Application of limonin in preparation of medicine for improving renal fibrosis

Abstract

The invention belongs to the technical field of biological medicines, and particularly relates to application of limonin in preparation of a medicine for improving renal fibrosis. The limonin used in the invention is also named evodia lactone, the CAS number of the limonin is 1180-71-8, the molecular formula of the limonin is C26H30O8, and the structure of the limonin compound is as shown in the formula I. The limonin compound can be applied to the field of pharmaceutical chemistry. According to the application, a unilateral ureteral obstruction model is constructed, a positive drug captopril is taken as a control group, and the application of limonin in improvement of renal fibrosis caused by acute kidney injury is verified. Results of embodiments show that limonin not only has low cytotoxicity and high safety, but also has anti-inflammatory and anti-fibrosis effects, and can inhibit proliferation and migration of renal fibroblasts and improve mitochondrial functions of renal epithelial cells.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine, and particularly relates to the application of limonin in the preparation of a drug for improving renal fibrosis. Background Art

[0002] Renal fibrosis is the common pathological basis for the progressive impairment of renal function caused by various reasons from acute renal injury to renal function, and is the main pathological change in almost all chronic kidney diseases and advanced renal diseases at the end stage. Its main characteristics are renal tissue damage, inflammation, extracellular matrix deposition and tubular epithelial-mesenchymal transition. Renal fibrosis affects about 10% of the world's population. The lifelong dialysis and kidney transplantation caused by the final renal failure bring serious economic burdens to patients, families and society. The pathogenesis of renal fibrosis is complex. In the early stage of acute renal injury, it is extremely important to protect the function of renal epithelial cells. However, in the process of progression after injury, the activation of renal fibroblasts induced by cell necrosis is the most important link in the development of chronic kidney disease. The core of its treatment is to reduce the deposition of extracellular matrix. At present, clinical treatment is mainly aimed at its primary disease, and there is no drug to reverse renal fibrosis. In clinical practice, glucocorticoids and renin-angiotensin-aldosterone signaling pathway blockers are mainly used to slow down renal dysfunction and delay the progression of late chronic kidney disease to the end stage. Sodium-glucose co-transporter 2 (SGLT2) inhibitors have been shown to have certain benefits in reducing renal inflammation, oxidative stress and fibrosis, but none of them are directly anti-renal fibrosis drugs. TGF-β blocker pirfenidone (PFD) is used for idiopathic pulmonary fibrosis, but it is still in Phase II clinical trials, and its efficacy is poor.

[0003] Therefore, it is urgent to develop new anti-renal fibrosis drugs with high safety and good efficacy. Summary of the invention

[0004] The purpose of the present invention is to provide a method for improving renal fibrosis caused by acute renal injury by using limonin. Limonin inhibits the activation and migration of renal fibroblasts and reduces the deposition of extracellular matrix, thereby improving renal function indicators and delaying the development of the kidney to end-stage renal failure.

[0005] In order to achieve the above object, the present invention provides the following technical solutions:

[0006] The present invention provides an application of limonin in the preparation of a drug for improving renal fibrosis, wherein the limonin has a CAS number of 1180-71-8 and a molecular formula of C 26 H 30 O8, molecular weight: 470.51; the structural formula of the limonin is shown in Formula I:

[0007]

[0008] Preferably, the limonin is derived from grapefruit seeds of the Rutaceae plant.

[0009] Preferably, the dosage of the limonin is 25-100 mg / kg.

[0010] Preferably, the dosage of limonin is suitable for all animals, preferably rats.

[0011] Preferably, the drug concentration of the limonin is 3.125-12.5 mg / ml.

[0012] The present invention also provides the use of the limonin in inhibiting the activation and migration of renal fibroblasts.

[0013] Preferably, the limonin inhibits the expression of TGF-β and reduces the migration of renal fibroblasts.

[0014] The present invention also provides the use of the limonin in reducing the deposition of extracellular matrix in kidney.

[0015] The present invention also provides the use of the limonin in improving renal function impairment caused by acute kidney injury.

[0016] Preferably, the limonin reduces blood creatinine and urea nitrogen levels and improves renal function.

[0017] Beneficial effects of the present invention:

[0018] The limonin of the invention has low cytotoxicity and high safety, can significantly improve renal function indicators, has anti-inflammatory and anti-fibrosis effects, inhibits the proliferation and migration of renal fibroblasts, and improves the mitochondrial function of renal epithelial cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative labor.

[0020] Figure 1 The morphological diagram of damaged kidneys after 3 weeks of treatment, from left to right are sham operation group, model group (UUO group), UUU + 25 mg / kg limonin (UUO + low-dose group), UUU + 50 mg / kg limonin (UUO + medium-dose group), UUU + 100 mg / kg limonin (UUO + high-dose group) group and UUU + 30 mg / kg captopril positive drug group (UUO + captopril group);

[0021] Figure 2 Effects of limonin treatment for 3 weeks on the morphology of damaged kidneys in renal obstruction caused by left ureteral stenosis (n=8, *: compared with the sham group; #: compared with the UUO group);

[0022] Figure 3 Effects of limonin treatment for 3 weeks on renal function biochemical indices in renal injury caused by left ureteral stenosis (n=8, BUN is serum urea nitrogen content, SCr is serum creatinine value; *: compared with the sham group; #: compared with the UUO group);

[0023] Figure 4 The effect of limonin treatment for 3 weeks on renal cortical pathology caused by UUO (n=8, the sample was collected from the renal cortex, and the staining was H&E staining and Masson staining, HE staining was pink, and Masson staining was positive for blue);

[0024] Figure 5 The graphs show the effect of 24h limonin treatment on the migration ability of renal fibroblasts induced by TGF-β and the migration ability of cells detected by Transwell assay (n=6, *: compared with the Control group; #: compared with the TGF-β group). DETAILED DESCRIPTION

[0025] The present invention provides the use of limonin in the preparation of a drug for improving renal fibrosis. The limonin used in the present invention is also known as Evodin, with a CAS number of 1180-71-8 and a molecular formula of C 26 H 30 O8, molecular weight: 470.51, extracted from grapefruit seeds of Rutaceae, pure white, bitter, crystalline. Limonin is easily soluble in ester-soluble organic solvents, difficult to dissolve in water, and has high solubility in methanol and ethanol. The drug has low toxicity. In the rat animal model, 100 mg / kg and 200 μmol / L at the cell level are non-cytotoxic. The drug is easy to extract and obtain, and is widely distributed in Rutaceae plants such as citrus, lemon and grape seeds.

[0026] The dosage of the limonin of the present invention is 25-100 mg / kg, and the drug concentration of the limonin is 3.125-12.5 mg / ml. The dosage and drug concentration are suitable for all animals, preferably rats.

[0027] Captopril is a synthetic non-peptide angiotensin converting enzyme (ACEI) inhibitor, which mainly acts on the renin-angiotensin-aldosterone system (RAAS system). The present invention uses captopril as a positive drug group, and uses the UUO model to set different experimental groups to verify that the limonin of the present invention not only has low cytotoxicity, high safety, and can significantly improve renal function indicators, but also has anti-inflammatory and anti-fibrosis effects, inhibits the proliferation and migration of renal fibroblasts, and improves the mitochondrial function of renal epithelial cells, and has the potential prospect of preparing anti-renal fibrosis drugs with high safety and good effects.

[0028] The UUO model (Unilateral Ureteral Obstruction model) used in the embodiment of the present invention is an animal model for studying renal interstitial fibrosis, which mainly induces renal injury and fibrosis by unilateral ureteral ligation. In the UUO model, the main purpose of the sham group (Sham group) is to serve as a control group to exclude the influence of the operation itself on the experimental results. The preparation principle of the UUO model is to cause obstruction by unilateral ureteral ligation, resulting in urine retention, compressing the renal tubules, and then causing progressive necrosis of renal tubular epithelial cells, interstitial acute and chronic inflammatory cell infiltration, and finally forming progressive renal interstitial fibrosis. This pathological feature is commonly used in evaluating the improvement of drugs.

[0029] The experimental object SPF-grade SD rats used in the embodiments of the present invention refer to Sprague Dawley rats that have been specially treated and are free of specific pathogens (SPF). Such rats are widely used in experimental animals, mainly in the fields of drug safety evaluation, medical research, etc.

[0030] Serum creatinine (Scr) and urea nitrogen (BUN) are two commonly used renal function indicators, which are clinically used to assess the health and function of the kidneys. Serum creatinine (Scr) is a sensitive indicator for detecting renal impairment, especially when renal function is severely impaired, serum creatinine levels will increase significantly. In the case of acute kidney injury, a rapid increase in serum creatinine levels can help with timely diagnosis and treatment. Urea nitrogen (BUN) is the main end product of protein metabolism, and its level is affected by protein intake, liver function, and kidney excretion capacity. In the case of renal insufficiency, the excretion of urea nitrogen is reduced, resulting in increased urea nitrogen levels in the blood.

[0031] In order to further illustrate the present invention, the technical solution provided by the present invention is described in detail below in conjunction with the accompanying drawings and embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0032] Limonin was purchased from Chengdu Jianteng Biotechnology Co., Ltd., with the number APC-105 and a purity of 98%; captopril was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd., with the CAS number 62571-86-2 and a purity of 98%; SPF-grade SD rats were obtained from the Animal Center of Southwest Medical University.

[0033] The production processes, experimental methods or detection methods involved in the embodiments of the present invention, unless otherwise specified, are all conventional methods in the prior art, and their names and / or abbreviations are all conventional names in the field, and are very clear and unambiguous in the relevant application fields. Technical personnel in the field can understand the conventional process steps based on the names and apply the corresponding equipment, and implement them according to conventional conditions or the conditions recommended by the manufacturer.

[0034] The various instruments, equipment, raw materials or reagents used in the embodiments of the present invention are not particularly limited in terms of their sources, and are all conventional products that can be purchased through regular commercial channels, or can be prepared according to conventional methods well known to those skilled in the art.

[0035] Example 1 Construction of a rat unilateral ureteral obstruction (UUO) model

[0036] 1. Construction of unilateral ureteral obstruction (UUO) model

[0037] SPF grade SD male rats aged 6-8 weeks were selected, and the body weight was controlled at about 260 g.

[0038] Rats were anesthetized with isoflurane gas, and the abdominal wall was cut open about 3-4 mm to the left of the midline of the upper abdomen to separate the left ureter.

[0039] The ureter was ligated and separated near the lower pole of the kidney, then double-ligated, and the ureter was cut between the two ligatures. The muscle layer and abdominal wall were sutured layer by layer, the sutured area was cleaned, and the rat was placed on a heating pad until it woke up.

[0040] 2. Drug preparation method

[0041] The dosage of limonin is: 25 mg / kg, 50 mg / kg and 100 mg / kg, and the administration volume is 8 mL / kg.

[0042] Captopril was used as a positive drug at a dose of 30 mg / kg.

[0043] Preparation method of limonin: prepare 0.5% sodium cellulose (CMC-Na, add 100 ml of normal saline to 0.5 g) with normal saline, add limonin after it is fully dissolved, and allow the limonin to be evenly dispersed in the sodium cellulose. When preparing the drug, the volume is controlled at 2 ml / rat according to the weight of the three groups of rats.

[0044] Preparation method of Captopril: Prepare 0.5% sodium cellulose (CMC-Na, add 100ml of normal saline to 0.5g) with normal saline, add captopril after it is fully dissolved, and allow captopril to be evenly dispersed in the sodium cellulose. When preparing the drug, the volume is controlled at 2ml / rat according to the body weight of the three groups of rats, and the concentration is 30mg / kg.

[0045] 3. Continuous intragastric administration began on the second day after surgery. Rats were divided into 6 groups, one of which was a sham operation group, and the other 5 groups were respectively given 2 ml of 0.5% sodium carboxymethyl cellulose, drug doses of limonin 25 mg / kg (low dose), 50 mg / Kg (medium dose) and 100 mg / Kg (high dose), and captopril 30 mg / kg, once a day, for 3 weeks (21 days). According to experimental needs, they were divided into a sham operation group, a model group (UUO group), UUU+25 mg / kg limonin (UUO+low dose group), UUU+50 mg / kg limonin (UUO+medium dose group), UUU+100 mg / kg limonin (UUO+high dose group) group and a UUU+30 mg / kg captopril positive drug group (UUO+captopril group).

[0046] Example 2 Renal function test

[0047] The changes in renal function were determined by kidney enlargement, ischemia, serum creatinine (Scr) and urea nitrogen (BUN) levels. The effects of the renal fibrosis model were evaluated by histopathological examination with HE staining and Masson staining. Renal fibrosis was further evaluated by renal tissue protein expression.

[0048] Experimental Figure 1-2 As shown, compared with the sham operation group, the UUU model group caused significant enlargement of the left kidney and showed severe ischemia of the left kidney, indicating that the model was successful;

[0049] Compared with the UUO group, the UUO+low-dose group can effectively improve the left kidney enlargement and left kidney ischemia caused by UUO, the UUO+medium-dose group can significantly improve the left kidney enlargement and left kidney ischemia caused by UUO, and the UUO+high-dose group has a more obvious effect. The UUO+captopril group can effectively improve the left kidney enlargement and left kidney ischemia caused by UUO, but its drug effect is weaker than that of the UUO+medium-dose group and the UUO+high-dose group, indicating that limonin can improve the renal morphological changes caused by acute kidney injury. It is also found that compared with the left and right kidneys in the same group, the difference in morphological changes of the left and right kidneys is gradually reduced with the increase of limonin concentration, which reflects the effectiveness of the limonin drug of the present invention.

[0050] After 3 weeks of drug treatment, the results of blood sampling from the heart to test renal function were as follows Figure 3As shown in the data, compared with the sham operation group, the UUO group could significantly increase the levels of blood creatinine and urea nitrogen, the UUO+low-dose group had a significant effect on reducing the urea nitrogen content, but had no obvious effect on blood creatinine, the UUO+low-dose group had a significant effect on reducing the levels of blood creatinine and urea nitrogen, while the positive drug captopril had no significant improvement on the increase of blood creatinine and urea nitrogen caused by UUO.

[0051] H&E staining and Masson staining were used to detect the changes in renal pathology. Figure 4 Compared with sham surgery, UUO can lead to obvious renal cortical inflammatory cell infiltration and proximal tubular structure loss, enlarged renal cystic cavity, and obvious positive Masson staining. Compared with the UUO group, the renal parenchymal inflammatory cell infiltration in the UUO+low-dose group was gradually improved, and the positive improvement of Masson staining was not obvious. The renal cortical inflammatory cell infiltration in the UUO+low-dose group and the UUO+high-dose group was significantly reduced, the glomerular cystic cavity was reduced, the renal tubular morphology was gradually restored, and the blue color of Masson staining was significantly reduced, and its effect was better than captopril.

[0052] Example 3 Inhibitory test of limonin on renal fibroblast migration

[0053] Transwell is a common in vitro assay for evaluating cell migration and invasion. The Transwell assay was used to examine the reversal effect of limonin on TGF-β-induced cellular fibrosis in human renal fibroblasts.

[0054] 1. Experimental Materials

[0055] Cells: Human kidney fibroblasts (Hkf)

[0056] Culture medium: DMEM medium containing 10% FBS

[0057] TGF-β: used to construct fibrosis model at the cellular level, dosage 10ng / ml

[0058] Culture conditions: conventional culture

[0059] 2. Experimental treatment

[0060] Prepare the upper culture chamber, first coat the basement membrane / hydrated basement membrane, then add DMEM medium containing 20% ​​FBS to the lower chamber, digest Hkf cells with 0.05% trypsin, prepare cell suspension, and promote cell proliferation and migration. Put the upper chamber into the lower chamber, count the Hkf cells, and then spread the cells in the upper chamber. The upper culture medium is DMEM medium containing 5% FBS.

[0061] The high concentration of fetal bovine serum in the lower culture medium promotes cell proliferation and migration, while the low concentration of fetal bovine serum in the upper culture medium prompts cells to migrate to the lower layer with high concentration.

[0062] The chambers were divided into 5 groups: control group, TGF-β treatment group, TGF-β + limonin 25μmol / L, TGF-β + limonin 50μmol / L, TGF-β + captopril 10μmol / L. After 24h, the cells in the upper chamber were wiped off with a cotton swab. To detect the Transwell chamber, the culture medium was removed, and the matrix gel and cells in the chamber were gently wiped with a cotton swab or cotton soaked in PBS. 600μl of 4% paraformaldehyde fixative was added to a clean well of a 24-well plate, and the chamber was placed in the fixation for 30 minutes. The fixative was discarded, and the inside and outside of the chamber were washed once with PBS. 600μl of crystal violet staining solution was added to a clean well of a 24-well plate, and the chamber was placed in the staining for 10 minutes. The chamber was removed, and the inside and outside of the chamber were washed with PBS for 3 times.

[0063] Observation and analysis: Observe the cells in the lower chamber wells using a microscope. Record the number, morphology, and migration of cells. Use image analysis software to analyze the extent of cell invasion.

[0064] The results are as follows Figure 5 As shown, compared with the control group, TGF-β10 ng / ml can significantly promote the migration of human renal fibroblasts, while limonin can inhibit the cell migration caused by TGF-β in a concentration-dependent manner. The anti-migration ability of limonin is better than that of captopril.

[0065] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. The use of limonin in the preparation of a drug for improving renal fibrosis, characterized in that: The CAS number of the limonin is 1180-71-8, and the molecular formula is: 26 H 30 O8, molecular weight: 470.51; the structural formula of the limonin is shown in Formula I:

2. The application according to claim 1, characterized in that: The limonin is derived from grapefruit seeds of the Rutaceae plant.

3. The application according to claim 1, characterized in that: The dosage of the limonin is 25-100 mg / kg.

4. The use according to claim 3, characterized in that: The drug concentration of the limonin is 3.125-12.5 mg / ml.

5. Use of limonin in inhibiting activation and migration of renal fibroblasts, characterized in that: The limonin is the limonin according to claim 1.

6. The use according to claim 5, characterized in that: The limonin inhibits the expression of TGF-β and reduces the migration of renal fibroblasts.

7. Use of limonin in reducing extracellular matrix deposition in kidney, characterized in that: The limonin is the limonin according to claim 1.

8. Use of limonin in improving renal function impairment caused by acute kidney injury, characterized in that: The limonin is the limonin according to claim 1.

9. The use according to claim 8, characterized in that: The limonin reduces blood creatinine and urea nitrogen content and improves renal function.

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