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. 2024 May 11;16(1):38.
doi: 10.1038/s41368-024-00305-z.

Sinensetin protects against periodontitis through binding to Bach1 enhancing its ubiquitination degradation and improving oxidative stress

Zhiyao Yuan #  1 , Junjie Li #  1 , Fuyu Xiao  1 , Yu Wu  1 , Zhiting Zhang  1 , Jiahong Shi  1 , Jun Qian  1 , Xudong Wu  2 , Fuhua Yan  3
Affiliations

Sinensetin protects against periodontitis through binding to Bach1 enhancing its ubiquitination degradation and improving oxidative stress

Zhiyao Yuan et al. Int J Oral Sci. .

Abstract

Periodontitis is a chronic inflammatory and immune reactive disease induced by the subgingival biofilm. The therapeutic effect for susceptible patients is often unsatisfactory due to excessive inflammatory response and oxidative stress. Sinensetin (Sin) is a nature polymethoxylated flavonoid with anti-inflammatory and antioxidant activities. Our study aimed to explore the beneficial effect of Sin on periodontitis and the specific molecular mechanisms. We found that Sin attenuated oxidative stress and inflammatory levels of periodontal ligament cells (PDLCs) under inflammatory conditions. Administered Sin to rats with ligation-induced periodontitis models exhibited a protective effect against periodontitis in vivo. By molecular docking, we identified Bach1 as a strong binding target of Sin, and this binding was further verified by cellular thermal displacement assay and immunofluorescence assays. Chromatin immunoprecipitation-quantitative polymerase chain reaction results also revealed that Sin obstructed the binding of Bach1 to the HMOX1 promoter, subsequently upregulating the expression of the key antioxidant factor HO-1. Further functional experiments with Bach1 knocked down and overexpressed verified Bach1 as a key target for Sin to exert its antioxidant effects. Additionally, we demonstrated that Sin prompted the reduction of Bach1 by potentiating the ubiquitination degradation of Bach1, thereby inducing HO-1 expression and inhibiting oxidative stress. Overall, Sin could be a promising drug candidate for the treatment of periodontitis by targeting binding to Bach1.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1
Fig. 1
Antioxidant and anti-inflammatory properties of Sin. a Sin cytotoxicity tested by CCK-8 assay (n = 3). b Detection of MDA content in PDLCs (n = 3). c Detection of GSH content in PDLCs (n = 3). d Measurement of ROS production in PDLCs by Flow cytometry (n = 3). e Statistics of mean fluorescence intensity (MFI) in d. f IL6 mRNA expression in PDLCs treated with Sin at different concentrations (n = 3). g IL10 mRNA expression in PDLCs treated with Sin at different concentrations (n = 3). Data are presented as the mean ± SD, when compared with control group, *P < 0.05, **P < 0.01, ***P < 0.001 in (A). When compared with control group, #P < 0.05, ##P < 0.01, when compared with TNF-α + IL-1β group, *P < 0.05, **P < 0.01 in b, c, e–g
Fig. 2
Fig. 2
Bach1 serves as a key target for Sin to exert its antioxidant effects. a Molecular docking of Sin with Bach1. b, c Sites prediction of Sin combined with Bach1. d The result of cellular thermal displacement assay (CETSA) of Bach1’s degradation in PDLCs without/with Sin. e Changes in the expression of Bach1 and HO-1 at different Sin concentrations (n = 3). f Confocal image of Bach1 under inflammatory conditions and Sin-treated conditions. g The binding of the promoter of the HMOX1 genes with Bach1 was investigated by ChIP‒qPCR. Data are presented as the mean ± SD, *P < 0.05, **P < 0.01 in f, g
Fig. 3
Fig. 3
Bach1 participates in the antioxidant capacity of Sin under inflammatory conditions. a–e Changes of oxidative stress markers, including ROS formation (a), SOD activity (b), GSH content (c), MDA production (d) after knockdown of Bach1 under inflammatory conditions without/with Sin. e Expression of Bach1 and HO-1 after knockdown of Bach1 under inflammatory conditions without/with Sin. f Expression of Bach1 and HO-1 after overexpression of Bach1 under inflammatory conditions without/with Sin. g–i Changes of oxidative stress levels, including GSH production (g), SOD activity (h), MDA content (i) after overexpression of Bach1 under inflammatory conditions without/with Sin. Data are presented as the mean ± SD, #P < 0.05, ##P < 0.01, ###P < 0.001, *P < 0.05, **P < 0.01, ***P < 0.001
Fig. 4
Fig. 4
Sin promotes ubiquitinated degradation of Bach1. a The cytoplasmic and intranucleus levels of Bach1 under normal and inflammation conditions with/without the treatment of Sin. b The level of Bach1 under inflammation conditions treated with Sin and inhibitors CQ, MG132. c Co-IP assays of Ubiquitin (Ub) and Bach1
Fig. 5
Fig. 5
The protective effect of Sin against periodontitis in vivo. a Micro CT imaging of bone resorption in periodontitis in mice. b H&E staining of periodontium. c IHC staining of TNF-α, IL-1β, and IL-6 in periodontium
Fig. 6
Fig. 6
Sin protects against periodontitis by targeting Bach1. a IHC staining of Bach1 and HO-1 of periodontium. b Sin promotes the ubiquitination degradation of Bach1 by binding to it, therefore improving oxidative stress and inflammatory levels, which eventually relieves periodontitis
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