WO2021114125A1 - Use of levistolide a in preparation of drugs for treating or preventing renal diseases - Google Patents

Abstract

The present invention falls within the field of medical technology, and relates to a drug for treating chronic kidney diseases, in particular to a pharmaceutical composition containing Levistolide A or Diligustilide (C21) and the use thereof. C21 can be used for preventing and treating chronic kidney system diseases caused by many factors, including but not limited to diabetes mellitus (DM), hypertension (HT), glomerulonephritis (GN), metabolic syndromes, urinary system diseases, etc.

Description

Use of angelica lactone A in preparing medicine for treating or preventing nephropathy Technical field

The present invention belongs to the technical field of medicine, and relates to medicines for treating chronic kidney disease, and particularly relates to the use of Levistolide A or Diligustilide (hereinafter referred to as C21) in the preparation of medicines for treating or preventing kidney disease.

Background technique

Chronic Kidney Disease (CKD) is a common clinical disease. The prevalence of adult CKD in my country is above 10%, which seriously endangers human health. In the process of chronic kidney disease, nephrons are gradually lost, glomeruli gradually harden, renal tubules atrophy, and interstitial fibrosis. There are various causes of CDK, including diabetes (DM), hypertension (HT), glomerulonephritis (GN), metabolic syndrome, and urinary system diseases. Among them, diabetes and hypertension are currently the main causes of CKD. Regardless of the cause, kidney disease continues to progress after the onset of CKD, and eventually it will inevitably enter end-stage renal disease (ESRD) until renal failure.

Large and mouse 5/6 nephrectomy (5/6 nephrectomy, 5/6 NX) animal models are widely used in the basic and clinical research of kidney diseases, and are currently recognized CDK models. The mouse genome has high homology with the human genome, and the tissue and organ structure and cell function are similar to humans. This animal model is more advantageous for CDK therapeutic drug discovery and evaluation. That is, when a drug acts on the 5/6 nephrectomy mouse model to improve the kidney pathological indicators of the model mouse, it will surely alleviate the symptoms of CDK and be used for kidney diseases caused by various factors such as hypertension and diabetes. Therapeutic effect.

Compound C21 (Chinese name: Angelica lactone A; English name: Levistolide A or Diligustilide) is mainly isolated from Umbelliferae Angelica, Ligusticum chuanxiong and Ligusticum chinense, and can also be obtained by organic synthesis from Ligustilide as raw material . Studies have shown that C21 has anti-tumor and gastric mucosal protection effects.

However, there is no report on the effect of C21 on the 5/6 nephrectomy mouse kidney injury model, improving the kidney pathological indicators of the model animal, and alleviating and treating chronic kidney injury. The role of C21 in the prevention or treatment of hypertensive nephropathy, diabetic nephropathy and other renal diseases has not been reported.

Summary of the invention

The inventors unexpectedly discovered in the research that C21 can be used to treat and prevent kidney diseases. C21 reduces blood creatinine (CRE) and urea nitrogen (BUN) levels in kidney injury model animals, reduces urine protein (mALB) levels, reduces urine microalbumin and urine creatinine ratio (ACR); improves the kidney of model animals The pathological changes of the injury prove that C21 can be used to prevent or treat chronic kidney injury disease or kidney disease caused by multiple causes, thus completing the present invention.

Therefore, the present invention includes the following aspects:

The present invention provides a pharmaceutical composition, which uses a compound represented by the following structural formula as an active ingredient:

The C21 in the present invention can be added in the form of a Chinese medicine extract, and the Chinese medicine extract contains 15% to 99.9% of C21 by weight. The traditional Chinese medicine is Angelica sinensis and/or Chuanxiong.

The present invention also provides the use of the compound represented by the general structural formula in the preparation of medicines, which can reduce the increase in blood creatinine (CRE) and urea nitrogen (BUN) levels caused by kidney injury, and reduce urine Protein level (mALB), reduce the ratio of microalbumin in urine to urine creatinine (ACR), and improve the pathological changes of the kidney caused by kidney injury.

The present invention also provides the use of the compound represented by the structural formula C21 in the preparation of a medicine, wherein the medicine is used for the treatment of kidney injury diseases.

In addition, the present invention also provides a method for treating diseases, which comprises administering to a patient the pharmaceutical composition, the active ingredients in the pharmaceutical composition can improve kidney damage.

The present invention provides a method for preventing and treating chronic kidney disease caused by various reasons including but not limited to diabetes (DM), hypertension (HT), glomerulonephritis (GN), metabolic syndrome, urinary system disease, etc. The method includes administering the pharmaceutical composition to the patient.

Description of the drawings

Figure 1 ¹ H NMR (600MHz, CDCl ₃ ) spectrum of compound C21.

Figure 2 ¹ H NMR (600MHz, CDCl ₃ ) spectrum low-field enlargement of compound C21.

Figure 3 ¹ H NMR (600MHz, CDCl ₃ ) spectrum high-field enlargement of compound C21.

Figure 4 ¹³ C NMR (600MHz, CDCl ₃ ) spectrum of compound C21.

Figure 5 ¹³ C NMR (600MHz, CDCl ₃ ) spectrum low-field enlargement of compound C21.

^{Figure 6 The 13} C NMR (600MHz, CDCl ₃ ) spectrum of compound C21 is a high-field enlarged view.

Figure 7 ESI-MS spectrum of compound C21.

Figure 8 The effect of C21 on urine output/drinking volume of mice with kidney injury. Blank control group (Con), sham operation control group (Sham) and model group (Veh) were treated with blank solvent; mice in the positive control Los group and Ena group were given losartan (3mg/kg) and enalapril, respectively Li (5mg/kg) positive control drug treatment; C21 drug treatment group was treated with 0.5mg/kg (C21L group), 1mg/kg (C21M group) and 2mg/kg (C21H group). After each group of mice were treated with drugs or blank reagents, the urine output and drinking water volume were monitored for 24 hours every 1 week. Compared with the normal group (Con) and the sham operation group (Sham), the ratio of urine volume/water consumption in the model group (Veh) was significantly higher; compared with the model group (Sham) in each treatment group (Los, Ena, C21L, C21M, C21H) ) The ratio of urine output/drinking water volume decreased to varying degrees, and it was time-dependent with drug treatment.

Figure 9 The effect of C21 on the urine microalbumin (mABL) content and the urine microalbumin to urinary inosine ratio (ACR) of mice with kidney injury. The mice in the blank control group (Con), sham operation control group (Sham) and model group (Veh) were treated with blank solvent; mice in the positive control Los group and Ena group were given Losartan (3mg/kg) and Enala respectively Prili (5mg/kg) treatment; C21 drug treatment group was treated with 0.5mg/kg (C21L group), 1mg/kg (C21M group) and 2mg/kg (C21H group). Mice in each group were intraperitoneally injected with drugs or blank reagents every other day for 4 weeks. Urine was collected for 24 hours, and the contents of microalbumin (A) and inosine in the urine were detected, and the ACR value (B) was calculated. The urine mABL content and ACR value of the blank control group (Con) and the sham operation group (Sham) were not significantly different; the urine mABL content and ACR value of the model group (Veh) were significantly higher than those of the Con and Sham groups, indicating that the kidney injury model was constructed Success; compared with the model group (Sham), the urine mABL content and ACR value of the C21 treatment group were significantly reduced, and showed a dose-dependent manner. ###P<0.001, compared with sham operation; *P<0.05, **P<0.01, ***P<0.001 compared with model group.

Figure 10 The effect of C21 on blood inosine (CRE) and urea nitrogen (BUN) in mice with kidney injury. The mice in the blank control group (Con), sham operation control group (Sham) and model group (Veh) were treated with blank solvent; mice in the positive control Los group and Ena group were given Losartan (3mg/kg) and Enala respectively Prili (5mg/kg) treatment; C21 drug treatment group was treated with 0.5mg/kg (C21L group), 1mg/kg (C21M group) and 2mg/kg (C21H group). The mice in each group were injected with drugs or blank reagents into the intraperitoneal cavity once every other day. After 4 weeks, the CRE (A) and BUN (B) levels in the blood of the mice in each group were detected. There was no significant difference in blood CRE and BUN contents between the blank control group (Con) and the sham operation group (Sham); the CRE and BUN contents of the model group (Veh) were significantly higher than those of the Con and Sham groups; compared with the model group (Veh), the CRE in the C21 treatment group And BUN content is significantly reduced. ###P<0.001, compared with sham operation; *P<0.05, **P<0.01, ***P<0.001 compared with model group.

Figure 11 The effect of C21 on kidney tissue pathology in mice with kidney injury. Mice in the blank control group (A), sham operation control group (B) and model group (C) were treated with blank solvent; mice in the positive control (D and E) groups were given losartan (3mg/kg) and Nazepril (5mg/kg) was treated as a positive control drug; the C21 drug treatment group was treated with 0.5 mg/kg (F), 1 mg/kg (G) and 2 mg/kg (H) respectively. The mice in each group were injected with drugs or blank reagents into the intraperitoneal cavity once every other day for 4 weeks, and then the kidneys of each group were taken to detect the histopathological changes of the kidneys of each group by HE staining. Obvious pathological changes occurred in the model group, the area of the glomerulus increased; the renal tubules and collecting ducts were dilated and cystic, the lumen was occluded or expanded, the structure of the renal tubules was destroyed, atrophy, collapsed in the official cavity, interstitial inflammatory cell infiltration, and fibrous tissue More hyperplasia. The above-mentioned pathological changes were significantly reduced after C21 drug treatment.

Figure 12 The effect of C21 on glomerular sclerosis in the kidney tissue of mice with kidney injury. Mice in the blank control group (A), sham operation control group (B) and model group (C) were treated with blank solvent; mice in the positive control (D and E) groups were given losartan (3mg/kg) and Nazepril (5mg/kg) was treated as a positive control drug; the C21 drug treatment group was treated with 0.5 mg/kg (F), 1 mg/kg (G) and 2 mg/kg (H) doses respectively. The mice in each group were injected with drugs or blank reagents into the intraperitoneal cavity once every other day for 4 weeks, and the kidneys of each group were taken to observe the glomerular sclerosis of the kidney tissues of the mice in each group by PAS staining. The glycogen deposition area was obvious in the model group, indicating glomerular sclerosis, and the glycogen deposition in the C21 treatment group was improved to varying degrees.

Figure 13 The effect of C21 on renal tissue fibrosis in mice with renal injury. Mice in the blank control group (A), sham operation control group (B) and model group (C) were treated with blank solvent; mice in the positive control (D and E) groups were given losartan (3mg/kg) and Nazepril (5mg/kg) was treated as a positive control drug; the C21 drug treatment group was treated with 0.5 mg/kg (F), 1 mg/kg (G) and 2 mg/kg (H) doses respectively. The mice in each group were injected with drugs or blank reagents into the intraperitoneal cavity once every other day for 4 weeks, and the kidneys of each group were taken to observe the fibrosis of the kidney tissues of the mice in each group by MASSON staining. The model group showed strong positive staining and showed severe fibrosis, and the degree of fibrosis in the C21 treatment group was significantly reduced.

Figure 14 The effect of C21 on the expression of α-smooth muscle actin (α-SMA), fibronectin (FN) and collagen type I (Collagen I) in the kidney tissue of mice with renal injury. Blank control group (Con), sham operation control group (Sham) and model group (Veh) mice were treated with blank solvent; mice in the positive control (Los and Ena) group were given Losartan (3mg/kg) and Nazepril (5mg/kg) was treated as a positive control drug; the C21 drug treatment group was treated with 0.5 mg/kg (C21L group), 1 mg/kg (C21M group) and 2 mg/kg (C21H group). Mice in each group were injected with drugs or blank reagents into the intraperitoneal cavity once every other day. After 4 weeks of treatment, kidney tissues were taken to extract proteins, and the expression of α-SMA, Collagen I and FN proteins in kidney tissues was detected by Western Blot technology. The relative protein expression statistics of α-SMA (B), Collagen Ⅰ (C) and FN (D) in the electrophoresis band (A) showed that compared with the blank control group and the sham operation group, the mouse kidney tissue in the model group The expression of α-SMA, Collagen I and FN protein increased significantly, indicating that the kidney of model mice has fibrotic damage. The expression of α-SMA, Collagen I and FN protein in the C21 treatment group was significantly lower than that of the model group, and showed a dose-dependent manner, indicating that C21 can significantly improve the kidney injury in mice. ###P<0.001, compared with sham operation; *P<0.05, **P<0.01, ***P<0.001 compared with model group.

Detailed ways

The technical solutions of the present invention will be described in detail below with reference to the drawings and embodiments, but the protection scope of the present invention includes but is not limited thereto.

The present invention provides an experimental basis for C21 in the prevention or treatment of chronic kidney disease.

The present invention also provides that C21 reduces blood creatinine (CRE) and urea nitrogen (BUN) levels in kidney injury model animals, reduces urine protein (mALB) levels, and reduces urine microalbumin and urine creatinine ratio (ACR). ) Experimental basis.

The invention provides the application of C21 in the preparation of medicines for treating kidney diseases. In the pharmaceutical composition containing C21 of the present invention, the dosage of the C21 compound as the active ingredient is 50 micrograms/kg body weight to 10 mg/kg body weight. The composition can be in any preparation form, for example, it can be an oral preparation or Injection preparations.

Based on the experimental results of the present invention and combined with the prior art, it can be inferred that the C21 compound of the present invention conforms to the above relationship, that is, to prevent or treat diabetic nephropathy, hypertensive nephropathy, glomerulonephritis and other kidney diseases caused by other causes.

1. Urine output mainly depends on glomerular filtration rate, renal tubular reabsorption, concentration and dilution functions, so it can reflect renal function to a certain extent. In the early stage of kidney injury, there will be staged polyuria, which can cause polyuria due to loss of polyuria. Polyuria can cause excessive load on the kidney and accelerate the damage of its function [Torres, VE; Bankir, L.; Grantham, JJ, A Case [Torres, VE; Bankir, L.; Grantham, JJ, A Case for Water in the Treatment of Polycystic Kidney Disease (the role of water in the treatment of polycystic kidney disease), Clinical Journal of the American Society of Nephrology, 2009, 4(6), 1140-1150.]. The change of 24-hour urine output is greatly affected by external factors such as daily water consumption, so the value of 24-hour urine output/drinking water volume is usually used as an indicator of renal function. The present invention proves through experiments that 5/6 nephrectomy mice can obviously reduce the ratio of urine volume/water volume and improve kidney function after applying C21 treatment.

2. Proteinuria appears in the early stage of kidney disease. Albumin is a common protein in the blood, a very small amount appears in urine under normal physiological conditions, but when renal function is impaired, protein will be abnormally leaked, so 24h urine albumin can be used as an indicator of renal function and urine albumin excretion Too much indicates abnormal kidney function [Hokamp, JA; Nabity, MB, Renal biomarkers in domestic species. (Renal function biomarkers of the local population). Veterinary Clinical Pathology 2016, 45, 28-56.]. The 24-hour urine output is relatively affected by environmental factors, so the ratio of albumin to creatinine (ARC) in urine more directly reflects renal function. The present invention proves that 5/6 nephrectomy mice can obviously reduce the ACR of mice with renal injury after being treated with C21, and can be used to treat renal system diseases.

3. Normally blood creatinine (CRE) and urea nitrogen (BUN) are excreted in urine, but when kidney is injured, the filtration capacity of the glomerulus decreases, which affects the urinary excretion of blood creatinine and blood urea nitrogen, causing blood Medium accumulation [Wasung, ME; Chawla, LS; Madero, M., Biomarkers of renal function, which and when? (Biomarkers of kidney function). Clinica Chimica Acta, 2015, 438, 350-357.]. The compensatory nature of the remaining kidney cannot fully meet the needs of the body's metabolism, and the concentration of blood creatinine and blood urea nitrogen continues to rise with the course of the disease. The present invention proves that C21 can significantly reduce the blood CRE and BUN contents of kidney injury mice after treating 5/6 nephrectomy mice.

4. After the onset of chronic kidney disease, as the course of the disease progresses, glomeruli sclerosis or degenerate, renal tubules atrophy, and ultimately lead to kidney atrophy, fibrosis, and renal failure [Hodgkins, KS; Schnaper, HW, Tubulointerstitial injury and the progression of Chronic kidney disease. (The relationship between tubular interstitial damage and the progression of chronic kidney disease. Pediatric Nephrology 2012, 27, 901-909.]. The present invention proves that after 5/6 nephrectomy mice are treated with C21, the renal tubular atrophy, occlusion or expansion of the official cavity of the mouse is reduced, the infiltration of interstitial inflammatory cells is reduced, the degree of fibrous tissue proliferation is significantly reduced, and the structure of the renal tubule is significantly restored. Therefore, C21 can be used to prevent or treat kidney disease.

5. Excessive deposition of extracellular matrix (ECM) in the renal interstitium, leading to interstitial fibrosis and glomerular sclerosis, α-smooth muscle actin (α-SMA), fibronectin (FN), type I collagen ( Collagen I) etc. are the main factors that induce the generation of ECM and are also the main components of ECM. Actin filaments are part of the cytoskeleton and play an important role in regulating cell morphology and movement. FN is an effect factor of renal fibrosis, which can be formed and widely deposited in the early stage of fibrosis. It is one of the intuitive indicators of renal fibrosis [Soylemezoglu, O.; Wild, G.; Dalley, AJ; MacNeil, S. ;Milford-Ward,A.;Brown,CB;el Nahas,AM,Urinary and serial type III collagen: markers of renal fibrosis. (Urine and serum type III collagen: a marker of renal fibrosis). Nephrol Dial Transplant 1997, 12, 1883-1889.]. The present invention proves that C21 can significantly reduce the expression of α-SMA, FN and Collagen I protein in kidney tissue after treating 5/6 nephrectomy mice, and improve kidney damage.

The present invention confirmed the above-mentioned effects of C21 through the following experiments.

Preparation Example 1.

5 kilograms (Kg) of chuanxiong decoction pieces were crushed and extracted three times with 95% ethanol under reflux, each extraction time was 2 hours (h), 1.5h, 1.5h, 15 liters (L) each time. The three ethanol extracts were combined and concentrated under reduced pressure at 45 degrees Celsius to obtain about 1.3Kg of brown viscous extract. The obtained extract was stirred and kneaded with water to 2.5L, and extracted 3 times with 2.5L ethyl acetate. The ethyl acetate extracts were combined, and concentrated under reduced pressure at 45 degrees Celsius to obtain 230 grams (g) of ethyl acetate extracts. After silica gel column chromatography (petroleum ether-ethyl acetate 100:1, 10:1, 4:1 gradient elution), Sephadex LH20 (methanol elution) to obtain compound C21. Identification by proton nuclear magnetic resonance spectrum (Figure 1-Figure 3), carbon spectrum (Figure 4-Figure 6) and ESI mass spectrum (Figure 7) and other methods, and compared with the literature, it is determined that the isolated C21 is Angelica lactone A (English name: Levistolide A or Diligustilide) pure product. [Kaouadji,M.; De Pachtere, F.; Pouget, C.; Chulia, AJ, Three additional phthalides derivatives an epoxymonomer and two dimmers from Ligusticum wallachii rhizomes The material includes an epoxy monomer and two dimers). Journal of natural products 1986,49(5),872-877; Wei,Y.; Huang,W.; Gu,Y.,Online isolation and purification of Four phthalide compounds from Chuanxiong rhizoma using high-speed counter-current chromatography coupled with semi-preparative liquid chromatography (on-line separation and purification of four phthalide compounds in Ligusticum chuanxiong by high-speed countercurrent chromatography-semi-preparative liquid chromatography). Journal of Chromatography. A 2013, 1284, 53-58.].

Angelica lactone A, white powder. ¹ H NMR (600MHz, CDCl ₃ ) δppm: 0.92 (3H, t, J = 7.4 Hz, H-11'), 0.93 (3H, t, J = 7.4 Hz, H-11), 1.30 (1H, m, H-5′a), 1.40(1H,m,H-4′a), 1.44(2H,m,H-10), 1.45(2H,m,H-10′), 1.53(1H,m,H -5a), 1.88(1H,m,H-5′b),1.93(1H,m,H-5b),2.03(1H,m,H-4′b),2.08(1H,m,H-4a ), 2.18(2H,m,H-9′),2.21(1H,m,H-4b),2.29(2H,q,J=7.8,H-9),2.55(1H,t,J=7.9Hz ,H-6), 2.99(1H,m,H-6′), 3.25(1H,d,J=8.8Hz,H-7),5.00(1H,t,J=7.5Hz,H-8′) ,5.07(1H,t,J=7.9Hz,H-8),7.35(1H,d,J=6.5Hz,H-7′)

¹³ C NMR(150MHz, CDCl ₃ )δ: 168.48(C-1), 164.92(C-1′), 155.00(C-3a), 150.42(C-3′), 147.97(C-3), 142.07( C-7′), 134.15(C-7′a), 126.51(C-7a), 112.19(C-8), 108.63(C-8′), 47.57(C-3′a), 41.54(C- 6′), 41.44(C-7′), 38.29(C-6), 31.02(C-4′), 28.94(C-5), 27.98(C-9), 27.46(C-9′), 25.75 (C-5′), 22.32(C-10), 22.27(C-10′), 19.73(C-4), 13.97(C-11), 13.85(C-11′).

ESI MS m/z:: [M+H] ⁺ 381.3, [M+Na] ⁺ 403.3 (compound formula: C ₂₄ H ₂₈ O ₄ ).

Preparation Example 2.

5Kg of Chinese angelica decoction pieces were crushed, and extracted with 95% ethanol under reflux three times for 2h, 1.5h, 1.5h, 15L each time. The three ethanol extracts were combined and concentrated under reduced pressure at 45 degrees Celsius to obtain a brown viscous extract about 1.4Kg. The obtained extract was stirred and dissolved with hot water at 45 degrees Celsius to 2.5L, and extracted with 2.5L ethyl acetate for 3 times. The ethyl acetate extracts were combined, and concentrated under reduced pressure at 45 degrees Celsius to obtain 190 g of ethyl acetate extract. The C21 compound was obtained by separation and purification in the same manner as in Preparation Example 1. Through ESI mass spectrometry, proton nuclear magnetic resonance spectroscopy and carbon spectroscopy, it was identified that the separated material was pure C21.

Angelica lactone A, white powder. ¹ H NMR (600MHz, CDCl ₃ ) δppm: 0.92 (3H, t, J = 7.4 Hz, H-11'), 0.93 (3H, t, J = 7.4 Hz, H-11), 1.30 (1H, m, H-5′a), 1.40(1H,m,H-4′a), 1.44(2H,m,H-10), 1.45(2H,m,H-10′), 1.53(1H,m,H -5a), 1.88(1H,m,H-5′b),1.93(1H,m,H-5b),2.03(1H,m,H-4′b),2.08(1H,m,H-4a ), 2.18(2H,m,H-9′),2.21(1H,m,H-4b),2.29(2H,q,J=7.8,H-9),2.55(1H,t,J=7.9Hz ,H-6),2.99(1H,m,H-6′), 3.25(1H,d,J=8.8Hz,H-7),5.00(1H,t,J=7.5Hz,H-8′) ,5.07(1H,t,J=7.9Hz,H-8),7.35(1H,d,J=6.5Hz,H-7′)

¹³ C NMR(150MHz, CDCl ₃ )δ: 168.48(C-1), 164.92(C-1′), 155.00(C-3a), 150.42(C-3′), 147.97(C-3), 142.07( C-7′), 134.15(C-7′a), 126.51(C-7a), 112.19(C-8), 108.63 (C-8′), 47.57(C-3′a), 41.54(C- 6′), 41.44(C-7′), 38.29(C-6), 31.02(C-4′), 28.94(C-5), 27.98(C-9), 27.46(C-9′), 25.75 (C-5′), 22.32(C-10), 22.27(C-10′), 19.73(C-4), 13.97(C-11), 13.85(C-11′).

ESI MS m/z:: [M+H] ⁺ 381.3, [M+Na] ⁺ 403.3 (compound formula: C ₂₄ H ₂₈ O ₄ ).

The compound prepared above was subjected to in vivo experiments in an animal model to determine the efficacy. In the following experimental example, 5/6 nephrectomy C57BL/6 mice are calculated at the dose of 25 g each. The pure C21 product used in the following experimental examples was obtained from Preparation Example 1.

Experimental example 1.

Effect of C21 on Urine and Blood Biochemical Indexes of 5/6 Nephrectomy C57BL/6 Mice (CDK Mice)

1 Experimental materials and methods

1.1 Preparation of test solution: 1.00 mg of pure C21 was dissolved in 100uL ethanol to prepare a stock solution, and the stock solution was added to 40% propylene glycol aqueous solution to prepare a C21 test solution of appropriate concentration. Take the same volume of ethanol and add it to a 40% propylene glycol aqueous solution to prepare a blank solvent. Prepare according to the above method before each use.

1.2 Experimental animals and methods:

1.2.1 Preparation, grouping and dosage of CKD mice

Under pentobarbital anesthesia (0.5%, 0.1ml/10g), open the lower left part of the back about 1cm, separate the kidney and peel off the renal capsule, clamp the renal blood vessel with hemostatic forceps, and remove 2/3 of the left kidney. Bleeding again, disinfect with iodophor, suture the inner skin and outer skin separately. In the sham operation group, only the renal capsule was dissected without removing the renal parenchyma, and the surgical procedure was the same. After 7 days, after the wound was healed, the entire right kidney was excised under the same anesthesia operation conditions as above to make a CKD mouse model. Control group, sham operation group, CKD mice were divided into model group, positive drug (Losartan and Enalapril) and low, medium and high treatment groups according to the random number table method, a total of 8 groups. Con group: normal control group (n=10); Sham group: sham operation control group (n=10); Veh group: operation model group (n=10); Los group: Losartan positive control group (n= 10); Ena group: enalapril positive control group (n=10); C21L group: study drug low-dose group (n=10); C21M group: study drug medium-dose group (n=10); C21H group: Study drug high-dose group (n=10).

Administration method: Con group, Sham group and Veh group intraperitoneal injection of blank solvent; positive control Los group, positive control Ena group, therapeutic administration low-dose group C21L group, medium-dose group C21M group and high-dose group C21H group, respectively, intraperitoneal injection The positive drug Losartan (3mg/kg), Enalapril (5mg/kg) or low (0.5mg/kg), medium (1mg/kg), high (2mg/kg) test drug C21 in different doses. Intraperitoneal injection or blank solvent, the frequency is once every other day, continuous administration for 4 weeks.

Dosage: The low, medium, and high doses of the treatment group were 0.5 mg/kg, 1 mg/kg, and 2 mg/kg, respectively. The positive drugs are losartan 3mg/kg and enalapril 5mg/kg. 40% 1,2 dihydroxypropanediol was used as the solvent.

1.2.2 Urine test: 24h urine of experimental mice in each group was collected with metabolic cage every week after modeling and after administration, and 24h drinking water and 24h urine output were recorded. Urine albumin (mALB), urea nitrogen (BUN) and creatinine (CRE) were tested with albumin ELISA kit and chemical kit, respectively, according to the instructions.

1.2.3 Blood test: at the end of the experiment (the 4th weekend), take the blood from the eyeballs, collect it with an EP tube, centrifuge at 3500r/min for 15min, take the supernatant serum and place it at -20°C for testing of blood biochemical indicators. The detection of blood urea nitrogen (BUN) and creatinine use the detection kit respectively, and operate according to the instructions.

2 experimental results

2.1 Urine output and water consumption: In this study, compared with the normal group and the sham operation group, the urine output/water consumption ratio of the model group was significantly increased; compared with the model group, the treatment groups decreased to varying degrees (Figure 8).

2.2 Urine microalbumin (mALB): The results show that compared with the normal control group, mALB in the sham operation group has no significant change, and the difference is not statistically significant (P>0.05); compared with the sham operation group, the mALB level in the model group is significant Increased (P<0.001); Compared with the model group, the mALB level of the positive control, low, medium, and high-dose treatment groups decreased (P<0.01 or P<0.001). The mALB expression level of each dose treatment group gradually decreased with increasing dose gradient (Figure 9A).

2.3 Microalbumin to creatinine ratio (ACR): 24h urine output is more affected by environmental factors, so ACR more directly reflects renal function. Compared with the normal control group and the sham operation group, the ACR value of the model group was significantly increased (P＜0.001); compared with the model group, the ACR value of the positive control, low, medium, and high dose treatment groups were significantly reduced (P＜0.01 or P<0.001) (B in Fig. 9).

2.4 Blood biochemical indicators CRE, BUN results

The results of the study showed that compared with the blank control group and the sham operation group, the blood CRE and BUN levels in the model group were significantly increased (P<0.001); the positive control group and the treatment groups were significantly reduced in blood CRE and BUN levels (P<0.05 or P<0.01) (Figure 10).

Experimental example 2.

Effect of C21 on the pathological changes of kidney tissue in C57BL/6 mice with 5/6 nephrectomy

1 Experimental materials and methods

1.1 Preparation, grouping and administration of CKD mice: same as experimental example 1.

1.2 Tissue collection: After 4 weeks of administration, take the remaining 1/6 of the kidney tissue of each group of mice, shake it gently in clean PBS, wash off the blood and weigh. The kidney was divided into 2 pieces, and one part was fixed in 4% paraformaldehyde (4% PFA) for pathological changes (HE staining, PAS staining, Masson staining) tissue sections and immunohistochemical paraffin sections. The other part of the kidney was separated from the renal cortex in a cryotube, and stored at -80°C after labeling for Western blot experiments.

1.3 Western blot experiment methods and reagents: detect the protein levels of mouse kidney α-SMA, FN and Col Ⅰ, select an appropriate amount of kidney tissue (30-40 mg), extract the total protein with 1 mL of tissue protein lysate, and measure the concentration by the BCA method. The prepared protein sample (45-60μg) was separated by electrophoresis and transferred to the PVDF membrane for immunoblotting reaction, and the gray value was analyzed by the gel image analysis system for statistical analysis. GAPDH and β-Actin antibodies were purchased from CST; α-SMA, FN and Collagen I antibodies were purchased from Abcam. The secondary antibody is CST, which is goat anti-rabbit antibody.

2 Results of histopathological changes

2.1 H&E staining results:

Blank control group and sham operation group: the structure of the renal tubules is basically normal, the mesangial cells and matrix are not proliferated, and the tubulointerstitium has no obvious lesions; the model group: the glomerular area is enlarged, and the mesangial area is enlarged. Renal tubules and collecting ducts are dilated and cystic, and the lumen is occluded or expanded. Some epithelial cells are deformed, necrosis, renal tubular structure destruction, atrophy, collapse of the official cavity, interstitial inflammatory cell infiltration, and more fibrous tissue proliferation. This shows that the method of removing the kidney in this experiment caused a successful mouse model of chronic renal failure. Compared with the model group, the renal tubule atrophy, occlusion or dilation of the official cavity in the C21 administration group was reduced, the infiltration of interstitial inflammatory cells was reduced, and the degree of fibrous tissue proliferation was reduced, indicating that C21 can significantly improve the symptoms of kidney injury in model animals ( Figure 11).

2.2 PAS staining results:

PAS staining (Periodic Acid-Schiff stain) is mainly used to detect carbohydrates in tissues in histology. Observe the kidney tissue structure and glomerular sclerosis. The blank control group and the sham operation group had no glomerular mesangial and basement membrane hyperplasia, and glomerular sclerosis; the model group glomerular mesangial hyperplasia, matrix increased, renal tubule swelling, accompanied by segmental glomeruli Sclerosis, collapse of glomerular vascular loops, and infiltration of inflammatory cells around the blood vessels. The mesangial proliferation and glycogen deposition of the C21 treatment group with different doses and the positive control group were improved to varying degrees (Figure 12).

2.3 Masson staining results:

Blank control group and sham operation group: renal tubular epithelial cells are tightly arranged without inflammatory cell infiltration in the renal interstitium; model group: renal tubular atrophy or expansion, renal interstitial area widening, inflammatory cell infiltration, and a significant decrease in the number of glomeruli. Compared with the model group, the renal tubular epithelial cells in the C21 treatment group with different doses are tightly arranged, inflammatory cells are reduced, and the interstitial collagen deposition is significantly reduced compared with the model group, and the vascular fibrosis is improved compared with the model group (Figure 13 ).

2.4 α-SMA, FN, Collagen I protein expression:

There was no significant difference in the expression of α-smooth muscle actin (α-SMA), fibronectin (FN), and collagen I (Collagen I) in the kidneys of the blank control group and the sham operation group. The expression of α-SMA, FN and Collagen I in the model group was significantly increased compared with the blank control group and the sham operation group (P＜0.01). Compared with the model group, the expression of α-SMA, FN, Collagen I in the positive control group was significantly decreased (P＜0.01). The expression of α-SMA, FN, Collagen I in the low, medium and high dose C21 treatment group was significantly lower than that of the model group (P＜0.05 or P＜0.01), and the high dose group was significantly better than the positive drug administration group (Figure 14).

Claims (7)

1. Use of the compound Angelica lactone A represented by the structural formula C21 in the preparation of a medicine for the treatment or prevention of nephropathy:

   
2. The use according to claim 1, wherein the kidney disease is chronic kidney disease.
3. The use according to claim 2, wherein the chronic kidney disease is a chronic kidney disease caused by one or more reasons selected from diabetes, hypertension, glomerulonephritis, metabolic syndrome and urinary system disease.
4. The use according to any one of claims 1 to 3, wherein the C21 is added in the form of a Chinese medicine extract, and based on the total weight of the Chinese medicine extract as 100%, the weight percentage of the Chinese medicine extract containing C21 is 15% To 99.9%.
5. The use according to claim 4, wherein the traditional Chinese medicine is Angelica sinensis and/or Chuanxiong.
6. The use according to any one of claims 1 to 5, wherein the medicine is an oral preparation or an injection preparation.
7. A method for treating nephropathy, the method comprising administering to a patient the angelica lactone A according to any one of claims 1-6 or a pharmaceutical combination containing the angelica lactone A according to any one of claims 1-6 Things.

Images