Use of raloxifene or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for the treatment of sepsis cardiomyopathy

Abstract

The application discloses application of raloxifene or a pharmaceutical salt thereof in preparation of a medicine for treating sepsis cardiomyopathy, and simultaneously discloses application of a STAT3 protein Tyr705 site as a sepsis cardiomyopathy treatment target, and belongs to the technical field of biological medicines. The application proves through in-vivo and in-vitro experiments that raloxifene can effectively improve body temperature drop and left ventricular systolic function reduction related to sepsis cardiomyopathy, reduce myocardial inflammatory infiltration, relieve myocardial oxidative stress, mitochondrial dysfunction and energy metabolism disorder, and play a definite myocardial protection role by inhibiting phosphorylation activation of the STAT3 protein Tyr705 site in myocardial tissue and cells. The application provides a novel targeted treatment strategy and drug selection for sepsis cardiomyopathy, and has a good clinical transformation application prospect.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to the use of raloxifene or its pharmaceutical salt in the preparation of a medicament for treating septic cardiomyopathy. Background Technology

[0002] Sepsis is a life-threatening organ dysfunction caused by a dysregulated host response to infection. Septic cardiomyopathy, also known as septic myocardial injury, is one of the serious complications of sepsis, mainly characterized by decreased left ventricular systolic function. About half of sepsis patients have varying degrees of myocardial depression in the early stages of the disease, and in severe cases, the short-term mortality rate can exceed 80%.

[0003] The pathogenesis of septic cardiomyopathy involves multi-layered interactions, with inflammation being a core component. Inflammation induces excessive reactive oxygen species (ROS), causing tissue damage, which is a common mechanism in multi-organ dysfunction in sepsis, and this damage mechanism is particularly prominent in organs with high energy demands. The heart is one of the most energy-consuming organs in the human body. As the main organelle for energy supply, the stability of the structure and function of mitochondria is crucial for maintaining cardiac function. Clinical studies have found that the cardiac tissue of patients with septic cardiomyopathy mainly exhibits disordered mitochondrial arrangement, significant swelling and rupture, and a reduced number of mitochondria, suggesting that cellular dysfunction is the main cause of septic cardiomyopathy, and mitochondria play a vital role in this process.

[0004] Current treatment for septic cardiomyopathy primarily focuses on anti-infection and hemodynamic support, which do not significantly improve prognosis, and specific therapeutic drugs for septic cardiomyopathy remain scarce. Given this situation, finding novel targeted therapies that can improve prognosis is a key need in the treatment of septic cardiomyopathy. However, to date, little is known about the effects of raloxifene or its pharmaceutically acceptable salts on septic cardiomyopathy. Summary of the Invention

[0005] In view of this, the purpose of the present invention is to provide the use of raloxifene or its pharmaceutical salt in the preparation of a medicament for treating septic cardiomyopathy, so as to solve the technical problem that the prior art medicaments for treating septic cardiomyopathy are not effective.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows: In a first aspect, the present invention provides the use of raloxifene or a pharmaceutical salt thereof in the preparation of a medicament for treating septic cardiomyopathy.

[0007] In a second aspect, the present invention provides the use of raloxifene or a pharmaceutical salt thereof in the preparation of a medicament for improving hypothermia associated with septic cardiomyopathy.

[0008] Thirdly, the present invention provides the use of raloxifene or its pharmaceutical salt in the preparation of a medicament for improving decreased left ventricular systolic function associated with septic cardiomyopathy.

[0009] Fourthly, the present invention provides the use of raloxifene or a pharmaceutically acceptable salt thereof in the preparation of a medicament for improving myocardial inflammatory infiltration associated with septic cardiomyopathy.

[0010] Fifthly, the present invention provides the use of raloxifene or its pharmaceutical salt in the preparation of a medicament for improving oxidative stress in myocardial tissue and myocardial cells associated with septic cardiomyopathy.

[0011] In a sixth aspect, the present invention provides the use of raloxifene or its pharmaceutical salt in the preparation of a medicament for improving myocardial tissue and myocardial cell mitochondrial dysfunction associated with septic cardiomyopathy.

[0012] In a seventh aspect, the present invention provides the use of raloxifene or a pharmaceutically acceptable salt thereof in the preparation of a medicament for improving myocardial energy metabolism disorders associated with septic cardiomyopathy.

[0013] Eighthly, the present invention provides the application of the Tyr705 site of STAT3 protein as a target in the preparation of a drug for treating septic cardiomyopathy. The drug exerts its therapeutic effect on septic cardiomyopathy by inhibiting the phosphorylation activation of STAT3 protein at the Tyr705 site in myocardial tissue and cardiomyocytes, thereby improving oxidative stress, and / or, improving mitochondrial dysfunction, and / or, improving myocardial energy metabolism disorders, and / or, improving myocardial inflammatory infiltration, and / or, improving decreased left ventricular systolic function, and / or, improving decreased body temperature.

[0014] As a further optimization of the present invention, the administration method of the drug includes at least one of oral administration and intravenous injection.

[0015] As a further optimization of the present invention, the dosage form of the drug includes at least one of tablets, powders, injections, granules, syrups, capsules, and solutions.

[0016] As a further optimization of the present invention, the pharmaceutical salt is a salt formed by raloxifene and an inorganic or organic acid.

[0017] This invention exhibits several significant technical effects: 1. This invention is the first to clearly define the novel pharmaceutical value of raloxifene or its pharmaceutical salt in the treatment of septic cardiomyopathy, filling the current industry gap of lacking specific therapeutic drugs for septic cardiomyopathy. Through in vitro and in vivo experiments, the target and multi-pathway cardioprotective effects of raloxifene in myocardial tissue and cells have been verified, providing a new targeted therapy strategy and drug selection for septic cardiomyopathy.

[0018] 2. This invention verifies that raloxifene can directly improve key symptoms of septic cardiomyopathy at the level of overall physiology and core organ function. Specifically: 1) Maintaining physiological homeostasis: It can significantly slow down the progressive decrease in body temperature in septic model mice as the disease progresses, correct the temperature abnormalities associated with septic cardiomyopathy, and maintain the body's normal physiological state. 2) Protecting left ventricular systolic function: It can effectively reverse the damage to left ventricular systolic function caused by sepsis, and significantly restore key left ventricular systolic function indicators: left ventricular ejection fraction (LVEF) and left ventricular shortening fraction (LVFS), directly addressing the pathological feature of septic cardiomyopathy with decreased left ventricular systolic function as its core. 3) Reducing myocardial tissue pathological damage: It can significantly alleviate pathological changes caused by sepsis, such as widening of myocardial interstitial spaces, interstitial edema, and perivascular inflammatory cell "cuff-like" infiltration, directly reducing sepsis-mediated myocardial structural damage at the tissue level.

[0019] 3. This invention systematically elucidates the pharmacological effects of raloxifene on the core pathogenesis of septic cardiomyopathy, blocking the progression of myocardial damage through three core pathways: inflammation, oxidative stress, and energy metabolism. The mechanism of action is clear and comprehensive, specifically: 1) Inhibiting the excessive inflammatory cascade of myocardium: It can significantly downregulate the mRNA expression levels of key pro-inflammatory factors such as IL-6, TNF-α, CCL2, and IL-1β in septic myocardial tissue, inhibiting myocardial inflammatory infiltration at the transcriptional level and blocking the vicious cycle of myocardial damage mediated by the inflammatory response. 2) Improving myocardial oxidative stress damage: It can significantly restore the expression of the key antioxidant protein GPX4 in septic myocardial tissue, enhancing the myocardium's endogenous antioxidant capacity; simultaneously, it significantly reduces the excessive ROS production in cardiomyocytes induced by lipopolysaccharide (LPS), clearing excess oxygen free radicals and alleviating oxidative stress-mediated myocardial and tissue damage. 3) Repairing mitochondrial dysfunction and myocardial energy metabolism disorders: Targeting the high-energy-consuming physiological characteristics of the heart, it repairs mitochondrial damage at both structural and functional levels, addressing the core pathogenesis of septic cardiomyopathy. First, it can significantly maintain the ATP content of septic myocardial tissue, reverse the myocardial energy metabolism disorder caused by sepsis, and ensure the energy supply for normal cardiac physiological function. Second, it can inhibit the phosphorylation activation of the key mitochondrial splitting protein DRP1 at the serine 616 (Ser616) site, reverse the downregulation of mitochondrial dynamics-related molecules MFN1 and OPA1 and the upregulation of FIS1, and restore the physiological homeostasis of mitochondrial fusion-fission. Third, it can restore the mitochondrial membrane potential of cardiomyocytes, inhibit LPS-induced mitochondrial network fragmentation, repair mitochondrial damage at the structural level, and improve cardiac dysfunction caused by mitochondrial dysfunction.

[0020] 4. This invention is the first to confirm that the Tyr705 site of the STAT3 protein is a key target for the treatment of septic cardiomyopathy, clarifying the core mechanism of action of raloxifene: by specifically inhibiting the phosphorylation activation of the STAT3 protein at the Tyr705 site in myocardial tissue and cardiomyocytes, it achieves multiple pharmacological effects including anti-inflammatory, antioxidant, mitochondrial function repair, and cardiac protection. This discovery not only lays a solid theoretical foundation for the development of new indications for raloxifene but also provides a novel target and intervention strategy for the development of targeted drugs for septic cardiomyopathy, possessing significant theoretical research and clinical application value. Attached Figure Description

[0021] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0022] Figure 1 The effect of Raloxifene on body temperature in septic mice at 0, 3, 6 and 12 hours. *** This indicates a significant difference between the two groups; *** P < 0.001.

[0023] Figure 2 The effect of Raloxifene on left ventricular systolic function in septic mice. Figure 2 A shows representative echocardiogram images of mice in each group; Figure 2 B represents the left ventricular systolic function parameters (LVEF and LVFS) of mice in each group. *** This indicates a significant difference between the two groups; *** P<0.001.

[0024] Figure 3 The effect of Raloxifene on myocardial inflammatory infiltration in septic mice. Figure 3 A shows HE-stained images of representative heart tissues from each group of mice (scale bar: 50 μm). Figure 3 B represents the expression levels of inflammatory factor mRNA in the myocardial tissue of each group of mice. * , ** and *** This indicates a significant difference between the two groups; * P<0.05, ** P<0.01, *** P < 0.001.

[0025] Figure 4 The study investigated the effects of Raloxifene on the viability of AC16 cardiomyocytes and LPS-induced activation of STAT3 phosphorylation in myocardial tissue and cells. Figure 4 A represents the Western blot analysis of the expression levels of p-STAT3 (Tyr705) and STAT3 proteins in the myocardial tissues of mice in each group. Figure 4 B is a quantitative graph showing the expression level of p-STAT3 (Tyr705) protein in the myocardial tissue of mice in each group. Figure 4 C represents the viability of AC16 cardiomyocytes treated with different concentrations of Raloxifene. Figure 4 D represents the Western blot analysis of p-STAT3 (Tyr705) and STAT3 protein expression levels in AC16 cardiomyocytes. Figure 4 E is a quantitative graph of the expression level of p-STAT3 (Tyr705) protein in AC16 cardiomyocytes. * , ** and *** This indicates a significant difference between the two groups; * P<0.05, ** P<0.01, *** P<0.001.

[0026] Figure 5 The effects of raloxifene on LPS-induced oxidative stress in myocardial tissue and AC16 cardiomyocytes were investigated. Figure 5 A represents the Western blot analysis of GPX4 protein expression levels in the myocardial tissues of mice in each group. Figure 5 B is a quantitative graph showing the expression level of GPX4 protein in the myocardial tissue of mice in each group. Figure 5 C represents a representative DHE staining image of AC16 cardiomyocytes. Figure 5 D represents the quantitative result of the fluorescence intensity of DHE staining in Image J. * and *** This indicates a significant difference between the two groups; * P<0.05, *** P<0.001.

[0027] Figure 6 The effects of raloxifene on LPS-induced mitochondrial function in myocardial tissue and AC16 cardiomyocytes were investigated. Figure 6 A represents the ATP content in the myocardial tissue of mice in each group. Figure 6 B represents the Western blot analysis of the expression levels of p-DRP1 (Ser616) and DRP1 proteins in the myocardial tissue of mice in each group. Figure 6C represents the mRNA expression levels of MFN1, OPA1, and FIS1, which are mitochondrial dynamics-related proteins, in the myocardial tissue of mice in each group. Figure 6 D is a representative JC-1 stained image of AC16 cardiomyocytes. Figure 6 E represents the quantitative result of Image J, which shows the ratio of fluorescence intensity of JC-1 aggregate to JC-1 monomer. Figure 6 F shows a representative Mito-Tracker staining image of AC16 cardiomyocytes. * , ** and *** This indicates a significant difference between the two groups; * P<0.05, ** P<0.01, *** P<0.001. Detailed Implementation

[0028] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0029] The present invention will be further illustrated by specific embodiments below, but these are not intended to limit the scope of the invention.

[0030] Example 1 Animal experimental drug preparation: (1) LPS (lipopolysaccharide) preparation: 10 mg LPS powder was dissolved in 5 mL of sterile physiological saline to prepare a stock solution with a concentration of 2 mg / mL; (2) Raloxifene preparation: 4.25 mg Raloxifene was dissolved in 220 μL of dimethyl sulfoxide (DMSO) solution until clear, then 880 μL of PEG300 solution was added and mixed until clear, then 110 μL of Tween-80 was added and mixed until clear, then 990 μL of sterile physiological saline was added. Drugs should be prepared and used immediately.

[0031] Establishment of mouse sepsis and treatment model: Six- to eight-week-old male C57BL / 6 mice were acclimatized in an SPF-grade barrier environment and then randomly divided into the following groups: (1) LPS + Raloxifene group: Raloxifene (15 mg / kg) was administered by gavage for 5 consecutive days, followed by intraperitoneal injection of LPS (10 mg / kg) on ​​day 5; (2) LPS group: An equal volume of solvent was administered by gavage for 5 consecutive days, followed by intraperitoneal injection of LPS (10 mg / kg) on ​​day 5; (3) CON group: An equal volume of solvent was administered by gavage for 5 consecutive days, followed by intraperitoneal injection of an equal volume of sterile saline on day 5. Relevant tests were performed 12 hours after intraperitoneal injection.

[0032] Body temperature was measured in mice at different time points (0, 3, 6, and 12 hours) after intraperitoneal injection of LPS. The results showed that LPS-induced body temperature gradually decreased over time, and Raloxifene could, to some extent, slow the temperature drop in septic mice. Figure 1 ).

[0033] Example 2 Hair was removed from the precordial region of mice using depilatory cream. Mice were then placed in an anesthesia jar filled with 2% isoflurane for induction of anesthesia. After induction, the mice were fixed in a supine position on a heated ultrasound examination table, and anesthesia was maintained with continuous inhalation at a ventilation rate of 2 L / min. The mouse heart rate was maintained between 350 and 450 beats / min. Cardiac function in mice was assessed using a VINNO 6 VET portable small animal ultrasound system (VINNO technology, Suzhou, China). Two-dimensional ultrasound images were obtained in the short-axis view of the left ventricle near the sternum at the level of the submitral papillary muscle. Left ventricular ejection fraction (LVEF) and left ventricular shortening fraction (LVFS) were measured after at least three independent cardiac cycles. LPS induced impaired left ventricular systolic function in mice, i.e., decreased LVEF and LVFS. Raloxifene could, to some extent, protect left ventricular systolic function in septic mice. Figure 2 A shows representative echocardiogram images of mice in each group; Figure 2 B represents the left ventricular systolic function parameters (LVEF and LVFS) of mice in each group.

[0034] Example 3 Mouse heart tissue was fixed in 10% formaldehyde at room temperature for 24 hours, then embedded and sectioned using a paraffin microtome. The sections were first baked in a 60°C oven for 1 hour to prevent detachment; then dewaxed by immersion in xylene for 5 minutes each time, three times in total; subsequently, they were immersed in 100%, 95%, and 70% alcohol for 1 minute each, followed by washing for several minutes; then the slides were shaken dry, stained with hematoxylin for 5 minutes, rinsed with double-distilled water to remove excess stain, differentiated with 1% hydrochloric acid ethanol for a few seconds, rinsed with running water to regain blue color, stained with eosin for 3 minutes, rinsed with double-distilled water to remove excess stain, and then immersed in 70%, 95%, and 100% alcohol for 30 seconds each; finally, they were immersed in xylene for clearing, 2 minutes each time, three times in total. After being mounted with resin, the slides were laid flat and allowed to air dry. Observation of the slides under an optical microscope revealed that LPS induced widening, loosening, and translucency of the interstitial spaces in mouse myocardial tissue, and that inflammatory cell infiltration formed a "cuff-like" change around blood vessels. Raloxifene could alleviate the degree of interstitial edema and inflammatory infiltration in the myocardial tissue of septic mice to some extent. Figure 3 Image A shows representative HE staining images of myocardial tissue from each group of mice.

[0035] The expression of IL-6, TNF-α, CCL2, and IL-1β in mouse myocardial tissue was detected at the transcriptional level using real-time quantitative polymerase chain reaction (RT-qPCR). LPS-induced changes significantly increased the mRNA expression levels of the inflammatory factors IL-6, TNF-α, CCL2, and IL-1β in mouse myocardial tissue. Raloxifene reduced the mRNA expression levels of these factors, suggesting that raloxifene can alleviate myocardial inflammatory factor infiltration in septic mice to some extent. Figure 3 B represents the statistical results of RT-qPCR analysis of the mRNA of inflammatory factors in myocardial tissue.

[0036] The mouse primer sequences used are shown in Table 1 below, and were normalized using the internal reference gene Rps18.

[0037] Table 1 RT-qPCR primer sequences Example 4 Mouse myocardial tissue preserved in liquid nitrogen was removed, and after adding an appropriate amount of RIPA lysis buffer containing phosphatase inhibitors and protease inhibitors, it was thoroughly homogenized into a tissue homogenate. The homogenate was then sonicated and lysed on ice for 40 minutes. After centrifugation at 12000g at 4℃ for 20 minutes, the supernatant was collected. The protein concentration in the supernatant was determined using the BCA method. An appropriate amount of ultrapure water was added to each sample to ensure uniform protein concentration, and 1 / 4 of the total volume of 5X protein buffer was added. The protein was heated at 100℃ for 5 minutes and then cooled on ice before use.

[0038] Western blot was used to detect the expression of STAT3 and its phosphorylation activation at the Tyr705 site in mouse myocardial tissue at the protein level. LPS induced increased phosphorylation of STAT3 at the Tyr705 site in myocardial tissue, i.e., increased expression of p-STAT3 (Tyr705), indicating STAT3 activation. Raloxifene reduced p-STAT3 (Tyr705) expression in the myocardial tissue of septic mice, suggesting that Raloxifene can inhibit STAT3 phosphorylation activation in the myocardial tissue of septic mice. Figure 4 A represents the Western blot analysis of the expression levels of p-STAT3 (Tyr705) and STAT3 proteins in the myocardial tissues of mice in each group; Figure 4 B is a quantitative graph showing the expression level of p-STAT3 (Tyr705) protein in the myocardial tissue of mice in each group.

[0039] Preparation of drugs for cell experiments: (1) LPS preparation: Dissolve 10mg LPS powder in 5mL sterile PBS solution to prepare a stock solution with a concentration of 2mg / mL, aliquot and store at -80℃; (2) Raloxifene preparation: Dissolve 1mg Raloxifene in 78.425μL DMSO solution to prepare a stock solution with a concentration of 25mM, aliquot and store at -80℃.

[0040] An appropriate amount of AC16 cardiomyocytes were seeded in 96-well plates. Each well was treated with 100 μL of DMEM medium containing 10% fetal bovine serum and 1% penicillin-streptomycin antibiotics. After 12 h of treatment with different concentrations (0 μM, 10 μM, 12.5 μM, 15 μM, 20 μM) of Raloxifene (i.e., DMSO solution and 0.8 μL, 1.0 μL, 1.2 μL, 1.6 μL 25 mM Raloxifene stock solution), each well was incubated with 10 μL of CCK8 reagent. After 2 h, the absorbance was measured at 450 nm to reflect cell viability. The results suggest that the maximum safe concentration of Raloxifene under these culture conditions is 15 μM. Figure 4 C represents the viability of AC16 cardiomyocytes treated with different concentrations of Raloxifene.

[0041] An appropriate amount of AC16 cardiomyocytes were seeded in six-well plates, and each well was given 2 mL of DMEM medium containing 10% fetal bovine serum and 1% penicillin-streptomycin antibiotics. The cells were grouped as follows: (1) CON group: 1 μL DMSO solution; (2) LPS group: 10 μg / mL LPS solution (i.e., 10 μL 2 mg / mL LPS stock solution); (3) LPS+Raloxifene 10 group: 10 μM Raloxifene + 10 μg / mL LPS solution; (4) LPS+Raloxifene 12.5 group: 12.5 μM Raloxifene + 10 μg / mL LPS solution; (5) LPS+Raloxifene 15 group: 15 μM Raloxifene + 10 μg / mL LPS solution; (6) LPS+Raloxifene 20 group: 20 μM Raloxifene + 10 μg / mL LPS solution. After treating cells with LPS solution for 12 hours, appropriate amounts of RIPA lysis buffer containing phosphatase inhibitors and protease inhibitors were added to each well. Cells were scraped off with a cell scraper, and the cell suspension was collected. After sonication, the cells were lysed on ice for 40 minutes. After centrifugation at 12000g at 4°C for 20 minutes, the supernatant was collected. The protein concentration in the supernatant was determined using the BCA method. Appropriate amounts of ultrapure water were added to each sample to ensure uniform protein concentration, and 1 / 4 of the total volume of 5X protein buffer was added. The protein was heated at 100°C for 5 minutes and then cooled on ice before use.

[0042] Western blot was used to detect the expression of STAT3 and its phosphorylation activation at the Tyr705 site in AC16 cardiomyocytes at the protein level. LPS induced an increase in p-STAT3 (Tyr705) expression in AC16 cardiomyocytes, indicating STAT3 activation. Raloxifene reduced LPS-induced p-STAT3 (Tyr705) expression in cardiomyocytes in a concentration-dependent manner, suggesting that raloxifene can inhibit LPS-induced STAT3 phosphorylation activation in cardiomyocytes. Figure 4 D represents the expression levels of p-STAT3 (Tyr705) and STAT3 proteins in cardiomyocytes as detected by Western blot. Figure 4 E is a quantitative graph of the expression level of p-STAT3 (Tyr705) protein in cardiomyocytes.

[0043] Example 5 Western blot was used to detect the expression of the antioxidant enzyme GPX4 in mouse myocardial tissue at the protein level. LPS induced a decrease in GPX4 expression in mouse myocardial tissue, suggesting a decline in the myocardial antioxidant capacity of septic mice. Raloxifene restored GPX4 expression in the myocardial tissue of septic mice, indicating that raloxifene can restore the myocardial antioxidant capacity and improve oxidative stress in septic mice. Figure 5 A represents the Western blot analysis of GPX4 protein expression levels in myocardial tissue of mice in each group; Figure 5 B is a quantitative graph showing the expression level of GPX4 protein in the myocardial tissue of mice in each group.

[0044] Preparation of Dihydroethidium (DHE) Stock Solution and Working Solution: Weigh 1 mg of superoxide anion fluorescent probe DHE powder, add 0.31 mL of DMSO solution, dissolve thoroughly to prepare a 10 mM stock solution, aliquot, and store at -80℃ protected from light. For staining, dilute the DHE stock solution to a 10 μM DHE working solution using DMEM medium, ensuring complete dissolution of the staining solution.

[0045] An appropriate amount of AC16 cardiomyocytes were seeded in six-well plates, and each well was given 2 mL of DMEM medium containing 10% fetal bovine serum and 1% penicillin-streptomycin antibiotics. The cells were grouped as follows: (1) CON group: 1 μL DMSO solution; (2) LPS group: 10 μg / mL LPS solution; (3) LPS+Raloxifene group: cells were treated with Raloxifene (based on the results of the cell experiments in Example 4, the drug concentration was selected as 12.5 μM) and 10 μg / mL LPS solution for 12 hours. After washing the cells three times with PBS, each well was incubated with 1 mL of DHE working solution at 37°C for 30 minutes. After washing the cells three times again with PBS, fresh medium pre-warmed at 37°C was added, and the cells were photographed under a fluorescence microscope. The red fluorescence in cardiomyocytes of the CON group was weak, while that of LPS-induced cardiomyocytes was significantly enhanced, indicating that LPS-induced ROS production in cardiomyocytes was elevated. Raloxifene could, to some extent, attenuate the intensity of LPS-induced red fluorescence, suggesting that Raloxifene could, to some extent, reduce the high ROS production level in cardiomyocytes induced by LPS and alleviate oxidative stress in cardiomyocytes. Figure 5 C is a representative DHE staining image of AC16 cardiomyocytes; Figure 5 D represents the quantitative result of the fluorescence intensity of DHE staining in Image J.

[0046] Example 6 Mouse myocardial tissue preserved in liquid nitrogen was removed, and an appropriate amount of ATP lysis buffer was added according to the ATP assay kit instructions. The tissue was thoroughly homogenized and lysed, and centrifuged at 12000g at 4℃ for 5 minutes. The supernatant was collected. Protein concentration in one portion of the supernatant was determined using the BCA method, while ATP content in the other portion was determined using chemiluminescence immunoassay according to the manufacturer's instructions. The ATP content per mg of protein was calculated, and the results were normalized to the CON group before comparisons between groups. LPS-induced ATP content in mouse myocardial tissue was significantly reduced, suggesting that LPS induces myocardial energy metabolism disorders. Raloxifene can maintain ATP content in myocardial tissue of septic mice to some extent, suggesting that raloxifene can protect myocardial energy metabolism in septic mice to some extent. Figure 6 A represents the ATP content in the myocardial tissue of mice in each group.

[0047] Western blot was used to detect the expression of DRP1 and its phosphorylation activation at the Ser616 site in mouse myocardial tissue and AC16 cardiomyocytes at the protein level. LPS-induced increased phosphorylation of DRP1 at the Ser616 site in myocardial tissue and AC16 cardiomyocytes, i.e., increased p-DRP1 (Ser616) expression, suggests DRP1 activation and excessive mitochondrial division. Raloxifene reduced p-DRP1 (Ser616) expression in myocardial tissue of septic mice or LPS-induced cardiomyocytes, suggesting that Raloxifene can inhibit DRP1 phosphorylation activation in myocardial tissue of septic mice or LPS-induced cardiomyocytes, thus inhibiting excessive mitochondrial division. Figure 6 B represents the expression levels of p-DRP1 (Ser616) and DRP1 proteins in the myocardial tissue of mice in each group.

[0048] The expression of mitochondrial dynamics-related proteins MFN1, OPA1, and FIS1 in mouse myocardial tissue was detected at the transcriptional level using RT-qPCR. LPS-induced mitochondrial dynamics balance significantly decreased the mRNA expression levels of MFN1 and OPA1 in mouse myocardial tissue, while significantly increasing the mRNA expression level of FIS1, suggesting that mitochondrial dynamics balance is more inclined towards division than fusion. Raloxifene can, to some extent, reverse the mRNA expression levels of MFN1, OPA1, and FIS1 in the myocardial tissue of septic mice, suggesting that Raloxifene can, to some extent, inhibit excessive mitochondrial division in the myocardium of septic mice. Figure 6 C represents the statistical results of RT-qPCR analysis of the mRNA of mitochondrial dynamics-related proteins MFN1, OPA1, and FIS1 in myocardial tissue.

[0049] The mouse primer sequences used are shown in Table 2 below, and were normalized using the internal reference gene Rps18.

[0050] Table 2 RT-qPCR primer sequences Preparation of JC-1 working solution: Take an appropriate amount of 200X JC-1 solution, dilute it by adding 1 mL of JC-1 staining buffer per 5 µL, and repeatedly pipette to mix well to obtain the JC-1 staining working solution.

[0051] Cells were treated for 12 hours according to the grouping method in Example 5. After washing the cells three times with PBS, each well was incubated with 1 mL of JC-1 working solution at 37°C for 20 minutes. After washing the cells three more times with PBS, fresh culture medium pre-warmed at 37°C was added, and red and green fluorescence were captured under a fluorescence microscope. In the CON group, cardiomyocytes showed obvious red fluorescence and weak green fluorescence. LPS-induced cardiomyocytes showed bright green fluorescence and a conversion from red to green fluorescence, indicating that LPS-induced mitochondrial membrane potential decreased. Raloxifene could inhibit the LPS-induced conversion from red to green fluorescence, suggesting that Raloxifene could restore the LPS-induced decrease in mitochondrial membrane potential in cardiomyocytes. Figure 6 D is a representative JC-1 staining image of AC16 cardiomyocytes; Figure 6 E represents the quantitative result of Image J, which shows the ratio of fluorescence intensity of JC-1 aggregate to JC-1 monomer.

[0052] Preparation of Mito-Tracker Red CMXRos stock solution and working solution: Take 50 μL of Mito-Tracker Red CMXRos mitochondrial red fluorescent probe stock solution with a concentration of 1 µg / µL, add 420 μL of DMSO solution and mix thoroughly to prepare a 200 μM stock solution. Aliquot and store at -80℃ protected from light. For staining, dilute the Mito-Tracker Red CMXRos stock solution to a final concentration of 20 nM using DMEM medium, ensuring complete dissolution of the staining solution.

[0053] Cells were treated in confocal culture dishes for 12 hours according to the grouping method in Example 5. After washing the cells three times with PBS, an appropriate amount of Mito-Tracker Red CMXRos working solution was added to each well, and the cells were incubated at 37°C for 15 minutes. After washing the cells three more times with PBS, fresh culture medium pre-warmed at 37°C was added, and images were taken under a confocal microscope. In the CON group, mitochondria in cardiomyocytes were distributed in a network pattern. LPS induced fragmentation of the mitochondrial network in cardiomyocytes, breaking the continuous tubular network into short rod-shaped isolated individuals. Raloxifene could inhibit the formation of mitochondrial fragmentation in cardiomyocytes, suggesting that Raloxifene can effectively inhibit the increase in LPS-induced mitochondrial fragmentation. Figure 6 F shows a representative Mito-Tracker staining image of AC16 cardiomyocytes.

[0054] The above are merely preferred embodiments of the present invention. It should be noted that the above preferred embodiments should not be considered as limitations on the present invention, and the scope of protection of the present invention should be determined by the scope defined in the claims. For those skilled in the art, several improvements and modifications can be made without departing from the spirit and scope of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. The use of raloxifene or its pharmaceutical salt in the preparation of a drug for the treatment of septic cardiomyopathy.

2. Use of raloxifene or its pharmaceutical salt in the preparation of a drug for improving hypothermia associated with septic cardiomyopathy.

3. Use of raloxifene or its pharmaceutical salt in the preparation of drugs for improving decreased left ventricular systolic function associated with septic cardiomyopathy.

4. Use of raloxifene or its pharmaceutical salt in the preparation of a medicament for improving myocardial inflammatory infiltration associated with septic cardiomyopathy.

5. The use of raloxifene or its pharmaceutical salts in the preparation of drugs for improving oxidative stress in myocardial tissue and cardiomyocytes associated with septic cardiomyopathy.

6. The use of raloxifene or its pharmaceutical salts in the preparation of drugs for improving myocardial tissue and mitochondrial dysfunction associated with septic cardiomyopathy.

7. Use of raloxifene or its pharmaceutical salt in the preparation of a medicament for improving myocardial energy metabolism disorders associated with septic cardiomyopathy.

8. The application of the Tyr705 site of STAT3 protein as a target in the preparation of a drug for treating septic cardiomyopathy, wherein the drug exerts its therapeutic effect on septic cardiomyopathy by inhibiting phosphorylation activation of STAT3 protein at the Tyr705 site in myocardial tissue and cardiomyocytes, thereby improving oxidative stress, and / or, improving mitochondrial dysfunction, and / or, improving myocardial energy metabolism disorders, and / or, improving myocardial inflammatory infiltration, and / or, improving decreased left ventricular systolic function, and / or, improving hypothermia.

9. The application according to any one of claims 1 to 8, characterized in that, The drug can be administered via at least one of oral administration or intravenous injection.

10. The application according to any one of claims 1 to 8, characterized in that, The dosage form of the drug includes at least one of tablets, powders, injections, granules, syrups, capsules, and solutions.

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