CN101732323A - Application of low-dose ursolic acid as medicament for treating diabetic early nephropathy - Google Patents

Abstract

The invention discloses the use of low-dose ursolic acid as a medicine for treating early diabetic nephropathy. The mode of administration of ursolic acid is oral, and the dosage is 50-70mg/kg body weight. The present invention confirms that oral low-dose ursolic acid has a significant inhibitory effect on the activation of ERK1/2 and JNK pathways in the MAPK pathway induced by diabetes and the phosphorylation level of STAT3 tyrosine, and can significantly reduce the level of phosphorylation induced by diabetes. The expression of iNOS in the renal cortex, more importantly, oral administration of ursolic acid can inhibit diabetes-induced glomerular hypertrophy and the accumulation of type IV collagen in the glomeruli, indicating that oral administration of ursolic acid has a significant effect on early diabetic nephropathy. treatment effect.

Description

Use of low-dose ursolic acid as a drug for the treatment of early diabetic nephropathy

technical field

The invention relates to the use of low-dose ursolic acid as a medicine for treating diabetic complications, in particular to the use of a medicine for treating early diabetic nephropathy.

Background technique

Diabetic nephropathy is one of the microvascular complications of diabetes, and is the main cause of chronic kidney disease and end-stage renal failure in diabetic patients. There are many reasons for diabetic nephropathy, including hemodynamic changes in renal tissue caused by diabetes (including the imbalance of blood flow regulation of renal vessels by renin-angiotensin and kinin-prostaglandin-thromboxane), diabetes-induced renal Biochemical metabolic disorders of tissues (including increased polyol pathway, increased protein non-enzymatic glycosylation, lipid metabolism disorders, etc.) and some diabetic patients also have symptoms of hypertension, etc. About 20-30% of diabetic patients suffer from nephropathy, which manifests as excretion of microalbumin in urine in the early stage, followed by massive proteinuria, and finally progresses to renal insufficiency, requiring dialysis or even kidney replacement to maintain life. Diabetes-induced renal failure is one of the leading causes of death among diabetic patients.

At present, the key to the treatment of diabetic nephropathy lies in early diagnosis and preventive treatment. Studies have shown that once patients have clinical symptoms of massive proteinuria, kidney damage caused by diabetes is often difficult to reverse. If measures can be taken before the occurrence of massive proteinuria in diabetic patients, the clinical symptoms and late renal failure of diabetic patients can be actively delayed. In view of the fact that the number of diabetic patients both type 1 and type 2 is increasing significantly all over the world, it is very urgent and important to find a way to treat diabetic nephropathy.

Ursolic acid is a class of triterpenoids, which are widely found in many natural plants such as hawthorn and Ligustrum lucidum. Studies in recent years have found that ursolic acid has a variety of biological activities, and has certain effects in anti-cancer, anti-cancer, liver protection, antibacterial, anti-virus, anti-oxidation, etc., and has a good prospect for development and utilization. At present, the application of ursolic acid in diabetes mainly focuses on the hypoglycemic effect of high-dose ursolic acid. At the animal level, especially without affecting non-fasting blood glucose levels, whether low-dose ursolic acid can significantly inhibit diabetes-induced glomerular hypertrophy and accumulation of type IV collagen in glomeruli and be used as a treatment for early diabetic nephropathy drugs that have not been reported.

Contents of the invention

The purpose of the present invention is to make up for the above-mentioned deficiencies in the prior art, and to provide a low-dose ursolic acid as a medicine for treating early diabetic nephropathy.

The technical solution for realizing the object of the present invention is: the use of low-dose ursolic acid as a drug for treating early diabetic nephropathy, the administration method is oral administration, and the administration dosage is 50-70mg/kg body weight.

Diabetic rodents are the main animal models used to study diabetes, and the streptozotocin-induced type 1 diabetes (insulin-dependent) rodent model has been very mature and has been widely used to study various diabetic complications. In this model, diabetic mice and clinical diabetic patients have similar early manifestations of renal lesions: pathological hypertrophy of glomeruli, hyperplasia of glomerular mesangial cells, thickening of basement membrane caused by glomerular matrix protein deposition And other important early pathological phenomena, and these are the important reasons that eventually lead to glomerulosclerosis and renal failure. It is generally believed that drugs that can inhibit these early pathological phenomena may delay or even inhibit the appearance of massive proteinuria and renal failure in the late stage of diabetic nephropathy.

The present invention studies the effect of oral low-dose ursolic acid on important early pathological phenomena such as pathological hypertrophy of glomeruli caused by diabetes, thickening of basement membrane caused by glomerular matrix protein deposition, and its effect on early kidney lesions related to diabetes. effect of pathways. Studies have confirmed that oral low-dose ursolic acid has a significant therapeutic effect on early diabetic nephropathy without reducing non-fasting blood sugar: it mainly inhibits glomerular hypertrophy caused by diabetes and collagen type IV in the glomeruli. These two phenomena are the most important pathological phenomena of early diabetic nephropathy. The present invention also proves that ursolic acid has a significant inhibitory effect on the activation of ERK1/2 and JNK pathways in the MAPK pathway induced by diabetes, and has a significant effect on the phosphorylation level of STAT3 tyrosine in the kidney induced by diabetes. Inhibition, and a significant reduction in the expression of iNOS in the kidney induced by diabetes. The activation and changes of these pathways have been shown to be associated with early kidney lesions in diabetes. In the present invention, the ursolic acid has a low dosage and has little side effects, and feeding the ursolic acid for 3 months has no significant influence on the physiological characteristics of the experimental mice.

Description of drawings

Figure 1 is a graph showing the results of oral administration of ursolic acid inhibiting glomerular hypertrophy caused by diabetes ( ^* p<0.05 compared with non-diabetic mice; ^** p<0.05 compared with diabetic mice).

Figure 2 is a graph showing the results of oral administration of ursolic acid to inhibit the accumulation of type IV collagen in glomeruli caused by diabetes ( ^* p<0.05 compared with non-diabetic mice; ^** p<0.05 compared with diabetic mice).

Fig. 3-A is the results of western blot detection of phosphorylated ERK1/2 and corresponding total protein expression levels in the renal cortex.

Figure 3-B is the quantification result of oral ursolic acid inhibiting the activation of the ERK1/2 pathway in the kidney cortex caused by diabetes ( ^* p<0.05 compared with non-diabetic mice; ^** p<0.05 compared with diabetic mice ).

Fig. 4-A is a graph showing the results of detecting the protein expression levels of phosphorylated JNK and total JNK in the renal cortex by Western blotting.

Figure 4-B is the quantification result graph of oral ursolic acid inhibiting the activation of JNK pathway in the kidney cortex caused by diabetes ( ^* p<0.05 compared with non-diabetic mice; ^** p<0.05 compared with diabetic mice).

Fig. 5-A is a graph showing the results of detecting the expression level of phosphorylated STAT3 in the renal cortex by Western blotting.

Figure 5-B is the quantification result of oral ursolic acid inhibiting the activation of STAT3 transcriptional activity in the kidney cortex caused by diabetes ( ^* p<0.05 compared with non-diabetic mice; ^** p<0.05 compared with diabetic mice) .

Fig. 6-A is a graph showing the results of detecting the expression level of iNOS in the kidney cortex by Western blotting.

Figure 6-B is the quantitative result graph of oral ursolic acid inhibiting the increase of iNOS expression in kidney cortex caused by diabetes ( ^* p<0.05 compared with non-diabetic mice; ^** p<0.05 compared with diabetic mice Compare).

Detailed ways

The present invention will be further described in detail below through specific examples.

The percentage concentrations used in the following examples are mass percentage concentrations unless otherwise specified.

The ursolic acid used in the examples of the present invention is produced by Nanjing Tesi Aimo Institute of Traditional Chinese Medicine. Thirty C57BL mice (purchased from the Animal Experiment Center of Wuhan University) were randomly divided into 3 groups: a type 1 diabetic mouse group, a ursolic acid-fed diabetic mouse group, and a non-diabetic mouse group, ten in each group.

The type 1 diabetic mouse model used was streptozotocin-induced:

1. Injection dose of streptozotocin: 60 mg streptozotocin/kg mouse body weight (streptozotocin was purchased from Amresco, USA).

2. Animal injection method: intraperitoneal injection in mice for 5 consecutive days.

3. Determination of diabetic mice: 7 days after the intraperitoneal injection of streptozotocin, blood was collected from the tail vein of the mice, and the blood glucose was measured with a OneTouch blood glucose meter. When the blood glucose was greater than 250 mg/dl, the mice were considered as diabetic mice.

Ursolic Acid Administration Mode and Dosage Used:

After ursolic acid is uniformly mixed with mouse powdered food by 0.01% mass/mass, it is extruded into blocks. The diabetic mice were fed without intake restriction, and the consumption of the administered food was recorded every week. It was calculated that the dose of ursolic acid administered to each mouse was 50-70 mg/kg body weight.

The index system used:

Detection of various physiological characteristics of experimental mice (including body weight, blood glucose, kidney weight and kidney weight/body weight); use PASH staining method to calculate glomerular volume; use type IV collagen immunohistostaining method to semi-quantify type IV Collagen accumulation in glomeruli; Western blot was used to quantify the activation levels of ERK and JNK pathways and STAT3 pathway in renal cortex; Western blot was used to quantify the expression of iNOS in renal cortex.

Example 1: Effects of Oral Ursolic Acid on Various Physiological Characteristics of Diabetic Mice

After feeding the diabetic mice with ursolic acid for 3 months, the body weight of the mice in each experimental group was measured, and blood was taken from the tail vein to determine the blood sugar level. The kidney weight of each group of mice was weighed, and the kidney weight/body weight was calculated, and the results are shown in Table 1.

Table 1. Experimental results of physiological characteristics of mice

Group (sample size) weight(g) Blood sugar (mmol/dl) Kidney weight (mg) Kidney weight/body weight (%) Diabetic group (10) 22.9±1.6 ^a 27.2±5.6 ^a 178.6±19.6 ^a 0.78±0.10 ^a Diabetic group + oral ursolic acid (10) 24.2±1.2 ^a 23.0±4.4 ^a 177.6±14.4 ^a 0.74±0.07 ^a Non-diabetic group (10) 27.5±2.3 11.2±1.4 163.8±20.3 0.60±0.07

Note: ^a in Table 1 means p<0.05, that is, there is a significant difference compared with non-diabetic mice.

The experimental results showed that compared with non-diabetic mice, streptozotocin-induced type 1 diabetic mice showed significant weight loss and high blood sugar levels, as well as increased kidney weight and kidney weight/body weight. However, oral administration of ursolic acid has no significant effect on these physiological characteristics of diabetic mice, indicating that the protective effect of oral ursolic acid on the kidneys of diabetic mice described below is not achieved by slowing down the severity of diabetes.

Embodiment 2: Inhibition of oral ursolic acid on glomerular hypertrophy caused by diabetes

The glomerulus is the most important ultrafiltration organ in the kidney. It maintains a low concentration of protein in the blood and urine to ensure the normal function of the body. In diabetes, the glomeruli are overloaded with ultrafiltration, resulting in pathological hypertrophy of the glomeruli.

After the kidneys of the mice in each experimental group were fixed with 10% neutral formaldehyde, they were routinely embedded in paraffin. Kidney sections were stained with PASH. 25-35 glomeruli were randomly selected from each mouse kidney section, and the area of each glomerulus was measured using ImagePlus 6.0 software after photographing with an Olympus BX60 microscope equipped with a CCD under a field of view of 400 times magnification. The volume of glomerulus is calculated by the following formula: volume of glomerulus (Vg)=β/k[area of glomerulus] ^3/2 , where β and k are both constants. After the calculation, the average volume and standard deviation of the glomerulus of each experimental group were taken, and the average volume of the glomerulus of the non-diabetic group was taken as a standard, which was set as 1. The results are shown in Figure 1.

The results showed that 3 months of diabetes caused a slight but significant hypertrophy of the glomeruli (37% increase compared to the non-diabetic group). Oral administration of ursolic acid significantly reduced the increase in glomerular volume caused by diabetes (65% reduction compared to the diabetic group).

Embodiment 3: The inhibitory effect of oral ursolic acid on the accumulation of type IV collagen caused by diabetes in glomeruli

The accumulation of extracellular matrix proteins in the glomerulus is one of the main causes of glomerulosclerosis and renal failure in post-diabetes. Type IV collagen is one of the major proteins of the kidney extracellular matrix protein. The expression of type IV collagen in the kidney was determined by immunohistochemical method. Conventional kidney paraffin sections were rehydrated with xylene and different concentrations of ethanol, and then 3% hydrogen peroxide was used to block the activity of endogenous peroxidase. After the sections were blocked with 2% goat serum, they were incubated overnight at 4°C with an antibody to type IV collagen (Rockland, USA). Washed with PBST, incubated with the corresponding secondary antibody at room temperature for 1 hour. The signal was amplified by the ABC kit (Vector Laboratories, USA), and the positive staining of type IV collagen was observed under a microscope after the color reaction of DAB substrate. 20-25 glomeruli were randomly selected from each mouse kidney section, and photographed with an Olympus BX60 microscope equipped with a CCD under a field of view of 400 times magnification. According to the degree of positive staining of type IV collagen, the expression level of type IV collagen in each glomerulus was divided into grades 0-4 (grade 0 means no positive staining). The expression of type IV collagen in all glomeruli was independently graded and identified by two researchers who did not know the grouping of the mice. Finally, take the mean value and standard deviation of the glomerular type IV collagen expression level of each experimental group, and take the average glomerular type IV collagen expression level of the non-diabetic group as the standard (set to 1), the results are shown in Fig. 2.

As shown in the results in Figure 2, compared with the non-diabetic group, after 3 months of diabetes, the mouse kidney showed a 3-fold increase in the expression level of glomerular type IV collagen. Oral administration of ursolic acid significantly reduced the diabetic-induced increase in glomerular collagen IV expression levels (75% reduction compared to the diabetic group).

Example 4: Oral ursolic acid inhibits the activation of the ERK1/2 pathway in kidney cortex caused by diabetes

The ERK1/2 pathway plays an important role in biological processes such as cell growth, differentiation, and inflammation. It has been reported that the ERK1/2 pathway plays a pivotal role in diabetic nephropathy. The activation of ERK pathway is regulated by the phosphorylation level of ERK1/2 protein. Therefore, this example quantitatively detects the degree of change of phosphorylated ERK1/2 and the corresponding total protein level in the renal cortex fraction by Western blotting.

The cortex of the kidney was excised under a dissecting microscope and placed in RIPA buffer (purchased from Beyond Company) for sonication. Total protein concentration was quantified by BCA protein quantification. After the same amount of protein was electrophoresed by SDS-PAGE, it was transferred to a cellulose membrane (Bio-rad, USA). Cellulose membranes were blocked with 2% skimmed milk and incubated with antibodies against phosphorylated ERK1/2 and total ERK1/2, respectively. Rinse the antibody-incubated cellulose membrane, and then add the corresponding secondary antibody for incubation. After reacting with ECL reagents, the protein bands recognized by the antibody can be displayed on the X-ray film after exposure, as shown in Figure 3-A. The protein bands were quantitatively analyzed with Quantity One 1-D analysis software (Bio-rad, USA). After calculating the gray scale of the protein band of phosphorylated ERK1/2, the mean value and standard deviation of the expression level of phosphorylated ERK1/2 in each experimental group were taken, and the mean value of the non-diabetic group was used as the standard (set to 1), the results are shown in Figure 3-B.

As shown in Figure 3-A, 3-B, compared with the non-diabetic group, the diabetic group significantly increased the expression of pho-ERK1/2 (2.5 times), while the expression of total ERK1/2 was not significantly Influence. Oral ursolic acid significantly inhibited diabetes-induced phosphorylation levels of ERK1/2 (60% reduction compared to diabetic group); however, there was no significant difference in total ERK1/2 expression in the oral ursolic acid group.

Embodiment 5: Oral ursolic acid inhibits the activation of the JNK pathway caused by diabetes in the kidney cortex

The JNK pathway plays an important role when cells are stimulated by external stress. It has been reported that the activation of the JNK pathway plays a pivotal role in pathological changes such as hyperplasia of glomerular mesangial cells and thickening of the basement membrane caused by glomerular matrix protein deposition in diabetes. In this example, Western blotting was used to quantitatively detect the degree of change in the protein levels of phosphorylated JNK and total JNK in the renal cortex.

After the same amount of protein was electrophoresed by SDS-PAGE, it was transferred to a cellulose membrane (Bio-rad, USA). Cellulose membranes were blocked with 2% skimmed milk and incubated with antibodies against phosphorylated JNK and total JNK, respectively. Rinse the antibody-incubated cellulose membrane, and then add the corresponding secondary antibody for incubation. After reacting with ECL reagents, the protein bands recognized by the antibody can be displayed on the X-ray film after exposure, as shown in Figure 4-A. The protein bands were quantitatively analyzed with Quantity One 1-D analysis software (Bio-rad, USA). After calculating the gray scale of the protein band of phosphorylated JN, take the mean value and standard deviation of the expression level of phosphorylated JNK in each experimental group, and take the mean value of the non-diabetic group as the standard (set to 1), the result See Figure 4-B.

As shown in Figures 4-A and 4-B, compared with the non-diabetic group, the diabetic group significantly increased the expression of pho-JNK (25-fold), but had no significant effect on the total JNK expression. Oral administration of ursolic acid significantly inhibited the phosphorylation levels of diabetes-induced JNK (73% reduction compared to the diabetic group); however, oral administration of ursolic acid had no significant difference on total JNK expression.

Example 6: Oral ursolic acid inhibits the activation of STAT3 transcriptional activity caused by diabetes in kidney cortex

STAT3 is a transcription factor that regulates cytokine signaling. Studies have shown that the transcriptional activity of STAT3 is regulated by the phosphorylation of its tyrosine 705 site. Recently, it has been reported that the transcriptional activity of STAT3 is closely related to the process of diabetic nephropathy. In this example, Western blotting was used to quantitatively detect the degree of change of phosphorylated STAT3 in the renal cortex.

After the same amount of protein was electrophoresed by SDS-PAGE, it was transferred to a cellulose membrane (Bio-rad, USA). Cellulose membranes were incubated with antibodies against phosphorylated STAT3 after blocking with 2% skimmed milk. Rinse the antibody-incubated cellulose membrane, and then add the corresponding secondary antibody for incubation. After reacting with ECL reagent, the phosphorylated STAT3 protein band recognized by the antibody can be displayed on the X-ray film after exposure, as shown in Figure 5-A. Quantity One 1-D analysis software (Bio-rad, USA) was used to quantitatively analyze the phosphorylated STAT3 protein bands. The mean value and standard deviation of the expression level of phosphorylated STAT3 in each experimental group were taken, and the mean value of the non-diabetic group was used as the standard (set as 1). The results are shown in FIG. 5-B.

As shown in Figure 5-A, 5-B, compared with the non-diabetic group, diabetes caused a significant increase (1.7 times) in the phosphorylation level of STAT3 tyrosine. Oral ursolic acid significantly inhibited the diabetes-induced increase in STAT3 tyrosine phosphorylation (63% reduction compared with the diabetic group).

Example 7: Oral ursolic acid inhibits the increase in the expression of iNOS induced by diabetes in the kidney cortex

Chronic inflammatory response is considered to be one of the causes of diabetic nephropathy. iNOS is one of the main regulatory proteins of inflammatory response. In this example Western blot was used to quantify iNOS expression levels in kidney cortex.

After the same amount of protein was electrophoresed by SDS-PAGE, it was transferred to a cellulose membrane (Bio-rad, USA). Cellulose membranes were incubated with anti-iNOS antibodies after being blocked with 2% skimmed milk. Rinse the antibody-incubated cellulose membrane, and then add the corresponding secondary antibody for incubation. After reacting with ECL reagent, the iNOS protein band recognized by the antibody can be displayed on the X-ray film after exposure, as shown in Figure 6-A. Quantity One 1-D analysis software (Bio-rad, USA) was used to quantitatively analyze the iNOS protein bands. The average value and standard deviation of the iNOS expression level of each experimental group were taken, and the average value of the non-diabetic group was taken as the standard (set as 1). The results are shown in FIG. 6-B.

As shown in Figures 6-A, 6-B, diabetes caused a significant increase (2.9-fold) in iNOS levels compared to the non-diabetic group. Oral administration of ursolic acid significantly inhibited the diabetes-induced increase in iNOS levels (73% reduction compared with the diabetic group).

In summary, the oral low-dose ursolic acid demonstrated by the present invention has a significant therapeutic effect on early diabetic nephropathy, and it is expected to be used as a basis for ursolic acid in the treatment of diabetic nephropathy or the alleviation of some symptoms in the future. Base.

Claims (1)

1. low-dose ursolic acid is as the purposes of treatment diabetic early nephropathy medicine, and administering mode is oral, and dosage is the 50-70mg/kg body weight.

Images