a. Cell Lines: Human umbilical vein endothelial cells (HUVECs, cat. no. C2519A; Lonza, Walkersville, MD) and dermal-derived Human Microvascular ECs were obtained from Lonza, Inc. (Walkersville, MD). Briefly, endothelial cells were plated at approximately 2,500 cells/cm2 in endothelial cell growth medium (EGM) followed by incubation at 37°C in 5% CO2. EGM was supplied with 10 µg/L human recombinant epidermal growth factor, 1.0 mg/L hydrocortisone, 50 mg/L gentamicin, 50 µg/L amphotericin B, 12 µg/L bovine brain extract, and 2% fetal bovine serum. No insulin was present in the media. Cells were grown in 25cm2 tissue culture flasks. The endothelial cells at the 6th passage (80% confluent) were serum-starved for 24 h before exposure to glucose or other reagents. The endothelial cells were cultured following the maximum aseptic procedure. Mycoplasma contamination was prevented by gentamicin and amphotericin B present in EGM. The morphology of the cells was checked by inverted phase contrast microscopy.
b. Reagents:
All reagents were obtained from Sigma-Aldrich (Oakville, ON, Canada) unless otherwise specified.
c. Incubation of HUVECs and HMECs with 5-Aza-Dc and ginseng before treatment with glucose:
HUVECs and HMECs were incubated with various concentrations of D- glucose ranging from 5 to 25 mmol/L for 24, 48, and 72 h. 5 mM D-gluocse represented euglycemia and 25 mM D-glucose represented hyperglycemia.
All the cell types were also incubated in an equiosmolar glucose medium as for the control (5 and 25mM L-glucose). 5-aza-2'-deoxycytidine (5-Aza-Dc, 5µM) (Sigma, St. Louis, USA) was dissolved in Dimethyl sulfoxide (DMSO) at room temperature for 30 m and added to HUVECs and HMECs 30 m prior to the addition of D-glucose. 5-Aza-Dc treated ECs along with their respective controls were collected at 24, 48, and 72 h for further experiments. The cell culture incubations were performed at 24, 48, and 72 h for investigating the rates of DNA methylation in a time dependent manner. The powdered extract of ginseng (2.5, 5.0, 7.5 and 10.0 µg/ml), in DMSO was added 30 m before incubation with glucose at various concentrations of the EGM. Control groups received equivalent amount of DMSO. The reagent concentrations are based on the previous studies from our laboratory (Sen et al. 2011).
d. Cell survival assay:
ECs were seeded onto 96-well plates at a density of 1.0 × 104 cells per well in 100 µl medium. Following exposure to appropriate reagents for 24 h, cell viability assay were performed. For such assay 10 µl of 4-[3-(4-Iodophenyl)-2- (4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene Disulfonate (WST-1, Roche Diagnostic Canada, Laval, Quebec, Canada) was added to each well and cells were incubated for 4 h at 37°C. A colorimetric determination (at 450 nm), based on the cleavage of the tetrazolium salt WST-1 by mitochondrial dehydrogenases in viable cells were carried out on a microplate reader (Bio- Rad Laboratories, Hercules, CA, USA). No difference in viability was observed in cells treated with low (5 mmol/l) or high glucose (25 mmol/l) up to 72 h. The cells in HG, however, showed a significantly lower proliferation rate compared with cells in low glucose (Chen et al. 2003). WST-1 assay was employed here because it has been reported to be more reliable than MTT or other cell viability a ssays in the cytotoxicity analysis of natural products such as ginseng and 5-aza- Dc. WST assay, has much lower cytotoxicity compared to MTT assay and is stable for up to 48 hours. Additionally, assays namely BrdU for cell proliferation and Propidium iodide staining for cellular apoptosis should have been performed for more broader assessment.
e. Animal experiments:
All animals were cared for according to the Guiding Principle in the Care and Use of Animals. All experiments were approved by the University of the Western council on the animal care committee [Protocol (AUP)-2010-001]. All experiments were performed following the ARRIVE Guidelines 2.0. Male C57BL/6 mice weighing 23–36 g were obtained from Charles River (Montreal, QC, Canada). The animals were maintained at 24–26°C, 60–80% relative humidity and on a 12-h light–dark cycle and were fed a standard diet and allowed free access to water. To create a model of T1DM, groups of animals received three i.p injections of STZ (50 mg/kg in citrate buffer, pH 5.6) on alternate days (Sen et al. 2012; Sen et al. 2013). The doses of 50–70 mg/kg of STZ produce long-lasting diabetes. Controls were injected with the same volume of citrate buffer. The animals were divided into 3 groups: Control: C, Diabetic (D1: 1 month and D2: 2 months, n = 5) and diabetic treated with the alcoholic extract of North American ginseng root extract [Ontario Ginseng Growers Association, OGGA, https://ofa.on.ca/federations/ontario-ginseng-growers-association/, 200 mg/Kg b.w for 1 month (DT1) and 2 months (DT2), (n = 5)] (Sen et al. 2012; Sen et al. 2013). Pre-clinical toxicological studies on oral administration of ginseng extracts have reported LD50 values of 750 mg/kg in rats and 200 mg/kg in mice respectively. Chronic studies in rats and mice treated with ginseng at doses up to 5000 mg/kg for 2 years did not show any toxic effects; furthermore, no increases in the incidence of cancer or non-neoplastic lesions were detected (National Toxicology Program 2011). The diabetic mice were implanted with insulin pellets that released small doses (0.5 units) of insulin to prevent glucosuria, ketonuria and hypoglycemia (1 U/day, LinShin Canada Inc., Toronto, ON, Canada). Insulin pellet implantation was necessary for the survival and long-term study of the T1DM mice for 1 and 2 months. The mortality rate in the STZ treated diabetic (D) and DT1 and DT2 was nil. After 1 and 2 months animals were sacrificed by cervical dislocation and kidneys were collected. The animals were sacrificed following the guidelines of the American Veterinary Medical Association. The serum glucose, body wt., and HBA1c levels of all the groups are presented in Table 1.
f. DNA isolation:
DNA from the ECs was isolated following the procedure as stated. Briefly, the culture medium was centrifuged at 3000g for 5 m to collect detached cells. Cells were lysed with a lysis buffer (10 mM Tris–HCl, pH 8.0, containing 10 mM EDTA, and 0.5% Triton X-100). RNA and proteins were digested using 0.1 mg/ml RNase at 37°C for 1 h, followed by 0.5 mg/ml proteinase K digestion at 37°C for 24 h. Total DNA was isolated by phenol/chloroform extraction and by absolute ethanol precipitation followed by ethanol (70%) wash. The supernatant was removed gently, and the DNA pellet was air-dried and dissolved in TE buffer (pH 8.0). DNA isolation from the mice kidney was performed by following the same protocol as mentioned above. The concentration of DNA was measured spectrophotometrically OD 260 nm/OD 280 nm = 0.92 nm.
g. Bisulfite conversion of genomic DNA:
Genomic DNA (2 µg) was treated with bisulfite using the Epitect DNA bisulfite treatment kit (Qiagen). The highly sensitive method utilizes innovative protection against DNA degradation that guarantees sensitive results, even from 1 ng DNA, and ensures high conversion rates of over 99%. Bisulfite conversion of the DNA solution was performed by a thermal cycler following the protocol as mentioned: Denaturation: 5 m − 95°C, Incubation: 25 m − 60°C, Denaturation: 5 m − 95°C, Incubation: 85 m − 60°C, Denaturation: 5 m − 95°C and Incubation: 175 m − 60°C. Converted DNA was quantitated as RNA using a UV spectrophotometer (NanoDrop) with Ab 260 nm 1.0 = 40 µg/ml. After the amplification process, the PCR product was digested with 2U Taq I restriction enzyme (Thermo Fisher Scientific, Waltham, MA, USA) in Taq I buffer (Thermo Fisher Scientific, Waltham, MA, USA) and incubated at 65°C overnight. The cut PCR product was separated on 2% agarose gel and followed by ethidium bromide staining. The intensities of the DNA fragments were quantified by gel densitometry.
h. Hot-start PCRs of Alu, B1, and LINE-1 elements of the bisulfite converted DNA derived from the ECs and kidney tissues:
Hot- start PCR of Alu, B1, and LINE-1 elements of the bisulfite converted DNA derived from the ECs and kidney tissues were performed. The superarray master mix of 50 µl for Alu and B1 elements contained PCR buffer: 12 µl, MgCl2: 1.5 mM, dNTPs: 200 µM, Taq pol (platinum taq) : 1.5 units, DNA: 2 µl (50ng) and remaining volume adjusted by adding dH2O. The concentration of the primers used was 50 pmoles. Sequences of the forward and reverse primers for Alu are: Forward: 5’-ATTTTAGTATTTTGGGAGGTCGAGGC-3' and Reverse: 5'--GCAATCTCGACTCACTACAAA CTCCG-3' (Erichsen et al. 2018). PCR condition for Alu: 94°C: 12 m Hot -start, 94°C: 30 s Denaturation, 55°C: 30 s Annealing, 72°C: 30 s Elongation, 72°C: 10 m Final elongation. Total: 42 cycles. PCR products were separated in 1.5% agarose gel. LINE-1 PCR of the bisulfite converted DNA isolated from the ECs was performed following the protocol as mentioned here. Each PCR mix contained the forward and reverse primer (each 0.4 µmol/L), 0.8 µmol/L of dNTPs, 1.5 mmol/L of MgCl2, 1× PCR buffer (Qiagen), 0.64 U of Taq pol (Qiagen), and 2 µl of bisulfite template DNA in a total volume of 20 µl. PCR conditions: initial denaturing at 95°C for 15 m; 45 cycles of 95°C for 20 s, 50°C for 20 s, and 72°C for 20 s; and final extension at 72°C for 5 m. The sequences of the forward and reverse primers are: Forward: 5'-GCGCGAGTCGAAGTAGGGC- 3' and Reverse: 5'-CTCCGACCAAATATAAAATATAATCTCG-3 (Erichsen et al. 2018). Amplification of an adjacent region without CpGs was used to normalize the amount of LINE-1 / Alu DNA methylation. The primers used for normalization were for LINE-1: forward: 5′-AGGTTTTATTTTTGGGGGTAGGGTATAG-3′; reverse: 5′-CCCCTACTAAAAAA TACCTCCCAATTAAAC-3′ and for Alu: forward: 5′-GGGTGGATTATGAGGTTAGGAGAT-3′; reverse: 5′-CATTCTCCTACCTCAACCTCCC-3′ (Erichsen et al. 2018). The primers were designed following the PrimerBLAST designing tool. The PCR products were separated in 2% agarose gel electrophoresis.
i. Quantification of methylated DNA in ECs by ELISA:
DNA methylation in the low glucose (5mM, LG), and high glucose (25 mM, HG) treated ECs and renal tissues were measured by the Imprint® Methylated DNA Quantification (Sigma-Aldrich). This is essentially a quantification protocol following the Deoxynucleoside triphosphate (dNTP) method. The control methylated DNA along with 100 ng of the isolated DNA from treated endothelial cells were incubated in the coated wells separately for 60 m at 37°C for adequate binding. The reaction was blocked thereafter by adding the block solution to each well for 30 m. This was followed by the addition of detection antibody and incubation for 15 m. Finally, the developing solution was added and absorbance was measured at 450 nm. Methylation was quantified from a standard plot of the increasing concentrations of methylated DNA supplied by the kit. DNA degradation experiment by agarose gel (1%) electrophoresis was performed before proceeding for ELISA.
j. Detection of the DNA methylation status of the FN1, VEGFa, and EDN1 genes:
DNA methylation status of FN1, VEGFa, and EDN1 genes was estimated by using the EpiTect Methyl II PCR Assays (Qiagen). The EpiTect Methyl II PCR Array system involves the differential cleavage of target sequences by two different restriction endonucleases whose activities require either the presence or absence of methylated cytosines in their respective recognition sequences. The real-time PCR quantifies the relative amount of DNA remaining after each enzyme digestion from which the methylation status of individual genes can be calculated. Briefly, equal amounts of each genomic DNA sample were added to the components of the EpiTect Methyl II DNA Restriction Kit to set up 4 different restriction digests: mock (Mo), methyl- sensitive (Ms), methyl-dependent (Md), and double (Msd). This is followed by digestion and heat inactivation of the enzymes. An aliquot of each digest was subjected to real-time PCR containing the appropriate RT2 SYBR Green Mastermix and the EpiTect Methyl II PCR assay following the recommended cycling program. The calculated CT values of each digest were then pasted into the correct excel-based data analysis template for the array format to calculate the percentage of methylated DNA.
k. Estimation of oxidative stress-related parameters in the ECs and renal tissue:
i) Superoxide anion generation:
The superoxide anion level was measured by using the chemiluminescence method (Superoxide anion detection kit, Calbiochem, EMD Chemicals, Inc.). The principle of this assay is based on, the superoxide anion generated causes oxidation of luminol leading to photon generation that is readily measurable by a luminometer. This kit utilizes an enhancer (phorbol-12- myristate-13-acetate, PMA) that increases the sensitivity of the assay by amplifying the chemiluminescence. After incubation of ECs with various reagents, cells were centrifuged and resuspended in 1 ml of supplemented growth medium and incubated for 30 m at 37◦C. The cells were then centrifuged and treated with superoxide anion assay medium. The light emission from each of the samples was recorded at regular intervals. Superoxide anion was measured in kidney tissues after extraction with dimethyl sulfoxide–tetrabutylammonium chloride (DMSO–TBAC) solution. TBAC–O2 •− complex was then detected by use of the luminol–EDTA–Fe enhanced chemiluminescence method (Yao et al. 2004).
ii) Mitochondrial ROS analysis:
Dichlorodihydrofluorescein diacetate (DCFH-DA) was used for determination of the mitochondrial ROS production (Biswas et al. 2007). DCFH-DA is able to readily penetrate the cell membrane and the diacetate esteric form can be rapidly de-esterified by the intracellular esterase to yield DCFH free form. DCFH is oxidized to yield the fluorescent DCF which correlates with the amount of ROS formed. ECs in 96-well plates pretreated with reagents were incubated with DCFH-DA, followed by washing with 10.0 mM HEPES buffer (pH 7.4) to remove the extracellular DCFH-DA. The fluorescence was measured at an excitation wavelength of 490 nm and emission wavelength of 525 nm using a multi-plate fluorescence plate reader (Molecular Devices, Sunnyvale, CA, USA). Control experiments were performed.
iii) Analysis of oxidative DNA damage:
ECs were analyzed for 8-OHdG expression, a marker for oxidative DNA damage using well-established methods in our laboratory (Chiu et al. 2009). Cells were plated on eight chamber-tissue culture slides and incubated for 24 h. Following treatment with specific reagents, cells was fixed with 1:1 methanol: acetone. The cells were stained with mouse monoclonal 8-OHdG antibody (1:50, SantaCruz Biotech) and FITC-conjugated goat anti-mouse antibody (1:200, invitrogen, Oregon, USA). Non-immune horse serum was used as the negative control. Slides were mounted in DakoCytomation Fluorescent Mounting Medium (DakoCytomation). Microscopy was performed by an examiner unaware of the identity of the sample using a Zeiss LSM410 inverted laser scan microscope equipped with fluorescein (Carl Zeiss Canada, North York, ON, Canada). The presence of 8-OHdG manifested as positively stained nuclei.
l) Nuclear protein extraction and NF-kappaB assay:
NF-kappaB p(65) estimation in the nuclear extracts was performed in all the groups (both treated and untreated) of ECs and renal tissue. The nuclear protein of ECs was prepared as described elsewhere, with some modifications (Schreiber et al. 1989). Briefly, octamer binding proteins with “mini-extracts” was prepared from a small number of cells. The cells were washed, resuspended in phosphate-buffered saline, and centrifuged (7000 × g for 15 s). The pellet was resuspended in 0.4 ml of cold buffer A [10 mmol/l HEPES, pH 7.9, 10 mmol/l KCl, 0.1 mmol/l EDTA, 0.1 mmol/l EGTA, 1 mmol/l 1,4- dithiothreitol (DTT), and 0.5 mmol/l PMSF] by gentle pipetting. The cells were allowed to swell on ice for 15 m. 25 µl of a 10% Igepal CA-630 were added, and cells were vortexed vigorously. The homogenate was centrifuged (10,000 g for 30 s). The nuclear pellet was resuspended in 50 µl of ice-cold buffer C (20 mmol/l HEPES, pH 7.9, 0.4 mol/l NaCl, 1 mmol/l EDTA, 1 mmol/l EGTA, 1 mmol/l DTT, and 1 mmol/l PMSF), and the tube was vigorously rocked at 4◦C for 15 m on a shaking platform. The nuclear extract was centrifuged at 4◦C (15,000 × g for 5 m), and the supernatant was frozen at − 70◦C. The protein concentrations were measured using the BCA protein assay, with BSA as a standard (Pierce, IL). NF-kappaB p(65) protein was estimated in the nuclear extracts of all groups of ECs by ELISA following the standard manufacturer’s (TransAM transcription assay kit, CA, USA) protocol. A similar protocol was followed for the estimation of NF-kappaB p65 in the renal tissues.
m) Statistical analysis:
The data are expressed as mean ± SE. Statistical significance was determined by analysis of variance (ANOVA) followed by the Bonferroni– Dunn test. Differences were considered to be statistically significant at values of p < 0.05.