Figure 1.
Scavenging performance of purified LFC and HFC polysaccharides against chemically-generated free radicals in vitro.
A) hydroxyl radical (site specific); B) hydroxyl radical (non- specific); C) hydrogen peroxide; D) singlet oxygen; E) superoxide radical anion (non-enzymatic); F) superoxide radical anion (enzymatic); G) DPPH radical, H) total antioxidant capacity (ABTS.+ scavenging); and I) total antioxidant capacity (β-carotene linoleate model). Results are representative of three independent experiments performed in triplicate and are represented as mean ± SD. Inhibition concentration (IC50) values were calculated from pharmacological dose-response curve fit (sigmoidal) the equation: Y = Bottom + (Top-Bottom)/[1+10∧{(LogIC50-X)* Hill slope}].
Figure 2.
Partial structure elucidation of HFC polysaccharide by NMR spectroscopy.
The purified samples were exchanged with deuterium by lyophilizing several times with D2O. The polysaccharide was dissolved in 0.7 mL of D2O (99.96%) at concentrations of 15 mg/mL (for 1H NMR) and 30 mg/mL (for 13C NMR). Spectra were run at a probe temperature of 40°C. A) in the 1H NMR spectrum, black dotted line indicating α and β proton anomers, and dotted circles displaying H2–H5 and H6 (–CH3 group) of the polysaccharide directed from downfield to upfield. B) in the 13C NMR spectrum, downfield dotted circle stands for α and β carbon anomers, black dotted line indicating C2–C5, and upfield dotted circle signifying C6 (–CH3 group) of the polysaccharide.
Figure 3.
The possible structure interpreted for HFC polysaccharide.
Figure 4.
The effect of HFC polysaccharide on H2O2-induced morphological changes in WI38 cells.
The cells were incubated in presence (100, 200 and 250 µg/mL) or in absence of HFC polysaccharide for 1 h followed by the treatment with 300 µM H2O2 in both the cases for varying periods of time (0–24 h). Cell morphology was observed under microscope in phase contrast mode. Results are representative of three independent experiments performed in triplicate. Indicated scale bars signify 50 µm distance and photographs were taken at 10× zoom.
Figure 5.
The protective effect of HFC polysaccharide on H2O2-induced DNA damage during cell cycle progression.
WI38 cells were incubated in presence (100, 200 and 250 µg/mL) or in absence of HFC polysaccharide for 1 h followed by the treatment with 300 µM H2O2 in both the cases for varying periods of time (0–24 h). The cell cycle progression was assayed by PI staining with flow-cytometry. The corresponding data are shown as bar graphs. Results are representative of three independent experiments performed in triplicate and are represented as mean ± SD. A one-way analysis of variance (ANOVA, Bonferroni corrections for multiple comparisons) was performed, where significant level stands for *p<0.05, **p<0.001, ***p<0.0001.
Figure 6.
Inhibitory effects of HFC polysaccharide on H2O2-induced apoptosis of WI38 cells.
WI38 cells were incubated in presence (200 and 250 µg/mL) or in absence of HFC polysaccharide for 1 h followed by the treatment with 300 µM H2O2 in both the cases for varying periods of time (0–24 h). Cellular apoptosis was assayed by annexin V-FITC and PI counterstaining and analyzed with flow cytometry. Camptothecin was used as positive control. Dual parameter dot plot of FITC fluorescence (x-axis) versus PI fluorescence (y-axis) is represented as logarithmic fluorescence intensity. Quadrants: upper left necrotic cells, lower left live cells, lower right apoptotic cells, and upper right necrotic or late phase of apoptotic cells. The corresponding data are shown as bar graphs. Results are representative of three independent experiments performed in triplicate and are represented as mean ± SD. A one-way analysis of variance (ANOVA, Bonferroni corrections for multiple comparisons) was performed, where significant level stands for ** p<0.001, *** p<0.0001.
Figure 7.
Stabilization of H2O2-induced intracellular ROS by HFC polysaccharide.
WI38 cells were incubated in presence (200 and 250 µg/mL) or in absence of HFC polysaccharide for 1 h followed by the treatment with 300 µM H2O2 in both the cases for varying periods of time (0–24 h). ROS levels were monitored by flow cytometry using H2DCF-DA. N-acetyl cysteine (NAC) was used as positive control. The mean fluorescence indices (MFI) are shown as bar graphs. Results are representative of three independent experiments performed in triplicate and are represented as mean ± SD. A one-way analysis of variance (ANOVA, Bonferroni corrections for multiple comparisons) was performed, where significant level stands for ** p<0.001, *** p<0.0001.
Figure 8.
The effects of HFC polysaccharide treatment on H2O2-induced regulation of mitochondrial functions.
The polysaccharide prevented H2O2-induced changes in the expression of Bcl2 family at both mRNA and protein level. WI38 cells were incubated in presence (200 and 250 µg/mL) or in absence of HFC polysaccharide for 1 h followed by the treatment with 300 µM H2O2 in both the cases for varying periods of time (0–24 h). A) Mitochondrial membrane potential (MMP) was monitored by DiOC6 staining with flow cytometry. The mean fluorescence indices (MFI) are shown as bar graphs. B) Protein level expression of Bcl2, Bcl-xl, and Bad was evaluated by immunoblotting. β-actin was used as loading control. Fold changes are represented as relative values of band densitometries normalized to control and are shown as numbers below the immunoblots. Results are representative of three independent experiments performed in triplicate and are represented as mean value. C) The ratio between Bax and Bcl2 were calculated from band densitometries of corresponding protein level expressions and are shown as bar graphs. D) Fold changes of Bcl2, Bcl-xl, Bad, Bax, and cytochrome c at mRNA level were calculated using real-time RT-PCR (SYBR green method). Fold changes are represented as relative values normalized to control and quantified in the terms of 2−ΔΔCt. GAPDH was used as internal control. Results are representative of three independent experiments performed in triplicate and are represented as mean ± SD. A one-way analysis of variance (ANOVA, Bonferroni corrections for multiple comparisons) was performed, where significant level stands for * p<0.05, ** p<0.001, *** p<0.0001.
Figure 9.
Translocation of Bax and cytochrome c induced by H2O2 and HFC polysaccharide during the stress and polysaccharide treatment period, respectively.
WI38 cells were incubated in presence (250 µg/mL) or in absence of HFC polysaccharide for 1 h followed by the treatment with 300 µM H2O2 in both the cases for varying periods of time (0–24 h). A) Protein level expression of Bax and cytochrome c in both cytosolic and mitochondrial fractions was observed by immunoblotting. β-actin and COX4 were used as loading control. Fold changes are represented as relative values of band densitometries normalized to control and are shown as numbers below the immunoblots. Results are representative of three independent experiments performed in triplicate and are represented as mean value. A one-way analysis of variance (ANOVA, Bonferroni corrections for multiple comparisons) was performed, where significant level stands for * p<0.05, ** p<0.001. B) H2O2-induced release of cytochrome c from mitochondria to cytosol and re-localization into mitochondria again during the polysaccharide treatment were monitored under fluorescence microscope using fluorescence-tagged (green florescence) specific antibodies. Similarly, mitochondrial translocation of Bax and their cytosolic re-localization was also tracked following the same procedure. The cells were treated with 100 nM MitoTracker Red (red florescence) for 30 min before cell-fixation for mitochondrial staining. Each image shown is representative of 20 random fields observed. Indicated scale bars signify 10 µm distance and photographs were taken at 100× zoom.
Figure 10.
Polysaccharide imposed protection against H2O2-induced intrinsic apoptosis of WI38 cells via caspase inhibition at protein level.
WI38 cells were incubated in presence (250 µg/mL) or in absence of HFC polysaccharide for 1 h followed by the treatment with 300 µM H2O2 in both the cases for varying periods of time (0–24 h). Protein level expression of various pro- and active forms of caspases (caspase-9, -3, and -7) and cleaved PARP was evaluated by immunoblotting. β-actin was used as loading control. Band densitometries are represented as a ratio between pro and active caspases in the form of bar graphs, Results are representative of three independent experiments performed in triplicate and are represented as mean ± SD. A one-way analysis of variance (ANOVA, Bonferroni corrections for multiple comparisons) was performed, where significant level stands for * p<0.05, ** p<0.001.
Figure 11.
Caspase inhibition assay using pan- and specific caspase inhibitors for intrinsic pathway.
The cells were pre-incubated for 1 h individually with 100 µM pan caspase-inhibitor Z-VAD-fmk, caspase-9-specific inhibitor Z-LEHD-fmk (50 µM), caspase-3 and -7-specific inhibitor Ac-DEVD-CHO (50 µM). Then the cells were incubated in presence (250 µg/mL) or in absence of HFC polysaccharide for 1 h followed by the treatment with 300 µM H2O2 in both the cases for varying periods of time (0–24 h). A) The expression of active forms of caspases (caspase-9, -3, and -7) in the presence of the inhibitors was evaluated by immunoblotting. B) Apoptosis was quantified by flow cytometry as described earlier. β-actin was used as loading control. Fold changes are represented as relative values of band densitometries normalized to control and are shown as numbers below the immunoblots. Results are representative of three independent experiments performed in triplicate and are represented as mean value. A one-way analysis of variance (ANOVA, Bonferroni corrections for multiple comparisons) was performed, where significant level stands for * p<0.05, *** p<0.0001.
Figure 12.
Stabilization of Nrf2/Keap1 signaling and their nuclear translocation induced by HFC polysaccharide.
WI38 cells were incubated in presence (250 µg/mL) or in absence of HFC polysaccharide for 1 h followed by the treatment with 300 µM H2O2 in both the cases for varying periods of time (0–24 h). A) Protein level expression of Nrf2 and Keap1 in both cytosolic and nuclear fractions was observed by immunoblotting. β-actin and Lamin A were used as loading control. Fold changes are represented as relative values of band densitometries normalized to control and are shown as numbers below the immunoblots. Results are representative of three independent experiments performed in triplicate and are represented as mean value. A one-way analysis of variance (ANOVA, Bonferroni corrections for multiple comparisons) was performed, where significant level stands for *p<0.05. B) H2O2-induced translocation of Nrf2 and Keap1 from cytosol to nucleus and re-localization into cytosol once again during the polysaccharide treatment were monitored under fluorescence microscope using fluorescence-tagged (red and green florescence, respectively, for Nrf2 and Keap1) specific antibodies. The cells were counterstained with DAPI (blue fluorescence) to visualize nuclear morphology. Each image shown is representative of 20 random fields observed. Indicated scale bars signify 10 µm distance and photographs were taken at 100× zoom.
Figure 13.
MAPK-mediated activation of Nrf2/Keap1 signaling during H2O2-induced apoptosis of WI38 cells.
The cells were incubated in presence (250 µg/mL) or in absence of HFC polysaccharide for 1 h followed by the treatment with 300 µM H2O2 in both the cases for varying periods of time (0–24 h). A) Protein level expression of phosphorylated JNK and p38, and cytosolic Nrf2 in the presence of JNK- and p38- specific inhibitors, SP600125 and SB203580, respectively. Before any other treatment, WI38 cells were pre-incubated with 10 µM inhibitors for 1 h separately. β-actin was used as loading control. Fold changes are represented as relative values of band densitometries normalized to control and are shown as numbers below the immunoblots. Results are representative of three independent experiments performed in triplicate and are represented as mean value. B) The mRNA level expression of Nrf2 and Keap1 was quantified using real-time RT-PCR. RNA was extracted from the treated and untreated WI38 cells and after enzymatic reverse transcription, the cDNA content was analyzed by electrophoresis in 2% agarose gel containing 0.1% ethidium bromide. Fold changes were calculated using real-time RT-PCR (SYBR green method). C) Similarly, the expression of HMOX1, NQO1, SOD1, GPX1, and GASTA2 at mRNA level was quantified. Fold changes are represented as relative values normalized to control and quantified in the terms of 2−ΔΔCt. GAPDH was used as internal control. Results are representative of three independent experiments performed in triplicate and are represented as mean ± SD. A one-way analysis of variance (ANOVA, Bonferroni corrections for multiple comparisons) was performed, where significant level stands for * p<0.05, ** p<0.001, *** p<0.0001.
Figure 14.
Plausible signaling cross-talk involved in the HFC polysaccharide treatment against H2O2-induced apoptosis of WI38 cells.