The decline of microglia in the dentate gyrus is a new phenomenon that may explain the pathogenesis of depression, and reversing this decline has an antidepressant effect. The development of strategies that restore the function of dentate gyrus microglia in under stressful conditions is becoming a new focus. Lymphocyte-activating gene-3 (LAG3) is an immune checkpoint expressed by immune cells including microglia. One of its functions is to suppress the expansion of immune cells. In a recent study, chronic systemic administration of a LAG3 antibody that readily penetrates the brain was reported to reverse chronic stress-induced hippocampal microglia decline and depression-like behaviors. We showed here that a single intranasal infusion of a LAG3 antibody (In-LAG3 Ab) reversed chronic unpredictable stress (CUS)-induced depression-like behaviors in a dose-dependent manner, which was accompanied by an increase in brain-derived neurotrophic factor (BDNF) in the dentate gyrus. Infusion of an anti-BDNF antibody into the dentate gyrus, construction of knock-in mice with the BDNF Val68Met allele, or treatment with the BDNF receptor antagonist K252a abolished the antidepressant effect of In-LAG3 Ab. Activation of extracellular signal-regulated kinase1/2 (ERK1/2) is required for the reversal effect of In-LAG3 Ab on CUS-induced depression-like behaviors and BDNF decrease in the dentate gyrus. Moreover, both inhibition and depletion of microglia prevented the reversal effect of In-LAG3 Ab on CUS-induced depression-like behaviors and impairment of ERK1/2-BDNF signaling in the dentate gyrus. These results suggest that In-LAG3 Ab exhibits an antidepressant effect through microglia-mediated activation of ERK1/2 and synthesis of BDNF in the dentate gyrus.

The role of peroxisome proliferator-activated receptor (PPAR)β/δ in hepatic fibrosis remains a subject of debate. Here, we examined the effects of a PPARβ/δ agonist on the pathogenesis of liver fibrosis and the activation of hepatic stellate cells (HSCs), the main effector cells in liver fibrosis, in response to the pro-fibrotic stimulus transforming growth factor-β (TGF-β). The PPARβ/δ agonist GW501516 completely prevented glucose intolerance and peripheral insulin resistance, blocked the accumulation of collagen in the liver, and attenuated the expression of inflammatory and fibrogenic genes in mice fed a choline-deficient high-fat diet (CD-HFD). The antifibrogenic effect of GW501516 observed in the livers CD-HFD-fed mice could occur through an action on HSCs since primary HSCs isolated from Ppard-/- mice showed increased mRNA levels of the profibrotic gene Col1a1. Moreover, PPARβ/δ activation abrogated TGF-β1-mediated cell migration (an indicator of cell activation) in LX-2 cells (immortalized activated human HSCs). Likewise, GW501516 attenuated the phosphorylation of the main downstream intracellular protein target of TGF-β1, suppressor of mothers against decapentaplegic (SMAD)3, as well as the levels of the SMAD3 co-activator p300 via the activation of AMP-activated protein kinase (AMPK) and the subsequent inhibition of extracellular signal-regulated kinase-1/2 (ERK1/2) in LX-2 cells. Overall, these findings uncover a new mechanism by which the activation of AMPK by a PPARβ/δ agonist reduces TGF-β1-mediated activation of HSCs and fibrosis via the reduction of both SMAD3 phosphorylation and p300 levels.

Ischaemia-reperfusion (IR)-associated acute kidney injury (AKI) is a severe clinical condition that lacks effective pharmacological treatments. Our recent research revealed that pretreatment with the angiotensin II type 2 receptor (AT2R) agonist C21 alleviates kidney damage during IR. Primary cilia are organelles crucial for regulation of epithelial cell homeostasis, which are significantly affected by IR injury. This study aimed to evaluate the impact of AT2R activation on cilia integrity during IR and to identify pathways involved in the nephroprotective effect of C21. Rats were subjected to 40 min of unilateral ischaemia followed by 24 h of reperfusion. Immunofluorescence analysis of the kidneys showed that the nephroprotective effect of C21 was associated with preservation of cilia integrity in tubular cells. AT2R agonists increased α-tubulin acetylation in primary cilia in tubular cells in vivo and in a cell model. Analysis of ERK phosphorylation indicated that AT2R activation led to diminished activation of ERK1/2 in tubular cells. Similar to AT2R agonists, inhibitors of α-tubulin deacetylase HDAC6 or inhibitors of ERK activation ameliorated IR-induced cell death and preserved cilia integrity. Immunofluorescence analysis of tubular cells revealed significant ERK localization at primary cilia and demonstrated that ERK inhibition increased cilia levels of acetylated α-tubulin. Overall, our findings demonstrate that C21 elicits a preconditioning effect that enhances cilia stability in renal tubular cells, thereby preserving their integrity when exposed to IR injury. Furthermore, our results indicate that this effect might be mediated by AT2R-induced inhibition of ERK activation. These findings offer potential insights for the development of pharmacological interventions to mitigate IR-associated AKI. KEY POINTS: The AT2R agonist C21 prevents primary cilia shortening and tubular cell deciliation during renal ischaemia-reperfusion. AT2R activation inhibits ERK1/2 in renal tubular cells. Both AT2R agonists and ERK1/2 inhibitors increase alpha-tubulin acetylation at the primary cilium in tubular cells. AT2R activation, ERK1/2 inhibition or inhibition of alpha-tubulin deacetylation elicit protective effects in tubular cells subjected to ischaemia-reperfusion injury.

UNASSIGNED: Endogenous sulfur dioxide (SO2) plays a crucial role in protecting heart from myocardial fibrosis by inhibiting the excessive growth of cardiac fibroblasts. This study aimed to investigate potential mechanisms by which SO2 suppressed myocardial fibrosis.
UNASSIGNED: Mouse model of angiotensin II (Ang II)-induced cardiac fibrosis and cell model of Ang II-stimulated cardiac fibroblast proliferation were employed. Our findings discovered that SO2 mitigated the aberrant phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2) induced by Ang II, leading to a reduction of fibroblast proliferation. Mechanistically, for the first time, we found that SO2 sulfenylated ERK1/2, and inhibited ERK1/2 phosphorylation and cardiac fibroblast proliferation, while a sulfhydryl reducing agent dithiothreitol (DTT) reversed the above effects of SO2. Furthermore, mutant ERK1C183S (cysteine 183 to serine) abolished the sulfenylation of ERK by SO2, thereby preventing the inhibitory effects of SO2 on ERK1 phosphorylation and cardiac fibroblast proliferation.
UNASSIGNED: Our study suggested that SO2 inhibited cardiac fibroblast proliferation by sulfenylating ERK1/2 and subsequently suppressing ERK1/2 phosphorylation. These new findings might enhance the understanding of the mechanisms underlying myocardial fibrosis and emphasize the potential of SO2 as a novel therapeutic target for myocardial fibrosis.

UNASSIGNED: Osteoarthritis (OA) is an inflammatory disorder of synovial joints which is mainly treated with therapeutic agents showing side effects associated with the gastrointestinal (GI) and metabolic system. Consequently, there is urgent need for a potent, safe and novel agent for treating OA and related disorders. Enoxolone is a pentacyclic triterpenoid obtained from the herb liquorice. Based on earlier findings, we postulated that enoxolone may produce chondroprotective activity by exerting anti-inflammatory, anti-catabolic and oxidative stress-decreasing effects.
UNASSIGNED: The chondrocytes were extracted from the femoral head articular cartilage of healthy rats. Immunofluorescence staining was done for identification of chondrocytes. Cell viability and proliferation studies were done using Cell Counting Kit-8. Apoptotic cells were identified by TUNEL assay and flow cytometry analysis. Autophagy was assessed by monodansylcadaverine assay. Western blot analysis was done for expression of proteins.
UNASSIGNED: In the present study we investigated the protective effect of enoxolone on interleukin 1β (IL-1β) treated Iry chondrocytes in vitro. Treatment with IL-1β resulted in a significant reduction in cell viability of cells in increasing dose and time. Treatment with enoxolone along with IL-1β caused a significant decrease in growth inhibition. Also, enoxolone inhibited the IL-1β mediated apoptosis and activation of caspase-3 in cells. We also observed that enoxolone elevated the levels of p-ERK1/2, light chain 3 (LC3)-II and Beclin-1 (autophagy markers) in chondrocytes. The expression of (LC3)-II and Beclin-1 was decreased when the cells were treated with U0126 (ERK1/2 inhibitor).
UNASSIGNED: Our findings demonstrate that enoxolone could suppress inflammatory signaling and apoptosis via the ERK1/2 pathway in chondrocytes.

Platelet concentrates (PC) are used to treat patients with thrombocytopenia and hemorrhage, but there is still the demand to find the optimal strategy for temperature-dependent storage of PC. Recently, we could show that cold storage for 1 h (short-term refrigeration) is sufficient to induce enhanced platelet responsiveness. The aim of this study was to investigate effects of cold storage on collagen-dependent activating signalling pathways in platelets from apheresis-derived PC (APC). APC on day 1 or day 2 of storage, were either continuously kept at room temperature (RT, 22 °C), or for comparison, additionally kept at cold temperature (CT, 4 °C) for 1 h. CD62P expression was determined by flow cytometry. Western Blot technique was used to analyze collagen-induced phosphorylation of p38, ERK1/2 or Akt/PKB and its inhibition by prostaglandin E1 (PGE1) or nitric monoxide donor. Adhesion of platelets on collagen-coated surfaces and intracellular phosphorylation of vasodilator-stimulated phosphoprotein (VASP) was visualized by immune fluorescence microscopy. CD62P expression was increased after short-term refrigeration. CT exposition for 1 h induced an elevation of basal ERK1/2 phosphorylation and an alleviation of PGE1- or DEA/NO-suppressed ERK1/2 phosphorylation in APC on day 1 and 2 of storage. Similar, but more moderate effects were observable for p38 phosphorylation. Akt/PKB phosphorylation was increased only in APC on day 2. Refrigeration for 1 h promoted platelet adhesion and reduced basal VASP phosphorylation in adherent platelets. The attenuation of inhibitory signalling in short-term refrigerated stored platelets is associated with enhanced reactivity of activating signalling pathways, especially ERK1/2. Functionally, these processes correlate with increased adhesion of refrigerated platelets on collagen-coated surfaces. The results help to further optimize temperature-dependent strategies for platelet storage.

UNASSIGNED: Herbal components, particularly sesquiterpenes, are progressively recognized as a crucial resource for developing effective therapeutic agents for breast cancer. In this study, the effect of a sesquiterpene lactone known as 8-O-dihydroxy-11a,13-dihydroeudesma-4(15)-en-12,6a-olide (persianolide- A) was examined in breast cancer cell lines.
UNASSIGNED: MDA-MB-231 and MCF-7 cancer cells were grown in DMEM solution with 10% FBS. Then, an MTT assay was performed to evaluate cell viability. Apoptosis was detected by annexin-PI staining. A caspase 3/7 activity assay kit was used to assess the activity of caspase-3 and caspase-7. Protein expression of Bcl-2, Bax, and p-ERK1/2 was determined by western blotting.
UNASSIGNED: This study showed that the IC50 values of the persianolide-A for MCF-7 and MDA-MB- 468 cells are 34.76 and 54.48 μM, respectively. In addition, persianolide-A showed a significant increase in apoptosis in both MDAMB-231 and MCF-7 breast cancer cell lines. Persianolide-A significantly increased the expression of the pro-apoptotic protein Bax and decreased the expression of the anti-apoptotic protein Bcl-2. Also, presinolide-A treatment led to a substantial increase in caspase activity with a ratio of 3/7 in both MCF- 7 and MDA-MB-231 cancer cells. In addition, the study showed that persianolide-A decreased the expression of p-ERK1/2 protein.
UNASSIGNED: The results of this study suggest that persianolide-A, sourced from Artemisia kopetdaghensis, induces cell apoptosis in breast cancer cell types. The molecular mechanisms could be implicated in the modulation of the ERK1/2 signaling pathway.

Prostaglandin-endoperoxide synthase 2 (PTGS2), a common biological macromolecule, is pivotal for innate immunity and pathogen recognition. In this study, we identified and characterized a CcPTGS2a-like gene in the common carp (Cyprinus carpio) with an open reading frame (ORF) of 1821 bp and epidermal growth factor and peroxidase domains. Our multiple sequence analysis revealed high homology between the amino acid sequence of CcPTGS2a-like and those of its homologs in other fish. CcPTGS2a-like mRNA and protein expressions were significantly upregulated in the spleen, head kidney, liver, and gill tissues upon exposure to Aeromonas hydrophila stimulation. CcPTGS2a-like protein recognized the conserved bacterial surface components and exhibited detectable bacterial binding activity. CcPTGS2a-like overexpression before exposure to A. hydrophila notably enhanced the survival rate of common carp, concomitant with decreased bacterial burden. The NF-κB/ERK signaling pathway initiated the immune response in common carp upon infection with A. hydrophila. CcPTGS2a-like overexpression or interference in the head kidney and Epithelioma papulosum cyprinid cells could modulate the p-NF-κB (p-p-65), p-IκBα, and p-ERK1/2 levels as well as the IL-1β and IL-6 mRNA expression. These results indicated potential CcPTGS2a-like involvement in the immune response of the common carp to bacterial infections through the NF-κB/ERK signaling pathway.

BACKGROUND: Acquired resistance to osimertinib limits its clinical efficacy in non-small cell lung cancer (NSCLC) with EGFR mutations. The widespread recognition of Taxus chinensis var. Mairei (Lemée et Lévl) Cheng et L.K. Fu (Chinese yew) as a natural anti-cancer medication is well-established. However, the specific contribution of Taxus chinensis var. Mairei (Lemée et Lévl) Cheng et L.K. Fu in addressing resistance to osimertinib is still uncertain.
OBJECTIVE: Based on the biological behaviors and lipid metabolism, we investigated whether aqueous extract of Taxus chinensis var. Mairei (Lemée et Lévl) Cheng et L.K. Fu (AETC) could enhance the antitumor effect of osimertinib in NSCLC with an investigation on the precise mechanisms.
METHODS: The effect of AETC on enhancing osimertinib sensitivity was assessed via cell viability measurements, levels of reactive oxygen species (ROS), apoptosis, and lipid levels. Western blotting was used to verify the mechanisms of AETC responsible for overcoming the resistance to osimertinib via ERK1/2 overexpression and knockdown models. In vivo validation was conducted using subcutaneous xenografts from osimertinib-resistant cells in nude mice.
RESULTS: Osimertinib-resistant cells exhibited altered cholesterol biosynthesis, which was induced by ERK1/2 activation. The combination of AETC and osimertinib can synergistically decrease the levels of ROS in cells, enhance apoptosis, and inhibit the growth of osimertinib-resistant cells. Mechanistic experiments demonstrated that AETC can downregulate the key regulators of cholesterol biosynthesis by regulating ERK1/2, inhibiting the endogenous synthesis rate of cholesterol, and suppressing the level of lipids in osimertinib-resistant cells and xenograft tumors when combined with osimertinib, ultimately reversing resistance to osimertinib.
CONCLUSIONS: The resistance to osimertinib is significantly influenced by cholesterol biosynthesis, highlighting its pivotal role in this context. AETC can enhance osimertinib sensitivity via ERK/SREBP-2/HMGCR-mediated cholesterol biosynthesis. These results provide a promising therapeutic target and potential treatment option for resistance to osimertinib.

Sleep/wake cycles intricately shape physiological activities including cognitive brain functions, yet the precise molecular orchestrators of sleep remain elusive. Notably, the clinical impact of benzodiazepine drugs underscores the pivotal role of GABAergic neurotransmission in sleep regulation. However, the specific contributions of distinct GABAA receptor subtypes and their principal scaffolding protein, gephyrin, in sleep dynamics remain unclear. The evolving role of synaptic phospho-proteome alterations at excitatory and inhibitory synapses suggests a potential avenue for modulating gephyrin and, consequently, GABAARs for sleep through on-demand kinase recruitment. Our study unveils the distinctive roles of two prevalent GABAA receptor subtypes, α1- and α2-GABAARs, in influencing sleep duration and electrical sleep activity. Notably, the absence of α1-GABAARs emerges as central in sleep regulation, manifesting significant alterations in both non-rapid eye movement (NREM) and rapid eye movement (REM) sleep during dark or active phases, accompanied by altered electroencephalogram (EEG) patterns across various frequencies. Gephyrin proteomics analysis reveals sleep/wake-dependent interactions with a repertoire of known and novel kinases. Crucially, we identify the regulation of gephyrin interaction with ERK1/2, and phosphorylations at serines 268 and 270 are dictated by sleep/wake cycles. Employing AAV-eGFP-gephyrin or its phospho-null variant (S268A/S270A), we disrupt sleep either globally or locally to demonstrate gephyrin phosphorylation as a sleep regulator. In summary, our findings support the local cortical sleep hypothesis and we unveil a molecular mechanism operating at GABAergic synapses, providing critical insights into the intricate regulation of sleep.