CN105560265A - Application of bupleurum polysaccharide in preparation of drugs used for preventing and treating diabetic nephropathy - Google Patents

Abstract

The invention belongs to the field of traditional Chinese medicines, and in particular relates to the use of Bupleurum polysaccharide in preparing anti-diabetic drugs. The Bupleurum polysaccharide extract has obvious prevention and treatment effect on mice diabetic nephropathy as confirmed by whole body and in vitro experiments. Bupleurum polysaccharides can improve the body weight of diseased animals, significantly reduce blood sugar and urine sugar levels, significantly promote insulin secretion, reduce serum creatinine levels, reduce urinary microalbumin secretion, reduce kidney index, and improve renal pathological symptoms, thereby achieving hypoglycemia and kidney disease. The role of protection. Bupleurum polysaccharide can also reverse TGF-β-induced renal tubular cell transdifferentiation. It can be used to prepare drugs for preventing and treating diabetes and kidney complications caused by diabetes.

Description

Application of Bupleurum polysaccharide in preparing medicine for preventing and treating diabetic nephropathy

technical field

The invention belongs to the field of traditional Chinese medicine, and relates to a new medicinal application of the traditional Chinese medicine Bupleurum polysaccharide. It specifically relates to the use of Bupleurum polysaccharide in the preparation of medicines for preventing and treating diabetic nephropathy

Background technique

Diabetes mellitus is a series of systemic metabolic disorder syndromes characterized by hyperglycemia and caused by absolute or relative insufficiency of insulin secretion. The statistical results of the Chinese Center for Disease Control and Prevention in 2010 show that the prevalence of diabetes among residents aged 18 and over in my country is 9.65%, and the estimated number is about 96.9 million. my country has become the country with the largest population of diabetes in the world (Kang Jihong, TiaoGuan et al. Translational Medicine Research, 2012,2(3):1-24.). With the advancement of urbanization and population aging in my country, the prevalence of diabetes is rising rapidly. The hazards of diabetes are mainly various microvascular complications caused by long-term high blood sugar. Diabetic Nephropathy (DN) is one of the main complications of diabetes. Currently, 15% to 25% of type 1 diabetes patients and 30% to 40% of type 2 diabetes patients have DN, and DN has become the primary cause of end-stage renal disease. cause. Long-term hyperglycemia can easily damage the filtering function of the kidneys, initially manifested as microalbuminuria, as the course of the disease prolongs, the urine protein increases, the ability of the kidneys to remove toxins in the blood gradually decreases, and eventually develops into end-stage renal disease (ESRD). Live on hemodialysis or a kidney transplant.

At present, the pathogenesis of diabetic nephropathy has not been fully elucidated, involving multiple mechanisms. How to prevent and treat diabetic nephropathy has become the focus of current medical circles. At present, the clinical treatment of diabetic nephropathy mainly adopts symptomatic treatment and combination drugs, and comprehensively lowers blood sugar, blood fat and blood pressure. There has been a lack of drugs specifically for this disease, so finding more effective drugs has become an urgent task.

Bupleurum is a common traditional Chinese medicine for treating exterior syndrome in my country, and it is a plant of the genus Bupleurum in the family Umbelliferae. Bupleurum bupleurum was first recorded in "Shen Nong's Materia Medica", which has the effects of relieving exterior fever, soothing the liver and relieving stagnation, and promoting Yang Qi (Pharmacopoeia Committee of the Ministry of Health of the People's Republic of China, Chinese Pharmacopoeia, Part One. Beijing: China Medical Science and Technology Press, 2010 :263), has anti-inflammatory, pain relieving, cooling effects, etc., and is often used as a compound to treat colds, headaches, fever, influenza, upper respiratory tract infections, etc.

Animal experiments have shown that Bupleurum polysaccharides can be used to treat inflammatory diseases (NavarroP, GinerRM et al. LifeSci68:1199-1206.) and autoimmune diseases (Yi-wenJiang, HongLi et al. Clinical and Developmental Immunology, 2012).

Taking a comprehensive view of the research at home and abroad, there is no report on the efficacy of Bupleurum polysaccharides in preventing and treating diabetic nephropathy.

Contents of the invention

The purpose of the present invention is to provide a new medicinal use of the traditional Chinese medicine Bupleurum polysaccharide; specifically, it relates to the use of Bupleurum polysaccharide in the preparation of medicines for preventing and treating diabetic nephropathy

The invention separates and extracts the total polysaccharide extract from the traditional Chinese medicine Bupleuri. Whole animal model tests have confirmed that it has a strong effect of preventing and treating experimental diabetic nephropathy, and in vitro experiments have confirmed that it has the effect of reversing TGF-β-induced renal tubular cell transdifferentiation.

The traditional Chinese medicine Bupleurum in the present invention includes Bupleurum chinense DC and Bupleurum smithiivar.parvifolium.

The present invention has carried out animal experiments, and the experimental results show that the Bupleurum polysaccharide has preventive and treatment effects on diabetic nephropathy. Animal experiments have confirmed that Bupleurum polysaccharides can significantly reduce blood sugar and urine sugar levels, significantly promote insulin secretion, lower serum creatinine levels, lower urinary microalbumin secretion, lower kidney index, improve kidney pathological damage, and show obvious hypoglycemic effects and renal protection; the results of in vitro cell experiments also confirmed that Bupleurum polysaccharide has a direct and obvious renal protective effect. In the TGF-β-induced renal tubular cell lesion model, Bupleurum polysaccharide reversed TGF-β-induced renal tubular cell Transdifferentiation. Furthermore, the Bupleurum polysaccharide can be used to prepare medicines for preventing and treating diabetic nephropathy.

The object of the present invention is achieved by the following methods:

1. Bupleurum polysaccharides used in the prevention and treatment of diabetic nephropathy test:

1) Body weight measurement

Mice were weighed and litter changed every two days. The results showed that, compared with the model group, each group of Bupleurum polysaccharides could improve the symptoms of emaciation in diabetic mice, increase the weight of the animals, and enhance the activity of the animals.

2) Determination of kidney index

The mice were weighed on the day of execution, which was the weight of the mice; after the mice were sacrificed, the kidneys on both sides were taken, and the surface blood was blotted dry with filter paper, and weighed to obtain the weight of the left and right kidneys, and the average was taken as the weight of the kidneys; Index = organ weight (g)/body weight (g)*1000. The results showed that: compared with the model group, each dose of Bupleurum polysaccharide could reduce the renal index, and the high dose had a significant difference (P<0.05).

3) blood sugar measurement

The mouse blood was collected and centrifuged to obtain serum. Glucose levels were determined according to the glucose oxidase method. The results showed that, compared with the model group, each dose of Bupleurum polysaccharide could significantly reduce the blood glucose level of the mice (P<0.05).

4) Determination of serum insulin content

The mouse blood was collected and centrifuged to obtain serum. Insulin levels were determined according to the instructions of the insulin ELISA kit. The results showed that, compared with the model group, each dose of Bupleurum polysaccharide could increase insulin levels and improve insulin secretion, and the high dose had a significant difference (P<0.01).

5) Determination of serum creatinine content

The mouse blood was collected and centrifuged to obtain serum. The creatinine content was determined according to the instructions of the creatinine kit. The results showed that, compared with the model group, each dose of Bupleurum polysaccharide could significantly reduce the creatinine level of mice (P<0.01).

6) Determination of urine sugar content

Collect urine samples from mice for 5 hours, centrifuge, take supernatant, and dilute 1:20. Urinary sugar was determined by the glucose oxidase method. The results showed that compared with the model group, each dose of Bupleurum polysaccharide could reduce the level of urine sugar, and the high dose had a significant difference (P<0.01).

7) Determination of urinary microalbumin

Collect urine samples from mice for 5 hours, centrifuge, take supernatant, and dilute 1:10. Urine samples were assayed according to the microalbumin ELISA kit instructions. The results showed that, compared with the model group, each dose of Bupleurum polysaccharide could reduce urinary microalbumin secretion, and the high dose had a significant difference (P<0.01).

8) Renal pathology

After the mice were sacrificed, the left kidney and pancreas were removed, fixed in 10% formalin, sectioned in 4 μm paraffin, and stained with hematoxylin-eosin. The results showed that, compared with the model group, each dose of Bupleurum polysaccharide could significantly reduce renal hypertrophy, glomerular matrix hyperplasia, renal tubular vacuolization and other renal pathological symptoms of DN.

2. Bupleurum polysaccharide used in TGF-β-induced renal tubular cell transdifferentiation test

When the body is in diabetic nephropathy, high glucose induces an increase in the expression of TGF-β (transforming growth factor β) in the kidney, which in turn causes renal tubular cells to transdifferentiate into fibroblasts, forming renal fibrosis. In the in vitro experiment, a TGF-β-induced renal tubular cell transdifferentiation model was established to simulate the environment of diabetic nephropathy in vivo. The results showed that the morphology of normal renal tubular epithelial cells was elliptical paving stone or polygonal; the renal tubular epithelial cells in the model group lost their intrinsic morphology and were fibroblast-like long spindles; compared with the model group, each dose of Bupleurum polysaccharide could Significantly reversed TGF-β-induced cell morphological changes.

Table 1 Body weight and kidney index of mice with diabetic nephropathy (X±SD)

Table 2 Serum and urine indicators of diabetic nephropathy mice (X±SD)

Among them, *P<0.05 compared with the model group; **P<0.01 compared with the model group; ***P<0.001 compared with the model group;

Description of drawings

Fig. 1 Results of body weight changes in mice with diabetic nephropathy.

Figure 2 The results of kidney index in mice with diabetic nephropathy.

Fig. 3 Measurement results of blood glucose level in mice with diabetic nephropathy.

Fig. 4 Determination results of serum insulin levels in mice with diabetic nephropathy.

Fig. 5 Determination results of serum creatinine level in mice with diabetic nephropathy.

Fig. 6 Measurement results of urine sugar level in mice with diabetic nephropathy.

Fig. 7 Measurement results of urinary microalbumin secretion in mice with diabetic nephropathy.

Figure 8 HE staining diagram of mouse kidney in normal group.

Fig. 9 HE staining diagram of kidney of mice in Bupleurum single-administration group.

Figure 10 HE staining of the kidneys of mice in the model group.

Fig. 1130 mg/kg Bupleurum polysaccharide administration group mouse kidney HE staining diagram.

Fig. 1260 mg/kg Bupleurum polysaccharide administration group mouse kidney HE staining diagram.

Fig. 137. HE staining diagram of kidney of mice in 2mg/kg glibenclamide administration group.

Fig. 14 Transdifferentiation experiment induced by TGF-β-morphological changes of renal tubular cells (aniline blue staining).

detailed description

Example 1

Take 100g of Bupleurum herb, extract by cold soaking with 95% ethanol, put the dregs in a ventilated place at room temperature to dry, then extract with hot water for 3 times, filter, combine the extracts, concentrate, centrifuge, and the supernatant is washed with trichloro Remove free protein with acetic acid, centrifuge, dialyze the supernatant with water for 3 days, concentrate the dialysate to a small volume, add ethanol to 80% alcohol content, centrifuge, precipitate and freeze-dry to obtain the total polysaccharide, the product yield is more than 3%, the polysaccharide content More than 65%.

The Bupleurum medicinal material can be selected from Bupleurum chinense DC or Bupleurumsmithiivar.parvifolium.

Example 2

Fifty-six C57BL/6 mice (8 weeks old) were randomly divided into 6 groups (groups A, B, C, D, E, and F) according to body weight: group A was the normal control group, and group B was given Bupleurum polysaccharide alone. Medicine group (normal mice were given Bupleurum polysaccharide, the dosage was 60mg·kg ^-1 ), group C was the model group, group D was the model group + Bupleurum polysaccharide 30mg·kg ^-1 dose group, group E was the model group+ Bupleurum polysaccharide 60mg·kg ^-1 dose group, group F was the model group + glibenclamide positive group (7.2mg·kg ^-1 ), with 8-12 rats in each group. Taking the day before modeling as Day-1, after the mice were fasted for 15 hours overnight, the models were established in groups C, D, E, and F, and the dose of STZ (Streptozotocin, streptozotocin) 100mg/kg was used for two consecutive days Intraperitoneal injection (0.1mL·10g ^-1 ). The mice in groups A and B were injected with the same amount of normal saline to simulate the modeling steps. After modeling, the mice were fed with normal diet, and the survival rate of the mice was observed within 72 hours. At 8 o'clock in the morning on Day9, the tail was cut to take blood, and the blood sugar of the animal was measured with a blood glucose meter. The mice with blood sugar ≥ 16.8mmol/L were selected as diabetic model mice. They were randomly divided into groups according to the blood sugar, and the drug group began to be given intragastric administration once a day. Lasts for 35 days (Day9-Day44). On Day 37-38, urine samples were collected for 5 hours using mouse metabolic cages, centrifuged at 800 g for 15 min, the supernatant was taken, weighed, and frozen at -20°C until testing. Urine samples are used to detect indicators such as urine sugar and urine microalbumin. On Day 44, mice were taken from eyeballs to collect blood. After centrifugation at 800g for 10 minutes, the blood was collected, subpackaged, and stored at -20°C until testing. Blood samples were used to detect indicators such as blood sugar and serum insulin. At the same time, the mouse kidney was taken out, weighed on a precision balance, recorded, and the organ index was calculated, organ index=organ weight (g)/body weight (g)*1000. The left kidney was cut along the renal pelvis with a knife and stored in 10% formalin solution for paraffin sectioning. The right kidney was wrapped in tin foil and stored in a -80°C refrigerator.

Example 3

The renal tubular cells were seeded, adhered to the wall overnight, and starved for 24 hours. The experiment was divided into 10 groups. They were: Normal group (normal control group); TGF-β group (model group); SB group (single plus TGF-β inhibitor); BPs20 group (single plus Bupleurum 20 μg/mL group); BPs40 group (single plus Bupleurum 40μg/mL group); BPs80 group (bupleurum 80μg/mL alone group); SB-TGF group; BPs20-TGF group; BPs40-TGF group; BPs80-TGF group. After 48 hours of intervention in each group, the cells were fixed with 4% paraformaldehyde, and the cell morphology was observed and photographed under an inverted microscope.

The results of animal experiments show that Bupleurum polysaccharides can improve the body weight of diabetic model animals, significantly reduce blood sugar and urine sugar levels, significantly improve insulin secretion, lower serum creatinine levels, reduce urine microalbumin secretion, and lower kidney index. The results of pathological examination showed that the glomerular mesangial cells in the model group increased significantly, the mesangial matrix hyperplasia, the cross-sectional area of the glomeruli increased significantly, a large number of renal tubular epithelial cells vacuolated, and some epithelial cell nuclei fell off. Compared with the model group, each dose of Bupleurum polysaccharide can significantly reduce the pathological symptoms of glomerular matrix hyperplasia and renal tubular vacuolization, and delay the progress of diabetic nephropathy. Experimental studies on isolated cells have shown that Bupleurum polysaccharides can reverse TGF-β-induced tubular cell transdifferentiation, suggesting that it can prevent renal tubular lesions caused by diabetes. Animal experiments and in vitro cell experiments together suggest that Bupleurum polysaccharide has two effects of lowering blood sugar and protecting kidney in mice with diabetic nephropathy.

Claims (3)

1. The use of Bupleurum polysaccharide in the preparation of medicines for treating diabetes. 2. The use of Bupleurum polysaccharide in the preparation of drugs for the prevention and treatment of diabetic nephropathy. 3. according to the described purposes of claim 1 or 2, it is characterized in that, described Bupleurum polysaccharide is prepared by following method: Take 100g of Bupleurum herb, extract by cold soaking with 95% ethanol, put the dregs in a ventilated place at room temperature to dry, then extract with hot water for 3 times, filter, combine the extracts, concentrate, centrifuge, and the supernatant is washed with trichloro Remove free protein with acetic acid, centrifuge, dialyze the supernatant with water for 3 days, concentrate the dialysate to a small volume, add ethanol to 80% alcohol content, centrifuge, precipitate and freeze-dry to obtain the total polysaccharide, the product yield is more than 3%, the polysaccharide content More than 65%; The Bupleurum medicinal material is selected from Bupleurum chinense DC or Bupleurumsmithiivar.parvifolium.