Blocking Smad2 signalling with loganin attenuates SW10 cell cycle arrest induced by TNF-α

Abstract

The activity of Schwann cells (SWCs) is very important in trauma-induced nerve repair, and tumour necrosis factor-α (TNF-α) produced during tissue injury inhibits the viability of SWCs, which delays the repair of peripheral nerves. Loganin is an iridoid glycoside that has been shown to alleviate a variety of cytotoxic effects. In the current study, we evaluated the potential efficacy and the mechanism of action of loganin in TNF-α-induced cytotoxicity in SW10 cells. The experimental results indicated that loganin blocked TNF-α-mediated Smad2 activation, downregulated the expression of the G1 phase cell cycle inhibitor p15IN4KB, and upregulated the expression of the G1 phase cell cycle activator cyclin D1-CDK4/6, which upregulated E2F-1-dependent survivin expression and relieved TNF-α-induced apoptosis in SW10 cells. The protective effect of loganin on SWCs has potential medicinal value in the promotion of peripheral nerve repair and is significant for studies in the field of tissue regeneration.

Fig 1. Loganin attenuated TNF-α-induced SW10 cell apoptosis.

Chemical structure of loganin (A). SW10 cells were incubated with 0, 2, 5, 10, 20 and 40 ng/mL TNF-α for 6, 12, 18, 24 and 48 h, and cell viability was determined in a CCK-8 assay (B). Schwann cells were pre-incubated with 10, 20 and 60 ng/mL loganin for 2 h followed by incubation with 10, 20 and 40 ng/mL TNF-α for 24 h, and cell viability was assessed (C). SW10 cells were pre-incubated with loganin for 2 h followed by TNF-α stimulation for 24 h, and the cells were then stained with DAPI (D). The expression levels of cleaved caspase-3, cleaved caspase-8, cleaved caspase-7, and cleaved PARP were measured using the ICW method (E). SW10 cells were incubated with TNF-α for 6 h, and the phosphorylation of FADD and RIP at various time points was detected by Western blotting (F). SW10 cells were pre-incubated with loganin followed by TNF-α stimulation for 30 min or 1 h before measuring p-RIP and p-FADD (G). Data are presented as the mean±S.D, *P<0.01 vs. the Control group (B, C, n = 2). *P<0.05 and **P<0.01 vs. the TNF-α group (E, n = 3).

Fig 2. Loganin blocked SW10 cell cycle activation by Smad2.

SW10 cells were incubated with loganin and TNF-α for 24 h, and changes in apoptotic signalling in SW10 cells were evaluated using a PathScan Apoptosis Signaling Antibody Array Kit (A). SW10 cells were stimulated with TNF-α for 24 h, and the level of p-Smad2 at different time points was measured; SW10 cells were stimulated with TNF-α (0, 2, 5, 10, 20, and 40 ng/mL) for 24 h; SW10 cells were pre-incubated with loganin for 2 h followed by TNF-α stimulation for 24 h, and p-Smad2 was detected by Western blotting (B). Construction of an siRNA plasmid for TNFR1 expression, transfection of SW10 cells, and Western blotting detection of interference efficiency and Smad2 activation (C). Three GV248-Smad2 shRNA plasmids targeting Smad2 expression were constructed. After 293T cells were transfected with lentiviruses carrying these plasmids, the culture supernatant from the 293T cells was collected and incubated with SW10 cells, and Smad2 expression in the SW10 cells was measured by Western blotting (D). SW10 cells were stained with propidium iodide, and the number of cells at each phase of the cell cycle was measured using flow cytometry (E). SW10 cells were incubated with loganin for 2 h or treated with GV248-Smad2 shRNA, followed by TNF-α stimulation for 24 h. The expression levels of p15^Ink4b, CDK4, CDK6, cyclin D1, and retinoblastoma (Rb) protein were measured using Western blotting (F).

Fig 3. Survivin attenuated TNF-α-induced SW10 cell apoptosis.

Schwann cells were incubated with 60 μg/mL loganin for 6, 12, 18, and 24 h, and survivin expression was detected by Western blotting; A SMAD2 RNAi plasmid was constructed and transfected into SW10 cells, and the survivin expression was detected by Western blotting; A GV218-Smad2-overexpressing plasmid was constructed and transfected into SW10 cells for 72 h and incubated with loganin for 24 h, the expression of survivin was measured (A). The loganin-treated cells were incubated with a green fluorescent protein-labelled survivin antibody, and the distribution of the fluorescence intensity in cells was observed using laser confocal microscopy (B). GV248-survivin shRNAs targeting 3 sites of survivin expression were constructed and transfected into 293T cells. The supernatant was collected and used to treat SW10 cells, and survivin expression was then measured (C). SW10 cells were separately exposed to survivin RNAi-3 and to GV218-Smad2-overexpressing plasmids for 72 h; the cells were then incubated with loganin and TNF-α, and the expression levels of cleaved caspase-3, cleaved caspase-8, cleaved caspase-7, and cleaved PARP were measured using ICW (D). Data are presented as the mean±S.D, *P<0.05 and **P<0.01 (F, n = 2).

Fig 4. Loganin activated E2F-1 to regulate survivin expression.

SW10 cells were incubated with 0, 10, 20, and 60 μg/mL loganin for 6, 12, 18, and 24 h, and the expression of the E2F-1 transcription factor was then measured; A Smad2 RNAi plasmid was constructed and transfected into SW10 cells, and the E2F-1 expression was detected by Western blotting; (A). An shRNA interference plasmid targeting E2F-1 expression was constructed and packaged into lentiviruses, and 293T cells were transfected with the plasmid. The cell culture supernatant was collected for SW10 interference, and E2F-1 was then detected by Western blotting (B). SW10 cells were treated with red fluorescent protein-labelled E2F-1 antibody, and the subcellular localisation of E2F-1 was observed using laser confocal microscopy (D). The E2F-1 RNAi-1 plasmid was transfected into SW10 cells. After 72 h, the transfected cells were incubated with loganin and the CDK4/6 inhibitor palbociclib, and the expression of survivin was measured (E). The luciferase reporter plasmid pG3L-mSurvivin was constructed and transfected into SW10 cells together with either GV218-Smad2 or E2F-1 RNAi. After the cells were incubated with loganin and palbociclib, changes in the fluorescence intensity of the cells were measured (F). Effect of blocking the SMAD2-CDK4/6-E2F-1-survivin signalling cascade on TNF-α-induced cell viability (G). Data are presented as the mean±S.D, *P<0.05 and **P<0.01 (F, n = 3). **P<0.01 vs. Control group, ##P<0.01 vs. TNF-α group, ππP<0.01 vs. loganin group (G).

Fig 5. Mechanism by which loganin attenuates TNF-α-induced SWC cytotoxicity.

Loganin blocks TNF-α-induced Smad2 signalling activation, activates the p15^Ink4b-cyclin D1-CDK4/6 pathway, and promotes EF2-1-dependent survivin expression to attenuate reductions in SW10 cell viability and SW10 cell apoptosis due to TNF-α released by inured tissue.