2 Department of Pharmacology of Chinese Materia Medica, China Pharmaceutical University, Nanjing 211198, China
Diabetes is a chronic, metabolic disease characterized by elevated level of blood glucose, which causes serious damage to heart, blood vessels, eyes, kidneys, and nerves [ref]. Diabetic nephropathy (DN) is one of the major microvascular complications of diabetes and the most common cause of end-stage renal disease affecting approximately 20% to 40% of diabetic patients . Oxidative stress plays an important role in the initiation and development of diabetic complications, including the DN . NADPH oxidase is one of the major sources for cellular reactive oxygen species (ROS), which can convert molecular oxygen to superoxide. The NADPH oxidase isoforms predominantly expressed in the renal system are Nox1, Nox2, and Nox4 [3-5]. Among them, Nox4 is the most abundant Nox isoform expressed in renal tubules, renal fibroblasts, glomerular mesangial cells, and podocytes [5-9]. Evidence suggests that Nox4 dehydrogenase domain exists in a conformation which allows the spontaneous transfer of electrons from NADPH to FAD , and Nox4-dependent ROS generation mediates glomerular hypertrophy and mesangial matrix accumulation . These events suggest that suppression of NADPH oxidases would be beneficial to the improvement of renal function.
The mitochondrion is a double membrane-bound organelle found in most eukaryotic cells and described as "the powerhouse of the cell" . Mitochondria are the major sites for ROS production and are particularly susceptible to oxidative damage . Enhanced oxidative phosphorylation in the mitochondrial electron transport chain increases the leakage of electrons, promoting ROS production through NADPH oxidases pathway, which in turn uncouples proteins and potentiates proton leakage through the adenine nucleotide translocator, ultimately leading to mitochondrial membrane injury through exacerbating oxidative stress . Glucose phosphorylation is catalyzed by the enzyme hexokinase (HK), of which four isozymes are present within mammalian tissue. HKⅡ is mainly located at the mitochondrial outer membrane , and mitochondrial bound hexokinase Ⅱ (mtHKⅡ) plays a major role in stabilizing mitochondrial membrane potential and controlling ROS production . PH values is important for mtHKⅡ dissociation, and when pH values < 7.0, the increase of HKⅡ dissociation induces the change of conformation of mitochondrial permeability transition pore and triggers mitochondrial ROS generation . With mtHKⅡ detachment and forward cycle of ROS production, the defect in mitochondrial integrity is responsible for mitochondrial malfunction and exacerbated oxidative stress.
Bombax ceiba L. (Bombacaceae) is widely distributed and cultivated in temperate Asia, possesses a strong ethnobotanical background, and extensively used as a famous folk medicine in the treatment of a wide range of diseases . In a previous research , we found that standard ethanol extract of Bombax ceiba leaves (BCE) was able to regulate glucose homeostasis and thus demonstrated its anti-oxidative activities in type 2 diabetic rats induces by high-fat diet and streptozotocin (STZ). Therefore, we assumed the potential of BCE for the treatment of diabetic complications. Considering mangiferin as the main composition of BCE, we supposed that it plays a major role in the efficacy of BCE. In the current study, the effects of BCE and its main constituent mangiferin on the protection of DN was investigated in vitro and in vivo, focusing on their regulatory effects on oxidative stress and mitochondrial function.Materials and Methods Materials
Mangiferin (purity, ≥ 98%) was purchased from Nanjing Guangrun Medical Technology Co., Ltd. (Nanjing, China). The standard Bombax ceiba leaves ethanol powder extract (BCE) was obtained as previously reported . HPLC-DAD analysis method was applied to determine the contents of mangiferin in BCE. The amount of mangiferin in BCE was determined to be 65.2% (W/W). Diphenyleneiodonium chloride (DPI) was purchased from Sigma Co. (St Louis, MO, USA). Metformin (MET) was provided by Beyotime Institute of Biotechnology (Shanghai, China). These agents were dissolved in dimethyl sulfoxide (DMSO) as a stock solution and the final working concentration of DMSO was < 0.1% (V/V). Streptozotocin (STZ) was purchased from Sigma Co.. The kits for measurement of fasting blood glucose (FBG), 24-h urinary albumin, serum creatinine (Scr), and blood urea nitrogen (BUN) were purchased from Jiancheng Bioengineering Institute (Nanjing, China).Experimental animals
ICR mice (male, five weeks old) were purchased from the Comparative Medicine Centre of Yangzhou University and were housed in colony cages in 12 h light/ 12 h dark cycles under standard room temperature (22 ± 2 ℃). The animal protocol was approved by Animal Ethics Committee of School of Chinese Materia Medica, China Pharmaceutical University. All the mice were allowed to access standard diet and tap water ad libitum.
The mice were injected intraperitoneally with 50 mg/kg body weight of STZ in 0.1 mol·L-1 citrate buffer (pH 4.4) for 5 consecutive days (after an overnight fast). The fasting blood glucose (FBG) was measured five days after the last STZ injection. The mice with the FBG of more than 11.1 mmol·L-1 were used for further study. BCE (40, 80, and 120 mg·kg-1), mangiferin (50 mg·kg-1) or metformin (200 mg·kg-1) were given daily by oral gavage to the animals in the experimental groups for 16 weeks. The mice in the control group were administered with the same volume of saline. At the end of experiment, the mice were placed in metabolic cages for 24 h to collect urine. Animals were killed on indicated time points, followed by collecting blood samples from retinal venous plexus. The fasting blood glucose, creatinine, and blood urea nitrogen were assayed using commercial enzyme kits. Besides, kidneys were processed for histology and immunostaining.Cell culture
SV40 MES 13 mouse mesangial (MES) cell line was purchased from Shanghai Baili Medical Technology Co., Ltd. (Shanghai, China) and maintained in Minimum Essential Medium supplemented with 10% FBS and antibiotics (100 U·mL-1 of penicillin G and 100 μg·mL-1 of streptomycin sulphate). Cultured cells were maintained at 37 ℃ in a humidified atmosphere of 5% CO2. After reaching 70%-80% confluence, the cells were washed with PBS twice and growth arrested with 0.5% FBS for 48 h, and then incubated with either normal glucose (5.5 mmol·L-1) or high glucose (25 mmol·L-1) for the indicated times.ROS and mitochondrial membrane potential (Δψm) analysis
For intracellular ROS detection, the MES cells were cultured to 80% confluency and treated with BCE (0.65 μg·mL-1), MGF (10 μmol·L-1) or DPI (1 μmol·L-1) in either 5.5 or 25 mmol·L-1 of glucose with 0.5% FBS for 48 h. The cells were washed with PBS twice and then kept with ROS specific fluorescent probe dye dihydroethidium (Beyotime Institute of Biotechnology, Shanghai, China) for 0.5 h at 37 ℃ in the dark. After washing, the cells were fixed in 4% paraformaldehyde (V/V) for 5 min at 4 ℃ and kept with DAPI (Sigma, St. Louis, MO, USA) for 5 min at 37 ℃ in the dark. At last, images were viewed by confocal scanning microscopy (Zeiss LSM 700) after cells were washed, and the fluorescence values were measured with Thermo Scientific Varioskan Flash microplate reader (excitation/emission at 510 nm/580 nm).
For Mitochondrial membrane potential (Δψm) assay, the MES cells were incubated with 500 nmol·L-1 of potentiometric dye TMRE (Abcam, Cambridge, MA, USA) for 30 min at 37 ℃ in the dark, washed with PBS, and fixed in 4% paraformaldehyde (V/V) for 5 min at 4 ℃. The cells (≥ 90 cells per dish) from each sample were viewed in several random fields by confocal scanning microscopy.Apoptosis analysis
Confluent cells were treated with BCE (0.65 μg·mL-1), MGF (10 μmol·L-1) or DPI (1 μmol·L-1) for 48 h, with or without 25 mmol·L-1 of glucose. After incubation, the cells were treated with 0.5% trypsin for 2 min at 37 ℃. After enrichment, centrifugation and re-suspension in ice-cold PBS, the cells were stained with Annexin V-FITC Apoptosis Detection Kit (KeyGEN Biotech Co., Ltd. Nanjing, China). Finally, 10 000 cells were counted for each sample and cellular fluorescence was imaged by flow cytometry analysis with a FACS Calibur Flow Cytometer (BD Biosciences, USA), according to manufacturer's instructions.Western blot analysis
For protein analysis, the cells were lysed in ice-cold RIPA buffer, incubated for 45 min, and then cleared by centrifugation at 12 000 g for 20 min at 4 ℃. The samples with equal amount of protein were electrophoresed on SDS-PAGE and transferred to polyvinylidene difluoride membranes, and the membranes were blocked at room temperature for 2 h. For immuno-blotting, the primary antibodies, including anti-cleaved caspase-3 (Cell Signaling Technology, Asp175), anti-caspase-3 (Bioworld Technology, BS5644), and anti-GAPDH (Bioworld Technology, AP0063), were used at 4 ℃ overnight, followed by incubating with the secondary antibody Goat Anti-Rabbit IgG (H + L) HRP (Bioworld, BS13278) at room temperature for 2 h or at 4 ℃ overnight. An enhanced ECL kit was used to detect signals and Image-Pro Plus 6.0 (IPP 6.0) software was used for densitometry.Immunofluorescence
The MES cells cultured on 35 mm glass bottom dish were washed with PBS, kept in the dark with Mito-Tracker Red (Beyotime Inc.) for 0.5 h at 37 ℃, washed with PBS, and then fixed with 4% paraformaldehyde for 20 min. The cells were permeabilized with 0.2% Triton X-100 and incubated with 5% BSA to block non-specific staining, and then incubated with specific primary antibodies (anti-Nox4, anti-HKⅡ) overnight at 4 ℃ in a humidified chamber. After washing, the cells were incubated with Alexa Fluor 488-labeled goat anti-rabbit IgG (H + L) antibody (Beyotime Inc.) for 1 h at 37 ℃, washed with PBS twice, and incubated in DAPI for 5 min at 37 ℃. The cells were mounted in mounting medium and visualized by confocal scanning microscopy.Immunohistochemical analysis
For immunohistochemical analysis, the kidney tissues were preserved by 4% paraformaldehyde and blocked by paraffin and cut to 5-μm sections for further analysis. The sections were heated in 10 mmol·L-1 sodium citrate buffer (pH 6.0) to retrieve antigens and the slides were incubated to reduce nonspecific background staining as endogenous peroxidase. The slides were then cooled after boiling in citrate buffer solution for 10 min and washed with PBS twice. Primary antibodies Nox4 and cleaved-caspase-3 were diluted for application at the incubation of tissues, followed by the secondary antibody. Elivison two-step method was performed for the immunohistochemical staining and microscopic assessments were taken under an Olympus DX45 microscope.Statistical analysis
The experimental results are expressed as means ± SD (standard deviation), and each experiment was performed a minimum of three times. The statistical analyses were performed using one-way analysis of variance (ANOVA), and P < 0.05 was considered statistically significant.Results BCE and MGF ameliorate the renal damage in STZ-induced DN mice
In order to evaluate the effects of BCE and MGF on DN damage, fasting blood glucose, serum creatinine, blood urea nitrogen and 24-h urine protein levels were measured. As illustrated in Fig. 1, BCE (80, 120 mg·kg-1) could significantly decrease FBG, Scr, BUN, and 24-h urine protein (P < 0.05). The treatment with MGF (50 mg·kg-1) and MET (200 mg·kg-1) were in accordance with the effect of BCE. The results suggested that BCE and MGF could improve the symptom of the DN mice and might have a protective effect on DN.
Histological studies (H & E staining) on STZ-induced diabetic kidneys showed increased glomerular size, congestion and hydropic changes in the proximal convoluted tubules (Fig. 2). These alterations were effectively prevented by oral administration of BCE and MGF. These results suggested the protective action of BCE and MGF in diabetic renal injury.
Immunohistochemical staining examination showed the enhanced protein expressions for Nox4 (Fig. 3A) and cleaved caspase-3 (Fig. 3B) in kidney tissues of the diabetic mice, whereas oral administration of BCE (80 and 120 mg·kg-1) or MGF (50 mg·kg-1) effectively attenuated the NOx4 and cleaved caspase-3 expression. As a positive control, anti-diabetic agent metformin also reduced Nox4 and cleaved caspase-3 expression in the kidneys. These results proved that BCE could down-regulate Nox4 protein expression and inhibit caspase-3 activity in the DN mice.
As Nox4 mediates superoxide generation, we assayed ROS production in MES cells with fluorescent probe dye dihydroethidium (DHE) specific for superoxide. High glucose stimulation induced intracellular ROS production, as indicated by the increase in red fluorescence, while BCE and MGF treatment reduced the red fluorescence intensity (Fig. 4). Meanwhile, ROS inhibitor DPI also reduced intracellular ROS generation demonstrated the effect on the regulation of oxidative stress and the relevance between BCE and MGF.
As intracellular ROS generation relies on the expression of NADPH oxidases, we investigated the Nox4 expression through immunofluorescence with confocal scanning microscopy. The Nox4 protein expression in the membrane was increased in response to high glucose stimulation (Fig. 5), while pretreatment of the MES cells with BCE suppressed the Nox4 induction. Similarly, high glucose induced alternations were reversed by MGF and ROS inhibitor DPI.
HKⅡ dissociation from mitochondria can trigger mitochondrial ROS generation. As pH value is important for mtHKⅡ dissociation, we first investigated the lactic acid, which influences pH values in cell culture medium, and found that high glucose stimulation induced lactic acid production, while BCE and MGF ameliorated this condition (Fig. 6A). Consistent with this, we observed mtHKⅡ dissociation from mitochondria in response to high glucose stimulation and secreted increase (Fig. 6B), while pretreatment of the MES cells with BCE and MGF suppressed HKⅡ dissociation from mitochondria membrane and lactic acid secretion.
Considering that mitochondria-dependent pathway is one of the mechanisms involved in apoptosis, we observed the influence of BCE and MGF on mitochondria-dependent cell apoptosis in the treated cells. We first assayed mitochondria membrane potential (Δψm) using TMRE staining. As shown in Fig. 7A, Δψm was greatly degraded by high glucose stimulation, backed by the reduction in red fluorescence, while BCE and MGF effectively prevented the collapse of Δψm as evidenced by increased red fluorescence intensity. Moreover, BCE also reduced high glucose induced cleaved caspase-3 activity (Fig. 7B). Consistent with this finding, flow cytometry analysis revealed that BCE and MGF effectively reduced apoptosis in the MES cells, demonstrating its protection of cell survival from high glucose insults (Fig. 7C).
Increasing evidence indicates a relationship between oxidative stress and diabetic nephropathy . In hyperglycemia, mitochondrial ROS generation can be drastically increased, causing damages to mitochondria membrane potential, which can trigger early events of cells apoptosis [20-21].
In the present study, a significant increase in plasma glucose level, BUN, creatinine, and urinary albumin indicated the progressive nephrotoxicity in STZ-induced diabetic mice. BCE and MGF, on the other hand, effectively reversed this pathophysiology by lowering plasma levels of glucose, BUN, and creatinine and urinary albumin level, and altering diabetes-induced oxidative stress in kidneys. On the other hand, although BCE demonstrated effective tendency following increased doses, the efficiency between 80 and 120 mg·kg-1 was not different; the possible cause might be relative to the narrow selection of the doses in the present study.
The primary catalytic function of NADPH oxidase is ROS generation, and this property determines its crucial role in oxidative stress. In view of the contribution of NADPH oxidase activation and ROS production to mitochondrial malfunction , the maintenance of NADPH oxidase protein expression is one of the key steps to induce mitochondrial dysfunctions. Even though the mechanisms by which high glucose induces Nox4 protein expression remain to be elucidated, it is reported that Nox4 protein expression and ROS generation are reversed by insulin treatment , confirming that hyperglycemia and hyperglycemia-induced mediators most likely account for these effects . Our work also proved that, in cultured mesangial cells, high glucose could trigger a rapid upregulation of Nox4 protein levels, associated with an increase in cellular ROS production. Evidence showed that Nox4-dependent ROS generation mediates glomerular hypertrophy and mesangial matrix accumulation . For this, we wondered if BCE and MGF contributed to suppressing NADPH oxidase activation and subsequent ROS production. In the present study, our in vitro and in vivo experiments demonstrated that BCE and MGF inhibited Nox4 protein expression markedly.
Although the mechanism is different from NADPH, mitochondrial bound hexokinase Ⅱ (mtHKⅡ) also plays a major role in stabilizing mitochondrial membrane potential and ROS production . HKⅡ expression levels and localization are highly regulated by physiology, hormones, and metabolic state , and acute hyperglycaemia has been shown to induce the detachment of HK from mitochondria [16, 25]. Hexokinase binds to mitochondria and regulate mitochondrial voltage-dependent anion channel, controlling mitochondrial pore formation [26-28]. Prevention of conformational change in molecular mPTP regulation complex can stabilize mitochondrial membrane potential and reduce ROS production . The present study proved that high glucose stimulation up-regulated lactic acid content and triggered mtHKⅡ dissociation, while BCE and MGF suppressed HKⅡ dissociation from mitochondria membrane, which demonstrated their antioxidant effect by reducing HKⅡ-related ROS generation.
Collectively, our work demonstrated that BCE and MGF prevented mitochondrial membrane potential collapse and ROS formation in the mesangial cells by regulation of hexokinase Ⅱ binding and Nox4 oxidase signaling. The present results from the studies with high glucose induced mesangial cells and STZ-induced DN mice suggested that BCE had the protective effects on diabetic caused kidney injury and that MGF, as the main component of BCE, might be a beneficial agent for the prevention and treatment of DN.
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