TWI421082B - Usage of 20-hydroxy ecdysone for manufacturing drug of kidney fibrosis and usage of a medication for manufacturing drug of kidney fibrosis - Google Patents

Description

Use of ecdysone for preparing medicine for treating renal fibrosis and pharmaceutical composition Use for preparing a medicament for treating renal fibrosis

The present invention relates to the use of an ecdysone, in particular to a pharmaceutical composition for the preparation of a medicament for treating renal fibrosis and for the treatment of renal fibrosis.

Please refer to Figure 1 for the chemical structure of 20-Hydroxy ecdysone (20HE). The molecular formula is C ₂₇ H ₄₄ O ₇ and its IUPAC is named (2β, 3β, 5β, 22R)-2,3. , 14, 20, 22, 25-hexa hydroxycholest-7-en-6-one. Ecdysone is a dehydrogenated phytosterol commonly found in plants or invertebrates, such as spinach or yam, and ecdysone is mainly used to regulate the molting and metamorphosis of insects.

Many studies have investigated the biological activity of ecdysone in vertebrates, confirming that ecdysone also has a variety of physiological activities in vertebrates. For example, ecdysone has the effects of lowering cholesterol, lowering blood sugar, having immune regulation, preventing arrhythmia, and protecting the liver; in addition, according to Yoshida et al. (1971), Effect of ecdysterone on hyper-glycemia in experimental animals. Pharmacol. (1971) 20: 3263-3268.) It was confirmed that ecdysone has an effect of regulating carbohydrate metabolism, lowering cholesterol, and inhibiting damage to the kidney caused by diabetes.

However, as of now, no research data has indicated that ecdysone has the function of treating renal fibrosis to avoid renal fibrosis and accelerate renal function loss in patients with kidney disease.

More specifically, Chronic Kidney disease, Referred to as CKD), the kidney is injured (such as hyperglycemia, high blood pressure, high proteinuria or other oxidative stress), which may lead to a decline in kidney function, or gradually evolve into renal fibrosis, which leads to loss of renal function.

The process of renal fibrosis can be divided into two phases:

(1) Early stage of renal fibrosis: When the kidney tissue receives different physiological pressures (including hyperglycemia, hypertension, high proteinuria or other oxidative stress), the kidney cells or their surrounding tissues release a series of inflammatory substances to make the kidneys The tissue continues to chronically inflame, promotes the loss of its intrinsic properties and changes in cell type, and then the kidney cells begin to release a series of growth factors, such as the first type of transforming growth factor β type I (TGF). -β1), connective tissue growth factor (CTGF) or epidermal growth factor (EGF), which leads to the proliferation and differentiation of renal interstitial fibroblasts and synthesis of extracellular matrix ( Extracellular matrix, referred to as ECM).

At this time, although the structure and function of the kidney have changed, the damaged kidney cells can still perform some functions. In the early stage of renal fibrosis, the damaged kidney cells can be restored to their original functions and blocked. The conversion of kidney cells into fibroblasts is a key link in blocking renal fibrosis. More specifically, a fibroblast cell line is a differentiated cell that loses its original cell marker, and a cell-like marker appears on the cell surface. This phenomenon is recognized as the beginning of cell transformation, and if the factor that induces transformation disappears, the cell It can still return to the type and characterization of the original cells. Therefore, blocking the transformation of kidney cells into fibroblasts is an important key to prevent renal fibrosis.

(2) Late stage of renal fibrosis: when kidney cells are punctured by inflammatory substances After being transformed into fibroblasts, it is continuously contacted with inflammatory substances, and the fibroblasts will be transformed into fibroblasts. At this time, the fibroblasts will proliferate without stimulating the inflammatory substances, and the synthesis will continue. Collagen type I and III cause ECM to accumulate rapidly, eventually leading to fibrosis of renal tubules and renal interstitium.

Since the development of renal fibrosis, the number of effective renal function units has gradually disappeared, and even if the kidneys are prevented from further fibrosis, the already fibrotic kidney tissue can no longer be repaired. When the renal function is completely lost, the kidney disease patient can only Maintain renal function by hemodialysis or peritoneal dialysis.

In academic studies, renal fibrosis is associated with epithelial-mesenchymal transition (EMT), an EMT phenomenon that occurs during embryonic development, fibrosis, and epithelial cancer metastasis. More specifically, when the kidney cells are stimulated by stress, they will secrete inflammatory substances, which will cause EMT in normal kidney cells, especially the kidney epithelial cells which are originally in the shape of a square, which are transformed into a fusiform type and have mobility. After the cells are detached from the original epithelial area, they are transformed into fibroblasts and secrete ECM to repair damaged kidney tissue, which eventually leads to hardening of the kidney fibers.

When EMT occurs in renal epithelial cells, the adhesion protein (E-cadherin) that originally binds the renal epithelial cells to each other is lost. Therefore, by measuring the amount of adhesion protein, it can be used as a criterion for judging the EMT phenomenon of renal epithelial cells ( Li et al ., 2006).

According to a 1998 study by Border et al., patients with renal fibrosis have more TGF-β1 than normal and kidneys. By activating the Smad signaling pathway (Smad signaling) by TGF-β1, the cells begin to express Snail transcription factors, which degrade the adhesion proteins of renal epithelial cells to cause EMT, and the above results lead to complete loss of function of kidney tissues.

There is currently no effective drug for inhibiting renal fibrosis. It can only slow the occurrence of renal fibrosis by controlling risk factors that cause renal fibrosis, such as controlling blood pressure, blood sugar or limiting protein intake. Among them, for patients with hypertension, The blood pressure is controlled by administering a blood pressure lowering drug, such as Angiotensin Converting Enzyme Inhibitor (ACE-I), to reduce the physiological stress caused by hypertension on kidney tissues.

Please refer to the master's thesis published by Wu Wenping at the China Medical University in the 98th year. It is indicated that the combination of Aliskiren and Valsartan is a blood pressure lowering drug for the treatment of renal fibrosis caused by unilateral ureteral obstruction. Efficacy, however, these antihypertensive drugs may be accompanied by side effects such as headache, dizziness, cough, diarrhea, abdominal pain, angioedema, and even kidney failure; in addition, there are other data indicating that Statin is a hypolipidemic drug. It also has the effect of treating renal fibrosis. However, in patients who are allergic to hypolipidemic drugs, side effects of rhabdomyolysis may occur.

The above drugs may be used only in patients with renal fibrosis suffering from hypertension or hyperlipidemia, except for the side effects that may cause discomfort to the user. For example, patients with renal fibrosis do not have the above-mentioned adaptive conditions. The drug is used as a treatment for renal fibrosis.

It can be seen from the above that if a natural compound can be provided as an active ingredient for treating renal fibrosis, and a renal fibrosis drug can be prepared for consumption by a mammal such as a human, the renal fibrosis of a patient with chronic kidney disease can be alleviated. Phenomena, to avoid the occurrence of renal function loss, and help to improve the current clinical medical treatment can not effectively control the occurrence of renal fibrosis.

The main object of the present invention is to provide a ecdysone for the preparation of a medicament for treating renal fibrosis, in particular, a phenomenon in which ecdysone inhibits the conversion of kidney cells into fibroblasts.

A second object of the present invention is to provide a pharmaceutical composition for the preparation of a medicament for treating renal fibrosis, which uses ecdysone as an active ingredient for inhibiting renal fibrosis to slow the conversion of kidney cells into fibroblasts.

In order to achieve the above object, the technical means used in the present invention include: a ecdysone for preparing a drug for treating renal fibrosis, and an ecdysone for inhibiting renal cells from being subjected to physiological or non-physiological stimulation. The role of renal fibrosis.

The use of the ecdysone of the present invention for the preparation of a medicament for treating renal fibrosis, the kidney cell line being stimulated by an induced fibrotic stimulator to activate the first type B transforming growth factor of the kidney cell (Transforming growth factor β Type I, referred to as TGF-β1) message transmission path and epithelial-mesenchymal transition (EMT) phenomenon.

The ecdysone of the present invention is used for the preparation of a medicament for treating a renal fibrosis which inhibits the expression of fibronectin in the kidney cells.

In the use of the ecdysone of the present invention for the preparation of a medicament for treating renal fibrosis, the ecdysone inhibits the expression of TGF-β1R I in the kidney cells.

The ecdysone of the present invention is used for the preparation of a medicament for treating a renal fibrosis which inhibits the expression of a Snail transcription factor of the kidney cell.

In the use of the ecdysone of the present invention for the preparation of a medicament for treating renal fibrosis, the ecdysone inhibits the hydrolysis of the intercellular adhesion protein of the kidney by activation of the Snail transcription factor.

The use of the ecdysone of the present invention for the preparation of a medicament for treating renal fibrosis, the ecdysone inhibiting the receptor-activated Smad protein of the kidney cell after stimulation of the kidney cell, in particular, Smad2/3 and pSmad2/3 The amount of performance.

The ecdysone of the present invention is used for the preparation of a medicament for treating a renal fibrosis which promotes the inhibitory Smad protein of the kidney cell after stimulation of the kidney cell, in particular, the expression amount of Smad7.

In the use of the ecdysone of the present invention for the preparation of a medicament for the treatment of renal fibrosis, the ecdysone is administered at a dose of 0.06 to 0.24 g (g) of interleukin 7 per kg of body weight.

A pharmaceutical composition for the preparation of a medicament for treating renal fibrosis, the pharmaceutical composition comprising: ecdysone as an active ingredient for inhibiting renal fibrosis; and at least one pharmaceutically acceptable drug adjuvant or drug carrier and The ecdysone binds to form the pharmaceutical composition.

The pharmaceutical composition of the present invention is for use in the preparation of a medicament for treating renal fibrosis, which is a parenteral or oral administration dosage form.

The pharmaceutical composition of the present invention is used for the preparation of a medicament for treating renal fibrosis, which is an injection, a sterile powder, a tablet, a capsule, a pill, a granule or a drop.

The pharmaceutical composition of the present invention is for use in the preparation of a medicament for treating renal fibrosis, which is administered to a subject by parenteral injection.

The pharmaceutical composition of the present invention is for use in the preparation of a medicament for treating renal fibrosis, which comprises 0.06 to 0.24 g (g) of ecdysone for administration to a biological individual per kg of body weight.

The above and other objects, features and advantages of the present invention will become more <RTIgt; For the purpose of preparing a medicament for treating renal fibrosis, please refer to Fig. 1 for the chemical structure of the ecdysone of the present invention. The ecdysone can be used as an active ingredient for the treatment of renal fibrosis, especially when the kidney cells are inhibited from being stimulated (e.g., After hyperglycemia, hypertension, high proteinuria or other stimulating factors such as oxidative stress, ecdysone can inhibit the fibrosis of kidney cells via the following pathways: (1) inhibiting fibronectin induced by the above-mentioned stimulating factors in kidney cells. (2) inhibiting the expression of Snail transcription factors in kidney cells, reducing the hydrolysis of intercellular adhesion proteins (E-cadherin), stabilizing the binding stability between kidney cells, and inhibiting the occurrence of EMT in renal cells due to stimulation. Phenomenon; (3) by regulating the activity of the TGF-β1 signaling pathway, For example, Receptor-activated Smads (R-Smads)-Smad2 and Smad3, or Inhibitory Smads (I-Smads)-Smad7 are activated to reduce EMT in kidney cells. Thus, the ecdysone of the present invention can effectively inhibit the path of fibrosis of kidney cells, and can be applied to the preparation of a medicament for treating renal fibrosis, and the ecdysone is combined with at least one medically acceptable drug adjuvant or drug carrier to form A pharmaceutical composition is administered to a vertebrate such as a human to achieve the effect of slowing or inhibiting the conversion of kidney cells into fibroblasts.

Ecdysone The present invention can be obtained various synthetic ways, derived or extracted from natural plants, e.g. Amaranthaceae (Amaranthaceae, such as spinach or Achyranthes aspera), duck extension Caoke (Commelinaceae, such as exposed plants), Podocarpaceae ( Podocarpaceae, such as cyan Days) or Dioscoreaceae (Dioscoreaceae, such as yam) and other plants; ecdysone present invention is a natural compound, thus ecdysone long-term use of liver and kidney toxicity caused by the lower body, the body will not Produce side effects. The ecdysone of this example was selected but not limited to the natural extracts purchased from Sigma.

In order to confirm that the ecdysone of the present invention has an effect of inhibiting the transformation of kidney cells into fibroblasts, in order to inhibit renal fibrosis of the kidney cells of the living body under physiological or non-physiological stimulation, (A) An in vitro model of renal cell fibrosis was established, and the fibrotic kidney cells were subjected to the following tests: (B) fibrin production rate of stimulated kidney cells, and (C) TGF-β1 of stimulated kidney cells Body volume, (D) activation of the TGF-β1 message transmission pathway in stimulated kidney cells, And (E) an EMT phenomenon of kidney cells and the like to confirm that the ecdysone of the present invention can inhibit the conversion of stimulated kidney cells into fibroblasts, thereby achieving the effect of inhibiting renal fibrosis.

The term "physiological stimulation" as used in the present invention refers to a situation in which the physiological condition of the living body exhibits hypertension, hyperglycemia, hyperlipemia or high urine protein; and the "non-physiological stimulation" as used in the present invention means that the organism is subjected to Exogenous stimuli such as viruses, bacteria or chemicals cause renal fibrosis in kidney cells via physiological or non-physiological stimuli.

This embodiment is selected but not limited to human proximal tubular epithelial cell line HK-2 (hereinafter referred to as renal epithelial cell HK-2), and the renal epithelial cell HK-2 is purchased from the American Type Culture Center (American Type). Culture Collection (ATCC), whose ATCC number is CRL-2190. In this embodiment, the renal epithelial cell HK-2 is cultured in a suitable medium for subculture, and the number of the renal epithelial cells HK-2 is expanded to seven to eight minutes of the culture container, and a buffer is taken to proliferate and culture. Renal epithelial cells HK-2 were flushed into the buffer from the wall of the culture vessel to facilitate cell counting of the renal epithelial cells HK-2.

More specifically, this example selects, but does not limit, the renal epithelial cell HK-2 in DMEM/F-12 medium (Dulbecco's modified Eagle medium nutrient mixture F-12 HAM (DMEM/) containing 10% fetal bovine serum albumin. Proliferation culture in F-12 = 1:1 mixture), Sigma-Aldrich Chemical, St Louis, MO), in which 100 units/ml of antibiotic (Penicillin, Biowest) was cultured per ml of DMEM/F-12 medium. 5% carbon dioxide gas, the temperature is 37 ° C, until the renal epithelial cells HK-2 grow to seven or eighty percent of the culture container, a commercial trypsin The culture vessel is repeatedly rinsed with enzyme enzyme buffer (Trypsin-EDTA, Gibco) to wash the adherent growth of renal epithelial cells HK-2 into the trypsin buffer, thereby completing activation of the renal epithelial cell HK-2. For cell counting and subsequent testing.

(A) In vitro model of renal cell fibrosis

In order to establish a fibrotic kidney cell in vitro mode, a kidney cell is contacted with an induced fibrotic stimulator to simulate renal fibrosis of the kidney cell, wherein the induced fibrotic stimulator can be selected as TGF-β1, β - hydroxybutyric acid (β-Hydroxybutyrate) or a saccharide compound (such as glucose); in this example, the above-mentioned activated renal epithelial cells HK-2 were transplanted to contain 0.5% fetal bovine serum and the concentration was 5 Ng/ In a milliliter (ng/ml) of TGF-β1 in DMEM/F-12 medium, after 24 hours of incubation with 5% carbon dioxide gas at a temperature of 37 ° C, the fibrotic kidney cell in vitro mode can be obtained ( Please refer to (a) and (b) of Figure 2).

In order to confirm that the ecdysone of the present invention can inhibit the fibrosis of kidney cells, in this example, five groups of renal epithelial cells HK-2 (each group containing at least 1×10 ⁵ cells/ml of renal epithelial cells HK-2) were taken. Conditional culture shown in Table 1, wherein the group A1 was cultured only for 48 hours in DMEM/F-12 medium containing 0.5% fetal bovine serum, and the group A2 was cultured with 0.5% fetal bovine serum and TGF-β1. After 48 hours of culture in DMEM/F-12 medium, groups A3 to A5 were cultured for 24 hours in DMEM/F-12 medium containing 0.5% fetal bovine serum and TGF-β1, and then added at concentrations of 12.5, 25 and 50, respectively. After the ecdysone concentration of micromolar (μM), the ecdysone was continued for 24 hours. After the culture was completed, each group of renal epithelial cells HK-2 was observed under a phase contrast microscope.

Referring to Figures (a) to (e) of Fig. 2, the phase difference micrographs of the renal epithelial cells HK-2 of Groups A1 to A5 of the present Example (magnifications are all 200×). Comparing the graphs (a) and (b), it can be seen that in the case of not being stimulated by TGF-β1 (group A1), the renal epithelial cell HK-2 line exhibits an elliptical shape (as indicated by the arrow in (a) Whereas), the renal epithelial cell HK-2 of group A2 is in a slender form (as indicated by the arrow in figure (b)), indicating that the renal epithelial cell HK-2 is stimulated by TGF-β1 After that, there is a tendency to fibrosis; and comparing the graphs (c) to (e), it is known that the group stimulated by TGF-β1 increases with the concentration of ecdysone, and the fibrosis phenomenon tends to slow down (especially The cell type of the group A4 and A5 is similar to that of the group A1, indicating that the renal epithelial cell HK-2 of the present embodiment is subjected to the ecdysone of the present invention. The effect is to inhibit the fibrosis of the renal epithelial cell HK-2. It can be seen that when the kidney cells are stimulated and fibrotic, the ecdysone of different concentrations is added, and the renal cell fibrosis is accompanied by ecdysone. The concentration of action is inhibited and it is shown that ecdysone does have an effect of inhibiting renal fibrosis in kidney cells.

(B) Fibrin production rate of stimulated kidney cells

Because kidney cells produce interstitial proteins such as Fibronectin, Collagen, Tenascin, or Laminin under stimulation, and secrete the mesenchymal protein to the kidneys. Extracellular, in particular, increases fibrin content between kidney cells. In order to confirm that the ecdysone of the present invention does have the effect of inhibiting the secretion of fibrin by the kidney cells, in this example, fibrin is induced in the kidney cells by an induced fibrotic stimulator, and the kidney is observed by Western blotting. The rate of fibrin production in the cells, the rate of fibrin production outside the kidney is observed by Enzyme-Linked ImmunoSorbet Assay (ELISA), and the stimulating hormone of the present invention is inhibited by the ecdysone of the present invention. The rate of fibrin production that is initiated.

For example, in the present embodiment, after the renal epithelial cell HK-2 is produced by TGF-β1 to produce fibrin, the cell lysate of the renal epithelial cell HK-2 is taken for Western blotting (for the B1-1 to B1). -5 groups), the kidney epithelial cells HK-2 supernatant was taken for ELISA (groups B2-1 to B2-5). In this example, five groups of renal epithelial cells HK-2 (each group containing at least 1 × 10 ⁵ cells/ml of renal epithelial cells HK-2) were cultured, and cultured under the conditions shown in Table 2, wherein, B1-1 The B2-1 group was cultured only for 48 hours in DMEM/F-12 medium containing 0.5% fetal calf serum, and the B1-2 and B2-2 groups were DMEM/F containing 0.5% fetal bovine serum and TGF-β1. After culturing for 48 hours in -12 medium, groups B1-3 to B1-5 and groups B2-3 to B2-5 were cultured for 24 hours in DMEM/F-12 medium containing 0.5% fetal bovine serum and TGF-β1. After that, ecdysone was added at a concentration of 12.5, 25 and 50 μM, respectively, and continued for 24 hours.

After the end of the culture, the B1-1 to B1-5 groups were taken from the cell culture medium of each group of renal epithelial cells HK-2, and the cells were lysed by lysis buffer, and then the Western blotting method was performed. The antibody was a commercial anti-fibrin (sc-9068) antibody (Santa Cruz Biotechnology, CA), and the muscle of each group of renal epithelial cells HK-2 was detected with a commercial actin antibody (Sigma, St. Louis, MO). Kinesin (β-actin, molecular weight 43KDa) was expressed as a control.

In group B2-1 to B2-5, the cell culture medium of each group of renal epithelial cells HK-2 was taken, and after centrifugation at 12000 rpm for 10 minutes, the supernatant of each group was obtained, and a commercial fibrin ELISA reagent group (Assaypro, St. Charles, MO) The absorbance of each group of supernatants at a wavelength of 450 nm was measured.

Please refer to Figures 3 and 4 for the Western blotting protein staining results of fibrin in the renal epithelial cell HK-2 and the fibrin expression quantification bar graph. This example is based on the absorbance value measured in the B1-1 group not stimulated by TGF-β1 (the fibrin expression amount of the renal epithelial cell HK-2 is defined as 100%), and is subjected to TGF-β1. The stimulated group B1-2 had a high fibrin production rate (about 190%), which was significantly different from the group B1-1 ( p < 0.005), indicating that the TGF-β1 stimulation in this example can indeed The amount of fibrin contained in renal epithelial cells HK-2 was increased; while the fibrin production rate in groups B1-4 and B1-5 was significantly different from that in group B1-2 ( p < 0.05), and The fibrin production rate of the B1-3 to B1-5 group decreased with the increase of the concentration of ecdysone. Among them, especially under the action concentration of 25 and 50 μM, the fibrin production rate was indeed reduced; The ecdysone concentration of the present invention is 25 and 50 μM, thereby inhibiting the fibrin production rate of the stimulated renal epithelial cell HK-2.

Please refer to Figure 5 for a quantitative bar graph of fibrin expression outside the renal epithelial cell HK-2. This example is based on the absorbance measured in the B2-1 group not stimulated by TGF-β1 (the fibrin expression of the renal epithelial cell HK-2 is defined as 100%), and is subjected to TGF-β1. The stimulated group B2-2 had a high fibrin production rate (about 180%), which was significantly different from the group B2-1 ( p < 0.01), indicating that the TGF-β1 stimulation in this example can indeed The amount of fibrin in renal epithelial cells HK-2 was increased; while the fibrin production rate in group B2-5 was significantly different from that in group B2-2 ( p < 0.05), and B2-3 to B2-5 The fibrin production rate of the group decreased with increasing concentration of ecdysone, and the fibrin production rate of renal epithelial cells HK-2 was indeed reduced under the action concentration of ecdysone at 25 and 50 μM.

In addition, in the present embodiment, the cell culture medium of the B2-1, B2-2, and B2-5 groups was subjected to immunofluorescence staining, and the fibrin distribution of the renal epithelial cell HK-2 was observed, wherein the immunofluorescence staining was performed. The method selects, but is not limited to, the detection of the distribution of the fibrin by a commercial anti-fibrin (ab23751) and a secondary antibody containing FITC; please refer to the figures (a) to (c) of Figure 6, The green fluorescence shown is a fibrin marker, the B2-1 group shows only a small amount of fibrin, and the B2-2 group shows a larger amount of fibrin than the B2-1 group, and the concentration is 50 μM. Ecdysone inhibits fibrin expression. From this, it is understood that the ecdysone concentration of the present invention is 25 and 50 μM, and does have an effect of suppressing the fibrin expression of the stimulated renal epithelial cell HK-2.

Thereby, the ecdysone of the present invention can effectively inhibit the production rate of fibrin produced by the stimulation of the renal cell by the induced fibrotic stimulator, thereby reducing the rate of renal fibrosis in the living body.

(C) TGF-β1 receptor expression in stimulated kidney cells

In order to confirm that the ecdysone of the present invention inhibits the activity of the TGF-β1 receptor, thereby inhibiting the renal cell from undergoing external stimulation, renal fibrosis occurs. More specifically, the kidney cell is stimulated by a high glucose environment. Increases the production of TGF-β1 by kidney cells and activates the TGF-β1 receptor (TGF-β1 receptor, TGF-β1R for short) that binds to this TGF-β1. Three TGF-β1 receptors are known (TGF, respectively) -β1R I, TGF-β1R II and TGF-β1R III).

In this embodiment, the expression level of TGF-β1R in kidney cells is observed by Western blotting; for example, this embodiment stimulates the kidney with TGF-β1. The skin cell HK-2 was taken from the cell lysate of the renal epithelial cell HK-2, and the Western blotting method was used to detect the expression of TGF-β1R I and TGF-β1R II in the renal epithelial cells.

In this example, 5 groups of renal epithelial cells HK-2 (each group contains 1×10 ⁵ cells/ml of renal epithelial cells HK-2), wherein the group C1 is only DMEM containing 0.5% fetal bovine serum. /F-12 medium was cultured for 48 hours, and group C2 was cultured for 48 hours in DMEM/F-12 medium containing 0.5% fetal bovine serum and 5 ng/ml of TGF-β1, and the C3 to C5 group was first containing 0.5. After 24 hours of incubation with % fetal bovine serum and TGF-β1 in DMEM/F-12 medium, ecdysone was added at concentrations of 12.5, 25 and 50 μM, respectively, and continued for 24 hours.

After the completion of the culture, the cell culture medium of each group of renal epithelial cells HK-2 was taken from the group C1 to C5, and the cells were lysed by the lysis buffer, followed by Western blotting method, in which two primary antibodies were used ( The performance of TGF-β1R I or TGF-β1R II of each group of renal epithelial cells HK-2 was detected by commercial anti-TGF-β1R I antibody and a commercial TGF-β1R II antibody purchased from Santa Cruz Biotechnology, respectively. The actin expression of HK-2 in each group of renal epithelial cells was detected as a control by a commercial actin antibody (Sigma).

Please refer to Figures 7 and 8a for the Western blotting protein staining results of TGF-β1R I (molecular weight 53KDa) in the renal epithelial cell HK-2 and its TGF-β1R I expression quantitative bar graph. The C1 group of this example was measured by the absorbance value of the group (control group) which was not co-cultured with ecdysone and was not stimulated by TGF-β1 (TGF-β1R I expression of the renal epithelial cell HK-2 in this group) The amount is defined as 100%); the C2 group is not co-cultured with ecdysone, and the group stimulated by TGF-β1, as shown in Fig. 8a, the expression of TGF-β1R I does increase, and Fig. 8a The C1 group was significantly different ( p <0.05); while the CGF to β1R I expression in the C3 to C5 group was inhibited as the concentration of ecdysone increased, and the ecdysone concentration was 25 μM or more. The expression of TGF-β1R I in renal cells was significantly decreased, and the C4 and C5 groups were significantly different from those in the C2 group ( p <0.01).

Please refer to Figures 7 and 8b for the Western blotting protein staining results of TGF-β1R II (molecular weight 70KDa) in the renal epithelial cells HK-2 and the TGF-β1R II expression quantitative bar graph. The group C1 of the present example was measured by the absorbance value of the group (control group) which was not co-cultured with ecdysone and was not stimulated by TGF-β1 (TGF-β1R II expression of the renal epithelial cell HK-2 in this group) The amount was defined as 100%), however, the amount of TGF-β1R II in the C2 to C5 group was not increased or decreased by stimulation with TGF-β1 or ecdysone, and the amount of TGF-β1R II in the C2 to C5 group was observed. There was no significant difference from Group C1.

From this, it is understood that the ecdysone of the present invention can reduce the expression of TGF-β1R I induced by TGF-β1-stimulated kidney cells, and can inhibit the renal fibrosis caused by stimulation of TGF-β1.

(D) Activation of the TGF-β1 message transmission pathway in stimulated kidney cells

In order to confirm the activation of a related protein in the TGF-β1 signaling pathway induced by stimulation of the renal cell by the induction of fibrotic stimuli, and the related protein activated in the TGF-β1 message transmission pathway, the ecdysone of the present invention is added. It is then inhibited to confirm that the ecdysone of the present invention can inhibit the action of renal fibrosis; more specifically, Smad2/3 in kidney cells and The pSmad2/3 line is a transcription factor involved in the promotion of renal fibrosis, and Smad7 is an inhibitory factor that inhibits the activation of Smad2/3, thereby inhibiting the occurrence of renal fibrosis.

For example, in the present embodiment, the renal epithelial cell HK-2 is stimulated by TGF-β1, and the cell lysate of the renal epithelial cell HK-2 is taken for Western blotting to detect Smad2/3 in the renal epithelial cell. Protein expression of phosphorylated Smad2/3 (pSmad2/3 for short) and Smad7.

In this example, 5 groups of renal epithelial cells HK-2 (each group contains 1×10 ⁵ cells/ml of renal epithelial cells HK-2), wherein the D1 group is only DMEM containing 0.5% fetal bovine serum. /F-12 medium was cultured for 48 hours, and group D2 was cultured for 48 hours in DMEM/F-12 medium containing 0.5% fetal bovine serum and 5 ng/ml of TGF-β1, and the D3 to D5 group was first contained at 0.5. After 24 hours of incubation with % fetal bovine serum and TGF-β1 in DMEM/F-12 medium, ecdysone was added at concentrations of 12.5, 25 and 50 μM, respectively, and continued for 24 hours.

After the end of the culture, the D1 to D5 groups were taken from the cell culture medium of each group of renal epithelial cells HK-2, and the cells were lysed by lysis buffer, followed by Western blotting method, in which primary antibodies were used. A commercial anti-Smad2/3 (sc-8332) antibody, a commercial anti-pSmad2/3 (sc-11769) antibody and a commercial anti-Smad7 (sc-11392) antibody were used to detect Smad2/ of each group of renal epithelial cells HK-2. 3. The performance of pSmad2/3 and Smad7, and the actin expression of HK-2 in each group of renal epithelial cells was detected as a control by a commercial actin antibody (Sigma, USA).

Please refer to Figures 9 and 10a for the Western blotting protein staining results of Smad2/3 (molecular weight 55~60KDa) in the renal epithelial cells HK-2 and the Smad2/3 performance quantitative bar graph. The D1 group of this example was not measured by the ecdysone and was not stimulated by TGF-β1 (control group). The absorbance value was measured (the Smad2/3 expression of the renal epithelial cell HK-2 in this group) It is defined as 100%); the D2 group is not co-cultured with ecdysone, and the group stimulated by TGF-β1, as shown in Fig. 10a, the Smad2/3 expression does increase, and the D1 of Fig. 10a There was a significant difference between the groups ( p <0.01); while the Smad2/3 expression in the D3 to D5 group was inhibited with the increase of the concentration of ecdysone, and the ecdysone concentration of 50 μM could make the Smad2/3 of the kidney cells. The performance was significantly reduced, and the D3 to D5 groups were significantly different from the D2 group ( p < 0.05).

Please refer to Figures 9 and 10b for the Western blotting protein staining results of pSmad2/3 (molecular weight 65KDa) in the renal epithelial cells HK-2 and the pSmad2/3 performance quantitative bar graph. The D1 group of this example was not measured by the ecdysone and was not stimulated by TGF-β1 (control group). The absorbance value was measured (the pSmad2/3 expression of the renal epithelial cell HK-2 in this group) It is defined as 100%); the D2 group is not co-cultured with ecdysone, and the group stimulated by TGF-β1, as shown in Figure 10b, the pSmad2/3 expression does increase, and the D1 of Figure 10b There was a significant difference between the groups ( p <0.01); while the pSmad2/3 expression levels in groups D3 to D5 were inhibited with increasing concentration of ecdysone, and the concentration of ecdysone was 50 μM to make pSmad2/3 of kidney cells. The performance was significantly reduced, and the D3 to D5 groups were significantly different from the D2 group ( p < 0.05).

Please refer to Figures 9 and 10c for the Western blotting protein staining results of Smad7 (molecular weight 51KDa) in the renal epithelial cells HK-2 and the quantitative bar graph of Smad7 expression. The D1 group of the present example was not measured by the ecdysone and was not stimulated by TGF-β1 (control group), and the absorbance value was determined as the reference (the amount of Smad7 expression of the renal epithelial cell HK-2 in this group was defined as 100%); Group D2 was not co-cultured with ecdysone, but with TGF-β1-stimulated group, it can be seen from Figure 10c that the amount of Smad7 in group D2 was not significantly different from that in group D1. TGF-β1 does not induce or inhibit the expression of Smad7; while the Smad7 expression of group D3 to D5 increases with the concentration of ecdysone, and when the concentration of ecdysone is 25μM, the Smad7 of kidney cells can be made. The amount of performance increased significantly, and the D4 and D5 groups were significantly different from the D2 group ( p <0.01).

It can be seen that the ecdysone of the present invention can inhibit the expression of transcription factors Smad2/3 and pSmad2/3 which induce renal fibrosis in kidney cells, and simultaneously activate the activity of Smad7 to effectively inhibit Smad2/3 and pSmad2/3. In this way, the kidney cells are inhibited from being affected by the stimulation of fibrotic stimuli.

(E) EMT phenomenon of kidney cells

In order to confirm that the ecdysone of the present invention inhibits the occurrence of EMT in kidney cells under stimulation, and thereby causes renal fibrosis in kidney cells, this embodiment analyzes the expression of Snail transcription factors in kidney cells by Western blotting. And the amount of adhesion protein between kidney cells and the expression of α-Smooth muscle actin (α-SMA). More specifically, the Snail transcription factor promotes the hydrolysis of renal intercellular adhesion proteins, thereby causing epithelial-mesenchymal transition (EMT) phenomenon in kidney cells, and the present embodiment demonstrates that the ecdysone of the present invention can inhibit the Snail transcription. Factor activity, avoiding water from the adhesion protein Solution to maintain the stability of binding between kidney cells.

For example, in the present embodiment, after the renal epithelial cell HK-2 is activated by TGF-β1 to activate the Snail transcription factor, the cell lysate of the renal epithelial cell HK-2 is taken for Western blotting, and the renal epithelium is detected. The Snail transcription factor of the cell HK-2 and the expression amount of the adhesion protein; more specifically, the adhesion protein is characteristic of epithelial cells, and the α-SMA is a characteristic of interstitial cells, and the present embodiment simultaneously detects The expression of adhesion proteins and smooth muscle actin in the kidney cells confirmed that the renal epithelial cells HK-2 did not change from epithelial cells to interstitial cells.

In this example, 5 groups of renal epithelial cells HK-2 (each group containing at least 1×10 ⁵ cells/ml of renal epithelial cells HK-2) were selected, wherein the group E1 was only containing 0.5% fetal bovine serum. The DMEM/F-12 medium was cultured for 48 hours, and the E2 group was cultured for 48 hours in DMEM/F-12 medium containing 0.5% fetal bovine serum and 5 ng/ml of TGF-β1, and the E3 to E5 group was first contained. After incubation with 0.5% fetal bovine serum and TGF-β1 in DMEM/F-12 medium for 24 hours, ecdysone at concentrations of 12.5, 25 and 50 μM were added, respectively, and incubated for 24 hours.

After the end of the culture, the E1 to E5 group was taken from the cell culture medium of each group of renal epithelial cells HK-2, and the cells were lysed by lysis buffer, followed by Western blotting method, in which the primary antibody (respectively A commercial anti-Snail (C15D3) antibody and a commercial anti-adhesion protein (sc-7870) antibody were used to detect the expression of Snail transcription factor, adhesion protein and smooth muscle actin (α-SMA) in each group of renal epithelial cells HK-2. In addition, a commercial actin antibody (Sigma, USA) was used to detect the expression of actin (beta-actin) of each group of renal epithelial cells HK-2 as a control.

Please refer to Figures 11 and 12 for the Western blotting protein staining results of the Snee transcription factor (molecular weight 29KDa) in the renal epithelial cell HK-2 and the quantitative bar graph of the Snail transcription factor. The E1 group of the present Example was measured by the absorbance value of the group (control group) which was not co-cultured with ecdysone and was not stimulated by TGF-β1 (the amount of Snail transcription factor of the renal epithelial cell HK-2 in this group) It is defined as 100%); the E2 group is not co-cultured with ecdysone, and the TGF-β1-stimulated group, as shown in Fig. 12, the Snail transcription factor does increase in the amount of expression, compared with the E1 group. Significant difference ( p <0.01); while the Snail transcription factor expression levels in groups E3 to E5 were inhibited with increasing concentration of ecdysone, and the ecdysone concentration of 25 μM significantly reduced the expression of Snail transcription factors in kidney cells. There was a significant difference between the E4 and E5 groups and the E2 group ( p <0.01).

In addition, in this example, the cell culture medium of the E1, E2, and E5 groups was subjected to immunofluorescence staining to observe the distribution of the Snail transcription factor of the renal epithelial cell HK-2, wherein the immunofluorescence staining method was selected but not The distribution of the Snail transcription factor is detected by a commercial anti-Snail transcription factor (ab85931) antibody (Abcam Biotechnology) and a secondary antibody containing FITC; please refer to the figures (a) to (c) of Fig. 13, The green fluorescent light shown in the figure is labeled with the Snail transcription factor, the E1 group shows only a few Snail transcription factors, and the E2 group shows a large amount of Snail transcription factor compared to the E1 group, and the concentration is 50 μM. Hormones inhibit the performance of Snail transcription factors. From this, it is understood that the ecdysone concentration of the present invention is 50 μM, and it does have an effect of suppressing the expression of the Snail transcription factor of the stimulated renal epithelial cell HK-2.

Please refer to Figures 14 and 15 for the Western blotting protein staining results of the renal epithelial cell HK-2 adhesion protein (molecular weight 135KDa) and the quantitative bar graph of the adhesion protein expression. The culture conditions of each group in this example are the same as those in the above E1 to E5 groups, and will not be described here. Among them, the absorbance value measured by the E1 group is the adhesion protein of the renal epithelial cell HK-2. The amount of performance was defined as 100%); the group E2 was not co-cultured with ecdysone, and the group stimulated with TGF-β1, as shown in Figure 15, the amount of adhesion protein did decrease, compared with group E1. There was a significant difference ( p <0.01); while the expression of adhesion protein in group E3 to E5 increased with the concentration of ecdysone, and the concentration of ecdysone was 25μM, which could significantly increase the expression of adhesion protein in kidney cells. There was a significant difference between the E4 and E5 groups compared with the E2 group ( p <0.01). It can be seen that the renal epithelial cell HK-2 can reduce the adhesion proteolysis caused by TGF-β1 stimulation by the influence of ecdysone.

Please refer to Figures 14 and 16 for the Western blotting protein staining results of α-SMA (molecular weight 43KDa) in the renal epithelial cells HK-2 and the quantitative bar graph of smooth muscle actin expression. The culture conditions of each group in the present embodiment are the same as those in the above E1 to E5 groups, and will not be described herein. Among them, the absorbance value measured in the E1 group is based on the α-the renal epithelial cells of the group - The SMA expression was defined as 100%); the E2 group was not co-cultured with ecdysone, and the TGF-β1-stimulated group, as shown in Fig. 16, the α-SMA expression did increase, and the E1 group There was a significant difference ( p <0.01); while the α-SMA expression in the E3 to E5 group was inhibited with the increase of the concentration of ecdysone, and the ecdysone concentration of 12.5 μM could make the α-SMA of the kidney cells. The performance was significantly lower, and the E4 and E5 groups were significantly different from the E2 group ( p < 0.01). From this, it can be seen that the renal epithelial cell HK-2 can reduce the formation of α-SMA caused by TGF-β1 stimulation by the influence of ecdysone.

In addition, in the present embodiment, the cell culture medium of the E1, E2, and E5 groups was subjected to immunofluorescence staining, and the distribution of the adhesion protein of the renal epithelial cell HK-2 and the smooth muscle actin (α-SMA) was observed. The immunofluorescence staining method selects, but is not limited to, a commercial anti-adhesion protein (ab53033) antibody and a commercial α-SMA (ab5694) antibody and a secondary antibody containing FITC to detect the adhesion protein and α-SMA, respectively. The distribution situation.

Please refer to the figure (a) to (c) in Figure 17. The green fluorescent light shown in the figure is the label of adhesion protein, the E1 group shows partial adhesion protein, and the E2 group shows significantly more than the E1 group. A small amount of adhesion protein, and the expression of adhesion protein can be enhanced by a concentration of 50 μM ecdysone; please refer to the figure (d) to (f) of Figure 17, where the green fluorescence is α - SMA marker, the E1 group showed only a little α-SMA, the E2 group showed significantly a large amount of α-SMA compared with the E1 group, and the expression of α-SMA was inhibited by the concentration of 50 μM ecdysone; The renal epithelial cell HK-2 enhances the binding stability by the ecdysone, so that the adhesion protein of the renal epithelial cell HK-2 is not reduced by TGF-β1 stimulation, and the α-SMA is not It will increase, and the EMT phenomenon does not occur in the renal epithelial cell HK-2 of the present embodiment.

It can be seen that the ecdysone of the present invention can reduce the Snail transcription factor induced by TGF-β1 stimulation, and increase the expression of adhesion protein and decrease the expression of α-SMA, thereby inhibiting the occurrence of EMT in kidney cells by molting. Hormone inhibits the kidney cells from being stimulated to produce EMT In order to achieve the effect of effectively inhibiting the occurrence of renal fibrosis.

According to the above, the ecdysone of the present invention can inhibit the expression of fibrin produced by stimulation of kidney cells, inhibit the expression of Snail transcription factors, and reduce the hydrolysis of adhesion proteins between kidney cells, thereby achieving stabilization of renal cells. The stability of binding between the two, and the ecdysis cells of the present invention can inhibit the expression of Smad2/3 and pSmad2/3 in the TGF-β1 signaling pathway, and promote the activity of Smad7 in renal cells. It does reduce the expression of Smad2/3 and pSmad2/3 in the kidney cells, thereby preventing the occurrence of EMT in kidney cells. It can be seen from the above that the ecdysone of the present invention does have a function of fibrosis of kidney cells to reduce the progress of renal fibrosis in the case where the kidney of the organism is stimulated; accordingly, the ecdysone of the present invention can be It is used to develop pharmaceutical compositions that inhibit renal fibrosis and has the effect of improving clinical medical quality.

The ecdysone of the present invention can be used as an active substrate for treating renal fibrosis, in various ways alone or in combination with at least one pharmaceutically acceptable drug adjuvant, drug carrier, other accessory ingredients, nutrition. a component or other pharmaceutically active ingredient, which forms ecdysone into the pharmaceutical composition and co-administers to various biological individuals, preferably by parenteral injection to a variety of biological individuals, the present embodiment is based on In 2007, Yang et al. published the paper "Safflower Extract: A Novel Renal Fibrosis Antagonist That Functions by Suppressing Autocrine TGF-β" in the Journal of Cellular Biochemistry, and it is preferred to convert the dose of the ecdysone to the organism of the present invention. 0.06~0.24g of ecdysone is administered per kilogram of body weight of the organism, preferably It is effective to inhibit the pathological characteristics of the renal fibrosis by administering 1 or 2 times a day; further, the ecdysone of the present invention can be manufactured into a parenteral using the technique known to those skilled in the art. Dosage forms for oral or oral administration, for example, injections, sterile powders, lozenges, capsules, pills, granules or drops, are preferably administered to a biological individual by injection to avoid gastrointestinal gastrointestinal damage The chemical structure of the ecdysone affects the effect of the ecdysone on the inhibition of the renal fibrosis.

In summary, the ecdysone of the present invention is used for the preparation of a medicament for treating renal fibrosis, in particular by using ecdysone as an active ingredient for inhibiting renal fibrosis, by (1) inhibiting fibrin expression of kidney cells; (2) It inhibits the expression of Snail transcription factors in kidney cells, reduces the hydrolysis of adhesion proteins between kidney cells, stabilizes the binding stability between kidney cells, and inhibits EMT in renal cells due to stimulation; (3) by regulating TGF- The activity of the factor of the β1 message transmission pathway specifically means inhibiting the expression of the activated Smad protein (Smad2/3) or promoting the expression of the inhibitory Smad protein (Smad7) to reduce the EMT phenomenon in the kidney cells. Achieve the effect of reducing the occurrence of renal fibrosis.

The pharmaceutical composition for treating renal fibrosis according to the present invention is particularly effective for inhibiting the activation of TGF-β1 signaling pathway and EMT phenomenon in kidney cells by ecdysone, so that kidney cells do not develop kidney under stimulation. Fibrosis has the effect of preventing kidney function loss caused by renal fibrosis in patients with chronic kidney disease.

While the invention has been described in connection with the preferred embodiments described above, it is not intended to limit the scope of the invention. The technical scope of the invention is protected, and therefore the scope of the invention is defined by the scope of the appended claims.

Fig. 1 is a schematic view showing the chemical structure of ecdysone of the present invention.

Fig. 2 is a photomicrograph of a phase difference of kidney cells of different culture conditions in accordance with a preferred embodiment of the present invention.

Fig. 3 is a graph showing the results of Western blotting protein staining of kidney cell fibrin in a preferred embodiment of the present invention.

Figure 4: Quantitative bar graph of intracellular fibrin expression of different concentrations of ecdysone on kidney cells in a preferred embodiment of the invention.

Figure 5: Quantitative bar graph of extracellular fibrin expression of ecdysone on kidney cells in a preferred embodiment of the invention.

Figure 6 is a photograph of immunofluorescence staining of kidney cell fibrin in different culture conditions in accordance with a preferred embodiment of the present invention.

Fig. 7 is a graph showing the results of Western blotting protein staining of kidney cells TGF-β1R I and TGF-β1R II in a preferred embodiment of the present invention.

Figure 8a: Quantitative bar graph of TGF-β1R I expression of different concentrations of ecdysone on kidney cells in a preferred embodiment of the invention.

Figure 8b is a quantitative bar graph showing the effect of different concentrations of ecdysone on renal cells in the preferred embodiment of the invention.

Fig. 9 is a graph showing the results of protein staining of Smad2/3, pSmad2/3 and Smad7 Western blotting methods of kidney cells according to a preferred embodiment of the present invention.

Figure 10a: Quantitative bar graph of Smad2/3 expression of quercetin at different concentrations of kidney cells in a preferred embodiment of the invention.

Figure 10b is a quantitative bar graph showing the effect of different concentrations of ecdysone on the expression of pSmad2/3 in kidney cells in a preferred embodiment of the invention.

Figure 10c: A quantitative bar graph of Smad7 expression of different concentrations of ecdysone on kidney cells in a preferred embodiment of the invention.

Figure 11 is a graph showing the results of protein staining of the Snail transcription factor western blot method of kidney cells according to a preferred embodiment of the present invention.

Figure 12: Quantitative bar graph of Snail transcription factor performance of different concentrations of ecdysone on kidney cells in a preferred embodiment of the invention.

Figure 13 is a photograph of immunofluorescence staining of kidney cells Snail transcription factors under different culture conditions in accordance with a preferred embodiment of the present invention.

Figure 14 is a graph showing the results of Western blotting protein staining of adhesion proteins of kidney cells in a preferred embodiment of the present invention.

Figure 15 is a quantitative bar graph showing the expression of adhesion proteins of different concentrations of ecdysone to kidney cells in a preferred embodiment of the invention.

Figure 16 is a graph showing the quantitative bar graph of α-SMA expression of different concentrations of ecdysone on kidney cells in the preferred embodiment of the present invention.

Figure 17 is a photograph of immunofluorescence staining of kidney cell adhesion proteins and α-SMA under different culture conditions in accordance with a preferred embodiment of the present invention.

Claims (16)

A ecdysone for the preparation of a medicament for treating renal fibrosis, which has the effect of inhibiting renal fibrosis by renal cells under physiological or non-physiological stimulation. The use of ecdysone according to item 1 of the patent application for the preparation of a medicament for treating renal fibrosis, wherein the kidney cell line is stimulated by an induced fibrotic stimulator to activate the first type B of the kidney cell Transforming growth factor β type I (TGF-β1) message transmission pathway and epithelial-mesenchymal transition (EMT) phenomenon. The use of ecdysone according to claim 1 of the patent application for the preparation of a medicament for treating renal fibrosis, wherein the ecdysone inhibits the expression of fibronectin in the kidney cells. The use of ecdysone according to the first aspect of the patent application for the preparation of a medicament for treating renal fibrosis, wherein the ecdysone inhibits the expression of TGF-β1R I of the kidney cells. The use of ecdysone according to item 1 of the patent application for the preparation of a medicament for treating renal fibrosis, wherein the ecdysone inhibits the expression of a Snail transcription factor of the kidney cell. The use of ecdysone according to claim 1 of the patent application for the preparation of a medicament for treating renal fibrosis, wherein the ecdysone inhibits the activation of the kidney cell adhesion protein (E-cadherin) by activation of the Snail transcription factor . Preparation of treatment by ecdysone according to item 1 of the patent application scope The use of a renal fibrosis drug, wherein the ecdysone inhibits the expression of the activated Smad protein of the kidney cell after the kidney cell is stimulated. The use of ecdysone according to item 7 of the patent application for the preparation of a medicament for treating renal fibrosis, wherein the receptor activates the Smad protein lines Smad2/3 and pSmad2/3. The use of ecdysone according to the first aspect of the patent application for the preparation of a medicament for treating a renal fibrosis, wherein the ecdysone promotes the expression of an inhibitory Smad protein of the kidney cell after the kidney cell is stimulated. The use of ecdysone according to claim 9 of the patent application for the preparation of a medicament for treating renal fibrosis, wherein the inhibitory Smad protein is Smad7. The use of ecdysone according to claim 1 of the patent application for the preparation of a medicament for treating renal fibrosis, wherein the ecdysone is administered in an amount of 0.06 to 0.24 g (g) of ecdysone per kg body weight of the individual. . Use of a pharmaceutical composition for preparing a medicament for treating renal fibrosis, comprising ecdysone as an active ingredient of renal fibrosis; and at least one pharmaceutically acceptable pharmaceutical adjuvant or drug carrier and ecdysone Combining to form the pharmaceutical composition. The pharmaceutical composition according to claim 12, wherein the pharmaceutical composition is a parenteral or oral dosage form. Use of the pharmaceutical composition according to claim 12 for the preparation of a medicament for treating renal fibrosis, wherein the pharmaceutical composition is injected Products, sterile powders, lozenges, capsules, pills, granules or drops. The pharmaceutical composition according to claim 12, for use in the preparation of a medicament for treating renal fibrosis, wherein the pharmaceutical composition is administered to a subject by parenteral injection. The pharmaceutical composition according to claim 12, wherein the pharmaceutical composition comprises 0.06 to 0.24 g of ecdysone to administer a biological individual per kilogram of body weight.