Artesunate ameliorates diabetic xerostomia in rats through regulating oral microbiota and metabolic profile in salivary gland based on NF-κB/NLRP3 signaling pathway

Background

Artemisia annua. L, as a valuable Chinese medicine, has been applied for millennia in China. Its major active ingredient, artemisinin, has demonstrated diverse pharmacological properties, including anti-inflammatory, antioxidant, and anti-diabetic effects. Recent studies suggest that artesunate (ART), an artemisinin derivative, exhibits promising therapeutic effects on diabetic complications. Nevertheless, the role and underlying mechanisms of ART in the treatment of diabetic xerostomia (DX) remain unclear.

Aim

This study aimed to elucidate the effects of ART on DX in a type 2 diabetes mellitus (T2DM) rat model, primarily from the perspective of oral microbiota and salivary gland (SG) metabolism, and to further explore potential mechanisms involved.

Methods

Various assessments including blood levels, insulin resistance (IR), saliva flow rate, as well as histological analyses through hematoxylin and eosin and Masson staining were performed to verify the reliability of DX model and protective effects of ART on the DX. Untargeted metabolomics and 16S rDNA sequencing were employed to respectively evaluate effects of ART on metabolite changes in SG and oral microbiota in the DX rats. Network pharmacology was employed to predict key pathways and targets with critical roles in ART's therapeutic effect on DX. Additionally, molecular docking and molecular dynamics (MD) simulations were utilized to evaluate interactions between ART and the identified key pathway targets. Surface plasmon resonance (SPR) experiment was performed to verify our computational predictions. Finally, molecular biology experiments were conducted to further validate the identified key pathway targets.

Results

ART treatment ameliorated the hyperglycemia, IR and hyposalivation, and ameliorated pathological changes and oxidative stress of SGs in the DX rats. Besides, 16S rDNA sequencing suggested that ART alleviated the perturbation of oral microbiota (such as Veillonella, Lactobacillus, Clostridium sensu stricto 1, Escherichia-Shigella, and Dubosiella). Untargeted metabolomics revealed that steroid hormone biosynthesis, taurine and hypotaurine metabolism of SGs in the DX rats were partially corrected by ART treatment. Correlation analysis demonstrated an obvious association between the oral microbiota species and SG metabolites. Network pharmacology analysis identified NF-κB pathway as a critical pathway of ART in treating DX. Meanwhile, molecular docking and MD simulation suggested stable binding of ART to NF-κB/NLRP3 pathway targets, particularly NLRP3. Furthermore, SPR confirmed a stable binding of ART to NLRP3, a key target in the NF-κB/NLRP3 pathway. Oxidative stress indicators involved in NF-κB pathway, including MDA and SOD levels, were significantly reduced after ART intervention. Western blotting and qRT-PCR experiments further revealed that ART inhibited increase of NF-κB/NLRP3 pathway related targets expression, including NF-κB, NLRP3, Caspase1, IL-1β, IL-18, TNF-α, and IL-6 in the SGs of DX rats.

Conclusion

ART exerted beneficial therapeutic effects on DX by modulating oral microbiota dysbiosis and restoring SG's metabolic profiles, and inhibiting activation of NF-κB/NLRP3 pathway, suggesting its potential novel application in DX treatment.