CN115252631B

CN115252631B - Application of pseudo-ginseng extract in preparation of medicine for treating diabetic nephropathy

Info

Publication number: CN115252631B
Application number: CN202210725233.2A
Authority: CN
Inventors: 桂定坤; 沈义兰; 薛瑞; 徐友华; 辛文锋; 林康鸿; 杨细飞; 汪年松
Original assignee: Shanghai Sixth Peoples Hospital
Current assignee: Shanghai Sixth Peoples Hospital
Priority date: 2022-06-24
Filing date: 2022-06-24
Publication date: 2024-05-14
Anticipated expiration: 2042-06-24
Also published as: CN115252631A

Abstract

The invention relates to an application of pseudo-ginseng extract in preparing a medicament for treating diabetic nephropathy; the notoginsenoside Fc can improve proteinuria and renal histopathology, reduce apoptosis of tubular cells, regulate expression of PTEN/PDK1/Akt/mTOR pathway proteins, reduce damage to DN glomerulus and mitochondria, reduce production of mitochondrial superoxide, and reduce loss of mitochondrial membrane potential; the notoginsenoside Ft1 can improve insulin resistance, lighten the expression of insulin receptor signaling pathway proteins p-IRS1/2, p-AKT and p-ERK1/2, can down regulate the protein expression of AGO-1 in kidney tissues of mice with two diabetic nephropathy models, restrain insulin resistance by regulating an AGO-1/TSP-1 pathway, improve metabolism and lighten DN kidney injury; the notoginsenoside Fc and notoginsenoside Ft1 can be used for preparing medicines for treating diabetic nephropathy.

Description

Application of pseudo-ginseng extract in preparation of medicine for treating diabetic nephropathy

Technical Field

The invention relates to the technical field of medicines, in particular to application of pseudo-ginseng extract in preparation of medicines for treating diabetic nephropathy.

Background

Diabetes is one of the most common chronic diseases in the world, and places a heavy burden on the whole society. It was counted that the prevalence of diabetes in the 20-79 year old population of 2021 was about 10.5% (5.366 billions), and increased to 12.2% (7.832 billions) by 2045. Diabetic nephropathy (Diabetic Nephropathy, DN) is one of the common microvascular complications of diabetes mellitus (Diabetes Mellitus, DM) and is also a main cause of Chronic kidney disease (Chronic KIDNEY DISEASE, CKD), the development of the diabetic nephropathy finally becomes End-stage kidney disease (End-STAGE RENAL DISEASE, ESRD), a patient can only carry out dialysis treatment, the pain of the body and the mind of the patient is caused, serious economic and social burden is caused, and the diabetic nephropathy is one of the great public health problems at present. With the rise in DN morbidity and prevalence, the proportion of chronic kidney disease associated with diabetes has exceeded that associated with glomerulonephritis, and blood glucose and blood pressure management is still the primary therapeutic strategy according to the kdig 2020 clinical practice guidelines, however, existing treatments for DN do not completely prevent disease progression, and therefore, there is an urgent need to explore methods for effectively slowing DN progression.

Notoginseng radix (Panax notoginseng) is a treasure in Chinese medicine, belongs to herbal plants of Araliaceae, is recorded in "Yuhua Jiedu", has effects of dredging collaterals, removing blood stasis, promoting blood circulation, removing blood stasis, detumescence and relieving pain, and can effectively relieve blood stasis symptom; the research shows that the effective components of the pseudo-ginseng are mostly saponin substances, wherein the total saponins (Panax Notognseng Saponins, PNS) of the pseudo-ginseng are main components in the saponin substances; however, the prevention and treatment of DN by pseudo-ginseng is rarely studied at present, and the action mechanism of the pseudo-ginseng is not completely clear.

Therefore, the study of the effect of pseudo-ginseng extract on diabetic nephropathy and the application of pseudo-ginseng extract in preparing medicaments for treating diabetic nephropathy is needed.

Disclosure of Invention

The invention aims at overcoming the defects in the prior art and provides application of pseudo-ginseng extract in preparing a medicament for treating diabetic nephropathy.

In order to achieve the above purpose, the technical scheme adopted by the invention is as follows:

Provides the application of the pseudo-ginseng extract in preparing the medicine for treating diabetic nephropathy.

Preferably, the pseudo-ginseng extract is pseudo-ginseng saponin.

More preferably, the notoginsenoside is notoginsenoside Fc or/and notoginsenoside Ft1.

More preferably, the molecular formula of notoginsenoside Fc is C ₅₈H₉₈O₂₆.

Preferably, the notoginsenoside Fc reduces the level of tubular apoptosis.

Preferably, the notoginsenoside Fc reduces oxidative stress levels in HK-2 cells.

Preferably, the notoginsenoside Fc improves mitochondrial function of HK-2 cells.

Preferably, the notoginsenoside Fc delays the progression of diabetic nephropathy by improving kidney function and structural abnormalities.

More preferably, the molecular formula of notoginsenoside Ft1 is C ₄₇H₈₀O₁₇, and the molecular weight is 917.13.

Preferably, the notoginsenoside Ft1 reduces the expression level of AGO-1 in kidney tissue of DN mice.

Preferably, the notoginsenoside Ft1 improves db/db mouse insulin resistance.

Preferably, the notoginsenoside Ft1 improves high fat diet + streptozotocin (HFD/STZ) mouse insulin resistance.

Preferably, the notoginsenoside Ft1 improves kidney function and abnormal structure of db/db mice, and delays the progress of diabetic nephropathy.

Preferably, the notoginsenoside Ft1 improves kidney function and structural abnormality of the HFD/STZ mice, and delays the progress of diabetic nephropathy.

Preferably, the medicament for treating diabetic nephropathy further comprises: one or more pharmaceutically acceptable auxiliary agents.

Preferably, the dosage form of the medicament for treating diabetic nephropathy comprises: decoction, pill, powder, unguent, pellet, medicated liquor, granule, oral liquid, capsule, tablet or injection.

Preferably, the administration route of the drug for treating diabetic nephropathy comprises: oral administration, sublingual administration, intramuscular or subcutaneous administration or intravenous administration.

Compared with the prior art, the invention has the following technical effects:

In vivo experiments are carried out on db/db mice and a high-fat diet plus Streptozotocin (STZ) -induced diabetic nephropathy model, and the treatment effect of notoginsenoside Fc and notoginsenoside Ft1 on diabetic nephropathy is discussed; the notoginsenoside Fc can improve proteinuria and renal histopathology, reduce renal tubular cell apoptosis, regulate PTEN/PDK1/Akt/mTOR channel protein expression and reduce DN glomerulus and mitochondrial injury, and further proves that the in vitro research can reduce the generation of mitochondrial superoxide and reduce Mitochondrial Membrane Potential (MMP) loss; the notoginsenoside Ft1 can improve insulin resistance, lighten the expression of insulin receptor signaling pathway proteins p-IRS1/2, p-AKT and p-ERK1/2, can down regulate the protein expression of AGO-1 in kidney tissues of mice with two diabetic nephropathy models, restrain insulin resistance by regulating an AGO-1/TSP-1 pathway, improve metabolism and lighten DN kidney injury; the notoginsenoside Fc and notoginsenoside Ft1 can be used for preparing medicines for treating diabetic nephropathy.

Drawings

FIG. 1 is a graph showing the effect of notoginsenoside Fc on blood and urine biochemistry and other indexes of a diabetic mouse in example 1; wherein, figure 1A is a graph of statistical analysis of urinary albumin/creatinine ratio (ACR) levels after 8 weeks of treatment; FIG. 1B is a graph of statistical analysis of Blood Urea Nitrogen (BUN) levels after 8 weeks of treatment; FIG. 1C is a graph of statistical analysis of kidney weight to body weight (KW/BW) levels after 8 weeks of treatment;

FIG. 2 shows the effect of notoginsenoside Fc on kidney morphology of diabetic mice in example 1 of the present invention; wherein, fig. 2A is a HE staining picture of kidney tissue of each group of mice; FIG. 2B is an immunohistochemical staining photograph of podocyte nephropathy protein (Nephrin) from kidney tissue of each group of mice; FIG. 2C is a semi-quantitative analysis of Nephrin expression changes;

FIG. 3 shows the effect of notoginsenoside Fc on apoptosis in diabetic mice in example 1 of the present invention; FIG. 3A is a Normal (Normal) group; FIG. 3B is a Model set; fig. 3C is a losartan (Losartan) treated group; FIG. 3D is a treatment group of notoginsenoside Fc (Notoginsenoside Fc);

FIG. 4 is a fluorescent image of the level of mitochondrial ROS in HK-2 cells of example 1 of the present invention;

FIG. 5 is a fluorescence image of the mitochondrial membrane potential level of HK-2 cells of example 1 of this invention;

FIG. 6 shows the effect of notoginsenoside Fc on the PTEN/PDK1/Akt/mTOR signaling pathway in diabetic mice in example 1 of the present invention; wherein, FIG. 6A is an immunohistochemical staining photograph of PTEN, PDK1, p-AKT and p-mTOR; FIG. 6B is a graph of the results of semi-quantitative analysis of immunohistochemistry for PTEN; FIG. 6C is a graph of the results of semi-quantitative analysis of immunohistochemistry for PDK 1; FIG. 6D is a graph showing the results of semi-quantitative analysis of p-AKT immunohistochemistry; FIG. 6E is a graph of the results of semi-quantitative analysis of p-mTOR immunohistochemistry;

FIG. 7 is a graph showing the high AGO-1 expression in kidney tissue of a diabetic mouse according to example 2 of the present invention; wherein FIG. 7A is the expression of AGO-1 protein; FIG. 7B is a semi-quantitative analysis of db/m and db/db mouse AGO-1 protein expression; FIG. 7C is a semi-quantitative analysis of HFD/STZ mouse AGO-1 protein expression; FIG. 7D is an immunohistochemical staining photograph of the AGO-1 protein;

FIG. 8 shows that notoginsenoside Ft1 in example 2 significantly reduces AGO-1 expression in kidney tissues of diabetic mice; wherein FIG. 8A is the expression of AGO-1 protein; FIG. 8B is an immunohistochemical staining photograph of the AGO-1 protein;

FIG. 9 shows improvement of db/db mouse insulin resistance by notoginsenoside Ft1 in example 2 of the present invention; wherein, fig. 9A is IPGTT level; FIG. 9B is IPITT horizontal; FIG. 9C is a graph comparing the IPGTT-AUC of mice in each group; FIG. 9D is a graph comparing IPITT-AUC for each group of mice; FIG. 9E is a graph comparing blood glucose in groups of db/db mice; fig. 9F is a PAS staining picture; FIG. 9G is a Western blot image of TSP-1 protein expression; FIG. 9H is a Western blot image of p-IRS-1, p-ERK 1/2 and p-AKT protein expression;

FIG. 10 shows improvement of HFD/STZ mouse insulin resistance by notoginsenoside Ft1 in example 2 of the present invention; wherein, fig. 10A is IPGTT level; FIG. 10B is IPITT horizontal; FIG. 10C is a graph comparing the IPGTT-AUC of mice in each group; FIG. 10D is a graph comparing IPITT-AUC for each group of mice; FIG. 10E is a graph comparing blood glucose in mice of STZ+HFD groups; FIG. 10F is a Western blot image of p-IRS-1, p-ERK 1/2 and p-AKT protein expression;

FIG. 11 shows improvement of db/db mouse kidney injury by notoginsenoside Ft1 in example 2 of the present invention; wherein, figure 11A is ACR levels after 8 weeks of treatment; FIG. 11B is blood urea nitrogen levels after 8 weeks of treatment; FIG. 11C is a 24 hour urine level after 8 weeks of treatment; FIG. 11D is a graph showing kidney weight to body weight (KW/BW) levels after 8 weeks of treatment; FIG. 11E is an HE, masson staining image; FIG. 11F is an electron microscope image; FIG. 11G is a Western blot image of fibrosis-associated protein α -SMA, vimentin;

FIG. 12A is the ACR levels after 8 weeks of treatment in example 2 of the present invention; FIG. 12B is blood urea nitrogen levels after 8 weeks of treatment; FIG. 12C is 24 hour urine volume level after 8 weeks of treatment; fig. 12D is the kidney weight to body weight (KW/BW) level after 8 weeks of treatment.

Detailed Description

The following description of the embodiments of the present invention will be made clearly and completely with reference to the accompanying drawings, in which it is apparent that the embodiments described are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.

It should be noted that, without conflict, the embodiments of the present invention and features of the embodiments may be combined with each other.

The invention is further described below with reference to the drawings and specific examples, which are not intended to be limiting.

Example 1

The embodiment provides application of notoginsenoside Fc in preparing medicines for treating diabetic nephropathy.

1. Experimental materials and methods

1.1 Pharmaceutical preparation

Notoginsenoside Fc (HPLC purity above 98%) was purchased from Anhui menstrual family limited (Shanghai, china); losartan is available from merck company; in animal experiments, notoginsenoside Fc and losartan are suspended in 0.5% methyl cellulose solution for intragastric administration; in the cell experiments, notoginsenoside Fc was dissolved in DMSO to prepare a mother liquor, which was diluted with medium to treat human renal proximal tubule epithelial cells (HK-2).

1.2 Animal experiments

The db/db mice used in this example were purchased from a sixth people hospital animal house agency affiliated to Shanghai university of transportation; the experiment and the operation thereof all conform to the national animal management regulations and the medical animal management implementation rules and are approved by the ethical committee of the animal of the sixth people hospital attached to Shanghai university of transportation; male db/db diabetic mice (C57 BLKS/J-LepRdb/LepRdb) and littermates male db/m wild type mice (C57 BLKS/J-LepRdb/+), 6 weeks old; 15 db/db mice, 5 db/m mice, were kept in SPF-grade environment at 18℃to 29℃with a relative humidity of 40% to 70% and given a standard clean diet and water;

db/m mice were considered as control group (Con), db/db mice were randomly divided into 5 groups:

(1) Model group: an equal volume of 0.5% sodium carboxymethyl cellulose solution was administered for intragastric administration;

(2) Losartan group (Losartan): losartan is suspended in a 0.5% sodium carboxymethyl cellulose solution and then irrigated with the stomach according to the dosage of 10 mg/kg/d;

(3) Notoginsenoside Fc low dose group: arasaponin Fc was suspended in a 0.5% sodium carboxymethylcellulose solution and then infused at a dose of 2.5 mg/kg/d;

(4) Dosage group in notoginsenoside Fc: the arasaponin Fc was suspended in a 0.5% sodium carboxymethylcellulose solution and then infused with stomach at a dose of 5 mg/kg/d;

(3) Notoginsenoside Fc high dose group: the arasaponin Fc was suspended in a 0.5% sodium carboxymethylcellulose solution and then infused with the stomach at a dose of 10 mg/kg/d;

body weight was monitored weekly and dosing was performed as described above for a total of 8 weeks.

1.3 Detection of Biochemical indicators of urine and blood

Urine was collected from the metabolism cage, centrifuged at 3500rpm for 15 minutes at 4 ℃ and urinary albumin and urinary creatinine in urine supernatant was measured with a full-automatic biochemical analyzer; urinary albumin excretion was calculated using the urinary albumin/creatinine ratio (ACR);

Blood was collected from the abdominal aorta, allowed to stand for 30 minutes or more, centrifuged at 3500rpm at 4℃for 15 minutes, and the blood Glucose (GLU) and Blood Urea Nitrogen (BUN) of the blood supernatant were measured by a full-automatic biochemical analyzer.

1.4 Detection of renal tissue pathology

4 Μm sections of paraffin-embedded kidney tissue were stained with Hematoxylin and Eosin (HE), dried for 30 minutes at 65 ℃ and dewaxed with xylene 2 times for 10 minutes each; sequentially rehydrating with 100% ethanol (I), 100% ethanol (II), 95% ethanol, 90% ethanol, 80% ethanol and deionized water for 10 minutes each time, and finally staining the slices with HE solution.

1.5 Immunohistochemical detection

Immunohistochemical detection was performed on 4 μm paraffin-embedded kidney tissue; after dewaxing and rehydration of the sections, antigen was extracted by boiling in citrate buffer, sections were blocked with 0.3% h ₂O₂ for 15 min and 5% bsa for 1 h; nephrin, PTEN, PDK1 incubation of p-AKT and p-mTOR primary antibody at 4deg.C overnight, then incubating the sections with secondary antibody at 37deg.C for 1 hour, finally, after counterstaining the sections with diaminobenzidine and hematoxylin, microphotographs were collected using an optical microscope and all microphotographs were analyzed by imageJ software.

1.6 Immunofluorescence and TUNEL method for detecting apoptosis

Placing the white mouse kidney tissue slices into a 60 ℃ oven for 2 hours to melt paraffin; dewaxing, fixing 4% paraformaldehyde, repairing antigen, punching a membrane by 0.3% Triton (Triton X-100), staining TUNEL marks, blocking by 5% BSA, staining by a first antibody, a fluorescent secondary antibody for light-proof incubation and a light-proof DAPI dye, sucking redundant liquid by using water-absorbing filter paper, dripping an anti-fluorescent quenching sealing tablet, covering tissues by using a cover glass slowly for sealing, fixing the cover glass by using transparent nail polish, and drying for 30 minutes in a fume hood; finally, observing by using a fluorescence microscope, shooting, and carrying out light-shielding at room temperature.

1.7 Cell culture

Human kidney proximal tubular epithelial cells (HK-2) were routinely cultured in DMEM medium containing 10% fetal bovine serum and 1% diabody at 37℃with 5% CO ₂;

HK-2 cells in log phase were selected and randomly divided into 6 groups:

(1) Normal glucose control group (Con): culturing the cells in a medium containing 5.5mmol/L glucose;

(2) High osmotic pressure group (HM): culturing the cells in a culture medium containing 5.5mmol/L glucose+24.5 mmol/L mannitol;

(3) High sugar group (HG): culturing the cells in a medium containing 30mmol/L glucose;

(4) Notoginsenoside Fc-L group: the cells are cultured in a culture medium containing 30mmol/L glucose and 10 mu mol/L notoginsenoside Fc;

(5) Notoginsenoside Fc-M group: the cells are cultured in a culture medium containing 30mmol/L glucose and 15 mu mol/L notoginsenoside Fc;

(6) Notoginsenoside Fc-H group: the cells are cultured in a culture medium containing 30mmol/L glucose and 20 mu mol/L notoginsenoside Fc;

Notoginsenoside Fc was dissolved in DMSO to prepare a mother solution, which was then diluted with medium and treated for 48 hours for each group.

1.8 Live cell imaging

Mitosox Red is a fluorescent probe specifically targeting live cell mitochondria, has cell membrane permeability, can rapidly and selectively combine with mitochondria, and is oxidized by superoxide once entering the mitochondria to generate Red fluorescence; mitochondrial superoxide production was measured using a MitoSOX Red mitochondrial superoxide indicator and Mitochondrial Membrane Potential (MMP) levels were detected by JC-1 detection kit.

1.9 Statistical analysis

Data were analyzed using GRAPHPAD PRISM and all data were expressed as mean ± Standard Deviation (SD), the differences between groups were statistically significant using one-way analysis of variance (ANOVA), P < 0.05.

2. Experimental results

2.1 Influence of notoginsenoside Fc on blood and urine Biochemical indexes of diabetic mice

Compared with the rats in the control group, the ACR level of the diabetic mice is obviously increased, but after 8 weeks of treatment, both notoginsenoside Fc and losartan can obviously reduce the ACR level of the diabetic rats (figure 1A); both sanchinoside Fc and losartan groups significantly reduced blood urea nitrogen levels compared to the diabetes model group (fig. 1B); the kidney weight/body weight ratio of db/db mice was significantly higher than that of normal db/m mice, and both notoginsenoside Fc and losartan reduced the kidney weight/body weight ratio of diabetic rats after 8 weeks of treatment (fig. 1C).

2.2 Effect of notoginsenoside Fc on kidney morphology in diabetic mice

Histologically, HE staining showed that diabetic mice had significant mesangial matrix deposition, tubular dilation and tubular vacuolation, both sanchinoside Fc and losartan significantly improved these pathological changes (fig. 2A); the Model group mice showed reduced levels of Nephrin in the kidneys compared to the control group (Normal), indicating reduced adhesion; whereas the renal Nephrin levels were relatively elevated in the losartan (Losartan) treated versus the notoginsenoside Fc (Notoginsenoside Fc) treated group compared to the Model group, indicating an elevated level of adhesion (fig. 2B-C); the results show that notoginsenoside Fc improves the abnormality of kidney morphology of diabetic rats.

2.3 Effect of notoginsenoside Fc on apoptosis of diabetic mice foot cells

In order to explore the influence of notoginsenoside Fc on the apoptosis level of kidney foot cells of db/db diabetic mice, the immunofluorescence detection of WT1 and TUNEL is carried out on frozen sections of the kidneys of the mice, and the apoptosis level of kidney foot cells of each group of mice is observed under a fluorescence microscope; the results are shown in FIGS. 3A-D, with elevated levels of apoptosis in the kidney foot cells in the Model mice compared to the Normal mice; the levels of apoptosis were reduced in mouse kidney podocytes in the losartan (Losartan) treated group compared to the Model group, and in the notoginsenoside Fc (Notoginsenoside Fc) treated group.

2.4 Effect of notoginsenoside Fc on the level of mitochondrial oxidative stress in HK-2 cells under high sugar levels

High sugar significantly increased production of mitochondrial superoxide in HK-2 cells and decreased production of mitochondrial superoxide after notoginsenoside Fc treatment.

2.5 Effect of notoginsenoside Fc on the mitochondrial Membrane potential level of HK-2 cells under high sugar action

JC-1 forms aggregates in the mitochondrial matrix when the mitochondrial membrane potential in the cell is high, and can generate red fluorescence, and conversely, JC-1 is a monomer and can emit green fluorescence when the potential is low because of incapability of aggregation; as shown in FIG. 5, high sugar can lead to loss of mitochondrial membrane potential in HK-2 cells, while notoginsenoside Fc can reverse mitochondrial membrane depolarization, suggesting that notoginsenoside Fc can restore mitochondrial function of HK-2 cells under high sugar.

2.6 Regulation of PTEN/PDK1/Akt/mTOR Signal pathway in diabetic mice by notoginsenoside Fc

As shown in fig. 6A-E, model group mice had reduced levels of kidney PTEN compared to Normal (Normal) group; whereas the renal PTEN levels were relatively elevated in the losartan (Losartan) treated group compared to the Model group, as compared to the notoginsenoside Fc (Notoginsenoside Fc) treated group; model (Model) group mice have elevated renal PDK1, p-AKT and p-mTOR levels compared to Normal (Normal) group; in contrast, the renal PDK1, p-AKT and p-mTOR levels were reduced in the losartan (Losartan) treated group compared to the Model group, and in the notoginsenoside Fc (Notoginsenoside Fc) treated group.

Example 2

The embodiment provides application of notoginsenoside Ft1 in preparing a medicament for treating diabetic nephropathy.

1. Experimental materials and methods

1.1 Pharmaceutical preparation

Notoginsenoside Ft1 (HPLC purity above 98%) was purchased from Anhui menstrual family biology limited; losartan is available from merck company; streptozotocin (STZ) was purchased from Sigma-Aldrich; in animal experiments, notoginsenoside Ft1 and losartan are suspended in 0.5% methyl cellulose solution for gastric administration.

1.2 Animal experiments

The db/m mice and db/db mice used in the example were purchased from Nanjing university model animal institute and were kept in SPF-grade animal house (temperature: 26 ℃ C., humidity: 55% -60%) of animal experiment center of university of double denier medical college, and the animal experiment operations were in accordance with the regulations of the ethical committee of animal experiment;

Mice were randomly divided into 6 groups (n=8/group), db/m mice served as control groups, db/db mice were randomly divided into 5 groups (n=8/group):

(2) Losartan positive control group: losartan is suspended in a 0.5% sodium carboxymethyl cellulose solution and then irrigated with stomach according to the dosage of 20 mg/kg/d;

(3) Pseudo-ginseng saponin Ft1 low dose group: the arasaponin Ft1 is suspended in 0.5 percent sodium carboxymethyl cellulose solution and then irrigated with stomach according to the dosage of 2.5 mg/kg/d;

(4) Dosage group of notoginsenoside Ft 1: the arasaponin Ft1 is suspended in 0.5 percent sodium carboxymethyl cellulose solution and then irrigated with stomach according to the dosage of 5 mg/kg/d;

(5) High dose group of notoginsenoside Ft 1: the arasaponin Ft1 is suspended in 0.5 percent sodium carboxymethyl cellulose solution and then irrigated with stomach according to the dosage of 10 mg/kg/d;

Body weight was monitored weekly and dosing was performed as described above for a total of 8 weeks;

The C57 mice used in the example were purchased from Nanjing university model animal institute and were bred in SPF-grade animal house (temperature: 26 ℃ C., humidity: 55% -60%) at animal experiment center of Kanji university pharmaceutical college, and the animal experiment operations were in accordance with the rules of the Ministry of ethics committee of animal experiment; mice at 4 weeks of age were fed a high fat diet 1 week later, 8 weeks later, fasted for 12 hours, and injected intraperitoneally with STZ (40 mg/kg/d) formulated with sterile 0.1mol/L citric acid-sodium citrate buffer, and the injection sites sterilized with alcohol for 5 days;

Under the condition of free drinking water and feeding, measuring normal blood sugar for 1 time before molding; after STZ administration, observing animal state every day, weighing 1 time every day, taking mice with fasting blood glucose of more than 16.7mmol/L and less than 2 times of blood glucose of a control group, and entering into a formal experiment;

mice were randomly divided into 6 groups (n=8/each group), normal mice served as control groups, and diabetic mice were randomly divided into 5 groups (n=8/each group):

(3) Pseudo-ginseng saponin Ft1 low dose group: the arasaponin Ft1 is suspended in 0.5 percent sodium carboxymethyl cellulose solution and then irrigated with stomach according to the dosage of 10 mg/kg/d;

(4) Dosage group of notoginsenoside Ft 1: the arasaponin Ft1 is suspended in 0.5 percent sodium carboxymethyl cellulose solution and then irrigated with stomach according to the dosage of 20 mg/kg/d;

(5) High dose group of notoginsenoside Ft 1: the arasaponin Ft1 is suspended in 0.5 percent sodium carboxymethyl cellulose solution and then irrigated with stomach according to the dosage of 30 mg/kg/d;

1.3 Detection of Biochemical indicators of urine and blood

Urine is collected from a mouse metabolism cage, the urine is placed at 4 ℃ and centrifuged at 3500rpm for 15 minutes, and urine microalbumin and urinary creatinine in urine supernatant are detected by a urine microalbumin enzyme-linked immunosorbent assay kit and a creatinine kit; urinary albumin excretion was calculated using the urinary albumin/creatinine ratio (ACR);

blood was taken through the orbit, allowed to stand for 30 minutes or more, centrifuged at 4500rpm at 4℃for 30 minutes, and serum creatinine and serum urea nitrogen were detected by creatinine kit and urea nitrogen kit.

1.4 Detection of renal tissue pathology

Paraffin-embedded 4 μm sections of kidney tissue were stained with Hematoxylin and Eosin (HE), masson and periodic acid-schiff (PAS), dried at 65 ℃ for 30min, and the sections were dewaxed in xylene 2 times, 10min each time, sequentially rehydrated with 100% ethanol (I), 100% ethanol (II), 95% ethanol, 90% ethanol, 80% ethanol and deionized water, 10min each time, followed by sections stained with HE, masson and PAS fluid, 20 pictures were randomly collected, quantified by two blind researchers, and the percentage of the mesangial matrix to occupy each glomeruli was calculated.

1.5 Electron microscope detection

Using a transmission electron microscope to observe glomeruli and podocyte Foot Processes (FP);

renal cortex was fixed with 2% glutaraldehyde and stained with uranyl acetate and lead citrate.

1.6 Immunohistochemical detection

Immunohistochemical detection was performed on 4 μm paraffin-embedded kidney tissue;

After dewaxing and rehydration of the sections, the antigen is boiled in citrate buffer to extract the antigen; sections were blocked with 0.3% H ₂O₂ for 15 min and 5% BSA for 1 hr; AGO-1 primary antibody was incubated overnight at 4 ℃, then sections were incubated with secondary antibody at 37 ℃ for 1 hour, finally sections were counterstained with diaminobenzidine and hematoxylin, microphotographs were collected using an optical microscope, and all microphotographs were analyzed by ImageJ software.

1.7 Western blot immunoassay (Western blot):

Western blot detects protein levels of kidney tissues and cells AGO-1, TSP-1, p-IRS1/2, p-AKT, AKT, p-ERK1/2, ERK1/2 and p-NFκ B p.

1.8 Detection of inflammatory factors

Serum and kidney tissue IL-1β, IL-6, IL-18 and TNF- α expression were detected by ELISA detection kit.

1.9 Statistical analysis:

All data metering data in this example are expressed as Mean ± standard error (Mean ± SEM); the differences between the groups were assessed using t-test (two-sample comparison, normal distribution compliance and variance alignment test satisfaction) or single factor analysis of variance (multiple-group sample comparison, normal distribution compliance and variance alignment test satisfaction), the above statistical treatment was performed using GRAPHPAD PRISM (GraphPad Software, usa) statistical software, and P <0.05 was the statistical difference significant.

2. Results

2.1 High AGO-1 expression in kidney tissue of diabetic mice

Through western blot detection, AGO-1 expression in kidney tissues of diabetic mice was found to be significantly increased compared to db/m mice and normal control C57 mice (FIGS. 7A-C); immunohistochemical results also showed high expression of AGO-1 in kidney tissue of db/db mice (fig. 7D).

2.2. Arasaponin Ft1 significantly reduces AGO-1 expression in kidney tissue of diabetic mice

Compared with a diabetic mouse, the western blot detection shows that the AGO-1 expression in kidney tissues of the diabetic mouse is obviously reduced after the treatment of notoginsenoside Ft1 for 8 weeks (figure 8A); the immunohistochemical results also showed that notoginsenoside Ft1 can significantly reduce the high expression of AGO-1 in kidney tissues (fig. 8B).

2.3 Notoginsenoside Ft1 improves db/db mouse insulin resistance

As shown in fig. 9A-E, after 30min of intraperitoneal injection of glucose into each group of mice, the blood glucose level rapidly increased to peak and then gradually decreased; the blood sugar of mice in the model group is obviously higher than that in the normal group (P < 0.05) at 30-120 min, and the IPGTT-AUC is obviously increased (P < 0.05) compared with that in the normal group, and after the intervention of notoginsenoside Ft1, the IPGTT level and the IPGTT-AUC are lower than those in the model group (P < 0.05) at 30-120 min; observed after intraperitoneal injection of insulin, db/db mice IPITT had significantly elevated blood glucose levels (P < 0.05) at 30-120 minutes and IPITT-AUC also showed an elevated trend (P < 0.05) compared to the normal group; compared with the model group, the notoginsenoside Ft1 treatment group can reduce 30-120 min blood glucose level (P < 0.05) and IPITT-AUC (P < 0.01), and the notoginsenoside Ft1 can regulate the IPGTT and IPITT levels of db/db mice, and enhance peripheral insulin sensitivity; blood sugar level can be obviously reduced after the treatment of the notoginsenoside Ft1 for 8 weeks, and although the treatment effect of the notoginsenoside Ft1 with low dosage has no obvious difference, the blood sugar level is also reduced to a certain extent; PAS staining of diabetic mouse kidneys found that notoginsenoside Ft1 significantly reduced renal glycogen deposition (FIG. 9F); thrombin-sensitive protein-1 (TSP-1) is a potent anti-angiogenic factor that inhibits vascular growth by preventing the stimulatory response of endothelial cells to various angiogenic factors, AGO-1 can decrease insulin sensitivity by activating TSP-1 expression, and as a result, it was shown that notoginsenoside Ft1 can significantly decrease TSP-1 expression levels in kidney tissue of db/db mice (fig. 9G); western blot further demonstrated that insulin signaling pathway associated proteins p-IRS-1, p-ERK 1/2 and p-AKT expression were significantly increased in kidney tissue of diabetic nephropathy mice treated with notoginsenoside Ft1 compared to model group mice, indicating activation of insulin associated signaling by fusion proteins (fig. 9H).

2.4 Notoginsenoside Ft1 improves HFD/STZ mouse insulin resistance

As shown in fig. 10A-E, after 30min of intraperitoneal injection of glucose in mice of each group, the blood glucose level rapidly increased to peak and then gradually decreased, the blood glucose level of the mice of the model group was significantly higher than that of the normal group (P < 0.05) at 30-120 min, and the IPGTT-AUC was significantly increased (P < 0.05) as compared with the normal group, and after intervention with notoginsenoside Ft1, the IPGTT level and IPGTT-AUC were lower than those of the model group (P < 0.05) at 30-120 min; the mice IPITT in the model group showed a significant increase in blood glucose levels (P < 0.05) at 30-120 minutes and a IPITT-AUC also showed an increasing trend (P < 0.05) when compared to the normal group after intraperitoneal injection of insulin; compared with the model group, the notoginsenoside Ft1 treatment group can reduce 30-120 min blood glucose level (P < 0.05) and IPITT-AUC (P < 0.01), and the notoginsenoside Ft1 can regulate IPGTT and IPITT levels of HFD/STZ mice and enhance peripheral insulin sensitivity; blood sugar level can be obviously reduced after the treatment of the notoginsenoside Ft1 for 8 weeks, and although the treatment effect of the notoginsenoside Ft1 with low dosage has no obvious difference, the blood sugar level is also reduced to a certain extent; western blot further demonstrated that insulin signaling pathway associated proteins p-IRS-1, p-ERK 1/2 and p-AKT expression were significantly increased in kidney tissue of diabetic nephropathy mice treated with notoginsenoside Ft1 compared to model group mice, indicating activation of insulin associated signaling by fusion proteins (fig. 10F).

2.5 Notoginsenoside Ft1 can improve kidney injury of db/db mice

ACR levels can be significantly reduced following treatment with notoginsenoside Ft1 in db/db mice, with blood urea nitrogen and 24 hour urine amounts significantly lower than untreated db/db mice (fig. 11A-B); the kidney index of the mice refers to the ratio of the bilateral kidney mass of the mice to the body weight of the mice, the relative weight change of the kidneys is reflected, the data show that the kidney index of the diabetic nephropathy mice is obviously increased compared with that of a control group, the kidney index of the diabetic mice can be obviously reduced after the treatment of the notoginsenoside Ft1 for 8 weeks (fig. 11C-D), the HE result shows that the mesangial cell proliferation of the diabetic nephropathy mice and the stroma are obviously increased, and the kidney pathological damage of db/db mice is obviously reduced after the treatment of the notoginsenoside Ft1 for 8 weeks (fig. 11E); electron microscopy found that compared with untreated diabetic nephropathy mice, the glomerular basement membrane thickening degree of diabetic nephropathy mice is reduced and podocyte podophyllo fusion degree is significantly improved after treatment by notoginsenoside Ft1 (fig. 11F), masson staining of kidney tissue of mice found that collagen deposition in kidney tissue of db/db mice is increased, and kidney collagen deposition level of diabetic mice can be significantly improved after treatment by notoginsenoside Ft1 (fig. 11E); western blotting results and quantitative statistical analysis thereof further show that notoginsenoside Ft1 can reduce the expression level of fibrosis related protein alpha-SMA and Vimentin in kidney tissues of diabetic nephropathy mice (FIG. 11G).

2.6 Notoginsenoside Ft1 can improve kidney injury of HFD/STZ mice

ACR levels can be significantly reduced following treatment with arasaponin Ft1 in HFD/STZ mice (fig. 12A), with blood urea nitrogen and 24 hour urine amounts significantly lower than untreated HFD/STZ mice (fig. 12B-C); the data show that the kidney index of diabetic nephropathy mice is significantly elevated compared to control; and the kidney index of the diabetic mice can be significantly reduced after 8 weeks of treatment with notoginsenoside Ft1 (FIG. 12D).

In conclusion, the invention discusses the therapeutic effect of notoginsenoside Fc and notoginsenoside Ft1 on diabetic nephropathy by carrying out in vivo experiments on db/db mice and a high-fat diet + Streptozotocin (STZ) -induced diabetic nephropathy model; the notoginsenoside Fc can improve proteinuria and renal histopathology, reduce renal tubular cell apoptosis, regulate PTEN/PDK1/Akt/mTOR channel protein expression and reduce DN glomerulus and mitochondrial injury, and further proves that the in vitro research can reduce the generation of mitochondrial superoxide and reduce Mitochondrial Membrane Potential (MMP) loss; the notoginsenoside Ft1 can improve insulin resistance, lighten the expression of insulin receptor signaling pathway proteins p-IRS1/2, p-AKT and p-ERK1/2, can down regulate the protein expression of AGO-1 in kidney tissues of mice with two diabetic nephropathy models, restrain insulin resistance by regulating an AGO-1/TSP-1 pathway, improve metabolism and lighten DN kidney injury; the notoginsenoside Fc and notoginsenoside Ft1 can be used for preparing medicines for treating diabetic nephropathy.

The foregoing description is only illustrative of the preferred embodiments of the present invention and is not to be construed as limiting the scope of the invention, and it will be appreciated by those skilled in the art that equivalent substitutions and obvious variations may be made using the description and illustrations of the present invention, and are intended to be included within the scope of the present invention.

Claims

1. Application of notoginsenoside Ft1 as unique active component in preparing medicine for treating diabetic nephropathy is provided.

2. The use according to claim 1, wherein the notoginsenoside Ft1 has a molecular formula of C ₄₇H₈₀O₁₇ and a molecular weight of 917.13.

3. The use according to claim 1, wherein the medicament for treating diabetic nephropathy further comprises: one or more pharmaceutically acceptable auxiliary agents.

4. The use according to claim 1, wherein the dosage form of the medicament for treating diabetic nephropathy comprises: decoction, pill, powder, unguent, pellet, medicated liquor, granule, oral liquid, capsule, tablet or injection.

5. The use according to claim 1, wherein the route of administration of the medicament for treating diabetic nephropathy comprises: oral administration, sublingual administration, intramuscular or subcutaneous administration or intravenous administration.