International Journal of Molecular Sciences

Chloride channel proteins (CLCs) are essential anion transporters involved in plant growth, osmotic regulation, and ion homeostasis. However, their genome-wide characterization in tetraploid alfalfa (Medicago sativa L.) remains unexplored. In this study, a total of 35 CLC family members were identified and underwent comprehensive bioinformatic analyses. Phylogenetic and structural analyses divided them into six subfamilies and two subclasses based on conserved residues such as GxGIPE. Members within the same subclass shared conserved domains and similar motif patterns. Analysis of duplication events indicated that 48 segmental duplications were the primary driving force behind the expansion of this gene family. Promoter analysis revealed abundant light, hormone, and stress-responsive cis-elements, suggesting multiple regulatory functions. Gene expression profiling demonstrated that salt, drought stress, and ABA treatment significantly induced the expression levels of some genes. Among them, MsCLC2 and MsCLC18 from Group c exhibited more than fivefold upregulation under both salt and drought stress, significantly higher than other members. Subcellular localization confirmed MsCLC18 on the plasma membrane, potentially regulating Cl⁻ efflux through a Cl⁻/H⁺ antiporter mechanism to alleviate Cl⁻ toxicity. These findings provide a theoretical foundation for the function study of CLC genes in alfalfa and offer new insights into the molecular evolution of polyploid plants under abiotic stress.

Algae-derived bioactives have emerged as promising nutraceuticals due to their ability to modulate key molecular pathways under physiological stress. Arthrospira platensis (Spirulina), a cyanobacterium widely recognized for its antioxidant and anti-inflammatory properties, is proposed as a functional supplement to preserve smooth muscle physiology. Progressive strength training (PST) can induce oxidative stress and disrupt electromechanical coupling in the uterus, potentially impairing female reproductive function. This study investigated whether supplementation with A. platensis prevents PST-induced uterine dysfunction and elucidated the molecular mechanisms involved. Virgin Wistar rats were divided into five groups: sedentary with saline (GS), sedentary with A. platensis (GAP100), adapted control (GC), PST-trained (GT), and PST-trained with A. platensis (GTAP100). An eight-week water-jump PST protocol was applied. Uterine contractile responses were recorded in isolated organ baths after cumulative KCl stimulation, in the absence or presence of pathway-specific inhibitors targeting nitric oxide synthase, cyclooxygenase, NADPH oxidase, or superoxide dismutase. Histological evaluations of uterine and ovarian tissues were also performed. PST increased contractile efficacy and myometrial thickness, associated with oxidative stress and activation of NO, COX, and NADPH oxidase pathways. Supplementation with A. platensis attenuated these alterations by enhancing NO signaling, stimulating relaxant prostanoids, and reducing superoxide production. These protective effects were abolished by inhibitors, confirming mechanistic involvement. Overall, our findings provide molecular evidence that A. platensis supplementation preserves uterine smooth muscle physiology under high-intensity resistance training, supporting its potential as a nutraceutical strategy for female reproductive health.

Biopolymer hydrogels are attractive matrices for localised enzyme and drug delivery owing to their intrinsic biocompatibility, biodegradability, and controlled release capacity. In this study, κ-carrageenan hydrogels were engineered as enzyme-delivery systems by reinforcing the matrix with cellulose nanocrystals (CNC) or chitin nanowhiskers (ChNW) and loading bromelain as a model enzyme. The objective was to evaluate how nanofiller chemistry and morphology influence network structure and release behaviour. Parallel fabrication under identical conditions enabled a direct CNC-ChNW comparison. CNC reinforcement compacted the network and reduced swelling, whereas ChNW produced more hydrated and open architectures. Both fillers enhanced surface wettability, while their concentration modulated bulk hydration and diffusivity. Bromelain release over 24 h followed diffusion-controlled kinetics, tunable by filler type and loading. Quantitative topography and pore-size mapping supported structure–function correlations between morphology and transport. All hydrogels were bio-based, biodegradable, and fully cytocompatible, highlighting their suitability for sustainable biomedical applications. Overall, this work provides a quantitative structure-property-function framework for designing enzyme-active κ-carrageenan systems for tunable bromelain release and related biomedical applications.