Fig 2: Inhibition of NF-?B nuclear translocation by isorhamnetin in LPS-stimulated BV2 microglial cells. (A) Cells were pretreated with 200 µM isorhamnetin for 1 h prior to treatment with 100 ng/ml LPS for 1 h. Nuclear and cytosolic proteins were prepared for western blot analysis using anti-NF-?B p65 and anti-I?Ba antibodies. Lamin B and actin were used as internal controls for nuclear and cytosolic fractions, respectively. (B) Bands were semi-quantified using ImageJ, normalized to lamin B and actin and the ratios were determined. Data are presented as the means ± standard deviation of three independent experiments. *P<0.05 compared with the control group; #P<0.05 compared with the LPS group. (C) Cells were pretreated with 200 µM isorhamnetin for 1 h prior to treatment with 100 ng/ml LPS for 1 h. Localization of NF-?B p65 was visualized by fluorescence microscopy (original magnification, ×400) following immunofluorescence staining with an anti-NF-?B p65 antibody (green). The cells were also stained with DAPI to visualize nuclei (blue). Results are representative of three independent experiments. ISO, isorhamnetin; LPS, lipopolysaccharide; NF-?B, nuclear factor-?B; I?Ba, inhibitor ?B-a; p, phosphorylated.

Fig 3: DPA inhibited the activation of NF-?B and MAPK p38 signaling pathways in LPS-activated BV2 cells. (A,H) Experimental procedure. (B–D) The representative images immunostained for p-p65, p65, and p-p38 (p-p65, p65, and p-p38 (red); DAPI (blue)). (E–G) p-p65, p65, and p-p38 expression per cell, quantified. (I) Changes in BV2 cell viability after treatment with SH at different concentrations for 48 h. (J) Changes in BV2 cell viability after treatment with 100 ng/mL LPS for 24 h with or without different concentrations of SH. (K) Changes in BV2 cell viability after treatment with 100 ng/mL LPS for 24 h with or without SH or/and DPA administration. (L,N) The representative images immunostained for p-p65 and Iba-1 (green) and DAPI (blue). (M,O) p-p65 and Iba-1 expression per cell, quantified. (P–R) NO, IL-1ß, and IL-1R1 concentrations in BV2 cell supernatant. The data are expressed as mean ± SEM (n = 5–6). ** p < 0.01 versus CT; ## p < 0.01 versus LPS. Results in Figure 3E–G were measured by one-way ANOVA followed by Tukey’s post hoc test; all other results were measured by two-way ANOVA.

Fig 4: BS-181 suppressed p65 nuclear translocation and phosphorylation of p65 in LPS-induced MH7A cells. (A) Western blot analysis of p65 protein level in nuclear and cytoplasm of LPS-induced MH7A cells treated with BS-181 for 48 hrs. The p65 protein level was down-regulated in nuclear and up-regulated in cytoplasm. ß-actin and histone 3 were used as a loading control. (B) Fluorescent immunocytochemistry analysis of p65 protein level in nuclear and cytoplasm of LPS-induced MH7A cells treated with BS-181 for 48 hrs. The nuclear translocation of p65 was reduced in LPS-induced MH7A cells treated with BS-181. Scale bar: 20 µm. (C) Western blot analysis for p-p65 in LPS-induced MH7A cells treated with BS-181 for 24 hrs. ß-actin was used as a loading control.

Fig 5: A proposed model for the mechanism of p16 in regulating intervertebral disc degeneration (IVDD).NF-?B-p65 activates p16 expression. p16 deficiency alleviates the reactive oxygen species (ROS) levels, senescence-associated secretory phenotype (SASP) and cellular senescence. Subsequently, p16 deficiency promotes the activity of cellular antioxidation, and the proliferation and stability of ECM, like aggrecan and collagen II. All of the pathways ultimately protect against the development of IVDD.