Team:Nanjing high school/Results
Results
Figure 1. SDS-PAGE assay
First of all, 6×His-PPM1A was
purified with a Ni-NTA column
followed by AKTA FPLC according to the protocol. Then we used SDS-PAGE to test the purity of 6×His-PPM1A. As
shown in Figure 1, the 6×His-PPM1A was purified successfully.
Figure 2. PPM1A enzyme activity
Next, we screened the PPM1A
activator in Lab in-house
compound library by phosphatase enzyme activity assay. The effect of compounds 5 (0.01, 0.1, 1, 5, 10, 20,
40, 100 μM) on the PPM1A was detected by phosphatase activity assay with pNPP as the substrate. All data
were presented as mean ± S.E.M (*P<0.05, **P< 0.01, ***P<0.001).
As indicated in Figure 2, among the
compounds, Compound 5
was finally selected for its highest enzymatic activity against PPM1A. Besides, it could also tell that
compound 5 dose-dependently enhanced PPM1A enzyme activity. These results thus implied that compound 5 was a
PPM1A enzymatic activator.
Figure 3. Compound 5 suppressed inflammation in BV-2 cells
Finally, the qPCR assay was further
carried out to verify
the inhibitive effect of compound 5 against
PPM1A. BV-2 cells were co-incubated with LPS and different concentrations (5, 10, 20 μm) of compound 5 for
24 h. Then the mRNA level of IL-1β and IL-6 were detected by the qPCR assay. All data were presented as
mean±S.E.M (*P<0.05, **P< 0.01, ***P< 0.001).
As shown in Figure 3, LPS effectively increased the mRNA level of IL-1β and IL-6,
and compound 5 suppressed
this increase effectively. Thus, these results confirmed that the suppressive effect of compound 5 against
inflammation in BV-2 cells.
Conclusion
In this project, Compound 5 was
demonstrated as a PPM1A
activator and its anti-inflammatory effect was determined. We will investigate the regulation of PPM1A
against inflammation and the diseases related to inflammation in future scientific research work.