signaling pathways. We hypothesized that if inhibition of a signaling pathway eliminated the effect of PAM3, PAM3 may be altering that pathway in some way to provide prosurvival effect against ischaemia. Cells were plated in a 96 well plate for overnight. To fix concentration for next experiment they were than treated with a wide range of concentrations (0.5, 1, 2.5, 5, 10 uM) of inhibitors for kinases and receptors with or without OGD.
We selected inhibitors, which are widely used previously. For p38 MAPK data demonstrated that SB203580 is the most extensively studied compound and specific for all isoform of p38 specially α and β (Lee et al. 1994). Regulation of genes greatly elucidate by SB203580, inhibitor of p38 MAPK (Ono and Han 2000). p38MAPK inhibition by SB203580 can attenuates OPC differentiation (Chew et al. 2010). U0126 reduced phospho-active ERK levels and effect was unaltered in normal tissues (Marampon et al. 2011).
U0126 can revert the slow Wallerian degeneration by inhibiting ERK1/2 MAPK (Evans et al. 2013). BAY 11-7085 can blocks NFκB pathway and known to inhibit IKK, and thereby inhibits IκB phosphorylation and NFκB activation (Manna, Manna and Sarkar 2007, Dai et al. 2005, Hammar et al. 2005).
Cells were seeded in 96-well plate, after overnight incubation cells were treated with or without OGD and inhibitor was added during and after OGD. SB203580 showed dose dependent effect on naïve cell and almost dose independent manner on OGD treated cell (Fig.4 A). 1 uM concentration was less toxic then others concentration. However, U0126 effect was dose dependent on naïve and OGD treated cells but was causing significant cell death on naïve cells even in 0.5 uM concentration (Fig.4 B). So U0126 considered as toxic for cell or as ERK1/2 is the upstream of all MAP pathways, so inhibiting this pathway might be harmful for cell. In case of NFκB inhibitor BAY11-7085 was not dose dependent but 1uM could be consider as less toxic for cell (Fig.4 C). So for future experiment I have
19 Figure 4: Dose dependent effect of inhibitors
Cells were treated with increasing concentrations of potent and selective inhibitors. Graph showing effects of different doses inhibitor of P38 MAPK, ERK1/2 and NFκB on OLs.
Cell was count by CCK assay. Graphs expressed as mean ± SEM. (N=5 from 3 to 4 culture). Graphs expressed as mean ± SEM. p < 0.05, p < 0.01 and p <0.001 indicated *, **
and ***.
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selected 1uM concentration for all inhibitors and treated cell simultaneously with PAM3, and measure cell viability and cytotoxicity by CCK and LDH assays.
PAM3 was then added to the cell during and after OGD with 1uM concentration of all inhibitors. CCK assay represent that SB203580 and BAY 11-7085 can reverse the effect of PAM3 significantly compare with the PAM3 only effect on OGD (Fig.5 B, C). All 3 inhibitor presenting significant cell death after OGD compare with control group both in CCK assay. Though U0126 decreased cell number in control condition significantly, I want to confirm relation between ERK1/2 and PAM3. But in OGD after addition of ERK1/2 inhibitor demonstrated significant cell death which can be moderate by PAM3 addition. It proves that ERK is not mediated by TLR2 (Fig. 5 A).
Significant cell death was caused by OGD with SB203580. After adding PAM3 result showing that cell number was significantly decreased compare with PAM3 even compare with OGD only group (Fig. 5 B). Same effect exerted by BAY 11-7085, NFκB inhibitor.
Addition of PAM3 could not provide protective effect and cell number was significantly decreased compare with PAM3 only group (Fig. 5 C). So these two inhibitor block PAM3 mediated survival which proved the relationship of these two pathways with TLR2 mediated cell survival.
Same effect like CCK assay was elucidated by LDH assay (Fig.6). After OGD, LDH level was significantly increased meaning significant cell death. PAM3 decreased the LDH level by providing protective effect. LDH level of PAM3 treatment in U0126, ERK1/2 inhibitor group was same like only PAM3 treated group (Fig. 6 A). Both group showing significant decreased of LDH. That indicate PAM3 can improve cell survival and ERK1/2 was not provide PAM3 mediated cell survival.
However, significantly high level of LDH even after adding PAM3 exerted by SB203580, p38 MAPK inhibitor and BAY 11 7085,NFκB inhibitor group (Fig. 6 B,C).
proved that in absence of p38 MAPK and NFκB, PAM3 not able to provide any protective effect. These results can confirm the involvement of these 2 pathways with PAM3.
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Figure 5: p38 MAPK or NFκB inhibitor block PAM3CSK4-mediated prosurvival effects on OLs under ischemic stress in CCK assay.
Quantification graphs demonstrated the PAM3 mediated effects of p38 qnd NFκB inhibitors on Ols. Cells were count by CCK assay. Here, * for compare to control, # for compare to OGD, ¶ for compare to OGD and PAM3. *,#; **,## and ***, ###, ¶¶¶ indicate p < 0.05, p < 0.01 and p < 0.001 by one-way ANOVA followed by Tukey’s posthoc analysis. (N=5 from 3 to 4 culture for each group).
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Figure 6: p38 MAPK or NFκB inhibitor block PAM3-mediated prosurvival effects on OLs under ischaemic stress in LDH assay.
Quantification graphs demonstrated the PAM3 mediated effects of p38 and NFκB inhibitors on OLs. Cells were count by CCK assay. Here, * for compare to control, # for compare to OGD, ¶ for compare to OGD and PAM3. p < 0.05 and p < 0.001 indicate *,#
and ***, ###, ¶¶¶ by one-way ANOVA followed by Tukey’s posthoc analysis. (N=5 from 3 to 4 culture for each group).
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5. Genetic knockdown of p38 MAPK and NFκB attenuates PAM3 mediated