Nobiletin alleviates brain injury in uremic mice and inhibits indoxyl sulfate-induced neurotoxicity in HT22 cells through the phosphatidylinositol 3-kinase/protein kinase B signaling pathway

Animals

C57BL/6J male mice aged 8 weeks and with a body weight of 20–25 g were sourced from Beijing HFK Biotechnology Co., Ltd., Beijing, China. They were housed in an SPF environment with a 12-h light/dark cycle, appropriate humidity, temperature, and cleanliness, and had free access to food and water. After a week of acclimatization, the mice were randomly assigned to four groups (n = 8): control, NOB, DDP, and DDP + NOB. They were administered with DDP (500 mg/kg/d, G5388, Sigma, St. Louis, MI, USA) or NOB (100 mg/kg/d, HY-N0155, MedChemExpress, Monmouth Junction, NJ, USA) through intraperitoneal injection.^[22] Blood, kidney, and brain tissue samples were collected after 1 week of treatment, and blood serum creatinine (SCr), urea nitrogen, kidney injury molecule 1 (Kim-1), and neutrophil gelatinase-associated lipocalin (Nagl) levels were measured using an automated biochemical analyzer (Cobas 8000 c702, Roche, Basel, Switzerland). LY294002 was purchased from MedChem Express (HY-10108, Monmouth Junction, NJ, USA). At the end of the experiment, the mice were euthanized by cervical dislocation under deep anesthesia induced by the intraperitoneal injection of 50 mg/kg sodium pentobarbital (P3761; Sigma-Aldrich; Merck KGaA). The animal experiments were approved by the Animal Ethics Committee of Guangzhou Miers Biotechnology Co., Ltd. (IACUC-MIS20230042) and were conducted in accordance with the Institutional and International Guidelines for Animal Care and use to ensure the ethical treatment of animals.

Hematoxylin and eosin (H&E) staining and tissue damage score

Kidney tissues were treated with 4% paraformaldehyde for fixation, followed by dehydration and embedding in paraffin. Sections of 4 μm thickness were cut and incubated overnight at 37°C, followed by dewaxing and rehydration. The kidney tissues were observed by H&E staining (C0105S, Beyotime, Shanghai, China). Consecutive fields were evaluated at a ×100 magnification under an optical microscope (IXplore Standard, Olympus, Tokyo, Japan).

Cell culture and grouping

HT22 cells were purchased from Procell (Wuhan, China, CL-0697) and authenticated by short tandem repeat profiling to confirm their origin. Mycoplasma contamination testing was also performed to ensure the authenticity and contamination-free status of the cell cultures. The HT22 mouse hippocampal neuronal cell line was cultured in a medium containing 10% FBS (A5256701, Gibco, Grand Island, NY, USA) and 1% antibiotics (15140148, Gibco, Grand Island, NY, USA) at 37°C with 5% carbon dioxide. The HT22 cells were plated and allowed to adhere, followed by treatment with DDP at concentrations ranging from 1 mm to 70 mm or NOB for 24 h. The cells were cocultured with DDP and NOB for 24 h to evaluate the effects of NOB (Sigma, St. Louis, MO, USA) on neurons. LY294002 (a specific PI3K inhibitor) was purchased from MedChemExpress (HY-10108, Shanghai, China). To investigate the effects of NOB on DDP-induced HT-22 cells, we treated the cells with NOB at concentrations of 25, 50, and 100 μM. The experimental groups were as follows: the untreated control group, the NOB (50 μM) alone group, the DDP (5 μM) alone group, the DDP + NOB 25 μM group, the DDP + NOB 50 μM group, and the DDP + NOB 100 μM group, totaling six groups. In Experiment 4, the PI3K inhibitor LY294002 was employed to examine the underlying mechanism of PI3K. The corresponding cell groups in this experiment included the control group, the NOB (50 μM) alone group, the DDP (5 μM) alone group, the DDP + NOB (50 μM) group, and the DDP + NOB (50 μM) + LY294002 (10 μM) group, also consisting of six groups in total.

Cell viability assay

Cell viability was assessed using CKK-8 assay (C0038, Beyotime, Shanghai, China) after treatment with various concentrations of DDP (0–20 mm) or NOB (0–100 mm) following the manufacturer’s instructions. Cell viability was measured according to the instructions of the cell counting kit-8 (CCK-8) assay kit. In brief, 100 μL of cell suspension was seeded in a 96-well plate and cultured overnight at 37°C. The next day, the medium was replaced, and different drugs were added for coculturing for 24 h. The medium was replaced with a fresh one, and 10 μL of CCK-8 solution was added to each well. The plate was then incubated for 30 min in the incubator. Absorbance at 450 nm was measured using a microplate reader (Thermo Scientific^™ Multiskan^™ FC) to assess cell viability.

ROS detection

Cell apoptosis was determined using the ROS assay kit (S0033S, Beyotime, Shanghai, China). The cells were pre-incubated at 37°C with 10 μM Dichlorodihydrofluorescein diacetate, followed by suspension and incubation in the dark for 20 min. After incubation, the cells were observed by fluorescence microscopy (FV4000, Olympus, Tokyo, Japan).

Western blot

Tissues or cells were homogenized in RIPA buffer (P0013 C, Beyotime, Shanghai, China) containing 1 mM PMSF (ST 507, Beyotime, Shanghai, China). After centrifugation (13,000 rpm, 10 min, 4°C), the supernatant was collected for Western blot analysis. The total protein concentration was measured using a BCA assay kit (P0012, Beyotime, Shanghai, China). In brief, 20 μg of proteins per lane were subjected to electrophoresis on a 10–15% SDS-PAGE gel and then transferred to a PVDF membrane (IPVH00010, Merck Millipore, Billerica, MA, USA). The equipment for electrophoresis and transfer was obtained from Bio-Rad (Hercules, CA, USA). The membrane was then blocked using 5% nonfat milk at room temperature for 1 h and subsequently incubated overnight at 4°C with primary antibodies targeting α-tubulin (2144), Bcl2 (15071), poly ADP-ribose polymerase (PARP) (9532), PI3K (4292), pyruvate dehydrogenase kinase 1 (PDK1) (3062), Akt (4691), p-PI3K (17366), p-PDK1 (3438), p-Akt (4060), and Bax (5023) (1:1000 each, Cell Signaling Technology, Danvers, MA, USA). Afterward, the membrane was incubated with HRP-conjugated anti-rabbit Immunoglobulin G (IgG) secondary antibody (1:2000, A0208, Beyotime, Shanghai, China), and HRP-conjugated anti-mouse IgG secondary antibody (1:2000, A0216, Beyotime, Shanghai, China). An appropriate amount of ECL (P0018S, Beyotime, Shanghai, China) (prepared by mixing Solution A and Solution B in a 1:1 volume ratio) was applied to the PVDF membrane to ensure that it was completely covered with the substrate. The membrane was then placed into a ChemiDoc XRS + chemiluminescence imaging system (Bio-Rad, Hercules, CA, USA) for detection. Semi-quantitative analysis of band intensity was performed using ImageJ software (version 1.5f, NIH, Maryland, USA).

Statistical analysis

The experimental data were analyzed using the Statistical Package for the Social Sciences (SPSS) 20.0 software (SPSS Inc., Chicago, IL, USA). All experiments were conducted in at least three replicates, and data from animal and cell model experiments were expressed as the mean ± Standard deviation. The normality of the data was assessed using the Shapiro–Wilk test. If the data in each group were normally distributed or approximately normal with homogeneous variances, comparisons between the two groups were performed using the independent samples t-test. For comparisons involving three or more groups, one-way analysis of variance was used. P < 0.05 was considered statistically significant.

NOB attenuates DDP-induced brain damage in uremic mice

To verify that DDP can induce uremia in mice, we first observed the overall condition of the mice. The mice in the DDP group exhibited significant weight loss, lethargy, rapid breathing, decreased body temperature, refusal to eat or drink, disheveled and dull fur, and minimal response to touch. By contrast, the mice in the DDP + NOB group showed reduced appetite, depressive behavior, and a sluggish response to touch, indicating that NOB can improve the overall health status of the mice. The changes in body weight are shown in Figure 1a. Throughout the experiment, the mice in the control and NOB groups tended to gain weight, and those in the DDP group tended to lose weight. In particular, the DDP + NOB group experienced minimal weight loss. The kidney coefficients in the mice are presented in Figure 1b. Compared with the control group, the DDP group showed a significant reduction in kidney coefficients (P < 0.001). However, the kidney coefficients of the DDP + NOB group were significantly higher than those of the DDP group, suggesting that NOB can alleviate the DDP-induced reduction in kidney coefficient in mice (P < 0.01).

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Figure 1:

NOB alleviates DDP-induced brain damage in uremic mice. (a) Body weight of mice. (b) Kidney coefficient of mice. (c-f) Serum levels of BUN, SCr, Kim-1, and Nagl in mice. (g) Representative images of hematoxylin & eosin staining morphological analysis of kidney tissue, showing renal injury features including cellular debris, tubular necrosis, and inflammatory cells ×100. Scale bar = 100 μm (h-k) WB analysis and quantification of Bcl-2, Bax, and PARP protein expression in mouse brain tissue. n = 3; ^✶✶✶P < 0.001, ^✶✶P < 0.01 versus Control, ###P < 0.001, ^##P < 0.01, ^#P < 0.05 versus DDP. The bar represents mean ± S.D. NOB: Nobiletin, BUN: Blood urea nitrogen, SCr: Serum creatinine, Bcl2: B-cell lymphoma-2, Bax: Bcl-2-associated X protein, PARP: Poly ADP-ribose polymerase, DDP: Cisplatin, WB: Western blot, S.D.: Standard deviation.

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Next, we assessed kidney function by measuring SCr, blood urea nitrogen (BUN), Kim-1, and Nagl levels. As shown in Figures 1c-f, the DDP-treated mice exhibited significantly higher levels of BUN in plasma and SCr, Kim-1, and Nagl in serum compared with the control group, indicating their impaired renal function due to DDP (P < 0.001). By contrast, the DDP + NOB group showed significantly lower levels of BUN, SCr, Kim-1, and Nagl than the DDP group, suggesting that NOB improves kidney function (P < 0.001). Examination of kidney tissues with H&E staining indicated that DDP treatment resulted in damage to the tubular interstitium, injury to tubular epithelial cells, and interstitial inflammation, all of which notably improved with NOB treatment [Figure 1g]. The above mouse renal injury phenotype results showed that DDP can induce uremia in mice, and NOB can improve the kidney injury of uremic mice. To assess cellular damage in the brains of uremic mice, we evaluated the expression of Bcl-2, Bax, and PARP (85 kDa) in brain tissues. Compared with the control group, the DDP-treated mice exhibited higher levels of Bax (P < 0.001) and PARP (P < 0.001) and lower levels of Bcl-2 (P < 0.01) in brain tissues [Figures 1h-k]. These levels indicated an increase in apoptosis, which was alleviated by NOB treatment (P < 0.05). Hence, brain cell damage in uremic mice possibly occurs through the enhancement of the apoptotic pathway, with NOB treatment potentially reducing this damage by influencing the expression of Bcl-2, Bax, and PARP.

Activation of PI3K/Akt signaling pathway by NOB

The regulation of apoptosis involves several signaling pathways, including the PI3K/Akt pathway that is involved in memory, learning, and the survival, differentiation, and axonal growth of neurons in the brain.^[23,24] To assess whether NOB alleviates DDP-induced brain tissue damage through the PI3K/Akt signaling pathway, we studied the activation of this pathway by conducting Western blot analysis. This method allowed us to measure the expression levels of key proteins involved in the PI3K/Akt signaling cascade, providing insights into the molecular mechanisms underlying the protective effects of NOB. Compared with that in the control group, DDP treatment caused a decline in p-PI3K (P < 0.01), p-PDK1 (P < 0.01), and p-Akt (P < 0.001) levels, but NOB treatment significantly restored their expression (P < 0.05) [Figures 2a-d]. These findings demonstrated that the protective effects of NOB against DDP-induced brain damage in uremic mice may be associated with the PI3K/Akt signaling pathway.

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Figure 2:

NOB activates the PI3K/Akt signaling pathway. (a-d) WB analysis and quantification of p-PI3K, p-PDK1, and p-Akt protein expression in mouse kidney tissue. n = 3; ^✶✶✶P < 0.001, ^✶✶P < 0.01 versus Control, ^###P < 0.001, ^##P < 0.01, ^#P < 0.05 versus DDP. The bar represents mean ± S.D. NOB: Nobiletin, PI3K: Phosphatidylinositol 3-kinase, PDK1: Pyruvate dehydrogenase kinase 1, Akt: Protein kinase B, DDP: Cisplatin, WB: Western blot.

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Anti-apoptotic effect of NOB on DDP-induced HT22 cells

To further investigate the mechanism by which NOB ameliorates brain damage in uremic mice, we treated HT22 mouse hippocampal neurons with DDP to simulate uremia-induced brain damage in vitro. HT22 cells were exposed to DDP at varying concentrations (0–20 mm) for 24 h, and cell viability was subsequently evaluated. Compared with that in the control, high DDP concentrations significantly reduced cell viability after 24 h (P < 0.001) [Figure 3a]. No significant differences in cell viability were observed when the HT22 cells were treated with NOB at concentrations ranging from 5 μM to 100 μM, indicating that NOB is nontoxic to these cells [Figure 3b]. When NOB was introduced to the DDP-treated group, their cell viability was markedly improved compared with that of the DDP-only group (P < 0.05), indicating that NOB can mitigate the toxicity caused by DDP in HT22 cells [Figure 3c].

We also examined the effects of NOB on DDP-induced apoptosis by measuring the expression of apoptosis-related proteins Bcl-2, Bax, and PARP (85 kDa). Compared with the control group, the group treated with DDP exhibited higher levels of Bax and PARP and lower levels of Bcl-2 (P < 0.001) [Figure 3d-g]. In contrast, NOB treatment markedly improved these apoptotic markers (P < 0.05) [Figure 3d-g]. Considering that ROS regulates apoptosis through oxidative stress, we measured the intracellular ROS levels. The findings revealed that NOB effectively decreased the oxidative stress induced by DDP (P < 0.01) [Figure 3h and i], verifying its potential to mitigate the harm to neurons resulting from uremic toxins.

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Figure 3:

NOB exhibits anti-apoptotic effects on DDP-induced HT22 cells. (a and b) CCK-8 assay evaluating the impact of DDP and NOB on HT22 cell viability. ^✶✶✶P < 0.0001, ^✶✶P < 0.001, ^✶P < 0.01 versus 0 (c) CCK-8 assay assessing the effect of various concentrations of NOB on DDP-induced HT22 cell viability. (d-g) WB analysis and quantification of Bcl-2, Bax, and PARP protein expression in HT22 cells across different groups. (h-i) DCFH-DA probe measurement of intracellular ROS levels in different groups. ×100 Scale bar = 100 μm. n = 3; ^✶✶✶P < 0.001, ^✶✶P < 0.01, ^✶P < 0.05 versus DDP; ^###P < 0.001 versus Control. The bar represents mean ± S.D. NOB: Nobiletin, DDP: Cisplatin, CCK-8: Cell counting kit-8, Bcl2: B-cell lymphoma-2, Bax: Bcl-2-associated X protein, PARP: Poly ADP-ribose polymerase, WB: Western blot, DCFH-DA: Dichlorodihydrofluorescein diacetate, ROS: Reactive oxygen species.

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NOB inhibits apoptosis through the PI3K/Akt signaling pathway

We further explored the mechanism by which NOB mitigates DDP toxicity by examining the expression changes of p-PI3K, p-PDK1, and p-Akt through WB analysis. Compared with those in the control group, the relative levels of p-PI3K, p-PDK1, and p-Akt were reduced by DDP but significantly restored by NOB treatment (P < 0.05) [Figure 4a-d]. To further confirm whether NOB protects against DDP-induced neurotoxicity by activating the PI3K/Akt signaling pathway, we treated HT22 cells with NOB and LY294002 (10 μM), a specific inhibitor of the PI3K/Akt pathway. Figures 4e-h show that after treatment with LY294002, the expression of the PI3K/Akt pathway was reduced (P < 0.05). According to the apoptotic-related proteins and ROS levels, the apoptosis inhibition induced by NOB was partially reversed after LY294002 treatment (P < 0.05) [Figure 4i-n]. Similarly, the CCK-8 assay showed that compared with the DDP + NOB group, the DDP + NOB + LY group exhibits decreased cell viability (P < 0.05) [Figure 4o]. These experimental results suggested that the protective effect of NOB on HT22 cells is partially blocked by the PI3K/Akt pathway inhibitor, indicating that NOB may alleviate DDP-induced HT22 cell apoptosis through the PI3K/Akt signaling pathway and exert a protective effect.

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Figure 4:

NOB inhibits apoptosis through the PI3K/Akt signaling pathway. (a-h) WB detection and quantitative analysis of the protein expression of p-PI3K, p-PDK1, and p-Akt in different groups of HT22 cells. (i-l) WB detection and quantitative analysis of the protein expression of Bcl2, Bax, and PARP in different groups of HT22 cells. (m and n) DCFH-DA probe measurement of intracellular ROS levels in different groups. ×100 Scale bar = 100 μm (o) CCK-8 assay assessing the effect of various LY concentrations on HT22 cell viability. n = 3; ^✶✶✶P < 0.001, ^✶✶P < 0.01, ^✶P < 0.05 versus DDP; ^##P < 0.01, #P < 0.05 versus DDP + NOB. The bar represents mean ± S.D. NOB: Nobiletin, DDP: Cisplatin, PI3K: Phosphatidylinositol 3-kinase, PDK1: Pyruvate dehydrogenase kinase 1, Akt: Protein kinase B, Bcl2: B-cell lymphoma-2, Bax: Bcl-2-associated X protein, PARP: Poly ADP-ribose polymerase, WB: Western blot, DCFH-DA: Dichlorodihydrofluorescein diacetate, ROS: Reactive oxygen species, LY: LY294002, CCK-8: Cell counting kit-8.

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