Figure 1.
WSM enhances alkaline phosphatase activity (ALP) in MC3T3-E1 cells.
A: Effect of WSM on cell viability percentage as measured by MTT assay indicating non-toxic nature of WSM for MC3T3-A1 cells. B: ALP activity measured by ALP assay kit, shows increases in ALP activity after WSM treatment. Data represent mean -SD. *p ≤ 0.001 (vs. control) where n= 6. C: Histochemical staining of MC3T3-A1 cells for ALP activity as observed under phase contrast microscope confirms that WSM increases ALP activity. D: One of the plates (out of triplicate) after ALP staining. Results are expressed as mean -SEM (n= 3). *p ≤ 0.05 (vs. control).
Figure 2.
WSM up-regulates osteoblast differentiation markers OCN and COL-1-A2 and accelerates bone nodule formation in MC3T3-A1 cells.
A (a-b): Detection of transcriptional levels of osteoblast differentiation marker genes viz. osteocalcin (OCN) and pro-alpha 2(I) collagen (COL-1A2) in RT-PCR of MC3T3-A1 cells, with and without WSM (0.025% w/v) treatment for 24 h. The expression of GAPDH, as housekeeping gene was qualitatively (a) detected by the presence of bands in all experiments. Significant increase in transcript (b) of both the marker genes was observed after WSM treatment. B (a-c): Alizarin red S staining of WSM treated (b) and control (a) MC3T3-E1 cells indicating WSM (0.025%, w/v) accelerates calcium deposition and enhances nodule formation. Black arrows indicate the mineral nodules. Quantification (c) of staining intensities were consistent with the visual observation. C (a-c): von Kossa-von Giesen staining of WSM treated (b) and control (a) MC3T3-E1 cells indicating WSM (0.025% w/v) enhances nodule formation. Black arrows indicate the mineral nodules deposition. Quantification (c) of staining intensities were consistent with the visual observation. Results are expressed as mean -SEM (n= 3). *p ≤ 0.05 (vs. control).
Figure 3.
WSM scavenges the ABTS and DPPH free radicals and inhibits lipid peroxidation
Percentage inhibition for scavenging capacity of WSM, assayed with three different methods. i.e. ABTS cation decolorization (at 734 nm), DPPH free radical decolorization (at 517 nm) and lipid peroxidation inhibition by measuring the TBARS production (at 532 nm). Results are expressed as mean -SEM (n= 6). *p ≤ 0.05 (vs. control).
Figure 4.
WSM reduces H2O2 and UV-B induced oxidative damage in HaCaT cells.
A: Effect of WSM on cell viability percentage as measured by MTT assay indicating non-toxic nature of WSM for HaCaT cells. B: Effect of WSM on proliferation of HaCaT cells exposed to H2O2 (0.25 and 0.5 mM) for 1 h. C: Effect of WSM on proliferation of HaCaT cells exposed to UV-B (30 and 40 mJ/ cm2). D: Effect of WSM on morphogenetic changes in HaCaT cells induced by H2O2 and UV-B and observed by phase contrast microscopy (200 X). Results are expressed as mean -SEM (n= 6). *p ≤ 0.05 (vs. control).