Compound C(Shahid Beheshti University)

Childhood obesity (CO) has become a global epidemic, leading to rising burden of many diseases and premature death. Thus, this study was conducted to screen the mitochondria-associated biomarkers for patients with CO, as well as the involved molecular mechanism.

After downloading GSE29718 and GSE104815 datasets from GEO database, differential expression analysis was conducted to screen the DEGs. Then the obtained DEGs were intersected with the mitochondrial-associated genes, and mitochondrial-related genes in CO were acquired, followed by key mitochondrial-related genes screening utilizing three machine learning algorithms. The qRT-PCR and western blot were employed to determine the expression of key genes. Gain-of-function experiment was applied to investigate the function of NIPSNAP3B in CO in vitro.

Total 364 DEGs were screened, then 18 mitochondrial-related genes in CO were obtained. These 18 mitochondrial-associated genes in CO enriched in pyruvate metabolism, arginine biosynthesis, and AMPK signaling pathway, etc. ACACB and NIPSNAP3B were considered as the key mitochondrial-related genes. Of note, NIPSNAP3B overexpression markedly reduced the TG level and the protein expression levels of PPARγ and C/EBPα in MDI-induced 3 T3-L1 cells. Also, ATP content, mitochondrial mass, MMP, and protein expression levels of PGC-1α, NRF1, and TFAM were changed after NIPSNAP3B upregulation in MDI-induced 3 T3-L1 cells. However, opposite results were observed after NIPSNAP3B downregulation. Compound C (AMPK inhibitor) or AMPK knockdown administration could reverse the effect of NIPSNAP3B on adipocyte lipid deposition and mitochondrial biogenesis.

NIPSNAP3B enhances mitochondrial biogenesis to attenuate lipid accumulation via AMPK pathway in CO.

Artificial insemination underpins both commercial swine production and the conservation of critically endangered breeds. The Wenchang pig, which is classified as a critically endangered breed, faces a genetic bottleneck due to its reduced breeding population. Current estimates suggest that the breeding population has become quite limited, highlighting the need for effective low-temperature semen preservation strategies. Here, we report that inclusion of 1 μM pinobanksin-a propolis-derived dihydroflavonol-in standard extender sustains the functionality and viability of Wenchang pig spermatozoa during 15 days of storage at 4 °C. Compared to control, pinobanksin maintained total and progressive motility and path velocities (VAP, VCL, VSL), preserved membrane and acrosomal integrity, stabilized mitochondrial membrane potential, and prevented ATP and Ca²⁺ depletion. Biochemically, treated sperm exhibited reduced reactive oxygen species and malondialdehyde levels, restored glutathione homeostasis, elevated activities of SOD and GSH-PX, and upregulated expression of NQO1, HO-1 and GPX4. Mechanistic interrogation revealed that pinobanksin elevates intracellular Ca²⁺, promotes phosphorylation of CaMKKβ and AMPK, and increases cellular Nrf2 abundance. Pharmacological blockade of CaMKKβ (STO-609) or AMPK (Compound C) ablated pinobanksin's antioxidant, anti-apoptotic and motility-preserving effects, whereas direct activation of Nrf2 (via TBHQ) reinstated sperm function under AMPK inhibition. These data delineate a coordinated signaling module involving CaMKKβ, AMPK and Nrf2 through which pinobanksin synchronizes redox and energy-metabolic defenses, establishing it as a dual-action cryoprotectant additive with significant promise for AI-based conservation of endangered porcine germplasm.