Chinese Journal of Natural Medicines  2017, Vol. 15Issue (8): 597-605  DOI: 10.3724/SP.J.1009.2017.00597
0

Cite this article as: 

XU Guang-Kai, SUN Chen-Yu, QIN Xiao-Ying, HAN Yu, LI Yi, XIE Guo-Yong, Qin Min-Jian. Effects of ethanol extract of Bombax ceiba leaves and its main constituent mangiferin on diabetic nephropathy in mice[J]. Chinese Journal of Natural Medicines, 2017, 15(8): 597-605.
[Copy]

Research funding

This work was supported by the Preponderant Discipline Construction Project for Traditional Chinese Medicines of Jiangsu Province

Corresponding author

Qin Min-Jian, Tel: 86-25-86185130, Fax: 86-25-8530-1528, E-mail: minjianqin@163.com

Article history

Received on: 17-Sep.-2016
Available online: 20 Aug., 2017
Effects of ethanol extract of Bombax ceiba leaves and its main constituent mangiferin on diabetic nephropathy in mice
XU Guang-Kai1 , SUN Chen-Yu1 , QIN Xiao-Ying2 , HAN Yu1 , LI Yi1 , XIE Guo-Yong1 , Qin Min-Jian1     
1 Department of Resources Science of Traditional Chinese Medicines, State Key Laboratory of Natural Medicines, China Pharmaceutical University, Nanjing 211198, China;
2 Department of Pharmacology of Chinese Materia Medica, China Pharmaceutical University, Nanjing 211198, China
[Abstract]: The present study was designed to explore the mechanism by which ethanol extract of Bombax ceiba leaves (BCE) and its main constituent mangiferin (MGF) affect diabetic nephropathy by combating oxidative stress. Oral administration of BCE and MGF to normal and streptozotocin (STZ)-induced diabetic mice were carried out. Fasting blood glucose, 24-h urinary albumin, serum creatinine, and blood urea nitrogen were tested, histopathology, and immunohistochemical analysis of kidney tissues were performed. Moreover, mesangial cells were treated with BCE and MGF for 48 h with or without 25 mmol·L-1 of glucose. Immunofluorescence, Western blot and apoptosis analyses were used to investigate their regulation of oxidative stress and mitochondrial function. BCE and MGF ameliorated biochemical parameters and restored STZ-induced renal injury in the model mice. In vitro study showed that high glucose stimulation increased oxidative stress and cell apoptosis in mesangial cells. BCE and MGF limited mitochondrial membrane potential (Δψm) collapse by inhibiting Nox4, mitochondrially bound hexokinase Ⅱ dissociation, and subsequent ROS production, which effectively reduced oxidative stress, cleaved caspase-3 expression and cell apoptosis. Our work indicated that BCE and MGF had protective effects on diabetic caused kidney injury and prevented oxidative stress in mesangial cells by regulation of hexokinase Ⅱ binding and Nox4 oxidase signaling.
[Key words]: Bombax ceiba     Mangiferin     Diabetic nephropathy     Mitochondrial function     Oxidative stress    
Introduction

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 [1]. Oxidative stress plays an important role in the initiation and development of diabetic complications, including the DN [2]. 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 [10], and Nox4-dependent ROS generation mediates glomerular hypertrophy and mesangial matrix accumulation [6]. 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" [11]. Mitochondria are the major sites for ROS production and are particularly susceptible to oxidative damage [12]. 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 [13]. 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 [14], and mitochondrial bound hexokinase Ⅱ (mtHKⅡ) plays a major role in stabilizing mitochondrial membrane potential and controlling ROS production [15]. 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 [16]. 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 [17]. In a previous research [18], 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 [18]. 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.

Figure 1 BCE and MGF ameliorate the chemical parameters in STZ-induced DN mice. The animals were treated with BCE (40, 80, and 120 mg·kg-1), MGF (50 mg·kg-1) or MET (200 mg·kg-1) by oral administration for 16 weeks. Fasting blood glucose (A), 24-h urine protein (B), serum creatinine (C), and blood urea nitrogen (BUN) (D) were assayed using commercial enzyme kits. The data are expressed as means ± SD (n = 8). *P < 0.05 vs the diabetic control group, #P < 0.05 vs the normal control group
BCE and MGF preventes renal injury in diabetic mice

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.

Figure 2 BCE and MGF restore the renal histology in the STZ-induced DN mice. The-induced DN mice were treated with BCE (40, 80, and 120 mg·kg-1), MGF (50 mg·kg-1) or MET (200 mg·kg-1) by oral administration for 16 weeks. Hematoxylin and eosin (HE) staining in kidneys were viewed (400 × magnification). Images were taken from one of the three independent experiments from three mice. Scale bar: 20 μm
BCE and MGF inhibits Nox4 and cleaved caspase-3 expression in kidneys of the STZ-induced DN mice

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.

Figure 3 BCE and MGF inhibit Nox4 and cleaved caspase-3 expression in the STZ-induced DN mice. The STZ-induced DN mice were treated with BCE (40, 80, and 120 mg·kg-1), MGF (50 mg·kg-1) or MET (200 mg·kg-1) by oral administration for 16 weeks. At the end of experiment, the kidney tissues were processed for immunostaining and Nox4 (A) and cleaved caspase-3 (B) protein expression in kidney were measured by immunohistochemistry, and the images were taken from one of the three independent experiments from three mice (400 × magnification). Scale bar: 20 μm
BCE and MGF reduces ROS generation in the MES cells

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.

Figure 4 BCE and MGF reduce intracellular ROS generation in the MES cells. The SV40 MES 13 mouse mesangial (MES) cells were treated with BCE (10 μmol·L-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. (A): Intracellular ROS production in MES cells was viewed by dihydroethidium staining labeling with confocal microscopy (original magnification 630 ×). (B): The fluorescence values were measured with Thermo Scientific Varioskan Flash microplate reader (excitation/emission at 510 nm/580 nm). Scale bar: 20 μm. Red: mitochondrial ROS. A minimum of 50 cells were analyzed for each experiment, and the results were obtained from 3 independent experiments. The values are means ± SD; *P < 0.05 vs the high glucose group; #P < 0.05 vs the indicated treatment
BCE and MGF suppresses Nox4 expression in the MES cells

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.

Figure 5 BCE and MGF suppress Nox4 expression in MES cells. The SV40 MES 13 mouse mesangial (MES) cells were treated with BCE (10 μmol·L-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. Nox4 location and expression in the membrane were viewed with the confocal scanning microscopy. Single cell fluorescence intensities were assessed from 50 cells of three different experiments. Scale bar: 20 μm
BCE and MGF decreases HKⅡ dissociation from mitochondrial membrane

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.

Figure 6 BCE and MGF decrease HKⅡ dissociation from mitochondria membrane. The SV40 MES 13 mouse mesangial (MES) cells were treated with BCE (10 μmol·L-1), MGF (10 μmol·L-1) or DPI (1 μmol·L-1) in either 5.5 or 25 mmol·L-1 glucose with 0.5% FBS for 48 h. (A): The lactic acid production was assayed using commercial enzyme kits. (B): HKⅡ location in the membrane was viewed with the confocal scanning microscopy. Single cell fluorescence intensities were assessed from 50 cells of three different experiments. Scale bar: 20 μm. The values are means ± SD (n = 8); *P < 0.05 vs the high glucose group; #P < 0.05 vs the indicated treatment
BCE and MGF reduces mitochondria-dependent cell apoptosis

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).

Figure 7 BCE and MGF reduced mitochondria-dependent cell apoptosis. SV40 MES 13 mouse mesangial (MES) cells were treated with BCE (10 μmol·L-1), MGF (10 μmol·L-1) or DPI (1 μmol·L-1) in 25 mmol·L-1 glucose with 0.5% FBS for 48 h. (A): mitochondrial membrane potential (1ψm) was viewed by TMRE labeling with confocal microscopy. Scale bar: 20 μm. A minimum of 50 cells were analyzed for each experiment, and the results were showed by comparing fold change in fluorescence intensity of each group versus 25 mmol·L-1 glucose group. (B): Cleaved caspase-3 expression was determined by Western blot. (C): The apoptosis assay was performed with AnnexinV/PI staining by flow cytometry analysis. The results were expressed as the mean ± SD of three independent experiments. *P < 0.05 vs the high glucose group; #P < 0.05 vs the indicated treatment
Discussion

Increasing evidence indicates a relationship between oxidative stress and diabetic nephropathy [19]. 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 [22], 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 [6], confirming that hyperglycemia and hyperglycemia-induced mediators most likely account for these effects [23]. 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 [6]. 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 [15]. HKⅡ expression levels and localization are highly regulated by physiology, hormones, and metabolic state [24], 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 [15]. 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.

References
[1]
Hakim FA, Pflueger A. Role of oxidative stress in diabetic kidney disease[J]. Med Sci Monitor, 2010, 16(16): RA37.
[2]
Taniguchi K, Xia L, Goldberg HJ, et al. Inhibition of Src kinase blocks high glucose-induced EGFR transactivation and collagen synthesis in mesangial cells and prevents diabetic nephropathy in mice[J]. Diabetes, 2013, 62(11): 3874-3886. DOI:10.2337/db12-1010
[3]
Bedard K, Krause K. The NOX family of ROS-generating NADPH oxidases: physiology and pathophysiology[J]. Physiol Rev, 2007, 87(1): 245-313. DOI:10.1152/physrev.00044.2005
[4]
Ravi N, Adam WC, Sowers JR. Redox control of renal function and hypertension[J]. Antioxid Redox Sign, 2008, 10(12): 2047-2089. DOI:10.1089/ars.2008.2034
[5]
Carlos S, O'Connor PM. NAD(P)H oxidase and renal epithelial ion transport[J]. Am J Physiol-Reg I, 2011, 300(5): 1023-1029. DOI:10.1152/ajpcell.00288.2010
[6]
Yves G, Karen B, James H, et al. Nox4 NAD(P)H oxidase mediates hypertrophy and fibronectin expression in the diabetic kidney[J]. J Biol Chem, 2005, 280(280): 39616-39626.
[7]
Bondi CD, Manickam ND. NAD(P)H oxidase mediates TGF-beta1-induced activation of kidney myofibroblasts[J]. J Am Soc Nephrol, 2010, 21(1): 93-102. DOI:10.1681/ASN.2009020146
[8]
Geiszt M, Kopp JB, Várnai P, et al. Identification of renox, an NAD(P)H oxidase in kidney[J]. P Natl Acad Sci USA, 2000, 97(14): 8010-8014. DOI:10.1073/pnas.130135897
[9]
Shiose A, Kuroda J, Tsuruya K, et al. A novel superoxide-producing NAD(P)H oxidase in kidney[J]. J Biol Chem, 2001, 276(2): 1417-1423. DOI:10.1074/jbc.M007597200
[10]
Nisimoto Y, Jackson HM, Ogawa H, et al. Constitutive NADPH-dependent electron transferase activity of the nox4 dehydrogenase domain[J]. Biochemistry, 2010, 49(11): 2433-2442. DOI:10.1021/bi9022285
[11]
Katrin H, William M. Evolutionary biology: essence of mitochondria[J]. Nature, 2003, 426(6963): 127-128. DOI:10.1038/426127a
[12]
Kornfeld OS, Hwang S, Disatnik MH, et al. Mitochondrial reactive oxygen species at the heart of the matter: new therapeutic approaches for cardiovascular diseases[J]. Circ Res, 2015, 116(11): 1783-1799. DOI:10.1161/CIRCRESAHA.116.305432
[13]
Zhang K, Kaufman RJ. From endoplasmic-reticulum stress to the inflammatory response[J]. Nature, 2008, 454: 455-462. DOI:10.1038/nature07203
[14]
Scott J, Weiss JN, Bernard R. Subcellular localization of hexokinases Ⅰ and Ⅱ directs the metabolic fate of glucose[J]. PLos One, 2011, 6(3): 150.
[15]
Rianne N, Otto E, Hollmann MW, et al. Targeting hexokinase Ⅱ to mitochondria to modulate energy me-tabolism and reduce ischaemia-reperfusion injury in heart[J]. Brit J Pharmacol, 2013, 171(8): 2067.
[16]
Wagner Seixas DS, Armando GP, Marietta Tuena GP, et al. Mitochondrial bound hexokinase activity as a preventive antioxidant defense: steady-state ADP formation as a regulatory mechanism of membrane potential and reactive oxygen species generation in mitochondria[J]. J Biol Chem, 2004, 279(38): 39846-39855. DOI:10.1074/jbc.M403835200
[17]
Jain V, Verma SK. Pharmacology of Bombax ceiba Linn[M]. Springer Science & Business Media, 2012.
[18]
Xu GK, Qin XY, Wang GK, et al. Antihyperglycemic, antihyperlipidemic and antioxidant effects of standard ethanol extract of Bombax ceiba leaves on high-fat diet and streptozotocin induced Type 2 diabetic rats[J]. Chin J Nat Med, 2017, 15(3): 168-177.
[19]
Baynes JW, Thorpe SR. Role of oxidative stress in dia-betic complications: a new perspective on an old paradigm[J]. Diabetes, 1999, 48(1): 1-9. DOI:10.2337/diabetes.48.1.1
[20]
Russell JW, Golovoy D, Vincent AM, et al. High glucose-induced oxidative stress and mitochondrial dysfunction in neurons[J]. Faseb J, 2002, 16(13): 1738-1748. DOI:10.1096/fj.01-1027com
[21]
Lemasters JJ, Qian T, Bradham CA, et al. Mitochondrial dysfunction in the pathogenesis of necrotic and apoptotic cell death[J]. J Bioenerg Biomembr, 1999, 31(4): 305-319. DOI:10.1023/A:1005419617371
[22]
Vendrov A, Vendrov K, Smith A, et al. NOX4 NADPH Oxidase-dependent mitochondrial oxidative stress in aging-associated cardiovascular disease[J]. Antioxid Redox Sign, 2015, 23(18): 1389-1409. DOI:10.1089/ars.2014.6221
[23]
Etoh T, Inoguchi T, Kakimoto M, et al. Increased expres-sion of NAD(P)H oxidase subunits, NOX4 and p22phox, in the kidney of streptozotocin-induced diabetic rats and its reversibity by interventive insulin treatment[J]. Diabetologia, 2003, 46(10): 1428-1437. DOI:10.1007/s00125-003-1205-6
[24]
Heikkinen S, Suppola S, Malkki M, et al. Mouse hexokinase Ⅱ gene: structure, cDNA, promoter analysis, and expression pattern[J]. Mamm Genome, 2000, 11(2): 91-96. DOI:10.1007/s003350010019
[25]
Pasdois P, Parker JE, Halestrap AP. Extent of mitochon-drial hexokinase Ⅱ dissociation during ischemia correlates with mitochondrial cytochrome c release, reactive oxygen species production, and infarct size on reperfusion[J]. J Am Heart Assoc, 2013, 2(1): e005645.
[26]
Rostovtseva TK, Tan W, Colombini M. On the role of VDAC in apoptosis: fact and fiction[J]. J Bioenerg Biomembr, 2005, 37(3): 129-142. DOI:10.1007/s10863-005-6566-8
[27]
Halestrap AP, Pereira GC, Pasdois P. The role of hexokinase in cardioprotection–mechanism and potential for translation[J]. Br J Pharmacol, 2015, 172(8): 2085-2100. DOI:10.1111/bph.12899
[28]
Pantic B, Trevisan E, Citta A, et al. Myotonic dystrophy protein kinase (DMPK) prevents ROS-induced cell death by assembling a hexokinase Ⅱ-Src complex on the mitochondrial surface[J]. Cell Death Dis, 2013, 4(5): 858-869.