Cinnamaldehyde attenuates kidney senescence and injury through PI3K/Akt pathway-mediated autophagy via downregulating miR-155.

BACKGROUND: To prove the internal connection, we deciphered the effect of cinnamaldehyde on kidney senescence through establishing animal and cell models.
METHODS: In vivo, a rat senescence model was constructed using D-galactose (D-gal), and the modeled rats were further treated with cinnamaldehyde. In vitro, rat renal tubular epithelial cells (NRK-52E) were transfected with miR-155 mimic or inhibitor and then treated with cinnamaldehyde, D-gal or PI3K inhibitor (LY294002). The serum levels of blood urea nitrogen (BUN) and serum creatinine (Scr) of the rats were measured by an automatic biochemical analyzer. Pathological changes of kidney were determined by hematoxylin-eosin staining. The senescence and viability of NRK-52E cells were assessed by SA-β-gal staining and CCK-8 assay, respectively. The levels of miR-155, p-PI3K/PI3K, p-Akt/Akt, LC3B (LC3-II and LC3-I) and Beclin1 were detected by qRT-PCR, immunohistochemistry, or western blot.
RESULTS: D-gal elevated the levels of BUN, Scr and miR-155 in the kidney, induced the renal pathological damage, inhibited the cell viability, increased the numbers of SA-β-gal-, LC3B- and Beclin1-positive cells and upregulated the levels of LC3-II/LC3-I and Beclin1 both in the kidney and cells. Cinnamaldehyde reversed D-gal-induced effects on the kidney and cells, and moreover, the cinnamaldehyde-induced anti-D-gal effects on cells could be suppressed by miR-155 mimic but promoted by miR-155 inhibitor. LY294002 potentiated D-gal-induced effects, and reversed cinnamaldehyde- and miR-155 inhibitor-caused impacts on the PI3K/Akt pathway and LC3-II/LC3-I level in D-gal-induced cells.
CONCLUSION: Cinnamaldehyde attenuates kidney senescence and injury through PI3K/Akt pathway-mediated autophagy via downregulating miR-155.

Introduction

Senescence is an inevitable process of retrogression of kidney tissue of patients with advancing age [1]. During senescence, renal structure and function change gradually, most of which can be reflected in cell cycle arrest, telomere shortening, senescence-associated β-galactosidase (SASP) activity increase, SASP secretion-induced inflammation, renal interstitial fibrosis, renal tubular atrophy and glomerulosclerosis [2-5]. Concomitantly, there also emerges a reduced rate of glomerular filtration, a critical stage generally in the assessment of kidney function, which affects the process of urine formation in the kidney [6-8].

Weakened kidney function with advancing age is probably attributed to multiple factors [1], among which autophagy is reported to play a fundamental role [9]. Autophagy is a multistep and dynamic process, during which highly regulated lysosomal proteins are degraded to recycle damaged or excess organelles and protein aggregates for maintaining intracellular homeostasis and cellular innovation [10]. Moreover, metabolic slowdown is a common feature of cell aging, bringing about negative situations that toxic wastes in the body fail to be excreted in a timely manner. As a result, toxic wastes can accumulate and cause long-term damage to tissues, cells, and protein functions, thereby reducing the survival rate of cells and affecting the lifespan of the body [11,12]. Autophagy works excellent in decomposing damaged proteins and cells, and using them repeatedly to extend the life of cells and delay the body’s aging, showing its potential in regulating cell senescence [12,13]. A previous study has shown that inhibition of autophagy in the kidney is associated with the aging-caused degeneration of proximal tubular cells [14]. Furthermore, several major nutrient-sensing pathways such as adenosine monophosphate-activated protein kinase (AMPK), sirtuin 1 and mammalian target of rapamycin (mTOR) are inferred to play regulatory roles in autophagy during kidney senescence [15-17].

Previous studies have discovered that cinnamaldehyde demonstrates a strong protective effect against aging-associated lipid peroxidation in rat kidney homogenates [18], and is able to counteract glomerular fibrosis and ameliorate renal dysfunction in leptin receptor-deficient mice [19]. Cinnamaldehyde, a natural compound existing in cinnamon and bay leaf, possesses multiple properties including anti-oxidation, anti-inflammation, anti-cytotoxicity, and anti-leishmaniasis [18]. A recent study exploring the antioxidative effect of cinnamaldehyde has pointed out that cinnamaldehyde can remarkedly attenuate ultraviolet irradiation-induced photoaging including wrinkle formation, epidermal hyperplasia, and dermal inflammatory cell infiltration in mice [20]. In addition, miRNAs including miR-21, miR-155 and miR-27a are reported to be involved in the role of cinnamaldehyde in different diseases [21,22]. Among them, miR-155 is recently proved to exert a regulatory effect on assorted diseases. For instance, tumor-derived exosomal miR-155 triggers cancer-associated cachexia to enhance tumor progression [23], miR-155 promotes macrophage pyroptosis via regulating NLRP3 inflammasome [24], and miR-155 downregulation can reduce the impaired autophagy in an experimental mouse with pancreatitis and further improve the prognosis of mouse [25]. More importantly, during kidney senescence, miR-155 is demonstrated to be dysregulated [26]. Meanwhile, cinnamaldehyde is uncovered to eliminate miR-155 abnormal upregulation to exert pharmaceutical effects against ulcerative colitis [21]. However, whether cinnamaldehyde impacts kidney senescence and whether miR-155 is involved in the effect of cinnamaldehyde protecting against kidney senescence are awaited to be studied.

Moreover, a study has put forward that the PI3K/AKT signaling pathway, one of the early and necessary signaling pathways of cell cycle progression [27], is implicated in the pharmaceutical effect of cinnamaldehyde [28]. The pathway can be activated by insulin as well as other growth factors and act as the upper stream of an autophagy regulator, mTOR pathway. The activation of P13K/Akt pathway exerts an inhibitory effect on autophagy in some types of breast cancer cells [27]. Thus, the PI3K/Akt signaling pathway was speculated to have a link with the anti-senescence effect of cinnamaldehyde in the kidney. The objective of this study was to explore the mechanism via which cinnamaldehyde ameliorates kidney senescence and injury through regulating the PI3K/Akt signaling pathway-mediated autophagy.

Materials and methods

Ethics statement

All animal experiments were performed in Jiaxing Hospital of Traditional Chinese Medicine in accordance with the guidelines of the China Council on Animal Care and Use. This study was approved by the Committee of Experimental Animals of Jiaxing Hospital of Traditional Chinese Medicine (approval number: ND20190789). Every effort was made to minimize pain and discomfort to the animals.

Animal experiments

D-gal (G0750, Sigma-Aldrich, St. Louis, MO, USA) and vitamin E (VE, 5.00862, Sigma-Aldrich, USA) used as positive controls were purchased from Sigma-Aldrich Chemical Co. LLC. Cinnamaldehyde purchased from Aladdin (C9H8O, purity: ≥99.5%, C108631-1g, Shanghai, China) was dissolved in 1% carboxymethyl cellulose (419273, Sigma-Aldrich, USA) to prepare a 2 mL/kg working solution for gavage administration. The animal experiments as well as the dosages of cinnamaldehyde and VE used in the experiment were both set according to the previous publication [29-32]. VE is a classic anti-aging drug [33,34], which was used by many scholars as a positive control in the research involving aging and injury [35,36]. Therefore, VE was selected as the positive control in this study.

Sprague-Dawley rats (male, weighing around 28–32 g) were procured from the Animal Center Affiliated to Nanjing Medical University (Nanjing, China). All the rats were kept at 20–24 °C, with 50% humidity in a 12 h (h) dark:12 h light cycle. They were fed with a standard rat chow and given free access to water in the Animal Center Affiliated to Jiaxing Hospital of Traditional Chinese Medicine. Prior to experiments, rats were acclimatized for 1 week. A total of 24 rats were evenly and randomly assigned into four groups (n = 6 per group): control group (rats without treatment), D-gal group (rats were injected with D-gal), D-gal + CA group (rats were injected with D-gal and received CA by gavage) and D-gal + VE group (rats were injected with D-gal and received CA by gavage). Rats in D-gal, D-gal + CA and D-gal + VE groups were subjected to subcutaneous injection of D-gal at a dose of 120 mg/kg/d for 6 weeks [29,30]. In the meantime, rats in D-gal + CA and D-gal + VE groups were additionally gavaged with cinnamaldehyde at a dose of 40 mg/kg/d or VE at a dose of 30 mg/kg/d for 6 weeks [31,32]. Six weeks later (rats were administrated with D-gal, cinnamaldehyde and VE every day during the 6 weeks), all rats were anesthetized by intraperitoneal injection with 5% pentobarbital sodium (P-010, Sigma-Aldrich, USA) and then sacrificed by cervical dislocation. Then, blood samples of the rats were collected by cardiac puncture for biochemical analyses and kidney specimens were harvested for the following experiments.

Automatic biochemical analyses

After the rats received injection of D-gal with or without cinnamaldehyde or VE by gavage, serum from all rats was obtained via centrifugation of the blood at 2000 × g for 20 minutes (min). The serum levels of blood urea nitrogen (BUN) and serum creatinine (SCr) of the rats were measured by an automatic biochemical analyzer (Labomed Inc FACA401, Fisher Scientific, Waltham, MA, USA).

Hematoxylin-eosin staining

Pathological changes of all rat kidney specimens were evaluated through hematoxylin-eosin staining using a staining kit (C0105M, Beyotime, Shanghai, China). Briefly, the kidney specimens were firstly fixed with 4% paraformaldehyde buffer (DF0134, Leagene Biotechnology, Beijing, China) for 24 h. Then, the tissue was transparentized with xylene (95682, Sigma-Aldrich, USA), dehydrated with gradient ethanol (E7023, Sigma-Aldrich, USA), and further embedded into paraffin (A55701, OKA, Beijing, China). After the tissue was cut into 4 μm-thick slices, the slices were dewaxed, stained with hematoxylin for 10 min and further incubated with hydrochloric acid alcohol (C0163M, Beyotime) for 2 seconds (s). Subsequently, the slices were dyed with eosin for 1 min and incubated with xylene and neutral gum (IH0265, Leagene Biotechnology). Finally, the tissue image was observed under an optical microscope (50-193-8115, Fisher Scientific, Waltham, MA, USA) at ×100 and ×400 magnification.

Immunohistochemistry assay

After the rats received an injection of D-gal with or without cinnamaldehyde or VE by gavage, the upper renal cortex and lower renal cortex (n = 6 per group) of the D-gal-treated rats were separately fixed in 2.5% pentanediol (260282, Sigma-Aldrich, USA), embedded in paraffin (1496904, Sigma-Aldrich, USA) and cut into 4 µm-thick sections. The sections were dewaxed by xylene and rehydrated by gradient ethanol (E7023, Sigma-Aldrich, USA), followed by being immersed in citrate (1613859, Sigma-Aldrich, USA) and boiled at 126 °C for 1–2 min for retrieval of antigens. H2O2 (3%) was used to block endogenous peroxidase for 20 min. Each section was incubated firstly with 50 µL goat serum (16210064, ThermoFisher, Waltham, MA, USA) at room temperature for 15 min, and then with 50 µL primary antibody against LC3B (ab48394, 1:200, abcam, UK) or Beclin1 (ab62557, 1:500, abcam, UK) in a wet box at 4 °C. Following the removal of the primary antibody, 50 µL secondary antibody Goat Anti-Rabbit IgG H&L (HRP) (ab205718, 1:2000, Abcam, UK) was added to cultivate each section. Positively stained cells were colored using 1 mL of 0.05% DAB solution (11718096001, Sigma-Aldrich, USA), followed by being stained with hematoxylin (H9627, Sigma-Aldrich, USA) for 3 min. After dehydration by gradient ethanol and hyalinization by xylene, the sections were analyzed by an optical microscope (50-193-8115, Fisher Scientific, Waltham, MA, USA) under ×100 and ×400 magnification for the observation of LC3B- and Beclin1-positive cells.

Cell culture

Rat proximal tubular epithelial cells (NRK-52E) were obtained from the Cell Bank Affiliated to Chinese Academy of Sciences (GNR 8, Beijing, China), and were cultured in Dulbecco’s modified Eagle’s medium/Ham’s F-12 (DMEM/F-12) (11320082, ThermoFisher, USA) supplemented with 5% fetal bovine serum (FBS; 10099141C, ThermoFisher, USA) at 37 °C with 5% CO2.

Cell transfection

MiR-155 mimic (M; miR1161214112154-1-5), mimic control (MC; miR1N0000001-1-5), miR-155 inhibitor (I; miR2160606012823-1-5) and inhibitor control (IC; miR2N0000001-1-5) were purchased from RIBOBIO (Guangzhou, China), which were separately used to transfect NRK-52E cells under the help of Lipofectamine 3000 transfection reagents (L3000015, ThermoFisher, USA). In a nutshell, NRK-52E cells were seeded in 96-well plates at a density of 1 × 104 cells/well. When the cell confluence reached 80%, miR-155 mimic, inhibitor and Lipofectamine 3000 transfection reagents were diluted into a mixed solution consisting of Opti-MEM (31985062, ThermoFisher, USA) and P3000 reagents. After the diluted plasmids were incubated with reagents at 37 °C for 10 min, gene-lipid complexes were obtained and added into the 96-well plates to culture the cells at 37 °C for 24 h.

Cell treatment

All the transfected or untransfected NRK-52E cells (5 × 105 cells/well) were inoculated in a 6-well plate (140644, ThermoFisher, USA) with the addition of 2 mL FBS into each well, and then allocated to ten groups: control group (cells without treatment), D-gal group (cells with D-gal treatment), D-gal + CA group (cells were treated with D-gal and CA), D-gal + CA + MC group (cells were transfected with miR-155 mimic control followed by D-gal and CA treatment), D-gal + CA + IC group (cells were transfected with miR-155 inhibitor control followed by D-gal and CA treatment), D-gal + CA + M group (cells were transfected with miR-155 mimic followed by D-gal and CA treatment), D-gal + CA + I group (cells were transfected with miR-155 inhibitor followed by D-gal and CA treatment), D-gal + LY294002 group (cells were treated with LY294002 and D-gal), D-gal + CA + LY294002 group (cells were treated with LY294002, D-gal and CA), and D-gal + CA + I+LY294002 group (cells were transfected with miR-155 inhibitor followed by LY294002, D-gal and CA treatment). Till the cells reached 60% confluence, the cells in the D-gal + CA, D-gal + CA + MC, D-gal + CA + M, D-gal + CA + IC, D-gal + CA + I, D-gal + CA + I+LY294002 and D-gal + CA + LY294002 groups were subjected to pretreatment with 20 μM cinnamaldehyde for 2 h and subsequent intervention with 100 mM D-gal for 24 h. Cells in D-gal group and D-gal + LY294002 group were treated with 100 mmol/L D-gal for 24 h [21]. In addition, for LY294002 (the PI3K inhibitor; HY-10108, MedChemExpress, Princeton, NJ, USA) treatment, the cells in the D-gal + LY294002 and D-gal + CA + LY294002 groups were treated with 10 μM LY294002 [37] which was added during 24-h D-gal treatment at 37 °C.

Quantitative reverse transcription polymerase chain reaction (qRT-PCR)

Rat kidney tissues were treated by a homogenizer (UH-48, Union-Biotech, Shanghai, China). Total miRNAs from the obtained homogenate and NRK-52E cells were extracted by TRIzol lysis buffer (15596018, ThermoFisher). First strand cDNAs from the isolated miRNAs were synthesized by Synthesis Kits (K1621, ThermoFisher, USA). QPCR reaction was performed on an Applied Biosystems PCR machine (7500 FAST real-time, Applied Biosystems, Foster City, CA, USA) with the TB Green Premix Ex Taq II (Tli RNaseH Plus) (RR820Q, TAKARA, China). The primers used were shown as follows: miR-155 (Forward: 5′-GGGTGTCGTATCCAGTGCAA-3′; Reverse 5′-GTCGTATCCAGTGCGTGTCG-3′) and U6 (Forward: 5′-CTCGCTTCGGCAGCACA-3′; Reverse: 5′-AACGCTTCACGAATTTGCGT-3′). Amplification conditions were set as follows: 95 °C for 10 min, followed by 40 cycles of 95 °C for 15 s and 60 °C for 1 min. The relative gene expression was normalized to U6 and quantified using the 2–ΔΔCT method [38]. This experiment was repeated three times.

Cell counting kit (CCK)-8 assay

The viability of NRK-52E cells was measured with the CCK-8 kit (C0037, Beyotime, China) according to the manufacturer’s instructions. The transfected or untransfected NRK-52E cells were digested by Trypsin-EDTA (25200072, ThermoFisher, USA), diluted to the density of 2 × 103 cells/well and exposed to D-gal for 24 h with or without cinnamaldehyde treatment for 2 h. Each well of a 96-well plate was introduced with 100 µL cell solution. The cells were mixed with CCK-8 solution at a ratio of 1: 10 and incubated at 37 °C for 1 h. The optical density was measured at a wavelength of 450 nm using a microplate reader (ELx808, BioTek, Winooski, VT, USA). This experiment was carried out in triplicate.

SA-β-gal staining assay

SA-β-gal staining on NRK-52E cells was conducted with SA-β-gal Staining Kit (C0602, Beyotime, China) as per the protocol. The transfected or untransfected NRK-52E cells were seeded at a density of 1 × 105 cells/well into a 24-well plate, with the addition of 1 mL DMEM/F-12. Thereafter, the cells were cultured at 37 °C with 5% CO2 to reach 70% cell confluence and exposed to D-gal for 24 h with or without cinnamaldehyde treatment for 2 h. Then, the cell supernatant was removed and replaced with 500 µL phosphate buffered saline (PBS; P7059, Sigma-Aldrich, USA). After the cells were washed with PBS twice, 250 µL fixing solution was added into each well and retained in the well for 15 min. Later, the cells were washed with PBS thrice, and incubated with 250 µL working solution (prepared in the darkness) in each well at 37 °C in CO2-free environment. Eight hours later, stained cells were observed using an optical microscopy. This experiment was performed three times.

Western blot

The proteins in rat kidney tissues and NRK-52E cells were extracted by RIPA Lysis and Extraction Buffer (89901, ThermoFisher, USA), and quantitated by a BCA Protein Assay Kit (23227, ThermoFisher, USA). The proteins were then separated by the sodium dodecyl sulfate-polyacrylamide gel electrophoresis gel (P0012A, Beyotime, Shanghai, China) and further transferred onto polyvinylidene fluoride membranes (FFP28, Beyotime, China) which were then blocked with 5% skim milk at room temperature for 1 h. The primary antibodies utilized to incubate the membranes at 4 °C overnight were those against PI3K (ab191606, 85 kDa, 1:1000, Abcam, Cambridge, MA, USA), p-PI3K (ab182651, 84 kDa, 1:1000, Abcam, USA), Akt (ab8805, 56 kDa, 1:500, Abcam, USA), p-Akt (ab38449, 56 kDa, 1:1000, Abcam, USA), LC3-I/LC3-II (ab48394, 19 kDa/17 kDa, 2 µg/ml, Abcam, USA), Beclin1 (ab62557, 52 kDa, 2 µg/ml, Abcam, USA) and GAPDH (ab8245, 36 kDa, 1:1000, Abcam, USA). After that, the secondary antibody Goat Anti-Rabbit IgG H&L (HRP) (ab205718, 1:2000, Abcam, USA) or Goat Anti-Mouse IgG H&L (HRP) (ab205719, 1:2000, Abcam, USA) was applied to cultivate the membranes at room temperature for 1 h. The obtained proteins in the membranes were photo-developed on an UVP BioSpectrum 810 Imaging System (UVP97062301, Fisher Scientific, USA) using Enhanced Chemiluminescent (ECL) Substrate Reagent Kit (WP20005, ThermoFisher, USA). The density of the protein bands was analyzed using Image J 1.52s (National Institutes of Health, Maryland, USA). Also, this experiment was conducted thrice.

Statistics

All the assessments were performed in triplicate. Some of the individual data were subjected to densitometric analyses. The data were expressed as means ± standard deviation (SD). The comparisons among the data from different groups were conducted using one-way analysis of variance (ANOVA). Statistically significant difference was recognized when p < 0.05.

Results

Cinnamaldehyde reversed D-gal-induced effects on kidney function indicators, histopathological changes, and PI3K/Akt pathway in rat kidney tissues

Kidney function indicators (BUN and Scr) and the protein and phosphorylation levels of PI3K and Akt were examined to mirror the degree of kidney function damage and the status of the affected pathway in D-gal-treated kidneys under cinnamaldehyde administration. The levels of BUN and Scr were notably elevated in D-gal group, compared to those in the control group (Figure 1(A,B), p < 0.001); while the levels of these indicators were evidently reduced in D-gal + CA group and D-gal + VE group when contrasted with those in D-gal group (Figure 1(A,B), p < 0.001). Next, the histopathological changes of rat kidney tissues were evaluated. According to Figure 1(C), the structure of the kidney in the control group was normal; the tissue pictures of D-gal group showed congestion of the capillaries between the glomeruli and the renal tubules, as well as the degeneration and necrosis of the renal tubules and the eosinophils in the renal tubules; and when compared with those in D-gal group, the tissue pictures of D-gal + CA group and D-gal + VE group displayed that the contours of the proximal and distal convoluted tubules were improved, the number of nuclear pyknosis was reduced, and the hyperemia was diminished. Subsequently, the PI3K/Akt signaling pathway, regarded as a regulatory target in protection against kidney injury, was also examined [39]. The ratio of p-P13K to P13K and the ratio of p-Akt to Akt were both lower in D-gal group than in the control group (p < 0.001), while the two ratios were elevated in D-gal + CA group and D-gal + VE group, compared to those in D-gal group (Figure 1(D–F), p <0.01, p < 0.001). These results collectively indicated that cinnamaldehyde and VE attenuated D-gal-induced impairment of kidney function and D-gal-induced inhibitory effects on the activation of the PI3K/Akt signaling pathway in rats.

Figure 1.

Cinnamaldehyde reversed D-gal-induced effects on kidney function indicators, histopathological changes, and the PI3K/Akt pathway in rat kidney tissues. (A,B). The levels of BUN and Scr in the serum of D-gal-treated rats with or without administration of cinnamaldehyde or VE were measured by an automatic biochemical analyzer. (C). The histopathological changes of rats were observed by hematoxylin-eosin staining (magnification: ×100 and ×400; scale: 50 µm and 15 µm). (D–F). The protein levels of phosphorylated (p)-PI3K, PI3K, p-Akt, and Akt in the kidney of D-gal-treated rats with or without administration of cinnamaldehyde or VE were analyzed by western blot, with GAPDH acting as a reference gene.  < 0.01; ***p or  < 0.001; *vs. Control; ^vs. D-gal (BUN: blood urea nitrogen, Scr: serum creatinine, D-gal: D-galactose, Cinnamaldehyde: CA, VE: vitamin E).

Cinnamaldehyde reversed D-gal-triggered autophagy activation and miR-155 upregulation in the kidney tubule tissues of rats

Autophagy is purposefully altered in the regulation of kidney diseases [40]. LC3B+ cells and Beclin1+ cells were pronouncedly multiplied by D-gal treatment in the kidney tubules, compared to those in the control group (p < 0.001; Figure 2(A–D)). However, under the co-administration of D-gal with cinnamaldehyde or VE, the kidney tubule tissues exhibited a smaller number of LC3B+ cells and Beclin1+ cells, compared to those only treated with D-gal (p < 0.01, p < 0.001; Figure 2(A–D)). In addition, the expressions of LC3B-I (called as LC3-I), LC3B-II (called as LC3-II) and Beclin1 in the kidney tubule tissues were detected by western blot. As shown in Figure 2(E–G), the expressions of LC3-II/LC3-I and Beclin1 in the tissues were upregulated by D-gal (p < 0.001), which, however, were then lessened by the administration of cinnamaldehyde or VE (Figure 2(E–G), p < 0.05, p < 0.01, p < 0.001). MiR-155 is found to be associated with kidney senescence [26]. Through qRT-PCR, a marked upregulation of miR-155 was uncovered in cells with the treatment of D-gal when compared with miR-155 level in cells without treatment (p < 0.001), while cinnamaldehyde and VE both eliminated this upregulation (Figure 2(H), p <0.001). These data implied that cinnamaldehyde and VE antagonized D-gal-induced promotive effects on autophagy in rats and this antagonistic effect may be related to miR-155.

Figure 2.

Cinnamaldehyde reversed D-gal-induced autophagy activation and miR-155 upregulation in the kidney tubule tissues of rats. (A–D). Representative photos of LC3B-positive cells and Beclin1-positive cells in the kidney tissues of D-gal-treated rats with or without administration of cinnamaldehyde or VE were presented via immunohistochemistry staining (magnification: ×100 and ×400; scale: 50 µm and 15 µm) (E–G). The expressions of LC3-I, LC3-II and Beclin1 in the kidney tubule tissues of D-gal-treated rats with or without administration of cinnamaldehyde or VE were analyzed by western blot, with GAPDH serving as an internal control. (H). The expression of miR-155 in the kidney tubule tissues of D-gal-treated rats with or without administration of cinnamaldehyde or VE was analyzed by qRT-PCR, with U6 serving as an internal control.  < 0.05;  < 0.01; ***p or  < 0.01; *vs. Control; ^vs. D-gal (D-gal: D-galactose, Cinnamaldehyde: CA, VE: vitamin E, qRT-PCR: quantitative reverse transcription polymerase chain reaction).

Cinnamaldehyde reversed D-gal-induced effects on cell viability, SA-β-gal activity, P13K/Akt signaling pathway, and autophagy in NRK-52E cells through regulating miR-155 expression

For exploring the mechanism of the anti-senescence effect of cinnamaldehyde in vitro, NRK-52E cells were selected, transfected with miR-155 mimic, and treated with D-gal and cinnamaldehyde. As shown in Figure 3(A), miR-155 expression was upregulated by D-gal (p < 0.001), but was reduced after co-treatment with D-gal and cinnamaldehyde (p < 0.001). The viability of NRK-52E cells was inhibited in D-gal group compared to that in the control group (p < 0.05, Figure 3(B)), while the viability of NRK-52E cells was strengthened in D-gal + CA group compared to that in D-gal group (p < 0.01, Figure 3(B)). Meanwhile, Figure 3(C) reflected that a great number of NRK-52E cells in D-gal group were positively stained with SA-β-gal compared to those in the control group, whilst the number of SA-β-gal+ cells was decreased in D-gal + CA group in contrast with that in D-gal group. The ratios of p-PI3K/PI3K and p-Akt/Akt were lower in D-gal group than in the control group (p < 0.001; Figure 3(D–F)), whereas the ratios of p-PI3K/PI3K and p-Akt/Akt were higher in D-gal + CA group than in D-gal group (p < 0.001; Figure 3(D–F)). Furthermore, the levels of LC3-II/LC3-I and Beclin1 were markedly upregulated in D-gal group compared to those in the control group, while the upregulated tendencies were reversed in D-gal + CA group (p < 0.001, p < 0.01; Figure 3(D,G,H)). These results collectively revealed that cinnamaldehyde antagonized D-gal-induced inhibitory effects on cell viability and the P13K/Akt signaling pathway, as well as D-gal-induced promotive effects on autophagy and senescence in NRK-52E cells. Furthermore, the role of miR-155 in cinnamaldehyde-induced antagonistic effects on nephrocyte senescence was investigated. In D-gal and cinnamaldehyde co-treated NRK-52E cells, all the above-mentioned cinnamaldehyde-induced antagonistic effects were reversed after transfection with miR-155 mimic as compared to those after transfection with miR-155 mimic control (p < 0.05, p < 0.01, p < 0.001; Figure 3(A–H)), which manifested that cinnamaldehyde downregulated miR-155 to impair D-gal-induced effects on cell viability, P13K/Akt signaling pathway and autophagy, thereby attenuating nephrocyte senescence.

Figure 3.

Cinnamaldehyde reversed D-gal-induced effects on viability, SA-β-gal activity, the P13K/Akt signaling pathway, and autophagy in NRK-52E cells through regulating miR-155 expression. (A). The expression of miR-155 in D-gal-treated NRK-52E cells with or without administration of cinnamaldehyde was evaluated by qRT-PCR, with U6 serving as an internal control. (B). The viability of D-gal-treated NRK-52E cells with or without administration of cinnamaldehyde was assessed by CCK-8 assay. (C). Representative photos of the senescence of D-gal-treated NRK-52E cells with or without administration of cinnamaldehyde were presented via SA-β-gal staining (magnification: ×200; scale: 100 µm). (D–H) The protein levels of p-PI3K, PI3K, p-Akt, Akt, LC3-II, LC3-I, and Beclin1 in D-gal-treated NRK-52E cells with or without administration of cinnamaldehyde were analyzed by Western blot, with GAPDH acting as a reference gene. *p or  < 0.05; or  < 0.01; ***p or or  < 0.001; * vs. Control; ^ vs. D-gal; # vs. D-gal + CA + miR-NC (D-gal: D-galactose, Cinnamaldehyde: CA, MC: mimic control, M: miR-155 mimic. qRT-PCR: quantitative reverse transcription polymerase chain reaction; CCK-8: cell counting kit-8).

The PI3K inhibitor reversed the effect of the cinnamaldehyde/miR-155 axis on cell viability, SA-β-gal activity, and autophagy in D-gal-induced NRK-52E cells

To further confirm whether the effect of cinnamaldehyde on D-gal-treated NRK-52E cells is fulfilled through regulating the PI3K/Akt signaling pathway-medicated autophagy, the inhibitor of PI3K (LY294002) was finally used (Figures 4 and 5). The cell viability was evaluated (Figure 4(A)), discovering that D-gal dwindled cell viability (p < 0.01), and cinnamaldehyde promoted while LY294002 inhibited the viability of D-gal-induced cells (p < 0.05, p < 0.001). Besides, the role of cinnamaldehyde in D-gal-induced cell viability was enhanced by miR-155 inhibitor and reversed by LY294002 (p < 0.05, p < 0.001) which also further offset the impact of miR-155 inhibitor on the viability of cells with co-treatment of D-gal and cinnamaldehyde (p < 0.01). As exhibited in Figure 4(B), D-gal augmented the number of SA-β-gal+ cells, which was reversed by cinnamaldehyde and enhanced by LY294002. In addition, the reversing effect of cinnamaldehyde was reinforced by miR-155 inhibitor and offset by LY294002 which also further weakened the impact of miR-155 inhibitor on the SA-β-gal+ cells co-treated with D-gal and cinnamaldehyde.

Figure 4.

The PI3K inhibitor reversed the effects of the cinnamaldehyde/miR-155 axis on the viability and SA-β-gal activity in D-gal-induced NRK-52E cells. (A). The viability of D-gal-treated NRK-52E cells with or without individual administration of cinnamaldehyde or LY294002 or co-administration of cinnamaldehyde and LY294002 was analyzed by CCK-8 assay. (B). Representative photos of the senescence of D-gal-treated NRK-52E cells with or without individual administration of cinnamaldehyde or LY294002 or co-administration of cinnamaldehyde and LY294002 were presented via SA-β-gal staining (magnification: ×200; scale: 200 µm). (C). The expression of miR-155 in D-gal-treated NRK-52E cells with or without individual administration of cinnamaldehyde or LY294002 or co-administration of cinnamaldehyde and LY294002 was analyzed by qRT-PCR, with U6 serving as an internal control. *p or or or  < 0.05; or or 0.01; ***p or or or  < 0.001; # vs. Control; * vs. D-gal; ^ vs. D-gal + CA; & vs. D-gal + LY294002; + vs. D-gal + CA + IC; vs. D-gal + CA + I. (D-gal: D-galactose, Cinnamaldehyde: CA, IC: inhibitor control, I: miR-155 inhibitor, qRT-PCR: quantitative reverse transcription polymerase chain reaction; CCK-8: cell counting kit-8).

Figure 5.

The PI3K inhibitor reversed the effect of the cinnamaldehyde/miR-155 axis on the autophagy in D-gal-induced NRK-52E cells. (A–E). The protein levels of p-PI3K, PI3K, p-Akt, Akt, LC3-II, LC3-I and Beclin1 in D-gal-treated NRK-52E cells with or without individual administration of cinnamaldehyde or LY294002 or co-administration of cinnamaldehyde and LY294002 were analyzed by Western blot, with GAPDH acting as a reference gene. or or &p < 0.05; or **p or or δδp < 0.01; ***p or or &&&p or or  < 0.001; # vs. Control; * vs. D-gal; ^ vs. D-gal + CA; & vs. D-gal + LY294002; + vs. D-gal + CA + IC; δ vs. D-gal + CA + I (D-gal: D-galactose, Cinnamaldehyde: CA, IC: inhibitor control, I: miR-155 inhibitor).

The expression of miR-155 (Figure 4(C)) was unveiled to be upregulated by D-gal (p < 0.01), and further downregulated by cinnamaldehyde (p < 0.001). MiR-155 inhibitor magnified the role of cinnamaldehyde in miR-155 expression of D-gal-induced cells (p < 0.001), while LY294002 barely affected miR-155 expression. In line with Figure 5(A–C), D-gal diminished ratios of p-PI3K/PI3K and p-Akt/Akt (p < 0.001), whereas cinnamaldehyde impaired and LY294002 strengthened the effect of D-gal (p < 0.01, p < 0.001). LY294002 reversed yet miR-155 inhibitor enhanced the role of cinnamaldehyde in the two ratios in D-gal-induced cells (p < 0.001). Also, LY294002 further reversed the role of miR-155 inhibitor in the ratios of p-PI3K/PI3K and p-Akt/Akt in cells co-treated with D-gal and cinnamaldehyde (p < 0.01). Finally, levels of LC3-II/LC3-I and Beclin1 were determined. As shown in Figure 5(A,D,E), D-gal increased the levels of LC3-II/LC3-I and Beclin1 (p < 0.01, p < 0.001). Cinnamaldehyde inhibited but LY294002 promoted the impacts of D-gal on those levels (p < 0.01, p < 0.001). Moreover, LY294002 reversed while miR-155 inhibitor enhanced the role of cinnamaldehyde in the levels of LC3-II/LC3-I and Beclin1 in D-gal-induced cells (p < 0.05, p < 0.01). Also, LY294002 further reversed the effect of miR-155 inhibitor on those levels in cells with the co-treatment of D-gal and cinnamaldehyde (p < 0.01). All these findings demonstrated that cinnamaldehyde regulated miR-155 expression and then activated the PI3K/Akt signaling pathway to inhibit autophagy in D-gal-induced NRK-52E cells.

Discussion

Prior studies have revealed that cinnamaldehyde antagonizes glomerular fibrosis and renal dysfunction in diabetic mice [19] and restores kidney enzyme activities and kidney function in metanil yellow-administrated rats [41], indicating a renoprotective role of cinnamaldehyde. Kidney senescence is a physiologic or pathological process that causes a higher risk for structural and functional changes in the kidney [1]. This study confirmed that cinnamaldehyde could exert similar anti-senescence and anti-injury effects on the kidney to almost the same extent as VE and it also generated an inhibitory effect on senescence-associated autophagy through downregulating miR-155 expression. Besides, the current study unearthed a novel signaling pathway, PI3K/Akt, the decreased phosphorylation of which could strengthen D-gal-induced autophagy as well as kidney senescence and injury in vivo and in vitro.

Senescence models are established in vitro and in vivo in the previous study mostly by administration with D-gal [29]. For example, D-gal triggers premature senescence of human lens epithelial cells by causing impairment to mitochondrial function and autophagy flux in vitro [42]. Also, D-gal is employed to create oxidative injuries in rats to simulate a natural aging process [43]. In Zheng’s study, subcutaneous injection of D-gal was performed to successfully establish a rat model of kidney senescence [35]. In this study, senescent kidneys were constructed in rats referring to the method in Zheng’s study and the in vitro senescent models were set up on NRK-53E cells by treatment with D-gal.

For evaluating the kidney damage caused by D-gal, the levels of BUN and Scr, generally accepted indices to indicate kidney injury, were measured. Significantly increased levels of BUN and SCr are perceived as hallmarks in severely injured kidney of patients with acute kidney injury (AKI) [44]. Similarly, a chronic kidney disease model of rats exhibits higher levels of BUN and SCr in the injured kidney, which surpasses the baseline for normal kidneys [45]. Consistent with the results above, this study demonstrated that D-gal treatment upregulated the levels of BUN and Scr, indicating that D-gal-induced senescent kidneys of rats present a manifestation of injury. Also, D-gal treatment causes a decrease in viability and an increase in SA-β-gal activity whose reduction indicates senescence of nephrocytes [35,46]. This study found that cinnamaldehyde and VE prominently suppressed the D-gal-induced upregulations of BUN and Scr, uncovered impaired kidney function, and restored viability and SA-β-gal activity of D-gal-induced NRK-53E cells, demonstrating protective effects of cinnamaldehyde and VE against D-gal-induced kidney senescence both in vitro and in vivo.

Furthermore, the AMPK-ULK1 signaling-mediated autophagy is discovered to be inhibited during VE-induced process of anti-senescence and anti-injury in the kidney [35]. This inhibited autophagy is identified by repressed conversion of LC3-II to LC3-I as well as Beclin1 level [35]. In the current study, an increased number of LC3B- and Beclin1-positive cells in kidney tubulars in vivo, and upregulations of LC3-II/LC3-I and Beclin1 in vitro were presented with D-gal treatment, implying an enhanced autophagy activity. Autophagy functions as a vital process conducing to maintaining kidney homeostasis and pathophysiology via the process where lysosomes are degraded to recycle damaged or excess organelles and protein aggregates [10, 47]. The absence of autophagy and proteasome pathways brings about development of glomerulosclerosis and proteinuria in senescing mice [48]. Therefore, D-gal treatment that stimulates senescence may trigger autophagy for renoprotection in response to aging injury. However, cinnamaldehyde and VE were discovered to inhibit the autophagy which had been promoted by D-gal treatment, contradicting the notion that autophagy induces renoprotection. Intriguingly, autophagy has been found to be capable of regulating oxidative stress and inflammation as well as its related signaling [49]. A study believed that reactive oxidative species can induce autophagy in the context of nutrient deprivation [50]. Under severe oxidative stress, the occurrence of lysosomal permeabilization can lead to autophagic impairment to further cause cell death [49]. Besides, research by Csiszar pointed out that vascular oxidative stress and inflammation occur generally during senescence [51]. Therefore, cinnamaldehyde and VE were surmised to protect nephrocytes from autophagic impairment caused by oxidative stress.

Activation of mTOR signaling pathway leads to the degeneration and recycling of intercellular components during autophagy [52]. The PI3K/Akt signaling pathway, acting as the upper stream of mTOR, participates in the modulation of autophagy, and kidney tubular epithelial inflammation can be induced by protease-activated receptor-2 via the PI3K/Akt/mTOR pathway activation-inhibited autophagy [53]. However, PI3K/Akt/mTOR pathway is activated by hydrogen sulfide to promote autophagy in LPS-mediated liver of chicken [54], and activation of Akt is found in accelerated senescence of mouse embryonic fibroblast cells with TXNIP deficiency under high glucose condition [55]. Also, a senescent kidney model of marmoset exhibits activated Akt pathway [56]. In contrast, this study proved that co-administration of cinnamaldehyde and VE reversed D-gal-induced inhibition on the phosphorylation of PI3K and Akt in vivo and in vitro, suggesting that the renoprotective effect of cinnamaldehyde and VE could be exerted via activating the PI3K/Akt signaling pathway.

MiRNAs, a class of small noncoding RNAs, are recorded to play modulatory roles in the kidney pathophysiological process including senescence [57]. A previous study has confirmed that the level of miR-155 is aberrantly increased during kidney senescence, and antiaging calorie restriction, a powerful antiaging regimen, can downregulate miR-155 level to restore PPARα expression and thus attenuate renal fibrosis during senescence [26]. This study unveiled that D-gal signally upregulated miR-155 level in the kidney, again implying that miR-155 is associated with kidney senescence. Moreover, by exogenously upregulating miR-155 expression to a higher level, it was discovered that cinnamaldehyde-induced restoration in viability, SA-β-gal activity, and PI3K/Akt signaling pathway was suspended, while cinnamaldehyde-induced inhibition on autophagy was eliminated. Besides, miR-155 downregulation had an opposite effect to miR-155 upregulation. Meanwhile, cinnamaldehyde was observed to reverse this senescence-associated miR-155 upregulation. Overall, the findings corroborated that miR-155 downregulation served as a key mechanism underlying cinnamaldehyde-induced protection against kidney senescence.

Ultimately, to ascertain whether the PI3K/Akt pathway activation-inhibited autophagy is causative to cinnamaldehyde-induced attenuation on kidney senescence, the inhibitor of PI3K was employed to treat the D-gal-induced nephrocyte senescence. Consequently, the data in this study verified the speculation by showing that the PI3K inhibitor reversed cinnamaldehyde- and miR-155 inhibitor-induced inhibition on autophagy in senescent nephrocytes.

In conclusion, this study demonstrated that cinnamaldehyde is pharmacologically similar to VE in our study, which attenuates D-gal-induced kidney senescence and injury by regulating miR-155 expression and then modulating PI3K/Akt signaling pathway activation-induced autophagy inhibition. This study proposes a novel therapeutic pathway for preventing senescence-associated kidney injury.