Physalin B reduce secretion of Aβ by inhibiting phosphorylation of STAT3 via down-regulated expression of β-secretase and γ-secretase

OBJECTIVE Extracellular amyloid-β(Aβ) plaques are one of the major pathological hallmarks of Alzheimer disease(AD). Therefore, decreasing Aβ levels is one strategyfor preventing the etiology of AD. Aβ peptides are generated from the cleavage of amyloid precursor protein(APP) by the β-secretase(BACE1) and γ-secretase(PS1). Inhibition of these secretases represents an obvious logical strategy to inhibit the generation of Aβ. In addition, signal transducer and activator of transcription 3(STAT3) is known to regulate many genes, and to significantly affect Aβ generation by controlling BACE1 and PS1 expression. Physalin B(PB), one of the major active steroidal constituents of solanaceaephysalis plants, possesses a wide variety of biological activities. PB down-regulates BACE1 and PS1 expression while it is unclear whether PB can regulate Aβ in N2 a/APPswe cells, and if so, whether it is by inhibiting the phosphorylation of STAT3. METHODS N2 a/APPswe cells were treated with PB in different concentrations for 24 h.(1) We used CCK8 method to detect the effects of different concentrations of PB on cell viability, and selected the best concentration for drug treatment to the cells.(2)The contents of Aβ40 and Aβ42 were determined by ELISA.(3) Western blotting was used to detect the expression levels of p-STAT3 and APP metabolism-related proteins, including APP, CTFα, CTFβ, BACE1,PS1, ADAM10 and so on.(4) RT-PCR was performed to detected the m RNA expression of BACE1 and PS1.(5) β-secretase activity Fluorometric assay kit was used to analyzed β-secretase activity.(6) In order to further explore the underlying mechanisms, N2 a/APPswe cells were pre-treated with 100 μmol·L-1 S3 I-201(a STAT3 inhibitor,can effectively prevent STAT3 phosphorylation) for 30 min and then treated with 3 μmol·L-1 PB incubated for 24 h. Then we evaluated the level of expression of STAT3 and p-STAT3 by Western blotting. RESULTS(1) CCK8 experiment results illustrated that PB did not show cytotoxicity at the applied concentration when cells were treated with PB(0, 0.3, 1 and 3 μmol·L-1). So, we used these concentrations in the following experiment.(2) ELISA results showed, compared to the control group, the contents of Aβ40 and Aβ42 decreased with the increasing of PB concentration.(3)According to Western blotting results, PB significant down-regulated the expression of BACE1, PS1, APP, CTFβ and p-STAT3 compared to the control group.(4) RT-PCR results indicated that PB can reduced the m RNA expression of BACE1 and PS1 effectively.(5) It turns out that PB can significantly inhibit BACE1 activity tested by BACE1 activity Fluorometric assay kit.(6) S3 I-201 had a similar manner to significantly inhibited STAT3 phosphorylation with PB through Western blotting. Moreover, co-treated with S3 I-201 and PB inhibited STAT3 phosphorylation much more than treatment with S3 I-201 alone. CONCLUSION These findings indicate that PB can effectively inhibit the expression of BACE1 and PS1 to reduce Aβ secretion by inhibiting the phosphorylation of STAT3.