4.3. In Vitro Assays

The L6 myoblast and the NRK epithelial cell lines were gifted by Prof. Parames Sil, Department of Molecular Medicine, Bose Institute, Kolkata, India. These cells were cultured in Dulbecco’s modified Eagle’s Medium (DMEM) supplemented with 10% FBS and antibiotics. The cells were maintained at 37 °C in a humidified atmosphere of 5% CO₂. The cells were passaged in every 3 days.

The L6 cells were exposed to high glucose (HG+) and glucose uptake assay was performed in the presence of Myr as per established protocol [26]. Briefly, cells (2 × 10⁴) were pre-incubated with Myr (10, 20, 30, and 50 μM) in a 96-well culture plate for 2 h followed by HG+ (30 mM) exposure for the following 20 h at 37 °C in a humidified atmosphere of 5% CO₂. A set of L6 cells cultured with 5.5 mM glucose served as the control (HG−). The glucose uptake was estimated using 6-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-2-deoxy-d-glucose (6-NBDG) following the established protocol [26]. The treated cells were incubated with a serum-free medium containing 6-NBDG (20 μM). After 30 min of incubation, cells were washed, lysed, and kept in the dark for 10 min. The cells were homogenized with 30 µL of DMSO and the plate was read immediately using a microplate reader at λ_excitation 466 nm/λ_emission 540 nm.

The L6 myoblast cells (2 × 10⁴) were pre-incubated with Myr (30 μM) in a 96-well culture plate for 2 h followed by HG+ (30 mM) exposure for the following 20 h at 37 °C in a humidified atmosphere of 5% CO₂. The concentration of Myr was calibrated on the basis of the glucose uptake assay. L6 cells that received different treatments were washed with cold PBS, lysed in the radio-immunoprecipitation assay (RIPA) buffer supplemented with protease and phosphatase inhibitors, and the protein samples were separated following the standard sequential fractionation process as described by Baghirova et al. [27]. Protein samples were quantified by ELISA (Bio-Rad, CA, USA). The sample proteins (20 μg) were resolved in 10% SDS-PAGE gel electrophoresis, and immunoblotting was performed as per the established protocol by our group [22]. The blot was developed by ECL substrate (Millipore, MA, USA) and the protein expression was detected in a ChemiDoc Touch imaging system (Bio-Rad, USA). The densitometric analysis was executed using Image Lab software (Bio-Rad, USA). The membranes were further subjected to mild stripping to detect the expressions of other proteins in the same membrane [28]. The expressions of PI3K (p85), P-IRS-1 (Tyr 895), total IRS-1, P-Akt (Ser 473), total Akt, and GLUT4 (in the membrane fraction) were studied. β-actin was used as a loading control for normalization.

To establish a model of diabetic nephropathy, the concentration and time-dependent toxic effect of D-glucose was measured. For measuring the concentration-dependent toxic effect of D-glucose, NRK cells (2 × 10⁴) were seeded in a 96-well culture plate. After 24 h, the cells were treated with D-glucose (5, 10, 20, 30, and 40 mM) and incubated for 72 h. The cell viability was measured using resazurin as per the protocol established by our group [21]. Briefly, 5 μL of 600 μM resazurin was added to the wells and incubated for 2 h, and the plate was read using a microplate reader at λ_excitation 535 nm/λ_emission 590 nm. To determine the time-dependent toxic effect of D-glucose, the cells were treated with D-glucose (30 mM), and the cell viability was measured at 6, 12, 24, 48, and 72 h using the resazurin-based assay. Based on the assays, D-glucose concentration of 30 mM and the incubation period of 48 h were optimized as the in vitro condition for diabetic nephropathy assay.

The NRK cells (2 × 10⁴) were seeded in a 96-well culture plate, and after 24 h, the cells were treated with Myr (30 μM) and the nephroprotective assay was performed in the presence of HG+ (30 µM). A set of NRK cells cultured with 5.5 mM glucose served as the control (HG−). A set of cells treated with HG+ served as the hyperglycemic control, and another set of cells treated with Myr (30 μM) in the HG−condition was kept to observe the effect of Myr (30 μM) in the normoglycemic cellular environment. The cells were incubated for 48 h at 37 °C in a humidified atmosphere of 5% CO₂.

The cell viability was measured using the resazurin-based assay [21]. The Hoechst staining was performed as per the protocol established by our group [21]. Briefly, cells under different sets were fixed with paraformaldehyde (4%) in phosphate buffer saline (PBS) of pH 7.4 for 20 min and were strained with Hoechst 33,258 (5 μg/mL in PBS) for 20 min. The cells were washed with PBS and counted under fluorescence microscope (Olympus-1 × 70, Japan, software-Metamorph).

The intracellular ROS production was measured employing a 2’,7’-dichlorofluorescein diacetate (DCFH-DA)-based assay, and DCF fluorescence was measured at λ_excitation 485 nm/λ_emission 525 nm under a fluorescence microscope (Olympus-1 × 70, Japan, software-Metamorph) [29]. Briefly, the cells that received different treatments were incubated with 10 mM DCFH-DA for 1 h at 37 °C in the dark. Then, the cells were washed and suspended in PBS, and fluorescence was measured. Firstly, DCFH-DA is deacetylated by viable cells to non-fluorescent DCFH, which in turn form fluorescent DCF by reacting with ROS [29]. NADPH oxidase level was measured as per the method described elsewhere [30]. Briefly, cells that received different treatments were detached by acutase and centrifuged at 2500× g for 5 min. the pellet was resuspended in PBS and the cells were incubated with NADPH (250 µM). NADPH consumption was checked by the decrease in absorbance at λ 340 nm for 10 min. For NADPH oxidase activity, the rate of NADPH consumption was inhibited by adding 10 µM diphenyleneiodonium 30 min prior to the assays. The amount of NADPH consumption was estimated using an absorption extinction coefficient of 6.22 mM⁻¹cm⁻¹. The cellular NO content was measured using a colorimetric assay kit and following the manufacturer’s protocol (Cayman Chemical Company, Ann Arbor, MI, USA). The lipid peroxidation index was measured by quantifying the TBARS concentration by following the protocol of Fraga and co-workers with little modification [31]. Briefly, 50 µL of cell extract was mixed with 50 µL SDS (3%), and the mixture was heated in a water bath after adding 200 µL of 0.1 N HCl, 30 µL of phosphotungstic acid (10%), and 100 µL of 2-TBA (0.7%). The TBRAS was extracted by n-butanol, and fluorescence was measured at λ_excitation 515 nm/λ_emission 555 nm under a fluorescence microscope (Olympus-1 × 70, Japan, software-Metamorph). The degree of protein carbonylation was assayed as per the established protocol [32]. Briefly, the sample was treated with an equal volume of 2,4-dinitrophenylhydrazine (1%) in 2 N HCl. After an hour, the mixture was treated with TCA (20%) and centrifuged. The precipitate was extracted with ethanol/ethyl acetate and dissolved in 8 M guanidine hydrochloride in 133 mM tris solution containing 13 mM EDTA. The absorbance was recorded at 365 nm. The extent of protein carbonylation was calculated using a molar extinction coefficient of 22,000 M⁻¹cm⁻¹. The levels of the CAT, SOD, GPx, and GST were assayed following methods described elsewhere [33]. In SOD assay, cell suspension containing 5 µg of protein was mixed with nitroblue tetrazolium (NBT), phenazine methosulphate, and sodium pyrophosphate, and the reaction was initiated by adding NADH. After 90 s, the reaction was terminated by adding glacial acetic acid, and the absorbance was measured at 560 nm. SOD activity was calculated as the enzyme concentration required inhibiting (µ-moles) of NBT-reduction/min. CAT activity was estimated spectrophotometrically by measuring the decomposition of 7.5 mM H₂O₂ at 240 nm for 10 min. CAT activity is defined as H₂O₂ consumption/min. In GPx estimation, H₂O₂ and NADPH were used as substrates, and NADPH to NADP⁺ conversion was estimated by measuring the changes in absorption intensity at 340 nm. GPx activity is defined as the amount of enzyme required to catalyze the conversion of 1 mol NADPH/minute. GST activity was measured spectrophotometrically at 340 nm based on the conjugation reaction with GSH in the first step of mercapturic acid synthesis. The reaction mixture comprises supernatant protein sample, EDTA, CDNB, KH₂PO₄ buffer, and GSH. The GST activity was measured as µmol of CDNB conjugate formed/min/mg protein. GSH and GSSG levels were estimated following the protocols developed by Hissin and Hilf [34]. In GSH estimation, the assay mixture comprising diluted cell extract, phosphate-EDTA buffer pH 8.0, and o-phthalaldehyde solution was incubated for 15 min. Fluorescence at 420 nm was measured with the activation at 350 nm. The GSH activity was expressed as nmol/mg of protein. In GSSG assay, diluted cell extract was mixed with 0.04 M N-ethylmaleimide and incubated at 30 min. Then, 0.1 N NaOH was added to the mixture. GSSG activity was measured following the procedure outlined above for GSH assay. The redox ratio was calculated as GSH/GSSG.

The cellular/subcellular protein samples (20 μg) obtained from NRK cells that received different treatments were resolved in 10% SDS-PAGE gel electrophoresis and immunoblotted as described earlier. The expressions of Bcl-2, Bad in the mitochondria, Cyt C in the cytosol, cleaved caspase 9, cleaved caspase 3, P-IκBα (Ser 32) in the cytosol, P-NF-κB p65 (Ser 536) in the nucleus, Keap1 in the cytosol, P-Nrf-2 (Ser40) in the nucleus, TGF-β1, P-Smad3 (Ser423/Ser425), Smad7, and collagen-IV were studied.