Application of serine protease inhibitor A3 in preparation of medicine for preventing and / or treating heart failure disease

Abstract

The invention discloses application of a serine protease inhibitor A3 in preparation of a medicine for preventing and / or treating heart failure diseases. Experimental results show that the SERPINA3 can improve cardiac function reduction, improve ventricular remodeling, reduce myocardial fibrosis, inhibit proliferation and migration ability of cardiac fibroblasts, inhibit differentiation of the cardiac fibroblasts to myofibroblasts and inhibit extracellular matrix proteins. The serine protease inhibitor A3 can inhibit cardiac fibroblast activation and myocardial fibrosis, and provides a new treatment strategy and means for clinical treatment of intractable heart failure.

Description

Technical Field

[0001] The present invention belongs to the field of medicine, and particularly relates to the use of serine protease inhibitor A3 in the preparation of medicines for preventing and / or treating heart failure. Background Art

[0002] Serine protease inhibitor A3 (SERPINA3), also known as alpha-1-antichymotrypsin (AACT), is a member of the serine protease inhibitor family (de Mezer M, Rogalinski J, Przewozny S, Chojnicki M, Niepolski L, Sobieska Mand Przystanska A. SERPINA3: Stimulator or Inhibitor of Pathological Changes. Biomedicines. 2023; 11; Baker C. SERPINA3 (aka alpha-1-antichymotrypsin). Frontiers in Bioscience A Journal & Virtual Library. 2007; 12). It is encoded by the SERPINA3 gene, located in the 14q32.13 region of chromosome 14, close to other serine protease inhibitor genes. SERPINA3 is 11.5 kbp in length and has five exons encoding three different mRNAs with the same 1271 nt coding sequence (NM_001085.5). The protein is translated into SERPINA3 protein, which consists of 423 amino acids with a molecular weight of 46 kDa and contains a DNA binding domain and a reactive center loop (RCL). The RCL extends outward from the main protein structure and directly binds to the target protease (e.g. Figure 1As shown in Figure 2 ). Due to subsequent post-translational modification at six glycosylation sites, its molecular weight increases by approximately 25% to 55–66 kDa. In addition, SERPINA3 mRNA may be alternatively spliced ​​(Fatima S, Gupta S, Khan AB, Rehman SU and Jairajpuri MA. Identification and validation of two alternatively spliced ​​novel isoforms of human alpha-1-antichymotrypsin. Biochem Biophys Res Commun. 2022; 628: 25-31). SERPINA3 is primarily produced by the liver and secreted into peripheral blood by hepatocytes, with a final concentration of 0.35-0.45 g / L in healthy individuals (Herman WA, Senko A, Korczowska I and Lacka K. Assessment of selected serum inflammatory markers of acute phase response and their correlations with adrenal androgens and metabolic syndrome in a population of men over the age of 40. Pol Arch Med Wewn. 2009; 119: 704-11). It was originally discovered as an acute phase plasma protease inhibitor that plays an important role in anti-inflammatory and antiviral responses (Sobieska M, Steiner I, Pucher B, Grzegorowski M and Samborski W. Glycosylation profile of selected acute phase proteins in children with chronic tonsillitis and allergic symptoms. Ann Acad Med Stetin. 2006; 52: 39-43).The main function of SERPINA3 is to inhibit serine proteases by forming a stable complex with chymotrypsins, cathepsin G, and mast cell chymase, inhibiting the hydrolytic activity of proteins and thus preventing changes in the extracellular matrix (ECM) (Rubin H, Wang ZM, Nickbarg EB, McLarney S, Naidoo N, Schoenberger OL, Johnson JL and Cooperman BS. Cloning, expression, purification, and biological activity of recombinant native and variant human alpha 1-antichymotrypsins. J Biol Chem. 1990; 265: 1199-207; Duranton J, Boudier C, Belorgey D, Mellet P and Bieth JG. DNA strongly impairs the inhibition of cathepsin G by alpha(1)-antichymotrypsin and alpha(1)-proteinase inhibitor. J Biol Chem. 1990; 265: 1199-207; Duranton J, Boudier C, Belorgey D, Mellet P and Bieth JG. DNA strongly impairs the inhibition of cathepsin G by alpha(1)-antichymotrypsin and alpha(1)-proteinase inhibitor. J Biol Chem. 2001; 265: 1199-207; Duranton J, Boudier C, Belorgey D, Mellet P and Bieth JG. DNA strongly impairs the inhibition of cathepsin G by alpha(1)-antichymotrypsin and alpha(1)-proteinase inhibitor. Chem. 2000; 275: 3787-92).

[0003] Heart failure triggers neuroendocrine activation, primarily manifested by overactivation of the sympathetic and renin-angiotensin-aldosterone systems (RAAS). Initially, activation of these two systems can compensatorily increase cardiac output, promote vasoconstriction, maintain blood pressure, and ensure perfusion of vital organs. Long-term, sustained activation increases myocardial energy consumption, accelerates myocardial death, and promotes cardiac and vascular remodeling. Therefore, the treatment of heart failure focuses on blocking activation of the neuroendocrine system to prevent and delay ventricular remodeling. Conventional drug therapies include angiotensin receptor / neprilysin inhibitors (ANRi), angiotensin-converting enzyme inhibitors (ACEIs), angiotensin II receptor blockers (ARBs), beta-blockers, aldosterone receptor antagonists, and sodium-glucose-coupled transporter-2 inhibitors (SGLT2i). ACEIs, ARBs, and ANRis relax blood vessels to lower blood pressure, improve blood flow, and reduce cardiac stress, but they also have a significant impact on blood pressure. For patients with advanced heart failure, myocardial contractility is significantly reduced, and baseline blood pressure is often low, significantly limiting their use. Furthermore, ACEIs, ARBs, and ANRis should not be used in patients with pregnancy, angioedema, bilateral renal artery stenosis, hyperkalemia, left ventricular outflow tract obstruction, or renal insufficiency. Beta-blockers can alleviate heart failure symptoms by slowing heart rate and lowering blood pressure, inhibiting sympathetic nerve activation, reducing cardiac workload, and improving prognosis. However, their significant negative inotropic effects can further exacerbate symptoms in heart failure patients with concurrent water and sodium retention. They should not be used in patients with bronchial asthma, second-degree or higher atrioventricular block, or severe bradycardia. Aldosterone antagonists can counteract the potassium loss caused by high-dose loop diuretics, but they may also cause hyperkalemia, especially when used concomitantly with ACE inhibitors, ARBs, and angiotensin receptor activators (ANRIs). Therefore, urine output and serum electrolyte levels should be closely monitored. SGLT2i drugs are used to treat diabetes to block glucose reabsorption, thereby excreting excess glucose in the urine and lowering plasma glucose levels. They can also improve the prognosis of patients with heart failure, possibly through diuresis and antihypertensive effects. However, SGLT2i drugs can cause weight loss and lower blood pressure, making them unsuitable for patients with severe weight loss or cachexia in the middle and late stages of heart failure. They are also inappropriate for patients with type 1 diabetes, hypotension, low glomerular filtration rate, or rapidly deteriorating renal function. Therefore, current heart failure treatments still primarily focus on improving symptoms, lacking effective means to improve ventricular remodeling and control myocardial hypertrophy and fibrosis during heart failure progression.

[0004] Current treatments for heart failure still primarily focus on symptom improvement. However, as heart failure progresses, pathological remodeling of the ventricles and the development of irreversible myocardial fibrosis are the primary pathophysiological basis of heart failure. Myocardial fibrosis is a common pathological hallmark of end-stage heart failure and its progression is irreversible. However, there are currently no effective drugs to inhibit the development and progression of myocardial fibrosis. Summary of the Invention

[0005] The purpose of the present invention is to provide a new medical use of serine protease inhibitor A3 (SERPINA3).

[0006] The novel medical use of the serine protease inhibitor A3 provided by the present invention is the use of SERPINA3 in the preparation of a drug for preventing and / or treating heart failure.

[0007] Specifically, the application is: use of SERPINA3 in the preparation of drugs for preventing and / or treating ventricular pathological remodeling and myocardial fibrosis.

[0008] More specifically, the application is: use of SERPINA3 in the preparation of a drug having at least one of the following functions:

[0009] 1) Improve decreased cardiac function;

[0010] 2) Improve ventricular remodeling;

[0011] 3) Reduce myocardial fibrosis;

[0012] 4) Inhibit the proliferation and migration of cardiac fibroblasts;

[0013] 5) Inhibit the differentiation of cardiac fibroblasts into myofibroblasts and extracellular matrix proteins;

[0014] 6) Inhibit cardiac fibroblast activation.

[0015] The decreased cardiac function and ventricular remodeling may specifically be decreased cardiac function and ventricular remodeling induced by pressure overload.

[0016] The experimental results of the present invention show that serine protease inhibitor A3 can inhibit the activation of cardiac fibroblasts and the occurrence of myocardial fibrosis, providing a new treatment strategy and means for the clinical treatment of refractory heart failure. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 This is the SERPINA3 protein structure.

[0018] Figure 2(A) is a representative echocardiographic image of the four groups of mice in Example 1 of the present invention; (B) M-ultrasound shows the left ventricular short axis shortening rate, ejection fraction, left ventricular posterior wall end-diastolic thickness, end-diastolic ventricular septal thickness, left ventricular end-diastolic diameter, left ventricular end-systolic diameter, and left ventricular mass of the four groups of mice after pressure load surgery; (C) is the ratio of heart weight to tibia length of the four groups of mice.

[0019] Figure 3 (A) is a paraffin section of mouse heart tissue stained with Masson's trichrome and picrosirius red in Example 2 of the present invention, with a scale bar of 2000 μm at 1.25X magnification and 50 μm at 40X magnification; (B) is the statistical results of fibrosis in four groups of mice.

[0020] Figure 4 (A) shows the situation in Example 3 of the present invention when the density of cardiac fibroblasts reached 80%. After scratching the bottom of the culture plate, the cells were pretreated with physiological saline and SERPINA3, and then treated with physiological saline or TGFβ for 24 hours. The migration of cells was recorded at 0 and 24 hours under an inverted microscope. (B) The scratch area was calculated using ImageJ software to calculate the scratch healing rate. Scale bar = 100 μm. (C) After pretreatment with physiological saline and SERPINA3, cardiac fibroblasts were treated with physiological saline or TGFβ for 24 hours. The cells were digested and counted and seeded in Transwell chambers. After chemotactic stimulation with 10% FBS in the lower layer for 4 hours, DAPI staining was performed, and 5 fields of view were photographed under an inverted fluorescence microscope, and the average value was calculated. (D) After pretreatment with physiological saline and SERPINA3 in a 96-well plate, the cardiac fibroblasts were treated with physiological saline or TGFβ for 24 hours. After that, 1000 μL of complete culture medium was replaced and 10 ul of culture medium was added. After 2 hours, the absorbance of each well was measured at 450 nm using a microplate reader.

[0021] Figure 5(A) shows the digestion and counting of cells in Example 4 of the present invention, the preparation of cell-containing gels, the observation of the relative contraction area of ​​the gels, and the detection of the contractility of the cells; (B) Statistical results of the relative contraction area of ​​the gels, n = 6; (C) Immunofluorescence staining of cardiac tissue, in which green fluorescence indicates α-SMA, red fluorescence indicates Phalloidin, and blue fluorescence indicates DAPI in the cell nuclei. Fluorescence images were taken with a confocal microscope using a 100x oil immersion lens, with a scale bar of 20 μm. (D) Fluorescence intensity was quantified using ImageJ software, n = 6. (E) 8-10-week-old C57BL / 6 mice underwent sham surgery or pressure overload (TAC) surgery, followed by intraperitoneal injection of saline or SERPINA3 recombinant protein (100 μg / kg) for 9 weeks. Left ventricular tissue was then harvested for protein immunoblotting to detect the protein expression of p-SMAD2 / 3, SMAD2 / 3, Col1a1, and CTGF, with GAPDH as an internal control. (F) Protein levels were quantitatively analyzed using ImageJ, n = 6. (G) Left ventricular tissue was harvested for RT-qPCR to detect the mRNA expression of Col1a1, Postn, CTGF, and α-SMA, n = 5. DETAILED DESCRIPTION

[0022] The present invention will be further described in detail below in conjunction with specific embodiments. The examples provided are only for illustrating the present invention and are not intended to limit the scope of the present invention. The examples provided below can serve as a guide for further improvements by those skilled in the art and are not intended to limit the present invention in any way.

[0023] Unless otherwise specified, the experimental methods in the following examples are conventional methods and were performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials and reagents used in the following examples, unless otherwise specified, were all commercially available.

[0024] Example 1: SERPINA3 improves pressure overload-induced cardiac function impairment in mice

[0025] 1. Establishment of mouse pressure load model

[0026] Male C57BL / 6 mice aged 8-10 weeks were selected and anesthetized by intraperitoneal injection according to body weight. The mice were immobilized, and the chest skin was depilated and disinfected with iodine. A homemade endotracheal guidewire was first inserted into the trachea, and then a cannula was gently and smoothly introduced into the trachea. After the guidewire was removed, the animal was connected to a small animal ventilator. A local thoracotomy was performed at the second intercostal space on the left side of the chest. The skin was incised parallel to the costal margin and dissected layer by layer. Under a stereoscopic microscope, simple tissue dissection was performed to expose the left common carotid artery, innominate artery (right brachiocephalic artery), and aortic arch. The aortic arch was located between the left common carotid artery and the innominate artery. Suture thread on a homemade threading hook was used to ligate the aortic arch to a 27G needle. A double knot was tied and the 27G needle was quickly withdrawn, taking care not to scratch the aortic wall. Excess thread was trimmed, the thymus was repositioned, the chest cavity was closed layer by layer, and the wound was sutured. Mice in the sham-operated group did not undergo aortic arch constriction. The remaining procedures were the same as those in the pressure-loaded (TAC) group. After surgery, the endotracheal tube was removed and the mouse was placed in a prone position in a warm cage and observed until it woke up.

[0027] 2. Cardiac ultrasound data measurement

[0028] First, use a mixture of oxygen and isoflurane (1%-2%) to induce the experimental mice into an anesthesia state. After they are completely anesthetized and the four mice are limp and without autonomous movement, use a hair removal machine to carefully clean the hair on the mouse's chest area. The cleaning range is about 2.5cm×3.5cm. After cleaning, use a hair removal cream to evenly rotate and apply it on the shaved area. After 3-5 minutes, use a wet wipe to wipe off the hair removal cream and residual hair to fully expose the skin of the chest area. Place the mouse in a supine position on the small animal ultrasound operating table, and use tape to stick the four limbs to the ECG recording electrodes at the four corners of the board. Place the mouse's mouth and nose in a small animal continuous gas anesthesia mask to maintain the mouse in an anesthesia state and observe its ECG condition. An appropriate amount of ultrasonic coupling gel was applied to the ultrasound probe and rat skin, and ultrasonic parameters of each cardiac cycle were measured. The left atrial diameter, systolic ventricular septal thickness, and diastolic ventricular septal thickness were measured from the parasternal left ventricular long-axis view. The left ventricular end-systolic diameter, left ventricular end-diastolic diameter, systolic left ventricular anterior wall thickness, diastolic left ventricular anterior wall thickness, systolic left ventricular posterior wall thickness, and diastolic left ventricular posterior wall thickness were measured from the parasternal left ventricular short-axis view. Vevo Lab 1.6.0 was used for cardiac ultrasound image analysis in rats.

[0029] 3. Experimental results

[0030] 8-10 week old C57BL / 6 mice were subjected to sham surgery or pressure loading (TAC) surgery. Three days after surgery, normal saline or SERPINA3 recombinant protein (100ug / kg) was injected intraperitoneally every three days. After nine weeks of treatment, cardiac ultrasound was performed to evaluate the cardiac function of the mice. Figure 2(A) Representative echocardiographic images of the four groups of mice. (B) M-ultrasound images of the four groups of mice after pressure loading surgery showed left ventricular fractional shortening, ejection fraction, left ventricular posterior wall end-diastolic thickness, end-diastolic interventricular septal thickness, left ventricular end-diastolic diameter, left ventricular end-systolic diameter, and left ventricular mass. (C) Heart weight / tibia length ratio of the four groups of mice. Experimental results are expressed as mean ± standard error. Intergroup comparisons were performed using two-way analysis of variance after Tukey's correction. n = 6. * represents P < 0.05; ** represents P < 0.01; *** represents P < 0.001. # Represents P < 0.0001.

[0031] Depend on Figure 2 The data show that SERPINA3 injection can inhibit the decrease in left ventricular short-axis shortening rate and ejection fraction of mice caused by pressure load surgery, the increase in left ventricular posterior wall end-diastolic thickness, end-diastolic ventricular septum thickness, left ventricular end-diastolic internal diameter, left ventricular end-systolic internal diameter, and the increase in left ventricular mass, heart weight and tibia length ratio, and improve pressure load-induced decreased cardiac function in mice.

[0032] Example 2: SERPINA3 improves pressure-overload-induced ventricular remodeling and reduces myocardial fibrosis in mice

[0033] With reference to the establishment of the mouse pressure load model in Example 1, 8-10 week old C57BL / 6 mice were subjected to sham surgery or pressure load surgery. Three days after the surgery, normal saline or SERPINA3 recombinant protein (100ug / kg) was intraperitoneally injected once every three days for 9 weeks. The mouse heart tissue paraffin sections were sliced ​​with Masson's trichrome ((1) Dewaxing and hydrating paraffin sections: paraffin sections of mouse heart tissue after sham surgery or pressure load surgery were taken out, dewaxed and hydrated, and placed in xylene I, xylene II, different concentrations of alcohol (100%, 90%, 80%, 70%), and deionized water in sequence. (2) Hematoxylin staining of cell nuclei: slides were taken out in sequence, 1-2 drops of hematoxylin staining solution were dripped on the tissue to evenly cover the entire tissue, stained for 5 minutes, and washed with deionized water. Dye solution. Add 1% hydrochloric acid alcohol to differentiate the tissue for a few seconds, rinse with deionized water for a few minutes to return to blue. (3) Ponceau fuchsin staining: shake off excess water on the glass slide, add 2-5 drops of Ponceau fuchsin stain solution to evenly cover the entire atrial tissue, stain for 5-10 minutes, and after satisfactory staining, wash off excess stain with deionized water. (4) Phosphomolybdic acid treatment: shake off the residual liquid, add 1-2 drops of phosphomolybdic acid solution to evenly cover the atrial tissue, and treat with phosphomolybdic acid for 5-10 minutes. (5) Aniline blue staining: Pour the phosphomolybdic acid solution, do not need to wash with water, add aniline blue solution directly on the atrial tissue, re-stain for 5-10 minutes, and use deionized water to wash off excess stain after staining. (6) Differentiation: Take out the glass slides one by one, add 1 drop of 1% glacial acetic acid and treat for 1 minute. (7) Dehydration and sealing: Put the slices into 95% alcohol I in turn. 5min; 95% alcohol II 5min; anhydrous ethanol I 5min; anhydrous ethanol II 5min; xylene I 5min; xylene II 5min dehydrated and transparent, take out the slices and dry them slightly, add a drop of neutral gum, and gently cover the coverslip to seal the slide. (8) After the slides are dried, they can be used for microscopic observation, image collection and analysis) and picrosirius red staining ((1) Take out the paraffin sections of mouse heart tissue after TAC sham operation or pressure load operation, dewax and hydrate them, and put them into xylene I, xylene II, different concentrations of alcohol (100%, 90%, 80%, 70%), and deionized water in sequence). (2) Hematoxylin staining of cell nuclei: Take out the slides in sequence, add 1-2 drops of hematoxylin staining solution on the tissue to evenly cover the entire tissue, stain for 5min, and wash off the staining solution with deionized water. Add 1% hydrochloric acid alcohol to differentiate the tissue for a few seconds, and rinse with deionized water for a few minutes to return to blue. (3) Sirius red staining: Add Sirius red staining solution to the slices and stain for 15-30 minutes. (4) Rinse: Gently rinse the surface of the slices with running water to remove excess stain. (5) Dehydration and transparency: Dehydrate the slices in 75% ethanol for 1 minute, then in 95% ethanol for 1 minute, then in anhydrous ethanol for 1 minute, and then in xylene for 3 times, 2 minutes each time, and finally seal with neutral gum. Figure 3 (A) shows paraffin sections of mouse heart tissue stained with Masson's trichrome and picrosirius red. Scale bar: 2000 μm at 1.25X magnification and 50 μm at 40X magnification. (B) Statistical results of fibrosis in four groups of mice. Experimental results are presented as mean ± standard error. Comparisons between groups were performed using two-way ANOVA after Tukey's correction. n = 6. # Represents P < 0.0001.

[0034] Depend on Figure 3 It can be seen that: Masson staining showed that SERPINA3 injection reversed the increase in fibrosis area in ventricular tissue of mice caused by pressure load surgery; Sirius red staining showed that SERPINA3 injection reduced the collagen content in ventricular tissue of mice increased by pressure load surgery, suggesting that SERPINA3 treatment can improve ventricular remodeling and fibrosis caused by pressure load surgery.

[0035] Example 3: SERPINA3 inhibits the proliferation and migration of cardiac fibroblasts

[0036] 1. Scratch test

[0037] 3-5 adult mice were killed by cervical dislocation. The hearts were immediately removed and placed in a culture dish containing cold PBS. In a sterile environment, the culture dish was placed on ice. The heart was cut open and the blood in the heart was cleaned. A new culture dish and clean PBS were replaced and the heart was washed twice. The heart was transferred to a new culture dish and cut into approximately 1 × 1 × 1 mm pieces using sterile tissue scissors. 3The chopped tissue pieces were transferred to a 50 mL centrifuge tube, 25 mL of digestion solution was added, and the tissue was digested in a 37°C biochemical incubator for 10 minutes. The mixture was allowed to stand for 1 minute, and the supernatant was collected and transferred to a new 50 mL centrifuge tube containing 2 mL of culture medium (containing 10% fetal bovine serum, penicillin, streptomycin, and DMEM). 25 mL of new digestion solution was added to the precipitated tissue pieces, and the digestion of the tissue pieces was repeated 7 to 10 times until the tissue pieces were basically digested. The supernatants collected from multiple times were centrifuged at 300 g for 5 minutes at 4°C. The cells were resuspended and the cell suspension was transferred to a 10 cm culture dish and placed in a 37°C incubator containing 5% CO2. After 2 hours, the viable fibroblasts attached to the wall appeared as small dots under the microscope. The cells were washed twice with preheated PBS, and the culture medium was replaced with new culture medium. Then, the culture dish was placed in a 37°C incubator containing 5% CO2. After 2 to 3 days, the fibroblasts reached a density of approximately 90% and could be passaged for subsequent experiments. After passage 4-5, when the density of cardiac fibroblasts reached more than 80%, a 200 μL sterile pipette tip was used to evenly streak the bottom of the culture plate vertically, washed 3 times with PBS to remove cell debris, and new complete culture medium was added. The cells were pretreated with SERPINA3 (100 ng / μL) for 1 hour (2 μL 100 ng / mL SERPINA3 recombinant protein was added to 2 mL of culture medium at a dilution of 1:1000), and then stimulated with normal saline and TGF-β (10 ng / μL, 2 μL 10 ng / mL TGF-β was added to 2 mL of culture medium at a dilution of 1:1000). The scratch width at 0 h was immediately recorded under an inverted microscope at 10 times magnification. After culturing in a cell culture incubator for 24 h, the scratch width was recorded again, and the scratch length was calculated using ImagePro plus software.

[0038] 2. Transwell experiment

[0039] The cells pretreated with TGFβ and SERPINA3 were digested with trypsin to prepare a cell suspension. The cells were washed 3 times with PBS and resuspended in 500 μL of 0.5% FBS culture medium. 300 μL of culture medium containing 10% FBS was added to the 24-well plate and placed in an 8 μm pore size Transwell chamber. 200 μL of culture medium containing 0.5% FBS was added to hydrate the permeable membrane. After the cell digestion and resuspension were completed, the 200 μL culture medium added in advance was discarded and 200 μL of 2.5×10 4After incubation at 37°C for 4-6 hours, the liquid in the Transwell chamber was discarded, the bottom of the chamber was placed in a well plate containing PBS and washed three times, and then the chamber was placed in a 24-well plate containing 4% paraformaldehyde for fixation for 1 hour. A moist cotton swab was used to wipe the cells in the inner layer of the chamber, and the lower layer of the chamber was placed in DAPI embedding solution. The cells were counted in five fields of view (upper, middle, lower, left, and right) of each well under an inverted fluorescence microscope at 20x, and analyzed with ImagePro plus software.

[0040] 3. CCK8 cell proliferation assay

[0041] Cardiac fibroblasts were plated in a 96-well plate. When the cell number reached about 70%, they were pretreated with SERPINA3 for 1 hour and then treated with TGFβ. After 24 hours, the old culture medium was removed with a pipette, and 100 μL of DMEM complete culture medium and 10 μL of enhanced CCK-8 solution were added to each well. The cells were incubated in a cell culture incubator at 37°C for 2 hours. The 96-well plate was removed and the cell solution turned yellow. The 96-well plate was placed in a microplate reader and the absorbance of each well was measured at 450 nm.

[0042] 4. Experimental results

[0043] Figure 4 (A) When cardiac fibroblasts reached a density of 80%, the bottom of the culture plate was scratched. After pretreatment with saline and SERPINA3, cells were treated with saline or TGFβ for 24 hours. Cell migration was recorded using an inverted microscope at 0 and 24 hours. (B) ImageJ software was used to analyze the scratch area and calculate the wound healing rate. Scale bar = 100 μm. Experimental results are expressed as mean ± standard error. Comparisons between groups were performed using two-way ANOVA with Tukey correction. n = 8. # =P < 0.0001. (C) Cardiac fibroblasts were pretreated with saline or SERPINA3 and then treated with saline or TGFβ for 24 hours. The cells were digested, counted, and seeded into Transwell chambers. 10% FBS was used as a chemotactic stimulus in the lower chamber for 4 hours. DAPI staining was performed, and five fields of view were photographed under an inverted fluorescence microscope. The average value was calculated. The results are expressed as mean ± standard error. Comparisons between groups were performed using two-way ANOVA with Tukey's correction. n = 6. #=P < 0.0001. (D) Cardiac fibroblasts grown in 96-well plates were pretreated with saline or SERPINA3 and then treated with saline or TGFβ for 24 hours. The culture medium was replaced with 1000 μL of complete medium and 10 μL of CCK8 solution was added. Two hours later, the absorbance of each well at 450 nm was measured using a microplate reader. Results are expressed as mean ± standard error. Comparisons between groups were performed using two-way ANOVA with Tukey's correction. n = 6. ** indicates P < 0.01; *** indicates P < 0.001.

[0044] Depend on Figure 4 It can be seen that both the scratch experiment and the Transwell experiment showed that compared with the control group, SERPINA3 treatment inhibited the abnormal migration of cardiac fibroblasts induced by TGFβ; the results of the Cell Counting Kit-8 (CCK8) cell proliferation experiment showed that compared with the control group, SERPINA3 treatment inhibited the abnormal proliferation of cardiac fibroblasts induced by TGFβ.

[0045] Example 4: SERPINA3 inhibits the differentiation of cardiac fibroblasts into myofibroblasts and the expression of extracellular matrix proteins

[0046] Gel shrinkage test

[0047] After treatment with SERPINA3 and TGF-β, the cells were trypsinized and centrifuged, then resuspended in DMEM containing 10% fetal bovine serum and counted to a final cell count of 3x10 in the control and experimental groups. 5 / ml; add 0.66ml of well-mixed cell suspension to a sterile centrifuge tube, add 0.33ml of 3mg / ml collagen solution to the cell suspension, quickly add an appropriate volume of 1M NaOH, and mix the solution. Take 500μL of the mixture to a 24-well plate and solidify at room temperature for 20 minutes. Gently add at least an equal amount (500uL) of cell culture medium to each well, gently separate the gel from the well wall with the tip of a pipette, and gently rotate the well plate to ensure that the gel is in a free suspension state. Place the 24-well plate in a cell culture incubator at 37°C and observe the contraction state of the two groups of cell gels every 12h, with 48h as the observation end point. After taking pictures with a camera, perform image analysis with Image J.

[0048] α-SMA immunofluorescence staining

[0049] (1) Dewaxing and hydrating paraffin sections: The dewaxing and hydrating steps are the same as those for HE staining.

[0050] (2) Antigen retrieval: Immerse the dewaxed atrial tissue slide in antigen retrieval solution and heat it in a microwave oven on high for 5 minutes until boiling. Let it stand for 5 minutes, then continue heating on medium-high for 5 minutes. Remove from the microwave and let it stand for about 30 minutes. After the solution and slides have reached room temperature, place the slides in PBS solution and wash them on a shaker at a constant speed for 3 times, 5 minutes each time.

[0051] (4) Permeabilization: Take out the tissue slides in order, shake off the residual liquid, use a histochemical pen to gently draw circles around the tissue, add 0.5% Triton X-100 just above the tissue, let it stand at room temperature for 30 minutes, and wash with PBS solution three times, each time for 5 minutes.

[0052] (5) Blocking: Shake off the liquid on the tissue slide, add 1% BSA solution, and block at room temperature for 1 hour.

[0053] (6) Primary antibody incubation: Discard the blocking solution, remove the residual liquid components as much as possible, and add the diluted primary antibody (α-SMA, 1:100) dropwise until the entire tissue is completely covered. Incubate at 4°C overnight.

[0054] (7) Binding of secondary antibodies: On the second day, the tissue slides were removed and washed three times with PBS for 5 minutes each time. The liquid components were aspirated and 20-50 μL of diluted fluorescent secondary antibody (1:200) and rhodamine-Phalloidin (1:200) were added dropwise. The slides were incubated at room temperature for 1 hour in the dark and washed three times with PBS for 5 minutes each time.

[0055] (8) DAPI mounting: Add DAPI mounting medium to the slide and let it stand for 30 minutes in the dark. After the slide is dry, use a confocal microscope to observe the image, save and analyze the image.

[0056] Western blotting experiments

[0057] (1) Prepare a 7.5% SDS-polyacrylamide gel (SDS-PAGE) according to the protein molecular weight and sample volume, and insert a 15-well upper gel comb;

[0058] (2) Protein electrophoresis was performed using the Bio-Rad mini-PROTEAN 3Cell system. 20 μg of protein sample and 2 μL of protein marker were added in the order of the pendulum pattern. The system was operated at a constant voltage of 80-120 V until bromophenol blue reached the bottom edge of the separation gel.

[0059] (3) Transfer: Remove the gel after electrophoresis and cut it vertically with a paddle. Adjust the size to a suitable size and place it on the transfer plate. Place the foam, filter paper, NC membrane, gel, filter paper, and foam on the transfer plate in order from top to bottom. Expel any excess bubbles and secure the transfer plate firmly before placing it on the transfer apparatus. Add ice cubes around the transfer apparatus to maintain low temperature. Set the transfer current to 220 mA and the transfer time to 2 h.

[0060] (4) Remove the NC membrane, block it with 5% skim milk powder, incubate it on a horizontal shaker at room temperature for 1 h, and wash off the remaining blocking solution with TBST membrane wash buffer;

[0061] (5) Dilute the primary antibody in a universal antibody diluent at a ratio of 1:1000, place the NC membrane containing the target protein in the corresponding primary antibody, and incubate with shaking at 4°C overnight;

[0062] (6) The next day, remove the NC membrane and place it in TBST washing solution. Place it on a shaker and adjust the speed to medium to wash the membrane for 10 minutes, washing three times in total.

[0063] (7) Dilute the secondary antibody with 5% skim milk powder at a ratio of 1:5000, place the NC membrane in the corresponding secondary antibody, and incubate on a horizontal shaker at room temperature for 1 hour;

[0064] (8) Remove the NC membrane and place it in TBST washing solution. Wash the membrane on a horizontal shaker at medium speed for 10 minutes, for a total of 3 washes.

[0065] (9) Mix chemiluminescent solution A and solution B in a ratio of 1:1. After the luminescent solution is evenly applied to the protein surface of the imprinted membrane, the NC membrane is neatly placed in the fully automatic chemiluminescent imaging system instrument for luminescent imaging.

[0066] (10) Select the bands with different exposure times and use Image J software to analyze the grayscale value and net optical density value of the target bands.

[0067] RT-qPCR

[0068] (1) Total RNA was extracted from left ventricular tissue using the RNA extraction kit from Shanghai Promega. The specific method was referred to the kit instructions.

[0069] (2) Measure the total RNA concentration using a Nanodrop One spectrophotometer. Wash the measuring point with mold-free sterile water and use enzyme-free sterile water as a blank control. Add 1 μL of the sample to be tested, measure the absorbance, and calculate the RNA concentration. An A260 / A280 ratio between 1.8 and 2.0 indicates good purity. Record the relevant data.

[0070] (3) The Beijing Quanshijin Biotechnology TransScript All-in-One First-Strand cDNA Synthesis SuperMix for qPCR reverse transcription kit was used for reverse transcription of total RNA. After the program was completed, the cDNA was placed in a -80℃ refrigerator for later use.

[0071] (4) According to the instructions of the PerfectStart Green qPCR SuperMix (Beijing Quanshijin) kit and the cDNA concentration, a 20 μL reaction system was prepared in an 8-well strip or 96-well plate, including: PerfectStart Green qPCR SuperMix, 10 μL; primer, 1 μL; enzyme-free sterile water and cDNA, a total of 9 μL.

[0072] (5) After thorough mixing, perform RT-qPCR reaction using an ABI 7500 fluorescence quantitative PCR instrument. After the reaction is completed, save the data and analyze the obtained data using the 2–ΔΔCt method.

[0073] We first pretreated cardiac fibroblasts with saline and SERPINA3 (100 ng / μL) for 1 hour (add 2 μL of 100 ng / mL SERPINA3 recombinant protein to 2 mL of culture medium at a dilution of 1:1000), and then treated them with saline or TGFβ (10 ng / μL, add 2 μL of 10 ng / mL TGF-β to 2 mL of culture medium at a dilution of 1:1000) for 24 hours. Figure 5(A) Cells were digested and counted, and cell-containing gels were prepared. The relative contraction area of ​​the gels was observed to test the contractility of the cells. (B) Statistical results of the relative contraction area of ​​the gels, n = 6. (C) Immunofluorescence staining of cardiac tissue. Green fluorescence indicates α-SMA, red fluorescence indicates phalloidin, and blue fluorescence indicates nuclei (DAPI). Fluorescence images were acquired using a confocal microscope with a 100x oil immersion lens. Scale bar, 20 μm. (D) Fluorescence intensity was quantified using ImageJ software. n = 6. (E) 8-10 week-old C57BL / 6 mice underwent sham surgery or pressure overload (TAC) surgery. Three days after surgery, they were intraperitoneally injected with saline or SERPINA3 recombinant protein (100 μg / kg). After 9 weeks of treatment, left ventricular tissue was harvested for Western blotting to measure the protein expression of p-SMAD2 / 3, SMAD2 / 3, Col1a1, and CTGF. GAPDH was used as an internal control. (F) Protein levels were quantitatively analyzed using Image J, n = 6; (G) Left ventricular tissue was collected for RT-qPCR to detect the mRNA expression of Col1a1, Postn, CTGF, and α-SMA, n = 5. Experimental results are expressed as mean ± standard error. Two-way ANOVA after Tukey correction was used for comparison between groups. ** represents P < 0.01; *** represents P < 0.001. # represents P < 0.0001, and ns represents no statistical difference.

[0074] Depend on Figure 5 Cardiac fibroblast gel contraction experiments showed that TGFβ treatment significantly reduced gel area and increased contractile force, a phenomenon inhibited by pretreatment with recombinant SERPINA3 protein. α-SMA immunofluorescence staining experiments showed that SERPINA3 inhibited the TGFβ-induced increase in α-SMA expression in cardiac fibroblasts. Western blotting experiments demonstrated that SERPINA3 inhibited the phosphorylation activation of SMAD2 / 3 and the increased expression of Col1a1 and CTGF proteins in cardiac tissue induced by pressure overload surgery. RT-qPCR experiments demonstrated that SERPINA3 inhibited the increased expression of Col1a1, CTGF, Postn, and α-SMAm RNA in cardiac tissue induced by pressure overload surgery. These experiments suggest that SERPINA3 inhibits the differentiation of cardiac fibroblasts into myofibroblasts and the production of extracellular matrix proteins.

[0075] The present invention has been described in detail above. For those skilled in the art, without departing from the purpose and scope of the present invention, and without the need to carry out unnecessary experimental conditions, the present invention can be implemented in a wide range under equivalent parameters, concentrations and conditions. Although the present invention provides specific embodiments, it should be understood that further improvements can be made to the present invention. In short, according to the principles of the present invention, this application is intended to include any changes, uses or improvements to the present invention, including changes that depart from the disclosed scope in this application and are made using conventional techniques known in the art.

Claims

1. Use of SERPINA3 in the preparation of a medicament for preventing and / or treating heart failure.

2. Use of SERPINA3 in the preparation of drugs for preventing and / or treating pathological ventricular remodeling.

3. Use of SERPINA3 in the preparation of drugs for preventing and / or treating myocardial fibrosis.

4. Application of SERPINA3 in the preparation of drugs for improving reduced cardiac function.

5. Application of SERPINA3 in the preparation of drugs for inhibiting the proliferation and migration ability of cardiac fibroblasts.

6. Application of SERPINA3 in the preparation of drugs for inhibiting the differentiation of cardiac fibroblasts into myofibroblasts and extracellular matrix proteins.

7. Application of SERPINA3 in the preparation of drugs for inhibiting cardiac fibroblast activation.

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