Sestrin2 (Sesn2) has been previously confirmed to be a stress-response molecule. However, the influence of Sesn2 on myogenic differentiation remains elusive. This study was conducted to analyze the role of Sesn2 in the myogenic differentiation of C2C12 myoblasts and related aspects in mdx mice, an animal model of Duchenne muscular dystrophy (DMD). Our results showed that knockdown of Sesn2 reduced the myogenic differentiation capacity of C2C12 myoblasts. Predictive analysis from two databases suggested that miR-182-5p is a potential regulator of Sesn2. Further experimental validation revealed that overexpression of miR-182-5p decreased both the protein and mRNA levels of Sesn2 and inhibited myogenesis of C2C12 myoblasts. These findings suggest that miR-182-5p negatively regulates myogenesis by repressing Sesn2 expression. Extending to an in vivo model of DMD, knockdown of Sesn2 led to decreased Myogenin (Myog) expression and increased Pax7 expression, while its overexpression upregulated Myog levels and enhanced the proportion of slow-switch myofibers. These findings indicate the crucial role of Sesn2 in promoting myogenic differentiation and skeletal muscle regeneration, providing potential therapeutic targets for muscular dystrophy.

BACKGROUND: Obesity and its associated complications raise significant public concern, revealing gender disparities in the susceptibility to metabolic disorders, with females often displaying greater resistance to obesity-related metabolic disorder than males. Sestrin2 is a crucial protein involved in metabolism and energy balance. This study seeks to explore whether Sesn2 knockout (KO) exacerbates high-fat diet (HFD) induced obesity in female mice.
METHODS: Female mice with wild-type (WT) and Sesn2 KO were subjected to a 12-week regimen of normal diet or HFD. Using a Body Composition Analyzer, body composition was gauged. Biochemical assays encompassed glucose, lipid, and liver function measurements, alongside 24-hour urine albumin excretion. Echocardiographic evaluation assessed cardiac function. Histopathological analysis of key metabolic tissues (liver, kidney, and heart tissues) were conducted. Western blotting or qRT-PCR evaluated key proteins and genes linked to inflammation, mitochondrial, and lipid metabolism in adipose tissues.
RESULTS: In comparison to mice fed a regular diet, those on a HFD exhibited significant increases in body weight and fat mass. Notably, Sesn2 KO further aggravated obesity, showcasing the most pronounced metabolic anomalies: elevated body weight, fat mass, impaired glucose tolerance, and insulin sensitivity, alongside heightened levels of free fatty acids and triglycerides. Additionally, KO-HFD mice displayed exacerbated multi-tissue impairments, including elevated hepatic enzymes, increased urinary albumin excretion, compromised cardiac function, and accumulation of lipids in the liver, kidney, and heart. Moreover, adipose tissue showcased altered lipid dynamics and function, characterized by enhanced triglyceride breakdown and modified adipokine levels. Browning was diminished, along with decreased Pgc1α and Sirt1 in KO-HFD mice.
CONCLUSIONS: Sesn2 KO exacerbates HFD-induced obesity and metabolic disorders in female mice. These findings underscore Sestrin2\'s novel role as a regulator of obesity in female mice.

OBJECTIVE: Endothelial-to-mesenchymal transition (EndoMT) is a significant biological phenomenon wherein endothelial cells undergo a loss of their endothelial traits and progressively acquire mesenchymal characteristics. Consequently, this transformation leads to both a compromised ability to maintain lumen permeability and alterations in vascular structure, which hampers the preservation of blood-brain barrier integrity. This study aimed to investigate inflammation-induced EndoMT and its etiology, with the goal of impeding the infiltration of peripheral inflammation into the central nervous system.
METHODS: Lipolysaccharide (LPS) was administered intraperitoneally to mice several times to establish a chronic inflammatory model. A cellular inflammatory model was established by LPS in human brain microvascular endothelial cells (HBMECs). The mRNA expressions of inflammatory cytokines interleukin-1β (IL-1β) and IL-6 were detected by real-time polymerase chain reaction (PCR). Immunofluorescence staining of platelet endothelial cell adhesion molecule-1 (CD31) and alpha smooth muscle actin (α-SMA) was conducted to assess the level of EndoMT. The expression levels of Occludin, zona occludens protein 1 (ZO-1), Sestrin2, microtubule-associated protein1 light chain 3 (LC3) and inducible nitric oxide synthase (iNOS) were detected by western blotting.
RESULTS: LPS treatment induced the downregulation of ZO-1 and Occludin, which was accompanied by the elevated expressions of iNOS, α-SMA, Sestrin2 and LC3-II in the mouse cortex and HBMECs. Mechanistically, the knockdown of Sestrin2 in HBMECs exacerbated the EndoMT induced by LPS treatment, while the overexpression of Sestrin2 inhibited this process. Moreover, the induction of autophagy by rapamycin rescued the EndoMT induced by Sestrin2 knockdown.
CONCLUSIONS: This study revealed that Sestrin2 inhibited endothelial inflammation and EndoMT via enhanced autophagy, which may provide a potential drug target for cerebrovascular inflammatory injury.

Abstract-Necrotizing enterocolitis (NEC) is a severe gastrointestinal disease in neonates, and effective strategies to prevent and treat NEC are still lacking. Studies have shown that N-acetylcysteine (NAC) has protective effects against NEC, however, the specific mechanism underlying its effects on intestinal functions remains unclear. Recently, NAC has been shown to suppress ferroptosis in many diseases, while it is unclear whether the beneficial effects of NAC on NEC are related to ferroptosis. In this study, we revealed that ferroptosis was significantly induced in intestinal samples from infants with NEC. NAC alleviated intestinal inflammation, barrier damage and ferroptosis in multifactorial NEC models in vivo and in vitro. Sestrin2 (SESN2) was identified as an important mediator of NAC-induced ferroptosis resistance in intestinal epithelial cells. Furthermore, SESN2 knockdown inhibited the inflammatory response, alleviated barrier damage and ferroptosis in intestinal epithelial cells and enhanced the protective effects of NAC to a certain extent. Conversely, cells overexpressing SESN2 showed the opposite changes. In summary, our study demonstrated that NAC attenuates NEC progression by decreasing SESN2 expression to inhibit ferroptosis in intestinal epithelial cells, suggesting that NAC might be an effective clinical treatment for NEC.

OBJECTIVE: Nitidine chloride (NC), a natural phytochemical alkaloid derived from Zanthoxylum nitidum (Roxb.) DC, exhibits multiple bioactivities, including antitumor, anti-inflammatory, and other therapeutic effects. However, the primary targets of NC and the mechanism of action (MOA) have not been explicitly defined.
METHODS: We explored the effects of NC on mTORC1 signaling by immunoblotting and fluorescence microscopy in wild-type and gene knockout cell lines generated by the CRISPR/Cas9 gene editing technique. We identified IGF2R as a direct target of NC via the drug affinity-responsive target stability (DARTS) method. We investigated the antitumor effects of NC using a mouse melanoma B16 tumor xenograft model.
RESULTS: NC inhibits mTORC1 activity by targeting amino acid-sensing signaling through activating transcription factor 4 (ATF4)-mediated Sestrin2 induction. NC directly binds to IGF2R and promotes its lysosomal degradation. Moreover, NC displayed potent cytotoxicity against various cancer cells and inhibited B16 tumor xenografts.
CONCLUSIONS: NC inhibits mTORC1 signaling through nutrient sensing and directly targets IGF2R for lysosomal degradation, providing mechanistic insights into the MOA of NC.

Sestrin2 is a highly conserved protein that can be induced under various stress conditions. Researches have revealed that the signaling pathway of the mammalian target of rapamycin (mTOR) is essential in modulating both glucose and lipid metabolism. However, the precise involvement of Sestrin2 in the hypothalamus, particularly in pro-opiomelanocortin (POMC) neurons, in control of energy homeostasis remains uncertain. In this study, we aimed to investigate the functional role of Sestrin2 in hypothalamic POMC neurons in regulation of energy balance, as well as revealing the underlying mechanisms. Therefore, cre-dependent AAV virus encoding or silencing Sestrin2 was injected into the hypothalamic ARC of pomc-cre transgenic mice. The results demonstrated that Sestrin2 overexpression in POMC neurons ameliorated high-fat diet (HFD)-induced obesity and increased energy expenditure. Conversely, Sestrin2 deficiency in POMC neurons predisposed mice to HFD induced obesity. Additionally, the thermogenesis of brown adipose tissue and lipolysis of inguinal white adipose tissue were both enhanced by the increased sympathetic nerve innervation in Sestrin2 overexpressed mice. Further exploration revealed that Sestrin2 overexpression inhibited the mTOR signaling pathway in hypothalamic POMC neurons, which may account for the alleviation of systematic metabolic disturbance induced by HFD in these mice. Collectively, our findings demonstrate that Sestrin2 in POMC neurons plays a pivotal role in maintaining energy balance in a context of HFD-induced obesity by inhibiting the mTOR pathway, providing new insights into how hypothalamic neurons respond to nutritional signals to protect against obesity-associated metabolic dysfunction.

Age-related hearing loss (ARHL) is an auditory disease characterized by gradual loss of high-frequency hearing sensitivity. Excessive reactive oxygen species trigger NLRP3-inflammasome activation that may be crucial for ARHL pathogenesis. The antioxidant factor Sestrin2 (SESN2) has been reported to be involved in the remission of oxidative stress and ARHL. However, the mechanism by which SESN2 protects auditory cells in the aging mouse cochlea remains unknown. Here, we observed that ectopic overexpression of SESN2 delayed ARHL, whereas SESN2 knockdown accelerated it. Importantly, we elucidated that SESN2 exerts a hearing-protective effect by inhibiting the production of NLRP3 by acting as a mitophagy agonist. Our study proposes a new theoretical basis for SESN2 prevention of ARHL and provides a novel therapeutic strategy for maintaining SESN2 activity in the aging cochlea.

UNASSIGNED: To explore the correlation of endoplasmic reticulum stress (ERS) and oxidative stress (OS), and the protective effect of Sestrin2 (SESN2) on human lens epithelial cells (HLECs).
UNASSIGNED: Tunicamycin (TM) was used to induce ERS in HLECs. 4-Phenylbutyric acid (4-PBA) was used to inhibit ERS. Eupatilin applied to HLECs as SESN2 agonist. SESN2 expression was knocked down via si-RNA in HLECs. The morphological changes of HLECs were observed by microscope. ER-tracker to evaluate ERS, ROS production assay to measure ROS, flow cytometry to calculate cell apoptosis rate. Immunofluorescence to observe Nrf2 translocation, and effects of TM or EUP on SESN2. Western blot and qPCR were used to evaluate the expression of GRP78, PERK, ATF4, CHOP, Nrf2, and SESN2 expression in HLECs with different treatment groups.
UNASSIGNED: ERS can elevate the expression of ROS and Nrf2 to induce OS. Upregulation of SESN2 was observed in ERS-mediate OS. Overexpression of SESN2 can reduce the overexpression of ERS-related protein GRP78, PERK, ATF4, proapoptotic protein CHOP, OS-related protein Nrf2, as well as ROS, and alleviate ERS injury at the same time. Whereas knockdown of SESN2 can upregulate the expression of GRP78, PERK, ATF4, CHOP, Nrf2, ROS, and deteriorate ERS damage.
UNASSIGNED: ERS can induce OS, they form a vicious cycle to induce apoptosis in HLECs, which may contribute to cataract formation. SESN2 could protect HLECs against the apoptosis by regulating the vicious cycle between ERS and OS.

Most central nervous diseases are accompanied by astrocyte activation. Autophagy, an important pathway for cells to protect themselves and maintain homeostasis, is widely involved in regulation of astrocyte activation. Reactive astrocytes may play a protective or harmful role in different diseases due to different phenotypes of astrocytes. It is an urgent task to clarify the formation mechanisms of inflammatory astrocyte phenotype, A1 astrocytes. Sestrin2 is a highly conserved protein that can be induced under a variety of stress conditions as a potential protective role in oxidative damage process. However, whether Sestrin2 can affect autophagy and involve in A1 astrocyte conversion is still uncovered. In this study, we reported that Sestrin2 and autophagy were significantly induced in mouse hippocampus after multiple intraperitoneal injections of lipopolysaccharide, with the elevation of A1 astrocyte conversion and inflammatory mediators. Knockdown Sestrin2 in C8-D1A astrocytes promoted the levels of A1 astrocyte marker C3 mRNA and inflammatory factors, which was rescued by autophagy inducer rapamycin. Overexpression of Sestrin2 in C8-D1A astrocytes attenuated A1 astrocyte conversion and reduced inflammatory factor levels via abundant autophagy. Moreover, Sestrin2 overexpression improved mitochondrial structure and morphology. These results suggest that Sestrin2 can suppress neuroinflammation by inhibiting A1 astrocyte conversion via autophagy, which is a potential drug target for treating neuroinflammation.

BACKGROUND: Allicin regulates macrophage autophagy and senescence, and inhibits hepatoma cell growth. This study investigated the mechanism by which allicin inhibits the growth of hepatoma cells.
METHODS: Hepa1-6 mouse hepatoma cells were subcutaneously injected into C57BL/6 J mice to construct a tumor transplantation model. Macrophages were cultured with the supernatant of hepatoma cells to construct a cell model. The levels of mRNA and proteins and the level of Sestrin2 ubiquitination were measured by RTqPCR, immunofluorescence and Western blotting. The levels of autophagy-related factors and the activity of senescence-associated β-galactosidase were determined by kits, and protein stability was detected by cycloheximide (CHX) tracking.
RESULTS: Data analysis of clinical samples revealed that RBX1 was highly expressed in tumor tissues, while Sestrin2 was expressed at low levels in tumor tissues. Allicin can promote the expression of the autophagy-related proteins LC3 and Beclin-1 in tumor macrophages and inhibit the expression of the aging-related proteins p16 and p21, thus promoting autophagy in macrophages and inhibiting cell senescence. Moreover, allicin can inhibit the expression of RBX1, thereby reducing the ubiquitination of Sestrin2, enhancing the stability of Sestrin2, activating autophagy in tumor macrophages and inhibiting senescence. In addition, allicin treatment inhibited the proliferation and migration of hepatoma carcinoma cells cocultured with macrophages and significantly improved the development of liver cancer in mice.
CONCLUSIONS: Allicin can affect the autophagy of macrophages and restrain the growth of hepatoma cells by regulating the ubiquitination of Sestrin2.