The past few years, the Kv1.3 channel has gradually become the target of many drug research and development due to its important role in autoimmune diseases, inflammation, and tumors7. At present, Kv1.3 blockers mainly include small molecule compounds and bioactive peptides; their development was limited owing to their low selectivity and toxic side effects. Bioactive polypeptides can bind with amino acids in the outer layer of potassium channels to produce a "pore-blocking" effect, thus making it an effective potassium channel blocker. Few relevant research has been done currently on the use of Kv1.3 blockers in the management of periodontitis.
This study's design idea is derived from BmKTX, which can specifically bind to Kv1.3 channels. BmKTX is an active peptide formed by the exchange of three pairs of disulfide bonds, mainly through intramolecular structures (α-Helix and β-Sheet) that specifically connect to Kv1.3 channels25. This blocking effect has been confirmed in animal models of autoimmune diseases such as multiple sclerosis, diabetes, inflammatory bowel disease, Sjogren's syndrome, and rheumatoid arthritis26, 27, 28–33. Therefore, under the condition of keeping its core skeleton structure unchanged, the residues on the BmKTX molecule were replaced and deleted to construct a peptide molecule (BmKTX-33) that specifically binds to Kv1.3 channels. Firstly, considering the design, synthesis, and stability of the short peptide, the non-backbone amino acids of BmKTX were deleted. Second, adding a positively charged residue (Arginine) to the front of the α-helix structure might combine with Kv1.3 channels to generate a salt bridge. To strengthen the polar interaction between the peptide and polar residues of Kv1.3 channels, we introduced two hydrophilic groups (Histidine and Threonine) in the β-sheet region. Studies have shown that10 the negatively charged amino acid 33 of BmKTX will lead to strong electrostatic repulsion between the β-Sheet domain and Kv1.3 channel; so cationic Histidine is used to replace Aspartic acid. Therefore, we designed a short peptide molecule (BmKTX-33) which specifically binds to Kv1.3 channels, thus inhibiting potassium ion efflux and achieving the antibacterial and anti-inflammatory functions to treat periodontitis. The findings demonstrated that BmKTX-33 might prevent potassium efflux; following BmKTX-33 therapy, inflammatory factor expression was markedly decreased.
The expression of the Kv1.3 channel was downregulated, according to RT-qPCR data after BmKTX-33 treatment. Furthermore, immunofluorescence and ELISA assays showed that the administration of BmKTX-33 controlled macrophage polarization and prevented the release of inflammatory mediators. Existing studies have shown that in the LPS-induced macrophage inflammation model, overexpression of Kv1.3 in macrophages can significantly promote the secretion of inflammatory factors by M1 macrophages34; the secretion level of inflammatory factors was significantly reduced after silencing the expression of Kv1.3 gene in macrophages35. These findings revealed that BmKTX-33, by downregulating Kv1.3 channel expression, was involved in macrophage polarization and the release of inflammatory factors. When macrophages were stimulated, Kv1.3 potassium channels mediated a large amount of potassium efflux, thus supplying the energy that propels calcium influx via CRAC [Calcium released-activated calcium] channels36. As secondary messengers, calcium ions influenced cytokine production and helped control signal transduction pathways37. Additionally, they activated tyrosine kinases to form complexes, which in turn triggers the MAPK ERK1/2 signaling pathway. ERK1/2 was then transferred into the nucleus to activate NF-kB; this series of activated signaling pathways was involved in the secretion of inflammatory factors. Therefore, Kv1.3 potassium channels can regulate macrophage polarization and participate in inflammatory response by affecting calcium influx. Meanwhile, research has demonstrated that NLRP3 expression may be increased by activating the NF-κB pathway38. Secondly, K+ efflux was the second messenger that induced NLRP3 complex assembly39. In this study, we confirmed that BmKTX-33 inhibited potassium efflux by detecting intracellular potassium fluorescence intensity; we verified that BmKTX-33 could inhibit the activation of the LPS-induced NLRP3 inflammasome by qPCR and immunofluorescence experiments. BmKTX-33 may prevent the LPS-induced NLRP3 inflammasome from becoming activated, according to qPCR and immunofluorescence studies.
After confirming that BmKTX-33 regulated the polarization of macrophages and improved the immune microenvironment, we also looked at whether BmKTX-33 might support MC3T3-E1 cells' early osteogenic activity, which included osteogenic differentiation. The findings demonstrated that MC3T3-E1 cells treated with BmKTX-33 for 7 days had greater expression levels of ALP, Runx, CoL1α1, and OCN than the LPS inflammatory induction group. The formation of mineralized nodules was another important indicator to measure the differentiation ability of osteoblasts40. After 21 days of osteogenic differentiation, alizarin red staining results also showed that BmKTX-33 promoted the maturation and calcification of MC3T3-E1 cells. Thus, we assume that BmKTX-33 aided in the MC3T3-E1 cells' osteogenic differentiation process, while the precise mechanism is yet unknown.
S. gordonii, F. nucleatum, and P. gingivalis are significant contributors to the onset and progression of periodontitis. Current study results showed that BmkTX-33 inhibited their biofilm-forming ability. The antibacterial effect of BmKTX-33 may be related to its positive charge (+ 8). Studies have shown11 that the initial interaction between antimicrobial peptides and bacterial cell membranes was mainly due to electrostatic interaction between positive and negative charges. Within a certain range, there was a positive correlation between the number of cationic charges and the bioactivity of antimicrobial peptides. Secondly, by Raman spectroscopy and circular dichroism detection, we found that BmKTX-33 had α-helix and two inverted parallel β-folded structures. Theα-helix structure played an important role in penetrating the cell membrane, thereby its integrity12. The β-sheet structure exposed more positive charges; wherein antimicrobial peptides can interact with LPS on the bacterial surface; this interfered with bacterial physiological functions13. In this study, the integrity of the bacterial cell membrane was observed under SEM. It was found that BmKTX-33 treatment had a significant damaging effect on the bacterial cell membrane after 24 hours, which further proved that BmKTX-33 exerted its antibacterial effect by destroying bacterial cell membranes.
Although this study clarified that BmKTX-33 had antibacterial properties, it also induced macrophages to polarize toward the M2 subtype, inhibited the excessive outflow of potassium ions, disrupted the production of NLRP3 inflammasomes, and promoted early bone formation. However, there were limitations which were as follows. In this study, only the docking site of BmKTX-33 and Kv1.3 channels was simulated by computer software; the action site of BmKTX-33 was not further confirmed by molecular dynamics simulation. Furthermore, this study analyzed intracellular potassium content only at a certain point in time to prove that BmKTX-33 can inhibit potassium outflow; the intracellular potassium ion dynamic change mechanism was not detected using the patch clamp approach. Furthermore, a rat model of periodontitis was not established in this work to investigate the precise function of BmKTX-33. Therefore, in the next experiment, we will prepare nanogel-containing BmKTX-33 powder and apply it to the periodontitis rat model to explore whether BmKTX-33 can improve the periodontic microenvironment and therefore promote alveolar bone regeneration.