Fig 2: Knock-down of Nrf2 reverses the effects of overexpressed CKIP-1 on cell viability, oxidative stress, inflammation and apoptosis of high-glucose treated HRECs. a Real-Time qPCR was used to detect relative Nrf2 mRNA levels (n = 3). b Western Blot was employed to detect relative Nrf2 protein expressions (n = 3). c Cell viability was evaluated by CCK-8 assay kit (n = 3). d MDA levels, SOD activity and GSH-PX activity were detected by MDA assay kit, SOD kit abd GSH-PX kit respectively (n = 3). Inflammation associated cytokines (TNF-α, IL-6 and IL-1β) were detected by ELISA (n = 3). e Calcein-AM/PI double stain kit was employed to detect cell apoptosis (Scale bar is 200 μm). f Apoptosis associated proteins (Bcl-2, Bax and Cleaved Caspase 3) were detected by Western Blot (n = 3). The data above in one experiments were repeated at least 3 times and performed as mean ± standard deviation (SD), *P < 0.05, **P < 0.01 and ***P < 0.001

Fig 3: The effects of overexpressed CKIP-1 on high-glucose treated HRECs in terms of cell viability, oxidative stress, inflammation and apoptosis. a Relative CKIP-1 mRNA levels were detected by Real-Time qPCR (n = 3). b Relative CKIP-1 protein levels were detected by Western Blot (n = 3). c CCK-8 assay kit was utilized to detect cell viability (n = 3). d MDA levels, SOD activity and GSH-PX activity were detected by MDA assay kit, SOD kit abd GSH-PX kit respectively (n = 3). Inflammation associated cytokines (TNF-α, IL-6 and IL-1β) were detected by ELISA (n = 3). e Calcein-AM/PI double stain kit was employed to detect cell apoptosis (Scale bar is 200 μm). f Apoptosis associated proteins (Bcl-2, Bax and Cleaved Caspase 3) were detected by Western Blot (n = 3). The data above in one experiments were repeated at least 3 times and performed as mean ± standard deviation (SD), *P < 0.05, **P < 0.01 and ***P < 0.001

Fig 4: Proposed mechanistic scheme for anti-inflammatory effects of CO liberated from CORM-2 in PM-treated HASMC models. In HASMCs, PM can induce VCAM-1, ICAM-1, MMP-2, and MMP-9 expression through the TLR2 and TLR4/NADPH oxidase/ROS/NF-κB/IL-6 pathway, thereby promoting monocyte adhesion and HASMC migration that are implicated in the development of inflammation. Importantly, CORM-2-liberated CO is able to prevent these inflammation-associated cellular events through the inhibition of the expression of relevant cell adhesion molecules and matrix metalloproteinases that is strictly regulated by a complex network of TLR2, TLR4, NADPH oxidase, ROS, NF-κB, and IL-6 signaling molecules.

Fig 5: P. gingivalis supernatant impairs the osteogenic potential of PDLSC.(A) IL-1 β, IL-6 and IL-8 production was measured using ELISA after PDLSCs were treated with P. gingivalis (P.g.) supernatant’s dilutions (1:500 and 1:50), or untreated cells as control simultaneously for 24 h. IL-1 β, IL-6 and IL-8 is expressed in pg/ml ( ± standard deviation). (B) Western blot analysis showed the protein expression of TLR2, TLR4 and GAPDH was used as the internal control. (C) Confluent PDLSC were stained with Alizarin Red after 21 days of cultivation in osteogenic medium containing P. gingivalis supernatant’s dilutions (1:500 and 1:50) and Alizarin Red was then extracted and measured for light absorbance at 405 nm. *p < 0.05 as determined by unpaired two-tailed t tests. Data from three biologically independent replicates.