonic agents for improving cardiac function or diuretics for reducing myocardial burden.

[0015] Furthermore, the present invention also provides the use of the pharmaceutical composition in the preparation of a drug for alleviating heart failure and improving myocardial damage.

[0016] Furthermore, the present invention also provides a pharmaceutical preparation containing high-purity natural borneol, which includes the pharmaceutical composition and is prepared in the form of tablets, capsules or injections, and is suitable for regulating the TGF-β / Smad signaling pathway, reducing the expression levels of TGF-β and Smad3, and increasing the expression level of Smad7, so as to achieve the effects of improving left ventricular ejection fraction, reducing the proportion of myocardial fibrosis and reducing myocardial cell apoptosis.

[0017] Furthermore, the present invention also provides the use of the pharmaceutical preparation in the preparation of a drug for alleviating heart failure and improving myocardial damage.

[0018] The present invention adopts the above-mentioned technical solution and utilizes high-purity natural borneol to alleviate heart failure and improve myocardial damage, thereby significantly achieving the following technical effects: 1. Improve cardiac function: High-purity natural borneol regulates the TGF-β / Smad signaling pathway, reduces the expression levels of TGF-β and Smad3, and increases the expression level of Smad7, thereby effectively improving the left ventricular ejection fraction (LVEF) and left ventricular fractional shortening (LVFS) in heart failure models, significantly improving the heart's pumping capacity, and improving the cardiac function of heart failure patients.

[0019] 2. Reduce the proportion of myocardial fibrosis: Experimental results show that high-purity natural borneol significantly reduces the fibrotic area in myocardial tissue, inhibits the transformation of fibroblasts into myofibroblasts, and reduces excessive collagen deposition, thereby reducing the proportion of myocardial fibrosis. This mechanism of action is closely related to the regulation of the TGF-β / Smad signaling pathway.

[0020] 3. Reduce myocardial cell apoptosis: The high-purity natural borneol in the present invention can effectively reduce the positive rate of myocardial cell apoptosis in heart failure models, reduce myocardial cell loss, protect myocardial cell structure and function, and significantly slow down the pathological process of heart failure.

[0021] 4. Reduce the level of myocardial injury markers: The pharmaceutical composition of the present invention significantly reduces the levels of myocardial injury markers (such as CK, LDH, HBDH, BNP and NT-proBNP) in serum, reflecting the protective effect on myocardial injury and the recovery effect on cardiac function.

[0022] In summary, the present invention reveals for the first time the protective mechanism of high-purity natural borneol against myocardial fibrosis and cell apoptosis by regulating the TGF-β / Smad signaling pathway, providing a theoretical basis and practical support for the development of innovative drugs for the treatment of heart failure. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 Effects of high-purity borneol on cardiac ultrasound imaging in mice with heart failure; Note: x±s, n=8-10. Compared with the normal group, "ΔΔ" p<0.01; compared with the model group, "*" p<0.05, "**" p<0.01.

[0024] Figure 2 These are the characteristic diagrams of cardiac ultrasound in each group of mice; A is the normal group, B is the model group, C is digoxin, D is high-dose borneol, E is medium-dose borneol, and F is low-dose borneol.

[0025] Figure 3 Effects of borneol on myocardial injury markers and myocardial fibrosis in rats with heart failure; Note: x±s, n=8-10. Compared with the normal group, "ΔΔ" p<0.01; compared with the model group, "*" p<0.05, "**" p<0.01.

[0026] Figure 4 The effect of borneol on myocardial fibrosis in mice with heart failure (Masson staining). Blue indicates fibrosis-positive areas. Aa: normal group; Bb: model group; Cc: digoxin; Dd: high-dose borneol; Ee: medium-dose borneol; Ff: low-dose borneol. AF: panoramic images; ad: fibrosis areas magnified 20×.

[0027] Figure 5The effect of borneol on cardiomyocyte apoptosis 5×, Tunel fluorescence staining, green indicates apoptosis-positive cells; Among them: A normal group, B model group, C digoxin, D high-dose borneol, E medium-dose borneol, F low-dose borneol.

[0028] Figure 6 The effect of borneol on cardiomyocyte apoptosis 20×, Tunel fluorescence staining, green indicates apoptosis-positive cells. Among them: A normal group, B model group, C digoxin, D high-dose borneol, E medium-dose borneol, F low-dose borneol.

[0029] Figure 7 The effect of high-purity borneol on the apoptosis rate of cardiomyocytes (n=8-10) and the relative expression of fibrosis regulatory factor genes (n=5) in heart failure mice (n=3); Note: x±s; compared with the normal group, "ΔΔ" p<0.01; compared with the model group, "*" p<0.05, "**" p<0.01.

[0030] Figure 8 The relative expression levels of smad3 and smad7 proteins in the heart tissue of heart failure mice treated with high-purity borneol; Note: x±s, n=3; compared with the normal group, “ΔΔ” p<0.01; compared with the model group, “*” p<0.05, “**” p<0.01. DETAILED DESCRIPTION

[0031] The following is a clear and complete description of the technical solutions in the embodiments of the present invention in conjunction with the embodiments of the present invention. Obviously, the embodiments described are only part of the embodiments of the present invention, not all of the embodiments. All other embodiments obtained by ordinary technicians in this field based on the embodiments of the present invention without making any creative efforts are within the scope of protection of the present invention.

[0032] 1. Experimental Materials 1.1 Experimental Reagents Isoproterenol hydrochloride, batch number 320230, Medchem Express. Digoxin tablets (0.25 mg / tablet), batch number 123230907, Shanghai SPH Xinyi Pharmaceutical Co., Ltd. High-purity borneol, batch number, manufacturer. CK (A032-1-1), LDH (A020-2-2), HBDH (E005-1-1), BNP (H166-1-2), and NT-proBNP (H334-1) assay kits were all purchased from Nanjing Jiancheng Bioengineering Co., Ltd. Nuclear and cytoplasmic protein extraction kits, batch number 20240427, KeyGen Biotech. BCA protein concentration assay kit, batch number 073333331020, was purchased from Beyotime. Simple WES kit, batch number 92780, was purchased from Protein Simple. Smad3 primary antibody, product number ab40854, was purchased from Abcom. GAPDH (Cat. No. 2118S) was purchased from Cellsignaling Technology. Smad7 primary antibody (Cat. No. MAB2029-SP) was purchased from Bio-Techne. TB Green Premix ExTaq II (Lot. No. AN41232A) was purchased from TAKARA. PrimeScript™ RT reagent Kit (Lot. No. AN90399A) was purchased from TAKARA. SteadyPure RNA Evaluation Kit (A5A2252) was purchased from Acridine Biotechnology Co., Ltd. Tunel Kit (Cat. No. A112) was purchased from Norvegian. Masson Tri-color Kit (Cat. No. BP0310) was purchased from Hubei Bios Biotechnology Co., Ltd.

[0033] 1.2 Experimental animals Sixty male C57BL / 6J mice, weighing 20 ± 3 g, were purchased from Hangzhou Medical College under production license number SCXK(Zhejiang)2019-0002 and maintained at the Experimental Animal Center of Zhejiang Academy of Traditional Chinese Medicine under use license number SYXK(Zhejiang)2019-0010. Mice were housed at 26°C and 60% CO with free access to water and food.

[0034] 1.3 Main instruments Small animal ultrasound system (M-mode ultrasound imaging system), Wes fully automated protein expression analysis system; tissue homogenizer (Tissue Lyser-48L, Shanghai Jingxin); multifunctional microplate reader (Varioskan LUX, Thermo Fisher); ABI Verit PCR instrument (Veriti 96, Applied Biosystems). Centrifuge (Eppendorf).

[0035] 2 Experimental methods 2.1 Animal grouping After one week of adaptive feeding, the mice were randomly divided into two groups: a normal group of 10 mice and a model group of 50 mice.

[0036] 2.2 Modeling method Except for the normal group, mice in the other model groups received subcutaneous injections of isoproterenol hydrochloride at a dose of 50 mg / kg / day for seven consecutive days to establish the model. Cardiac ultrasounds were performed during the modeling period, and a decrease in the left ventricular ejection fraction below 49% was considered a successful model.

[0037] 2.3 Administration After successful modeling, the model group mice were randomly divided into a model control group, a digoxin group, a low-dose borneol group, a medium-dose borneol group, and a high-dose borneol group, with 10 mice in each group. The drug-treated mice were given the corresponding drug treatment every day. The digoxin group was given a dose of 0.18 mg / kg, and the borneol doses were 0.2g / kg, 0.4g / kg, and 0.8g / kg for 7 consecutive days, during which the modeling was continued.

[0038] 2.4 Detection indicators 2.4.1 Cardiac ultrasound examination Seven days after administration, the mice were subjected to echocardiographic examination: the mice were anesthetized by isoflurane inhalation, and M-mode echocardiography was performed using a small animal ultrasound instrument. The left ventricular internal diameter at end-diastole (LVIDd), left ventricular internal diameter at end-systole (LVIDs), left ventricular ejection fraction (LVEF), and left ventricular ejection fractionalshortening (LVFS) of the mice in each group were measured.

[0039] 2.4.2 Sample Collection The mice were fasted for 4 h before blood collection. After echocardiography, the eyeballs were removed and blood was collected. The collected blood was allowed to stand for 30 min and then centrifuged in a high-speed centrifuge (2000 r / min) at 4°C for 15 min. The supernatant in the tube was aspirated, repackaged and numbered, and stored in a -80°C refrigerator for later use.

[0040] After blood collection, the mouse chest was opened to expose the heart. The right atrial appendage was cut open, and the heart was perfused with 1× PBS at a flow rate of 5.8 r / min through the left ventricle until the liver turned grayish white. The heart was removed and placed on ice. The tissue was quickly transferred to liquid nitrogen and then to a -80°C freezer. The apical portion of the heart tissue was fixed in formalin and stained with Masson's stain for histopathological changes.

[0041] 2.4.3 ELISA detection of myocardial injury markers The concentration changes of myocardial injury biological indicators CK, LDH, HBDH, BNP, and NT-proBNP were detected in mouse serum.

[0042] 2.4.4 Myocardial fibrosis Mouse heart tissue was stained with Masson staining, and images were taken from 10 randomly selected fields of view under an upright fluorescence microscope (×200). Statistical analysis was performed using ImageJ. Fibrosis percentage (%) was calculated as: collagen area / total area × 100%.

[0043] 2.4.5 Apoptosis rate of mouse cardiac cells After dewaxing the paraffin sections of the heart, the Tunel test was used to detect the apoptosis rate of cardiac cells, and the fluorescence staining was used to observe the positive rate of cell apoptosis. Five fields of view were randomly selected using CaseViewer software for statistical analysis of green fluorescent cells to calculate the positive cell rate.

[0044] 2.4.6 Detection of myocardial fibrosis marker gene expression in cardiac tissue by qPCR Total RNA was extracted from heart tissue and reverse transcribed into cDNA. Real-time quantitative PCR was used to detect the expression of TGFβ, smad3 and smad7 genes in heart tissue.

[0045] 2.4.7 Weston blot detection of myocardial fibrosis marker protein expression in cardiac tissue Total protein was extracted from heart tissue, and protein concentration was determined by BCA. The expression levels of Smad3 and Smad7 proteins in heart tissue were detected using Simplewes kit.

[0046] 2.5 Statistical methods All data were expressed as mean ± standard deviation (x ± s). SPSS 19.0 statistical software was used for data analysis. When the variance was homogeneous, the t-test was used for comparison between two groups, and one-way ANOVA was used for comparison between multiple groups. The rank sum test was used for statistical analysis of non-normal or unequal variance data. p The difference was statistically significant when the value was <0.05.

[0047] 3 Experimental Results 3.1 Effects of high-purity borneol on cardiac ultrasound parameters in heart failure mice The experimental results are as follows Figure 1-2 As shown, heart failure mice can significantly increase left ventricular LVIDd and LVIDs ( p <0.01), reduced left ventricular FS and EF ( p <0.01). Positive drugs and borneol treatment can significantly shorten the left ventricular LVIDd and LVIDs ( p <0.01, p <0.05), increased left ventricular FS and EF ( p <0.01).

[0048] 3.2 Effects of high-purity borneol on myocardial injury markers in heart failure mice Depend on Figure 3 (AE) It can be seen that the myocardial injury markers in the serum of heart failure mice were significantly higher than those in normal mice ( p <0.01), the positive drug digoxin can significantly reduce the content of myocardial injury markers in the serum of heart failure mice ( p <0.01), high, medium and low doses of borneol can significantly reduce the levels of various myocardial injury markers in the serum of heart failure mice ( p <0.01, p <0.05).

[0049] 3.3 Effects of high-purity borneol on myocardial fibrosis in mice with heart failure According to Masson's results, ( Figure 3 F. Figure 4 ), normal mice showed no myocardial fibrosis, while heart failure mice showed more obvious and larger blue fibrosis areas. The fibrosis area in the digoxin group was less than that in the model group ( p <0.01), the myocardial fibrosis areas in the high-, medium- and low-dose borneol groups were smaller than those in the model group ( p <0.01, p <0.05).

[0050] 3.4 Effect of high-purity borneol on cardiac cell apoptosis in heart failure mice Myocardial apoptosis in each group of mice was observed by immunofluorescence. Figure 5-6 , Figure 7 A) No apoptotic cells were found in the myocardial tissue of the normal group mice, while diffuse apoptosis was observed in the myocardial tissue of the model group mice ( p <0.01), the apoptosis rate in the myocardial tissue of the mice in the positive group was significantly reduced ( p <0.01), the positive rates of myocardial cell apoptosis in the three borneol dose groups were also lower than that in the model group ( p <0.01).

[0051] 3.5 Effect of high-purity borneol on the expression of myocardial fibrosis marker genes in heart tissue of heart failure mice The experimental results show that ( Figure 8 The relative expression levels of TGFβ and smad3 genes in the heart tissue of the model group mice were significantly increased ( p <0.01), while smad7 was significantly decreased ( p <0.01). Each drug-treated group can significantly reduce the relative expression of TGFβ and smad3 genes ( p＜ 0.01 ，p＜ 0.05), and increased the relative expression of smad7 ( p <0.01, p <0.05).

[0052] 3.6 Effect of high-purity borneol on the expression of myocardial fibrosis marker proteins in heart tissue of heart failure mice The experimental results show that ( Figure 8 EF), smad3 protein was highly expressed in the heart tissue of heart failure mice in the model group ( p <0.01), low expression of smad7 ( p <0.01). After treatment with high-purity borneol in heart failure mice, the relative expression of smad3 protein in heart tissue was significantly reduced ( p <0.01, p <0.05), high dose of borneol can significantly increase the relative expression of smad3 protein in heart tissue ( p <0.05).

[0053] 4 Discussion Cardiac ultrasound plays a crucial role in screening for impaired cardiac function. It can assess cardiac structure and function, diagnose cardiac impairment, monitor cardiac recovery, and evaluate cardiac workload and hemodynamic status. It offers the advantages of being non-invasive, convenient, and providing real-time imaging. Therefore, cardiac ultrasound is an indispensable tool for screening for impaired cardiac function. It clearly displays the structure of the heart's four chambers (left atrium, left ventricle, right atrium, and right ventricle) and the heart valves. In the setting of heart failure, cardiac ultrasound can reveal features such as cardiac dilatation and weakened ventricular wall motion, which are pathophysiological manifestations of heart failure. By measuring the ejection fraction (EF), which is the percentage of left ventricular stroke volume to ventricular end-diastolic volume, cardiac ultrasound can assess the heart's pumping function. The literature "Mote K, Verbrugge FH, Borlaug BA. Heart Failure with Preserved Ejection Fraction: Mechanisms and Treatment Strategies. Annu Rev Med. 2022;73:321-337." suggests that a reduced ejection fraction is one of the key hallmarks of heart failure. The experimental results of this application show that high-purity borneol can significantly shorten the left ventricular end-diastolic and end-systolic diameters, increase the left ventricular short-axis shortening rate and ejection fraction, suggesting that borneol treatment can improve the pumping function of mice with heart failure.

[0054] Although cardiac ultrasound plays an important role in the examination of heart failure, it cannot completely replace other examination methods. For example, blood tests for indicators such as brain natriuretic peptide (BNP) are also one of the important means of diagnosing heart failure. This application detected the concentrations of heart failure biomarkers CK (creatine kinase), LDH (lactate dehydrogenase), HBDH (α-hydroxybutyrate dehydrogenase), BNP (B-type natriuretic peptide), and NT-proBNP (amino-terminal B-type natriuretic peptide precursor) in the serum of heart failure mice. The literature "Gencbay M, Degertekin M, Basaran Y, et al. Microbubbles associated with mechanical heart valves: their relation with serum lactic dehydrogenase levels. Am Heart J. 1999;137(3):463-468." suggests that in patients with heart failure, because myocardial cells may be damaged to varying degrees, CK, LDH, and HBDH may be released into the blood, resulting in elevated blood levels, which may indicate damage to the myocardium or other muscle tissues. The literature "Castiglione V, Aimo A, Vergaro G, Saccaro L, Passino C,Emdin M. Biomarkers for the diagnosis and management of heart failure. HeartFail Rev. 2022;27(2):625-643." suggests that BNP is mainly synthesized and secreted by the ventricular wall cells of the heart, and has diuretic, natriuretic and vasodilatory effects, which helps to reduce cardiac load and improve cardiac function. NT-proBNP is a precursor of BNP, which exists stably in the blood and is not easily degraded. It has similar physiological functions and pathological changes as BNP. Elevated BNP is one of the important biomarkers of heart failure, which helps in the early diagnosis, risk stratification, treatment effect monitoring and prognosis evaluation of heart failure. The detection of NT-proBNP also has important clinical significance and can be used for the diagnosis, risk stratification, treatment effect monitoring and prognosis evaluation of heart failure. Compared with BNP, NT-proBNP is more stable in the blood and is less affected by interference factors, so it may sometimes be more suitable for the monitoring and evaluation of heart failure. In summary, blood levels of CK, LDH, HBDH, BNP, and NT-proBNP each have distinct significance in the diagnosis of heart failure. Changes in these indicators can reflect the degree of myocardial cell damage, the impairment of cardiac function, and the severity and prognosis of heart failure.The research results of this application show that borneol treatment can significantly reduce the concentrations of the above heart failure markers in the blood of mice with heart failure, suggesting that borneol has a certain therapeutic effect on mice with heart failure.

[0055] Myocardial fibrosis refers to the pathological process in which diseased myocardial cells hypertrophy, degeneration and necrosis, and fibers in the diseased myocardial interstitium proliferate, transform and migrate, leading to massive proliferation, reduced degradation and uneven distribution of collagen in the myocardial interstitium, and surrounding and segmenting myocardial cells. Simply put, myocardial fibrosis is the result of an imbalance between collagen synthesis and degradation. The literature "Paulus WJ, Zile MR. From Systemic Inflammation to Myocardial Fibrosis: The Heart Failure With Preserved Ejection Fraction Paradigm Revisited. Circ Res. 2021;128(10):1451-1467" reveals that excessive extracellular matrix (ECM) deposition can lead to myocardial stiffness, arrhythmias and decreased cardiac function, and is an important pathological basis for cardiac remodeling in ischemic heart disease, hypertension, cardiomyopathy, etc., and its ultimate outcome is heart failure. Therefore, inhibiting excessive ECM deposition is an important measure to prevent myocardial fibrosis and avoid heart failure, and is also one of the current research hotspots in the cardiovascular field. The experimental results showed that after borneol treatment of heart failure mice, the area of cardiac fibrosis in the mice was significantly reduced and the positive rate of cell apoptosis was significantly reduced. It can be seen that borneol has a certain protective effect on the development of cardiac fibrosis and myocardial cell necrosis in heart failure mice.

[0056] The paper "Meng L, Lu Y, Wang X, et al. NPRC deletion attenuates cardiac fibrosis in diabetic mice by activating PKA / PKG and inhibiting TGF-β1 / Smad pathways. Sci Adv. 2023;9(31):eadd4222." reveals that the TGF-β / smad signaling pathway is a recognized myocardial fibrosis signaling pathway. Activation of the TGF-β / smad signaling pathway can promote fibroblast proliferation, increase collagen production and the expression of MMPs-related proteins, leading to reduced collagen degradation, thereby promoting the development of myocardial fibrosis. TGF-β is an important factor in the TGF-β / smad signaling pathway regulating myocardial fibrosis, and is involved in regulating fibroblast proliferation, transformation, migration, and extracellular matrix production. The literature "Jia J, Zhao XA, Tao SM, et al. Icariin improves cardiac function and remodeling via the TGF-β1 / Smad signaling pathway in rats following myocardial infarction. EurJ Med Res. 2023;28(1):607." reveals that Smads are downstream molecules of TGF-β. Currently, 8 types of mammalian Smads have been found, of which Smad3 and Smad7 are both involved in the regulation of the TGF-β signaling pathway. The literature "Huang Y, Qi Y, Du JQ, Zhang DF. MicroRNA-34a regulates cardiacfibrosis after myocardial infarction by targeting Smad4. Expert Opin TherTargets. 2014;18(12):1355-1365." reveals that TGF-β activates the Smad2 / 3 complex by binding to its receptor, which then forms a hetero-oligomeric complex with Smad4 and is transported to the cell nucleus. After binding to DNA, it regulates the expression of related genes, promotes the transformation of fibroblasts into myofibroblasts, and thereby increases collagen production.The literature "Luo H, Fu L, Wang X, Yini Xu, Ling Tao, Shen X. Salvianolic acid B ameliorates myocardialfibrosis indiabetic cardiomyopathy by deubiquitinating Smad7. Chin Med. 2023;18(1):161." reveals that Smad7 is an inhibitory Smad protein (I-Smad) of the Smad protein family, which can bind to activated type I receptors and inhibit the signal transduction of the TGF-β family. In summary, TGF-β and Smads play an important role in myocardial fibrosis through complex signal transduction and regulation, and are key factors in myocardial damage. Therefore, this application observed the effect of borneol on fibrosis regulatory factors by detecting the relative gene expression levels of TGF-β, Smad3, and Smad7 in the hearts of heart failure mice and the relative expression levels of Smad3 and Smad7 proteins. The experimental results showed that borneol treatment could significantly reduce the gene expression of TGF-β and the relative gene and protein expression of Smad3, while increasing the gene and protein expression of Smad7, suggesting that the mechanism of action of borneol in protecting heart failure mice may be related to the TGF-β / smad signaling pathway.

Claims

1. Application of high-purity natural borneol in the preparation of drugs for alleviating heart failure and improving myocardial damage.

2. The use according to claim 1, characterized in that The high-purity natural borneol reduces the expression levels of TGF-β and Smad3 and increases the expression level of Smad7 by regulating the TGF-β / Smad signaling pathway, thereby improving the left ventricular ejection fraction, reducing the proportion of myocardial fibrosis and reducing myocardial cell apoptosis.

3. The use according to claim 1, characterized in that The usage of the high-purity natural borneol is 0.2 g / kg to 0.8 g / kg.

4. A pharmaceutical composition for alleviating heart failure and improving myocardial damage, characterized in that: The pharmaceutical composition contains high-purity natural borneol, which regulates the TGF-β / Smad signaling pathway, reduces the expression levels of TGF-β and Smad3, and increases the expression level of Smad7, thereby achieving the effects of improving left ventricular ejection fraction, reducing the proportion of myocardial fibrosis, and reducing myocardial cell apoptosis.

5. The pharmaceutical composition according to claim 4, characterized in that The usage of the high-purity natural borneol is 0.2 g / kg to 0.8 g / kg.

6. The pharmaceutical composition according to claim 4 or 5, characterized in that The pharmaceutical composition is administered by subcutaneous injection or oral administration.

7. The pharmaceutical composition according to claim 4, characterized in that The pharmaceutical composition further comprises auxiliary ingredients, including a cardiotonic agent for improving cardiac function or a diuretic for reducing myocardial burden.

8. Use of the pharmaceutical composition according to any one of claims 4 to 7 in the preparation of a medicament for alleviating heart failure and improving myocardial damage.

9. A pharmaceutical preparation containing high-purity natural borneol, characterized in that: The preparation comprises the pharmaceutical composition according to any one of claims 4 to 7, prepared in the form of tablets, capsules or injections, and is suitable for regulating the TGF-β / Smad signaling pathway, reducing the expression levels of TGF-β and Smad3, and increasing the expression level of Smad7, so as to achieve the effects of improving left ventricular ejection fraction, reducing the proportion of myocardial fibrosis and reducing myocardial cell apoptosis.

10. Use of the pharmaceutical preparation according to claim 9 in the preparation of a medicament for alleviating heart failure and improving myocardial damage.

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