Synthesis, Activity, and Application of Fluorescent Analogs of [D1G, Δ14Q]LvIC Targeting α6ß4 Nicotinic Acetylcholine Receptor.

α6ß4* nicotinic acetylcholine receptor (nAChR) (* represents the possible presence of additional subunits) is mainly distributed in the central and peripheral nervous system and is associated with neurological diseases, such as neuropathic pain; however, the ability to explore its function and distribution is limited due to the lack of pharmacological tools. As one of the analogs of α-conotoxin (α-CTx) LvIC from Conus lividus, [D1G, Δ14Q]LvIC (Lv) selectively and potently blocks α6/α3ß4 nAChR (α6/α3 represents a chimera). Here, we synthesized three fluorescent analogs of Lv by connecting fluorescent molecules 6-carboxytetramethylrhodamine succinimidyl ester (6-TAMRA-SE, R), Cy3 NHS ester (Cy3, C) and BODIPY-FL NHS ester (BDP, B) to the N-terminus of the peptide and obtained Lv-R, Lv-C, and Lv-B, respectively. The potency and selectivity of three fluorescent peptides were evaluated using two-electrode voltage-clamp recording on nAChR subtypes expressed in Xenopus laevis oocytes, and the potency and selectivity of Lv-B were almost maintained with the half-maximal inhibition (IC50) of 64 nM. Then, we explored the stability of Lv-B in artificial cerebrospinal fluid and stained rat brain slices with Lv-B. The results indicated that the stability of Lv-B was slightly improved compared to that of native Lv. Additionally, we detected the distribution of the α6ß4* nAChR subtype in the cerebral cortex using green fluorescently labeled peptide and fluorescence microscopy. Our findings not only provide a visualized pharmacological tool for exploring the distribution of the α6ß4* nAChR subtype in various situ tissues and organs but also extend the application of α-CTx [D1G, Δ14Q]LvIC to demonstrate the involvement of α6ß4 nAChR function in pathophysiology and pharmacology.

Potential Mechanisms of Metformin-Induced Apoptosis in HeLa Cells.

Metformin is a traditional antidiabetic drug that also shows potential antitumor effects in cervical cancer. However, some of its apoptosis-related mechanisms are still unclear. In this study, flow cytometry, western blotting, and RNA sequencing (RNA-seq) were used to evaluate the molecular mechanisms of metformin in HeLa cells. The results showed that metformin inhibited cell viability and promoted apoptosis, the protein expression level of Caspase-3 (CASP3) was increased and that of BCL-2 was decreased in HeLa cells treated with metformin. The RNA-seq results indicated a total of 239 differentially expressed genes between the metformin and control check (CK) groups, with 136 genes upregulated and 103 genes downregulated, and 14 of them were found to be associated with apoptosis signaling pathways. The DDIT3 and HRK genes were robustly upregulated in HeLa cells by the endoplasmic reticulum (ER) stress and the mitochondrial pathway of apoptosis. Metformin also affects the expression of PPP2R5C, PPP2R5A, and RRAGA, which participate in biological processes such as PI3K-AKT, mTOR, and AMPK signaling pathways. Metformin mediates the expression of related genes to induce apoptosis.