Coumarin derivates have been proposed as a potential treatment for metabolic-dysfunction-associated fatty liver disease (MAFLD). However, the mechanisms underlying their beneficial effects remain unclear. In the present study, we explored the potential of the coumarin derivate esculetin in MAFLD, focusing on hepatocyte lipotoxicity and lipid accumulation. Primary cultures of rat hepatocytes were exposed to palmitic acid (PA) and palmitic acid plus oleic acid (OA/PA) as models of lipotoxicity and lipid accumulation, respectively. Esculetin significantly reduced oxidative stress in PA-treated hepatocytes, as shown by decreased total reactive oxygen species (ROS) and mitochondrial superoxide production and elevated expression of antioxidant genes, including Nrf2 and Gpx1. In addition, esculetin protects against PA-induced necrosis. Esculetin also improved lipid metabolism in primary hepatocytes exposed to nonlipotoxic OA/PA by decreasing the expression of the lipogenesis-related gene Srebp1c and increasing the expression of the fatty acid β-oxidation-related gene Ppar-α. Moreover, esculetin attenuated lipid accumulation in OA/PA-treated hepatocytes. The protective effects of esculetin against lipotoxicity and lipid accumulation were shown to be dependent on the inhibition of JNK and the activation of AMPK, respectively. We conclude that esculetin is a promising compound to target lipotoxicity and lipid accumulation in the treatment of MAFLD.

STRA8 (Stimulated By Retinoic Acid Gene 8) is a retinoic acid (RA) induced gene that plays vital roles in spermatogonial proliferation, differentiation and meiosis. The SETD8 and STRA8 protein interaction was discovered using the yeast two‐hybrid technique using a mouse spermatogonial stem cell (SSC) cDNA library. The interaction of these two proteins was confirmed using co‐immunoprecipitation and identification of key domains governing the protein: protein complex. STRA8 and SETD8 showed a mutual transcriptional regulation pattern that provided evidence that SETD8 negatively regulated transcriptional activity of the STRA8 promoter. The SETD8 protein directly bound to the proximal promoter of the STRA8 gene. STRA8 increased the transcriptional activity of SETD8 promoter in a dose‐dependent manner. For the first time, we have discovered that STRA8 and SETD8 display a cell cycle‐dependent expression pattern in germline cells. Expression levels of SETD8 and H4K20me1 in S phase of STRA8 overexpression GC1 cells were different from that previously observed in tumour cell lines. In wild‐type mice testis, SETD8, H4K20me1 and PCNA co‐localized with STRA8 in spermatogonia. Further, our studies quantitated abnormal expression levels of cell cycle and ubiquitination‐related factors in STRA8 dynamic models. STRA8 and SETD8 may regulate spermatogenesis via Cdl4‐Clu4A‐Ddb1 ubiquitinated degradation axis in a PCNA‐dependent manner.

Arctigenin, a natural lignan from Arctium lappa L., exhibits potent antifibrotic activity, yet its molecular mechanisms remain unclear. Endoplasmic reticulum (ER) stress is known to promote hepatic stellate cell (HSC) activation and liver fibrosis. This study investigates the therapeutic potential of arctigenin in HSC activation through ER stress modulation. Primary rat HSCs were activated (3-7 days) and treated with tunicamycin (ER stress inducer) or 4-PBA (ER stress inhibitor). Arctigenin attenuated ER stress markers (e.g., GRP78) and suppressed the expression of fibrotic marker α-SMA in ER stress-challenged activating (day 3) and activated (day 7) HSCs. Arctigenin restored lipid homeostasis by modulation of both lipogenesis (via Dgat2 and Ppar-γ upregulation) and lipolysis (suppression via ATGL inhibition). ER stress activated ER-associated degradation (ERAD), triggering the formation of small lipid droplets (LD). Arctigenin normalized the ERAD activity, thereby rescuing LD integrity and suppressing HSC activation. Our findings demonstrate that arctigenin mitigates HSC activation by suppressing ER stress and restoring lipid homeostasis via modulating ERAD-mediated lipid dysregulation. As a dietary and medicinal compound, arctigenin emerges as a promising therapeutic candidate for liver fibrosis.

The prevalence of metabolic dysfunction-associated fatty liver disease (MAFLD) is increasing rapidly worldwide due to the obesity epidemic. Advanced stages of the MAFLD, such as non-alcoholic steatohepatitis (NASH) with advanced fibrosis or cirrhosis are affecting global health. Extracellular vesicles (EVs) are released by all cell types and are important in cell-to-cell communication and maintaining homeostasis, but they also play a role in the pathogenesis of various diseases. EVs contain biological information such as lipids, proteins, messenger RNAs (mRNAs), small RNAs, and DNA, and they act on (distant) target cells. The cargo of EVs is dependent on the type and the state of the releasing cell. EVs have been proposed as biomarkers, prognostic, and even therapeutic agents, also in the context of liver diseases. This review aims to give an overview of the current knowledge on EVs in MAFLD, including the role and interaction of EVs with different cell types in the liver. Several aspects of EVs, including their origin, characteristics, cargo, and functions are reviewed. Moreover, the potential of EVs as targets for the treatment of MAFLD is discussed.

Gene expression analyses have revealed that there are >2,300 testis-enriched genes in mice, and gene knockout models have shown that a number of them are responsible for male fertility. However, the functions of numerous genes have yet to be clarified. The aim of the present study was to identify the expression pattern of testis-expressed protein 33 (TEX33) in mice and explore the role of TEX33 in male reproduction. Reverse transcription-polymerase chain reaction and western blot assays were used to investigate the mRNA and protein levels of TEX33 in mouse testes during the first wave of spermatogenesis. Immunofluorescence analysis was also performed to identify the cellular and structural localization of TEX33 protein in the testes. Tex33 knockout mice were generated by CRISPR/Cas9 gene-editing. Histological analysis with hematoxylin and eosin or periodic acid-Schiff (PAS) staining, computer-assisted sperm analysis (CASA) and fertility testing, were also carried out to evaluate the effect of TEX33 on mouse spermiogenesis and male reproduction. The results showed that Tex33 mRNA and protein were exclusively expressed in mouse testes and were first detected on postnatal days 21-28 (spermiogenesis phase); their expression then remained into adulthood. Immunofluorescence analysis revealed that TEX33 protein was located in the spermatids and sperm within the seminiferous tubules of the mouse testes, and exhibited specific localization to the acrosome, flagellum and manchette during spermiogenesis. These results suggested that TEX33 may play a role in mouse spermiogenesis. However, Tex33 knockout mice presented no detectable difference in testis-to-body weight ratios when compared with wild-type mice. PAS staining and CASA revealed that spermatogenesis and sperm quality were normal in mice lacking Tex33. In addition, fertility testing suggested that the Tex33 knockout mice had normal reproductive functions. In summary, the findings of the present study indicate that TEX33 is associated with spermiogenesis but is not essential for sperm development and male fertility.

Stimulated by retinoic acid 8 (Stra8), one of genes induced by retinoic acid (RA), is required for the meiotic initiation of male spermatogenesis. The present study found that Stra8 inhibited apoptosis in male Stra8-knockout mice, and in mice with vitamin A deficiency and vitamin A recovery in vivo. This phenotype was also verified in GC1 spermatogonia (spg) cells overexpressing Stra8. In addition, microarray analysis identified that there were nine differentially expressed genes (DEGs) in the Stra8-overexpressed GC1 spg cells compared with the control groups; the expression of these nine genes was verified via mRNA expression levels. The DEGs were as follows: Phosphatidylinositol-dependent kinase 1 (PDK1), a key gene upstream of protein kinase B (AKT); angiopoietin 2, a B-cell lymphoma 2 (Bcl-2)-inhibited gene; transcription factor 4, glutathione S-transferase P91 and ubiquitin-specific protease 33, mitogen-activated protein kinase (MAPK)-related genes; oxidative stress induced growth inhibitor 1, related to the P53 pathway; Bcl-2, P53, ERK (MAPK1/3), c-Jun N-terminal kinase (MAPK8/9), and P38 (MAPK14), all of which are key genes involved in the AKT signaling pathway. Therefore, the present study further verified these genes and found that the mRNA and protein expression levels of PDK1, AKT, Bcl-2 and ERK were increased. Although the mRNA expression level of P53 was decreased, there was no significant difference in the protein expression level in Stra8-overexpressing GC1 spg cells compared with controls. In addition, Caspase 3, one of the executioner caspases, was decreased in Stra8-overexpressing GC1 spg cells compared with the control groups. Therefore, it was suggested that Stra8 may directly or indirectly inhibit caspases through the AKT signaling pathway and ultimately exert an anti-apoptotic effect in the male reproductive system.