CN115944738A - Application of complement C3 cracking inhibitor in preparation of medicine for treating diabetic cardiomyopathy - Google Patents

Abstract

The invention provides application of a complement C3 cracking inhibitor in preparing a medicament for treating diabetic cardiomyopathy, and also provides application of a receptor antagonist of a complement C3 cracking product C3a in preparing a medicament for treating diabetic cardiomyopathy. Wherein, the complement C3 lysis inhibitor CP40-KK and the receptor antagonist C3aRA of the C3a can improve the myocardial function of a diabetic mouse by inhibiting a C3a-C3aR pathway, and provide a potential treatment target point for the diabetic cardiomyopathy.

Description

Application of complement C3 cracking inhibitor in preparation of medicine for treating diabetic cardiomyopathy

Technical Field

The invention relates to the technical field of biological medicines, in particular to application of a complement C3 lysis inhibitor in preparation of a medicine for treating diabetic cardiomyopathy.

Background

Diabetes Mellitus (DM) is a chronic disease characterized by hyperglycemia that seriously harms human health, and its prevalence and incidence are on an increasing trend year by year. Diabetic Cardiomyopathy (DCM) is one of the leading causes of death in Diabetic patients. DCM pathogenesis involves many aspects such as metabolic disorders, inflammation, oxidative stress and fibrosis, however its specific mechanism has not yet been elucidated.

The Complement System (CS) is composed of plasma, membrane and intracellular proteins, and is involved in the formation of the immune system, which is one of the defense lines of the body against invading pathogens. Complement plays an important role in the immune system, elevated levels of complement are detected in a variety of disease injuries, and complement activation often occurs in inflammatory and oxidative stress states — studies have found that Advanced Glycation Endproducts (AGEs), hyperglycemia-induced Reactive Oxygen Species (ROS), and immune complex deposition all activate the complement system, while DCM development is associated with AGEs deposition and oxidative stress, suggesting that complement system activation also occurs in DCM.

In recent years, numerous studies have also shown that, for example, complement system activation is closely related to the development of diabetes, and complement C3 is a key molecule for complement activation. Increased complement C3 levels have been shown to be associated with insulin resistance and the onset of diabetes. In addition, it has been found that the C3 cleavage product C3a plays a pathogenic role in diabetic nephropathy and that urinary C3a levels are associated with glomerulopathy severity, whereas the C3a receptor (C3 aR) antagonist C3aRA is able to reduce renal fibrosis, alleviate podocyte mitochondrial dysfunction and ameliorate diabetic nephropathy by inhibiting the TGF β/Smad3 pathway. High levels of C3 have also been found in other studies relating to diabetic complications to be associated with increased risk of diabetic retinopathy, nephropathy and neuropathy. However, little has been done on the role of complement C3 and its cleavage product C3a in DCM.

Disclosure of Invention

The invention aims to provide an application of a complement C3 cracking inhibitor and a receptor antagonist of a complement C3 cracking product C3a in preparing a medicament for treating diabetic cardiomyopathy, and the application can be seen that the complement C3 cracking inhibitor CP40-KK and the receptor antagonist C3aRA of the C3a can improve the myocardial function of a diabetic mouse by inhibiting a C3a-C3aR pathway, and provide a potential treatment target for the diabetic cardiomyopathy.

In order to achieve the above object, the present invention provides the following technical solutions:

the invention provides application of a complement C3 cracking inhibitor in preparation of a medicament for treating diabetic cardiomyopathy.

Preferably, the complement C3 cleavage inhibitor comprises CP40-KK.

The invention provides application of a receptor antagonist of a complement C3 cleavage product C3a in preparing a medicament for treating diabetic cardiomyopathy.

Preferably, the receptor antagonist for C3a comprises C3aRA.

Preferably, the C3aR a inhibits phosphorylation of ERK downstream of C3 aR.

Preferably, the C3aRA functions by inhibiting the expression of NLRP3-Caspase-1-IL-1 β/IL-18 pathway related molecules.

By adopting the technical scheme, the invention has the following beneficial effects:

1. the CP40-KK can be combined with complement C3 of human and mice, inhibit C3 lysis and play a role in the generation and development of diabetic cardiomyopathy through a C3a-C3aR pathway.

2. The invention discovers that C3a is a main effector molecule of the mouse, and both CP40-KK and C3a receptor antagonists can improve the cardiac function of a diabetic mouse by inhibiting a C3a-C3aR pathway, relieve the degree of myocardial hypertrophy, reduce the degree of cardiac fibrosis and play a role in protecting diabetic cardiomyopathy.

3. According to the invention, the activation and oxidative stress of NLRP3 induced by high glucose and C3a are mutually promoted and participate in the development of diabetic cardiomyopathy. The C3a receptor antagonist can inhibit NLRP3-Caspase-1-IL-1 beta/IL-18 pathway to improve diabetic cardiomyopathy; can also reduce NADPH pathway and mitochondrial pathway oxidative stress level, improve mitochondrial function, reduce apoptosis, and protect diabetic cardiomyopathy.

Drawings

FIG. 1 is a schematic diagram of a diabetic cardiomyopathy mouse model;

fig. 2 shows the random blood glucose, body weight and heart-weight-tibia length ratio of mice (in the figure, a is the ratio of the control to the random blood glucose of diabetic mice, B is the ratio of the control to the body weight of diabetic mice, and C is the ratio of the heart-weight-tibia length of control to the heart-weight-tibia length of diabetic mice (n =6, × P < 0.01));

fig. 3 shows the cardiac ultrasound results of the control group and the diabetic mice (in the figure, a is the Left Ventricular Ejection Fraction (LVEF) of the control group and the diabetic mice, B is the left ventricular shortening fraction (LVFS) of the control group and the diabetic mice, C is the E/a ratio (n =6, × P < 0.01) of the control group and the diabetic mice, D is the M-type echocardiogram of the control group and the diabetic mice, and E is the E peak and the a peak typical of the control group and the diabetic mice);

FIG. 4 shows the result of mouse left ventricular hemodynamics (A in the figure is the dP/dt of mice in the control group and the diabetic group _max (maximum rate of rise of left ventricular pressure), reflecting the contraction function of left ventricle, B is control group and diabetic mice-dP/dt _max (maximum rate of decline of left ventricular pressure), reflecting left ventricular diastolic function (n =6, # P)<0.01))；

FIG. 5 is a schematic view of a tissue dehydration step;

fig. 6 shows the results of HE and WGA staining of heart tissue sections of mice in the control group and the diabetic group (in the figure, a is an image of hematoxylin-eosin staining (scale =50 μm) of myocardial tissue, B is a statistic of cross-sectional area size of cardiomyocytes stained with hematoxylin-eosin, C is a WGA staining (scale =20 μm) image of myocardial tissue, and D is a statistic of cross-sectional area size of cardiomyocytes stained with WGA (n =6, × P < 0.01));

fig. 7 shows sirius red and Masson staining results of cardiac tissue sections of control and diabetic mice (in the figure, a represents sirius red staining (upper, red is collagen fiber) and Masson staining (lower, blue is collagen fiber) of cardiac muscle tissue, typical pictures (scale =100 μm), and B represents area statistics of sirius red and Masson staining positive regions (n =6, P < 0.01));

FIG. 8 is the plasma C3 concentrations of control and diabetic mice;

FIG. 9 shows the expression level of C3 protein in heart tissue of control and diabetic mice;

figure 10 is a quantitative analysis of C3 western blot band gray values (Mean ± SD, n =6, × P < 0.01) for control and diabetic mouse heart tissue;

figure 11 shows control and diabetic mouse heart tissue C3 and C3a expression levels (in the figure, a is heart tissue C3a (scale =100 μm) and C3aR (scale =200 μm) immunohistochemical staining, B is C3a and C3aR immunohistochemical staining positive area comparison (Mean ± SD, n =6,. Star.p < 0.01));

FIG. 12 shows the effect of high glucose (HG, 30 mmol/L) intervention on the expression levels of C3, C3a and C3aR in H9C2 cells (in the figure, A is Westernblot band for total protein in H9C2 cells, B is C3 Westernblot band quantitative assay for gray scale, C is C3aWesternblot band quantitative assay for gray scale, and D is C3aRwesternblot band quantitative assay for gray scale (Mean. + -. SD, n =6,. P <0.01,. P < 0.05));

FIG. 13 shows the effect of hyperglycosemia intervention on the localization of C3aR nuclear plasma in H9C2 cells (A in the figure is the result of Westernblot of H9C2 nuclear plasma protein after hyperglycosemia intervention (NG: normal sugar, HG: hyperglycosemia) and B is the result of C3aR immunofluorescence staining of H9C2 cells after hyperglycosemia intervention (ruler =50 μm));

FIG. 14 is a schematic diagram of the modeling and intervention of a diabetic cardiomyopathy mouse;

fig. 15 is a graph of mouse blood glucose, body weight and heart-weight-tibia length ratio (in the graph, a is mouse random blood glucose concentration, B is mouse body weight, and C is mouse heart-weight-tibia length ratio (n =6,. P <0.05,ns, non-significant));

FIG. 16 shows the mouse left ventricular hemodynamics (A is dP/dt in the figure) _max (maximum rate of rise of left ventricular pressure), B is-dP/dt _max (maximum rate of decrease of left ventricular pressure) (n =6, # P<0.01,ns,non-significant))；

Fig. 17 shows the results of echocardiography of mice (in the figure, a is M-type echocardiography, B is the comparison of left ventricular ejection fraction of mice, C is the comparison of left ventricular minor axis shortening rate of mice, D is the images of E peak and a peak of blood flow of mitral valve of mice, E is the statistical plot of blood flow E/a of mitral valve of mice (n =6,. About.p <0.01,ns, non-significant));

fig. 18 shows mRNA expression levels of cardiac injury indicators ANP, BNP, and β -MHC (n =6, P <0.01, P <0.05, ns, non-significant);

fig. 19 shows mouse heart tissue HE and WGA staining (in the figure, a indicates the heart tissue HE staining (scale 50 μm) and the statistical result of the cross-sectional area of cardiomyocytes, and B indicates the WGA staining (scale 20 μm) and the statistical result of the cross-sectional area of cardiomyocytes (n =6, × P <0.01, ns, non-significan));

fig. 20 sirius red and Masson staining of mouse heart tissue sections (in the figure, a is cardiac muscle tissue sirius red staining (red is collagen fiber, scale =100 μm) and statistics of staining positive area, B is Masson staining (blue is collagen fiber, scale =100 μm) and statistics of staining positive area (n =6, { P <0.01,ns, non-significan));

FIG. 21 is a SPR sensorgram showing the binding of human and mouse C3 to CP40-KK (A is a SPR sensorgram showing the binding of human complement C3 to CP40-KK, and B is a SPR sensorgram showing the binding of mouse complement C3 to CP 40-KK));

figure 22 is mouse plasma anaphylatoxin C3a concentrations (n =6, # P <0.01, # P <0.05,ns, non-significan);

FIG. 23 shows the protein expression level of C3 fragment in mouse myocardial tissue (A in the figure is Western blot band of C3 cleavage fragment in mouse cardiac tissue, B is Westernblot grayscale quantitative analysis of C3 cleavage fragment (n =6,. Times.P < 0.01));

FIG. 24 shows ERK phosphorylation levels of mouse heart tissue (in the figure, A is a band of mouse heart tissue phosphorylated ERK1/2 and total ERK1/2Westernblot, and B is a ratio of gray values of phosphorylated ERK1/2 and total ERK1/2 bands (n =6,. Times.P <0.01, ns, non-significan));

FIG. 25 levels of indicators of inflammation in mouse heart tissue (in the figure, A is Western blot band of mouse heart tissue, B is Westernblot grayscale quantitation of NLRP3, C is Westernblot grayscale quantitation of Caspase-1, D is Westernblot grayscale quantitation of IL-1 β, and E is Westernblot grayscale quantitation of IL-18 (n =6,. About.P <0.01, ns, non-significan));

FIG. 26 is a graph showing the effect of C3a and C3aRA on cardiomyocytes C3aR (in the graph, A is the result of Western blot on total proteins of cardiomyocytes AC16, B is the result of phosphorylation ERK of AC16 cells, and quantitative analysis of bands of total ERK Western blot (n =3,. About.P <0.01,. About.P <0.05,ns, non-significan));

FIG. 27 shows verification of anti-inflammatory effect of C3aRA (in the figure, A is the Westernblot result of AC16 total protein of cardiomyocytes, B is the Western blot banding quantitative analysis of NLRP3, C is the Westernblot gray scale quantitative analysis of Caspase-1, D is the Western blot gray scale quantitative analysis of IL-1 beta, and E is the Western blot gray scale quantitative analysis of IL-18 (n =3,. About.P <0.01,. P <0.05, ns, non-significan));

FIG. 28 shows the expression levels of NADPH oxidase catalytic subunit in murine cardiac tissue (A in the figure is the Western blot result for NADPH oxidase catalytic subunit in murine cardiac tissue, B is the P47-phox immunohistochemical staining of a section of murine cardiac tissue, C is the Western blot band grayscale quantitative analysis for gp91-phox, D is the Western blot band grayscale quantitative analysis for P67-phox, and E is the Westernblot band grayscale quantitative analysis for P47-phox (n =6,. Times.P <0.01,. P <0.05, ns, non-significan));

FIG. 29 shows the expression levels of AC16 NADPH oxidase subunit and SOD2 in cardiomyocytes (A in the figure is the result of Western blot on NADPH oxidase subunit and SOD2 in cardiomyocytes, B is the quantitative analysis of Western blot bands at gp91-phox by grayscale scan, C is the quantitative analysis of Westernblot bands at P67-phox by grayscale scan, D is the quantitative analysis of Westernblot bands at P47-phox by grayscale scan, and E is the quantitative analysis of SOD2 by Western blot bands by grayscale scan (n =3,. Times.P <0.01,. Times.P <0.05, ns, non-significan));

figure 30 is MitoSOX staining of cardiomyocytes (scale =200 μm, red MitoSOX, reflecting intracellular ROS levels, blue nucleus);

figure 31 is cardiomyocyte JC-1 staining (scale =50 μm, green for JC-1 monomer, indicating a decrease in mitochondrial membrane potential, red for JC-1 multimer, indicating normal mitochondrial membrane potential);

FIG. 32 shows cardiomyocyte DHE staining (scale =100 μm, DHE in red, reflecting intracellular ROS levels, hochest in blue, representing the nucleus);

figure 33 is TUNEL staining of mouse heart tissue with white arrows indicating apoptotic cells (scale =100 μm, green fluorescence is TUNEL staining, indicating apoptotic nuclei, blue fluorescence indicates nuclei);

FIG. 34 is a graph of C3aRA reduced AC16 cardiomyocyte apoptosis (A in the graph is the cardiomyocyte AC16 total protein apoptosis indicator Westernblot band, B is the Bax/Bcl-2 gray scale quantification (n =3,. About.P <0.01,. About.P <0.05, ns, non-significant), C is the sheared Caspase-3 band gray scale quantification (n =3,. About.P <0.01, ns, non-significant), D is the AC16 cardiomyocyte TUNEL staining (scale =200 μm));

figure 35 is a graph of MCC950 decreasing ROS expression (a in the graph is AC16 cardiomyocyte DHE (staining scale =50 μm) and B is a statistical graph of the number of positive cells stained for DHE (n =3, × P <0.01,ns, non-significant));

fig. 36 shows NAC effects on NLRP3 expression (a in the figure is cardiomyocyte AC16 DHE staining (scale =50 μm), B is NLRP3Western blot band, C is a statistical plot of the number of positive cells stained by AC16 DHE (n =3, × P <0.01, ns, non-significant), and D is a statistical plot of the Western band grayscale scan of NLRP3 protein (n =3, × P <0.01, × P <0.05, ns, non-significant)).

Detailed Description

The technical solutions provided by the present invention are described in detail below with reference to examples, but they should not be construed as limiting the scope of the present invention.

Example 1 diabetic cardiomyopathy mouse model establishment

(I) diabetic cardiomyopathy mouse modeling

1. Laboratory animal

The mice used in the experiment are male C57BL/6J mice of 7 weeks old, purchased from Beijing Wintonli Hua corporation, and bred in SPF-level animal house of the experimental animal center of Tongji hospital affiliated to Tongji medical college of Huazhong university of science and technology, the breeding environment temperature is set to be 20-26 ℃, the daily light is 12 hours, the dark is 12 hours, so as to circulate, and sufficient water and food are provided. Animal experiments have been approved by the ethical committee of the experimental animal center of college of peer-to-peer medical college of science and technology, huazhong.

2. Experimental methods

Wild type male C57BL/6J mice, 7 weeks old, were purchased, delivered to the animal house and acclimated for 1 week, and given a 60mg/kg dose of streptozotocin (STZ, sigma-Aldrich, USA) for 5 consecutive days i.p. injections, and the control group was injected with an equal volume of solvent (0.05 mol/L sodium citrate buffer, pH 4.5), and the molding procedure is shown in FIG. 1. Tail vein blood was taken 2 weeks after the last injection to determine random blood glucose, and mice were included in the diabetes model group when random blood glucose was higher than 16.7 mmol/L. Mice were kept for 26 weeks of age, 2 times a week for mice with blood glucose >27.7mmol/L, each time with 0.5U long-acting insulin injected subcutaneously to maintain their survival, to 34 weeks of age.

3. Results

Mice were weighed at 34 weeks of age and tested for random blood glucose. Compared with the prior art, the DCM mice show a remarkable increase in random blood sugar (A in figure 2), a remarkable decrease in body weight (B in figure 2), and successful diabetes modeling. After sacrifice, the heart weight was weighed, the tibia length was measured, and the heart-to-tibia length ratio (HW/TL) was calculated, and the results showed that the heart-to-tibia length ratio of the diabetic mice significantly increased, suggesting the possible presence of myocardial hypertrophy (C in fig. 2).

(II) detection of cardiac function of diabetic cardiomyopathy mice

1. Echocardiography examination of mice

Mice were subjected to echocardiography prior to sacrifice. The hair of the precordial region of the mouse is removed by dipping a cotton swab in the depilatory cream in advance, the mouse is fixed on a test board in a supine position by using an adhesive tape, and a small amount of ultrasonic coupling agent is coated on the four limbs of the mouse, so that the good contact between the mouse and the electrode of the test board is ensured. After the fixation, isoflurane is used for continuous gas anesthesia with the assistance of a breathing machine, the dosage of the anesthetic is adjusted, the heart rate of the mouse is stabilized by 500-600bpm, a proper amount of ultrasonic coupling agent is coated on the precordial region, and the two-dimensional ultrasonic cardiogram and the M-type ultrasonic cardiogram of the long axis and the short axis are recorded. And moving the probe to the apex of the heart, adjusting the anesthetic dose, controlling the heart rate of the mouse to be 500bpm, and recording the E peak and the A peak of the blood flow of the mitral valve. The results are shown in fig. 3, which reflects that various indexes of the heart function of the mice, such as Left Ventricular Ejection Fraction (LVEF), left ventricular shortening fraction (LVFS), E/a and the like, are obviously reduced in the diabetic mice, and indicate that the heart contraction and relaxation functions of the diabetic mice are damaged.

2. Left ventricular hemodynamic examination

The mice were anesthetized with 1% sodium pentobarbital by intraperitoneal injection (4 μ L/g), fixed in the supine position, and a longitudinal incision was made in the center of the neck of the mice, with the incision range as large as possible, to facilitate the subsequent operation. The muscles were bluntly separated with forceps, and the right common carotid artery was exposed and isolated. The distal end is then ligated with a 4-0 surgical suture, the thread ends are pulled outward and fixed, and after the artery is full, the proximal end of the artery is clamped with an artery clamp. And (3) cutting a small opening at the position close to the ligation position by using a microsciscope under the direct vision of a body type microscope, delivering a Millar catheter into an artery by using an insulin needle for assistance, loosening an artery clamp, inserting the catheter into the left ventricle, and collecting a hemodynamic signal.

Calculating the maximum rate of decrease-dP/dt of the left chamber pressure after the measurement is completed _max And maximum rate of rise dP/dt of left chamber pressure _max . As a result, mouse left ventricular hemodynamic index dP/dt _max and-dP/dt _max It also indicates that the left ventricle contraction and relaxation functions of the diabetic mouse are reduced (see figure 4), and the diabetic cardiomyopathy model is successfully modeled.

3. Tissue paraffin embedding section

(1) Material taking: after the mice are sacrificed, fresh heart tissues are taken, transverse cutting is carried out at the position 2-3mm above the apex of the heart, a ventricular ring with the width of 2mm is cut, and 4% paraformaldehyde is placed for fixing for more than 24 hours.

(2) And (3) dehydrating: the tissue was removed from the fixative and placed in a dehydration box and placed in a dehydrator for gradient alcohol dehydration in sequence according to the steps of fig. 5.

(3) Embedding: embedding with embedding machine, adding melted wax in the embedding frame, taking out tissue from the dehydration box before wax solidification, placing the heart with transverse section facing upwards in the embedding frame, and waiting for solidification.

(4) Slicing: slicing the tissue with paraffin slicer to thickness of 4 μm, flattening in warm water, taking out with glass slide, and baking in 60 deg.C oven.

WGA and HE staining

The obtained sections were stained with hematoxylin-eosin (HE) and wheat lectin (WGA), and the morphological changes of the cardiomyocytes were observed. As can be seen by comparing the morphology of the two groups, the cross-sectional area of the myocardial cells of the mice in the diabetic group is significantly larger than that of the control group (see FIG. 6).

5. Sirius red staining and Masson staining

The degree of cardiac fibrosis was examined by sirius red and Masso staining of mouse heart tissue. The degree of fibrosis around the heart vessels of the mice in the diabetic group was significantly higher than that in the control group, as determined by two staining experiments (see FIG. 7).

Example 2C 3, C3a and C3aR expression in diabetic cardiomyopathy mice

(I) the expression level of C3 in blood plasma and heart tissue of diabetic mice

1. Determination of serum complement C3 concentration

The concentration of serum complement C3 is measured by using a complement C3 detection kit (product number E032-2) of Nanjing institute of bioengineering, and the measurement principle is an immunoturbidimetry.

The determination process comprises the following steps: preparing a reaction system shown in table 1; gently oscillating the well plate, incubating at 37 ℃ for 5 minutes, and reading the absorbance A1 at 340 nm; add 50. Mu. L R2 to each well; gently oscillating the well plate, incubating at 37 ℃ for 5 minutes, and reading the absorbance A2 at 340 nm; calculating delta A = A2-A1, drawing a standard curve by using different concentrations of the standard substance and corresponding delta A, and substituting for measurement Kong A to calculate the concentration. The results are shown in FIG. 8.

TABLE 1 complement C3 concentration determination reaction System

	Blank hole	Standard hole	Assay well
				Distilled water	2μL	—	—
Standard liquid	—	2μL	—
				Sample(s)	—	—	2μL
R1	250μL	250μL	250μL

2. Total protein extraction from cardiac tissue

(1) Taking out the frozen tissue from an ultra-low temperature refrigerator at minus 80 ℃, quickly transferring the frozen tissue to a mortar containing a proper amount of liquid nitrogen, grinding the tissue into powder, putting 1/2 of the frozen tissue into an EP tube precooled in the liquid nitrogen for extracting the total protein of the heart tissue, using 1/3 of the frozen tissue for extracting the nucleoprotein and the plasma protein of the heart tissue, and using the remaining 1/6 of the frozen tissue for extracting the RNA of the tissue. (2) 500. Mu.L of RIPA lysate (to which protease and phosphatase inhibitors have been added in advance) was added to the tubes containing the tissue and incubated on ice for more than 15 minutes. (3) After the lysis, the mixture was centrifuged at 12000g for 15 minutes in a centrifuge cooled to 4 ℃ in advance, and the obtained supernatant was total protein. (4) The total protein concentration was measured by diluting the protein 20-fold (with PBS) because the tissue protein concentration was high. The BCA protein concentration determination kit of the doctor Wuhan organism is used for measuring the concentration, a standard curve is required to be set each time, and the standard curve is prepared by adopting a multiple dilution method and is shown in Table 2. Preparing a BCA working solution, reagent A: reagent B =50, ready for use, mixed well. (6) Add 95. Mu.L BCA working solution and 5. Mu.L sample to be tested to each well, shake for 30 seconds, mix well, incubate for 30 minutes at 37 ℃. Two replicates were set for each sample. (7) And taking out the plate, cooling to room temperature, detecting absorbance by using an enzyme-labeling instrument, and setting the wavelength to 562nm. (8) And drawing a standard curve according to the concentration of the standard substance and the corresponding absorbance, calculating the protein concentration of the sample, adding a proper amount of RIP lysate into the sample with higher concentration, and adjusting the concentration of all the samples to be 1 mu g/mu L. Adding 200 mul of 5 Xreduction type protein loading buffer solution into 800 mul of protein, boiling for 10 minutes, sub-packaging in a plurality of spiral sharp-bottomed tubes after boiling, freezing at-80 ℃ and avoiding repeated freeze thawing.

TABLE 2 preparation table of standard curve for protein concentration determination

In addition, the total protein of the extracted heart tissue of the mouse is detected by Western blotting (Western Blot), and as a result, the expression of complement C3 in the heart tissue of the mouse is also found, and the level of complement C3 in the heart tissue of the diabetic mouse is obviously higher than that of a control group (see fig. 9 and fig. 10).

(II) C3a and C3aR expression level in heart tissue of diabetic mouse

Further immunohistochemical staining (IHC) of C3a and C3a receptors (C3 aR) was performed on paraffin sections of cardiac tissue. The heart tissue section staining results (see a in fig. 11) show that the protein expression levels of heart tissues C3a and C3aR of the diabetic mice were significantly increased, and the staining positive areas were concentrated around the cell nuclei. Quantitative analysis of the staining positive regions using Image J picture analysis software revealed that the areas of the staining positive regions were significantly higher in the diabetic group than in the control group (see B in fig. 11), and there were statistical differences between the two groups. That is, it is demonstrated that in the diabetic state, both C3 production and lysis are increased, resulting in elevated local C3a levels in cardiac tissue. Increased C3a further induces increased local C3aR production in cardiac tissue. C3a regulates downstream molecules by binding to C3aR locally in the heart, playing a role in DCM.

Example 3 high sugar stimulation affected the expression of C3, C3a and C3aR in H9C2 cells

(I) high sugar intervention promotes the expression of C3, C3a and C3aR of cardiac muscle cells

Validation was also performed on the rat cardiomyocyte line H9c2 (purchased from American Type Culture Collection (ATCC)). Rat cardiomyocyte line H9c2 was cultured in low-sugar DMEM medium (glucose concentration 5 mmol/L) containing 10% FBS, which was subjected to complement inactivation (56 ℃,30 min) prior to use. Starvation treatment is carried out for 12 hours by changing a serum-free culture medium when the cell density is about 50%, the complete culture medium is changed back after the treatment, total cell protein is extracted after 48 hours of high-sugar (the final concentration is 30 mmol/L) intervention, and the expression quantity of C3, C3a and C3aR in the myocardial cells is detected by Westernblot.

As can be seen from fig. 12, cardiomyocytes H9C2 were able to express C3, C3a and C3aR, and hyperglycemic intervention stimulated increased expression.

(II) high-sugar intervention promotes myocardial cell C3aR to transfer from nucleus to cytoplasm

Immunohistochemical staining of heart tissue showed that C3aR staining was concentrated near the cardiac nuclei, and western blot results of H9C2 cellular protein showed expression of C3aR in cardiac cells. In order to explore the influence of high-sugar stimulation on myocardial cell C3aR, H9C2 cells are subjected to high-sugar stimulation for 48 hours, nuclear plasma protein is extracted after intervention is completed, cell immunofluorescence staining is carried out, and the expression amount and position of C3aR are determined.

As can be seen from FIG. 13, the expression level of C3aR in H9C2 cytoplasm is increased and the expression level in nucleus is decreased after hyperglycosemia intervention, which indicates that C3aR in cells is transferred from nucleus to cytoplasm by hyperglycosemia stimulation.

Example 4C3 involvement in the development of diabetic cardiomyopathy by the C3a-C3aR pathway

Compstatin family derivatives are targeted inhibitors of complement C3, which prevent C cleavage by sterically hindering C3 from binding to its convertase. CP40 is one of its improved products, formed by D-tyrosine substituting the N-terminal acetyl group of Compstatin. The polypeptide CP40-KK selected in the research is formed by adding two hydrophilic lysine residues on the basis of CP40, and compared with CP40, the polypeptide CP40-KK has higher solubility and stronger binding force with C3. The C3aR antagonist C3aRA (SB 290157) is a selective C3a receptor antagonist and can block the activation of signal molecules such as ERK and the like at the downstream of a C3a receptor.

(I) C3aR antagonist and CP40-KK intervention can improve cardiac function of diabetic mice

CP40-KK and C3aRA intervention is respectively given to a mouse model with diabetic cardiomyopathy, and the action of C3 in the diabetic cardiomyopathy and the main action path thereof are determined by comparing a diabetic group, a mouse inhibiting C lysis and a mouse only inhibiting a C3a-C3aR pathway.

1. Diabetic mice were randomized at 26 weeks of age into 3 groups: DCM group, DCM + CP40-KK group, DCM + C3aRA group. The DCM group was given daily injections of an equal volume of saline, and the latter two groups were given daily intraperitoneal injections of CP40-KK (2 mg/kg/d in saline) and C3aRA (30 mg/kg/d in 10% ethanol/distilled water), respectively, for 8 weeks. Echocardiography and left ventricular hemodynamics tests were performed at 34 weeks of age of the mice, sacrificed and blood and tissue were removed (see figure 14).

CP40-KK polypeptide sequence:

DTyr-Ile-[Cys-Val-Trp(Me)-Gln-Asn-Trp-Sar-Ala-His-Arg-Cys]-mIle-Lys-Lys-NH ₂ (CP 40-KK) was synthesized by Shanghai Gill Biochemical Co., ltd.

After intervention is completed, the blood sugar, the weight and the length of shin bone of the mouse are measured, and the result shows that the blood sugar of the diabetic mouse is obviously increased and the weight of the diabetic mouse is obviously reduced, while the intervention of C3aRA and CP40-KK does not obviously improve the blood sugar and the weight of the diabetic mouse. However, the ratio of cardiac-to-tibial length was significantly higher in the diabetic mice than in the control, whereas both the C3aRA and CP40-KK interventions reduced the ratio of cardiac-to-tibial length and improved the degree of cardiac hypertrophy, and the ratio of cardiac-to-tibial length was not significantly different between the two drug-dried groups (see fig. 15).

Mice were also subjected to left ventricular hemodynamics (figure 16) and echocardiography (figure 17). dP/dt in diabetic mice _max 、-dP/dt _max The absolute value, left Ventricular Ejection Fraction (LVEF), left ventricular minor axis shortening fraction (LVFS), E/A and the like reflect a significant decrease in cardiac function, while C3aRA or C3aRA is administeredThere was a clear improvement in cardiac function following CP40-KK intervention and no clear difference was seen between the two intervention groups C3aRA and CP40-KK, indicating that C3 plays a role in diabetic cardiomyopathy primarily through the C3a-C3aR pathway.

2. Enzyme linked immunosorbent assay. Mouse serum C3a elisa kit was purchased from warrior gmbh.

3. Cardiac tissue RNA extraction

(1) After sacrifice, 1/6 of the ground tissue was taken and placed into an EP tube pre-cooled in liquid nitrogen. (2) Add 500. Mu.L Trizol to the EP tube, mix well with shaking, then let stand at room temperature for 5 minutes. (3) The EP tube was opened in a fume hood, 100. Mu.L of chloroform was added thereto, shaken for 15 seconds using a vortex shaker, and then allowed to stand at room temperature for 5 minutes. (4) When standing, precooling the low-temperature centrifuge to 4 ℃, setting the centrifugal force to be 10000g, and centrifuging for 15 minutes at 4 ℃. (5) And taking out the E tube after the centrifugation is finished, and placing the E tube in an ice box, wherein the liquid in the tube is layered at the moment, the upper layer is a colorless water phase, the RNA mainly exists in the layer, the middle layer is milky white, and the lower layer is a pink organic phase. Transfer the top colorless aqueous phase to a new EP tube, taking care to avoid wicking to the middle layer. (6) An equal volume of isopropanol was added to the aqueous phase, mixed by inversion and incubated at room temperature for 10 minutes. (7) 10000g, centrifuged at 4 ℃ for 15 minutes, and the supernatant was discarded. (8) Add 500. Mu.L of 75% ethanol (in DEPC water) to the tube and shake vigorously. (9) 7500g, 5 min at 4 deg.C, discard the supernatant and see a very small amount of white gelatinous precipitate at the bottom of the tube. (10) The centrifugation of step 9 was repeated, the residual liquid was aspirated off with a 10 μ L micropipette, the EP tube was inverted on clean paper and dried at room temperature. (11) After air drying, observing the precipitation amount, adding 20-40 mu L of non-enzyme water to dissolve the precipitate, and uniformly mixing the precipitate by using a vortex oscillator. After mixing, the RNA concentration and OD260/280 of each sample were measured using a ultramicro nucleic acid analyzer.

4. Reverse transcription. The cDNA of the product obtained by using a reverse transcription kit of the Biotech Co., ltd, botek, egyhan was stored in an ultra-low temperature freezer at-80 ℃.

5. Real-time fluorescence quantitative PCR, and detecting mRNA expression levels of heart injury indexes ANP, BNP and beta-MHC. The SYBR Green Fast qPCR Mix kit from Biotech Limited, botach, wuhan was used. The primer sequences are shown in Table 3.

TABLE 3 mouse primer sequence Listing

As can be seen from FIG. 18, the myocardial injury index of the mice in the diabetes group is remarkably increased, while the C3aRA and CP40-KK intervention group are remarkably reduced, which suggests that the C3aRA or CP40-KK can improve the heart injury caused by diabetes.

(II) C3aR antagonist and CP40-KK intervention can improve myocardial cell hypertrophy of diabetic mice

Mice were sacrificed at 34 weeks of age and heart tissue was left. The part of the ventricle 2-3mm above the apex of the heart is cut and sectioned by paraffin embedding. The paraffin sections were stained with hematoxylin-eosin (HE) stain and wheat agglutinin (WGA) to clarify the size and morphological changes of cardiomyocytes, and the results are shown in fig. 19.

Statistical analysis is carried out on the cross sectional area of the myocardial cells, the myocardial cell hypertrophy and disorganization of a diabetic mouse are found, the myocardial cell hypertrophy condition of a C3aRA and CP40-KK intervention group is obviously improved, and the statistical difference is found compared with that of the diabetic mouse, but the cross sectional area of the myocardial cells of the C3aRA and CP40-KK intervention group mouse is compared, the statistical difference is not found between the two mouse, and the C3a-C3aR pathway plays an important role in diabetic cardiomyopathy compared with the C3b pathway.

(III) C3aR antagonist and CP40-KK intervention can improve degree of cardiac tissue fibrosis of diabetic mice

Further, sirius red and Masson staining is carried out on heart tissues of mice to observe the fibrosis degree of the heart tissues, and results show that (shown in figure 20) more collagen fibers are deposited around heart blood vessels of the mice in a diabetic group, the fibrosis degree is obviously higher than that of a control group, and C3aRA or CP40-KK intervention can improve the fibrosis degree around the blood vessels and plays a protective role in diabetic cardiomyopathy.

Example 5 validation of the Effect of C3 inhibitors CP40-KK and C3aR antagonists

(I) CP40-KK binds to complement C3 and inhibits C3 cleavage

Prior to administration, the kinetic affinity of the synthetic CP40-KK for binding to mouse complement C3 was determined by Surface Plasmon Resonance (SPR). The results show (see FIG. 21) that human complement C3 binds CP40-KK with an affinity constant (KD) of 1.53. Mu.M, whereas mouse complement C3 binds CP40-KK with an affinity constant of 26.7. Mu.M.

TABLE 4CP40-KK kinetic affinity assay for human and mouse complement C3

After intervention, the plasma of the mouse is left, the level of the plasma C3a is detected, the heart tissue protein of the mouse is extracted, and the protein expression level of the C3 cracking segment in the heart tissue is detected. The results show that the plasma C3a level of the diabetic mice is obviously higher than that of the control group, and the plasma C3a concentration of the CP40-KK intervention group mice is obviously reduced, which indicates that the CP40-KK can inhibit the lysis of the complement C3 of the mice (see figure 22); western blot results showed that the protein levels of the C3 cleaved fragments of CP40-KK interfering group were significantly lower than those of DCM group and C3aRA group, again demonstrating that CP40-KK inhibits complement C3 cleavage (see FIG. 23).

(II) C3aR antagonists inhibit C3aR activation in mouse cardiac tissue

Research shows that activation of ERK1/2 can be directly promoted after C3aR is activated, so that the activation level of C3aR is reflected by detecting the level of phosphorylated ERK1/2 at the downstream of the C3aR, and the result is shown in figure 24, the phosphorylation of heart ERK1/2 of diabetic mice is increased, and the phosphorylation level of heart ERK1/2 of diabetic mice can be obviously reduced through C3aRA intervention, so that the C3aRA can effectively play the role of antagonist and inhibit the activation of C3 aR.

Example 6 mechanism of C3aR antagonists to ameliorate diabetic cardiomyopathy

Based on the above results, the plasma C3 and C3a levels of diabetic mice were significantly increased and the expression of C3, C3a and C3aR in cardiomyocytes was up-regulated, and it was speculated that C3a, an anaphylatoxin derived from circulating or locally produced heart, might act by binding to C3aR in cardiomyocytes. After the C3aR is activated, inflammatory reaction is enhanced through signal molecules such as downstream ERK and the like, the oxidative stress level is improved, and further cardiac injury is promoted. In vivo and in vitro studies were also carried out to determine the specific regulatory mechanism of C3a on cardiomyocytes.

(I) C3aR antagonists ameliorate diabetic cardiomyopathy by inhibiting the inflammatory-small NLRP3-Caspase-1-IL-1 beta/IL-18 pathway

c3aR antagonists reduce the level of inflammation of cardiac tissue in diabetic cardiomyopathy mice

In order to determine the activation level of inflammatory corpuscle NLRP3 downstream pathway of mouse heart tissue, we reserve part of heart tissue, extract tissue protein, and detect the expression level of NLRP3-Caspase-1-IL-1 beta/IL-18 pathway related protein by Westernblot, and the result is shown in figure 25, the expression level of inflammatory corpuscle NLRP3, activated Caspase-1, activated IL-1 beta and IL-18 of diabetic mouse heart tissue is obviously higher than that of a control group, and after C3aRA intervention is given, the expression level of the inflammatory indicator protein is obviously reduced, which indicates that NLRP3 and the downstream signal pathway thereof are activated in DCM mouse heart tissue, and the C3aRA intervention can reduce the inflammatory level of heart tissue and improve diabetic cardiomyopathy by blocking NLRP3-Caspase-1-IL-1 beta/IL-18 pathway.

C3aR antagonist can block myocardial cell C3a-C3aR channel and reduce inflammation level induced by C3a

(1) To verify the anti-inflammatory effect of C3aRA, a related experiment was performed on the human cardiac muscle cell line AC16 (available from Shanghai Chunhai Biotech Ltd.). The cell experiments are divided into four groups of control, C3aRA, C3a and C3a + C3aRA. Starvation treatment is carried out for 12 hours by changing a serum-free culture medium before intervention, a complete culture medium is changed after treatment, pretreatment is carried out for half an hour by adding C3aRA (1 mmol/L), stimulation is carried out by adding C3a (0.5 mmol/L), and total cell protein is extracted after 48 hours. The results of verification of the activation effect of C3a on myocardial cell C3aR and the antagonism effect of C3aR show (FIG. 26) that exogenous C3a intervention can activate C3aR of myocardial cell AC16, directly results in increased downstream ERK phosphorylation, and C3aRA can also act on AC16, play the role of antagonist and inhibit the activation of C3 aR.

(2) The inflammation-inducing effect of C3a on cardiomyocytes AC16 and the protective effect of C3aRA were further investigated. Cardiomyocytes AC16 were cultured on high sugar medium (25 mmol/L glucose) and plated onto six-well plates, four groups of controls, C3aRA, C3a + C3aRA at the time of intervention. The serum-free culture medium is replaced with a complete culture medium for starvation treatment for 12 hours before intervention, C3aRA (1 mmol/L) is added for pretreatment for 30 minutes, then C3a (0.5 mmol/L) is added for stimulation, total cell protein is extracted after 48 hours, and the expression conditions of NLRP3, caspase-1, IL-1 beta and IL-18 are detected by Westernblot. The cell experiment result is consistent with the animal experiment, the expression level of the NLRP3 inflammatory corpuscle protein of the cell cultured by high sugar is not different among various groups, and the C3a intervention obviously increases the activation of Caspase-1, IL-1 beta and IL-18, which suggests that under the high sugar environment, C3a can play a second signal participating in the activation of NLRP3, stimulates the shearing activation of Caspase-1 and IL-1 beta, and C3aRA can antagonize the proinflammatory effect of C3a, reduces the activation level of downstream effector molecules, and shows that the C3a-C3aR axis plays an important role in the activation process of the inflammatory corpuscle (see figure 27).

(di) C3aR antagonists for ameliorating diabetic cardiomyopathy by reducing myocardial cell oxidative stress levels

C3aR antagonists are able to reduce the level of DCM mouse heart tissue NADPH oxidase subunit expression

The mouse heart tissue is reserved, tissue protein is extracted, the expression level of NADPH oxidase subunit is detected by Westernblot and immunohistochemical staining, and the result (figure 28) shows that the expression levels of gp91-phox, p67-phox and p47-phox of the mouse heart tissue in the diabetic group are obviously higher than those of a control, which indicates that NADPH oxidase is activated in the heart tissue of the diabetic mouse, and the expression level of the NADPH oxidase can be reduced to a certain extent by C3aRA intervention, so that the protection effect is exerted.

C3aR antagonists inhibit myocardial cell NADPH oxidase activity

To verify the conclusion of the animal experiments, the same experiments were performed on the human cardiac muscle cell line AC 16. Cellular intervention was also divided into four groups, control, C3aRA, C3a + C3aRA, and total cellular protein was extracted 48 hours after stimulation with C3a (0.5 mmol/L) and NADPH oxidase subunit expression was examined using Westernblot (FIG. 29). C3a intervention increases the expression of subunits gp91-phox, p67-phox and p47-phox of NADPH oxidase of myocardial cells, and reduces the expression of superoxide dismutase SOD2, which indicates that C3a intervention increases the activity of NADPH oxidase of myocardial cells, reduces the oxidation resistance, and C3aRA intervention can reduce the oxidative stress level.

c3aR antagonists reduce C3 a-induced mitochondrial damage and ameliorate mitochondrial oxidative stress

The effects of C3a and C3aRA on mitochondrial oxidative stress were explored. The same intervention was performed on cardiomyocytes AC16, and MitoSOX staining and JC-1 staining of the cells detected changes in mitochondrial ROS accumulation levels and mitochondrial membrane potential. Since MitoSOX can target to mitochondria, bind to anionic superoxide in mitochondria, bind to mitochondrial DNA after oxidation, and fluoresce red, mitoSOX staining was used to reflect mitochondrial oxidative stress levels, as a result see fig. 30, mitochondrial ROS levels significantly increased in cells after C3a intervention, while C3aRA intervention inhibits its elevation.

When the mitochondrial membrane potential is normal, JC-1 exists in a polymer form and emits red fluorescence, and when the mitochondrial membrane potential is reduced or eliminated, JC-1 exists in a cytoplasm in a monomer form and emits green fluorescence. Thus, JC-1 staining can reflect whether mitochondria are damaged and changes in their membrane potential. JC-1 staining result shows (figure 31), the positive control CCCP acts on cells for 30 minutes to enable the mitochondrial membrane potential to disappear, the mitochondrial membrane potential is reduced after C3a intervenes, the staining is mainly green fluorescence, and C3aRA has a certain improvement effect on mitochondrial damage.

In conclusion, C3a can cause mitochondrial damage, reduce mitochondrial membrane potential and increase ROS production, and C3aRA intervention can relieve mitochondrial damage to a certain extent, reduce mitochondrial oxidative stress level and protect myocardial cells.

c3aR antagonists exert protective effects in diabetic cardiomyopathy by lowering intracellular ROS levels, reducing apoptosis

(1) The total intracellular ROS levels were explored. The staining of the myocardial cells subjected to C3a and C3aRA intervention with superoxide anion fluorescent probe (DHE) shows that (figure 32), the number of DHE staining positive cells in one visual field of the C3a intervention group is obviously greater than that of the control group, and the C3aRA intervention can block the induction of the C3a on oxidative stress to a certain extent.

(2) The effect of C3aRA on apoptosis was explored. TUNEL staining of mouse heart tissue was performed and the results (fig. 33) show that diabetic mice show increased cardiomyocyte apoptosis and that C3aRA has some protective effect.

(3) Further validation was performed on cardiomyocytes AC16 cultured in vitro. The AC16 myocardial cells cultured by high sugar are divided into 4 groups, the intervention is performed before, the total cell protein is extracted after the intervention is completed, the apoptosis index is detected by Westernblot, and TUNEL staining is carried out, and the result (figure 34) shows that the Bax/Bcl-2 and the sheared Caspase-3 level of the C3a intervention group are obviously increased, and the C3aRA intervention has a certain improvement effect. The TUNEL staining result is consistent with that of an animal tissue section, the number of TUNE staining positive cells of the C3a intervention group is obviously higher than that of the other three groups, and the suggestion is that C3a can increase the ROS accumulation of myocardial cells and promote apoptosis under a high-sugar environment, and the pathogenic effect can be blocked by C3aRA.

(III) the high-sugar and C3 a-induced NLRP3 activation and ROS generation influence each other and are jointly involved in the pathogenesis of diabetic cardiomyopathy

NLRP3 inhibitor MCC950 can reduce intracellular ROS level

The NLRP3 inhibitor MCC950 was selected to intervene in AC16 cardiomyocytes and cells were DHE stained to observe the effect of intervention on intracellular ROS levels. The results show (figure 35) that the number of DHE staining positive cells was significantly higher for cells that intervened with both high sugars and C3a than for controls, whereas the intracellular ROS levels decreased significantly after inhibition of NLRP3 with MCC950, i.e. inhibition of the NLRP3 pathway decreased intracellular ROS accumulation.

ROS inhibitor N-acetylcysteine can inhibit NLRP3 expression up-regulation

The ROS inhibitor N-acetylcysteine (NAC) was selected to intervene in cardiomyocytes AC16 and intracellular ROS and NLRP3 levels were examined. The results are shown in fig. 36, and the DHE staining positive cell number of the NAC stem-control group is significantly lower than that of the C3a + HG stem-control group, which proves that NAC can really inhibit ROS. Furthermore, westernblot results showed that the expression level of NLRP3 protein in NAC intervention group was comparable to that in control group and significantly lower than that in C3a + HG intervention group, suggesting that inhibition of intracellular ROS levels could prevent C3a + HG-induced elevation of NLLRP3 expression.

As can be seen from the above examples, C3a is the main effector molecule, and C3aR can be used as a therapeutic target for chronic complications of diabetes. C3a induces activation of NLRP3-Caspase-1-IL-1 beta/IL-18 pathway and accumulation of ROS of intracellular NADPH oxidase pathway and mitochondrion pathway of the myocardial cells through acting on C3aR of the myocardial cells, so that the oxidative stress level is increased and the apoptosis is increased. Inflammation and oxidative stress induced by C3a are causally and complementarily involved and participate in the development of diabetic cardiomyopathy. The C3aR antagonist can inhibit activation of NLRP3 channel, reduce oxidative stress level and reduce myocardial cell apoptosis so as to play a protective role in diabetic cardiomyopathy.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.

Claims (6)

1. Application of complement C3 cracking inhibitor in preparing medicine for treating diabetic cardiomyopathy is disclosed.

2. The use of claim 1, wherein the inhibitor of complement C3 cleavage comprises CP40-KK.

3. The application of receptor antagonist of complement C3 cleavage product C3a in preparing medicine for treating diabetic cardiomyopathy.

4. The use according to claim 3 wherein the receptor antagonist of C3a comprises C3aRA.

5. The use according to claim 4, wherein said C3aRA inhibits phosphorylation of ERK downstream of C3 aR.

6. The use according to claim 4 wherein the C3aRA acts by inhibiting the expression of NLRP3-Caspase-1-IL-1 β/IL-18 pathway related molecules.

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