Contributed equally
The aim of the present study was to investigate the renoprotective effects of isorhamnetin (ISO) in type 2 diabetic rats and its effects on the nuclear factor-κB (NF-κB) signaling pathway, which is associated with diabetic nephropathy. The type 2 diabetic rat model was established by a high-fat diet plus streptozocin injection and the rats were subsequently treated with two dosages of ISO, respectively. The levels of blood glucose were determined. Urinary osteopontin, kidney injury molecule-1 (KIM-1) and albumin were measured to evaluate the renal function of the rats. Renal NF-κB signaling activity was assessed by measuring the levels of NF-κB p65, phospho-NF-κB p65, inhibitor of NF-κB (IκBα) and phospho-IκBα, and the NF-κB p65 DNA-binding activity. Downstream inflammatory mediators [tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), IL-6, intercellular adhesion molecule-1 (ICAM-1) and transforming growth factor-β1 (TGF-β1)] of the NF-κB signaling pathway were investigated to evaluate the renal inflammatory response. Renal levels of malondialdehyde and total superoxide dismutase were detected to access the oxidative stress. Furthermore, glomerular mesangial cells (GMCs) were treated with lipopolysaccharide and ISO. In the cellular experiment, the NF-κB signaling activity, levels of TNF-α, IL-1β, IL-6, ICAM-1 and TGF-β1, and oxidative stress were also investigated. The results showed that ISO decreased the levels of urinary osteopontin, KIM-1 and albumin. ISO also inhibited the NF-κB signaling activity, decreased the production of inflammatory mediators and attenuated oxidative stress in diabetic rats and GMCs. The present investigations revealed that ISO had ameliorative effects on diabetes-induced renal damage and the activity may be associated with the negative regulation of NF-κB signaling pathway.
As a leading cause of clinical end-stage renal disease, diabetic nephropathy (DN) is the second most prevalent diabetes-associated complication (
Accumulating evidence indicates that inflammation is one of the major factors that has significant roles in the initiation and progression of DN (
Excessive production of reactive oxygen species has been found in diabetic animals and patients (
Considering the crucial roles of inflammation and oxidative stress in the pathogenesis of DN, antioxidant therapy and anti-inflammation therapy have been tested in DN. Cumulative evidence suggests that these managements can preserve renal function and prevent or slow the progression of DN (
Sprague-Dawley rats (8 weeks old and 180–220 g) obtained from the SLAC Lab Animal Center (Shanghai, China) were used in this study. Procedures used in the current study were approved by the Ethics Committee of Affiliated Hospital of Weifang Medical College (Weifang, Shandong, China). Animals were housed 5/cage in a controlled environment (22±1°C, 12-h light/dark cycle).
Type 2 diabetes mellitus (DM) was induced according to the method described in the literature (
The diabetic rats were randomly divided into 3 groups (n=10 in each group): DM, 50 mg/kg ISO (ISO-50) and 150 mg/kg ISO (ISO-150) groups. Rats in the ISO-50 and ISO-150 groups were orally administered ISO (50 and 150 mg/kg/day, respectively) after grouping for consecutive 12 weeks. Rats in the control group (n=10 in each group) and the DM group were orally administrated with the same volume of saline.
On the last day of ISO treatment, the rats were placed in metabolic cages and 24-h urine was collected. The kidney was removed and rinsed with ice-cold saline. A section of kidney tissue was homogenized (100 mg renal tissue/ml saline) and centrifuged at 1,050 × g for 10 min. The supernatant was collected and further centrifuged at 10,000 × g for 10 min.
Levels of urinary osteopontin and kidney injury molecule-1 (KIM-1) were measured using ELISA kits according to the instructions provided by the manufacturer (Boster Biological Technology, Ltd., Wuhan, Hubei, China). The levels of urinary albumin were measured using an automatic biochemistry analyzer. Fasting blood glucose (FBG) was measured using OneTouch®Ultra machine.
Rat glomerular mesangial cells (GMCs) (Chinese Center for Typical Culture Collection, Wuhan, Hubei, China) were maintained at 5% CO2 and 37°C in Dulbecco's modified Eagle's medium (Wisent Bioproducts, St. Bruno, Quebec, Canada) containing fetal bovine serum, 100 µg/ml streptomycin 100 U/ml penicillin. GMCs were randomly divided into the following 4 groups: Normal control (NC), LPS [with the presence of 10 nmol/ml LPS (Sigma-Aldrich, St. Louis, MO, USA) in the medium], ISO-5 (10 nmol/ml LPS and 5 µM ISO in the medium) and ISO-10 (10 nmol/ml LPS and 10 µM ISO in the medium) groups. The cells in each group were cultured for 72 h and the medium was collected. Cells were lysed with lysis buffer and the supernatants of the cell lysates were collected after centrifugation at 18,000 × g for 10 min at 4°C.
The levels of NF-κB p65, phospho-NF-κB p65, IκBα and phospho-IκBα in the cell lysate and in the renal homogenate were measured using ELISA kits, according to the manufacturer's protocols. NF-κB p65 ELISA kits were purchased from Cusabio Biotech Co., Ltd. (Wuhan, China), phospho-NF-κB p65 and phospho-IκBα ELISA kits were purchased from Cell Signaling Technology, Inc. (Beverly, MA, USA), and the IκBα ELISA kits were purchased from Jining Co. (Shanghai, China).
Nuclear protein was extracted from the kidney and rat GMCs. NF-κB DNA-binding activity was measured using the NF-κB p65 transcription factor ELISA assay kits (Cayman Chemical Co., Ann Arbor, MI, USA) according to the manufacturer's protocol.
The levels of the NF-κB downstream inflammatory mediators, TNF-α, IL-1β, IL-6, ICAM-1 and TGF-β1, in the cell culture medium and in the renal homogenate were measured using ELISA kits from R&D Systems, Inc. (Minneapolis, MN, USA) according to the manufacturer's protocol.
Total RNA was isolated from fresh kidney tissue and GMCs using TRIzol reagents (Invitrogen, Thermo Fisher Scientific, Inc., Waltham, MA, USA). cDNA was synthesized using the cDNA synthesis kit (Takara Bio, Inc., Kyoto, Japan) and amplified by reverse transcription-quantitative polymerase chain reaction (RT-qPCR). The primer sequences were: TNF-α forward, 5′-TGATCGGTCCCAACAAGGA-3′ and reverse primer, 5′-TGCTTGGTGGTTTGCTACGA-3′; IL-1β forward, 5′-ACTATGGCAACTGTCCCTGAAC-3′ and reverse primer, 5′-GTGCTTGGGTCCTCATCCTG-3′; IL-6 forward, 5′-AGTTGCCTTCTTGGGACTGA-3′ and reverse primer 5′-CAGAATTGCCATTGCACAAC-3′; NF-κB p65 forward, 5′-TGCAGGCTCCTGTGCGAGTG-3′ and reverse primer, 5′-TCCGGTGGCGATCGTCTGTGT-3′; ICAM-1 forward, 5′-CGTGGCGTCCATTTACACCT-3′ and reverse primer, 5′-TTAGGGCCTCCTCCTGAGC-3′; TGF-β1 forward, TGGCGTTACCTTGGTAACC and reverse primer, GGTGTTGAGCCCTTTCCAG; and β-actin forward, 5′-AGGCCCCTCTGAACCCTAAG-3′ and reverse primer, 5′-CCAGAGGCATACAGGGACAAC-3′. The PCR program involved 95°C for 30 sec and 40 PCR cycles (95°C for 5 sec and 60°C for 30 sec). The PCR reactions were perforemd with an iQ5 Real-Time PCR Detection system (Bio-Rad, Hercules, CA, USA).
Oxidative stress activity was assessed by measuring the levels of malondialdehyde (MDA) in the cell culture medium and in the renal homogenate using chemichromatometry kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China). Antioxidant activity was assessed by measuring levels of total superoxide dismutase (T-SOD) using chemichromatometry kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China).
Statistical analyses were carried out using SPSS software 14.0 (SPSS, Inc., Chicago, IL, USA). Data are reported as mean ± standard deviation and were analyzed using one-way analysis of variance followed by Student-Newman-Keuls test for multiple comparisons. For all the tests, P<0.05 was considered to indicate a statistically significant difference.
The DM group had higher levels of urinary osteopontin, KIM-1 and albumin compared to the control group (P<0.05). The ISO-50 and ISO-150 groups had lower levels of these renal damage markers compared to the DM group (P<0.05), and those levels of the ISO-150 group were lower compared to the ISO-50 group (P<0.05), indicating that ISO dose-dependently improved the renal function in the diabetic rats (
No significant differences in the FBG levels were observed among the DM, the ISO-50 and the ISO-150 groups (P>0.05). All three groups had higher levels of FBG compared to the control group (P<0.05) (
In the
In the
The changes (protein and mRNA) of TNF-α, IL-1β, IL-6, ICAM-1 and TGF-β1, which are the downstream inflammatory mediators of NF-κB signaling pathway, were investigated. In the
The levels of MDA and T-SOD were investigated. In the
The prevalence of DN is rapidly increasing worldwide due to a rise in the prevalence of type 2 DM (
ISO is a plant flavonoid abundant in herbal medicinal plants, such as
Regulation of blood glucose is believed to contribute to a reduced renal risk. The blood glucose levels of the animals were evaluated and the DM and ISO groups had comparable levels of blood glucose. The results indicated that the recovery of renal damage did not benefit from blood glucose reduction.
Among the complex mechanisms leading to DN, inflammation has a key role (
In addition to mediating an inflammatory response, NF-κB also triggers the oxidative reactions. NF-κB has an important role between the interaction of oxidative stress and inflammation. Oxidative stress is involved in numerous disorders (
In order to confirm the anti-inflammatory and antioxidative activity of ISO, rat GMCs were cultured with the presence of LPS or LPS+ISO. LPS is extensively used to induce an inflammatory state
Collectively, ISO had renoprotective effects in type 2 diabetic rats. The recovery of renal damage may be associated with the inhibition of the NF-κB signaling pathway.
Effects of ISO treatment on renal damage parameters and FBG.
Treatment | Osteopontin, pg/ml | KIM-1, pg/ml | Albumin, mg/24 h | FBG, mmol/l |
---|---|---|---|---|
Control | 10.69±2.17 | 8.10±1.34 | 0.41±0.08 | 5.36±0.88 |
DM | 48.10±7.07 |
25.17±3.05 |
4.12±0.70 |
11.68±2.01 |
ISO-50 | 35.08±5.33 |
19.44±2.28 |
3.25±0.51 |
10.91±2.55 |
ISO-150 | 25.05±4.04 |
14.27±2.11 |
1.83±0.38 |
10.04±2.69 |
Data are expressed as mean ± standard deviation. P<0.05 vs.
control
DM; and
ISO-50 groups. FBG, fasting blood glucose; KIM-1, kidney injury molecule-1; DM, diabetes mellitus; ISO-50, 50 mg/kg/day isorhamnetin; ISO-150, 150 mg/kg/day isorhamnetin.
Effects of ISO treatment on renal NF-κB signaling.
Treatment | NF-κB p65, pg/ml | phospho-NF-κB p65, pg/ml | IκBα, pg/ml | phospho-IκBα, pg/ml | NF-κB p65 DNA-binding activity, OD |
---|---|---|---|---|---|
Control | 35.17±6.33 | 13.50±2.63 | 30.09±4.36 | 8.66±1.05 | 0.25±0.03 |
DM | 98.09±12.51 |
65.28±8.11 |
29.82±4.22 | 21.05±3.17 |
0.89±0.13 |
ISO-50 | 71.20±9.07 |
48.79±6.90 |
31.07±5.34 | 15.10±2.02 |
0.65±0.11 |
ISO-150 | 55.83±6.86 |
30.83±5.04 |
33.10±5.8 | 11.34±1.61 |
0.48±0.08 |
Data are expressed as mean ± standard deviation. P<0.05 vs.
control
DM; and
ISO-50 groups. NF-κB, nuclear factor-κB; IκBα, inhibitor of NF-κB; OD, optical density; DM, diabetes mellitus; ISO-50, 50 mg/kg/day isorhamnetin; ISO-150, 150 mg/kg/day isorhamnetin.
Effects of ISO treatment on NF-κB signaling in the glomerular mesangial cells.
Treatment | NF-κB p65, pg/ml | phospho-NF-κB p65, pg/ml | IκBα, pg/ml | phospho-IκBα, pg/ml | NF-κB p65 DNA-binding activity, OD |
---|---|---|---|---|---|
NC | 17.55±3.04 | 6.11±1.03 | 15.66±3.05 | 6.14±1.02 | 0.22±0.03 |
LPS | 56.07±7.11 |
40.73±6.35 |
16.11±1.64 | 12.28±1.54 |
0.93±0.15 |
ISO-5 | 41.26±4.88 |
28.05±4.19 |
16.80±2.55 | 9.31±1.07 |
0.62±0.09 |
ISO-10 | 30.10±4.14 |
14.66±3.71 |
17.13±3.10 | 7.03±1.10 |
0.40±0.08 |
Data are expressed as mean ± standard deviation. P<0.05 vs.
control
LPS; and
ISO-5 groups. NF-κB, nuclear factor-κB; IκBα, inhibitor of NF-κB; OD, optical density; NC, normal control; LPS, lipopolysaccharide; ISO-5, 10 nmol/ml LPS and 5 µM isorhamnetin; ISO-10, 10 nmol/ml LPS and 10 µM isorhamnetin.
Effects of ISO treatment on the renal inflammatory mediators.
Treatment | TNF-α, pg/ml | IL-1β, pg/ml | IL-6, pg/ml | ICAM-1, pg/ml | TGF-β1, pg/ml |
---|---|---|---|---|---|
Control | 34.07±5.44 | 51.16±8.10 | 25.03±4.15 | 41.05±6.32 | 67.71±9.45 |
DM | 90.11±11.20 |
132.07±16.39 |
65.34±8.27 |
96.17±12.85 |
212.53±30.36 |
ISO-50 | 66.19±8.07 |
100.29±12.24 |
50.37±6.01 |
73.54±8.35 |
152.96±21.88 |
ISO-150 | 49.58±6.21 |
76.44±9.89 |
38.03±4.14 |
56.62±6.98 |
117.51±15.30 |
Data are expressed as mean ± standard deviation. P<0.05 vs.
control
DM; and
ISO-50 groups. TNF-α, tumor necrosis factor-α; IL, interleukin; ICAM-1, intercellular adhesion molecule-1; TGF-β1, transforming growth factor-β1; DM, diabetes mellitus; ISO-50, 50 mg/kg/day isorhamnetin; ISO-150, 150 mg/kg/day isorhamnetin.
Effects of ISO treatment on the inflammatory mediators in the glomerular mesangial cells.
Treatment | TNF-α, pg/ml | IL-1β, pg/ml | IL-6, pg/ml | ICAM-1, pg/ml | TGF-β1, pg/ml |
---|---|---|---|---|---|
NC | 12.82±2.12 | 23.03±4.57 | 14.08±1.99 | 8.01±1.09 | 20.31±4.75 |
LPS | 43.58±5.33 |
71.79±9.04 |
45.31±6.05 |
21.22±3.15 |
51.12±7.08 |
ISO-5 | 32.04±4.97 |
52.21±5.93 |
33.20±4.52 |
16.38±2.01 |
39.63±4.11 |
ISO-10 | 20.15±3.06 |
36.48±5.09 |
23.94±3.03 |
12.09±1.71 |
30.34±4.62 |
Data are expressed as mean ± standard deviation. P<0.05 vs.
control
LPS; and
ISO-5 groups. TNF-α, tumor necrosis factor-α; IL, interleukin; ICAM-1, intercellular adhesion molecule-1; TGF-β1, transforming growth factor-β1; NC, normal control; LPS, lipopolysaccharide; ISO-5, 10 nmol/ml LPS and 5 µM isorhamnetin; ISO-10, 10 nmol/ml LPS and 10 µM isorhamnetin.
Effects of ISO treatment on the renal mRNA expression levels of the inflammatory mediators.
mRNA expression level | ||||||
---|---|---|---|---|---|---|
Treatment | NF-κB p65 | TNF-α | IL-1β | IL-6 | ICAM-1 | TGF-β1 |
Control | 1.00±0.16 | 1.00±0.08 | 1.00±0.11 | 1.00±0.08 | 1.00±0.12 | 1.00±0.11 |
DM | 2.65±0.31 |
3.03±0.36 |
2.76±0.29 |
2.89±0.31 |
2.93±0.36 |
3.43±0.47 |
ISO-50 | 2.01±0.25 |
2.34±0.31 |
2.01±0.25 |
2.16±0.27 |
2.20±0.26 |
2.56±0.40 |
ISO-150 | 1.53±0.18 |
1.65±0.27 |
1.44±0.20 |
1.51±0.20 |
1.69±0.21 |
1.85±0.28 |
Data are expressed as mean ± standard deviation. P<0.05 vs.
control
DM; and
ISO-50 groups. NF-κB, nuclear factor-κB; TNF-α, tumor necrosis factor-α; IL, interleukin; ICAM-1, intercellular adhesion molecule-1; TGF-β1, transforming growth factor-β1; DM, diabetes mellitus; ISO-50, 50 mg/kg/day isorhamnetin; ISO-150, 150 mg/kg/day isorhamnetin.
Effects of ISO treatment on mRNA expression of inflammatory mediators in the glomerular mesangial cells.
mRNA expression level | ||||||
---|---|---|---|---|---|---|
Treatment | NF-κB p65 | TNF-α | IL-1β | IL-6 | ICAM-1 | TGF-β1 |
NC | 1.00±0.11 | 1.00±0.098 | 1.00±0.13 | 1.00±0.14 | 1.00±0.12 | 1.00±0.11 |
LPC | 3.69±0.52 |
3.25±0.41 |
2.91±0.30 |
3.21±0.44 |
2.43±0.21 |
2.68±0.24 |
ISO-5 | 2.45±0.41 |
2.12±0.29 |
2.17±0.32 |
2.52±0.35 |
1.95±0.15 |
2.05±0.11 |
ISO-10 | 1.67±0.25 |
1.51±0.23 |
1.60±0.19 |
1.73±0.21 |
1.53±0.16 |
1.61±0.12 |
Data are expressed as mean ± standard deviation. P<0.05 vs.
control
LPS; and
ISO-5 groups. NF-κB, nuclear factor-κB; TNF-α, tumor necrosis factor-α; IL, interleukin; ICAM-1, intercellular adhesion molecule-1; TGF-β1, transforming growth factor-β1; NC, normal control; LPS, lipopolysaccharide; ISO-5, 10 nmol/ml LPS and 5 µM isorhamnetin; ISO-10, 10 nmol/ml LPS and 10 µM isorhamnetin.
Effects of ISO treatment on the renal oxidative stress markers.
Treatment | MDA, mmol/g protein | T-SOD, U/mg protein |
---|---|---|
Control | 0.61±0.11 | 73.53±10.39 |
DM | 1.58±0.20 |
33.14±5.32 |
ISO-50 | 1.19±0.14 |
45.28±6.27 |
ISO-150 | 0.93±0.15 |
59.95±6.09 |
Data are expressed as mean ± standard deviation. P<0.05 vs.
control
DM; and
ISO-50 groups. MDA, malondialdehyde; T-SOD, total superoxide dismutase; DM, diabetes mellitus; ISO-50, 50 mg/kg/day isorhamnetin; ISO-150, 150 mg/kg/day isorhamnetin.
Effects of ISO treatment on the oxidative stress markers in the glomerular mesangial cells.
Treatment | MDA, mmol/g protein | T-SOD, U/mg protein |
---|---|---|
NC | 0.15±0.02 | 30.12±5.04 |
LPS | 0.62±0.10 |
14.38±2.37 |
ISO-5 | 0.45±0.05 |
19.85±2.86 |
ISO-10 | 0.30±0.05 |
25.41±2.91 |
Data are expressed as mean ± standard deviation. P<0.05 vs.
control
LPS; and
ISO-5 groups. MDA, malondialdehyde; T-SOD, total superoxide dismutase; NC, normal control; LPS, lipopolysaccharide; ISO-5, 10 nmol/ml LPS and 5 µM isorhamnetin; ISO-10, 10 nmol/ml LPS and 10 µM isorhamnetin.