Journal: Cells

Article Title: Salidroside Derivative SHPL-49 Exerts Anti-Neuroinflammatory Effects by Modulating Excessive Autophagy in Microglia

doi: 10.3390/cells14060425

Figure Lengend Snippet: SHPL-49 exerts anti-inflammatory effects by suppressing the NF-κB signaling pathway. ( A ) Immunohistochemical staining images of NF-κB and IL-6 in brain tissue sections from rats subjected to pMCAO and treated with SHPL-49 (15 mg/kg) and ED (7.5 mg/kg) for three days, Scale = 100 μm. ( B ) The IODs for NF-κB and IL-6 immunohistochemistry in brain tissue sections for each treatment group. ( C ) Representative Western blot of NF-κB in the nucleus and cytoplasm of OGD-BV2 cells after treatment with SHPL-49 (200 μM). ( D ) Quantification of the Western blot results. ( E ) Immunofluorescence staining pattern of NF-κB in OGD-BV2 cells following treatment with SHPL-49 (200 μM); Scale = 10 μm. ( F ) Quantification of the average fluorescence intensity of NF-κB in the nucleus of BV2 cells. ( G ) Representative Western blot of p-IKKβ, IKKβ, p-IκBα, and IκBα proteins in OGD-BV2 cells after treatment with SHPL-49 (200 μM). ( H ) Quantification of the Western blot results. ( I ) The protein expression levels of inflammatory factors IL-6, IL-1β, and iNOS in the supernatant of OGD-BV2 cells following treatment with SHPL-49 (200 μM). Data are presented as means ± SD with n = 6 per group. *** p < 0.001 vs. Ctrl; ## p < 0.01, ### p < 0.001 vs. OGD.

Article Snippet: Membranes were incubated with 5% nonfat milk (Beyotime, Shanghai, China, P0216) or BSA (Beyotime, Shanghai, China, ST023) followed by overnight incubation at 4 °C with the following primary antibodies: NF-κB (1:1000, Cell Signaling Technology, MA, USA, D14E12), p-IKKβ (1:1000, UpingBio, Hangzhou, Zhejiang, China, YP-Ab-14443), IKKβ (1:1000, Boster, Wuhan, China, BM4875), p-IκBα (1:1000, UpingBio, Zhejiang, China, YP-Ab-01253), IKBα (1:1000, UpingBio, Zhejiang, China, YP-Ab-01830), P62/SQSTM1 (1:1000, Boster, Wuhan, China, BM4385), LC3B (1:200, PTMab, Zhejiang, China, PTM-6384), ATG5 (1:1000, Cell Signaling Technology, MA, USA, D5F5U), Bclin1 (1:1000, Cell Signaling Technology, MA, USA, D40C5), LAMP2 (1:200, UpingBio, Zhejiang, China, YP-Ab-14094), Bax (1:1000, Cell Signaling Technology, MA, USA, 14796), Bcl-2 (1:1000, AiFang biologic, Hunan, China, AF0060), Cleaved caspase-3 (1:1000, Cell Signaling Technology, MA, USA, 9661S), and Cleaved caspase-9 (1:1000, Cell Signaling Technology, MA, USA, 9507S).

Techniques: Immunohistochemical staining, Staining, Immunohistochemistry, Western Blot, Immunofluorescence, Fluorescence, Expressing