CN107335051A - A kind of pharmaceutical composition for protecting chronic heart failure renal function - Google Patents

Abstract

The invention discloses a pharmaceutical composition for protecting renal function of chronic heart failure. The pharmaceutical composition is composed of spironolactone and relaxin at a mass ratio of 15000:1. The present invention has determined the renal protective effect of combining spironolactone and relaxin in chronic heart failure. The research results show that in the model of myocardial fibrosis, the combined program can significantly improve cardiac function, myocardial hypertrophy, fibrosis, and protect the kidneys. , the effect is better than monotherapy. Cardiorenal syndrome (Cardio‑renal syndrome, CRS) means that when the damage to another organ cannot be compensated by the heart or kidney, mutual causality forms a vicious circle, which eventually accelerates the joint damage and failure of the heart and kidney function. The process of chronic heart failure is accompanied by kidney damage. Therefore, the pharmaceutical composition mixed with spironolactone and relaxin proposed by the present invention provides a new idea for kidney protection in the process of chronic heart failure.

Description

A pharmaceutical composition for protecting renal function of chronic heart failure

technical field

The invention relates to the field of medicine, in particular to a pharmaceutical composition for protecting renal function of chronic heart failure.

Background technique

Chronic heart failure (CHF) is the final stage of many cardiovascular diseases such as myocardial infarction, cardiomyopathy, hypertensive heart disease, and the occurrence of heart failure is related to the gradual fibrosis of the heart. Myocardial fibrosis refers to excessive accumulation of collagen fibers in the normal tissue structure of the myocardium, a significant increase in collagen concentration and collagen volume fraction in cardiac tissue, or changes in collagen composition. Collagen fibers reduce the compliance of the ventricular wall, increase stiffness, and limit diastolic filling; fibrosis of the coronary artery makes the wall thicker, the lumen is narrowed, the elasticity is reduced, and the blood supply of myocardial cells is reduced; myocardial cell necrosis and scarring Formation damages the contractile function of the myocardium. In addition, the electrical conduction of myocardial tissue is abnormal, leading to the occurrence of arrhythmia. Chronic heart failure is the final stage of the development of most cardiovascular diseases, and it is one of the major health-threatening problems in the world. Its drug research has broad prospects for application.

Cardiorenal syndrome (Cardio-renal syndrome, CRS) is that when the heart or kidney cannot compensate for the damage to another organ, mutual causality forms a vicious circle, which eventually accelerates the joint damage and failure of the heart and kidney function. Cardiorenal syndrome type 1 (CRS1) is characterized by acute kidney injury (AKI) and renal insufficiency in patients with acute deterioration of cardiac function. Acute kidney injury and dysfunction can result from direct or indirect effects of heart failure. Type II is chronic renal insufficiency caused by chronic cardiac insufficiency. Impairment of renal function can increase the short-term and long-term mortality of patients, and this change is closely related to the fibrosis of the heart and kidney. Acute and chronic injuries of the heart and kidney organs can increase the expression of cell signaling proteins in immune cells, promote the infiltration of inflammatory cells, and activate fibroblasts to promote the deposition of type I and III collagen, eventually leading to an increase in extracellular matrix and irreversible damage to the heart and kidney. fibrosis. Therefore, the research on the mechanism of cardiorenal syndrome and the development of effective anti-fibrosis drugs are the difficulties and hot spots in the field of heart failure and renal failure research.

Circulatory and local renin-angiotensin-aldosterone systems are involved in the myocardial fibrotic response. In an animal model of renovascular hypertension, ligation of one side of the renal artery can lead to a significant increase in the circulatory RAS system and fibrosis in both the left and right ventricles. However, in partial ligation of the inferior abdominal aorta, this type of hypertension is characterized by increased pressure load without significant activation of the RAS system, and no early fibrotic response was observed. This shows that the RAS system plays an important role in the occurrence of myocardial fibrosis. In the RAS system, angiotensin II and aldosterone have different mechanisms of action on myocardial fibrosis. Myocardial fibrosis does not begin to appear until 3 weeks after infusion of aldosterone, but only 3-4 days after infusion of angiotensin II. muscle fibrosis. It is speculated that the effect of aldosterone on myocardial fibrosis is not direct. Further studies have found that elevated circulating angiotensin II, whether exogenous (via the injection route) or endogenous (in response to renal ischemia), is associated with early myocardial necrosis followed by reparative fibrosis. The application of aldosterone appears late repair fibrosis, which itself does not directly lead to myocardial cell necrosis. In addition, after subcutaneous infusion of aldosterone and salt feeding in rats for one month, in the models of renovascular hypertension and aldosterone infusion-induced hypertension, the aldosterone receptor antagonist spironolactone still has anti-fibrotic effects without lowering blood pressure. role of transformation. This shows that angiotensin II and aldosterone have different mechanisms of action in causing reparative fibrosis (essentially cardiomyocyte necrosis). There may also be differences in their metabolic effects on fibrillar collagen.

In recent years, the role of the renin-angiotensin-aldosterone system (RAAS) in the study of the mechanism of heart failure has been widely recognized, and the intervention of the overactivated RAAS system in the occurrence and development of heart failure has achieved success. However, the suppression of aldosterone by sufficient converting enzyme inhibitors is also temporary, and up to 40% of patients develop aldosterone escape during anti-angiotensin II therapy. Aldosterone stimulates protein synthesis, and its increased collagen is an important factor in myocardial fibrosis and cardiovascular remodeling. Aldosterone escape reduces the therapeutic effect of heart failure, and the use of its antagonists can further reduce the overall mortality of heart failure. Therefore, anti-aldosterone therapy has an important role in the treatment of heart failure.

Spironolactone has a similar chemical structure to aldosterone, and the two compete in the cortex of the distal convoluted tubule and collecting duct, and have direct antagonism at the level of mineralocorticoid receptors on the cell membrane, thereby antagonizing the action of aldosterone . Studies have found that spironolactone has an anti-myocardial fibrosis effect and can reduce the mortality rate of patients with heart failure. It is currently the most commonly used aldosterone receptor antagonist in clinical practice.

Several studies have demonstrated the importance of aldosterone in oxidative stress, inflammatory response, and fibrotic processes in renal disease. During the first month of treatment with spironolactone in patients with early-stage chronic kidney disease, spironolactone was found to be safe with close monitoring of renal function and electrolytes. In rat models of chronic heart failure, spironolactone reduces renal inflammatory cell infiltration, reduces fibrosis, and protects the kidney.

In addition, spironolactone is cheap, has potassium-sparing and diuretic effects, is widely used clinically and is often used in combination with other drugs.

Relaxin was first discovered by Frederick Hisaw in 1926 when he was studying the changes of the pelvic girdle during pregnancy. It can cause obvious relaxation of the pubic ligament, hence the name. It has been clear that relaxin not only plays an important role in pregnancy, but also has important effects on the structure and function of blood vessels in non-pregnant animals. Studies have found that relaxin can stimulate the synthesis of nitric oxide and cAMP, and has a series of biological functions such as dilating blood vessels, degrading collagen, regulating fluid balance and hindering platelet aggregation.

In the atrial and ventricular myocardium of patients with congestive heart failure, endogenous relaxin can sustain high expression. Relaxin expression was increased 3-5 fold in the hypertrophic left ventricle of mice with chronic aortic arch coarctation. In the isoproterenol-induced rat myocardial ischemia injury model, the levels of relaxin in the myocardium and plasma, as well as the mRNA expression of myocardial relaxin were significantly increased. The mRNA and protein levels of relaxin in the heart of spontaneously hypertensive rats (SHR) were significantly increased. The role of relaxin in the cardiovascular system has been extensively studied. It has the effect of positive frequency conversion and variable force on the left atrium, dilates blood vessels and lowers blood pressure, protects and regulates myocardial hypertrophy, and participates in the repair and regeneration of myocardial infarction. . Relaxin has been shown to reduce left ventricular fibrosis in various models of cardiomyopathy (eg, defect in the relaxin gene; cardiac overexpression of β2 adrenergic receptors and hypertension). Recently, relaxin was also shown to attenuate cardiac fibrosis in a high-dose isoproterenol-induced model of myocardial ischemic injury. These studies demonstrate that relaxin is a potent anti-fibrotic factor.

The protective effect of relaxin on the kidney has been confirmed in different experimental models. Knockout of the relaxin gene in mice leads to renal hypertrophy, dysfunction, and fibrosis, and exogenous relaxin treatment ameliorates glomerular and tubular fibrosis. Relaxin improves glomerulosclerosis and reduces renal impairment in aged rats. Relaxin plays an anti-fibrotic role in renal papillary necrosis model, angiotensin II-induced hypertension model and glomerular basement membrane disease model. In addition, relaxin also has the effect of increasing renal blood flow and improving glomerular filtration. Overrate effect.

The previous research of our research group confirmed the anti-fibrosis effect of relaxin and the protective effect on the kidney, and proved that the anti-fibrosis effect was dose-dependent. Relaxin is expected to be used in the treatment of human cardiac fibrotic diseases while protecting the kidneys. At present, relaxin is expensive, and drug combination therapy should be considered to reduce the dosage of relaxin. Therefore, the combined treatment of spironolactone and relaxin provides a new idea for renal protection in the treatment of chronic heart failure.

Recently, endothelial-to-myofibroblast transition or endothelial-to-mesenchymal transition (Endothelial-to-mesenchymal transition, EndMT) has been shown to promote fibrosis. Similar to the transformation of endothelial myofibroblasts, during the EndMT process, endothelial cells gradually lose their own characteristics, and the cell morphology becomes elongated and spindle-shaped, and gradually acquires the phenotypic characteristics of mesenchymal cells such as proliferation, migration, and collagen synthesis . The process of EndMT involves changes in many specific biochemical factors, mainly including endothelial cell markers such as platelet-endothelial cell adhesion molecule-1 (CD31), vascular endothelial cadherin (VE-cadaerin) , downregulation of von Willebrand factor (vWF), mesenchymal markers such as α-smooth muscle actin (α-SMA), vimentin and fibroblast-specific The expression of protein (Fibroblast specific protein1, FSP1) is up-regulated, and the existence of EndMT can be confirmed by the changes of these cell markers. Transforming growth factor-β (Transcription grower factor-β, TGF-β) can promote and induce the phenomenon of EndMT. EndMT has been found to be involved in the occurrence and development of fibrosis in various diseases such as pulmonary fibrosis, liver fibrosis, corneal fibrosis, intestinal fibrosis, wound healing, cardiac fibrosis and renal fibrosis. Zeisberg et al. first confirmed that EndMT can lead to renal fibrosis. They found in three models of renal fibrosis that a considerable part of myofibroblasts could express the endothelial cell marker CD31 (also known as platelet endothelial cell adhesion molecule-1) And mesenchymal cell markers α-SMA, fibroblast-specific protein-1. Li et al. also confirmed that EndMT exists in the early stage of diabetic renal fibrosis and can promote the production of myofibroblasts. Therefore, EndMT provides a new direction for the study of the mechanism of cardiorenal syndrome.

Contents of the invention

The purpose of the present invention is to provide a pharmaceutical composition for protecting renal function of chronic heart failure for the protection of kidney in the process of chronic heart failure. Spironolactone has protective effects on the heart and kidneys, is cheap, and is widely used clinically. Relaxin also has protective effects on the heart and kidneys, and its anti-fibrosis effect is dose-dependent. At present, relaxin is expensive, and drug combination therapy should be considered to reduce the dosage of relaxin. The pharmaceutical composition for protecting renal function of chronic heart failure provided by the present invention is composed of spironolactone and relaxin mixed at a mass ratio of 15000:1.

The therapeutic effect of the pharmaceutical composition of the invention is better than that of monotherapy. In the model of myocardial fibrosis, spironolactone and relaxin combined application have anti-myocardial fibrosis effect.

The beneficial effects of the present invention are: the pharmaceutical composition of the present invention is composed of spironolactone and relaxin mixed at a mass ratio of 15000:1, which is a combination of effective doses of spironolactone and relaxin in a small dose range. The pharmaceutical composition has cardiac function protective effect and renal protective effect, protects renal function by inhibiting the EndMT mechanism, and provides a new idea for the treatment of cardiorenal syndrome.

Description of drawings

Figure 1. The pharmaceutical composition composed of spironolactone and relaxin can increase LVSP, +dp/dtmax, -dp/dtmax and reduce LVEDP in the chronic heart failure model, and the effect is better than that of monotherapy; experimental grouping: Blank group (Control); model group (Iso); spironolactone group (Iso+SP); relaxation group (Iso+RLX); combined treatment group (Iso+SP+RLX).

Figure 2. The pharmaceutical composition of spironolactone and relaxin reduces left ventricular mass index (LVWI), right ventricular mass index (RVWI) and kidney mass index (KWI) in chronic heart failure model (P<0.05 ), the effect was better than monotherapy (P<0.05).

Fig. 3, the pharmaceutical composition of spironolactone and relaxin reduces myocardial tissue interstitial hyperplasia, myocardial arrangement disorder, myocardial cell hypertrophy, vacuole formation, and inflammatory cell infiltration in chronic heart failure model (Fig. 3A), It can reduce vacuolar degeneration, fibrous proliferation and inflammatory cell infiltration of renal tubular epithelial cells in kidney tissue (Fig. 3B), and the effect is better than monotherapy.

Figure 4, the pharmaceutical composition formed by combining spironolactone and relaxin reduces the area of blue fiber tissue in myocardial tissue (Figure 4A, C) and the area of blue fiber tissue in kidney tissue (Figure 4B) in chronic heart failure model , D) (P<0.01), decreased the contents of collagen I and collagen III in myocardial tissue (Fig. 4E) and the contents of collagen I and collagen III in kidney tissue (Fig. 4F) (P<0.01). The effect of combination therapy was better than monotherapy (P<0.05).

Figure 5. The pharmaceutical composition composed of spironolactone and relaxin increases renal platelet-endothelial cell adhesion molecule fluorescence signal (Platelet endothelial cell adhesion molecule-1, CD31) and reduces α-smooth muscle in the chronic heart failure model Actin (α-smooth muscle actin, α-SMA) fluorescence signal (Fig. 5A, B) (P<0.01), increased CD31 protein expression, decreased α-SMA and TGF-β protein expression (Fig. 5C, D and E ) (P<0.01), suggesting that the pharmaceutical composition inhibits renal endothelial-to-mesenchymal transition (Endotherial-to-mesenchymal transition, EndMT), and the effect is better than monotherapy (P<0.01).

detailed description

The present invention will be further described in detail below in conjunction with the embodiments and the accompanying drawings, but the embodiments of the present invention are not limited thereto.

The invention confirms the kidney protection effect of the pharmaceutical composition composed of spironolactone and relaxin in chronic heart failure through experiments.

1. The pharmaceutical composition composed of spironolactone and relaxin protects heart function, and the effect is better than that of monotherapy.

(1) The present invention utilizes isoproterenol to establish an animal model of cardiac fibrosis in chronic heart failure.

(2) Animal grouping: the present invention selects 50 male SD rats and randomly divides them into 5 groups after one week of adaptive feeding: control group, model group, spironolactone group, relaxin group and combined treatment group.

(3) Cardiac function evaluation of each group: In the present invention, rats were intubated in the right carotid artery to record and analyze LVSP, LVEDP, +dp/dtmax and -dp/dtmax. The results showed that: in the chronic heart failure model group, LVSP, +dp/dtmax, -dp/dtmax decreased, and LVEDP increased. The drug combination improved the effect of isoproterenol on cardiac function more than monotherapy (Figure 1).

(4) Measurement of left and right ventricular mass index: In the present invention, rats were measured with left ventricular mass index (LVWI) and right ventricular mass index (RVWI). The results showed that LVWI and RVWI increased in the chronic heart failure model group. The drug combination ameliorated the effects of isoproterenol on left and right ventricular mass index more effectively than monotherapy (Fig. 2A,B).

(5) Myocardial histological observation of each group: the myocardial tissue in the present invention was stained with HE. The results showed that in the chronic heart failure model group, extensive interstitial hyperplasia, myocardial arrangement disorder, cardiomyocyte hypertrophy, vacuole formation, and inflammatory cell infiltration were seen. The drug composition improved the myocardial histological changes caused by isoproterenol, and the effect was better than that of monotherapy (Fig. 3A).

(6) Evaluation of left ventricular fibrosis in each group:

In the present invention, Masson'trichome staining is used to measure the area of left ventricular fibrosis under a light microscope. The results showed that a large number of blue fibrotic tissues were seen in the chronic heart failure model group. The drug combination reduced the area of left ventricular fibrosis more effectively than monotherapy (Fig. 4A,C).

In the present invention, the contents of collagen I and collagen III in myocardial tissue are determined. The results showed that: the content of collagen Ⅰ and collagen III in the myocardial tissue of the chronic heart failure model group increased. The drug composition reduced the contents of collagen I and collagen III in myocardial tissue in the chronic heart failure model, and the effect was better than that of single drug therapy ( FIG. 4E ).

2. The pharmaceutical composition composed of spironolactone and relaxin protects renal function, and the effect is better than that of monotherapy.

(1) Measurement of kidney mass index: In this experiment, both kidneys were weighed, and the kidney mass index (KWI, mg/g) was calculated. The results showed that the kidney mass index increased in the chronic heart failure model group. The drug combination reduced renal mass index changes in a chronic heart failure model more effectively than monotherapy (Fig. 2C).

(2) Histological observation of kidneys in each group: In this experiment, HE staining was used to observe the histological changes of kidneys. The results showed that: in the chronic heart failure model group, the renal tubular epithelial cells showed vacuolar degeneration, fibrous proliferation and inflammatory cell infiltration. The drug combination reduced renal histological changes in a chronic heart failure model more effectively than monotherapy (Fig. 3B).

(3) Evaluation of renal fibrosis in each group:

The area of renal fibrosis was measured under a light microscope with Masson'trichome staining. The results showed that a large number of blue fibrotic tissues were seen in the kidneys of the chronic heart failure model group. The drug combination reduced the area of renal fibrosis in the chronic heart failure model more effectively than monotherapy (Fig. 4B, D).

The contents of collagen Ⅰ and collagen III in kidney tissue were determined by ELISA. The results showed that the contents of collagen Ⅰ and collagen III in the renal tissue of the chronic heart failure model group increased. The drug composition reduced the contents of collagen I and collagen III in kidney tissue in the chronic heart failure model, and the effect was better than that of single drug therapy ( FIG. 4F ).

3. The pharmaceutical composition composed of spironolactone and relaxin inhibits the renal EndMT phenomenon in the chronic heart failure model, and the effect is better than single drug therapy.

(1) The expression of CD31 and α-SMA in renal tissues of each group was observed by fluorescence immunofluorescence: the expressions of α-SMA (fibrosis marker protein) and CD31 (endothelial marker protein) in endothelial cells were detected by immunofluorescence method to observe the phenomenon of EndMT. The results showed that: in the chronic heart failure model group, the expression of CD31 in kidney tissue decreased, and α-SMA increased, suggesting the existence of EndMT phenomenon. The drug combination improved the EndMT phenomenon in the chronic heart failure model, and the effect was better than that of monotherapy (Fig. 5A, B).

(2) Western blot method was used to detect the expression of TGF-β, CD31 and α-SMA in kidney tissue of each group. The results showed that: in the chronic heart failure model group, the expression of CD31 in kidney tissue decreased, and TGF-β and α-SMA increased, suggesting the existence of EndMT phenomenon. The drug combination ameliorates the EndMT phenomenon in a chronic heart failure model more effectively than monotherapy (Figures C, D, and E).

The specific implementation is as follows:

1) Experimental animals and experimental environmental conditions.

50 healthy male SD rats (about 6 weeks), weighing 200g-220g, were fed with standard rodent diet. The experimental environment fully complies with the standards stipulated by the International Society for the Protection of Animals and meets the basic space requirements necessary for the life of small animals.

2) Establishment of animal model: A rat model of chronic heart failure was established by the isoproterenol method.

3) Grouping of animals: 50 clean-grade Wistar male rats with a body mass of (220±10) g were randomly divided into 5 groups. Model group: Subcutaneous injection of isoproterenol on the back of rats, 5 mg/kg/d, for 7 days in total. The control group was given the same amount of normal saline. Spironolactone group: rats were given 30 mg/kg/d by intragastric administration for 21 days in total. Relaxin group: Rats were injected subcutaneously at 2ug/kg/d for a total of 21 days. Combined treatment group: rat spironolactone administered intragastrically, 30mg/kg/d, relaxin administered subcutaneously 2ug/kg/d (spironolactone and relaxin by mass ratio 15000:1), a total of 21 sky.

4) Cardiac function evaluation in each group: After the rats were weighed, they were anesthetized by intraperitoneal injection of 1% sodium pentobarbital. Rats were fixed on the mouse board, the neck was depilated and skinned, the right carotid artery was separated, and a catheter filled with 0.05% heparin saline was inserted into the left ventricle. LVEDP, +dp/dtmax and -dp/dtmax.

5) Measurement of left and right ventricular mass index: after blood collection, the left ventricle of the rat was perfused with 4°C pre-cooled normal saline until the heart and kidneys became pale, and then the heart was taken out and weighed. The left ventricular mass divided by body weight was the left ventricular mass index (LVWI), and the right ventricular mass divided by body weight was the right ventricular mass index (RVWI).

6) Myocardial histological observation in each group: Myocardial tissue was stained with HE to observe its pathological changes. A small amount of tissue (about 80 mg) from the apex of the left ventricle was taken, fixed in formaldehyde, routinely dehydrated, and embedded in paraffin. The remaining part was put into a sterile cryopreservation tube and immediately put into liquid nitrogen for quick freezing, and then transferred to a -80°C refrigerator for other testing items.

Paraffin embedding process:

(1) Fix at 4°C with 4% paraformaldehyde for 24 hours;

(2) Rinse with water;

(3) 50% ethanol for 30 minutes;

(4) 75% ethanol for 30 minutes;

(5) 85% ethanol for 30 minutes;

(6) 95% ethanol for 40 minutes;

(7) 95% ethanol for 40 minutes;

⑻100% ethanol Ⅰ 40min;

⑼100% ethanol II 40min;

⑽ Soak in xylene I for 30 minutes; soak in xylene II for 30 minutes;

⑾ Paraffin I, soak in wax for 2 hours; Paraffin II, soak in wax for 2 hours;

⑿ Specimen embedding;

HE staining steps:

⑴ Paraffin section production, the thickness is about 4μm;

(2) Conventional dewaxing and hydration (100% xylene solution I, II dewaxing each for 15 minutes, 100% ethanol I, II, 95% ethanol I, II, 90%, 80% ethanol hydration each for 5 minutes, ultrapure water washing for 2 minutes* 2 times);

(3) Hematoxylin staining for 3 minutes;

(4) Washing with ultrapure water for 5 minutes;

(5) 1% hydrochloric acid alcohol differentiation for about 5 seconds;

(6) Rinse with ultrapure water and examine under the microscope to observe the staining of cell nuclei;

⑺Wash with ultrapure water for 5 minutes;

⑻ Eosin staining for 5 minutes;

⑼Ultrapure water washing;

⑽Dehydrated, transparent, and sealed;

7) Detection of myocardial tissue collagen area in each group:

The paraffin sections of myocardial tissue were stained by Masson. Under Masson's staining, the cardiomyocytes were red, the collagen was blue, and the nuclei were black. Myocardial interstitial fibrosis was observed under a microscope, and 5 non-repetitive fields of view were randomly selected under a 200-fold field of view and photographed. Using Image-Pro Plus software, the myocardial collagen area was measured, and the average value was taken. The larger the value, the more obvious the fibrosis.

Masson staining steps:

⑴ Paraffin section production, the thickness is about 4μm;

(2) Conventional dewaxing and hydration;

(3) Fix at 60°C for 2 hours;

⑷Wash with ultrapure water for 5 minutes*2 times;

⑸Hematoxylin staining for 5 minutes;

⑹Wash with ultrapure water for 5 minutes*2 times;

⑺ Ponceau red acid fuchsin dyeing for 3 minutes;

⑻Wash with ultrapure water for 3 minutes;

⑼10 minutes of dyeing in 1% phosphomolybdic acid;

⑽Put directly into the aniline blue solution for 10 minutes without washing with water, and observe the color of the fibrous tissue under a microscope;

⑾Wash with ultrapure water for 3 minutes;

⑿Quickly dehydrated, transparent in xylene, and sealed;

8) Determination of collagen I and collagen III content in myocardial tissue of each group: 100 mg of myocardial tissue was chopped and added to 1 ml of PBS (PH7.4), fully homogenized. Centrifuge for about 20 minutes (3000 rpm). Collect the supernatant. Determine according to the ELISA kit instructions:

⑴Preparation: Take out the kit from the refrigerator, rewarm and balance at room temperature for 30 minutes;

(2) Add the standard and the sample to be tested: set the standard well, the blank control well and the sample well to be tested, add 50 μL of the standard to the standard well; add 10 μL of the sample to be tested to the sample well to be tested, and then add 40 μL of the sample diluent (that is, the sample is diluted 5 times); the blank control well is not added;

(3) Incubation: Incubation in an incubator for 30 minutes;

⑷Washing the plate: wash the washing liquid repeatedly for 4-6 times, and print on the filter paper to dry;

(5) Add enzyme-labeled working solution: add 50 μL of enzyme-labeled working solution to each well, and do not add to the blank control well;

⑹ Incubation: Incubation in an incubator for 30 minutes;

⑺ washing board: same as above;

(8) Color development: add 50 μL of color reagent A solution to each well, then add 50 μL of color developer B solution, shake and mix gently for 30 seconds;

(9) Termination: Add 50 μL of stop solution to each well to stop the reaction;

⑽Determination: Set the blank well to zero, and within 30 minutes after the reaction is terminated, measure the absorbance value of each well with a wavelength of 450nm;

⑾Calculation: Calculate the linear regression equation of the standard curve according to the concentration of the standard substance and the corresponding OD value, and calculate the concentration of the sample using the regression equation according to the OD value of the sample; the final concentration is the actual measured concentration multiplied by the dilution factor;

9) Evaluation of kidney mass index in each group: weigh both kidneys, and the ratio of the average value (mg) to body weight (g) is the kidney mass index (mg/g).

10) Kidney histological observation of each group: HE staining method was used to observe the changes of renal histology. A small amount of kidney tissue (about 80 mg) was taken, fixed in formaldehyde, routinely dehydrated, and embedded in paraffin. The remaining part was put into a sterile cryopreservation tube and immediately put into liquid nitrogen for quick freezing, and then transferred to a -80°C refrigerator for other testing items.

HE staining steps:

⑴ Paraffin section production, the thickness is about 4μm;

(2) Conventional dewaxing and hydration (100% xylene solution I, II dewaxing each for 15 minutes, 100% ethanol I, II, 95% ethanol I, II, 90%, 80% ethanol hydration each for 5 minutes, ultrapure water washing for 2 minutes* 2 times);

(3) Hematoxylin staining for 3 minutes;

(4) Washing with ultrapure water for 5 minutes;

(5) 1% hydrochloric acid alcohol differentiation for about 5 seconds;

(6) Rinse with ultrapure water and examine under the microscope to observe the staining of cell nuclei;

⑺Wash with ultrapure water for 5 minutes;

⑻ Eosin staining for 5 minutes;

⑼Ultrapure water washing;

⑽Dehydrated, transparent, and sealed;

11) Detection of collagen area of kidney tissue in each group: Kidney paraffin sections were subjected to Masson staining, the area of kidney collagen was measured, and the average value was taken. The larger the value, the more obvious the fibrosis.

12) Determination of collagen I and collagen III contents in kidney tissue of each group. Cut 100mg of kidney tissue into pieces and add 1ml of PBS (PH7.4) to fully homogenate. Centrifuge for about 20 minutes (3000 rpm). Collect the supernatant. Determination was performed according to the instructions of the ELISA kit.

13) Evaluation of renal endothelial cell interstitialization in each group:

(1) Fluorescence immunofluorescence observation of CD31 and α-SMA fluorescence signals in kidney tissue of each group. In this study, immunofluorescence was used to detect the expression of α-sma (fibrosis marker protein) and CD31 (endothelial marker protein) in endothelial cells to observe the phenomenon of EndMT.

Immunofluorescence steps:

① Paraffin sections are made, with a thickness of about 4 μm;

②Baking paraffin sections at 60°C for 2 hours;

③Conventional dewaxing and hydration;

④Citrate antigen retrieval solution (pH=6.0) for high-pressure antigen retrieval for 3 minutes, cool naturally, wash with 0.1M PBS for 2 minutes*3 times;

⑤Incubate with 3% H2O2 for 10min, wash with 0.1M PBS for 2min*3 times;

⑥Incubate with 10% fetal bovine serum at room temperature for 30 minutes, then shake dry;

⑦ Add the mixed primary antibody (α-sma antibody, Boster, dilution concentration 1:100; CD31 antibody, Abcam, dilution concentration 1:30), overnight at 4°C, wash with 0.1M PBS for 5min*3 times;

⑧ Add the mixed fluorescent secondary antibody (1:300) dropwise, incubate at 37°C in the dark for 2 hours, wash with 0.1M PBS for 5 minutes*6 times, and seal with anti-fluorescence quenching mounting medium.

(2) The expressions of α-sma, CD31 and TGF-β were measured by WB.

Main solution preparation:

①6% separating gel 5ml

Ultrapure water 2.0ml

30% Acr-Bis (29:1) 1.0ml

1M Tris,pH8.8 1.9ml

10% SDS 0.05ml

10% ammonium persulfate 0.05ml

TEMED 0.004ml

Use immediately after mixing

②10% glue 5ml

Ultrapure water 1.3ml

30% Acr-Bis (29:1) 1.7ml

1M Tris,pH8.8 1.9ml

10% SDS 0.05ml

10% ammonium persulfate 0.05ml

TEMED 0.002ml

Use immediately after mixing

③5% stacking gel 2ml

Ultrapure water 1.4ml

30% Acr-Bis(29:1)0.33ml

1M Tris,pH6.8 0.25ml

10% SDS 0.02ml

10% ammonium persulfate 0.02ml

TEMED 0.002ml

Use immediately after mixing

④ Electrophoresis buffer:

Tris 3.03g

Glycine 14.4g

SDS lg

Dissolve in ultrapure water to 1000ml.

⑤Transfer buffer:

Tris 3g

SDS lg

Glycine 14.4g

Methanol 200mL

Dilute the ultrapure water to 1000mL and reuse it up to 4 times.

⑥5XTBS buffer:

Tris 12.1g

NaCl 40g

Dissolve in ultrapure water, adjust the pH value to about 7 with concentrated HCl, and dilute to 1000mL.

⑦TBST buffer

20% Tween20 1ml

TBS 100ml

It can be used after mixing, and it is best to prepare it immediately.

⑧5% skimmed milk

Skimmed milk powder 5g

TBST 100ml

Store at 4°C after dissolution. When in use, return to room temperature and filter for one-time use.

WB experimental steps

⑴Total protein extraction from tissue:

①Take 100mg tissue block and put it in the spherical part of 1-2ml homogenizer, and cut the tissue as much as possible with scissors.

②Add 1000μl lysate and 10ul PMSF to a homogenizer, place on ice for full homogenization.

③ Lyse on ice for 1 hour, transfer to a 1.5ml centrifuge tube, centrifuge at 12,000 rpm for 5 minutes at 4°C, take the supernatant and put it in a 0.5ml centrifuge tube and store at -80°C.

(2) Determination of total protein content by BCA method

(3) SDS-PAGE electrophoresis

Prepare 6% or 10% SDS-PAGE separating gel for different protein detection, configure 4% stacking gel, slowly perfuse the gel, and let it stand at room temperature to solidify. Fix the prepared gel glass plate on the gel electrophoresis instrument, and add the electrophoresis buffer into the upper and lower grooves of the electrophoresis instrument respectively. Carefully remove the sample comb. Calculate the volume of the solution containing 50 μg protein as the loading volume. Add 5*SDS loading buffer to a final concentration of 1*, and bathe in boiling water for 10 minutes to fully denature the protein. Add each sample and protein molecular weight marker respectively, and balance the sample volume with 1*sample buffer. Initially, 70V constant voltage electrophoresis was used until the maker protein layered and then changed to 120V constant voltage electrophoresis to speed up the electrophoresis speed. Electrophoresis until bromophenol blue runs out of the gel.

⑷Transfer film: Take out the rubber plate and cut off the stacking gel. Cut the PVDF membrane with the same size as the glue, place it in the splint of the protein transfer tank in the order of cathode-filter paper-glue-PVDF membrane-filter paper-anode, and conduct electroporation with a constant current of 300mA for 90min.

⑸Sealing: 5% skimmed milk was closed for 1h.

⑹Immune reaction: After blocking, wash the membrane with TBST for 10min*3 times, move the membrane into a clean antibody incubation box, add primary antibody diluent (CD31 antibody, Santa, dilution concentration 1:1000; dilution concentration 1:1000; α -SMA antibody, Wuhanboshide, dilution concentration 1:1000; TGF-β1 antibody, Bioworld Technology, dilution concentration 1:1000; GAPDH antibody, Bioworld Technology, dilution concentration 1:5000), cover the box, seal with tape, shake at 4°C Incubate overnight; wash the membrane with TBST for 10 min*3 times; move the PVDF membrane into a clean antibody incubation box again, and add diluted horseradish peroxidase-labeled secondary antibody (horseradish peroxidase-labeled goat anti-rabbit IgG, Bioworld Technology, dilution concentration 1:5000—1:10000), incubate at room temperature for 2h; wash the membrane with TBST for 15min*3 times; place the PVDF membrane in ECL luminescent solution, and shake at room temperature for 5min.

⑺Development: Gel imaging analysis system detects and records PVDF membrane imaging

⑻Image analysis:

Use Image-Pro Plus software to analyze the grayscale of the target bands in the scanned images.

14) Statistical processing

SPSS 16.0 statistical analysis software was used for data processing, and the obtained data were average ± standard deviation Said, univariate measurement data sample means were compared using one-way ANOVA (One-way ANOVA), P<0.05 means the difference is statistically significant, P<0.01 means the difference is very significant.

The above-mentioned embodiments are used to illustrate the present invention, rather than to limit the present invention. Within the spirit of the present invention and the protection scope of the claims, any modification and change made to the present invention will fall into the protection scope of the present invention.

Claims (3)

1. A pharmaceutical composition for protecting renal function in chronic heart failure, characterized in that it is composed of spironolactone and relaxin mixed in a mass ratio of 15000:1. 2. The pharmaceutical composition for protecting renal function in chronic heart failure according to claim 1, characterized in that the pharmaceutical composition protects cardiac function. 3. The pharmaceutical composition for protecting chronic heart failure renal function according to claim 1, characterized in that the pharmaceutical composition protects renal function by inhibiting endothelial-to-mesenchymal transition (Endothelial-to-mesenchymal transition, EndMT) mechanism .