Use of acetaldehyde dehydrogenase 1 family member a3 and inhibitors thereof in the preparation of a medicament for chronic heart failure

Abstract

The present application relates to acetaldehyde dehydrogenase 1 family member A3, and particularly to the application of acetaldehyde dehydrogenase 1 family member A3 and its inhibitor in the preparation of a drug for chronic heart failure. The present application discloses the application of acetaldehyde dehydrogenase 1 family member A3 as a drug target in the preparation of a drug for chronic heart failure. The present application first discloses the correlation between acetaldehyde dehydrogenase 1 family member A3 and chronic heart failure, and confirms that the vascular endothelial injury can be improved by specific knockdown or interference of ALDH1A3 to treat chronic heart failure, which indicates that ALDH1A3 can become a new target for treating chronic heart failure or cardiovascular diseases. Therefore, acetaldehyde dehydrogenase 1 family member A3 can be used as a target for screening a drug for preventing, relieving and / or treating chronic heart failure.

Description

TECHNICAL FIELD

[0001] The present application relates to acetaldehyde dehydrogenase 1 family member A3, and in particular to the use of acetaldehyde dehydrogenase 1 family member A3 and its inhibitors in the preparation of a drug for chronic heart failure BACKGROUND

[0002] Heart failure (HF), simply heart failure, is a disease characterized by the inability to produce enough cardiac output to meet the body's metabolic needs [1] , with ischemia, hypertension and vascular disease being the most common causes. According to foreign statistics, the prevalence of heart failure in the population is about 1.5%-2.0%, and up to 6%-10% in people over 65 years of age. The prevalence of heart failure in China is 1.3%, with about 10 million patients, and about 500,000 new cases each year, with a 5-year mortality rate of about 50%-80%. Heart failure patients have poor quality of life and poor prognosis, which seriously threatens people's health and life [2] . There are currently several drugs for treating heart failure in clinical practice:

[0003] (1) Diuretics

[0004] For heart failure patients with fluid retention, diuretics are the only drug that can fully control and effectively eliminate fluid retention, and are an essential component of standard heart failure treatment, but diuretics alone cannot maintain long-term clinical stability. Rational use of diuretics is one of the key factors for the success of other drugs for treating heart failure.

[0005] (2) ACEI

[0006] ACEI is the first drug proven to reduce mortality in heart failure patients, and is the drug with the most accumulated evidence-based medical evidence. It is recognized as the cornerstone and first choice for treating heart failure.

[0007] (3) ARB

[0008] Basically the same as ACEI, recommended for patients who cannot tolerate ACEI. It can also be used for symptomatic heart failure patients who have improved clinical status after treatment with diuretics, ACEI and beta-blockers, but cannot tolerate aldosterone receptor antagonists.

[0009] (4) Angiotensin Receptor Neprilysin Inhibitor (ARNI)

[0010] ARNI has the effects of ARB and neprilysin inhibitor, which can increase the levels of natriuretic peptide, bradykinin and adrenomedullin and other endogenous vasoactive peptides. The representative drug of ARNI is sacubitrilat, which can improve symptoms, improve prognosis and reduce mortality in heart failure.

[0011] (5) Beta-blockers

[0012] Due to long-term persistent over-activation and stimulation of sympathetic nervous system, myocardial β1 receptor down-regulation and impaired function in patients with chronic heart failure, β receptor blocker therapy can restore the normal function of β1 receptor, so that it is up-regulated. Clinical trials have confirmed that long-term application of β receptor blocker (such as metoprolol succinate, bisoprolol and carvedilol) in patients with HFrEF can improve symptoms and quality of life, and reduce the risk of death, hospitalization and sudden death. The target dose or maximum tolerated dose of β receptor blocker therapy for heart failure should be achieved.

[0013] (VI) aldosterone receptor antagonists

[0014] When ACEI or ARB is used for a long time, aldosterone is reduced at first, and then the "escape phenomenon" occurs. The addition of aldosterone receptor antagonists can inhibit the harmful effects of aldosterone, which is beneficial to patients with heart failure.

[0015] (VII) Ivabradine

[0016] It is suitable for HFrEF patients with sinus rhythm. When ACEI / ARB / ARNI, β receptor blocker, aldosterone receptor antagonist are used, and the target dose or maximum tolerated dose of β receptor blocker has been reached, the heart rate is still ≥ 70 times / min, and the symptoms persist [New York Heart Association (NYHA) class II-IV], Ivabradine can be added. Ivabradine can also be used for patients with heart rate ≥ 70 times / min, who are contraindicated or intolerant to β receptor blockers.

[0017] (VIII) Digitalis drugs

[0018] Digitalis drugs can increase intracellular Na+ levels by inhibiting Na+ / K+-ATPase in failing myocardial cell membranes, promote Na+-K+ exchange, and increase intracellular Ca+ levels, thereby exerting positive inotropic effects. Studies have shown that the use of digoxin can improve symptoms and exercise tolerance in patients with heart failure. Meta-analysis shows that long-term use of digoxin in patients with heart failure has a neutral effect on mortality, but can reduce the risk of hospitalization [3] .

[0019] Heart failure is the end stage of various heart diseases, and although various traditional drug standard treatments are used, the prognosis is still not ideal, and new drugs and updates and improvements in treatment methods are urgently needed. However, there are fewer drugs developed specifically for heart failure with preserved ejection fraction (HFpEF), and many have failed due to negative phase III clinical trials, with no significant improvement in total mortality [4] .

[0020] Therefore, finding or discovering new targets or drugs is the key to solving the above problems.

[0021] The aldehyde dehydrogenase (ALDH) family is the most important aldehyde metabolic enzyme system in cells, and plays a key catalytic role in the conversion of aldehyde substances into alcohol or corresponding carboxylic acid substances. Interestingly, the enzyme activity of ALDH in the serum of patients with pancreatic cancer is higher. In addition, the catalytic activity of ALDH can be used as a marker for identifying and separating normal stem cells and tumor stem cells, and is involved in the regulation of tumor occurrence, development and chemotherapy resistance and various malignant biological behaviors, and is related to the prognosis of various tumor patients. Aldehyde dehydrogenase 1 family member A3 (ALDH1A3) is one of the important members of the ALDH family, which can oxidize all-trans-retinal to retinoic acid, and plays an important role in the development process of vertebrates by regulating the synthesis of retinoic acid. In addition, in the presence of NAD(P)+, ALDH1A3 can oxidize acetaldehyde to acetic acid, thereby participating in the detoxification process of aldehyde substances. More importantly, the expression level of ALDH1A3 in various tumor tissues is high, which can be used as a marker for breast cancer, non-small cell lung cancer and other tumor stem cells, and is closely related to tumor metastasis [5] , has an important function in the formation of breast cancer, glioblastoma, gastric cancer and other tumors, mainly in participating in tumor cell autophagy, maintaining cell cycle and promoting cell proliferation.

[0022] However, there is no report on ALDH1A3 and treatment of heart failure at present. SUMMARY

[0023] OBJECTIVE

[0024] The purpose of the present application is to provide a new target for treating heart failure, i.e. to find that aldehyde dehydrogenase 1 family member A3 and its inhibitor can treat chronic heart failure.

[0025] TECHNICAL SCHEME

[0026] The application of aldehyde dehydrogenase 1 family member A3 as a drug screening target in the preparation of a drug for treating chronic heart failure.

[0027] The application of an aldehyde dehydrogenase 1 family member A3 inhibitor in the preparation of a drug for treating chronic heart failure.

[0028] The application of an aldehyde dehydrogenase 1 family member A3 inhibitor in the preparation of a drug for treating chronic heart failure, characterized in that the aldehyde dehydrogenase 1 family member A3 inhibitor is siRNA, which is any one of the following sequences:

[0029]

[0030] In particular, the present application provides:

[0031] Firstly, to investigate the key function and mechanism of acetaldehyde dehydrogenase 1 family member A3 (ALDH1A3) in vascular endothelial injury of chronic heart failure, heart-specific ALDH1A3 knockdown mice were constructed. After three weeks of routine feeding, the chronic heart failure vascular endothelial injury model of mice was induced by CAL. After five weeks, samples were taken, and the key function of ALDH1A3 in CAL-induced chronic heart failure vascular endothelial injury was investigated by ELISA, echocardiography, TTC staining, HE, Masson staining, Western blot and other techniques. Serum biochemical results showed that specific knockdown of ALDH1A3 could significantly reduce the content of brain natriuretic peptide (BNP), high-sensitivity C-reactive protein (hs-CRP), tumor necrosis factor (TNF-α) and nitric oxide (NO) and other heart failure and inflammation-related indicators in the serum of chronic heart failure mice. Echocardiography results showed that specific knockdown of ALDH1A3 could significantly improve the left ventricular ejection fraction, left ventricular shortening fraction and stroke volume of chronic heart failure mice, and improve the heart function of chronic heart failure mice. TTC results showed that specific knockdown of ALDH1A3 could significantly reduce the myocardial infarct size of chronic heart failure mice. At the same time, pathological detection results also showed that specific knockdown of ALDH1A3 could reduce the inflammatory infiltration and myocardial fibrosis of chronic heart failure mice, and improve the myocardial pathological injury. ELISA, Western blot and immunohistochemistry results showed that the content of ALDH1A3, ESM1 and adhesion molecule proteins in chronic heart failure model mice was significantly increased, and the content of endothelial injury markers ESM1, ICAM-1 and VCAM-1 proteins in heart failure mice was significantly reduced after specific knockdown of ALDH1A3. The above results suggest that inhibition of ALLDH1A3 expression can significantly improve vascular endothelial injury and thus improve chronic heart failure.

[0032] Further, the model of HUVECs endothelial cell injury induced by oxygen-glucose deprivation was used to verify the key function of ALDH1A3 in vascular endothelial injury in vitro. First, the MTT colorimetric method was used to detect the activity of HUVECs endothelial cells to determine the modeling time of OGD of HUVECs. Then, the expression of ESM1 and adhesion molecule protein related to vascular endothelial injury in HUVECs before and after siRNA interference and plasmid overexpression of ALDH1A3 was investigated by Western blot and immunofluorescence. The results of Western blot and immunofluorescence showed that the contents of ALDH1A3, ESM1, ICAM-1 and VCAM-1 proteins related to endothelial injury in OGD-induced HUVECs were significantly increased, while the contents of ESM1, ICAM-1 and VCAM-1 proteins related to endothelial injury in endothelial cells were significantly reduced after interfering ALDH1A3, indicating that inhibiting the expression of ALDH1A3 can significantly improve the OGD-induced vascular endothelial injury of HUVECs. At the same time, the overexpression of ALDH1A3 can significantly increase the contents of ESM1, ICAM-1 and VCAM-1 proteins related to endothelial injury in HUVEC cells compared with the OGD group, indicating that the increase of ALDH1A3 can promote the formation of vascular endothelial injury. This is consistent with the results in vivo, which indicates that ALDH1A3 is a key regulatory protein of chronic heart failure vascular endothelial injury.

[0033] Based on the key regulatory protein ALDH1A3 of chronic heart failure vascular endothelial injury, the molecular docking virtual technology was used to select the active ingredients in the clinical blood-activating and stasis-removing drugs that may interact with it. First, the complete structure of ALDH1A3 was downloaded from the PDB protein structure database, and then the main active ingredients in blood-activating and stasis-removing drugs such as tanshinone IIA, cryptotanshinone, hydroxysafflor yellow A, etc. were obtained from TCMID, HMDB and other databases. Autodock vina was used to analyze the binding between compounds and regulatory proteins, and the results showed that cryptotanshinone (CTS) had strong binding ability with ALDH1A3. Then, SPR was used to verify the interaction between CTS and ALDH1A3, and the results showed that the binding constant between them was 5.536 μM, which had strong binding affinity.

[0034] A mouse model of chronic heart failure vascular endothelial injury was induced by coronary artery ligation, and after 2 weeks of modeling, different doses of CTS (10 mg / kg, 20 mg / kg, 40 mg / kg) were administered by gavage, once a day, and after 3 weeks of sampling, the improvement effect of CTS on CAL-induced chronic heart failure vascular endothelial injury was detected by ELISA, echocardiography, TTC staining, HE, Masson staining, Western blot and other techniques. The serum biochemical results show that CTS can significantly reduce the content of BNP, hs-CRP, TNF-alpha and NO and other heart failure and inflammation related indicators in the serum of chronic heart failure mice. The echocardiography results show that CTS can significantly improve the left ventricular ejection fraction, left ventricular shortening fraction and stroke volume of chronic heart failure mice, and improve the heart function of chronic heart failure mice. The TTC results show that CTS can significantly reduce the myocardial infarction area of chronic heart failure mice. At the same time, the pathological detection results also show that CTS can reduce the inflammatory infiltration and myocardial fibrosis of the heart of heart failure mice, and improve the pathological structure damage of the myocardium. ELISA, Western blot and immunohistochemical results show that CTS can significantly reduce the content of ALDH1A3 and its downstream endothelial injury markers ESM1, ICAM-1 and VCAM-1, and improve the CAL-induced chronic heart failure vascular endothelial injury. The above results show that CTS has a significant improvement effect on the heart function weakening, myocardial infarction, pathological structure damage and the like of CAL-induced heart failure mice, which may be through inhibiting the expression of ALDH1A3 to improve the vascular endothelial injury. The above results again prove that ALDH1A3 can be used as a target for screening heart failure drugs.

[0035] Key points of the present application

[0036] The technical key point of the present application is that specific knockdown or interference of ALDH1A3 can improve vascular endothelial injury and thus treat chronic heart failure

[0037] Beneficial effects

[0038] The present application first discovers the relevance of acetaldehyde dehydrogenase 1 family member A3 and chronic heart failure, confirms that specific knockdown or interference of ALDH1A3 can improve vascular endothelial injury and thus treat chronic heart failure, and suggests that ALDH1A3 can become a new target for treating chronic heart failure or cardiovascular diseases. Therefore, acetaldehyde dehydrogenase 1 family member A3 can be used as a target for screening drugs for preventing, relieving and / or treating chronic heart failure. BRIEF DESCRIPTION OF DRAWINGS

[0039] Figure 1Inhibition efficiency of ALDH1A3 specific knockdown in chronic heart failure mice. ALDH1A3 heart-specific knockout model was constructed by in situ injection of ALDH1A3-KD or AAV-NC, and sham operation and CAL operation were performed after 3 weeks. After feeding for 5 weeks, (A) Western blot representative image of ALDH1A3 (B) Immunohistochemical representative image of ALDH3A3 (n=3). The results are expressed as mean ± standard deviation. # p<0.05, ## p<0.01 vs. Sham group, ** p<0.01 vs. CAL group.

[0040] Figure 2 Changes in serum biochemical indicators of heart failure mice after specific knockdown of ALDH1A3. ALDH1A3 heart-specific knockout model was constructed by in situ injection of ALDH1A3-KD or AAV-NC, and sham operation and CAL operation were performed after 3 weeks. After feeding for 5 weeks, serum samples were collected, and ELISA was used to detect (A) BNP, (B) hs-CRP, (C) TNF-α and (D) NO in serum (n=6). The results are expressed as mean ± standard deviation. # p<0.05, ## p<0.01 vs. Sham group, ** p<0.01 vs. CAL group. Figure 3 Effect of specific knockdown of ALDH1A3 on cardiac function of chronic heart failure mice. ALDH1A3 heart-specific knockout model was constructed by in situ injection of ALDH1A3-KD or AAV-NC, and sham operation and CAL operation were performed after 3 weeks. After feeding for 5 weeks, echocardiography analysis was performed. (A) Representative image of M-mode echocardiogram. (B) Left ventricular ejection fraction. (C) Left ventricular fractional shortening. (D) Stroke volume (n=6). The results are expressed as mean ± standard deviation. ## p<0.01 vs. Sham group, ** p<0.01 vs. CAL group.

[0041] Figure 4 Effect of specific knockdown of ALDH1A3 on myocardial infarct size in chronic heart failure mice. ALDH1A3 heart-specific knockout model was constructed by in situ injection of ALDH1A3-KD or AAV-NC, and sham operation and CAL operation were performed after 3 weeks. After feeding for 5 weeks, (A) heart sections were stained with 1% TTC solution, and the white area of the heart was infarct tissue; (B) infarct size was analyzed by ImageJ (n=3). The results are expressed as mean ± standard deviation. ##p<0.01 vs. Sham group, **p<0.01 vs. CAL group.

[0042] Figure 5Effect of specific knockdown of ALDH1A3 on the histopathology of chronic heart failure mice. ALDH1A3 heart-specific knockout model was constructed by in situ injection of ALDH1A3-KD or AAV-NC, and sham operation and CAL operation were performed after 3 weeks. After 5 weeks of feeding, the heart was taken for HE staining (n=3). A is the pathological photo, and B is the statistical result, which is expressed as mean ± standard deviation. ##p<0.01 vs. Sham group, **p<0.01 vs. CAL group.

[0043] Figure 6 Effect of specific knockdown of ALDH1A3 on myocardial fibrosis in chronic heart failure mice. ALDH1A3 heart-specific knockout model was constructed by in situ injection of ALDH1A3-KD or AAV-NC, and sham operation and CAL operation were performed after 3 weeks. After 5 weeks of feeding, the heart was taken for Masson staining (n=3). A is the pathological photo, and B is the statistical result, which is expressed as mean ± standard deviation. ##p<0.01 vs. Sham group, **p<0.01 vs. CAL group.

[0044] Figure 7 Effect of specific knockdown of ALDH1A3 on ESM1, an endothelial cell-specific molecule, in serum and heart of chronic heart failure mice. ALDH1A3 heart-specific knockout model was constructed by in situ injection of ALDH1A3-KD or AAV-NC, and sham operation and CAL operation were performed after 3 weeks. Samples were collected after 5 weeks of feeding. (A) EMS1 serum ELISA results (n=6); (B) ESM1 immunohistochemical results (n=3). The results are expressed as mean ± standard deviation. ##p<0.01 vs. Sham group, **p<0.01 vs. CAL group.

[0045] Figure 8 Effect of specific knockdown of ALDH1A3 on ICAM-1 and VCAM-1 in serum and heart of chronic heart failure mice. ALDH1A3 heart-specific knockout model was constructed by in situ injection of ALDH1A3-KD or AAV-NC, and sham operation and CAL operation were performed after 3 weeks. Samples were collected after 5 weeks of feeding. (A) Serum ELISA results of ICAM-1 (n=6), (B) Serum ELISA results of VCAM-1 (n=6), and (C) Immunohistochemical results of ICAM-1 and VCAM-1 (n=3). The results are expressed as mean ± standard deviation. ##p<0.01 vs. Sham group, **p<0.01 vs. CAL group.

[0046] Figure 9 Cell survival of HUVEC cells under different hypoxic times. MTT method was used to detect the viability of HUVEC cells under different hypoxic times (n=6). The results are expressed as mean ± standard deviation.** p<0.01 vs. Control group.

[0047] Figure 10 siRNA-ALDH1A3 sequence screening. After transfection of corresponding siRNA for 48 hours, the inhibition efficiency of siRNA on ALDH1A3 was detected by Western blot (n=3). A is the Western blot detection photo, and B is the statistical result, which is expressed as mean ± standard deviation. ##p<0.01 vs. NC group.

[0048] Figure 11 Effect of interfering ALDH1A3 on ESM1 in OGD-induced HUVECs vascular endothelial injury. After transfection of siRNA-ALDH1A3 for 48 hours and OGD modeling for 18 hours, the expression of ESM1 in HUVECs cells was detected by immunofluorescence (n=3). A is the photo, and B is the statistical result, which is expressed as mean ± standard deviation. **p<0.01 vs. Control group, $$p<0.01 vs. OGD group.

[0049] Figure 12 Effect of interfering ALDH1A3 on ICAM-1 and VCAM-1 in OGD-induced HUVECs vascular endothelial injury. After transfection of siRNA-ALDH1A3 for 48 hours and OGD modeling for 18 hours, (A) the expression of ICAM-1 and VCAM-1 was detected by immunofluorescence, (B) the statistical result of ICAM-1 and (C) the statistical result of VCAM-1 (n=3). The results are expressed as mean ± standard deviation. **p<0.01 vs. Control group, $$p<0.01 vs. OGD group.

[0050] Figure 13 ALDH1A3 plasmid overexpression efficiency investigation. A is the photo, and B is the statistical result; after transfection of HUVECs cells with ALDH1A3 overexpression plasmid for 48 hours, the Western blot analysis results of ALDH1A3- were obtained (n=3). The results are expressed as mean ± standard deviation. ##p<0.01 vs. vector group.

[0051] Figure 14 Effect of overexpressing ALDH1A3 on ESM1 in OGD-induced HUVECs vascular endothelial injury. A is the photo, and B is the statistical result; after transfection of ALDH1A3 overexpression plasmid for 48 hours and OGD modeling for 18 hours, the expression of ESM1 in HUVECs cells was detected by immunofluorescence (n=3). The results are expressed as mean ± standard deviation. ** p<0.01 vs. vector group, $$ p<0.01 vs. OGD group.

[0052] Figure 15 Effect of overexpression of ALDH1A3 on ICAM-1 and VCAM-1 in OGD-induced HUVECs vascular endothelial injury. HUVECs cells were transfected with ALDH1A3 overexpression plasmid for 48 hours, and the expression of ICAM-1 and VCAM-1 was detected after 18 hours of OGD modeling. (A) Representative images of immunofluorescence, (B) statistical results of ICAM-1 and (C) statistical results of VCAM-1 (n = 3). The results are expressed as mean ± standard deviation. **p < 0.01 vs. vector group, $$p < 0.01 vs. OGD group.

[0053] Figure 16 Interaction mode between ALDH1A3 and the top 3 compounds with binding affinity. (A) Interaction with cryptotanshinone, (B) interaction with tanshinone IIA, and (C) interaction with salvianolic acid A.

[0054] Figure 17 Binding affinity of ALDH1A3 to CTS,

[0055] Figure 18 Effect of CTS on ALDH1A3 protein content in chronic heart failure mice. A is a photograph, and B is a statistical result. After successful modeling of CAL, different doses of CTS (10 mg / kg, 20 mg / kg, 40 mg / kg) and Met (5.14 mg / kg) were administered by gavage. ALDH1A3 immunohistochemical detection results (n = 3). The results are expressed as mean ± standard deviation. #p < 0.05, ##p < 0.01 vs. Sham group, **p < 0.01 vs. CAL group.

[0056] Figure 19 Effect of CTS on cardiac function in chronic heart failure mice. After successful modeling of CAL, different doses of CTS (10 mg / kg, 20 mg / kg, 40 mg / kg) and Met (5.14 mg / kg) were administered by gavage. (A) Representative images of M-mode echocardiogram. (B) Left ventricular ejection fraction. (C) Left ventricular fractional shortening. (D) Stroke volume (n = 6). The results are expressed as mean ± standard deviation. ##p < 0.01 vs. Sham group, **p < 0.01 vs. CAL group.

[0057] Figure 20Effect of CTS on serum biochemical indicators in mice with chronic heart failure. After successful modeling of CAL, different doses of CTS (10 mg / kg, 20 mg / kg, 40 mg / kg) and Met (5.14 mg / kg) were administered by gavage. The contents of (A) BNP, (B) hs-CRP, (C) TNF-a and (D) NO in serum were detected by ELISA method (n=6). The results were expressed as mean ± standard deviation. ## p<0.01 vs. Sham group, ** p<0.01 vs. CAL group.

[0058] Figure 21 Effect of CTS on myocardial infarct size in mice with chronic heart failure. After successful modeling of CAL, different doses of CTS (10 mg / kg, 20 mg / kg, 40 mg / kg) and Met (5.14 mg / kg) were administered by gavage. (A) Heart sections were stained with 1% TTC solution, and the white area of the heart was infarct tissue. (B) Infarct size was analyzed by Image J (n=3). The results were expressed as mean ± standard deviation. ##p<0.01 vs. Sham group, **p<0.01 vs. CAL group.

[0059] Figure 22 Effect of CTS on histopathology in mice with chronic heart failure. A is a photo, and B is a statistical result; after successful modeling of CAL, different doses of CTS (10 mg / kg, 20 mg / kg, 40 mg / kg) and Met (5.14 mg / kg) were administered by gavage. The heart was taken for HE staining analysis (n=3). The results were expressed as mean ± standard deviation. ## p<0.01 vs. Sham group, ** p<0.01 vs. CAL group.

[0060] Figure 23 Effect of CTS on myocardial fibrosis in mice with chronic heart failure. A is a photo, and B is a statistical result; after successful modeling of CAL, different doses of CTS (10 mg / kg, 20 mg / kg, 40 mg / kg) and Met (5.14 mg / kg) were administered by gavage. The heart was taken for Masson staining analysis (n=3). The results were expressed as mean ± standard deviation. ##p<0.01 vs. Sham group, **p<0.01 vs. CAL group.

[0061] Figure 24Effect of CTS on ESM1 in serum and heart of mice with chronic heart failure. After successful modeling of CAL, different doses of CTS (10 mg / kg, 20 mg / kg, 40 mg / kg) and Met (5.14 mg / kg) were respectively administered by gavage. (A) ESM1 serum ELISA results (n=6), (B) ESM1 immunohistochemical results (n=3). The results are expressed as mean ± standard deviation. ## p<0.01 vs. Sham group, * p<0.05, ** p<0.01 vs. CAL group.

[0062] Figure 25 Effect of CTS on ICAM-1 and VCAM-1 in serum and heart of mice with chronic heart failure. After successful modeling of CAL, different doses of CTS (10 mg / kg, 20 mg / kg, 40 mg / kg) and Met (5.14 mg / kg) were respectively administered by gavage. (A) Serum ICAM-1 enzyme-linked immunosorbent assay results (n=6), (B) Serum VCAM-1 enzyme-linked immunosorbent assay results (n=6), (C) ICAM-1 and VCAM-1 immunohistochemical results (n=3). The results are expressed as mean ± standard deviation. ##p<0.01 vs. Sham group, *p<0.05, **p<0.01 vs. CAL group. DETAILED DESCRIPTION

[0063] Example 1 Key function of ALDH1A3 in CAL-induced vascular endothelial injury in chronic heart failure

[0064] I. Experimental materials

[0065] 1. Experimental animals

[0066] SPF male ICR mice (8 weeks, 19-22 g) were purchased from the Comparative Medicine Center of Yangzhou University, license number: 202137997. The mice were free to drink and eat, and were raised in a SPF level animal experiment room with a temperature of 27°C and a relative humidity of 40%-70%. The mice were adaptively raised for one week before the experiment, and all animal experiment procedures were performed in accordance with the "Guide for the Care and Use of Laboratory Animals", and the experimental program was approved by the Animal Ethics Committee of China Pharmaceutical University.

[0067] 2. Experimental reagents

[0068]

[0069]

[0070] 3. Experimental equipment

[0071]

[0072] II. Experimental Methods

[0073] 1. Construction of Adeno-Associated Virus

[0074] Shanghai Jiman Biotechnology Co., Ltd. was commissioned to construct a specific knockdown virus based on the ALDH1A3 gene information from human sources. The vector used for the specific knockdown adeno-associated virus was Flag-T2A-eGFP-WPRE, carrying the ALDH1A3 gene (ALDH1A3-cKD) or negative control (AAV-NC). Mice were injected with ALDH1A3-cKD or AAV_NC (x1011 pfu / Ml) through the myocardium (2 sites, 10 μL / site). The insert sequence in the recombinant clone was identical to the target sequence, so the plasmid construction was successful. After 8 weeks, the inhibition efficiency of the adeno-associated virus was detected by Western blot.

[0075] 2. Animal grouping and administration

[0076] 48 male ICR mice were randomly divided into 4 groups:

[0077] Sham operation group (Sham+AAV-NC): Adeno-associated virus blank vector was injected in situ, and after 3 weeks of regular feeding, modeling was performed, and then feeding was continued for 5 weeks.

[0078] Sham operation knockdown group (Sham+ALDH1A3-cKD): Adeno-associated virus specific to knockdown ALDH1A3 was injected in situ, and after 3 weeks of regular feeding, modeling was performed, and then feeding was continued for 5 weeks.

[0079] Model group (Model+AAV-NC): Adeno-associated virus blank vector was injected in situ, and after 3 weeks of regular feeding, modeling was performed, and then feeding was continued for 5 weeks.

[0080] Model knockdown group (Model+ALDH1A3-cKD): Adeno-associated virus specific to knockdown ALDH1A3 was injected in situ, and after 3 weeks of regular feeding, modeling was performed, and then feeding was continued for 5 weeks.

[0081] 3. Preparation of chronic heart failure model in mice

[0082] Mice were anesthetized by intraperitoneal injection of 1% sodium pentobarbital, placed in a supine position with limbs fixed, and connected to a ventilator (tidal volume 3 mL, respiratory ratio 2:1, heart rate 110). Hair was removed from the left chest, and the area was disinfected with povidone-iodine. A longitudinal incision was made in the skin on the left side of the sternum, and the chest muscles were dissected layer by layer to expose the fourth rib. The thoracic cavity was punctured in the intercostal space with curved forceps to expose the heart. The heart was squeezed out by gently pressing on the thoracic cavity, and a 6 / 0 suture was quickly ligated 3 mm below the origin of the left anterior descending coronary artery. The heart was then retracted, and after completely expelling the air, the suture was quickly closed. In the sham-operated group, only the suture was inserted without ligation; the remaining procedures were the same as in the model group. Mice were sacrificed after five weeks of standard feeding.

[0083] 4. Serum biochemical index detection

[0084] Blood samples were collected immediately after euthanasia of mice. After standing at room temperature for 1 hour, the samples were centrifuged at 3000 rpm for 15 minutes at 4°C to separate serum. The supernatant was stored at -80°C until further analysis. The levels of BNP, hs-CRP, TNF-α, ESM1, ICAM-1, VCAM-1, and NO in the serum were determined according to the kit instructions.

[0085] 5. Echocardiography

[0086] The Vevo 3100LT system was used to evaluate cardiac function in mice. Each mouse was secured to a heated imaging platform with 3M tape, anesthetized with 2.5% isoflurane, and then a coupled-adhesive ultrasound probe was used to reach the left sternum to capture cardiac motion images in M-mode. The following parameters were obtained using the PSLAX method: left ventricular ejection fraction (LV EF), left ventricular fractional shortening (LV FS), and stroke volume (SV) as indicators of cardiac function.

[0087] 6. TTC staining

[0088] Eight weeks after ligation, the mouse heart was rapidly removed and frozen at -70°C. The ventricular tissue was cut into five layers perpendicular to the long axis of the heart. The layers were incubated in 24-well plates containing 1% triphenyltetrazolium chloride (TTC) solution at 37°C in the dark for 15 minutes, and photographed using a digital camera. The white areas represent the infarcted regions, and the infarct area was measured using a computer-aided planar measurement method. The infarct area was calculated as infarct area / left ventricular area, expressed as a percentage of the total left ventricular area.

[0089] 7. Pathological and histological examination

[0090] After euthanasia, the heart was removed from the mice, washed with pre-cooled PBS solution, fixed in 4% chloral hydrate for 24 hours, embedded in paraffin, and then cut into thin sections of about 4-5 μm for HE and Masson staining. The sections were scanned using a digital pathology slide scanner and reviewed and scored by pathologists.

[0091] 8. Immunohistochemical staining

[0092] The heart was removed after euthanasia of the mouse, soaked in 4% paraformaldehyde for 2 days, paraffin-embedded, and the section thickness was 4 μm. Then the section was deparaffinated and hydrated under the gradient elution of xylene, ethanol, 85% ethanol and distilled water. The section was incubated with blocking solution at room temperature for 1 hour. The section was added with the corresponding primary antibody, stored at 4°C for 24 h, then incubated with secondary antibody for 1 h, and stained with DAB and hematoxylin. The dehydrated section was observed under an optical microscope, and finally imaged under a 400X magnifying lens. In order to avoid the influence of subjective factors on the experimental results, the personnel interpreting the results of immunohistochemical staining adopted a double-blind method.

[0093] 9. Western blot assay of protein expression

[0094] The heart tissue was ground by an electrically driven tissue homogenizer at 60 Hz for 2 min, and was allowed to stand for 0.5 h. Then, the protein concentration was determined according to the manufacturer's instructions using a BCA kit, and the sample was stored at -80°C for subsequent experiments. The protein was added to an SDS-PAGE gel for electrophoresis, transferred to a PVDF membrane, and then the membrane was blocked with a fast blocking buffer. The corresponding primary antibody was incubated at 4°C overnight. The next day, the corresponding secondary antibody was added and incubated at room temperature for 2 h (all antibodies were diluted according to the instructions and using the corresponding antibody diluent). The protein signal was detected using the ECL method, and the protein band intensity was measured using Image Lab software, with GAPDH as the standard protein. The relative expression level of the protein = gray value of the target protein / gray value of the internal reference.

[0095] 10. Statistical analysis of experimental results

[0096] All data are expressed as mean ± SEM. The data were statistically analyzed using GraphPad Prism 8 software. Student's two-tailed t-test was used for comparison between two groups. One-way ANOVA, Bonferroni post-hoc test, Kruskal-Wallis post-hoc test, Dunn post-hoc test were used for comparison among multiple groups as appropriate. A p value of less than 0.05 was considered statistically significant.

[0097] III. Experimental results

[0098] 1. Investigation of the inhibition efficiency of specific knockdown of ALDH1A3 in mice

[0099] The results of detection of the inhibition efficiency of specific knockdown of ALDH1A3 in mouse myocardium are shown in Table 1, and the inhibition efficiency of ALDH1A3 reached the requirement, so that subsequent experiments can be carried out. Figure 1

[0100] 2. Effect of specific knockdown of ALDH1A3 on serum biochemical indicators in chronic heart failure mice ​

[0101] The serum of each group of mice was isolated and determined for BNP, hs-CRP, TNF-α and NO. As shown in Figure 2 The content of BNP, hs-CRP, TNF-α and NO in the serum of CAL group mice was significantly higher than that of Sham group, indicating that CAL operation caused significant myocardial injury. After specific knockdown of ALDH1A3, the content of BNP and NO in the serum of sham+ALDH1A3-cKD group mice did not change significantly compared with sham+NC group, while the content of hs-CRP and TNF-α decreased significantly. Compared with CAL group, the content of BNP, hs-CRP, TNF-α and NO in the heart of CAL+ALDH1A3-cKD group mice was significantly reduced, suggesting that specific knockdown of ALDH1A3 in the heart can significantly improve chronic heart failure and inflammatory response.

[0102] 3. Effect of specific knockdown of ALDH1A3 on heart function of chronic heart failure mice

[0103] As shown in Figure 3 Compared with Sham group, the left ventricular ejection fraction, left ventricular fractional shortening and stroke volume of CAL group mice were significantly reduced, indicating that coronary ligation can significantly affect the heart function of mice. After specific knockdown of ALDH1A3, the left ventricular ejection fraction, left ventricular fractional shortening and stroke volume of CAL+ALDH1A3-cKD group mice were significantly higher than those of CAL group, while specific knockdown of ALDH1A3 did not significantly affect the heart function of sham-operated mice.

[0104] 4. Effect of specific knockdown of ALDH1A3 on myocardial infarct size of chronic heart failure mice

[0105] The results of heart TTC staining are shown in Figure 4 The myocardial tissue of Sham group and Sham+ALDH1A3-cKD group mice showed normal red color, and the heart of CAL group mice showed a large area of white infarct area, with an infarct area of 34%. After specific knockdown of ALDH1A3, the myocardial infarct size of heart failure mice was significantly reduced.

[0106] 5. Effect of specific knockdown of ALDH1A3 on myocardial histopathology of chronic heart failure mice

[0107] The results of HE staining are shown in Figure 5As shown, the myocardial structure of the Sham group mice was complete, the myocardial cells were arranged in order, the outline was clear, the cytoplasm was rich and uniform, the interstitium was normal, and no cell necrosis was observed; the myocardial cells of the CAL group were arranged in disorder, part of the myocardial cells were vacuolated, nuclear loss occurred, and inflammatory cell infiltration and interstitial congestion were observed. After specific knockdown of ALDH1A3, compared with the Sham group, no obvious difference was observed in the Sham+ALDH1A3-cKD group. Compared with the CAL group, the myocardial cells of the CAL+ALDH1A3-cKD group were arranged in order, and the inflammatory infiltration was obviously recovered.

[0108] 6. Effect of specific knockdown of ALDH1A3 on myocardial fibrosis in chronic heart failure mice

[0109] Masson staining results are shown in Figure 6 As shown, compared with the Sham group, the heart tissue of the model mice showed obvious fibrosis, collagen deposition was obvious, and the percentage of collagen protein increased significantly. After specific knockdown of ALDH1A3, compared with the CAL group, the degree of myocardial fibrosis in the CAL+ALDH1A3-cKD group was obviously reduced.

[0110] 7. Effect of specific knockdown of ALDH1A3 on endothelial cell specific molecule ESM1 in serum and heart of chronic heart failure mice

[0111] ELISA and immunohistochemical results are shown in Figure 7 As shown, the content of ESM1 in the CAL group mice was obviously increased compared with the Sham group, indicating that the CAL operation caused obvious vascular endothelial injury. After specific knockdown of ALDH1A3, compared with the sham+NC group, the content of ESM1 in the serum of the sham+ALDH1A3-cKD group mice was obviously reduced, while the content of ESM1 in the heart did not change obviously. Compared with the CAL group, the content of ESM1 in the serum and heart of the CAL+ALDH1A3-cKD group mice was significantly reduced, indicating that specific knockdown of ALDH1A3 in the heart can significantly improve the vascular endothelial injury.

[0112] 8. Effect of specific knockdown of ALDH1A3 on endothelial adhesion molecules ICAM-1 and VCAM-1 in serum and heart of chronic heart failure mice Figure 8 As shown, the content of ICAM-1 and VCAM-1 in the CAL group mice was obviously increased compared with the Sham group, indicating that the CAL operation induced the activation of endothelial adhesion molecules. After specific knockdown of ALDH1A3, compared with the CAL group, the content of ICAM-1 and VCAM-1 in the serum and heart of the CAL+ALDH1A3-cKD group mice was significantly reduced, indicating that specific knockdown of ALDH1A3 can significantly inhibit the activation of endothelial adhesion molecules.

[0113] Example 2A Key function research of ALDH1A3 in OGD-induced HUVECs vascular endothelial injury

[0114] I. Experimental materials

[0115] 1. Experimental cell strain

[0116] HUVEC human umbilical vein endothelial cells were purchased from Shanghai Institute of Cell Biology, Chinese Academy of Sciences.

[0117] 2. Experimental reagents

[0118]

[0119]

[0120] Other reagents refer to Example 1.

[0121] 3. Experimental apparatus

[0122]

[0123] Other experimental apparatus refer to Example 1.

[0124] 4. Preparation of reagents

[0125] (1) Cell cryopreservation solution

[0126] The cell cryopreservation solution was prepared by FBS and DMSO in a ratio of 9:1.

[0127] (2) MTT solution

[0128] In the dark, weigh 50 mg of MTT and dissolve it in 10 mL of PBS solution. Stir the MTT solution to fully dissolve, then filter it in a clean bench and divide it into brown centrifuge tubes, store it at -20°C for standby, and dilute it with double-antibody-free DMEM medium at a ratio of 1:9 when used.

[0129] II. Experimental methods

[0130] 1. Cell culture

[0131] HUVEC human umbilical vein endothelial cells were cultured in DMEM high-sugar medium containing 10% FBS and 1% penicillin-streptomycin. The culture conditions were 37°C, 5% CO2 in a constant temperature and humidified incubator, and the culture medium of the cells was replaced every 48h. Endothelial cells grow mostly adherent, and when the cell growth density reaches 80%-90%, they are passaged or subjected to subsequent experiments.

[0132] 2. siRNA-ALDH1A3 sequence construction and synthesis

[0133] Nantong Jin Xu Biotechnology Co., Ltd. was commissioned to design and synthesize the following three siRNA sequences for Human-ALDH1A3 gene:

[0134]

[0135] 3. Construction of overexpression plasmid

[0136] Shanghai Jiman Biotechnology Co., Ltd. was commissioned to construct a specific overexpression ALDH1A3 plasmid according to the ALDH1A3 gene information from human sources. The vector of the overexpression plasmid was PGMLV-CMV-MCS-3xFlag-EF1-ZsGreen1-T2A-Puro, which carried the overexpression ALDH1A3 gene (Overexpression-ALDH1A3) or negative control (vector). After transfection of HUVEC cells for 48 h, the overexpression efficiency was detected by Western blot.

[0137] 4. Cell grouping transfection and OGD model preparation

[0138] HUVECs in the logarithmic growth phase were inoculated into 100 mm dishes or 60 mm dishes, and fresh DMEM high glucose medium containing 10% fetal bovine serum was added and cultured for 12 h. The density should not exceed 50%. Then the cells were divided into the following eight groups:

[0139] Control+siNC group: After transfection of HUVEC endothelial cells with NC empty vector for 48 h, the DMEM high glucose medium containing 0.1% FBS was replaced, and the normal culture was continued at 37°C, 5% CO2 in the incubator for 18 h.

[0140] OGD+siNC group: After transfection of HUVEC endothelial cells with NC empty vector for 48 h, the DMEM medium without sugar was replaced, and the hypoxic culture was carried out in the hypoxic box containing 1% O2, 5% CO2, 94% N2 for 18 h.

[0141] Control+siALDH1A3 group: After transfection of HUVEC endothelial cells with siRNA-ALDH1A3 for 48 h, the DMEM high glucose medium containing 0.1% FBS was replaced, and the normal culture was continued at 37°C, 5% CO2 in the incubator for 18 h.

[0142] OGD+siALDH1A3 group: After transfection of HUVEC endothelial cells with siRNA-ALDH1A3 for 48 h, the DMEM medium without sugar was replaced, and the hypoxic culture was carried out in the hypoxic box containing 1% O2, 5% CO2, 94% N2 for 18 h.

[0143] Control+vector group: HUVEC endothelial cells transfected with empty plasmid for 48 hours, then replaced with DMEM high-sugar medium containing 0.1% FBS, and continued to be cultured normally at 37°C, 5% CO2 in the incubator for 18 hours.

[0144] OGD+vector group: HUVEC endothelial cells transfected with empty plasmid for 48 hours, then replaced with sugar-free DMEM medium, and cultured in a hypoxic chamber with 1% O2, 5% CO2, and 94% N2 for 18 hours.

[0145] Control+overexpression-ALDH1A3 group: HUVEC endothelial cells transfected with overexpression plasmid for 48 hours, then replaced with DMEM high-sugar medium containing 0.1% FBS, and continued to be cultured normally at 37°C, 5% CO2 in the incubator for 18 hours.

[0146] OGD+overexpression-ALDH1A3 group: HUVEC endothelial cells transfected with overexpression plasmid for 48 hours, then replaced with sugar-free DMEM medium, and cultured in a hypoxic chamber with 1% O2, 5% CO2, and 94% N2 for 18 hours.

[0147] 5. Cell viability detection

[0148] Logarithmic growth phase HUVECs endothelial cells were trypsinized and seeded in 96-well plates at a density of 5x103 per well, and placed in the corresponding condition in the incubator for normal or hypoxic culture. After treatment, MTT was added and incubated in the dark for 3-4 hours. The OD value of each well was measured by enzyme marker (measurement wavelength 570nm, reference wavelength 650nm), and the cell activity was calculated.

[0149] 6. Cell immunofluorescence

[0150] HUVECs entering logarithmic growth phase for experiment were inoculated into a confocal dish, and after 24 hours of culture in fresh DMEM high-sugar medium containing 10% fetal bovine serum, OGD modeling was performed. After modeling, the medium was discarded, washed with PBS, and 200-300μL of methanol was added to fix the cells for 30 minutes. Then, immunostaining blocking solution was added for blocking. Then, the corresponding primary antibody (ALDH1A3 / ESM1 / ICAM-1 / VCAM-1) diluted with antibody diluent was added, and incubated at 4°C overnight. The next day, the corresponding rabbit-488 / mouse-594 / sheep-594 secondary antibody was incubated in the dark at room temperature for 1.5-2 hours, DAPI was added, and finally, anti-fluorescence quenching mounting solution was added, and the laser confocal microscope was photographed.

[0151] 7. Western blot method for detecting protein expression

[0152] After OGD modeling, the culture dish was taken out, the medium was poured off, the residual was sucked, and it was placed on ice. Each dish was added with 1 mL of 1x PBS (4°C pre-cooling), 10 μL of PMSF was added in each 1 mL of lysis solution, 40 μL of lysis solution was added in each dish, and it was placed in a -80°C refrigerator for 5 min to freeze and break the cell membrane. The cells were scraped off on ice with a scraper and transferred to an EP tube, and were lysed at 4°C for 30 min; centrifuged at 12000 rpm at 4°C for 15 min. The supernatant was taken, which was the desired protein, and was quantified. The remaining sample was stored at -80°C for subsequent experiments. The remaining operations refer to Example 1.

[0153] 8. Statistical analysis

[0154] See Example 1.

[0155] III. Experimental results

[0156] 1. Cell survival of HUVEC cells under different hypoxic times

[0157] HUVEC endothelial cells were cultured for different hypoxic times (6, 12, 18, and 24 h), and the cell survival rate was detected by MTT method. The results are shown in Figure 9 The longer the hypoxic time, the lower the cell survival rate. After 18 h of hypoxia, the survival rate of HUVEC endothelial cells was 60%, which was significantly changed, so 18 h was selected as the modeling time of HUVECs.

[0158] 2. Screening of siRNA-ALDH1A3 sequence

[0159] To further explore the key role of ALDH1A3 in OGD-induced endothelial injury at the cellular level, siRNA interference and plasmid overexpression technology were used for subsequent research. The highest expression inhibition efficiency of ALDH1A3 was screened by Western blot method, and the overexpression efficiency of the overexpression plasmid was investigated. The results are shown in Figure 10 The highest efficiency of siRNA-1 was about 53%, the inhibition efficiency of siRNA-2 was 34%, and the inhibition efficiency of siRNA-3 was 43%, so siRNA-1 was selected for subsequent experiments.

[0160] 3. Effect of interfering ALDH1A3 on ESM1, an endothelial cell-specific molecule, in OGD-induced HUVECs vascular endothelial injury

[0161] As shown in Figure 11As shown, compared with the control group, the ESM1 content in HUVEC cells in the OGD-stimulated model group was significantly increased, indicating that OGD stimulation can aggravate endothelial damage in HUVEC cells. However, after inhibiting ALDH1A3, the ESM1 content in endothelial cells of the OGD+siRNA-ALDH1A3 group was significantly decreased.

[0162] 4. Effects of ALDH1A3 interference on endothelial adhesion molecules ICAM-1 and VCAM-1 in OGD-induced vascular endothelial injury of HUVECs

[0163] like Figure 12 As shown, compared with the control group, the levels of ICAM-1 and VCAM-1 in HUVEC cells in the OGD-stimulated model group were significantly increased, indicating that OGD stimulation can activate adhesion factors in HUVEC cells. After interfering with ALDH1A3, the levels of ICAM-1 and VCAM-1 in the endothelial cells of the model group were significantly decreased. This indicates that inhibiting ALDH1A3 can significantly inhibit OGD-induced activation of adhesion factors.

[0164] Statistical results for (C)VCAM-1 (n=3). Results are expressed as mean ± standard deviation. ** p<0.01 vs. Control group $$ p<0.01 vs. OGD group.

[0165] 5. Evaluation of the efficiency of ALDH1A3 plasmid transfection

[0166] Plasmid exosphere efficiency, such as Figure 13 As shown, the plasmid transfection efficiency was 100% 48 hours after transfection, and the plasmid transfection effect met the requirements.

[0167] like Figure 14 As shown, compared with the control group, the ESM1 content in HUVEC cells in the Model+vector group was significantly increased after OGD stimulation, indicating that OGD stimulation can induce endothelial damage in HUVEC cells. After ALDH1A3 overexpression, the ESM1 content in the vector+Overexpression-ALDH1A3 group was slightly increased, but the difference was not statistically significant. The ESM1 content in endothelial cells in the OGD+Overexpression-ALDH1A3 group was significantly increased, indicating that overexpression of ALDH1A3 can significantly aggravate OGD-induced endothelial cell damage.

[0168] 7. Effects of ALDH1A3 on endothelial adhesion molecules ICAM-1 and VCAM-1 in OGD-induced vascular endothelial injury of HUVECs

[0169] like Figure 15As shown, compared with the blank group, the content of ICAM-1 and VCAM-1 in HUVEC cells in the model group after OGD stimulation was significantly increased. After overexpression of ALDH1A3, the content of ICAM-1 and VCAM-1 in the vector + Overexpression-ALDH1A3 group was slightly increased, but there was no significant difference, and the content of ICAM-1 and VCAM-1 in the endothelial cells in the OGD + Overexpression-ALDH1A3 group was significantly increased. It is indicated that overexpression of ALDH1A3 can promote the activation of adhesion factors.

[0170] Example 3 Screening of potential active ingredients in blood-activating and stasis-removing drugs interacting with ALDH1A3 based on molecular docking

[0171] I. Experimental materials

[0172] 1. Experimental reagents

[0173]

[0174] 2. Experimental instruments

[0175]

[0176] II. Experimental methods

[0177] 1. Selection of active ingredients in blood-activating and stasis-removing drugs

[0178] The main active ingredients in blood-activating and stasis-removing drugs were mainly obtained from Traditional Chinese Medicine Integrated Database (TCMID), Traditional Chinese Medicine System Pharmacology Database and Analysis Platform (TCMSP), and Bioinformatics Analysis Tool for Molecular Mechanism of Traditional Chinese Medicine (BATMAN-TCM).

[0179] 2. Molecular docking and interaction analysis

[0180] Protein-ligand docking simulations were performed using AutoDock Tools 1.5.6 and Pymol for the initial screening. The three-dimensional structure of ALDH1A3 was downloaded from the Protein Data Bank (PDB chain ID: 5FHz, https: / / www.rcsb.org / ) and homologous aggregates and ligands were removed using Pymol software. Potential monomeric compounds from blood-activating and stasis-removing drugs were derived from the integrated Traditional Chinese Medicine Database (TCMID, http: / / www.megabionet.org / tcmid / ) and Traditional Chinese Medicine Pharmacology Database (http: / / tcmspw.com / tcmsp.php). The structures of these compounds were downloaded from SciFinder (http: / / scifinder.cas.org) in the format “MOL2”. The structures were then pre-processed and docked using AutoDock Tools 1.5.6 and the spatial conformation of the compound with the highest docking score was used. The analysis of the interactions between the protein and the ligand was performed in PLIP (https: / / plip-tool.biotec.tu-dresden.de / ) using the online interaction analysis.

[0181] 3. Surface plasmon resonance (SPR)

[0182] The Biacore T200 system was used to analyze the affinity between ALDH1A3 and CTS. The soluble protein was diluted as immobilized ligand in 10 mM sodium acetate buffer at pH 5.5. ALDH1A3 was immobilized on the Fc2 sample channel of the sensor chip (CM5) using the amino coupling kit. The final ALDH1A3 immobilization level was approximately 11000 RU. A similar procedure was performed on the reference Fc1 channel, but without the injection of the ligand. Subsequently, CTS was used as the analyte at different concentrations, and 1.05 x PBS-P containing 5% DMSO was used as the running buffer. For the binding study, the analyte was applied at the corresponding concentration in the flow buffer at a flow rate of 30 μL / min, with a contact time of 60 s and a dissociation time of 120 s. The chip platform was cleaned with flow buffer and 50% dimethyl sulfoxide. The data were analyzed by curve fitting using a 1:1 binding model using the Biacore T200 evaluation software.

[0183] III. Experimental results

[0184] 1. Molecular docking and interaction analysis

[0185] The molecular docking results show that the top three compounds most stable in binding with ALDH1A3 are Cryptotanshinone (-9.3 kcal / mol), Tanshinone IIA (-9.2 kcal / mol), and Salvianolic acid A (-9.1 kcal / mol). The interaction analysis results show that the binding mode of small molecule compounds with ALDH1A3 is mainly hydrogen bonding and hydrophobic interaction. The specific binding sites of ALDH1A3 with the top three small molecule compounds are shown in Figure 16

[0186] 2. Interaction analysis of ALDH1A3 with Cryptotanshinone

[0187] To further understand the formation of signal receptor complexes, we used surface plasmon resonance (SPR) to study the binding affinity of CTS with ALDH1A3 receptor. As shown in Figure 17 The binding of ALDH1A3 with CTS showed a fast association-dissociation process with a binding constant of 5.536 μM, indicating strong binding affinity.

[0188] Example 4. Study on the effect of Cryptotanshinone inhibiting ALDH1A3 to improve CAL-induced chronic heart failure vascular endothelial injury

[0189] I. Experimental materials

[0190] 1. Experimental animals

[0191] See Example 1.

[0192] 2. Experimental reagents

[0193]

[0194] The remaining reagents are described in Example 1, Example 2, and Example 3.

[0195] 3. Experimental equipment

[0196] See Example 1 and Example 2.

[0197] II. Experimental methods

[0198] 1. Animal grouping and drug administration

[0199] 70 male ICR mice were randomly divided into 7 groups:

[0200] Sham group: 2 weeks after the operation, the mice were given i.g. equal volume of physiological saline containing 0.1% carboxymethylcellulose sodium once / day, for three consecutive weeks

[0201] ​Model group (CAL): 2 weeks after operation, intragastric administration (i.g.) of physiological saline containing 0.1% carboxymethylcellulose sodium once a day for three weeks.

[0202] Low-dose cryptotanshinone group (CAL+CTS 10 mg / kg): 2 weeks after operation, intragastric administration (i.g.) of CTS once a day for three weeks.

[0203] Medium-dose cryptotanshinone group (CAL+CTS 20 mg / kg): 2 weeks after operation, intragastric administration (i.g.) of CTS once a day for three weeks.

[0204] High-dose cryptotanshinone group (CAL+CTS 40 mg / kg): 2 weeks after operation, intragastric administration (i.g.) of CTS once a day for three weeks.

[0205] Metoprolol group (CAL+Met 5.14 mg / kg): 2 weeks after operation, intragastric administration (i.g.) of Met once a day for three weeks.

[0206] 2. Preparation of chronic heart failure mouse model

[0207] See Example 1.

[0208] 3. Detection of serum biochemical indicators

[0209] See Example 1.

[0210] 4. Echocardiography detection

[0211] See Example 1.

[0212] 5. TTC staining

[0213] See Example 1.

[0214] 6. Pathological histological examination

[0215] See Example 1.

[0216] 7. Immunohistochemical staining

[0217] See Example 1.

[0218] 8. Western blot detection of protein expression

[0219] See Example 1.

[0220] 9. Statistical analysis of experimental results

[0221] See Example 1.

[0222] III. Experimental results

[0223] 1. Effect of CTS on ALDH1A3 protein content in chronic heart failure mice

[0224] The immunohistochemical results are shown in Figure 18 The content of ALDH1A3 in the blank group mice was inhibited after administration of cryptotanshinone. The content of ALDH1A3 in the CAL group mice was significantly higher than that in the sham group. After treatment with CTS (10 mg / kg, 20 mg / kg and 40 mg / kg) and Met (5.14 mg / kg), the content of ALDH1A3 in the CAL treatment group mice was significantly decreased, indicating that CTS can be used as an inhibitor of ALDH1A3 and can be used for further experiments.

[0225] 2. Effect of CTS on heart function in chronic heart failure mice

[0226] As shown in Figure 19 , coronary ligation caused a significant decrease in left ventricular ejection fraction, left ventricular fractional shortening and stroke volume in mice. However, after treatment with CTS at different doses (10 mg / kg, 20 mg / kg and 40 mg / kg) and Met (5.14 mg / kg), the left ventricular ejection fraction, left ventricular fractional shortening and stroke volume index of chronic heart failure mice were improved, while CTS had no significant effect on the heart function of sham-operated mice.

[0227] 3. Effect of CTS on serum biochemical indicators in chronic heart failure mice

[0228] The serum obtained from each group of mice was determined by ELISA kit. The results are shown in Figure 20 The BNP, hs-CRP and TNF-α in the serum of the model group mice were significantly higher than those in the sham group, indicating that CAL operation caused significant chronic heart failure. However, after administration, the heart failure indicators of the CTS (10 mg / kg, 20 mg / kg and 40 mg / kg) and Met (5.14 mg / kg) groups were significantly decreased compared with the model group, and the decrease in the high-dose (40 mg / kg) CTS group was the most obvious.

[0229] 4. Effect of CTS on myocardial infarct size in chronic heart failure mice

[0230] The TTC staining results are shown in Figure 21 The myocardial tissue of the sham-operated mice showed normal red color, while the myocardial tissue of the model mice showed a large area of white infarct area after coronary artery ligation for 5 weeks, with an infarct area of up to 33%. Treatment with CTS (10 mg / kg, 20 mg / kg and 40 mg / kg) and Met (5.14 mg / kg) significantly reduced the myocardial infarct size in chronic heart failure mice, and the high-dose (40 mg / kg) CTS showed the best improvement.

[0231] 5. Effect of CTS on the histopathology of the heart tissue of chronic heart failure mice

[0232] The HE staining results are shown in Figure 22 The myocardial structure of the mice in the Sham group was complete, the myocardial cells were arranged in an orderly manner, the outline was clear, the cytoplasm was rich and uniform, the interstitium was normal, and no cell necrosis was observed. The myocardial cells of the mice in the CAL group were arranged in a disordered manner, some of the myocardial cells were vacuolated, nuclear loss occurred, and inflammatory cell infiltration and interstitial congestion were observed. After the administration of CTS (10 mg / kg, 20 mg / kg and 40 mg / kg) and Met (5.14 mg / kg), the cells were arranged more orderly than in the model group, the area of inflammatory cell infiltration was reduced, and the degree of myocardial cell edema and necrosis was significantly reduced.

[0233] 6. Effect of CTS on myocardial fibrosis of the heart of chronic heart failure mice

[0234] The Masson staining results are shown in Figure 23 Compared with the mice in the Sham group, the mice in the model group showed obvious fibrosis, obvious collagen deposition, and a significant increase in the percentage of collagen protein. However, the mice in the model group showed significant improvement after CTS treatment, and the percentage of myocardial collagen protein decreased with increasing CTS dose, and the highest dose of CTS (40 mg / kg) had the best therapeutic effect. Metoprolol can also significantly inhibit myocardial fibrosis and reduce collagen deposition.

[0235] 7. Effect of CTS on ESM1 in the serum and heart of chronic heart failure mice

[0236] The effects of CTS on ESM1 in the serum and heart of chronic heart failure mice were investigated by ELISA and immunohistochemical methods. The experimental results are shown in Figure 24 Compared with the mice in the Sham group, the mice in the CAL group showed a significant increase in ESM1 expression in the serum and heart. Compared with the mice in the CAL group, the mice treated with CTS (10 mg / kg, 20 mg / kg and 40 mg / kg) and Met (5.14 mg / kg) showed a significant decrease in ESM1 expression in the serum and heart.

[0237] 8. Effect of CTS on ICAM-1 and VCAM-1 in the serum and heart of chronic heart failure mice

[0238] The effects of CTS on ICAM-1 and VCAM-1 in the serum and heart of chronic heart failure mice were investigated by ELISA and immunohistochemical methods. The experimental results are shown in Figure 25The results showed that the protein levels of ICAM-1 and VCAM-1 in the CAL group were significantly higher than those in the Sham group. Compared with the CAL group, the protein levels of ICAM-1 and VCAM-1 were significantly decreased in the CTS (10 mg / kg, 20 mg / kg and 40 mg / kg) and Met (5.14 mg / kg) treatment groups. The above results showed that CTS could significantly inhibit the activation of adhesion factors induced by CAL.

[0239] References

[0240] 1. Dridi H, Kushnir A, Zalk R, et al. Intracellular calcium leak in heart failure and atrial fibrillation: a unifying mechanism and therapeutic target [J]. Nat Rev Cardiol. 2020, 17, 732-747.

[0241] 2. Witman MA, Garten RS, Gifford JR, et al. Further Peripheral Vascular Dysfunction in Heart Failure Patients With a Continuous-Flow Left Ventricular Assist Device: The Role of Pulsatility [J]. JACC Heart Fail. 2015, 3, 703-11.

[0242] 3. Primary Care Reasonable Drug Use Guide. Chinese Medical Association, Chinese Medical Association Clinical Pharmacy Branch, Chinese Medical Association Journal Society, Chinese Medical Association General Medicine Branch, Chinese General Practitioner Journal Editorial Committee, Primary Health Care Reasonable Drug Use Guide Writing Expert Group. Chinese General Practitioner Journal, 2021, 20(1): 42-49. DOI: 10.3760 / cma.j.cn114798-20201118-01166.

[0243] 4. Zang YX, Fan YZ, Li KQ, et al. Progress in Drug Treatment of Heart Failure [J]. Chinese Journal of Cardiovascular Disease (Network Edition), 2021, 04(1): 1-6. DOI: 10.3760 / cma.j.cn116031.2021.1000086.

[0244] 5.Cai J,Li F,Duan J J,Yu S C.Human aldehyde dehydrogenase family 1 member A3 regulates the invasion of pancreatic cancer cells[J].Journal of the Third Military Medical University,2019,41(17):1642-1648.

Claims

1. Use of an acetaldehyde dehydrogenase 1 family, member A3 inhibitor for the preparation of a medicament for the treatment of chronic heart failure, characterized in that, The acetaldehyde dehydrogenase 1 family member A3 inhibitor is siRNA, and the nucleotide sequence thereof is: Sense strand: GCAACCAAUACUGAAGUUCAAdTdT Antisense strand: UUGAACUUCAGUAUUGGUUGCdTdT.

Images