Protective Effects of Swertiamarin against Methylglyoxal-Induced Epithelial-Mesenchymal Transition by Improving Oxidative Stress in Rat Kidney Epithelial (NRK-52E) Cells

Abstract

Increased blood glucose in diabetic individuals results in the formation of advanced glycation end products (AGEs), causing various adverse effects on kidney cells, thereby leading to diabetic nephropathy (DN). In this study, the antiglycative potential of Swertiamarin (SM) isolated from the methanolic extract of E. littorale was explored. The effect of SM on protein glycation was studied by incubating bovine serum albumin with fructose at 60 °C in the presence and absence of different concentrations of swertiamarin for 24 h. For comparative analysis, metformin was also used at similar concentrations as SM. Further, to understand the role of SM in preventing DN, in vitro studies using NRK-52E cells were done by treating cells with methylglyoxal (MG) in the presence and absence of SM. SM showed better antiglycative potential as compared to metformin. In addition, SM could prevent the MG mediated pathogenesis in DN by reducing levels of argpyrimidine, oxidative stress and epithelial mesenchymal transition in kidney cells. SM also downregulated the expression of interleukin-6, tumor necrosis factor-α and interleukin-1β. This study, for the first time, reports the antiglycative potential of SM and also provides novel insights into the molecular mechanisms by which SM prevents toxicity of MG on rat kidney cells.

Keywords: AGE-inhibitor; advanced glycation end product (AGE); diabetic nephropathy; epithelial to mesenchymal transition; oxidative stress; swertiamarin.

Figure 1

(a) TLC profile: Lane 1, 2: Fractions containing SM along with impurities. Lane 3: Standard Lane 4: Methanolic extract of E. littorale Lane 5: Fraction containing SM in the TLC mobile phase (Chloroform: methanol in 8:2 v/v proportion) (b) The HPLC chromatogram of SM isolated in the lab (b1) and of the standard SM (b2) at 1 mg/mL using acetonitrile: water (10:90) as the mobile phase. (c) An overlay of FT-IR spectrum of standard SM (red) and SM isolated in the lab (blue). (d) The mass spectrum (LC-MS) of isolated swertiamarin showing characteristic m/z of 375.1.

Figure 2

The intensity of fluorescence of AGEs in the presence and absence of swertiamarin (SM) and its comparison with metformin (M) at different concentrations measured using an excitation wavelength of 370 nm and an emission wavelength of 440 nm. The results are Mean ± SD of 3 individual experiments. ^#### p < 0.0001 represents comparison between bovine serum albumin (BSA) negative and BSA + Fructose (Fru) group. **** p < 0.0001 represents the statistical significance w.r.t the BSA + Fru group. ^aaaa p < 0.0001 represents the comparison between SM and M.

Figure 3

Antiglycative effect of swertiamarin at 100 µg/mL (SM 100) as shown by absorbance at 280 nm using UV-spectrophotometer (a), the structural changes as seen by the shift in the peaks due to glycation (b), suggesting hyperchromicity (c). The results are Mean ± SD of 3 individual experiments. **** p < 0.0001, ** p < 0.054241.

Figure 4

Estimation of carbonyl content. As compared to the native BSA, glycated BSA showed remarkably higher level of carbonyl content. The treatment with SM showed reduction in the carbonyl content, as compared to the glycated BSA. (*** p < 0.001 and ** p < 0.01). Results are mean ± SD of 3 individual experiments.

Figure 5

FTIR spectra of BSA (black line), glycated BSA (red line) and the glycated BSA treated with SM (blue line).

Figure 6

(a) The effect MG on the cell viability in NRK-52E cells. NRK-52E cells when treated with different concentrations of MG caused cell death in a dose-dependent manner as compared to control. (b) The cytotoxicity of SM was checked on NRK-52E cells. The treatment with SM did not show any toxicity on the NRK-52E cells at various concentrations. Results are Mean ± SD of 3 individual experiments.

Figure 7

Morphology of NRK-52E cells in each treatment group at 24 h. The untreated NRK-52E cells showed its characteristic epithelial morphology (a). The treatment with 200 µM MG changed the epithelial morphology of NRK-52E cells to elongated fibroblast like morphology, indicated with arrows (b). Cotreatment with 100 µg/mL SM prevented MG—induced morphological changes in NRK-52E cells (c). Above images are representative microscopy images of each group under 10× objective.

Figure 8

Detection of argpyrimidine levels. As compared to the control group, treatment with 200 µM MG in the NRK-52E cells after 24 h resulted in the formation of argpyrimidine by MG-induced modifications of arginine which could be prevented in MG and 100 µg/mL SM cotreatment group (* p < 0.05). Results are Mean ± SD of 3 individual experiments.

Figure 9

(a) HPLC chromatograms of MDA measured from NRK-52E cells after DNPH derivatization. The treatment with 200 µM MG in NRK-52E cells increased the levels of MDA measured using ODS2 reverse phase column in the presence of acetonitrile and milliQ water containing 0.2% acetic acid, with a ratio 38:62 respectively as the mobile phase. Treatment with 100 µg/mL SM in the presence of MG attenuated the production of MDA. (b) The levels of ROS were also measured using fluorescence spectroscopy with excitation and emission wavelengths 495 nm and 529 nm, respectively. Treatment with SM in the presence of MG could inhibit the elevation of ROS significantly (** p < 0.01) as shown in MG treated group (** p < 0.01), proving the antioxidative characteristic of SM. Results are Mean ± SD of 3 individual experiments.

Figure 10

mRNA expression of ER stress genes (a) CHOP and (b) Grp-78 using qRT-PCR. The expression of CHOP and Grp78 was significantly upregulated in MG-exposed cells as compared to the untreated control NRK-52E cells after 24 h. The treatment with 100 µg/mL SM along with 200 µM MG could alleviate the upregulation of CHOP and Grp78, indicating the protection against ER stress. Results are Mean ± SD of 3 individual experiments analysed by ANOVA. The symbol *** p < 0.001 indicates the comparison of MG group w.r.t control and ^# p < 0.05, ^### p < 0.001 indicates the comparison of SM group w.r.t. MG group.

Figure 11

qRT-PCR analysis results of (a) RAGE (b) NADPH oxidase (c) iNOS (d) IL-6 (e) TNF-α (f) IL-1β (g) ICAM-1, and (h) MCP-1 in MG-treated NRK-52E cells alone and SM for 24 h. The gene expression levels were calculated after normalizing against housekeeping 18S rRNA and are presented as relative mRNA expression units. Values represent mean ± SD of 3 individual experiments. The symbol * p < 0.05, ** p < 0.01, **** p < 0.0001 indicate the comparison of MG group w.r.t control and ^# p < 0.05, ^## p < 0.01, ^#### p < 0.0001 indicate the comparison between MG and SM group.

Figure 12

qRT-PCR analysis results of (a) p38 MAPK (b) TGF-β (c) α-SMA (d) Fibronectin-1 (e) E-cadherin (f) HO-1 and (g) Nrf-2 in MG-treated NRK-52E cells alone and SM for 24 h. The gene expression levels were calculated after normalizing against housekeeping 18S rRNA and are presented as relative mRNA expression units. Values represent mean ± SD of 3 individual experiments. The symbol ** p < 0.01, *** p < 0.001, **** p < 0.0001 indicate the comparison of MG group w.r.t control and ^## p < 0.01, ^### p < 0.001, ^#### p < 0.0001, indicate the comparison between MG and SM group.

Figure 13

Protein levels of TGF-β and HO-1 in NRK-52E measured using ELISA. The treatment with 200 μM MG for 24 h, up-regulated the levels of TGF-β proteins which was prevented by the co-treatment with 100 μg/mL SM. The levels of HO-1 were declined in the MG treated group after 24 h which was alleviated by SM, proving the role of SM in inhibiting TGF-β expression by upregulating HO-1. Values represent mean ± SD of 3 individual experiments. **** p < 0.0001 indicate the comparison of MG group w.r.t control and ^### p < 0.001, ^#### p < 0.0001, indicate the comparison between MG and SM group.

Figure 14

Chemical structure of swertiamarin and Argpyrimidine.

Figure 15

(a) Binding of SM with RAGE (left) and the amino acids involved in the interaction (right panel). (b) Binding of Argpyrimidine with RAGE (left) and the amino acid involved in the interaction (right panel).