International Journal of Molecular Sciences

The Dof (DNA binding with one finger) transcription factor family is a plant-specific group of transcription factors that play critical roles in plant growth and development, stress response, and the regulation of secondary metabolism. Prunella vulgaris (P. vulgaris) has attracted considerable attention due to its medicinal value, with rosmarinic acid being one of its key bioactive components. However, the systematic identification of the Dof transcription factor family in P. vulgaris and its regulatory role in rosmarinic acid biosynthesis remains poorly understood. In this study, based on the whole-genome data of P. vulgaris, we identified 48 Dof transcription factor genes distributed across 14 chromosomes using bioinformatics approaches. Physicochemical analysis revealed that the encoded proteins have molecular weights ranging from 15,482.44 to 55,875.53 Da, amino acid lengths between 142 and 509, and theoretical isoelectric points from 4.84 to 10.2. All proteins were predicted to be hydrophilic and localized in the nucleus. Phylogenetic analysis classified them into four subfamilies, and multiple sequence alignment confirmed that all members contain a conserved C2-C2-type zinc finger domain. Analysis of cis-regulatory elements in the promoter regions identified numerous elements related to light responsiveness, hormone response, and development. Transcriptomic expression profiling demonstrated distinct tissue-specific expression patterns of Dof genes, with some showing high expression in spikes and seeds. Correlation analysis between gene expression and rosmarinic acid content identified three candidate genes potentially involved in the regulation of rosmarinic acid biosynthesis, which were further validated by RT-qPCR. Moreover, protein–protein interaction network predictions indicated 242 interactions among 23 Dof proteins. This study provides the first systematic identification of the Dof transcription factor family in P. vulgaris, offering important insights into the transcriptional regulation of rosmarinic acid biosynthesis and presenting potential genetic targets for enhancing rosmarinic acid production through genetic engineering.

This study evaluated the effectiveness and safety of mechanical debridement (MD) in treating experimental peri-implantitis (EPI) in rats with osseointegrated implants, specifically those treated with high-dose zoledronate. Senescent Wistar rats underwent the extraction of their upper incisor, followed by immediate implant placement. After 8 weeks, the implants were exposed, and a transmucosal component was placed. The animals were divided into four groups: Control (C), ZOL, ZOL-EPI, and ZOL-EPI-MD. In the 9th week, drug treatment commenced, consisting of the administration of 0.45 mL of a vehicle (for group C) or zoledronate (for groups ZOL, ZOL-EPI, and ZOL-EPI-MD) every 4 days over 10 weeks. After 5 weeks of drug treatment, a cotton bandage was placed around the implants to induce EPI in the ZOL-EPI and ZOL-EPI-MD groups. In the ZOL-EPI-MD group, the ligature was removed at week 16, and local treatment was performed using MD. Euthanasia was conducted at week 19. Histological sections were obtained and stained with hematoxylin–eosin for histopathological and histometric analyses, such as the percentage of total bone tissue (B.Ar/T.Ar) and the percentage of non-vital bone tissue (NVB.Ar/B.Ar). Immunohistochemical reactions were performed to detect TNFα, IL-1β, VEGF, OCN, and TRAP. In the peri-implant connective tissue, mild, intense, and moderate inflammatory infiltrates were observed in the ZOL, ZOL-EPI, and ZOL-EPI-MD groups, respectively. Immunolabeling for TNFα and IL-1β correlated with these histopathological findings. The ZOL and ZOL-EPI-MD groups showed lower immunolabeling for VEGF compared to the control group. There was a reduction in TRAP-positive cells and lower immunolabeling for OCN in the groups treated with zoledronate, with the ZOL-EPI-MD group displaying even lower levels of OCN compared to the ZOL group. While there was no significant difference in B.Ar/T.Ar across the groups, both the ZOL, ZOL-EPI, and ZOL-EPI-MD groups exhibited higher levels of NVB.Ar/B.Ar, with the ZOL-EPI-MD group showing the highest NVB.Ar/B.Ar compared to ZOL and the other groups. In conclusion, MD, as a standalone treatment, showed neither effectiveness nor safety in the management of EPI in rats that received high doses of zoledronate.

Blood–brain barrier (BBB) breakdown is a hallmark of several neurological disorders, including multiple sclerosis (MS). NX210c, a novel therapeutic peptide, has shown promise in restoring BBB integrity, in both preclinical and clinical settings, offering potential for use in MS populations and across various central nervous system conditions with overlapping mechanisms. In this study, we evaluated the therapeutic potential of NX210c in patients with relapsing–remitting MS (RRMS) using a previous quantitative systems pharmacology (QSP) model currently redesigned to capture the dynamic interplay between BBB integrity and immune system activity. We validated the QSP model using both preclinical and clinical datasets, and generated virtual populations representing healthy individuals and RRMS patients for in silico testing. NX210c was assessed as both a monotherapy and in combination with established MS treatments. Simulations predicted time course changes in key BBB integrity markers, including tight junction protein (TJP) expression and transendothelial electrical resistance (TEER), under various dosing regimens. NX210c treatment was associated with a significant attenuation of BBB degradation compared to untreated controls (~7–8% higher TJP expression and BBB electrical resistance). Furthermore, we investigated the long-term impact of NX210c on clinical outcomes such as relapse rates. Both 5 and 10 mg/kg doses (single cycle [thrice-weekly for 4 weeks]) induced improvement in disease activity in RRMS patients, as well as a 10 mg/kg dose (single or repeated 4-week cycles every 6 months) in highly active patients. Particularly when administered alongside one of five commonly used MS therapies (interferon β-1a, teriflunomide, cladribine, natalizumab, ocrelizumab), in the highly active subpopulation, the model on average predicted a reduction in relapse frequency in the 10 mg NX210c-treated group versus untreated group from four to no relapses over two years. These findings suggest that NX210c may enhance therapeutic efficacy in RRMS by promoting BBB restoration and modulating immune responses, offering a promising avenue for combination treatment strategies.