Application of total saponins of Gynostemma pentaphyllum in the preparation of drugs for preventing and / or treating diabetic renal fibrosis

Abstract

The present invention provides the use of total saponins of gypenosides in the preparation of a drug for preventing and / or treating diabetic renal fibrosis, and specifically belongs to the field of medical technology. The present invention provides the use of total saponins of gypenosides in the preparation of a drug for preventing and / or treating diabetic renal fibrosis. Compared with the positive drug dapagliflozin (DATA) for treating DKD, total saponins of gypenosides (Gyp) not only have the effects of lowering blood sugar and slowing down renal fibrosis, but also can protect liver damage, have the effects of lowering blood lipids and slowing down liver lipid accumulation.

Description

Technical Field

[0001] The invention belongs to the field of medical technology, and specifically relates to the application of total saponins of Gynostemma pentaphyllum in preparing medicines for preventing and / or treating diabetic renal fibrosis. Background Art

[0002] Diabetic nephropathy (DKD) is one of the main causes of end-stage renal failure (ESRD). The main characteristics of diabetic nephropathy include pathological changes such as decreased glomerular filtration rate, glomerular sclerosis, podocyte damage and renal fibrosis, which eventually lead to gradual renal failure.

[0003] At present, the main method for treating DKD is to use drugs such as dipeptidyl peptidase-4 (DPP-4) inhibitors, sodium-glucose cotransporter-2 (SGLT-2) inhibitors and angiotensin II receptor antagonists (ARBs), but long-term use can cause side effects in patients. Traditional Chinese medicine mostly uses natural animal, plant or mineral ingredients, and has mild medicinal properties. Its multiple targets produce synergistic effects and have great advantages in treating complex diseases. Therefore, it is of great clinical significance to find new therapeutic drugs in traditional Chinese medicine.

[0004] The TCM syndrome differentiation and treatment method plays a unique role in the prevention and treatment of diabetic nephropathy. At present, TCM believes that the main pathogenesis of diabetic nephropathy is deficiency of both qi and yin, accompanied by dampness, turbid toxins, heat and other solid evils. The 2005 "Diabetes Nephropathy Diagnosis and Treatment Program of TCM" pointed out: "Long-term illness enters the collaterals, and turbid toxins damage the kidneys. Long-term diabetes mellitus, sugar toxicity and lipid toxicity, damage the kidney collaterals, damage the kidney body, and make the kidney function dysfunctional, and turbid toxins stagnate inside." Therefore, to prevent and treat diabetic nephropathy, it is necessary not only to nourish qi and yin, but also to pay attention to clearing away heat and detoxification to prevent changes, and activating the body's positive energy is the key to solving the occurrence and development of diabetic nephropathy.

[0005] The Chinese medicine Gynostemmapentaphyllum (Thub.) Makino is the dried aerial part of a perennial creeping herbaceous plant of the Cucurbitaceae family. It is recorded in the National Compilation of Chinese Herbal Medicines as an excellent natural health food with "food and medicine of the same origin", and is known as the "Southern Ginseng". According to the theory of traditional Chinese medicine, it is bitter, slightly sweet, cold, and enters the lung, spleen, and kidney meridians. It has the effects of invigorating qi and nourishing yin, clearing away heat and detoxifying, and relieving cough and expectoration. It is commonly used in the clinical treatment of chronic tracheitis, gastroenteritis, and cardiovascular disease. Modern studies have shown that the active ingredient of Gynostemma pentaphyllum, Gynostemma pentaphyllum total saponin (Gyp), has a variety of pharmacological properties including anti-inflammatory, antioxidant and neuroprotective effects. At the same time, some researchers have pointed out that Gyp can also improve pulmonary fibrosis, but there are few reports at home and abroad on whether it can improve diabetic renal fibrosis. Summary of the invention

[0006] The purpose of the present invention is to provide the use of total saponins of gypenostomum pentaphyllum in the preparation of a drug for preventing and / or treating diabetic renal fibrosis. Total saponins of gypenostomum pentaphyllum can achieve the treatment of renal fibrosis.

[0007] The present invention provides the use of total saponins of Gynostemma pentaphyllum in preparing medicine for preventing and / or treating diabetic renal fibrosis.

[0008] The present invention also provides the use of total saponins of Gynostemma pentaphyllum in the preparation of medicines for preventing and / or treating diabetes, diabetic liver damage and diabetic renal fibrosis.

[0009] The present invention also provides the use of total saponins of Gynostemma pentaphyllum in the preparation of drugs for lowering blood sugar, lowering blood lipids, improving liver damage, and improving kidney damage; the improvement of liver damage includes improving liver fatty lesions and slowing down liver lipid accumulation; the improvement of kidney damage includes improving kidney fibrosis.

[0010] The present invention provides the use of total saponins of gypenostomum pentaphyllum in the preparation of a drug for preventing and / or treating diabetic renal fibrosis. Compared with traditional Chinese medicine, western medicine has large side effects. The total saponins of gypenostomum pentaphyllum of the present invention have the effect of preventing and / or treating diabetic renal fibrosis. The test results show that compared with the positive drug dapagliflozin (DATA) for treating DKD, total saponins of gypenostomum pentaphyllum (Gyp) not only has the effect of lowering blood sugar and slowing down renal fibrosis, but also can protect liver damage, have the effect of lowering blood lipids and slowing down liver lipid accumulation. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] 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.

[0012] Figure 1 This is a graph showing the hypoglycemic effect of total saponins of Gynostemma pentaphyllum provided by the present invention on a DKD mouse model;

[0013] Figure 2 This is a result diagram of the lipid-lowering effect of total saponins of Gynostemma pentaphyllum provided by the present invention on a DKD mouse model;

[0014] Figure 3 This is a graph showing the effect of the total saponins of Gynostemma pentaphyllum provided by the present invention on inhibiting liver fatty lesions in a DKD mouse model;

[0015] Figure 4 This is a graph showing the effect of the total saponins of Gynostemma pentaphyllum provided by the present invention on alleviating renal damage in a DKD mouse model;

[0016] Figure 5This is a graph showing the effect of total saponins of Gynostemma pentaphyllum provided by the present invention on improving early renal fibrosis in a DKD mouse model. DETAILED DESCRIPTION

[0017] The present invention provides the use of total saponins of gypenostomum pentaphyllum in the preparation of a drug for preventing and / or treating diabetic renal fibrosis. The present invention does not specifically limit the source of total saponins of gypenostomum pentaphyllum, and conventional commercial products can be used. The total saponins of gypenostomum pentaphyllum described in the present invention can prevent and / or treat diabetic renal fibrosis.

[0018] The present invention also provides the use of total saponins of gypenostomum pentaphyllum in the preparation of drugs for preventing and / or treating diabetes, diabetic liver damage and diabetic kidney fibrosis. The present invention finds that total saponins of gypenostomum pentaphyllum not only has a hypoglycemic effect, but also can slow down diabetic kidney fibrosis and reduce diabetic liver damage.

[0019] The present invention also provides the use of total saponins of Gynostemma pentaphyllum in the preparation of drugs for reducing blood sugar, reducing blood lipids, improving liver damage, and improving kidney damage; the improvement of liver damage includes improving liver fatty lesions and slowing down liver lipid accumulation; the improvement of kidney damage includes improving kidney fibrosis. The test results show that total saponins of Gynostemma pentaphyllum can improve liver damage by improving liver fatty lesions and slowing down liver lipid accumulation. At the same time, total saponins of Gynostemma pentaphyllum can reduce blood sugar, reduce blood lipids, and improve kidney damage.

[0020] To further illustrate the present invention, the application of total saponins of Gynostemma pentaphyllum provided by the present invention in the preparation of drugs for preventing and / or treating diabetic renal fibrosis is described in detail below in conjunction with the accompanying drawings and examples, but they should not be construed as limiting the scope of protection of the present invention.

[0021] Example 1

[0022] The present invention uses 6-8 week old male C57BL / 6 mice for the experiment. All animal experiments were conducted in accordance with the Guide for the Care and Use of Laboratory Animals proposed by the National Institutes of Health (NIH) of the United States and approved by the Animal Ethics Committee of Zhejiang University.

[0023] Grouping and drug administration

[0024] Healthy 6-8-week-old male mice weighing 18-20g were placed in an environment with a temperature of 21-23℃ and a light cycle of 12h, and were allowed free access to food and water. The mice were then randomly divided into 5 groups according to their body weight, including a control group (Control), a DKD model, a low-dose group (Gyp-L) of total saponins of Gynostemma pentaphyllum (purchased from Chengdu Lemeitian Pharmaceutical Technology Co., Ltd.), a high-dose group (Gyp-H) of total saponins of Gynostemma pentaphyllum, and a positive drug group (DATA). The control group was fed with ordinary food, and the mice in other groups were fed with a high-fat diet (HFD: 67.5% ordinary feed, 10% lard, 20% sucrose, 0.5% pig bile salt, and 2% cholesterol) until the end of the experiment. After 6 weeks, the experimental mice (intraperitoneal injection) were injected with 100mg / kg STZ diluted in citrate buffer solution (0.1mol / LpH4.5), while normal mice were injected with citrate buffer alone. After 72 hours, fasting was performed and blood was collected from the tail tip to measure fasting blood glucose (FBG). The mice with a measured value greater than or equal to 16.7 mmol / L were evaluated as diabetic mice. After the diabetic model was established, total saponins of Gynostemma pentaphyllum and dapagliflozin were dissolved in 0.5% sodium carboxymethylcellulose (CMC-Na), respectively, and the low-dose group of total saponins of Gynostemma pentaphyllum 50 mg / kg (Gyp-L), the high-dose group of total saponins of Gynostemma pentaphyllum 100 mg / kg (Gyp-H) and the positive drug dapagliflozin 2 mg / kg (DATA) were gavaged, and the control group was given an equal volume of CMC-Na solution. After administration, body weight and fasting blood glucose were measured every 2 weeks, and the treatment was continued for 8 weeks.

[0025] Sample collection

[0026] Mouse urine sample collection: At the end of the 8-week treatment, mice were placed in separate metabolic cages to collect urine for 24 hours. The collected urine was divided into EP tubes and labeled. The EP tubes were placed in a centrifuge and centrifuged at 3500 rpm for 10 minutes at 4°C. The supernatant was collected and transferred to a -80°C refrigerator for testing.

[0027] Mouse liver and kidney sample collection: Anesthetize the mouse, collect blood, and perfuse physiological saline into the systemic blood vessels until the kidneys turn white. Then quickly remove the kidneys and liver, remove the surrounding fat tissue and blood vessels and other accessory structures, and gently absorb the surface liquid with pre-weighed filter paper or non-woven fabric, and quickly weigh the wet weight of the tissue. Then put half of it into a centrifuge tube containing 4% paraformaldehyde and mark it for subsequent paraffin embedding and frozen section preparation. The other half is divided into cryopreservation tubes, marked and quickly placed in liquid nitrogen, and transferred to a -80℃ refrigerator after freezing.

[0028] Calculation of renal index

[0029] To calculate the renal index, the weight of the mouse must be accurately recorded and the ratio of kidney wet weight to mouse body weight must be calculated, usually expressed as kidney wet weight (unit: mg) divided by body weight (unit: g), expressed in the form of mg / g.

[0030] Mouse fasting blood glucose (FBG) detection

[0031] Before the experiment, mice were fasted but allowed free access to water. At the beginning of the test, the mice were manually fixed, the tail tip was wiped with a 75% alcohol cotton ball, and then the capillary at the end of the tail tip was lightly punctured with a blood collection needle. The blood that flowed out was dripped onto the blood-absorbing area of ​​the test paper inserted into the blood glucose meter in advance, and the blood glucose meter was waited for to automatically read and record the blood glucose value. After the blood collection was completed, the tail tip was gently pressed with a cotton ball to stop the bleeding. After observing that there was no bleeding, the mice were put back in the cage, and it was ensured that there was no risk of infection at the blood collection site of the tail.

[0032] Oral glucose tolerance test (OGTT) in mice

[0033] The mice in each group were fasted for more than 16 hours. During the fasting period, the mice maintained normal drinking water, and the required glucose solution dosage (usually 2g / kg body weight, dissolved in sterile saline) was calculated according to body weight. The basal blood glucose level of mice in the fasting state was measured with a blood glucose meter and recorded as the 0min blood glucose value. Subsequently, the glucose solution was gavaged (OGTT), and the timing was started from the completion of gavage, and the blood glucose value of each mouse at each time point was measured at 15min, 30min, 60min, 90min, and 120min. After the experiment, the data were sorted and the glucose tolerance curve was drawn, with the blood glucose value as the vertical axis and the time as the horizontal axis to evaluate the trend of blood glucose change over time. In addition, the area under the curve (AUC) can be calculated to quantify the glucose tolerance of mice.

[0034] Serum biochemical index detection

[0035] The collected blood samples were centrifuged (normally set at 3000 rpm for 10-15 min), and the separated serum was quickly transferred to a new sterile centrifuge tube for testing. According to the manufacturer's instructions, the levels of various indicators such as alanine aminotransferase (ALT), aspartate aminotransferase (AST), total cholesterol (TC), triglycerides (TG), low-density lipoprotein cholesterol (LDL-C) and serum creatinine (Scr) in the serum samples were determined using a fully automatic biochemical analyzer (FDC7000i, Fujifilm Co, Tokyo, Japan). At the same time, the BUN content in the serum was detected according to the instructions of the BUN test kit.

[0036] Detection of 24-hour urine microalbumin (mALB) in mice

[0037] Before the experiment, the sample was warmed to room temperature and diluted if necessary to ensure that the sample concentration was within the detection range. The operation was performed according to the instructions of the urine microalbumin test kit, and finally the absorbance value of each well was measured on an ELISA reader at a wavelength of 450nm. A standard curve was drawn based on the absorbance of the standard to calculate the concentration of urine microalbumin in the sample.

[0038] Mouse histopathology

[0039] The collected kidney and liver tissues were fixed overnight in 4% paraformaldehyde solution, then embedded in paraffin and cut into 4 μm sections. Tissue sections were subjected to staining protocols including hematoxylin and eosin (H&E), Masson and PAS staining. Sections were observed using an optical microscope (CS2, Leica Microsystem, Wetzlar, Germany).

[0040] Oil Red O

[0041] Freshly prepared kidney tissues were embedded in OCT complex and stained with Oil Red O at 5 μm. Sections were rinsed with isopropanol and stained with Oil Red O (Sigma-Aldrich) working solution for 30 min according to the manufacturer's instructions. Nuclei were counterstained with hematoxylin for 5 min and examined under light microscopy.

[0042] Immunofluorescence staining (IF)

[0043] The SP kit (ZSGB-BIO; OriGene Technologies, Inc., Beijing, China) was used. The sections were stained with the following antibodies overnight: collagen I (Col-Ⅰ; 1:300, dilution, Proteintech, Chicago, USA) and α-smooth muscle actin (α-SMA; 1:500, dilution, Proteintech, Chicago, USA) at 4°C overnight. The sections were further incubated with secondary antibodies (1:2000 dilution, Proteintech, Chicago, USA) for 1 h, and the images were observed under a fluorescence microscope.

[0044] Statistical analysis

[0045] In this example, GraphPad Prism software (GraphPad Holdings, San Diego, CA, USA) was used to analyze the data, and the values ​​were expressed as mean ± SD (mean ± SD; N = 6 mice per group). The data satisfied the normal distribution, and the mean values ​​between groups were compared using one-way analysis of variance (ANOVA) and Dunnett's test. The difference was considered statistically significant when p value < 0.05.

[0046] result

[0047] Gynostemma pentaphyllum total saponins reduce blood glucose levels in DKD mouse models.

[0048] Figure 1 The results of the effect of total saponins of Gynostemma pentaphyllum on blood glucose in DKD mouse model are shown in Figure 1, including A) fasting blood glucose level, B) glucose tolerance level OGTT, C) area under the blood glucose curve AUC and D) liver PAS staining; # Indicates p<0.05vs control group, ## Indicates p<0.01vs control group, ### Indicates p<0.001vs control group, * Indicates p<0.05vs model group, ** Indicates p<0.01vs model group, *** Indicates p<0.001 vs model group. Figure 1 As shown in Figure 2, the FBG level in DKD mice was significantly higher than that in the control group (P<0.001). However, after 8 weeks of Gyp treatment, the FBG level was significantly decreased (P<0.01, Figure 1 In OGTT, the glucose levels in the DKD group at 15min, 30min, and 60min were significantly higher than those in the Control group (P<0.001). Compared with the DKD group, the blood glucose levels in the Gyp group at 60min and 90min were significantly lower (P<0.01, Figure 1 After glucose loading, the area under the curve (AUC) of diabetic mice increased significantly, while that of the Gyp group decreased significantly (P<0.001, Figure 1 C in the figure). In addition, the present invention uses PAS staining to evaluate whether Gyp can increase liver glycogen storage in DKD mice. The results showed that the liver cells of the control group mice had normal morphology and high glycogen content; the livers of the DKD model group mice showed disordered liver cell structure and low glycogen content; and after 8 weeks of administration, the livers of the Gyp group mice increased glycogen storage to varying degrees ( Figure 1 The above results indicate that Gyp has a hypoglycemic effect in the DKD rat model.

[0049] Gynostemma pentaphyllum total saponins reduce the level of blood lipids in DKD mice.

[0050] Figure 2 The results of the effect of total saponins of Gynostemma pentaphyllum on blood lipids in DKD mouse model are shown in Figure 1, including A) serum total cholesterol content (TC), B) serum triglyceride content (TG), and C) low-density lipoprotein cholesterol (LDL-C). # Indicates p<0.05vs control group, ## Indicates p<0.01vs control group, ###Indicates p<0.001vs control group, * Indicates p<0.05vs model group, ** Indicates p<0.01vs model group, *** Indicates p<0.001 vs model group. Figure 2 Compared with the control group, the levels of TC, TG and LDL-C in the serum of DKD model mice were significantly increased, and Gyp significantly reduced TC (P<0.001, Figure 2 A), TG (P < 0.001, Figure 2 B) and LDL-C (P<0.001, Figure 2 C), but the TC level of mice in the DATA group did not decrease significantly ( Figure 2 The above results show that Gyp can reduce the blood lipid level of DKD model mice, and its effect of reducing TC and LDL-C is better than that of positive drugs.

[0051] Effect of total saponins from Gynostemma pentaphyllum on the inhibition of hepatic fatty lesions in the DKD mouse model.

[0052] Figure 3 The results of the effect of total saponins of Gynostemma pentaphyllum on liver fatty lesions in the DKD mouse model are shown in Figure 1. A) serum alanine aminotransferase (ALT) content, B) serum aspartate aminotransferase (AST) content, C) liver HE staining, and D) liver oil red staining. # Indicates p<0.05vs control group, ## Indicates p<0.01vs control group, ### Indicates p<0.001vs control group, * Indicates p<0.05vs model group, ** Indicates p<0.01vs model group, *** Indicates p<0.001 vs model group. Figure 3 Compared with the control group, the levels of ALT and AST in DKD model mice were significantly increased (P<0.001). Figure 3 A) and AST (P<0.001, Figure 3 The levels of ALT and AST in the DATA group were not significantly reduced. The results showed that Gyp can slow down liver damage, but the effect of positive drugs on liver damage was not obvious.

[0053] In order to further prove the protective effect of Gyp on the liver, the present invention observed the pathological sections of the liver by HE, and found that the control group had regular liver lobule structure and a large number of hepatocytes with clear nuclei, and the DKD group had a large number of vacuoles in the liver tissue, which was inferred to be lipid droplet accumulation. However, compared with the DKD group, the administration of high-dose Gyp to diabetic mice reduced the number and size of lipid droplets ( Figure 3 C in the figure). At the same time, the liver lipid droplets were stained with oil red, and the results were consistent with those of HE staining, proving that Gyp can reduce the accumulation of liver lipid droplets ( Figure 3 The above results indicate that Gyp can protect the liver by reducing lipid accumulation in the liver, and the effect of Gyp-H is better than that of positive drugs.

[0054] Effect of total saponins from Gynostemma pentaphyllum on improving renal injury in DKD mouse model.

[0055] Figure 4 The results of the effect of total saponins of Gynostemma pentaphyllum on renal injury in DKD mouse model are shown in the figure; A) mouse body weight, B) kidney HE staining, C) kidney index, D) 24h urine microalbumin content (mALB), E) serum urine creatinine content (Scr), and F) serum urea nitrogen content (BUN). # Indicates p<0.05vs control group, ## Indicates p<0.01vs control group, ### Indicates p<0.001vs control group, * Indicates p<0.05vs model group, ** Indicates p<0.01vs model group, *** Indicates p<0.001 vs model group. Figure 4 Gyp administration had no significant effect on the body weight of DKD mice ( Figure 4 A in the figure). Obvious renal pathological changes were observed by H&E staining of DKD kidneys, such as glomerular hypertrophy, basement membrane thickening, mesangial expansion, and tubular dilatation and cavitation. However, this tissue structural abnormality improved with Gyp treatment ( Figure 4 The results of kidney index calculation showed that kidney hypertrophy was inhibited after Gyp treatment compared with the DKD group (P<0.001, Figure 4 In addition, the present invention also detected the relevant indicators of renal insufficiency in DKD mice. As shown in the results, Gyp reduced 24h urinary microalbumin (P<0.001, Figure 4 D in), serum creatinine (P<0.001, Figure 4 E) and urea nitrogen (P<0.001, Figure 4 The above results show that renal function improved after 8 weeks of Gyp treatment.

[0056] Effect of total saponins from Gynostemma pentaphyllum on alleviating early renal fibrosis in the kidney of DKD mouse model.

[0057] Figure 5 The results of the effect of total saponins of Gynostemma pentaphyllum on renal fibrosis in the DKD mouse model are shown in Figure 2; A) renal fluorescence staining, B) renal MASSON staining.

[0058] like Figure 5 As shown. The present invention evaluates kidney fibrosis by immunofluorescence staining. Immunofluorescence staining of renal tissue sections showed that compared with the control group, the expression of α-SMA and Col-Ⅰ in DKD mice was significantly increased, and Gyp administration significantly decreased ( Figure 5 Masson staining showed that treatment with Gyp resulted in a significant reduction in collagen deposition in the kidneys ( Figure 5 The above results indicate that Gyp has the effect of slowing down renal fibrosis in DKD mice.

[0059] 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. Use of total saponins of Gynostemma pentaphyllum in the preparation of drugs for preventing and / or treating diabetic renal fibrosis.

2. Application of total saponins of Gynostemma pentaphyllum in the preparation of drugs for preventing and / or treating diabetes, diabetic liver damage and diabetic renal fibrosis.

3. Application of total saponins of Gynostemma pentaphyllum in the preparation of drugs for lowering blood sugar, lowering blood lipids, improving liver damage, and improving kidney damage; the improvement of liver damage includes improving liver fatty lesions and slowing down liver lipid accumulation; the improvement of kidney damage includes improving renal fibrosis.

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