TWI407953B - Pharmaceutical composition for modulating complement factor b (cfb) expression - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for modulating complement factor B (CFB) expression in cells, comprising a tannic acid.

Description

Pharmaceutical composition for regulating complement factor B (CFB) expression

The present invention relates to a pharmaceutical composition for regulating the expression of complement factor B (CFB) in a cell.

In recent years, the destruction of the ozone layer has increased the level of ultraviolet radiation in the sun, threatening human health. UV radiation has a wavelength of 200-400 nm. UV radiation can be divided into three categories according to wavelength: long-wavelength UVA (320-400 nm), intermediate wavelength UVB (280-320 nm), short-wavelength UVC (200-280). Nm). Ultraviolet radiation can damage the tissues of the eye (Galichanin K, L Fgren S, Bergmanson J, S Derberg P. Evolution of damage in the lens after in vivo close to threshold exposure to UV-B radiation: cytomorphological study of apoptosis. Exp Eye Res. 2010 Sep;91(3):369-77) and phototoxic effects Retinal pigment epithelial (RPE) cells (Patton WP, Chakravarthy U, Davies RJ, Archer DB. Comet assay of UV-induced DNA damage in retinal pigment epithelial cells. Invest Ophthalmol Vis Sci 1999; 40: 3268-3275). Near-ultraviolet light (300-400 nm) radiation causes major damage in the retinal pigment epithelium, and damage caused by ultraviolet light may be associated with age-related macular degeneration (AMD) (Szaflik JP, Janik-Papis K, Synowiec E, et al) DNA damage and repair in age-related macular degeneration. Mutat Res 2009;669:169-176).

Among the elderly, age-related macular degeneration is the most common cause of irreversible blindness (Almeida LN, Carolino RM, Sperandio DC, Nehemy MB, De Marco LA. The role of molecular genetic factors in age-related macular degeneration. Arq Bras Oftalmol 2009; 72: 567-572). Previous studies have shown that complement factor B (CFB) and complement factor H (CFH) play an important role in the pathogenesis of AMD in more than 50% of cases (Maller J, George S, Purcell S, et al. Common Variation in three genes, including a noncoding variant in CFH, strongly influences risk of age-related macular degeneration. Nat Genet 2006;38:1055-1059;Gold B,Merriam JE,Zernant J,et al. Variation in factor B(BF And complement component 2 (C2) genes are associated with age-related macular degeneration. Nat Genet 2006; 38: 458-462). Complement factor B and complement factor H are involved in human immune and inflammatory responses. For several ocular diseases, proinflammatory cytokines are important mediators of cellular activity. For example, the proinflammatory cytokine-interleukin-6 (IL-6) is involved in inflammation of retinal pigment epithelial cells (Qin S , Ni M, De Vries GW. Implication of S-adenosylhomocysteine hydrolase in inhibition of TNF-alpha- and IL-1beta-induced expression of inflammatory mediators by AICAR in RPE cells. Invest Ophthalmol Vis Sci 2008;49:1274-1281). Ultraviolet B (UVB) induces the expression of IL-6, which causes intracellular signals in cultured human pterygium epithelial cells (Di Girolamo N, Wakefield D, Coroneo MT. UVB-mediated induction Cytokines and growth factors in pterygium epithelial cells. Invest Ophthalmol Vis Sci 2006; 47: 2430-2437). IL-6 binds to its receptor (eg, gp130) to activate pathways of Janus kinase (JAK), signal transducer, and activator of transcription 3 (STAT-3) to induce expression of several downstream target genes (Murray PJ) The JAK-STAT signaling pathway: input and output integration. J Immunol 2007;178:2623-2629). According to reports, IL-6 increases the rate of CHB mRNA and protein synthesis in fibroblasts (Katz Y, Revel M, Strunk RC. Interleukin 6 stimulates synthesis of complement proteins factor B and C3 in human skin fibroblasts. Eur J Immunol 1989; 19: 983-988).

U.S. Patent No. 2010/0247434 discloses a method of reducing or preventing angiogenesis in a mammal comprising administering to the mammal a therapeutically effective amount of gallic acid or a derivative thereof. Although the publication states that angiogenesis-related diseases include age-related macular degeneration, and derivatives of gallic acid include tannic acid, the examples thereof do not show the effect of tannic acid on the treatment of macular degeneration. In addition, this publication does not disclose the effect of tannic acid on the regulation of complement factor B.

It is still unclear that UVB-induced IL-6 signaling pathways are involved in the retinal pigment epithelium.

The present invention investigates whether the IL-6/JAK2/STAT3 signaling pathway can be activated by ultraviolet B (UVB) and whether activation of this pathway in retinal pigment epithelial (RPE) cells in turn leads to up-regulation of complement factor B (CFB). Tannic acid was also tested as a protective agent for UVB damage to RPE cells. In the present invention, the anti-inflammatory mechanism of tannic acid is also explored.

The present inventors have found that UVB induces IL-6 protein expression, which in turn activates signal transduction in the IL-6/JAK2/STAT3/CFB pathway, resulting in upregulation of CFB (Fig. 6). This signal pathway explains the pathogenesis of UV-induced age-related macular degeneration (AMD). Because the increase in CFB expression in RPE cells is known to be involved in the development of AMD (Maller J, George S, Purcell S, et al. Common variation in three genes, including a noncoding variant in CFH, strongly influences risk of age-related macular Degeneration. Nat Genet 2006;38:1055-1059;Gold B,Merriam JE,Zernant J,et al.Variation in factor B(BF)and complement component 2(C2)genes is associated with age-related macular degeneration. Nat Genet 2006; 38: 458-462), the results of the present invention provide a deeper understanding of UV-induced AMD. The present invention further demonstrates that tannic acid can effectively prevent UVB damage by inhibiting IL-6 production, phosphorylation of STAT-3, and upregulation of complement factor B. Thus, the present invention illustrates the deleterious mechanism of UVB on retinal pigment epithelial cells and also shows the potential utility of tannic acid to prevent UVB injury. The present inventors have also found that tannin can reduce the phosphorylation of STAT3 and the expression of complement factor B even without UV radiation, which enhances the use of tannic acid in preventing the development of AMD.

CFB is associated with an acute inflammatory response and its serum levels vary significantly during inflammation. CFB is an important central component of the alternative complement pathway. Activation of the alternative pathway results in the cleavage of CFB into Ba and Bb fragments. It has been reported that both Ba and Bb fragments exhibit various biological activities. Several diseases are known to be associated with CFB, such as age-related macular degeneration, complement factor B deficiency, hemolytic uremic syndrome, atherosclerosis, schizophrenia, glomerulonephritis, autoimmune diseases (including Not limited to Behçet's syndrome, recurrent oral ulcers and Crohn's disease, uveitis, infectious diseases or fungal infections.

Unless otherwise defined, the terms used herein have their meaning as commonly understood by those of ordinary skill in the art. As used throughout this application, the following terms shall have the following meanings:

The term "UVB" or "ultraviolet B" means one of the three types of invisible light emitted by the sun. The other two are UVA and UVC.

The term "disease, disorder, or medical condition associated with complement factor B (CFB) performance" means a disease, disorder, or medical condition that is primarily (or partially) caused by inappropriate expression of CFB. The disease, disorder, or medical condition associated with complement factor B (CFB) performance may include, but is not limited to, age-related macular degeneration, complement factor B deficiency, hemolytic uremic syndrome, atherosclerosis, schizophrenia, and small kidney. Spherical nephritis, autoimmune disease (such as Bethel's syndrome (Beh Et's syndrome), recurrent oral ulcers and Crohn's disease, uveitis, infectious disease or fungal infection.

Accordingly, the present invention provides a pharmaceutical composition for regulating the expression of complement factor B (CFB) in a cell, comprising tannic acid. In a preferred embodiment, the composition is for the treatment or prevention of a disease, disorder or medical condition associated with complement factor B (CFB) performance. The disease, disorder or medical condition is preferably selected from the group consisting of age-related macular degeneration, complement factor B deficiency, hemolytic uremic syndrome, atherosclerosis, schizophrenia, glomerulonephritis, autoimmune disease, and eye. Pigmentitis, infectious disease or fungal infection. More preferably, the disease, disorder or medical condition is age-related macular degeneration. Preferably, the expression of complement factor B is induced by ultraviolet light; more preferably, the ultraviolet light is ultraviolet light B. In a preferred embodiment, the cell line is retinal pigment epithelial cells. In a preferred embodiment, the pharmaceutical composition modulates the expression of complement factor B by modulating phosphorylation of STAT3; more preferably, the pharmaceutical composition modulates the phosphorylation of STAT3 by modulating IL-6 protein production. Chemical.

The invention may be embodied in different forms and is not limited to the examples mentioned below. The following examples are merely representative of the various aspects and features of the present invention.

Example 1 Cell culture and reagents

ARPE-19 cells (CRL-2302, human retinal pigment epithelial cells, American Type Culture Collection, Manassas, VA) were cultured in 1:1 DMEM medium at 37 ° C, 5% CO 2 and 90% relative humidity. 10% fetal calf serum in a mixture of HAM F-12 nutrient mixtures. The fused cells (90% confluence) were used in all experiments. The culture material was obtained from Gibco-BRL (Rockville, MD). Tannic acid, AG490 and all other reagents were obtained from Sigma Chemical Company (St. Louis, MO).

UVB radiation

The source of UVB radiation is the EL series (UVP Inc., San Gabriel, CA, USA) using UV lamps. In a lamp, 100% of the energy emission wavelength was 302 nm, the radiation intensity was 1.04 mW/cm ² , and a target distance of 15 cm was measured using a UVX Digital Radiometer (UVP Inc.).

Trypan blue exclusion analysis

Trypan blue exclusion analysis was used to determine the effect of treatment on ARPE-19 cell growth and survival. The cells were cultured for 18-24 hours after placing 200,000 ARPE-19 cells in a 3 cm culture plate. The cells were exposed to different UVB radiation doses for 24 hours. At 24 hours, cells were excised and collected using trypsin in the same tube. The cell pellet was washed with 300 μl of PBS (pH 7.4) and resuspended. Trypan blue was added to the same volume (μl) of cell suspension and the number of cells was counted using a hemocytometer, and each sample was repeated twice.

Real-time quantitative reverse transcription polymerase chain reaction (RT-PCR)

All RNA exposed to UVB radiation (5-15 mJ/cm ² ) and ARPE-19 cells after 24 hours was isolated using TRIzol reagent (invitrogen). 1 μg of all RNA was taken out using RT-PCR kit for reverse transcriptase RT-PCR (Applied Biosystems). The primers for detecting IL-6 mRNA are 5'-CCTGCAAG ACCATCGACATG-3' (progress, SEQ ID NO. 1) and 5'-CTGGCGAGCCTTAGTTTGGA-3' (reverse, SEQ ID NO. 2); The primers for detecting CFB were 5'-TGGTTTGGGAACACAGGAAGGGTA-3' (forward, SEQ ID NO. 3) and 5'-TCCCTTTGAAGGGCGAATGACTGA-3 (reverse, SEQ ID NO. 4); the primer for detecting STAT3 was 5 '-GATCCAGTCCGTGGAACCAT-3' (forward, SEQ ID NO. 5) and 5'-ATAGCCCATGATGATTTCAGCAA-3' (reverse, SEQ ID NO. 6); the primer for detecting JAK2 is 5'-GCTCAGTGGCGGCATGAT-3' (forward, SEQ ID NO. 7) and 5'-CACTGCCATCCCAAGACATTC-3' (reverse, SEQ ID NO. 8); the primer for detecting GAPDH is 5'-AACAGCGACACCCATCCTC-3' (forward, SEQ ID NO. 9) and 5'-CATACCAGGAAATGAGCTTGACAA-3' (reverse, SEQ ID NO. 10). Instant PCR is in a Gene Amp 7900 Sequence Detection System SDS Execution (Applied Biosystems, Foster City, CA, USA) and use Green PCR Master Mix Applied Biosystems. The relative amount of gene expression was analyzed by comparative Ct method. Each sample was normalized based on the formula GADH content according to the formula 2-ΔΔCT.

Immunotransfer method

All cell lysates were electrophoresed on a 10% SDS-PAGE film, transferred to a membrane and blocked, and then probed for phosphorylated STAT3 (Tyr705) and STAT3 (Cell Signaling Technology, Danvers, MA) )antibody. In order to control the loading of the protein, all ink dots were also detected using GAPDH (Millipore, Billerica, MA). Streaks were detected using an enhanced chemiluminescence system (Millipore, Billerica, MA).

Enzyme Linked Immunosorbent Assay (ELASA)

Approximately 200,000 ARPE-19 cells were cultured on a 3 cm culture dish for 18-24 hours. The cells were exposed to UVB radiation for 24 hours, and 1 ml aliquots of the cell culture supernatant were withdrawn and frozen at -80 °C. ELISA was performed using the OptEIA Human IL-6 ELISA (BD Pharmingen) kit according to the manufacturer's instructions. The supernatant sample was placed in a 96-well plate and two replicate assays were performed. Freshly diluted rIL-6 (available as standard control) was used in each plate analysis to generate a standard curve. The value of the sample pan was read at a wavelength of 450 nm using a spectrophotometric plate reader. The raw data was corrected for blank holes and converted to pg/ml using a standard curve. The number of cells per sample was normalized.

Statistical Analysis

Data for continuous variables are expressed as mean ± standard deviation (SD). The average between the two groups was compared using an unpaired t-test. A P value of less than 0.05 is considered to be statistically significant.

result Dose effect of UVB radiation on the growth of ARPE-19 cells

After 24 hours of irradiation, UVB radiation dose-dependently (0-25 mJ/cm2) reduced cell proliferation (Fig. 1). Since cells had a survival rate of 74-54% when the UVB radiation dose was 5-15 mJ/cm ² , it was decided to use a UVB dose of up to 15 mJ/cm ² in subsequent studies.

Effect of UVB radiation on mRNA expression in IL-6/JAK2/STAT3 pathway

IL-6 binds to cell surface receptors to induce signal transduction in the JAK2-STAT3 pathway (Sherman CT, Brasier AR. Role of signal transducers and activators of transcription 1 and -3 in inducible regulation of the human angiotensinogen gene by interleukin -6. Mol Endocrinol 2001; 15:441-457). The mRNA abundance of IL-6, JAK2 and STAT3 after UVB reaction was detected by real-time quantitative RT-PCR. UVB radiation dose-dependently (0-15 mJ/cm ² ) increased IL-6 mRNA at 24 hours (Fig. 2A). Consistent with the cell viability assay, it was found here that 10 mJ/cm ² of UVB radiation induced the maximum mRUA expression of the JAK2 and STAT3 genes, and at 24 hours after 15 mJ/cm ² UVB radiation, the amount would be slightly Drop (Figures 2B and 2C).

Effect of tannic acid on UVB-induced IL-6 protein production

The present invention determines whether UVB can induce production of IL-6 protein in the supernatant of ARPE-19 cell culture by ELISA. It was found that UVB radiation increased the performance of IL-6 protein (Fig. 3A). The effect of tannic acid on IL-6 showed that tannic acid reduced about 40% of UVB-induced IL-6 protein production in ARPE-19 cells (Fig. 3B).

Effect of tannic acid on UVB-induced STAT3 phosphorylation

IL-6 binding to its receptor gp130 leads to Tyr705 phosphorylation of STAT3 (Sherman CT, Brasier AR. Role of signal transducers and activators of transcription 1 and -3 in inducible regulation of the human angiotensinogen gene by interleukin-6. Mol Endocrinol 2001; 15:441-457). The phospho-STAT3 ^Tyr705 protein will dimerize and form an activated nuclear transcriptional complex, which in turn metastasizes to the nucleus and regulates the target gene (Sherman CT, Brasier AR. Role of signal transducers and activators of transcription 1 and -3 in inducible regulation of the human angiotensinogen gene by interleukin-6. Mol Endocrinol 2001; 15:441-457). Since UVB can induce IL-6 expression, it is further tested whether UVB can induce phosphorylation of STAT3. Using immunotransfer, it was found that UVB radiation slightly increased the phosphorylation of Tyr705 on STAT3. This result also shows that tannic acid significantly attenuates UVB-induced STAT3 phosphorylation in RPE (Fig. 4).

Effect of tannic acid or JAK2 inhibitor on UVB-induced CFB mRNA

It was determined whether UVB can induce the expression of CFB mRNA. The results showed that the maximum CFB mRNA expression was detected under 10 mJ/cm ² UVB radiation (Fig. 5A). However, the amount of CFB mRNA expression decreased slightly after 15 hours of UVB irradiation at 15 mJ/cm ² (Fig. 5A). To determine whether UVB-induced CFB up-regulation is through the IL6/JAK2/STAT3 pathway, the specific JAK2 inhibitor AG490 was used to observe the performance of the CFB gene. AG490 has been shown to inhibit JAK/STAT signals and prevent retinal degeneration after intense light exposure (Samardzija M, Weuzel A, Aufenberg S, Thiersch M, Reme C, Grimm C. Differential role of Jak-STAT signaling in retinal degenerations. FASEB J 2006;20:2411-2413). Using immunotransfer, it was found that the dose of AG490 (40 μM) inhibited the phosphorylation of STAT3 in Tyr705 (data not shown), and AG490 also partially reduced UVB-induced CFB mRNA expression (Fig. 5B). It was also noted that tannic acid reduced UVB-induced CFB mRNA expression in ARPE-19 cells (Fig. 5C).

A person skilled in the art will readily appreciate that the present invention can be easily accomplished with the results and advantages and those present in the present invention. The compositions of the present invention and the processes and methods for their manufacture are representative of the preferred embodiments, which are exemplary and not limited to the field of the invention. Those skilled in the art will be aware of the modifications and other uses therein. These modifications are intended to be within the spirit of the invention and are defined in the scope of the claims.

The present invention has been described in detail with reference to the embodiments of the present invention, and the invention may be Spirit and scope.

All patents and publications mentioned in the specification are subject to the general skill of the art in the field of the invention. All patents and publications are hereby incorporated by reference to the same extent as if each individual publication is specifically and individually indicated.

The invention as exemplified herein may be practiced in the absence of any element, or a plurality of elements, limitations, or limitations. The nouns and expressions used are as a description and not a limitation of the description, and are not intended to be used to exclude any nouns and expressions that are equivalent to the features or parts thereof shown and described, but Various changes are possible within the scope of the patent application of the invention. Therefore, it is to be understood that the present invention has been disclosed and described herein in accordance with the preferred embodiments and the features of the present invention. Within the scope.

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Figure 1 shows the effect of UVB radiation on cell viability. The ARPE-19 cells (2x10 ⁵ cells) were cultured in 3 cm culture dishes for 24 hours. The cells were treated with 0 mJ/cm ² (blank bars) or 5-25 mJ/cm ² UVB (grey bars) and then cultured for 24 hours. The number of cells was calculated by trypan blue exclusion assay. The results shown are the average of three independent experiments. * P <0.05 vs. 0 mJ/cm ² UVB.

Figure 2 shows that UVB radiation upregulates IL-6/JAK2/STAT3 mRNA expression. ARPE-19 cells were cultured for 24 hours at 0 mJ/cm ² (blank bars) or 5-15 mJ/cm ² UVB (grey bars). The relative expression level of IL-6/JAK2/STAT3 mRNA was detected by real-time RT-PCR. The results normalized by GAPDH are shown as the average of three independent experiments. * P < 0.05, ** P < 0.01, *** P < 0.001 vs. 0 mJ/cm ² UVB.

Figure 3 shows that tannic acid (TA) reduces UV-induced up-regulation of IL-6. ARPE-19 cells were cultured for 24 hours at 0 mJ/cm ² (blank bars) or 5-15 mJ/cm ² UVB (grey bars). The protein expression level of IL-6 was measured by ELISA. (A) UVB dose-dependently reduces IL-6 protein production. (B) Tannic acid (TA) (25 μM, bar 4) reduced UVB (strip 2) induced IL-6 protein production. The results are shown as the average of three independent experiments. * P <0.05 and ** P <0.01 vs. 0 mJ/cm ² UVB; ^# P<0.05 vs. 10 mJ/cm ² UVB.

Figure 4 shows that tannic acid inhibits STAT3 phosphorylation induced by UVB on Try705. The ARPE-19 cells (5x10 ⁵ cells) were cultured in 6 cm culture dishes for 24 hours. Cells were cultured for 24 hours after exposure to the indicated doses of UVB radiation. Phospho-STAT3 ^{Tyr705 was} detected by immunotransfer. UVB increased the degree of phospho-STAT3 ^Tyr705 (runway 2) and TA (25 μM) inhibited UV-induced phosphorylation-STAT3 ^Tyr705 (Runway 4).

Figure 5 shows that tannic acid (TA) and JAK2 inhibitors reduce UVB-induced CFB mRNA. ARPE-19 cells were cultured for 24 hours at 0 mJ/cm ² (blank bars) or 5-15 mJ/cm ² UVB (grey bars). The performance of CFB mRNA was detected by real-time RT-PCR. (A) UVB induces CFB mRNA. (BC) TA (25 μM) and AG490 (40 μM in 0.2% DMSO) reduced UVB-induced CFB mRNA. Relative performance data is presented after standardization by GAPDH. The results shown are the average of 3 independent experiments. * P <0.05 ** P <0.01 and *** P <0.001 vs. 0 mJ/cm ² UVB; ^# P <0.05 and ^## P<0.01 vs. 10 mJ/cm ² UVB.

Figure 6 is a schematic representation of the signal pathway involved in UVB-induced RPE inflammation.

Claims (7)

A use of tannic acid for the preparation of a pharmaceutical composition that modulates the expression of interleukin-6 (IL-6), transcriptional activator-3 (STAT-3) and complement factor B (CFB). The use according to claim 1, wherein the pharmaceutical composition is for treating or preventing a disease, disorder or medical condition associated with the expression of complement factor B. According to the use of the second aspect of the patent application, wherein the disease, disorder or medical condition is selected from the group consisting of age-related macular degeneration, complement factor B deficiency, hemolytic uremic syndrome, atherosclerosis, schizophrenia, glomerulus Nephritis, autoimmune disease, uveitis, infectious disease or fungal infection. The use according to item 2 of the scope of the patent application, wherein the disease, disorder or medical condition is age-related macular degeneration. According to the use of the first aspect of the patent application, wherein the expression of complement factor B is induced by ultraviolet rays. According to the use of the fifth aspect of the patent application, wherein the ultraviolet light is ultraviolet light B. The use according to the first aspect of the patent application, wherein the cell line is a retinal pigment epithelial cell.