Application of crocin A in the preparation of drugs for treating IgA nephropathy

Abstract

This invention relates to the field of biomedical technology, and particularly to the application of crotonin A in the preparation of drugs for treating IgA nephropathy. Pharmacodynamic experiments conducted in vivo on a Thy1 rat model showed that crotonin A effectively reduced 24-hour urinary protein and the urinary protein-to-creatinine ratio in an IgA nephropathy model—an anti-Thy1 rat nephritis model—and inhibited the expression of mRNAs of genes related to renal cortical inflammation, proliferation, and fibrosis, including Cyclin E, α-SMA, FN, CCL2, NF-κB, and IL-6, further improving renal pathology. In in vitro cell models, crotonin A inhibited the proliferation of rat mesangial cells and suppressed the expression of mRNAs of TGF-β, Cyclin E, α-SMA, CCL2, NF-κB, and IL-6, indicating that crotonin A has significant anti-inflammatory, anti-cell proliferation, and anti-fibrotic effects. Brucella oleracea has considerable therapeutic potential, providing a potential new drug option for the clinical prevention and treatment of IgA nephropathy, and is of great significance for the development of safe and effective drugs for the treatment of IgA nephropathy.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, and in particular to the application of crotonin A in the preparation of drugs for treating IgA nephropathy. Background Technology

[0002] IgA nephropathy is the most common primary glomerular disease in the world. The etiology and pathological mechanism of IgA nephropathy are unknown. Its main pathological feature is the deposition of immune complexes formed by the polymerization of IgA1 and abnormally glycosylated IgA1 in the glomerular mesangial area. Clinical manifestations of IgA nephropathy are diverse and vary considerably. Most patients have an insidious onset. Clinically, approximately 40%-45% of patients present with isolated hematuria (microscopic or gross hematuria), 35% present with hematuria accompanied by mild to moderate proteinuria, and some present with isolated proteinuria. Epidemiological studies worldwide show significant geographical differences in the incidence of IgA nephropathy. In Asia, the incidence rate is as high as 60%, while in Europe and North America, the reported incidence rates are 30% and 10%, respectively. Approximately 80% of IgA nephropathy patients are children and young adults (20-30 years old). Although the clinical progression of IgA nephropathy is highly heterogeneous, most cases exhibit a chronic, progressive development, with 15-20% of patients progressing to end-stage renal disease within 10 years of initial onset. ESRD (extracorporeal membrane oxygenation disease) affects 30-40% of patients, who progress to ESRD within 20-30 years of onset, eventually leading to dialysis or kidney transplantation. This places a huge socioeconomic burden on individuals and the global health economy.

[0003] With the extensive research on the mechanism of IgA nephropathy in recent years, the medical community now believes that IgA nephropathy is an autoimmune disease caused by multiple factors, and has proposed the "quadruple blow" theory: (1) a significant increase in the synthesis of low-glycosylated IgA1 (Gd-IgA1) in the blood circulation; (2) the production of autoantibodies against Gd-IgA1 molecules; (3) the formation of circulating immune complexes containing pathogenic Gd-IgA1; (4) the deposition of complexes in the glomerular mesangial area, leading to activation of glomerular mesangial cells and glomerular damage. The main differences between IgA nephropathy and diabetic nephropathy are: (1) different pathogenic factors. Most patients develop IgA nephropathy mainly due to infectious factors, such as upper respiratory tract infection and intestinal infection, which leads to immune system disorder and causes immune complex deposition in the kidneys. The pathogenic factors of diabetic nephropathy are closely related to persistent hyperglycemia, insulin resistance, unreasonable diet, and renal microvascular lesions; (2) different symptoms. After diabetic nephropathy occurs, the first symptom is elevated blood sugar, followed by hypertension, edema, and massive proteinuria. After IgA nephropathy occurs, in addition to hypertension and edema, patients will also experience visible hematuria, increased foamy urine, gradually decreasing urine volume, lower back pain, and increased frequency of urination at night; (3) Different treatment plans. Treatment for IgA nephropathy generally involves choosing drugs to control infection and proteinuria. If hypertension occurs, antihypertensive drugs can be taken. The treatment for diabetic nephropathy is mainly through hypoglycemic drugs and antihypertensive drugs. Therefore, IgA nephropathy is widely recognized as a complex disease involving multiple genes and factors. In recent years, numerous genome-wide association studies (GWAS) have suggested that susceptibility to IgA nephropathy is related to certain genetic variations in the mucosal immune system, complement replacement pathway, antigen processing, and presentation pathway. To date, there is still no effective cure for IgA nephropathy. Currently, commonly used clinical treatments include hormones, cyclophosphamide, azathioprine, anticoagulants, etc., which have certain effects in alleviating symptoms, reducing proteinuria, and inhibiting active lesions, but they cannot effectively delay or stop the progression of chronic kidney disease (CKD). Moreover, their clinical application is limited due to the frequent serious adverse reactions or toxic side effects. Summary of the Invention

[0004] To address the aforementioned problems, based on existing drugs for treating IgA nephropathy, this invention provides the application of crotonin A in the preparation of drugs for treating IgA nephropathy.

[0005] On the one hand, the present invention provides the use of crotonin A in the preparation of medicaments for treating IgA nephropathy.

[0006] Furthermore, the drug for treating IgA nephropathy prepared from crocin A can inhibit the mRNA expression of genes related to renal cortical inflammation, proliferation, and fibrosis, such as Cyclin E, α-SMA, CCL2, NF-κB, IL-6, and FN.

[0007] Furthermore, drugs prepared from crocin A for treating IgA nephropathy can inhibit the proliferation of glomerular mesangial cells.

[0008] Furthermore, drugs prepared from crotonin A for treating IgA nephropathy can reduce proteinuria and improve kidney pathology.

[0009] Furthermore, drugs for treating IgA nephropathy prepared from crotonin A can be oral preparations, sublingual tablets, pills, or injections.

[0010] Furthermore, oral preparations include tablets, capsules, granules, pills, drops, fruit juices, or syrups.

[0011] Furthermore, the injection is an injection solution, a powder injection, or a lyophilized powder injection.

[0012] On the other hand, the present invention also provides a medicament for treating IgA nephropathy, comprising crotonin A and pharmaceutically acceptable excipients.

[0013] Furthermore, pharmaceutically acceptable excipients include any one or a combination of several of the following: disintegrants, humectants, binders, fillers, absorption enhancers, solvents, lubricants, surfactants, flavorings, sweeteners, antioxidants, preservatives, or colorings.

[0014] Compared with the prior art, the beneficial effects of the present invention are:

[0015] 1. Pharmacodynamic experiments conducted in the Thy1 rat model in vivo demonstrated that crotonin A can effectively inhibit the mRNA expression of genes related to renal cortical inflammation, proliferation, and fibrosis, as well as Cyclin E, α-SMA, CCL2, NF-κB, IL-6, and FN, in the Thy1 rat model of IgA nephropathy. It also reduced 24-hour urinary protein and the urinary protein-to-creatinine ratio, improving renal pathology. In an in vitro cell model, crotonin A inhibited the proliferation of rat mesangial cells and suppressed the expression of mRNAs such as TGF-β, Cyclin E, α-SMA, CCL2, NF-κB, and IL-6, indicating that crotonin A has significant anti-inflammatory, anti-cell proliferation, and anti-fibrotic effects. The significant therapeutic efficacy of crotonin A in treating IgA nephropathy demonstrates considerable therapeutic potential, providing a potential new drug option for the clinical prevention and treatment of IgA nephropathy. This research is of significant implications for exploring safe and effective drugs for treating IgA nephropathy and further investigating their underlying renal protective mechanisms.

[0016] 2. This invention confirms the definite efficacy of crotonin A monomer in the treatment of IgA nephropathy. Compared with traditional Chinese medicine compound formulations containing croton, this invention provides a clearer treatment and prevention of IgA nephropathy and reduces the occurrence of side effects to a certain extent. Attached Figure Description

[0017] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0018] Figure 1 The chemical structural formula of crocin A is shown below.

[0019] Figure 2 Example 1 of the present invention illustrates the effects of crocin A on the antagonistic effects on the urine protein-to-creatinine ratio, 24-hour urine protein, and kidney pathology in Thy1 model rats;

[0020] Figure 3 Example 1 of this invention illustrates the effect of crocin A on the antagonistic activity of CCL2, IL-6, NF-κB, α-SMA, FN, and Cyclin E mRNA in the renal cortex of Thy1 model rats.

[0021] Figure 4 Example 2 of this invention illustrates the effect of crocin A on LPS-induced mesangial cell proliferation;

[0022] Figure 5 Example 2 of the present invention describes the effects of crocin A on LPS-induced proliferation of related mesangial cells and the expression of inflammatory factors TGF-β, α-SMA, Cyclin E, IL-6, CCL2, NF-κB mRNA, as well as cell cycle distribution. Detailed Implementation

[0023] The technical solution of the present invention will be clearly and completely described below with reference to specific embodiments. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the protection scope of the present invention.

[0024] The crocin A (BA) described in this invention is a quassin-like compound extracted from the dried fruit of *Brucea javanica*, CAS: 25514-31-2, with the molecular formula C0.05. 26 H 34 O11 It has a molecular weight of 522.53, contains one oxygen-containing heterocycle and one lactone ring, and its chemical structure is as follows: Figure 1 As shown. This invention does not specifically limit the source of the crotonin; commercially available crotonin A, commonly used in the art, is sufficient.

[0025] Example 1: The role of crotonin A in the prevention and treatment of IgA nephropathy in vivo.

[0026] 1.1 Materials:

[0027] Male Wistar animals aged 6-8 weeks (all SPF grade) were purchased from Zhejiang Vital River Laboratory Animal Technology Co., Ltd.; crocin A was purchased from Nanjing Chunqiu Bioengineering Co., Ltd.

[0028] 1.2 Animal husbandry and grouping:

[0029] The experimental rats were housed in the experimental animal barrier system of the Guangdong Provincial Institute of Traditional Chinese Medicine, fed with ordinary feed, and given free access to food and water. The lighting was alternating for 12 hours a day. The experiment has been approved by the Animal Experiment Ethics Committee.

[0030] Thirty-six male Wistar rats were randomly divided into six groups according to body weight after 7 days of acclimatization, with six rats in each group. These groups were designated as a healthy control group, a model group, a low-dose crotonin A administration group, a medium-dose crotonin A administration group, a high-dose crotonin A administration group, and an olmesartan positive control group (OM).

[0031] 1.3 Dosing regimen:

[0032] The control group and the model group were injected intraperitoneally with the corresponding volume of physiological saline. The low, medium and high dose groups of crotonin A were injected intraperitoneally with 0.5 mg / kg, 1 mg / kg and 1.5 mg / kg crotonin A solution, respectively, once every other day for 7 consecutive days. The olmesartan positive control group was given 10 mg / kg olmesartan by gavage once a day for 7 consecutive days.

[0033] 1.4 Detection Indicators

[0034] 1.4.1 Rat urine protein detection:

[0035] Urine was collected from rats on days 3 and 7 after drug administration using metabolic cages. Rats were placed in metabolic cages for 24 hours without food or water, and urine was collected and recorded. The change in the urine protein-to-creatinine ratio was detected using a fully automated biochemical analyzer, and the 24-hour urine protein quantification was calculated.

[0036] 1.4.2 PCR detection of rat renal cortex: The expression changes of important inflammatory factors in the kidney, such as CCL2, NF-κB, IL-6, α-SMA, Cyclin E, and FN mRNA, were detected.

[0037] 1.4.3 Detection of rat kidney pathology: HE and PAS staining were used to observe the improvement of rat kidney pathology.

[0038] 1.5 Results

[0039] Three days after administration, medium- and high doses of crotonin A effectively reduced the urinary protein-to-creatinine ratio and 24-hour urinary protein quantification in rats. Seven days after administration, low, medium, and high doses of crotonin A all reduced the urinary protein-to-creatinine ratio and 24-hour urinary protein quantification in rats, as shown in the following results. Figure 2 As shown in A-2B, pathological evaluation with PAS staining revealed partial glomerular hypertrophy in the model group, with significant mesangial cell proliferation and mesangial matrix deposition, accompanied by capillary loop compression, capillary lumen narrowing, and isolated structural damage, vascular disappearance, and the appearance of nodules and clumps of solid areas, exhibiting a diffuse finger-like distribution. Administration of crotonin A effectively improved mesangial cell proliferation and mesangial matrix deposition, improving renal pathology, as shown in the results. Figure 2 As shown in C-2F, PCR experiments were performed on RNA extracted from rat renal cortex. The results showed that the mRNA levels of CCL2, IL-6, NF-κB, FN, α-SMA, and Cyclin E were significantly increased in the model group compared to the healthy control group. Administration of crotonin A significantly reduced the expression of these mRNAs. The results are as follows... Figure 3 As shown in Figure A, the above results all indicate that crotonin A can reduce renal inflammation, decrease mesangial cell proliferation and mesangial matrix deposition, improve renal pathology, and prevent glomerular sclerosis, thereby playing a role in the prevention and treatment of IgA nephropathy.

[0040] Example 2: The effect of crocin A in treating LPS-induced mesangial cell proliferation and inflammation models

[0041] 2.1 Materials:

[0042] Rat glomerular mesangial cells HBZY-1 were purchased from the Cell Center of the Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences; crotonin A was purchased from Nanjing Chunqiu Biotechnology Co., Ltd.; LPS was purchased from Sigma-Aldrich, USA; BCA protein concentration assay kit was purchased from Thermo Scientific, USA; Trizol reagent (15596-018) was purchased from Life Technologies, USA; 2×EasyTaq PCR SuperMix (AS111) was purchased from Beijing TransGen Biotech Co., Ltd.

[0043] 2.2. Cell culture, modeling, grouping, and drug treatment:

[0044] HBZY-1 rat mesangial cells were cultured in DMEM complete medium containing 10% (v / v) FBS, 100 U / ml penicillin, and 100 μg / ml streptomycin at 37°C in a sterile incubator with 5% CO2. After cell attachment, the cells were synchronized with serum-free medium for 24 hours and then divided into control, model, crotonin A, and olmesartan positive control groups. Mesangial cells were stimulated with 100 ng / ml LPS for 48 hours to induce mesangial cell proliferation and an inflammation model. Subsequently, different concentrations of crotonin A (30 nM, 60 nM, and 120 nM) were used to stimulate mesangial cells for 24 hours, and relevant tests were performed.

[0045] 2.2.1 The expression levels of mRNA of related genes such as TGF-β, α-SMA, Cyclin E, CCL2, NF-κB, and IL-6 were detected by qPCR. HBZY-1 cells in logarithmic growth phase were used for qPCR at a rate of 2 × 10⁻⁶ mRNA. 5 Cells were seeded per well in 6-well cell culture plates. After full cell adhesion, the cells were synchronized with serum-free DMEM medium for 24 h. The model group and the crotonin A treatment group were stimulated with 100 ng / ml LPS for 48 h, followed by stimulation with different concentrations of crotonin A (30 nM, 60 nM, 120 nM) for 24 h. Total RNA was extracted from the cells using the Trizol method, and cDNA was obtained by reverse transcription PCR using 5×PimeScriptRTmastermix. Then, using rat TGF-β, α-SMA, Cyclin E, CCL2, and NF-κB primers (primer sequences are shown in Table 1) and 2×SYBR Green Master as materials, the total system was 10 μl. The reaction conditions were: 94℃, 5 min pre-denaturation; 94℃, 30 s denaturation; 60℃, 30 s annealing; 72℃, 1 min extension, for 40 cycles. The expression fold was calculated using △△CT, where △△CT = (CT(target gene in test sample) - CT(internal standard gene in test sample)) - (CT(target gene in control sample) - CT(internal standard gene in control sample)), to obtain the mRNA expression level of the relevant genes in each group.

[0046] 2.2.2 Colony formation experiment: HBZY-1 rat mesangial cells were seeded at a density of 500 cells / well in 6-well plates. After the cells adhered, they were divided into a model group, a control group, a positive control group (olmesartan), and a group receiving crotonin A (30 nM, 60 nM, and 120 nM crotonin A). The medium was changed every 3 days, and the observation was carried out for a total of 14 days. The number of colonies formed in each culture well was calculated.

[0047] 2.2.3 EDU Cell Proliferation Assay: HBZY-1 rat mesangial cells were seeded at a density of 10,000 cells / well in 12-well plates. After cell adhesion, the cells were synchronized with serum-free medium for 24 hours and divided into a model group, a control group, a positive control group (olmesartan), and a crotonin A group (30 nM, 60 nM, and 120 nM crotonin A). The model group and the crotonin A group were stimulated with 100 ng / ml LPS for 48 hours. The proliferation of mesangial cells was observed for 24 hours after drug administration.

[0048] 2.2.4 Flow Cytometry Cell Cycle Experiment: HBZY-1 rat mesangial cells were seeded at a density of 100,000 per well in 6-well plates. After the mesangial cells adhered, they were synchronized with serum-free medium for 24 h and then divided into model group, control group, olmesartan group, and crotonin A group. The model group and crotonin A group were stimulated with 100 ng / ml LPS for 48 h, and then treated with different concentrations (30 nM, 60 nM, and 120 nM crotonin A) for 24 h. The cells were then collected, fixed overnight with pre-cooled 70% anhydrous ethanol, centrifuged at 3000g for 10 min the next day to remove the anhydrous ethanol, added 500 μL of propidium iodide staining solution (PI), and incubated at 37℃ in the dark for 30 min. The cells were then analyzed by flow cytometry.

[0049] 2.3 Results

[0050] CCK8 cell proliferation experiments showed that 100 ng LPS treatment for 48 h stimulated mesangial cell proliferation, while different concentrations of crocin A inhibited the proliferation of HBZY-1 mesangial cells. The results are as follows: Figure 4 As shown in A and 4B, this can simultaneously reduce the number of clones formed, such as Figure 4 As shown in C-4D, the EDU cell proliferation assay revealed that different concentrations of BA could inhibit mesangial cell replication and reduce mesangial cell proliferation. Figure 4 As shown in E-4F, further flow cytometry experiments revealed that stimulation with 100 ng / ml LPS induced more cells to enter the S phase, while reducing the number of cells in the G0G1 phase. However, the addition of different concentrations of crotonin A significantly increased the number of cells in the G0G1 phase and decreased the number in the S phase, inducing G0G1 phase arrest and thus inhibiting mesangial cell proliferation. This suggests that crotonin's inhibition of mesangial cell proliferation may be related to its induction of cell cycle arrest. The results are as follows... Figure 5As shown in A-5B, crocin A had an effect on LPS-induced expression of IL-6, CCL2, NF-κB, TGF-β, α-SMA, and Cyclin E mRNA in HBZY-1 cells. IL-6, CCL2, NF-κB, TGF-β, α-SMA, and Cyclin E are important cytokines in IgA nephropathy and are closely related to the development and progression of the disease. qPCR results showed that different doses of crocin A could inhibit LPS-induced expression of IL-6, CCL2, NF-κB, TGF-β, α-SMA, and Cyclin E mRNA. Figure 5 As shown in Figure C, in vitro experiments have shown that crotonin A has significant anti-inflammatory effects and inhibits mesangial cell proliferation, and can be used to prevent and treat IgA nephropathy.

[0051] The present invention has been further described above with reference to specific embodiments. However, it should be understood that the specific description herein should not be construed as limiting the nature and scope of the present invention. Various modifications made to the above embodiments by those skilled in the art after reading this specification are all within the scope of protection of the present invention.

Claims

1. Application of crotonin A in the preparation of drugs for treating IgA nephropathy.

2. The application according to claim 1, characterized in that, The drug is an oral preparation or an injection.

3. The application according to claim 2, characterized in that, The oral preparations are tablets, capsules, granules, pills, drops, or syrups.

4. The application according to claim 2, characterized in that, The injection is either a liquid injection or a powder injection.

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