Cycloastragenol-flower type lactose nanoparticle and new application thereof

Abstract

The invention discloses cycloastragenol-flower-shaped lactose nanoparticles and a new application thereof. The cycloastragenol-flower-shaped lactose nanoparticles can effectively inhibit myocardial fibrosis caused by inflammatory cell infiltration and inflammatory factor gene expression level increase. And myocardial collagen volume fraction increase caused by myocardial fibers, myocardial fibrosis marker gene expression level increase and ANF gene expression level up-regulation in myocardial tissues are inhibited. The cycloastragenol-flower-shaped lactose particle can obviously improve the degree of myocardial fibrosis of an experimental mouse induced by CVB3 infection, and has a good application prospect in prevention and treatment of myocardial fibrosis.

Description

Technical Field

[0001] The present invention relates to a new use of cyclocarya palida alcohol-flower-shaped lactose nanoparticles (CAG-FL), and particularly relates to the use of cyclocarya palida alcohol-flower-shaped lactose nanoparticles (CAG-FL) in the preparation of a drug for treating myocardial fibrosis caused by inflammation. Background Art

[0002] Myocardial fibrosis (MF) is a cardiac interstitial remodeling characterized by excessive proliferation of myocardial interstitial fibroblasts, excessive deposition and abnormal distribution of collagen fibers, and a significant increase in collagen volume fraction caused by various factors. Myocardial fibrosis is closely related to a variety of cardiovascular diseases including dilated cardiomyopathy (DCM), and viral myocarditis is one of the most common causes of secondary DCM. Clinically, about 20% of patients may develop DCM; in primary dilated cardiomyopathy, about 60% of patients can detect viral infection, suggesting that the inflammatory response caused by infection may be an important initiating factor for DCM. The inflammatory response is an important factor in the occurrence and development of myocardial fibrosis, and the NLRP3 inflammasome plays an important role. After the NLRP3 inflammasome is activated, it further activates Caspase-1 to promote the secretion of downstream inflammatory factors and amplify the inflammatory response. The recruitment of inflammatory cells and the expression of inflammatory factors induce excessive proliferation and phenotypic transformation of myocardial fibroblasts; at the same time, cardiac microvascular endothelial cells can be transformed into activated fibroblasts through Endo-MT under the stimulation of inflammatory factors to participate in myocardial remodeling. TGF-β1 is the most important regulatory factor for Endo-MT, and inflammatory stimulation can significantly up-regulate the expression of TGF-β1, inducing phenotypic and functional changes of cardiac microvascular endothelial cells through the TGF-β1 / smad signaling pathway. Cardiac fibroblasts are the main cells secreting extracellular matrix in myocardial tissue, and type I and type III collagens are the main components of the extracellular matrix. Under pathological conditions, the synthesis of extracellular matrix increases and the degradation decreases, and the change in its content can be used to evaluate the degree of myocardial fibrosis. The fibrotic myocardium has increased stiffness and decreased compliance, directly activating the renin-angiotensin-aldosterone system and stimulating atrial cells to secrete an increased level of atrial natriuretic factor (ANF) to promote diuresis and natriuresis, controlling blood volume and blood pressure.

[0003] Currently, there is no specific treatment for myocardial fibrosis in clinical practice. Generally, angiotensin-converting enzyme inhibitors (ACEIs), β-blockers, aldosterone receptor antagonists (ARBs), etc. are used. However, there are problems such as large side effects, many complications, and poor patient tolerance. Clinical studies have found that traditional Chinese medicine has played a positive role in the prognosis of patients, especially showing good therapeutic effects in the treatment of some severe or critically ill patients. Cycloastragenol is the active sapogenin after the glycosyl group of astragaloside IV, the active ingredient of astragalus, and belongs to the cyclolanostane-type tetracyclic triterpenoid compound( Figure 1 ). Research shows that cycloastragenol has anti-aging, antiviral, anti-inflammatory, and immunomodulatory effects. However, its water-insoluble property limits its clinical use. The research team of this invention has recently prepared medicinal flower-shaped carrier lactose microparticles with nanostructured pores( Figure 2 , Figure 3 ), and preliminarily explored its adsorption and desorption capabilities for cycloastragenol( Figure 4 ). On this basis, cycloastragenol-flower-shaped lactose preparations were prepared, significantly improving the water solubility and bioavailability of the drug and reducing the drug toxicity. This new type of nano-preparation has obtained an invention patent authorization (Patent No. ZL202111026322.X). Astragalus is a traditional Chinese medicine for "tonifying qi". Astragalus and its active ingredients have been clinically applied in the fields of immunomodulation, antiviral, and anti-organ fibrosis. However, there is currently no report on the treatment of myocardial fibrosis with cycloastragenol. The clinical and basic research on using nano-carriers to transport cycloastragenol for the prevention and treatment of myocardial fibrosis is even blank. Summary of the Invention

[0004] Objective of the Invention: The objective of this invention is to provide the use of cycloastragenol-flower-shaped lactose nanoparticles in the preparation of drugs for the treatment of myocardial fibrosis.

[0005] This invention discovers that cycloastragenol-flower-shaped lactose particles have a significant improvement effect on a mouse myocardial fibrosis model induced by coxsackievirus B3 (CVB3) infection.

[0006] Technical Solution: On the one hand, this invention provides cycloastragenol-flower-shaped lactose particles, whose active ingredient is cycloastragenol and the carrier is flower-shaped lactose particles.

[0007] On the other hand, this invention provides the use of cycloastragenol-flower-shaped lactose nanoparticles in products for the prevention and / or treatment of myocardial fibrosis.

[0008] Furthermore, it is any one of the following:

[0009] (1) Preparation of products for inhibiting myocardial fibrosis caused by myocardial inflammatory cell infiltration;

[0010] (2) Preparation of products for inhibiting the increase in myocardial collagen volume fraction caused by myocardial fibrosis;

[0011] (3) Prepare a product for inhibiting the participation of TGF-β1-mediated endothelial-mesenchymal transition in the process of myocardial fibrosis.

[0012] (4) Prepare a product for inhibiting the up-regulation of the expression of myocardial fibrosis marker genes in myocardial tissue caused by myocardial fibrosis;

[0013] (5) Prepare a product for inhibiting myocardial fibrosis caused by the up-regulation of the expression of inflammatory factors in myocardial tissue;

[0014] (6) Prepare a product for inhibiting the up-regulation of the expression of ANF gene in myocardial tissue caused by myocardial fibrosis and / or cardiac insufficiency;

[0015] The present invention also protects a product, the active ingredient of which is cycloastragenol and the carrier is flower-shaped lactose; the uses of the product are any one of the following:

[0016] (1) Inhibit myocardial fibrosis caused by infiltration of cardiac inflammatory cells;

[0017] (2) Inhibit the increase in myocardial collagen volume fraction caused by myocardial fibrosis;

[0018] (3) Inhibit the participation of TGF-β1-mediated endothelial-mesenchymal transition in the process of myocardial fibrosis.

[0019] (4) Inhibit the up-regulation of the expression of myocardial fibrosis marker genes in myocardial tissue caused by myocardial fibrosis;

[0020] (5) Inhibit the up-regulation of the expression of ANF gene in myocardial tissue caused by myocardial fibrosis and / or new cardiac insufficiency;

[0021] (6) Inhibit myocardial fibrosis caused by the up-regulation of the expression of inflammatory factors in myocardial tissue;

[0022] (7) Inhibit TGF-β1-mediated endothelial-mesenchymal transition in myocardial tissue.

[0023] Any of the above-mentioned myocardial fibrosis marker genes may specifically be Collagen1 gene, Collagen3 or TGF-β1 gene.

[0024] Any of the above-mentioned inflammatory factors in myocardial tissue may specifically be Caspase-1 gene, IL-6 gene, IL-18 gene, NLRP3 gene.

[0025] Any of the above-mentioned products may be a drug, food or health product.

[0026] Furthermore, the drug effect has dose dependence, and the dose is preferably 5 mg / kg, 10 mg / kg, 20 mg / kg.

[0027] The present invention also protects a drug for preventing myocardial fibrosis, whose active ingredient is cyclocophorol and whose carrier is flower-shaped lactose particles.

[0028] The present invention also protects a drug for treating myocardial fibrosis, whose active ingredient is cyclocophorol and whose carrier is flower-shaped lactose particles.

[0029] Any of the above-mentioned drugs can be introduced into the body by means of oral administration, injection, nasal dropping, eye dropping, nasal spraying, eye spraying, jetting, osmosis, absorption, physical or chemical mediation. Any of the above-mentioned drugs can be made into various forms such as injection solution, suspension, powder, tablet, granule, etc. The drugs in the above various dosage forms can be prepared according to the conventional methods in the pharmaceutical field.

[0030] Compared with the prior art, the present invention has the following beneficial effects:

[0031] The present invention proves through experiments that cyclocophorol-flower-shaped lactose particles can effectively inhibit myocardial fibrosis caused by the infiltration of inflammatory cells and the increase in the gene expression level of inflammatory factors; inhibit the increase in myocardial collagen volume fraction, the increase in the gene expression level of myocardial fibrosis markers and the up-regulation of the gene expression level of ANF in myocardial tissue caused by myocardial fibrosis. In summary, cyclocophorol-flower-shaped lactose particles can significantly improve the degree of myocardial fibrosis induced by CVB3 infection in experimental mice and have good application prospects in the prevention and treatment of myocardial fibrosis. Description of the Drawings

[0032] Figure 1 is the structural formula of cyclocophorol;

[0033] Figure 2 is the medicinal flower-shaped carrier lactose microparticle containing nanostructured pores Figure 1 ;

[0034] Figure 3 is the medicinal flower-shaped carrier lactose microparticle containing nanostructured pores Figure 2 ;

[0035] Figure 4 is the adsorption and desorption ability curve of the medicinal flower-shaped lactose carrier to cyclocophorol;

[0036] Figure 5 is the HE staining of paraffin sections of mouse hearts ( Figure 5 A) and the pathological score ( Figure 5 B);

[0037] Figure 6 is the Sirius red staining of paraffin sections of mouse hearts ( Figure 6 A) and the results of collagen volume fraction ( Figure 6 B);

[0038] Figure 7Results of relative expression levels of CollagenI, CollagenIII, TGF-β1, and ANF genes in mouse myocardial tissues by Real TimePCR;

[0039] Figure 8 Results of relative expression levels of NLRP3, Caspase-1, IL-18, and IL-6 genes in mouse myocardial tissues by Real Time PCR. Specific implementation scheme

[0040] To make the objectives, technical solutions, and advantages of the present invention clearer, the technical solutions of the present invention will be further described below.

[0041] The preparation method of cyclocophorol-flower-shaped lactose particles in the following experiments adopts the method of patent number ZL202111026322.X.

[0042] The raw material of cyclocophorol is purchased from Nanjing Daosifu Biotechnology Co., Ltd., with a purity greater than 98%;

[0043] CVB3 virus (Nancy strain) is stored in this laboratory for scientific research work.

[0044] Balb / C mice are purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.

[0045] Example 1: Role of cyclocophorol-flower-shaped lactose nanoparticles in the preparation of drugs for treating myocardial fibrosis.

[0046] The specific method is as follows:

[0047] 1. Pharmacological experiments of cyclocophorol-flower-shaped lactose nanoparticles (CAG-FL)

[0048] (1) Establishment of a mouse model of myocardial fibrosis induced by CVB3 infection.

[0049] Male purebred Balb / C mice at 4-6 weeks of age are raised in an SPF-class animal room at a room temperature of 20°C ± 2°C, with free access to water and food, a 12-hour day-night cycle, and a relative humidity of 50% ± 10%. All experimental procedures involving animals have been approved by the Ethics Review Committee of the Experimental Animal Science Department of Fudan University.

[0050] Coxsackievirus B3 (CVB3) Nancy strain (stored in this laboratory) is passaged and replicated on human embryonic kidney cells, frozen and thawed 3 times, and the TCID 50 = 10 6.7 is measured on Wish cells, and 50 μl is used. The above virus is diluted and inoculated into mice by intraperitoneal injection under sterile conditions, and they are raised conventionally. Seven days after the first inoculation of CVB3, CVB3 virus is inoculated again once, and the mice are raised conventionally until the end of the modeling on the 14th day.

[0051] (2) Experimental grouping. The experimental mice were randomly divided into the following 5 groups (6 mice in each group) for the experiment.

[0052] Normal control group: Intragastric administration with normal saline (0.2 ml / mouse) once a day for 14 consecutive days.

[0053] Model control group: Model establishment by intraperitoneal inoculation with CVB3, and at the same time, intragastric administration with normal saline (0.2 ml / mouse) once a day, and raised until the 14th day.

[0054] High-dose CAG-FL group: Model establishment by intraperitoneal inoculation with CVB3. CAG-FL was dissolved in normal saline, and the intragastric administration dose was 20 mg / kg (calculated based on the effective ingredient of astragaloside IV), and continuously intragastric administered and raised until the 14th day.

[0055] Medium-dose CAG-FL group: Model establishment by intraperitoneal inoculation with CVB3. CAG-FL was dissolved in normal saline, and the intragastric administration dose was 10 mg / kg (calculated based on the effective ingredient of astragaloside IV), and continuously intragastric administered and raised until the 14th day.

[0056] Low-dose CAG-FL group: Model establishment by intraperitoneal inoculation with CVB3. CAG-FL was dissolved in normal saline, and the intragastric administration dose was 5 mg / kg (calculated based on the effective ingredient of astragaloside IV), and continuously intragastric administered and raised until the 14th day.

[0057] On the 15th day, the mice in each group were sacrificed after blood collection from the eyeballs, and the hearts of the mice were completely isolated. A part of the myocardial tissue was fixed with 10% neutral formaldehyde and used for pathological staining examination; another part of the tissue was quickly frozen in an -80°C refrigerator for Real time PCR detection.

[0058] (3) The myocardial tissue fixed with 10% neutral formaldehyde was paraffin-embedded, sectioned, and then stained with HE. Scoring was performed according to the degree of inflammatory cell infiltration and tissue lesions, with scores ranging from 0 to 4: 0 points: no lesions and inflammatory cell infiltration; 1 point: mild degree of lesions or inflammatory cell infiltration; 2 points: moderate degree of lesions or inflammatory cell infiltration; 3 points: severe degree of lesions or inflammatory cell infiltration; 4 points: very severe degree of lesions or inflammatory cell infiltration. The results were as Figure 5 shown: Compared with the normal control group, focal lesions and a large number of inflammatory cell infiltrations were visible in the myocardial tissue of the CVB3 model control group; compared with the CVB3 model control group, the degree of myocardial tissue lesions and inflammatory cell infiltrations in the low-dose, medium-dose, and high-dose CAG-FL groups were significantly improved. The results suggest that the low-dose (5 mg / kg), medium-dose (10 mg / kg), and high-dose (20 mg / kg) CAG-FL can significantly improve the myocardial tissue lesions and inflammatory cell infiltrations in the chronic stage after CVB3 infection.

[0059] (4) The paraffin-embedded and sectioned myocardial tissues fixed with 10% neutral formaldehyde were subjected to Sirius red staining, and the staining results were used for quantitative analysis of myocardial fibrosis using Image J software. The results are as Figure 6 shown: Figure 6 A shows the results of Sirius red staining, Figure 6 B shows the quantitative results of fibrosis degree. Compared with the normal control group, the myocardial collagen volume fraction of the CVB3 model control group was significantly increased as detected by Sirius red staining (p < 0.05); compared with the CVB3 model control group, the myocardial collagen volume fractions of the low-dose (5 mg / kg), medium-dose (10 mg / kg), and high-dose (20 mg / kg) CAG-FL groups were significantly decreased (p < 0.05). The results suggest that the low-dose (5 mg / kg), medium-dose (10 mg / kg), and high-dose (20 mg / kg) CAG-FL groups can significantly improve myocardial fibrosis in the chronic stage of CVB3 infection.

[0060] (5) Total RNA was extracted from the homogenized myocardial tissues stored at -80 °C and reverse transcribed into cDNA. Using the cDNA as a template, the gene expression levels of ANF, CollagenI, CollagenIII, TGF-β1, NLRP3, Caspase-1, IL-6, and IL-18 in the myocardial tissues of mice in each group were detected.

[0061] The ANF gene is the gene shown in GenBank: NM_009930; the CollagenI gene is the gene shown in GenBank: NM_007742; the CollagenIII gene is the gene shown in GenBank: NM_009930; the TGF-β1 gene is the gene shown in GenBank: NM_011577; the NLRP3 gene is the gene shown in GenBank: NM_145827; the Caspase-1 gene is the gene shown in GenBank: NM_009807; the IL-6 gene is the gene shown in GenBank: NM_001314054; the IL-18 gene is the gene shown in GenBank: NM_008360.

[0062] The primer sequences for detecting the ANF gene are as follows:

[0063] ANF-F: 5’-GCTTCCAGGCCATATTGGAG-3’ (15);

[0064] ANF-R: 5’-GGGGGCATGACCTCATCTT-3’ (16).

[0065] The primer sequences for detecting the CollagenI gene are as follows:

[0066] CollagenI-F: 5’-GCTCCTCTTAGGGGCCACT-3’(1);

[0067] CollagenI-R: 5’-CCACGTCTCACCATTGGGG-3’(2).

[0068] The primer sequences for detecting the CollagenIII gene are as follows:

[0069] CollagenIII-F: 5’-CTGTAACATGGAAACTGGGGAAA-3’(3);

[0070] CollagenIII-R: 5’-CCATAGCTGAACTGAAAACCACC-3’(4).

[0071] The primer sequences for detecting the TGF-β1 gene are as follows:

[0072] TGF-β1-F: 5’-CTCCCGTGGCTTCTAGTGC-3’(5);

[0073] TGF-β1-R: 5’-GCCTTAGTTTGGACAGGATCTG-3’(6).

[0074] The primer sequences for detecting the NLRP3 gene are as follows:

[0075] NLRP3-F: 5’-ATTACCCGCCCGAGAAAGG-3’(7);

[0076] NLRP3-R: 5’-TCGCAGCAAAGATCCACACAG-3’(8).

[0077] The primer sequences for detecting the Caspase-1 gene are as follows:

[0078] Caspase-1-F: 5’-ACAAGGCACGGGACCTATG-3’(9);

[0079] Caspase-1-R: 5’-TCCCAGTCAGTCCTGGAAATG-3’(10).

[0080] The primer sequences for detecting the IL-6 gene are as follows:

[0081] IL-6-F: 5’-CCAAGAGGTGAGTGCTTCCC-3’(11);

[0082] IL-6-R: 5'-CTGTTGTTCAGACTCTCTCCCT-3' (12).

[0083] The primer sequences for detecting the IL-17 gene are as follows:

[0084] IL-18-F: 5'-GACTCTTGCGTCAACTTCAAGG-3' (13);

[0085] IL-18-R: 5'-CAGGCTGTCTTTTGTCAACGA-3' (14).

[0086] Using the mouse β-actin gene as an internal reference, the primer sequences are as follows:

[0087] β-actin-F-F: 5'-GGCTGTATTCCCCTCCATCG-3';

[0088] β-actin-R-F: 5'-CCAGTTGGTAACAATGCCATGT-3'.

[0089] The gene expression results of CollagenI, CollagenIII, TGF-β1, and ANF are shown in Figure 7 as follows. Compared with the normal control group, the expressions of CollagenI, CollagenIII, and TGF-β1 in the myocardial tissue of the model control group were all significantly up-regulated (p < 0.001 or p < 0.01). Compared with the model control group, the expressions of CollagenI, CollagenIII, and TGF-β1 in the myocardial tissue of the low, medium, and high-dose groups of CAG-FL were all significantly decreased (p < 0.05 or p < 0.01). The results suggest that the high-dose group (20 mg / kg), medium-dose group (10 mg / kg), and low-dose group (5 mg / kg) of CAG-FL all have inhibitory effects on the up-regulation of the expression levels of genes related to myocardial fibrosis mediated by CVB3 infection to varying degrees.

[0090] Compared with the normal control group, the expression level of the ANF gene in the myocardial tissue of the model control group of mice was significantly increased (p < 0.001). Compared with the model control group, the expression levels of the ANF gene in the myocardial tissue of the high, medium, and low-dose groups of CAG-FL were all significantly decreased (p < 0.05). The results suggest that CAG-FL granules can have a significant inhibitory effect on the up-regulation of the expression level of the ANF gene caused by myocardial fibrosis in mice.

[0091] As Figure 8The expression levels of NLRP3 gene, Caspase-1 gene, IL-18 gene and IL-6 gene in mouse myocardium are shown. Compared with the normal control group, the expression levels of NLRP3, Caspase-1, IL-18 and IL-6 genes in the myocardial tissues of the model control group mice were significantly increased (p<0.05 or p<0.01). Compared with the model control group, the high, medium and low dose groups of CAG-FL could significantly reduce the expression levels of NLRP3, Caspase-1, IL-17 and IL-6 genes (p<0.05 or p<0.01). The results suggest that CAG-FL has an obvious inhibitory effect on the high expression of inflammatory factor genes mediated by the activation of NLRP3 inflammasome in mouse myocardial tissues after CVB3 infection.

[0092] The above are only the preferred embodiments of the present invention and do not impose any limitation on the present invention. Any person skilled in the art, without departing from the scope of the technical solution of the present invention, makes any form of equivalent substitution or modification and other changes to the technical solution and technical content disclosed by the present invention, all of which belong to the content of the technical solution of the present invention and are still within the protection scope of the present invention.

Claims

1. A cycloastragenol-flower-shaped lactose nanoparticle, characterized in that: The active ingredient is cycloastragenol and the carrier is flower-shaped lactose particles.

2. Use of the cycloastragenol-floral lactose nanoparticles according to claim 1 in the preparation of products related to the prevention and / or treatment of myocardial fibrosis.

3. The use according to claim 2, characterized in that: is any of the following, (1) Preparing a product for inhibiting myocardial fibrosis caused by myocardial inflammatory cell infiltration; (2) preparing a product for inhibiting the increase in myocardial collagen volume fraction caused by myocardial fibrosis; (3) preparing products for inhibiting TGF-β1-mediated endothelial-mesenchymal transition involved in myocardial fibrosis; (4) preparing a product for inhibiting the upregulation of myocardial fibrosis marker gene expression in myocardial tissue caused by myocardial fibrosis; (5) Preparation of products for inhibiting myocardial fibrosis caused by up-regulation of inflammatory factors in myocardial tissue; (6) preparing products for inhibiting up-regulation of ANF gene expression in myocardial tissue caused by myocardial fibrosis and / or heart failure; (7) Prepare products for inhibiting myocardial fibrosis caused by cardiac inflammatory cell infiltration.

4. The use according to claim 3, characterized in that: The myocardial fibrosis marker gene may specifically be Collagen1 gene, Collagen3 or TGF-β1 gene.

5. The use according to claim 4, characterized in that: The primer sequences used to detect the CollagenI gene are as follows: CollagenI-F: 5'-GCTCCTCTTAGGGGCCACT-3'(1); CollagenI-R: 5'-CCACGTCTCACCATTGGGG-3'(2); The primer sequences used to detect the CollagenIII gene are as follows: CollagenIII-F: 5'-CTGTAACATGGAAACTGGGGAAA-3'(3); CollagenIII-R: 5'-CCATGCTGAACTGAAAACCACC-3'(4); The primer sequences used to detect the TGF-β1 gene are as follows: TGF-β1-F: 5'-CTCCCGTGGCTTCTAGTGC-3'(5); TGF-β 1-R: 5'-GCCTTAGTTTGGACAGGATCTG-3'(6).

6. The use according to claim 3, characterized in that: The inflammatory factors in the myocardial tissue may specifically be Caspase-1 gene, IL-6 gene, IL-18 gene, and NLRP3 gene.

7. The use according to claim 6, characterized in that: The primer sequences used to detect the NLRP3 gene are as follows: NLRP3-F: 5'-ATTACCCGCCCGAGAAAGG-3'(7); NLRP3-R: 5'-TCGCAGCAAAGATCCACACAG-3'(8); The primer sequences used to detect Caspase-1 gene are as follows: Caspase-1-F: 5'-ACAAGGCACGGGACCTATG-3'(9); Caspase-1-R: 5'-TCCCAGTCAGTCCTGGAAATG-3'(10); The primer sequences used to detect the IL-6 gene are as follows: IL-6-F: 5'-CCAAGAGGTGAGGTGCTTCCC-3'(11); IL-6-R: 5'-CTGTTGTCAGACTCTCTCCCT-3'(12); The primer sequences used to detect the IL-17 gene are as follows: IL-18-F: 5'-GACTCTTGCGTCAACTTCAAGG-3'(13); IL-18-R: 5'-CAGGCTGTCTTTTGTCAACGA-3'(14).

8. The use according to claim 2, characterized in that: The product is a medicine, food or health product.

9. The use according to claim 8, characterized in that: The drug effect is dose-dependent, and the dose is preferably 5 mg / kg, 10 mg / kg, or 20 mg / kg.

10. The use according to claim 3, characterized in that: The primer sequences used to detect the ANF gene are as follows: ANF-F: 5'-GCTTCCAGGCCATATTGGAG-3'(15); ANF-R: 5'-GGGGGCATGACCTCATCTT-3'(16).

Images