TWI386218B - The use of the king of the line of ethanol extracted products to treat diabetic nephropathy caused by renal fibrosis - Google Patents

Description

Treatment of renal fibrosis caused by diabetic nephropathy using the ethanol-extracted product of Wang Wuliu

The present invention relates to the use of ethanol-extracted products of one of Vaccaria segetalis (Neck.) Grack to treat renal fibrosis caused by diabetic nephropathy.

Diabetic nephropathy is a chronic kidney disease that causes renal fibrosis and eventually leads to diabetes mellitus (DM), which currently affects about 10% of the world's population. Such disease damage affects many organs, including the eyes, nerves, blood vessels, heart and kidneys. In the United States, diabetes is the most common cause of kidney failure.

Renal fibrosis comes from long-term inflammation and tissue damage in the kidney. It is the common pathway and main pathological basis for various kidney diseases to progress to chronic renal failure. Pathological symptoms include proteinuria, hypertension, ketonuria, hypoglycemia, usually leading to glomerular sclerosis. Renal tubular atrophy, abnormal extracellular matrix and excessive deposition, etc., ultimately rely on dialysis and kidney transplantation to maintain life, and the pathological response of renal fibrosis is mainly extracellular matrix accumulation, hyperglycemia, hyperlipidemia and hypertension .

Many studies have pointed out that the first type of transforming growth factor-β1 (TGF-β1) is a recognized index of renal fibrosis. TGF-β1 induces extracellular matrix accumulation and ultimately increases the condition of diabetic nephropathy (Gunwar, S., F. Ballester, et al. (1998). Glomerular basement membrane. J Biol Chem 273, 8767-8775). Fibrin is one of the most relevant target proteins in diabetic nephropathy and is an indicator for assessing pathological status in early diabetic nephropathy (Efstratiadis, G., M. Divani, et al. (2009). Renal fibrosis. Hippokratia 13, 224- 229.).

Type I B transforming growth factor (TGF-β1) is an induced cell fibrosis factor associated with diabetic nephropathy (Shi, Y. and J. Massague (2003). Mechanisms of TGF-beta signaling from cell membrane to the Nucleus. Cell 113, 685-700), although many profibrotic factors are involved in the progression of renal fibrosis, TGF-β1 and its downstream Smad signaling pathway play an important role. TGF-β1 can regulate cell proliferation, cell differentiation, cell migration and cell fibrosis. The Smad protein family is a messenger responsible for the transmission of TGF-β1-stimulated messages to the nucleus. When the TGF-β1 receptor on the cell membrane is stimulated by TGF-β1, the Smad protein is phosphorylated. The resulting Smad protein transmits the TGF-β1 message from the cell membrane into the nucleus, which in turn triggers the operation of downstream genes.

The Smad protein family can be divided into three types: (1) receptor-activated Smads (including Smad2, Smad3), named R-Smads; (2) common mediator Smads (including Smad4), named Co-Smads; (iii) inhibitory Smads (including Smad7), named I-Smad. TGF-β1 signaling is the use of phosphorylated Smad2, Smad3 and Smad4 to transport to the nucleus, control the transcription of TGF-β1 sensitive genes, and stimulate cell fibrosis, in which Smad4 can form a complex with Smad2/3 protein. The body, which effectively achieves the task of delivery, is in a critical position. On the contrary, the inhibitory effect produced by Smad7 blocks the TGF-β1-induced fibrotic message transmission. Therefore, it can effectively prevent or slow down renal cell fibrosis by inhibiting or blocking the signaling of TGF-β1.

In many countries, natural or organic plant products are often considered as an alternative to scientific medicine. Because of the lack of precise scientific research support, Chinese herbal medicine is often classified as an alternative treatment, and it is excluded from the main treatment. outer.

Vaccaria segetalis (Neck.) Grack, commonly known as burdock, is widely distributed in the grasslands of Europe, the United States, Canada, and Asia. It is often used to promote blood circulation and promote milk secretion, slowing erythema, Chinese herbal medicines that improve menstruation and treat breast infections. In the Chinese herbal medicine and herbal medicine literature, Wang Buliu's water extract can promote blood circulation and can be used as an effective biological response modifier to enhance the circulation balance in the host.

Accordingly, it is an object of the present invention to provide a pharmaceutical composition for treating renal fibrosis caused by diabetic nephropathy comprising an ethanol-extracted product of one of Vaccaria segetalis (Neck.) Grack.

The pharmaceutical composition according to the present invention can be manufactured into a dosage form suitable for parenteral, topical or oral administration using techniques well known to those skilled in the art, including, but not limited to, injection products (injection). (eg, sterile aqueous solutions or dispersions), sterile powders, tablets, troches, pellets, capsules, and the like.

The pharmaceutical composition according to the present invention may be administered by a parenteral route selected from the group consisting of: intraperitoneal injection, subcutaneous injection, intramuscular injection. And intravenous injection.

The pharmaceutical composition according to the present invention may further comprise a pharmaceutically acceptable carrier which is widely used in pharmaceutical manufacturing techniques. For example, the pharmaceutically acceptable carrier can comprise one or more agents selected from the group consisting of excipients, solvents, emulsifiers, suspending agents, decomposers, binders, stabilizers, chelating agents, diluents, gums Coagulants, preservatives, lubricants, absorption delaying agents, liposomes, and the like.

In a preferred embodiment of the present invention, the pharmaceutical composition comprises a pharmaceutically acceptable solvent, and the solvent is any one of the following: water, physiological saline, phosphate buffered physiological saline, sugar-containing solution, and An aqueous solution of alcohol.

The present invention also provides the use of an ethanol-extracted product of Vaccaria segetalis (Neck. Grack) for the preparation of a medicament for treating renal fibrosis caused by diabetic nephropathy.

According to the present invention, the dosage of the ethanol-extracted product of Vaccaria segetalis (Neck.) Grack and the number of administrations vary depending on factors such as the severity of the disease to be treated, the route of administration, and Body weight, age, physical condition and response of the individual being treated.

The above and other technical contents, features and advantages of the present invention will be apparent from the following detailed description of the preferred embodiments.

It is to be understood that all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art of the invention, unless otherwise defined.

In research and development of a Chinese herbal medicine for the treatment of renal fibrosis caused by diabetic nephropathy, the inventors found that Vaccaria segetalis (Neck.) Grack has a good effect in inhibiting renal cell fibrosis, but so far There have been no reports on the efficacy of Wang Buliu's ethanol extraction products for the treatment of renal fibrosis in diabetic nephropathy.

The effect of inhibiting the expression of TGF-βRI, pSmad2/3, Smad2/3 and Smad4 in cells by inhibiting the expression of Smad7 in cells can greatly reduce the intracellular and cellular cells of NRK-49F cells. The expression of fibronectin can actually block or delay the cell fibrosis signal transmission of NRK-49F cells, so Wang does not leave the ethanol extract product for the treatment of renal fibrosis, and it can be further applied. A drug for the treatment of renal fibrosis.

A preferred embodiment of the use of the present invention for the preparation of a medicament for the treatment of renal fibrosis caused by diabetic nephropathy is described below, but it should be understood that this embodiment is for illustrative purposes only, and It should not be construed as limiting the implementation of the invention.

[Examples]

A. Biological materials

The cell line used was normal rat kidney cells (NRK-49F), a fibroblasts purchased from the American Type Culture Collection (American Type Culture Collection, ATCC), the accession number is CRL-1570. NRK-49F cells were cultured in 25T flasks (25T flask) using 10% calf serum (BCS), 100 U/mL penicillin G, 100 μg/ mL streptomycin, and 5.5 mM glucose in DMEM (Dulbecco modified Eagle medium, purchased from GIBCO), and cultured in 37 ° C and 5% CO ₂ incubator, every 3 days to replace the fresh medium .

B. Wang does not leave the ethanol extraction product

The stem segment that Wang did not stay in was purchased from Taiwan in September 2009. The extraction method of Wang Wuliuxing extract product is shown in Figure 1. After soaking 10 g of dried Wang Buliu stem segments in 100 mL of 60% ethanol, the ethanol solution is heated to 95 ° C and heated for 6 hours. . Then, the temperature was lowered to 65 ° C, and ultrasonic waves were shaken for 90 minutes to prepare an extraction mixture. Then, the residue in the extraction mixture is filtered out with a filter paper having a pore size of 0.22 μm to obtain a desired ethanol extract, and the ethanol extract is heated and dried (65 ° C) to form a powdery extract. The measured king did not leave the weight of the powdery extract. Then, the obtained king powder-free extract product was redispersed in Milli-Q water to prepare a Vaccaria segetalis (Neck. Garcke extract, VSE) at a concentration of 10 mg/mL.

C. Cell viability test

The effect of different doses of VSE on TGF-β1-induced NRK-49F cell growth was tested. The NRK-49F cells were first inoculated into a 25 cm ² culture flask for 24 hours, and the cell volume was about 2.7× ^{10 5} . The culture medium contained 5% calf serum (BCS), 100 U/mL penicillin (penicillin) G, 100 μg/mL streptomycin in DMEM (Dulbecco modified Eagle medium, DMEM). Next, continue to culture with a small amount of serum (0.5%), let NRK-49F cells starve for 24 hours, and then change the culture medium containing 0.1% BCS, 100 U/mL penicillin G and 100 μg/mL. Streptomycin in DMEM and TGF-β1 (purchased from R&D, USA) and VSE, respectively. Among them, VSE and TGF-β1 were not added to the control group, and TGF-β1 (5 ng/mL) and VSE were added to the experimental group. The VSE concentrations were 0, 25, 50 and 100 μg/mL, respectively, and then continued to culture for 24 hours. The cell morphology was observed with a 100-fold microscope.

After the above groups were cultured for 24 hours, the culture medium in the flask was removed, washed twice with PBS, and then 1 mL of trypsin-EDTA was added. After the cells were detached, 2 mL of fresh medium was added and trypsin-EDTA activity was added. The mixture was taken to a 15 mL centrifuge tube for centrifugation (1200 rpm, 5 minutes), the supernatant was removed, an appropriate amount of fresh medium was added, and the cell pellet was sufficiently broken to prepare a cell suspension. 50 μL of the cell suspension was thoroughly mixed with an equal volume of trypan blue to form a mixed solution, and then 10 μL of the aforementioned mixture was placed on a Hemocytometer, and counts of viable cells and dead cells were counted.

Cell viability (%) = (a / b) × 100%

a: Number of cells not stained by trypan blue (number of viable cells)

b: total number of cells

D. TGF-β1 induces in vitro renal cell fibrosis test

The in vitro renal cell fibrosis test was carried out in the above control group and the experimental group with a VSE dose of 100 μg/mL. After the preparation of the above control group and the experimental group, the culture was continued for 24 hours, and at the 8th hour of the culture process, At 16 hours, 20 hours and 22 hours, 50 μg/mL cycloheximide (CHX) was added to one of the experimental groups to inhibit protein production, that is, each experimental group. The imines lasted for 16, 8, 4, and 2 hours, respectively. After the above-mentioned 24 hours of culture, the cell culture solution of each experimental group was separately centrifuged (2000 rpm, 5 minutes), and the supernatant was collected to measure the expression of extracellular fibronectin.

The amount of extracellular fibronectin expressed was determined using human Fibronectin EIA KIT. The above supernatant (100 μL) was added to a 96-well culture dish, and the surface of each well was pre-coated with mouse monoclonal anti-human fibronectin antibodies. After homogenization, the reaction was carried out at 37 ° C for 1 hour. Then, the liquid in each well was removed and washed three times with 400 μL of washing buffer (PBS containing 0.1% Tween 20), and then the liquid in the plate was emptied.

Next, 100 μL of secondary antibody against peroxidase (POD) anti-human extracellular fibrin was added to each well and mixed well, and the reaction was carried out at 37 ° C for 1 hour. Next, the liquid in each well was removed and washed four times with 400 μL of wash buffer, which in turn drained the liquid in each well. Then, 100 μL of Substrate Solution was added to each well, and the reaction was carried out at room temperature for 15 minutes, followed by 100 μL of Stop Solution (1N H ₂ SO ₄ ). Go to each well to stop the reaction. Finally, the absorbance (OD450) of each well was measured by an ELISA reader (ELISA reader, Bio-Rad, Model 1550) at a wavelength of 450 nm within one hour after the addition of the stop solution.

In addition, the absorbance (OD450) of fibronectin standard solutions (concentrations of 400 ng/mL, 200 ng/mL, 100 ng/mL, 50 ng/mL, 25 ng/mL, and 12.5 ng/mL) was used as a control. Group, draw a standard curve.

E. Western blotting analysis

(1) Preparation of protein samples and measurement of protein concentration:

First, after the control group and the experimental group were cultured for 24 hours, the culture solution in the culture plate was removed, and the cells were washed twice with PBS, and the residual liquid in the culture plate was blotted dry, and then an appropriate amount of trypsin-EDTA was added and 37 The action was carried out at °C for 5 minutes. After the cells were detached from the bottom of the dish, fresh DMEM (containing 10% BCS, 100 U/mL penicillin G and 100 μg/mL streptomycin) was added to neutralize trypsin-EDTA activity, and The resulting mixture was centrifuged (4 ° C, 1,200 rpm, 5 minutes), the supernatant was removed, and 400 μL of cell lysis buffer was added and mixed, and ultrasonic waves were taken in an ice bath. The ultrashonicator shatters the cells under an operating condition of 5 seconds for the oscillation, 1 second for the stop, and 10 cycles. Subsequently, centrifugation (13,000 rpm, 10 minutes) was carried out at 4 ° C, and the supernatant was collected, and the supernatant was measured for absorbance at 595 nm by Bio-Rad DC Protein Assay. Value, the concentration of the protein is determined.

In addition, a portion of the supernatant was used for the following SDS-polyacrylamide gel electrophoresis (SDS-PAGE) analysis.

(2) Western blotting analysis:

Collect 350 μL of the above (1) Preparation of Protein Samples and Protein Concentration Measurements to collect the supernatant, and 350 μL of 2X sample buffer (containing 0.125 M Tris-HCl, 4% SDS, After 20% glycerol was uniformly mixed with 10% mercaptoethanol (pH 6.8), it was heated in a water bath at 100 ° C for 5 minutes. Next, after centrifugation (4 ° C, 13,000 rpm, 10 minutes), the supernatant was collected for use, and the supernatant was used as a protein sample.

15 μg of protein sample was taken for sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). After the electrophoresis is completed, the separated protein on the gel sheet is transferred to a polyvinylidene difluoride membrane (PVDF membrane). Next, the transferred PVDF membrane was taken out and blocked with 10% skim milk (with TBST, Tris-Buffered Saline/Tween-20) at room temperature, followed by washing with TBST for 10 minutes.

Then, a TBST-diluted primary antibody containing 5% low-fat milk was added, and the primary antibodies used were rabbit anti-Smad7, anti-Smad4, anti-fibronectin, anti-TGF-βRI, anti-Smad2/3, and anti-pSmad2/ A multi-strain antibody (rabbit anti Smad7 polyclonal antibody, Santa Cruz Inc., USA) was allowed to act uniformly for 1 hour at room temperature. After the primary antibody was reacted, it was washed 6 times with TBST for 10 minutes each time. Next, a TBST diluted secondary antibody containing 5% low fat milk was added, and the secondary antibody used was goat anti rabbit IgG antibody (CALBIOCHEM, Cat. No. 401315) at room temperature. After 1 hour, the lower action was washed 6 times with TBST for 10 minutes each time.

In addition, β-actin (β-actin) was used as an internal control, and rabbit anti-β-actin antibody (Sigma, USA) was used as a primary antibody to goats. The same experiment was carried out as an anti-rabbit IgG antibody (CALBIOCHEM, Cat. No. 401315) as a secondary antibody.

Next, the PVDF membrane was treated with Chemilucent ECL Detection System (Cat. No. 2600, Millipore, USA) and subjected to Smad7, Smad4, fibronectin, TGF-β RI, Smad2/3 and pSmad2/ 3 protein expression analysis.

In the present example, the amount of Smad7 protein expression of each group was normalized by the amount of β-actin protein expression, and then the normalized Smad7 protein expression amount of the control group was regarded as 100%, and the other groups were The Smad7 protein expression was normalized to the normal control group, and the protein expression levels of Smad4, fibronectin, TGF-β RI, Smad2/3, and pSmad2/3 were all used.

F. Immunofluorescence staining (Immunofluorescence)

The expression of fibronectin in the cells was observed by immunofluorescence analysis. NRK-49F cells were seeded in an 8-well plate of chamber slid at 4 × 10 ³ cells/well, and cultured in a 37 ° C incubator for 24 hours, and then replaced with fresh DMEM. The culture medium (5% BCS) was cultured for 24 hours, then replaced with fresh DMEM medium (containing 0.1% BCS), and a predetermined concentration of TGF-β1 (5 ng/ml) and VSE (50, 100 μg) were added in groups. /mL) and continue to culture for 24 hours.

Next, cell immunofluorescence staining was performed. The old culture solution was removed, washed twice with PBS, then fixed with 4% paraformaldehyde for 30 minutes, washed 4 times with PBS, and then added with 0.1% Triton X-100/in PBS for 5 minutes to produce NRK-49F cell membrane. The pores allowed the dye to enter and were washed 4 times with PBS. Then, using 1% BSA (bovine serum albumin)/PBS for 60 minutes at room temperature, rabbit anti-human fibronectin antibody was applied overnight at 4 ° C, and then washed 5 times with PBS, followed by 5 times. The cells were treated with FITC-conjugated goat anti-mouse IgG antibody for 60 minutes at room temperature and then washed 5 times with PBS. Finally, the mounting was carried out by adding Mounting Medium and photographing using a fluorescent microscope.

G. Statistical Analysis

In the above embodiment, the experiments of each group were repeated 3 times, and the experimental data was expressed by the standard deviation (S.D.). The difference between the two sets of experimental data was assessed by unpaired Student's t-test, and the difference between the three sets of experimental data was analyzed by one-way ANOVA and unpaired Student's t-test. Evaluation. If the obtained statistical alignment result is p < 0.05, it represents statistical significance.

【test results】

A. Effect of VSE on survival rate of TGF-β1-induced NRK-49F cells

As shown in Figures 2 to 4, NRK-49F cells still have a high survival rate under the stimulation of TGF-β1 (5 ng/mL) and VSE (25, 50 and 100 μg/mL), which is shown in TGF. Under the co-stimulation of β1 and VSE, even if the VSE concentration is increased to 100 μg/mL, it will not cause NRK-49F cell death or promote NRK-49F cell growth.

B. Effect of VSE on TGF-β1-stimulated NRK-49F cells

The enzyme-linked immunosorbent assay (ELISA) was used to analyze the fibrin expression of NRK-49F cells induced by VSE antagonizing TGF-β1, thereby confirming the role of VSE in anti-fibrosis. As shown in Figure 5, the expression of extracellular fibronectin in NRK-49F cells was significantly increased under stimulation with only TGF-β1 (5 ng/mL), but when treated with VSE at the same time, extracellular fibronectin The amount of performance decreased significantly. When the VSE concentration increased above 50 μg/mL, the expression of fibronectin was much lower than that of the control group (without TGF-β1 and VSE treatment), confirming that VSE can significantly inhibit TGF- 11 induced fibronectin secretion performance.

Effects of VSE on TGF-β1 (TGF-β1 receptor protein) and Smad expression in TGF-β1-stimulated NRK-49F cells

As shown in Figure 6, TGF-β1 induced a significant increase in TGF-βRI expression in NRK-49F cells, but when NSE-49F cells were simultaneously treated with VSE, VSE was found to significantly reduce TGF-βRI protein. And the performance of fibronectin.

As shown in Figures 7 and 8, the effect of TGF-β1 and VSE on the signal molecule expression of the Smad protein of NRK-49F cells was stimulated by TGF-β1 (5 ng/mL) alone, pSmad2/3, The performance of Smad2/3 and Smad4 was significantly increased, but the expression of pSmad2/3, Smad2/3 and Smad4 decreased significantly when treated with VSE at the same time. This result indicates that VSE may be through the inhibition of TGF-β1 downstream information. The TGF-β1-induced cell fibrosis was reversed.

As shown in Figure 9, on the other hand, when only TGF-β1 (5 ng/mL) was stimulated, the expression of inhibitory Smad protein (Smad7) was rather insignificant, but at the same time as VSE, Smad7 expression That is, it is greatly improved, and when the VSE additive amount is only 25 μg/mL, it is enough to induce the Smad7 expression to increase by several tens of times, which reverses the reduced Smad7 protein expression induced by TGF-β1.

Therefore, based on the above results, it was shown that VSE can actually delay or block TGF-β1-induced intracellular cells by increasing the amount of Smad7 expression while inhibiting the expression of TGF-βRI, pSmad2/3, Smad2/3 and Smad4. Fibrosis message delivery, in other words, VSE should be used to improve renal cell fibrosis symptoms.

D. Effect of cycloheximide and VSE on the degradation of TGF-βRI

Figure 10 shows that NRK-49F cells were stimulated with TGF-β1 (5 ng/mL) alone, and stimulated simultaneously with TGF-β1 (5 ng/mL) and VSE at different time points (8, 16, 20). TGF-βRI expression of cycloheximide (50 μg/mL) was added to 22 hours), and it was shown that VSE did reduce the TGF-βRI protein expression of NRK-49F cells.

E. Immunofluorescence analysis of fibronectin expression

TGF-β1 induces renal fibrosis in patients with glomerulonephritis and diabetic nephropathy, and TGF-β1 induces extracellular and intracellular fibronectin production in the kidney as shown in Figs. NRK-49F cells were stimulated with TGF-β1 (5 ng/mL) alone after 24 hours of starvation, and co-stimulated with VSE (50, 100 μg/mL) with TGF-β1 (5 ng/mL). The results showed that TGF-β1 did increase the expression of intracellular and extracellular fibronectin in NRK-49F cells, but under the stimulation of VSE, not only the expression of fibronectin in cells decreased, but also the expression of extracellular fibronectin. At the same time, it was confirmed that VSE significantly inhibited TGF-β1-induced fibronectin expression in NRK-49F cells.

In summary, the results of the above analysis showed that VSE (100 μg/mL) did not cause harm to NRK-49F cells, and it was confirmed that VSE was stimulated by TGF-β1 at the same time. NRK-49F cells can significantly reduce the intracellular and extracellular fibers of NRK-49F cells by inhibiting the expression of TGF-βRI, pSmad2/3, Smad2/3 and Smad4 in cells and relatively inducing the expression of Smad7. Connexin expression can indeed block or delay the cell fibrosis signal transmission of NRK-49F cells. Therefore, the present invention does not retain the extract (VSE) for the treatment of renal fibrosis caused by diabetic nephropathy. It can be further applied to the preparation of a drug for treating renal fibrosis caused by diabetic nephropathy. Therefore, the object of the present invention can be achieved.

The above is only the preferred embodiment of the present invention, and the scope of the invention is not limited thereto, that is, the simple equivalent changes and modifications made by the scope of the invention and the description of the invention are All remain within the scope of the invention patent.

BRIEF DESCRIPTION OF THE DRAWINGS Fig. 1 is a flow chart showing the extraction and preparation of the extract of the king of the present invention; Fig. 2 is a view showing the morphology of NRK-49F cells stimulated by TGF-β1 and VSE; and Fig. 3 shows that TGF-β1 and VSE are stimulated to NRK-49F cells. Effect of Survival; Figure 4 shows the effect of TGF-β1 and VSE stimulation on the number of cells in NRK-49F cells; Figure 5 (a) shows the fibronectin standard curve; Figure 5 (b) shows VSE stimulation of TGF-β1 The effect of fibronectin expression on NRK-49F cells; Figure 6(a) shows the TGF-βRI band image of NRK-49F cells treated with TGF-β1 and VSE after Western blot analysis; Figure 6(b) shows VSE inhibited TGF-β1-stimulated TGF-βRI expression in TRK-β1 cells; Figure 7(a) shows that NRK-49F cells were treated with TGF-β1 and VSE, and Western blot analysis of pSmad2/3 and Smad2/3 The image of the zone; Figure 7(b) shows that VSE inhibits the expression of pSmad2/3 and Smad2/3 in TRK-β1-stimulated NRK-49F cells; Figure 8(a) shows that NRK-49F cells are treated with TGF-β1 and VSE After that, the Smad4 region was imaged by Western blot analysis; Figure 8(b) showed that VSE inhibited the expression of Smad4 and fibronectin in TGF-β1-stimulated NRK-49F cells; Figure 9(a) shows NRK-49F TGF-β1 cells after treatment with the VSE, Smad7 by Western blot analysis of the region with an image; FIG. 9 (b) show VSE The Smad7 expression of TRK-β1-stimulated NRK-49F cells was induced; Figure 10 (a) shows the TGF-βRI band image of NRK-49F cells treated with TGF-β1 and VSE after Western blot analysis; Figure 10 ( b) shows the effect of VSE on the degradation of TGF-βRI; Figure 11 shows the effect of VSE on the expression of intracellular fibronectin in NRK-49F cells; and Figure 12 shows the effect of VSE on the expression of extracellular fibronectin in NRK-49F cells. .

Claims (10)

A pharmaceutical composition for treating renal fibrosis caused by diabetic nephropathy, comprising a product extracted by ethanol, one of Vaccaria segetalis (Neck.) Grack. The pharmaceutical composition for treating renal fibrosis caused by diabetic nephropathy according to claim 1, further comprising a pharmaceutically acceptable carrier. A pharmaceutical composition for treating renal fibrosis caused by diabetic nephropathy according to claim 2, wherein the pharmaceutically acceptable carrier comprises one or more reagents selected from the group consisting of Excipients, solvents, emulsifiers, suspending agents, decomposers, binders, stabilizers, chelating agents, diluents, gelling agents, preservatives, lubricants, absorption delaying agents, and liposomes. A pharmaceutical composition for treating renal fibrosis caused by diabetic nephropathy according to claim 3, wherein the pharmaceutically acceptable carrier comprises a solvent selected from the group consisting of water, Phosphate buffered saline, sugar containing solution, and aqueous solution containing alcohol. The pharmaceutical composition for treating renal fibrosis caused by diabetic nephropathy according to claim 1, wherein the pharmaceutical composition is in a dosage form for oral administration. The pharmaceutical composition for treating renal fibrosis caused by diabetic nephropathy according to claim 5, wherein the dosage form is selected from the group consisting of a solution, a suspension, an emulsion, a powder, Tablets, pills, syrup, buccal tablets, tablets, chewing gum, thick paste and capsules . The pharmaceutical composition for treating renal fibrosis caused by diabetic nephropathy according to claim 1, wherein the pharmaceutical composition is in a form for parenteral administration. An ethanol-extracted product of Vaccaria segetalis (Neck.) Grack is used for the preparation of a medicament for treating renal fibrosis caused by diabetic nephropathy. The ethanol-extracted product of Vaccaria segetalis (Neck.) Grack according to claim 8 of the patent application scope is for the purpose of preparing a medicament for treating renal fibrosis caused by diabetic nephropathy, Among them, the pharmaceutical product is prepared in a dosage form for oral administration. The ethanol-extracted product of Vaccaria segetalis (Neck.) Grack according to claim 8 of the patent application scope is for the purpose of preparing a medicament for treating renal fibrosis caused by diabetic nephropathy, Among them, the pharmaceutical product is made into a dosage form for parenteral administration.