Is astaxanthin beneficial for apoptosis in rat renal tubular epithelial cells?

 With the development of diagnostic, interventional and radiologic techniques in coronary artery disease, the use of contrast media is increasing. Contrast-induced acute kidney injury (CI-AKI) is becoming a common cause of medical renal failure [1]. Meanwhile, CI-AKI, in-stent thrombosis and post-stent restenosis are the three major complications after percutaneous coronary intervention (PCI) [2].The pathogenesis of CI-AKI is not yet fully understood, and direct tubular cytotoxicity, contrast-induced oxygen radical damage and renal hemodynamic changes are the important mechanisms [3,4]. Astaxanthin (AST) is a kind of carotenoid, has anti-inflammatory, antioxidant, regulation of glucose and lipid metabolism, reduce hepatic and renal damage and other effects [5]. Currently, most of the studies on CI-AKI are focused on animal level and clinical studies. This experiment aimed to establish an in vitro model of CI-AKI, study the role of apoptosis in rat CI-AKI and the protection of AST against apoptosis, and explore its possible mechanisms.

 

1 Materials and Methods

1.1 Main reagents and instruments: AST (Sigma, purity>99%), iodohydrol (I, Yangzijiang Pharmaceutical Co., Ltd.), fetal bovine serum (Sijiqing, China), DMEM/F12 (Hyclone, USA), nicotinamide (Dalian Meilun Biotechnology Co., Ltd., China), bicuculline carboxylic acid (BCA) protein kit, annexin V-FITC/propidium iodide (PI) apoptosis kit (Nanjing Kaiji Biotechnology Development Co., Ltd.), mitochondrial membrane electric voltage kit (Nanjing Kaiji Biotechnology Development Co., Ltd.), mitochondrial membrane electrode kit (Nanjing Kaiji Biotechnology Development Co. Annexin V-FITC/Propidium Iodide (PI) Apoptosis Kit (Nanjing Kaiji Biotechnology Development Co., Ltd.), Mitochondrial Membrane Potential Assay Kit (JC-1, Jiangsu Biyuntian Biotechnology Co., Ltd.), β-actin antibody (Protein-tech, Inc., USA), Silent Information Regulatory Factor 2-related Translocator (SIRT) 1 antibody (Absin Biotechnology Co., Ltd.), Silent Information Regulatory Factor 2 (SIRT) 1 antibody (Absin Biotechnology Co., Ltd.), Silent Information Regulator 2 (SIRT) 1 antibody (Absin Biotechnology Co., Ltd.). Absin Biotechnology Co., Ltd.), P53 antibody (Abclonal Biotechnology Co., Ltd.), Bax antibody (Proteintech, Inc., USA), Bcl-2 antibody (Santa Cruz, Inc., USA), CO2 incubator (Heraeus Corp., Inc.), triple-vapor incubator (Thermo Corp., Inc.), and enzyme labeling instrument (Bó Lè Company, Inc., USA), and protein electrophoresis and electrophoresis. The protein electrophoresis and electrotransfer device (Bio-Rad) was used for the analysis of the proteins.

 

1.2 Cell culture and grouping    

Rat renal tubular epithelial cell line (NRK-52E) was cultured in DMEM/F12 medium containing 10% fetal bovine serum, 0.1% penicillin and streptomycin, and the cells were digested and passaged with 0.25% trypsin when they had grown to 80%. After passaging, the cells were randomly grouped into blank control group, solvent control (DMSO) group (0.1% DMSO), I group (50 g I/L), AST pretreated (AST+I) group (50 g I/L I +20 μmol/L AST), AST pretreated + SIRT1 inhibitor nicotinamide (AST+I+NA) group (50 g I/L I + 20 μmol/L AST), and AST+I+NA group (50 g I/L I +20 μmol/L AST). 20 μmol/L AST+20 mmol/L NA), I+SIRT1 inhibitor (I+NA) group (50 g I/L I+20 mmol/L NA).

 

1.3 4,6-Diamidino-2-phenylindole (DAPI) fluorescence staining observation Nucleus morphology   

Rat renal tubular epithelial cells grown in 24-well culture plates were washed three times with phosphate buffer solution (PBS), fixed with 4% paraformaldehyde (dissolved in 0.1 mol/L phosphate buffer (PB), pH 7.4) for 15 min at room temperature, washed three times with PBS for 5 min, and stained with 3 ml of DAPI (dissolved in 0.2% Triton PBS) for 5~10 min, and washed with PBS three times for 5 min. The staining was observed on an Olympus fluorescence microscope at 400 nm as the excitation light and 455 nm as the emission light. DAPI (dissolved in PBS containing 0.2% Triton) was added for 5~10 min, and washed with PBS for 3 times × 5 min. The staining was observed on an Olympus fluorescence microscope with the excitation light at 400 nm and the emission light at 455 nm.

 

1.4 Annexin V-FITC/PI double staining flow cytometry for cell apoptosis detection   

The cells were gently moistened with 2 ml of PBS in the culture dish and the PBS was removed. Resuspend the cells in the previous medium or pre-cooled 1× binding buffer to a concentration of 1×106 cells/ml. Take 500 μl of cell suspension (5×105 cells) into a centrifuge tube, centrifuge at 1,000 r/min for 5 min, remove supernatant, add 500 μl of PBS to wash the cells, centrifuge, and remove supernatant. Add 500 μl of 1× binding buffer to wash the cells, centrifuge, and remove the supernatant; add 100 μl of 1× binding buffer to resuspend the cells, and then add 5 μl of Annexin V-APC to react the cells for 15 min at room temperature and protected from light. Wash the cells with 500 μl of 1× binding buffer, centrifuge and remove the supernatant; add 200 μl of 1× binding buffer to resuspend the cells and add 5 μl of PI. The samples were analyzed by flow cytometry within 1 hour.

 

1.5 Apoptosis assay   

Discard the medium in the 6-well plate, wash the plate with PBS three times, add 1 ml of JC-1 staining buffer, mix thoroughly, and incubate for 20 min at 37 ℃ in a cell culture incubator. During the incubation period, an appropriate amount of JC-1 staining buffer (1×) was prepared by adding 4 ml of distilled water for every 1 ml of JC-1 staining buffer (5×) and incubated at 37℃ in an ice bath. After incubation at 37℃, the supernatant was aspirated, and the cells were washed twice with JC-1 staining buffer (1×), and 2 ml of the cell culture medium was added, and the incubated cells were observed under the fluorescence microscope. The excitation light was set at 490 nm and the emission light was set at 530 nm for the detection of JC-1 monomer; the excitation light was set at 525 nm and the emission light was set at 590 nm for the detection of JC-1 polymer.

 

1.6 Protein Expression Levels by Western Blotting   

The total protein was extracted, and the protein concentration was determined by BCA method, then subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), which was closed at room temperature after membrane transfer, and then immunohybridized with specific antibody, β-actin was used as the internal reference, and the chemiluminescent agent developed the color, and then finally, the target bands were subjected to the scanning densitometric analysis. Each group was repeated 3 times.

 

1.7 Statistical methods    

Analysis of variance (ANOVA) and q-test were performed using Graphpad Prism 5.0 software.

 

2 Results

2.1 Morphologic changes in apoptosis in each group    

In the Control group and DMSO group, the nuclei were uniformly stained and no apoptotic cells were seen.In group I, there were consolidated nuclei and deep staining of nuclei, and the consolidated nuclei were highlighted, and some of the cells were seen to be nuclear cleavage. Compared with group I, group AST+I showed improved nuclear consolidation and deep staining, and fewer apoptotic cells.In group AST+I+NA, there were more consolidated and highlighted nuclei, and more apoptotic cells compared with group AST+I. Compared with AST+I+NA group, apoptotic vesicles were further increased in I+NA group, and cell damage was aggravated. See Figure 1.

 

2.2 Comparison of apoptosis rate among groups    

Compared with the Control group, the difference of apoptosis rate in DMSO group was not statistically significant (P >0.05); the apoptosis rate in group I was significantly higher (P <0.05), and the apoptosis rate in AST+I group was significantly lower (P <0.05) than that in group I. The apoptosis rate in AST+I group was significantly higher (P <0.05) than that in AST+I group. The apoptosis rate of AST+I+NA group was significantly higher than that of AST+I group (P<0.05). Compared with AST+I+NA, the difference was statistically significant in I+NA group (P<0.05), and there was no significant difference in I group (P>0.05). See Table 1.

 

2.3 Comparison of JC-1 fluorescence intensity among groups    

The difference in JC-1 fluorescence intensity between the DMSO group and the Control group was not statistically significant (P>0.05), and the JC-1 fluorescence intensity in the I group was significantly lower than that in the Control group (P<0.05). JC-1 fluorescence intensity in AST+I group was significantly higher than that in I group (P<0.05). The fluorescence intensity of JC-1 was significantly higher in AST+I group (P<0.05) and lower in I+NA group (P<0.05) compared with AST+I+NA group (P<0.05).There was no statistically significant difference in the fluorescence intensity of JC-1 between the AST+I+NA group and the I group (P>0.05). See Table 1 and Figure 2.

 

2.4 Changes in protein expression in renal tubular epithelial cells in each group    

Compared with the Control group, there was no difference in protein expression in the DMSO group (P >0.05), SIRT1 and Bcl-2 protein expression in the I group was significantly reduced, and P53 and Bax expression were significantly increased (P < 0.05). SIRT1 and Bcl-2 protein expression in cells of the AST+I group was significantly increased compared with that of the I group, and P53 and Bax expression were significantly reduced (P<0.05). (In the AST+I+NA group, compared with the AST+I group, the protein content of SIRT1 and Bcl-2 was significantly reduced, and the expression of P53 and Bax was significantly increased (P<0.05). Compared with the AST+I+NA group, the expression of SIRT1 and Bcl-2 proteins in the I+NA group was significantly reduced, and the expression of Bax and P53 proteins was significantly increased (P<0.05). See Table 1,Figure 3.1 ~ 6: Control group, DMSO group, I group, AST+I group, AST+I+NA group, I+NA group.

 

3 Discussion

AST is a naturally occurring red carotenoid derivative that is widely found in many animals and plants. Natural AST has many potentials, such as antioxidant, anti-inflammatory, immunomodulatory, tumor growth inhibition, anti-aging, and prevention of cardiovascular and cerebral vascular diseases, etc. Guo et al. showed that AST can reduce renal tubular cell apoptosis by regulating the pro-apoptotic proteins of mitochondrial pathway, and improve the acute renal injury in rats with severe burns. The results of this experiment confirm that AST pretreatment can significantly reduce contrast-induced apoptosis, and its nephroprotective effect is closely related to the activation of SIRT1, stabilization of mitochondrial membrane potential, and regulation of apoptosis-related proteins, P53, Bax, Bcl-2 expression.

 

Apoptosis is an autonomous, programmed cell death that is stimulated and genetically regulated [8], and the Bcl-2 family is the key to apoptosis in the mitochondrial pathway, where anti-apoptotic proteins such as Bcl-2 and pro-apoptotic proteins such as Bax can activate a series of downstream target molecules to regulate mitochondrial membrane permeability and integrity, and then regulate the release of pro-apoptotic factors [9], which mediate apoptosis. The release of mitochondrial pro-apoptotic factors is then regulated [9]. The target genes of P53 encode a variety of pro-apoptotic proteins, such as P53-induced gene proteins (PIGs), apoptosis protein 1 (Fas), apoptosis enzyme-activating factor (Apaf)-1, Bax, and P53 up-regulation of apoptosis regulator (PU-MA), etc. P53 acts as a kind of transcription factor to regulate the expression of downstream apoptosis-related proteins, and also regulates the release of mitochondrial pro-apoptotic factors [10]. As a transcription factor, P53 regulates the expression of downstream apoptosis-related proteins, contributing to the occurrence of apoptosis [11]. P53 can also directly bind to Bak and activate it, inducing the oligomerization of Bak, which in turn induces the release of cytochrome (Cyt)c and ultimately leads to apoptosis [12,13].

 

SIRT1 is a nicotinamide adenine dinucleotide (NAD+)-dependent class III histone deacetylase [14], and its downstream effectors include histones, P53, nuclear factor (NF)-κB, peroxisome proliferator-activated receptor (PPAR)γ and coactivator (PGC-1α), forkhead proteins (FOXOs), hypoxia-inducible factor (HIF)1, etc. By deacetylating them, it regulates glycolipid metabolism, suppresses oxidative stress and inflammation, reduces apoptosis, and attenuates aging, among others. By deacetylating PPARγ and coactivating factors (PGC-1α), forkhead protein transcription factors (FOXOs) and hypoxia-inducible factors (HIF)1, AST can regulate glycolipid metabolism, inhibit oxidative stress and inflammation, reduce apoptosis, and attenuate the aging process, and thus it plays an important role in tumors, regulation of lipid metabolism, cardiovascular diseases, and other diseases [15,16]. The present results suggest that AST protects NRK-52E cells by activating SIRT1 signaling and that SIRT1 acts upstream of these molecules. In addition, compared with the AST+I+NA group, the I+NA group lacked the protection of AST, and the expression of SIRT1 and Bcl-2 proteins was reduced, while the expression of P53 and Bax was increased, which confirmed the protective effect of AST.

 

In conclusion,AST can improve CI-AKI,its main mechanism is to activate the SIRT1 pathway,regulate the mitochondrial membrane potential,down-regulate the expression of pro-apoptotic proteins P53 and Bax,and up-regulate the expression of anti-apoptotic protein Bcl-2 to exert the anti-apoptotic effect.AST has provided a new choice for the prevention and treatment of contrast-induced nephropathy (CIN),but the specific mechanism of the nephroprotective effect of AST has not been fully elucidated,and still needs to be further researched. However, the specific mechanism by which AST exerts its nephroprotective effect has not been fully elucidated, and further studies are needed.

 

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