TWI415621B - Usage of interleukin-7 for manufacturing drug of kidney fibrosis and usage of a medication for treatment of kidney fibrosis - Google Patents

Abstract

Usage of interleukin-7 for manufacturing drug of kidney fibrosis, wherein interleukin-7 inhibits the kidney fibrosis when the kidney cells stimulated by physiological or non-physiological stimulation. A medication of kidney fibrosis, comprises interleukin-7 as active element, and at least one medical acceptable adjuvant or carrier, which combines with interleukin-7 to form the medication.

Description

Use of interleukin 7 for preparing medicine for treating renal fibrosis and pharmaceutical composition Use for preparing a medicament for treating renal fibrosis

The present invention relates to a use of interleukin 7, and more particularly to a pharmaceutical composition for the use of interleukin 7 for the preparation of a medicament for the treatment of renal fibrosis and for the treatment of renal fibrosis.

Interluekin-7 (IL-7) is a multifunctional cytokine with a molecular weight of about 25KDa, which is produced by bone marrow stromal cells or thymic stromal cells. The interleukin-7 mainly has stimulating lymphocytes. Including the physiological activities of maturation, growth, differentiation and survival of B cells, T cells and NK cells, especially for B cells and T cells, one of the important cytokines controlling cell development; interleukin In addition to regulating immune function, 7 organisms also have potential chemotactic properties, regulate monocyte involvement in inflammation, and promote platelet formation.

According to a study published by Huang et al. in 2002 (IL-7 Inhibits Fibroblast TGF-β Production and Signaling in Pulmonary Fibrosis, J. Clin. Invest. 109: 931-937), it was confirmed that the interleukin 7 system has an inhibitory state. The role of Idiopathic pulmonary fibrosis, especially by inhibiting Smad7 to inhibit the first type of transforming growth factor β (I) of a pulmonary fibrosis mother cell (Transformed growth factor β type I, referred to as TGF-β1) Excessive performance, and achieve the effect of delaying the course of patients with pulmonary fibrosis.

However, as of now, there is no research data indicating that interleukin 7 can also be used to treat renal fibrosis to avoid renal fibrosis and accelerate kidney disease. The patient's kidney function is lost.

Chronic kidney disease (CKD) refers to kidney damage (such as hyperglycemia, high blood pressure, high proteinuria or other oxidative stress), leading to a decline in kidney function, or gradually evolve into renal fibrosis, and then kidney Loss of function.

The formation of renal fibrosis can be divided into two stages:

(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 TGF-β1, Connective Tissue Growth Factor (CTGF) or Epidermal growth factor (EGF), etc., which leads to the proliferation and differentiation of renal interstitial fibroblasts, and the synthesis of extracellular matrix (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 the kidney cells are stimulated by inflammatory substances and converted into fibroblasts, they are continuously contacted with inflammatory substances, and the fibroblasts will be transformed into fibroblasts. At this time, the fibroblasts will not. If you need the stimulation of inflammatory substances, you can proliferate yourself and continue to synthesize collagens of type I and III, so that ECM can accumulate rapidly, eventually leading to fibrosis of renal tubules and renal interstitial.

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., renal fibrosis In patients, renal cells exhibit more TGF-β1 than normal, and renal cells activate Smad signaling pathway by TGF-β1, which begins to express Snail transcription factors and degrades adhesion proteins of renal epithelial cells. The EMT phenomenon occurs and the above results lead to complete loss of function of the kidney tissue.

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, a renal fibrosis drug can be prepared for human use. When it is ingested by mammals, it can slow down the renal fibrosis in patients with chronic kidney disease, 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 use of interleukin 7 for the preparation of a medicament for treating renal fibrosis, in particular, the phenomenon of inhibiting the transformation of kidney cells into fibroblasts by interleukin 7.

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 interleukin 7 as an active ingredient for inhibiting renal fibrosis to slow down the conversion of kidney cells into fibroblasts. .

In order to achieve the foregoing object, the technical means used in the present invention comprises: a use of interleukin 7 for the preparation of a medicament for treating renal fibrosis, and the interleukin 7 system inhibits renal cells from being subjected to physiological or non-physiological stimulation. In the case of renal fibrosis.

The use of the interleukin 7 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 use of the interleukin 7 of the present invention for the preparation of a medicament for treating renal fibrosis inhibits the expression of fibrin in the kidney cells (Fibronectin).

In the use of the interleukin 7 of the present invention for the preparation of a medicament for treating renal fibrosis, the interleukin 7 inhibits the expression of the TGF-β1 receptor of the kidney cells.

In the use of the interleukin 7 of the present invention for the preparation of a medicament for treating renal fibrosis, the TGF-β1 is subjected to the system TGF-β1R I and TGF-β1R II.

In the use of the interleukin 7 of the present invention for the preparation of a medicament for treating renal fibrosis, the interleukin 7 inhibits the expression of the Snail transcription factor of the kidney cell.

The use of the interleukin 7 of the present invention for the preparation of a medicament for treating renal fibrosis inhibits the expression of the renal cell Slug transcription factor.

In the use of the interleukin 7 of the present invention for the preparation of a medicament for treating renal fibrosis, the interleukin 7 inhibits the hydrolysis of the renal intercellular adhesion protein by activation of the Snail transcription factor or the Slug transcription factor.

In the use of the interleukin 7 of the present invention for the preparation of a medicament for treating renal fibrosis, the interleukin 7 is stimulated by the kidney cells to inhibit the receptor-activated Smad protein of the kidney cells, in particular, Smad2/3 And the amount of performance of pSmad2/3.

In the use of the interleukin 7 of the present invention for the preparation of a medicament for treating renal fibrosis, the interleukin 7 is stimulated by the kidney cells to promote the inhibitory Smad protein of the kidney cells, in particular, the expression amount of Smad7 .

In the use of the interleukin 7 of the present invention for the preparation of a medicament for treating renal fibrosis, the dose of the interleukin 7 is administered in an amount of 2.8 to 56 μg (microgram) of interleukin 7 per kg of body weight.

A pharmaceutical composition for preparing a medicament for treating renal fibrosis, the pharmaceutical composition comprising interleukin 7 as a protein for inhibiting renal fibrosis An active ingredient, the pharmaceutical composition further comprising at least one pharmaceutically acceptable pharmaceutical adjuvant or drug carrier in combination with interleukin 7.

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.

In the use of the pharmaceutical composition of the present invention for the preparation of a medicament for treating renal fibrosis, the pharmaceutical composition comprises 2.8 to 56 μg/ml of interleukin 7 to be administered to a biological individual per kilogram of body weight.

The above and other objects, features and advantages of the present invention will become more <RTIgt;

The present invention provides a use of interleukin 7 for the preparation of a medicament for treating renal fibrosis, and the interleukin 7 system can be used as an active ingredient for treating renal fibrosis, particularly when inhibiting stimulation of kidney cells (such as hyperglycemia and hypertension). After high-proteinuria or other stimulating factors such as oxidative stress, interleukin-7 can inhibit renal cell fibrosis via the following pathways: (1) inhibiting the expression of fibronectin induced by the above-mentioned stimulating factor in kidney cells; (2) Inhibition of Snail transcription factor and Slug transcription factor in kidney cells The performance of the kidney cells to reduce the hydrolysis of E-cadherin to stabilize the binding stability between kidney cells, inhibit the EMT phenomenon of kidney cells stimulated; (3) by regulating TGF-β1 signaling Pathway factor activity, such as receptor-activated Smads (R-Smads)-Smad2 and Smad3, or Inhibitory Smads (I-Smads)-Smad7, to reduce renal cellogenesis EMT phenomenon. Thus, the interleukin 7 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 interleukin 7 and at least one medically acceptable drug adjuvant or The drug carrier combines to form a pharmaceutical composition for use by humans and other vertebrates to thereby slow or inhibit the conversion of kidney cells into fibroblasts.

The interleukin 7 of the present invention can be obtained by various artificial synthesis methods, such as synthesis by recombinant interleukin-7 gene translation; the interleukin 7 of the present invention is a naturally occurring compound in vivo, and therefore, long-term use of interleukin It is less toxic to the liver and kidney caused by the human body 7 and does not cause side effects on the human body. This example was chosen but not limited to testing with interleukin 7 from R&D Corporation (USA).

In order to confirm that the interleukin 7 of the present invention has an effect of inhibiting the transformation of kidney cells into fibroblasts, it is possible to inhibit renal fibrosis caused by physiological or non-physiological stimulation of kidney cells of the living body (A) First, establish an in vitro model of renal cell fibrosis, and then perform the following tests on the fibrotic kidney cells: (B) fibrin production rate of stimulated kidney cells, and (C) TGF of stimulated kidney cells. Β1 message transmission pathway, (D) downstream of the TGF-β1 message transmission pathway of stimulated kidney cells Tests such as activation of agglutination factor and (E) EMT phenomenon of kidney cells to confirm that the interleukin 7 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); the in vitro model of renal cell fibrosis in this example simulates a cell present in a hyperglycemic environment (hereinafter referred to as a high glucose environment), taking the above The activated renal epithelial cell HK-2 was transfected into DMEM/F-12 medium containing 0.1% fetal bovine serum, and glucose was added to the DMEM/F-12 medium to make the DMEM/F-12 medium glucose (Sigma). -Aldrich, St.Louis, MO, USA) The final concentration is 27.5 micromolar (mM) to simulate the hyperglycemic state of diabetic patients, cultured under conditions of 5% carbon dioxide gas and 37 ° C. After 48 hours, the renal epithelial cell HK-2 was induced to undergo renal fibrosis, and the fibrotic kidney cell in vitro mode was obtained (Jau-Shyang Huang, 2007), in which the fibrotic kidney cells were induced by a high glucose environment. The number of cells will decrease, and Patterns kidney cells than normal cells tend patterns of elongated.

In order to confirm that the interleukin 7 of the present invention can inhibit the fibrosis of renal cells, in this example, six groups of renal epithelial cells HK-2 (each group containing at least 5 × 10 ⁵ cells/ml of renal epithelial cells HK-2) were taken. The culture was carried out under the conditions shown in Table 1, wherein the group A1 was cultured only for 48 hours in DMEM/F-12 medium containing 0.1% fetal bovine serum, and the group A2 was contained with 0.1% fetal bovine serum and 27.5 mM. After incubation for 48 hours in DMEM/F-12 medium of glucose, groups A3 to A6 were cultured for 24 hours in DMEM/F-12 medium containing 0.1% fetal bovine serum and 27.5 mM glucose, respectively, and then added at a concentration of 10, 50, respectively. After 100 and 200 Ng/ml (ng/ml) of interleukin-7, the cells were incubated for 24 hours. After the culture was completed, each group of renal epithelial cells HK-2 was observed under a phase contrast microscope.

Please refer to Fig. 2, which is a bar graph of the percentage of cells in groups A1 to A6, based on the number of cells in group A1 (the cell percentage is defined as 100%), and stimulated in a high glucose environment. In the A2 group, the cell percentage decreased to 60%, and the cells in the A3 to A6 group had a positive trend with the concentration of interleukin-7.

Please refer to Fig. 1 (a) to (f) for the phase difference micrograph of the renal epithelial cells HK-2 of group A1 to group A6 of the present embodiment (magnification The rate is 200×). Comparing (a) and (b), it can be seen that in group A1 which is not stimulated by high glucose environment, the renal epithelial cell HK-2 line has an elliptical shape (as indicated by the arrow in (a) figure). The renal epithelial cell HK-2 of group A2 showed a slender shape (as indicated by the arrow in (b), indicating that the renal epithelial cell HK-2 tends to occur after being stimulated by a high glucose environment. The phenomenon of fibrosis; and comparing (c) to (f), it can be seen that the group stimulated by the high glucose environment increases with the concentration of interleukin 7, and the fibrosis phenomenon tends to slow down (especially by slender shape). The type of cells in the A5 and A6 groups is similar to that of the group A1, indicating that the renal epithelial cells HK-2 of the present embodiment are subjected to the present invention. The effect of interleukin 7 inhibits the fibrosis of the renal epithelial cell HK-2.

It can be seen that when the kidney cells are stimulated and fibrotic, and the different concentrations of interleukin 7 are added, the renal cell fibrosis is inhibited as the concentration of interleukin 7 increases, indicating interleukin 7 does have the effect of inhibiting the occurrence of renal fibrosis in kidney cells.

(B) Fibrin production rate of stimulated kidney cells

Because kidney cells produce many interstitial proteins, such as Fibronectin, Collagen, Tenascin, or Laminin, and secrete these interstitial proteins. In addition to the kidney cells, in particular, increase the fibrin content between kidney cells. In order to confirm that the interleukin 7 of the present invention does have an effect of inhibiting the secretion of fibrin by kidney cells, this example induces fibrin production in kidney cells in a high glucose environment, and observes the kidney by Western blotting. The rate of fibrin production in cells is determined by Enzyme-Linked ImmunoSorbet Assay. ELISA) The rate of fibrin production outside the kidney was observed, and the rate of fibrin production caused by the high glucose environment was inhibited by the interleukin 7 of the present invention.

For example, in the present embodiment, after the renal epithelial cell HK-2 is produced to produce fibrin in a high glucose environment, the cell lysate of the renal epithelial cell HK-2 is taken for Western blotting (for the B1-1 to B1). Group -6), the kidney epithelial cells HK-2 supernatant was taken for ELISA (groups B2-1 to B2-6).

In this example, six groups of renal epithelial cells HK-2 (each group containing at least 5 × 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 0.1% fetal calf serum, and the B1-2 and B2-2 groups were DMEM/F containing 0.1% fetal bovine serum and TGF-β1. After culturing for 48 hours in -12 medium, groups B1-3 to B1-6 and groups B2-3 to B2-6 were cultured for 24 hours in DMEM/F-12 medium containing 0.1% fetal bovine serum and 27.5 mM glucose. After that, the interleukin 7 at concentrations of 10, 50, 100 and 200 ng/ml were added separately and continued for 24 hours.

After the end of the culture, the B1-1 to B1-6 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 a group of renal epithelial cells HK-2 actin was detected with a commercial actin antibody (Sigma, USA). -actin, molecular weight 43 KDa) was used as a control.

Group B2-1 to B2-6 were taken from the cell culture medium of each group of renal epithelial cells HK-2, and after centrifugation at 12000 rpm for 10 minutes, the supernatant of each group was obtained as a commercial fibrin ELISA reagent group (Biomedical, MA, USA) The absorbance of the supernatant of each group 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 Group B1-1 which is not stimulated by a high glucose environment (the fibrin expression of the group of renal epithelial cells HK-2 is defined as 100%), and is subjected to a high sugar environment. The stimulated group B1-2 had a high fibrin production rate (about 500%), which was significantly different from the group B1-1 ( p < 0.05), indicating that the present example was able to stimulate with a high sugar environment. The amount of fibrin contained in renal epithelial cells HK-2 was increased; while the fibrin production rate in groups B1-3 to B1-6 was significantly different from that in group B1-2 ( p < 0.05), and The fibrin production rate of the B1-3 to B1-6 groups decreases with the increase of the concentration of interleukin-7, and in particular, when the concentration of interleukin 7 is 10 ng/ml or more, fibrin production can be reduced. From this, it can be seen that the concentration of the interleukin 7 of the present invention is 10 to 200 ng/ml, thereby suppressing the effect of 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 value measured in Group B2-1 which is not stimulated by a high glucose environment (the fibrin expression of the group of renal epithelial cells HK-2 is defined as 100%), and is subjected to a high sugar environment. The stimulated group B2-2 had a high fibrin production rate (about 150%), which was significantly different from the group B2-1 ( p < 0.05), indicating that the present example was able to stimulate with a high sugar environment. The amount of fibrin in renal epithelial cells HK-2 was increased; while the fibrin production rate in groups B2-4 to B2-6 was significantly different from that in group B2-2 ( p < 0.05), and B2-4 The rate of fibrin production in the B2-6 group decreases with the increase of the concentration of interleukin-7. In particular, the concentration of interleukin 7 is 50 ng/ml or more, which can reduce the fibrin of renal epithelial cells HK-2. Production rate.

In addition, in the present embodiment, the cell culture medium of the B2-1, B2-2, and B2-6 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, a commercial anti-fibrin (ab23751) and a secondary antibody containing FITC to detect the distribution of the fibrin; please refer to Fig. 6 (a) to (c), The green fluorescent light was labeled with fibrin, the B2-1 group showed only a small amount of fibrin, and the B2-2 group showed significantly more fibrin than the B2-1 group, and the concentration was 200 ng/ml. Interleukin 7 inhibits the performance of fibrin. From this, it is understood that the concentration of the interleukin 7 of the present invention is 50 to 200 ng/ml, and it does have an effect of suppressing the fibrin expression of the stimulated renal epithelial cell HK-2.

Thereby, the interleukin 7 of the present invention can achieve effective inhibition of kidney cells The rate of fibrin production by stimulation of the induced fibrotic stimuli, thereby reducing the rate of renal fibrosis in the organism.

(C) TGF-β1 message transmission pathway in stimulated kidney cells

In order to confirm that the interleukin 7 of the present invention inhibits the fibrosis of the renal cells by inhibiting the signal transduction pathway of the renal cell TGF-β1 stimulated by the high glucose environment, in more detail, the renal cells are highly exposed. After stimulation by the sugar environment, the production of TGF-β1 is increased by the kidney cells, and the TGF-β1 receptor (TGF-β1 receptor, TGF-β1R for short) is activated, and three TGF-β1 receptors are currently known. (TGF-β1R I, TGF-β1R II and TGF-β1R III, respectively). In this example, the production rate of TGF-β1 in kidney cells was measured by ELISA, and the expression level of TGF-β1R was detected by Western blotting method.

In this embodiment, the renal epithelial cell HK-2 is stimulated by a high glucose environment, and the cell supernatant of the renal epithelial cell HK-2 is taken for ELISA to detect the TGF-β1 production rate of the renal epithelial cells, and the western ink is used. Point method was used to detect the protein expression of TGF-β1R I and TGF-β1R II in the renal epithelial cells.

In this example, 6 groups of renal epithelial cells HK-2 (each group contains 5×10 ⁵ cells/ml of renal epithelial cells HK-2), wherein the group C1 is only DMEM containing 0.1% 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 0.1% fetal bovine serum and 27.5 mM glucose, and the C3 to C6 group was first containing 0.1% fetal bovine serum. After incubation with DMEM/F-12 medium of 27.5 mM glucose for 24 hours, the concentrations of 10, 50, 100 and 200 ng/ml of interleukin 7 were added, respectively, and the cells were incubated for 24 hours.

After the end of the culture, groups C1 to C6 were taken from each group of renal epithelial cells. The cell culture medium of HK-2 was taken, the cell supernatant was taken for ELISA, and the cells were lysed by lysis buffer, and the cell lysate was taken for Western blotting.

The ELISA of this example selects, but is not limited to, the absorbance of the supernatant of each group at a wavelength of 450 nm as measured by a commercial TGF-β1 ELISA reagent set (R&D Biomedical, MA, USA).

Referring to Fig. 7, the TGF-β1 expression of the renal epithelial cell HK-2 was quantified. This example is based on the absorbance value measured in Group C1 which is not stimulated by a high glucose environment (the TGF-β1 production rate of the renal epithelial cell HK-2 is defined as 100%), and is stimulated by a high glucose environment. Group C2 had a high TGF-β1 production rate (about 230%), which was significantly different from that of Group C1 ( p < 0.05), indicating that the stimulation of high glucose environment in this example did improve renal epithelial cells. The TGF-β1 production rate of HK-2; and the TGF-β1 production rate of the C4 to C6 group was significantly different from that of the C2 group ( p < 0.05), and the TGF-β1 production rate of the C4 to C6 group. The system decreases with the increase of the concentration of interleukin-7, especially when the concentration of interleukin 7 is 10 ng/ml or more, which can reduce the TGF-β1 production rate of renal epithelial cells HK-2.

The Western blotting method of this example detects each group of renal epithelial cells HK- with two primary antibodies (commercially available from SANTA CRUZ Biotechnology, a commercial anti-TGF-β1R I antibody and a commercial TGF-β1R II antibody). 2 The expression of TGF-β1R I or TGF-β1R II was additionally measured by a commercial actin antibody (Sigma) as actin expression of each group of renal epithelial cells HK-2.

Please refer to Figures 8 and 9a 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 the present example was not correlated with the interleukin 7 and was not stimulated by a high glucose environment (control group), and the absorbance value was measured (TGF-β1R of the renal epithelial cell HK-2 of the group). The amount of I expression is defined as 100%); the group C2 is not co-cultured with interleukin 7, and the group stimulated by high glucose environment, as shown in Fig. 9a, the amount of TGF-β1R I does increase. Compared with the C1 group in Fig. 9a, there was a significant difference ( p <0.05); while the TGF-β1R I expression in the C3 to C5 group was inhibited as the concentration of interleukin 7 was increased, interleukin The concentration of TGF-β1R I in kidney cells was significantly lower at 7 concentrations above 250 ng/ml, and there was a significant difference between groups C4 and C5 compared with group C2 ( p <0.01).

Please refer to Figures 8 and 9b 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 performance quantitative bar graph. The C1 group of the present example was not correlated with the interleukin 7 and was not stimulated by a high glucose environment (control group), and the absorbance value was measured (TGF-β1R of the renal epithelial cell HK-2 of the group). The expression level of II is defined as 100%); the group C2 is not co-cultured with interleukin 7, and the group stimulated by high glucose environment, as shown in Fig. 9b, the expression of TGF-β1R II does increase. Compared with group C1 of Figure 9b, there was a significant difference ( p <0.05); while the amount of TGF-β1R II in group C3 to C5 was inhibited with the increase of the concentration of interleukin 7, the effect of interleukin The concentration of TGF-β1R II in kidney cells was significantly lower at 7 concentrations above 250 ng/ml, and there was a significant difference between groups C3 to C5 and group C2 ( p < 0.01).

It can be seen that the interleukin 7 of the present invention can promote the production of kidney cells. TGF-β1, and the TGF-β1R I and TGF-β1R II expression of renal cells stimulated by the TGF-β1, the interleukin 7 of the present invention can inhibit the TGF-β1 message transmission pathway of kidney cells, slow down renal cells The effect of renal fibrosis occurs.

(D) Downstream signaling factor activation of TGF-β1 signaling pathway in stimulated kidney cells

In order to confirm that the kidney cells are stimulated by a high glucose environment, the activation of the related proteins downstream of the TGF-β1 message transmission pathway, and the related proteins activated in the TGF-β1 message transmission pathway, the addition of the interleukin 7 of the present invention It was later inhibited to confirm that the interleukin 7 of the present invention can inhibit the action of renal fibrosis; more specifically, the Smad2/3 and pSmad2/3 lines in kidney cells and the transcription factors involved in promoting renal fibrosis, Smad7 It is used to inhibit the inhibitory factor of Smad2/3 activation, thereby inhibiting the occurrence of renal fibrosis, while the Smad4 line cooperates with the activated Smad protein to co-regulate the Smad protein (Common-mediator Smads, referred to as Co-Smads).

For example, in this embodiment, the renal epithelial cell HK-2 is stimulated by a high glucose environment, 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), Smad7 and Smad4.

In this example, 6 groups of renal epithelial cells HK-2 (each group contains 5×10 ⁵ cells/ml of renal epithelial cells HK-2), wherein the D1 group is only DMEM containing 0.1% 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 0.1% fetal bovine serum and 27.5 mM glucose, and the D3 to D6 group was first containing 0.1% fetal bovine serum. After incubation with DMEM/F-12 medium of 27.5 mM glucose for 24 hours, the concentrations of 10, 50, 100 and 200 ng/ml of interleukin 7 were added, respectively, and the cells were incubated for 24 hours.

After the end of the culture, the D1 to D6 groups were taken from the cell culture medium of each group of renal epithelial cells HK-2, and the cells were lysed with lysis buffer, and then Western blotting method was carried out, in which the primary antibody was used. Commercial anti-Smad2/3 (sc-8332) antibody, a commercial anti-pSmad2/3 (sc-11769) antibody, a commercial anti-Smad7 (sc-11392) antibody and a commercial anti-Smad4 (sc) from SANTA CRUZ Biotechnology -7154) Antibody) The expression of Smad2/3, pSmad2/3, Smad7 and Smad4 of each group of renal epithelial cells HK-2 was detected, and each group of renal epithelial cells was detected by a commercial actin antibody (Sigma, USA). The actin of HK-2 was shown as a control.

Please refer to Figure 10 for Smad2/3 (molecular weight 55~60KDa), pSmad2-3 (molecular weight 58KDa), Smad4 (molecular weight 61KDa) and Smad7 (molecular weight 51KDa) in the renal epithelial cell HK-2. Western blotting protein staining results, Figure 11a is a quantitative bar graph of Smad2/3, 11b is a quantitative bar graph of pSmad2/3, and 11c is a quantitative bar graph of Smad7, 11d The Smad4 representation is a quantitative bar graph.

The D1 group of the present example was not correlated with the interleukin 7 and was not stimulated by a high glucose environment (control group), and the absorbance value was measured (the group of renal epithelial cells HK-2 in Fig. 9a). The Smad2/3 expression was defined as 100%, the pSmad2/3 expression of the group of renal epithelial cells HK-2 in Fig. 11b was defined as 100%); the D2 group was not co-cultured with interleukin 7, but In the group with high glucose environmental stimuli, it can be seen from Figures 11a and 11b that the Smad2/3 expression or pSmad2/3 expression did increase, and the D2 group in Fig. 11a was significantly different from the D1 group ( p <0.01), and the D2 group in Figure 11b was significantly different from the D1 group ( p <0.01); while the Smad2/3 performance and the pSmad2/3 performance in the D3 to D6 groups were associated with The concentration of leucorrhea 7 was inhibited and increased. The D3 to D6 group was significantly different from the D2 group ( p < 0.05). Among them, the medium interleukin 7 concentration of 50 ng/ml or more could make the Smad2/ of the kidney cells. 3 The amount of expression and the amount of pSmad2/3 expression were significantly reduced to the state of group D1 which was not stimulated by a high glucose environment.

Please refer to Figures 10 and 11c 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 correlated with the interleukin 7 and was not stimulated by a high glucose environment (control group), and the absorbance value was measured (the amount of Smad7 of the renal epithelial cell HK-2 in this group) It is defined as 100%); the D2 group is not co-cultured with interleukin 7, but in the group stimulated by high glucose environment, it can be seen from Fig. 11c that the amount of Smad7 in group D2 is significantly different from that in group D1. ( p <0.01), indicating that the high glucose environment does not induce or inhibit the expression of Smad7; while the Smad7 expression in the D3 to D6 group increases with the concentration of interleukin-7, when interleukin 7 The concentration of Smad7 in kidney cells was significantly increased at a concentration of 250 ng/ml, and there was a significant difference between the D3 and D6 groups compared with the D2 group ( p < 0.01).

Please refer to Figures 10 and 11d for the Western blotting protein staining results of Smad4 (molecular weight 61KDa) in the renal epithelial cells HK-2 and the Smad4 expression quantitative bar graph. The D1 group of the present example was measured by the absorbance value of the group (control group) which was not co-cultured with interleukin 7 and was not stimulated by a high glucose environment (the amount of Smad4 expression of the renal epithelial cell HK-2 in this group) It was defined as 100%). However, the D2 to D6 groups were not stimulated by high glucose environment or stimulated with interleukin-7. The Smad4 expression in groups D2 to D6 was less than that in group D1. Significant difference ( p < 0.05).

It can be seen that the interleukin 7 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, thereby inhibiting renal fibrosis caused by stimulation of the renal cells by the induction of fibrotic stimuli.

(E) EMT phenomenon of kidney cells

In order to confirm that the interleukin 7 system 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 Snail transcription factors in kidney cells by western blotting method. And the amount of Slug transcription factor expression, as well as the amount of adhesion protein between kidney cells and the expression of α-Smooth muscle actin (α-SMA). More specifically, the Snail transcription factor and the Slug transcription factor promote hydrolysis of renal intercellular adhesion proteins, thereby causing epithelial-mesenchymal transition (EMT) phenomenon in kidney cells, and the present invention is illustrative of the interleukin 7 of the present invention. The system can inhibit the activity of the Snail transcription factor and the Slug transcription factor, avoid hydrolysis of the adhesion protein, and maintain the binding stability between the 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 and the Slug transcription factor, the cell lysate of the renal epithelial cell HK-2 is taken for Western blotting and detection. Measuring the kidney Snail transcription factor and Slug transcription factor of HK-2 and the expression of the adhesion protein; more specifically, the adhesion protein is characteristic of epithelial cells, and the α-SMA is characterized by interstitial cells, The examples simultaneously detected the adhesion protein and smooth muscle actin expression of the kidney cells, and confirmed that the renal epithelial cell HK-2 did not change from epithelial cells to interstitial cells.

In this example, 6 groups of renal epithelial cells HK-2 (each group containing at least 5×10 ⁵ cells/ml of renal epithelial cells HK-2) were selected, wherein the group E1 was only containing 0.1% 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 0.1% fetal bovine serum and 27.5 mM glucose β1, and the E3 to E6 group was firstly contained with 0.1% fetal. After 24 hours of culture in bovine serum and 27.5 mM glucose in DMEM/F-12 medium, the concentrations of interleukin 7 at concentrations of 10, 50, 100 and 200 ng/ml were added, respectively, and the cells were incubated for 24 hours.

After the end of the culture, the E1 to E6 group was taken from the cell culture medium of each group of renal epithelial cells HK-2, and the cells were lysed with lysis buffer, and then Western blotting method was performed, in which the primary antibody was used. A commercial anti-Snail (C15D3) antibody, a commercial anti-Slug (C19G7) antibody, a commercial anti-adhesion protein (sc-7870) antibody, and a commercial α-SMA (sc-32251) antibody were used to detect each group of renal epithelial cells HK -2 Snail transcription factor, Slug transcription factor, adhesion protein and α-SMA, and a commercial actin antibody (Sigma, USA) was used to detect actin of HK-2 in each group of renal epithelial cells (β- Actin) performance as a control.

Please refer to Figures 12 and 13a 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 this example was not measured with the interleukin 7 and was not stimulated by a high glucose environment (control group). The absorbance value was measured (the Snail transcription factor of this group of renal epithelial cells HK-2) The amount of performance was defined as 100%); the group E2 was not co-cultured with interleukin 7 and the group stimulated by high glucose environment, as shown in Fig. 13a, the amount of Snail transcription factor did increase, with E1 There was a significant difference between the groups ( p <0.01); while the Snail transcription factor expression levels of the E3 to E6 groups were inhibited with the increase of the concentration of interleukin-7, and the concentration of interleukin 7 was 50 ng/ml. The expression of Snail transcription factors in kidney cells was significantly decreased, and the expression of Snail transcription factors in groups E3 to E6 was significantly different from that in group E2 ( p < 0.05).

Please refer to Figures 12 and 13b for the Western blotting protein staining results of the renal epithelial cell HK-2, the Slug transcription factor (molecular weight 30KDa) and the Slug transcription factor expression quantitative bar graph. 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 values measured in the E1 group (the expression of the Slug transcription factor of the renal epithelial cell HK-2 in this group) The amount is defined as 100%); the E2 group is not co-cultured with interleukin 7, but the group stimulated by high glucose environment, as shown in Fig. 13b, the amount of the Slug transcription factor does increase, and the group E1 Compared with the significant difference ( p <0.01), the expression levels of Slug transcription factors in the E3 to E6 groups were inhibited with the increase of the concentration of interleukin-7, and the concentration of interleukin-7 was 50 ng/ml. The expression of Slug transcription factors in the cells was significantly decreased, and the expression of Slug transcription factors in the E3 to E6 groups was significantly different from that in the E2 group ( p < 0.05).

Please refer to Figures 12 and 13c 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 E6 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 expression was defined as 100%); the group E2 was not co-cultured with interleukin 7, and the group stimulated by high glucose environment, as shown in Fig. 13c, the amount of adhesion protein did decrease, and group E1 There was a significant difference ( p <0.01); while the adhesion protein expression in the E3 to E6 group increased with the concentration of interleukin-7, and the concentration of interleukin 7 was 100 ng/ml. The expression of adhesion proteins was significantly increased, and the E3 to E6 groups were significantly different from 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 12 and 13d for the Western blotting protein staining results of α-SMA (molecular weight 42KDa) 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 E6 groups, and will not be described here. Among them, the absorbance value measured by 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 group stimulated by high glucose environment, as shown in Fig. 10, the α-SMA expression did increase, and the E1 group Compared with the significant difference ( p <0.01), the α-SMA expression in the E3 to E6 group was inhibited with the increase of the concentration of ecdysone, and the ecdysone concentration of 10 nM or more could make the α of the kidney cells. The SMA performance was significantly reduced, and the E3 to E6 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 this example, the cell culture medium of the E1, E2, and E5 groups was subjected to immunofluorescence staining, and the distribution of Snail transcription factor, Slug transcription factor, adhesion protein, and smooth muscle actin of the renal epithelial cell HK-2 was observed. Wherein, the immunofluorescence staining method selects, but is not limited to, a commercial anti-Snail transcription factor (ab85931) antibody (Abcam Biotechnology), a commercial anti-Slug transcription factor antibody, a commercial anti-adhesion protein (ab53033) antibody, and a commercial The α-SMA (ab5694) antibody detects the distribution of the Snail transcription factor, the Slug transcription factor, the adhesion protein and the α-SMA in renal cells, respectively, with a secondary antibody comprising FITC. Please refer to the table corresponding to Table 3 (a) to (l) for the transcription factor or protein of the fluorescent marker.

Please refer to Figure 14 (a) to (l) for the adhesion protein expressed by the suprarenal spleen cell HK-2 of group E1 ((a)), smooth muscle actin (d) Figure), Snail transcription factor ((g) map) and Slug transcription factor ((j) The results of the immunofluorescence staining of the E2 and E5 groups were compared with the E1 group to confirm the expression of the adhesion protein, smooth muscle actin, Snail transcription factor and Slug transcription factor of the present example. In line with the results of the Western dot method.

More specifically, the amount of adhesion protein in group E2 ((b) is significantly lower than that in group E1, and the amount of adhesion protein in group E5 ((c)) is significantly higher than that in group E2. The group, even larger than the adhesion protein expressed in the E1 group, inhibited the expression of adhesion proteins by a concentration of 500 ng/ml of interleukin-7.

The α-SMA performance of the E2 group ((e) is significantly higher than that of the E1 group, while the α-SMA performance of the E5 group ((f)) is significantly higher than that of the E2 group. The α-SMA expression amount inhibited the expression of α-SMA by a concentration of 500 ng/ml of interleukin-7.

The Snail transcription factor expression in group E2 ((h) is significantly higher than that in group E1, while the Snail transcription factor in group E5 (i) is significantly higher than group E2. The Snail transcription factor expression amount inhibited the expression of the Snail transcription factor by a concentration of 500 ng/ml of interleukin-7.

The amount of Slug transcription factor in group E2 ((k)) was significantly higher than that in group E1, while the amount of Slug transcription factor in group E5 ((1) was significantly higher than that in group E2). The expression of Slug transcription factor inhibited the expression of Slug transcription factors by a concentration of 500 ng/ml of interleukin-7.

It can be seen that the interleukin 7 of the present invention can indeed reduce the Snail transcription factor and Slug transcription factor induced by high glucose environment stimulation in kidney cells. And the expression of smooth muscle actin, and increase the expression of adhesion proteins produced by kidney cells, thereby inhibiting the occurrence of EMT in kidney cells, thereby inhibiting the stimulation of the kidney cells to produce EMT, thereby effectively inhibiting the occurrence of renal fibrosis. The effect.

As described above, the interleukin 7 system of the present invention can inhibit the expression of fibrin produced by stimulation of kidney cells, inhibit the expression of Snail transcription factor and Slug transcription factor, and reduce the hydrolysis of adhesion proteins between kidney cells. In order to achieve stable binding stability between kidney cells, and the ecchymocytes of the present invention can inhibit the expression of Smad2/3 and pSmad2/3 in the TGF-β1 signaling pathway after stimulation of renal cells, and promote renal cells. The activity of Smad7 actually reduces 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 interleukin 7 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 living body is stimulated; accordingly, the present invention Baisu 7 can be applied to the development of pharmaceutical compositions for inhibiting renal fibrosis, and has the effect of improving clinical medical quality.

The interleukin 7 system 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, and other accessory ingredients. , a nutrient component or other pharmaceutically active ingredient, the interleukin 7 is formed into the pharmaceutical composition, and co-administered to various biological individuals, preferably by a parenteral injection method to give various biological individuals an appropriate dose, The examples are published in the Endocrinology Journal "Bone Morphogenetic Protein-2 Antagonizes Renal" by Yang et al., 2009. In the paper of Interstitial Fibrosis by Promoting Catabolism of Type I Transforming Growth Factor-β Receptors, the dosage of the interleukin 7 administered to the organism of the present invention is preferably 2.8 to 56 μg of interleukin per kg of body weight. 7, preferably 1 or 2 times a day, can effectively inhibit the pathological characteristics of the renal fibrosis; in addition, the interleukin 7 of the present invention can be manufactured by using techniques well known to those skilled in the art. In a dosage form suitable for parenteral or oral administration, for example, an injection, a sterile powder, a tablet, a capsule, a pill, a granule or a drop, preferably administered to a biological individual by injection to avoid The digestive juice of the gastrointestinal tract destroys the chemical structure of the interleukin 7, and affects the effect of the interleukin 7 on inhibiting the renal fibrosis.

In summary, the use of the interleukin 7 of the present invention for the preparation of a medicament for treating renal fibrosis, in particular, the use of interleukin 7 as an active ingredient for inhibiting renal fibrosis, by (1) inhibiting fibrin of kidney cells (2) inhibiting the expression of Snail transcription factors in kidney cells, reducing the hydrolysis of adhesion proteins between kidney cells, stabilizing the stability of binding between kidney cells, and inhibiting EMT phenomenon caused by stimulation of kidney cells; (3) The activity of a factor that regulates the TGF-β1 signaling pathway, in particular, inhibits the expression of the receptor-activated Smad protein (Smad2/3) or promotes the expression of the inhibitory Smad protein (Smad7) to reduce EMT in renal cells. Phenomenon, thereby achieving the effect of reducing the occurrence of renal fibrosis.

The use of the pharmaceutical composition of the invention for preparing a medicament for treating renal fibrosis, in particular, the interferon 7 effectively inhibits the activation of the TGF-β1 message transmission pathway and the EMT phenomenon in the kidney cells of the living body, so that the kidney cells are stimulated In case, no renal fibrosis will occur, and it will be slow to avoid The effect of renal function loss in patients with renal disease due to renal fibrosis.

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 photomicrograph of a phase difference of kidney cells in different culture conditions according to a preferred embodiment of the present invention.

Fig. 2 is a bar graph showing the percentage of cells in the kidney cells of Groups A1 to A6 of the preferred embodiment of the present invention.

Fig. 3 is a graph showing the results of Western blotting protein staining of intracellular fibrin in which different concentrations of interleukin 7 are applied to kidney cells in a preferred embodiment of the present invention.

Figure 4: Quantitative bar graph of intracellular fibrin expression of different concentrations of interleukin 7 on renal cells in a preferred embodiment of the invention.

Fig. 5 is a quantitative bar graph showing the expression of extracellular fibrin acting on kidney cells by a preferred embodiment of the present 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.

Figure 7 is a quantitative bar graph showing the effect of different concentrations of interleukin 7 on renal cells in a preferred embodiment of the invention.

Figure 8: Preferred embodiments of the present invention different concentrations of interleukin 7 on the kidney Western blotting protein staining results of TGF-β1R I and TGF-β1R II by cell action.

Figure 9a: Quantitative bar graph of TGF-β1R I expression of different concentrations of interleukin 7 on renal cells in a preferred embodiment of the invention.

Figure 9b: Quantitative bar graph of TGF-β1R II expression of different concentrations of interleukin 7 on renal cells in a preferred embodiment of the invention.

Fig. 10 is a graph showing the results of Western blotting of Smad2/3, pSmad2/3, Smad4 and Smad7 protein samples of kidney cells according to a preferred embodiment of the present invention.

Figure 11a: Quantitative bar graph of Smad2/3 expression of different concentrations of interleukin-7 on renal cells in a preferred embodiment of the invention.

Figure 11b is a quantitative bar graph showing the pSmad2/3 expression of different concentrations of interleukin 7 on renal cells in a preferred embodiment of the invention.

Figure 11c: A quantitative bar graph of the Smad7 expression of different concentrations of interleukin 7 on kidney cells in a preferred embodiment of the invention.

Figure 11d: Quantitative bar graph of Smad4 expression of different concentrations of interleukin 7 on renal cells in a preferred embodiment of the invention.

Fig. 12 is a graph showing the results of protein staining of western blotting proteins of Snail transcription factor, Slug transcription factor, adhesion protein and smooth muscle actin of kidney cells according to a preferred embodiment of the present invention.

Figure 13a: Quantitative bar graph of Snail transcription factor expression of different concentrations of interleukin 7 on kidney cells in a preferred embodiment of the invention.

Figure 13b: A quantitative bar graph showing the effect of different concentrations of interleukin 7 on renal cells in a preferred embodiment of the invention.

Figure 13c: A quantitative bar graph showing the expression of adhesion proteins of different concentrations of interleukin 7 against kidney cells in a preferred embodiment of the invention.

Figure 13d: Quantitative bar graph of smooth muscle actin expression of different concentrations of interleukin 7 on kidney cells in a preferred embodiment of the invention.

Figure 14 is a photograph of immunofluorescence staining of kidney cells Snail transcription factor, Slug transcription factor, adhesion protein and smooth muscle actin in different culture conditions according to a preferred embodiment of the present invention.

Claims (18)

An interleukin 7 is used for the preparation of a medicament for treating renal fibrosis, and the interleukin 7 system has the effect of inhibiting renal fibrosis by renal cells under physiological or non-physiological stimulation. The use of interleukin 7 according to claim 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 of the kidney cell Transforming growth factor β type I (TGF-β1) message transmission pathway and epithelial-mesenchymal transition (EMT) phenomenon. The use of interleukin 7 according to claim 1 of the patent application for the preparation of a medicament for treating renal fibrosis, wherein the interleukin 7 inhibits the expression of fibronectin in the kidney cells. The use of interleukin 7 according to claim 1 of the patent application for the preparation of a medicament for treating renal fibrosis, wherein the interleukin 7 inhibits the expression level of the TGF-β1 receptor of the kidney cell. The use of interleukin 7 according to claim 1 of the patent application for the preparation of a medicament for treating renal fibrosis, wherein the TGF-β1 is subjected to the system TGF-β1R I and TGF-β1R II. The use of interleukin 7 according to claim 1 of the patent application for the preparation of a medicament for treating renal fibrosis, wherein the interleukin 7 inhibits the expression of the Snail transcription factor of the kidney cell. The use of interleukin 7 according to claim 1 of the patent application for the preparation of a medicament for treating renal fibrosis, wherein the interleukin 7 inhibits the kidney Expression of the cell Slug transcription factor. The use of interleukin 7 according to claim 1 of the patent application for the preparation of a medicament for treating renal fibrosis, wherein the interleukin 7 inhibits the renal intercellular adhesion protein (E-cadherin) from the Snail transcription factor Or hydrolysis by activation of the Slug transcription factor. The use of interleukin 7 according to claim 1 of the patent application for the preparation of a medicament for treating renal fibrosis, wherein the interleukin 7 is stimulated by the kidney cells to inhibit the activation of Smads of the kidney cells. The amount of protein expressed. The use of interleukin 7 according to claim 9 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 interleukin 7 according to claim 1 of the patent application for the preparation of a medicament for treating renal fibrosis, wherein the interleukin 7 is stimulated by the kidney cells to promote the inhibitory Smad protein of the kidney cells The amount of performance. The use of interleukin 7 according to claim 11 of the patent application for the preparation of a medicament for treating renal fibrosis, wherein the inhibitory Smad protein is Smad7. The use of interleukin 7 according to item 1 of the patent application for the preparation of a medicament for treating renal fibrosis, wherein the dose of the interleukin 7 is administered in an amount of 2.8 to 56 micrograms per kilogram of body weight (μg ) The white pigment 7 A pharmaceutical composition for preparing a medicament for treating renal fibrosis, the pharmaceutical composition comprising interleukin 7, as an active ingredient for inhibiting renal fibrosis, the pharmaceutical composition further comprising at least one pharmaceutically acceptable The drug adjuvant or drug carrier binds to the interleukin. The pharmaceutical composition according to claim 14, wherein the pharmaceutical composition is a parenteral or oral administration. The use of the pharmaceutical composition according to claim 14 for the preparation of a medicament for treating renal fibrosis, wherein the pharmaceutical composition is an injection, a sterile powder, a tablet, a capsule, a pill, a granule or a drip Agent. Use of the pharmaceutical composition according to claim 14 of the patent application for the preparation of a medicament for the treatment of renal fibrosis, wherein the pharmaceutical composition is administered to a subject by parenteral injection. The use of the pharmaceutical composition according to claim 14 for the preparation of a medicament for treating renal fibrosis, wherein the pharmaceutical composition comprises 2.8 to 56 μg of interleukin 7 for administration of a biological individual per kilogram of body weight .