Fig 2: Catalpol activated Keap1/Nrf2/ARE pathway in H2O2-treated RPE cells. (a and b) ARPE-19 cells were pretreated with or without catalpol (10, 20 and 40 μM) for 24 h, and then treated with 400 μM H2O2 for 6 h. The expressions of HO-1, NQO1, Keap1, Total-Nrf2 and Nuclear-Nrf2 were measured with Western blot assay. Histone H3 was used as an internal control for nucleoprotein, while β-actin was used as an internal control for total protein. (c) The effect of catalpol on the formation of Keap1/Nrf2 complex was determined using co-immunoprecipitation assay. (d) Treatment with ML385, a Nrf2 inhibitor, blocked the antioxidant protective effects of catalpol on increases in the expression of Nrf2 protein in H2O2-treated ARPE-19 cells. (e) Changes in gene expression of NQO1 were assayed with qRT-PCR. Data are presented as mean ± S.D. of three independent experiments (* p < 0.05 vs. control group, # p < 0.05 vs. H2O2 (400 μM)-treated group).

Fig 3: ROS participates in the effect of miRNA-200a-3p on inflammation in vitro. (A and B) ROS levels were assessed in cells treated with miR-200a-3p plasmid and ROS inhibitor (magnification, ×200). (C) Caspase-1 and (D) NLRP3 protein expressions were determined by (E) western blotting analysis. (F) IL-1β and (G) IL-18 levels were also determined. Data are presented as the mean ± standard error of the mean. ##P<0.01 vs. negative group; **P<0.01 vs. miR-200a-3p overexpression group. Negative, negative group; miR-200a-3p, miR-200a-3p overexpression group; ROS inhibitor, overexpression of miR-200a-3p and ROS inhibitor group; miR, microRNA; NLRP3, NLR family pyrin domain containing 3; Keap1, Kelch like ECH associated protein-1; Nrf2, nuclear factor erythroid 2 like 2; HO-1, heme oxygenase; ROS, reactive oxygen species; IL, interleukin.

Fig 4: Knockdown of MALAT1 attenuates neuroinflammation caused by LPS/ATP treatment in BV2 cells. After the BV2 cells were treated with LPS/ATP, LPS/ATP + sh-NC, and LPS/ATP + sh-MALAT1, a the expression of MALAT1 was analysed by qPCR. b The analysis of the cytokines TNF-α, IL-1β and IL-18 through ELISA for the different groups. c The mRNA levels of TNF-α, IL-1β and IL-18 were measured by qPCR in different groups. d The ROS levels in different groups were determined by fluorescence imaging of DCFH-DA. e The western blot analysis of cleaved caspase 1, NLRP3, ASC, Nrf2 and HO-1 levels in different groups. f The mRNA levels of HO-1, NQO-1, and SOD2 in different groups were measured by qPCR. The data in the graph are presented as the means ± SD as the relative levels from three replications. *p < 0.05, **p < 0.01, ***p < 0.001

Fig 5: BC-1901S increases NRF2-dependent gene transcription and decreases inflammation. A) RT-qPCR of GPx2 and HO-1 in Beas-2B cells treated with BC-1901S at 0, 0.1, 1, and 10 μM. Fold change (RQ) is normalized to endogenous GAPDH expression. Data and mean ± SD of 3 independent experiments. B) Beas-2B cells were transfected with an ARE-reporter (pGL4.37[luc2P/ARE/Hygro]) vector and treated with BC-1901S at various doses. Luminescence signals were collected and quantified. Data and mean ± SD of 3 independent experiments. C) NRF2, HO-1, GPx-1/2, and β-Actin immunoblots after NRF2 dsiRNA knockdown with or without BC-1901S treatment. D, E) PBMCs were treated with LPS or LPS and BC-1901S at various doses for 18h. Cell culture supernatants were then assayed for TNF (D) and IL-1β (E) using ELISA. Data and mean ± SD of 3 independent experiments. F) Immunoblot of NRF2 in MLE-12 after treatment with BC-1901S at indicated time points. G) MLE were treated with LPS or LPS and BC-1901S at various doses for 18h. Cell culture supernatants were then assayed for IL-6 using ELISA. Data and mean ± SD of 3 independent experiments. P < 0.05, *; P < 0.01, **; P < 0.001, ***; P < 0.0001, ****; by one-way ANOVA with adjusted P-value for multiple comparisons (Dunnett's) compared to “0”.