Acta Pharmacologica Sinica最新文献

While immune checkpoint inhibitors (ICIs) are promising in the treatment of metastatic melanoma, about half of patients do not respond well to them. Low levels of human leukocyte antigen-DR (HLA-DR) in tumors have been shown to negatively influence prognosis and response to ICIs. Lysophosphatidic acid (LPA) is produced in large amounts by melanoma and is abundantly present in the tumor microenvironment. LPA induces the release of various cytokines and chemokines from tumor cells, which affect cancer development, metastasis, and tumor immunity. In the present study, we investigated the role of LPA-induced IL-10 release in regulating HLA-DR expression and the underlying mechanisms in human melanoma cells. We showed that LPA (0.001-10 μM) dose-dependently increased DR6 transcript levels through activating LPAR1 in HEK293T cells. Knockdown of NF-κB1 abrogated the LPA-increased DR6 expression without affecting basal DR6 expression in both A2058 and A375 melanoma cell lines. LPA (10 µM) significantly increased IL-10 transcripts in A2058 and A375 melanoma cells, the effect was abolished by pharmacological inhibition of LPAR1 or knockdown of DR6. We found a statistically significant correlation between the expression of LPAR1, DR6 and IL-10 in human melanoma tissue and an association between increased expression of LPAR1 and reduced effectiveness of ICI therapy. We demonstrated that LPA (10 µM) markedly suppressed HLA-DR expression in both A375 and A2058 melanoma cells via activating the LPAR1-DR6-IL-10 pathway. These data suggest that the LPAR1-DR6-IL-10 autocrine loop could constitute a novel mechanism used by tumor cells to evade immunosurveillance by decreasing HLA-DR expression.

Pulmonary fibrosis (PF) is a chronic, progressive and irreversible interstitial lung disease characterized by unremitting pulmonary myofibroblasts activation, extracellular matrix (ECM) deposition and inflammatory recruitment. PF has no curable medication yet. In this study we investigated the molecular pathogenesis and potential therapeutic targets of PF and discovered drug lead compounds for PF therapy. A murine PF model was established in mice by intratracheal instillation of bleomycin (BLM, 5 mg/kg). We showed that the protein level of pulmonary protein phosphatase magnesium-dependent 1A (PPM1A, also known as PP2Cα) was significantly downregulated in PF patients and BLM-induced PF mice. We demonstrated that TRIM47 promoted ubiquitination and decreased PPM1A protein in PF progression. By screening the lab in-house compound library, we discovered otilonium bromide (OB, clinically used for treating irritable bowel syndrome) as a PPM1A enzymatic activator with an EC₅₀ value of 4.23 μM. Treatment with OB (2.5, 5 mg·kg^-1·d^-1, i.p., for 20 days) significantly ameliorated PF-like pathology in mice. We constructed PF mice with PPM1A-specific knockdown in the lung tissues, and determined that by targeting PPM1A, OB treatment suppressed ECM deposition through TGF-β/SMAD3 pathway in fibroblasts, repressed inflammatory responses through NF-κB/NLRP3 pathway in alveolar epithelial cells, and blunted the crosstalk between inflammation in alveolar epithelial cells and ECM deposition in fibroblasts. Together, our results demonstrate that pulmonary PPM1A activation is a promising therapeutic strategy for PF and highlighted the potential of OB in the treatment of the disease.

Our previous study shows that activation of pregnane X receptor (PXR) exerts hepatoprotection against lithocholic acid (LCA)-induced cholestatic liver injury. In this study we investigated whether PXR activation could inhibit hepatocyte pyroptosis, as well as the underlying mechanisms. Male mice were treated with mouse PXR agonist pregnenolone 16α-carbonitrile (PCN, 50 mg·kg^-1·d^-1, i.p.) for 7 days, and received LCA (125 mg/kg, i.p., bid) from D4, then sacrificed 12 h after the last LCA injection. We showed that LCA injection resulted in severe cholestatic liver injury characterized by significant increases in gallbladder size, hepatocellular necrosis, and neutrophil infiltration with a mortality rate of 68%; PCN treatment significantly inhibited hepatocyte pyroptosis during LCA-induced cholestatic liver injury, as evidenced by reduced serum lactic dehydrogenase (LDH) levels, TUNEL-positive cells and hepatocyte membrane damage. Furthermore, PXR activation suppressed both the NOD-like receptor protein 3 (NLRP3) inflammasome-induced canonical pyroptosis and the apoptosis protease activating factor-1 (APAF-1) pyroptosome-induced non-canonical pyroptosis. Inhibition of the nuclear factor kappa B (NF-κB) and forkhead box O1 (FOXO1) signaling pathways was also observed following PXR activation. Notably, dual luciferase reporter assay showed that PXR activation inhibited the transcriptional effects of NF-κB on NLRP3, as well as FOXO1 on APAF-1. Our results demonstrate that PXR activation protects against cholestatic liver injury by inhibiting the canonical pyroptosis through the NF-κB-NLRP3 axis and the non-canonical pyroptosis through the FOXO1-APAF-1 axis, providing new evidence for PXR as a prospective anti-cholestatic target.

Abdominal aortic aneurysm (AAA) is a degenerative disease that caused mortality in people aged >65. Senescence plays a critical role in AAA pathogenesis. Advances in AAA repair techniques have occurred, but a remaining priority is therapies to limit AAA growth and rupture. Our Previous study found cyclic nucleotide phosphodiesterase 1C (PDE1C) exacerbate AAA through aggravate vascular smooth muscle cells (VSMCs) senescence by downregulating Sirtuin1 (SIRT1) expression and activity. Vinpocetine as a selective inhibitor of PDE1 and a clinical medication for cerebral vasodilation, it is unclear whether vinpocetine can rely on SIRT1 to alleviate AAA. This study showed that pre-treatment with vinpocetine remarkably prevented aneurysmal dilation and reduced aortic rupture in elastase-induced AAA mice. In addition, the elastin degradation, MMP (matrix metalloproteinase) activity, macrophage infiltration, ROS production, collagen fibers remodeling, and VSMCs senescence were decreased in AAA treated with vinpocetine. While these effects were unable to exert in VSMCs-specific SIRT1 knockout AAA mice. Accordingly, we revealed that vinpocetine suppressed migration, proliferation, and senescence in VSMCs. Moreover, vinpocetine reduced SIRT1 degradation by inhibiting lysosome-mediated autophagy. In conclusion, this study indicated that vinpocetine may be as a potential drug for therapy AAA through alleviate VSMCs senescence via the SIRT1-dependent pathway.

Carnitine O-acetyltransferase (CRAT) is a crucial enzyme involved in mitochondrial energy metabolism. Alterations in CRAT activity have emerged as significant contributors to the pathogenesis of Leigh syndrome and related mitochondrial disorders. In this study we employed an integrated approach combining in silico docking analysis and virtual screening of chemical libraries with subsequent in vitro validation to identify small molecule modulators of the activity of the wild type (WT) CRAT and the p.Tyr110Cys (Y110C) variant associated to an early onset case of Leigh syndrome. Through 3D molecular modeling, docking simulations, and virtual screening of chemical libraries, potential CRAT modulators were prioritized based on their predicted binding affinities and interactions with the 3D models of the WT-CRAT and of the p.Tyr110Cys-CRAT mutant. The performed in silico analyses were validated through in vitro assays on the purified recombinant CRAT proteins and cell-lysates from control fibroblasts and the fibroblasts of a patient with genetic diagnosis of CRAT-deficiency, carrying the compound heterozygous missense variants in the CRAT gene, namely p.Tyr110Cys and p.Val569Met. Based on the above screening by applying the indicated filtering strategy and mentioned criteria, 3 commercially available approved drugs (also known for their possible interactions with mitochondria) namely glimepiride, artemisinin and dorzolamide, as well as suramin (already known for its ability to interact with mitochondrial proteins) were tested in in vitro assays. We found that suramin (1-1000 μM) dose-dependently inhibited the activity of both WT-CRAT and p.Tyr110Cys_CRAT variant. Artemisinin (0.1-200 μM) dose-dependently stimulated the activity of the recombinant p.Tyr110Cys CRAT mutant, whereas glimepiride and dorzolamide did not change the activity of these proteins towards acetyl-CoA. This study demonstrates the effectiveness of this combined approach in identifying novel compounds for modulating CRAT enzyme activity, providing valuable insights for potential therapeutic interventions targeting CRAT-related disorders.

Abnormal activation of NLRP3 inflammasome causes the progression of gout, and no small-molecule inhibitor of NLRP3 has been approved yet for clinical use. In this study we established a nigericin-induced inflammasome activation cell model for screening of a natural product library by measuring IL-1β secretion in cell supernatants. Among 432 compounds tested, we found that hypocrellin A (HA), one of the major active components of a traditional ethnic medicinal fungus Hypocrella bambusea in the Northwest Yunnan of China, exhibited the highest inhibition on IL-1β production (IC₅₀ = 0.103 μM). In PMA-primed THP-1 cells or bone marrow derived macrophages (BMDMs) treated with multiple stimuli (nigericin, ATP or MSU), HA dose-dependently suppressed the activation of NLRP3 inflammasome, reducing the subsequent release of inflammatory cytokines and LDH. Furthermore, the suppression of inflammasome activation by HA was specific to NLRP3, but not to AIM2 or NLRC4. In LPS-primed BMDMs treated with nigericin, HA inhibited ASC oligomerization and speckle formation, and blocked the NLRP3-NEK7 interaction during inflammasome assembly without influencing the priming stage. Moreover, we demonstrated that HA directly bound to the NACHT domain of NLRP3, and that Arg578 and Glu629 were the critical residues for HA binding to NLRP3. In MSU-induced peritonitis and acute gouty arthritis mouse models, administration of HA (10 mg/kg, i.p., once or twice daily) effectively suppressed the inflammatory responses mediated by NLRP3 inflammasome. We conclude that HA is a broad-spectrum and specific NLRP3 inhibitor, and a valuable lead compound to develop novel therapeutic inhibitors against NLRP3-driven diseases. This study also elucidates the anti-inflammation mechanisms and molecular targets of HA, a major active component in medicinal fungus Hypocrella bambusea that has been long used by Chinese ethnic groups.

Paclitaxel is one of the main chemotherapic medicines against triple-negative breast cancer (TNBC) in clinic. However, it has been perplexed by paclitaxel resistance in TNBC patients, resulting in a poor prognosis. Abnormal protein glycosylation is closely related to the occurrence and progression of tumors and malignant phenotypes such as chemotherapy resistance. CD24 is a highly glycosylated membrane protein that is highly expressed in TNBC, leading to tumorigenesis and poor prognosis. In this study we investigated the relationship between abnormal glycosylation of CD24 and paclitaxel susceptibility in TNBC and the molecular mechanisms. We showed that CD24 protein levels were significantly up-regulated in both TNBC tissues and cells, and CD24 protein was highly glycosylated. Genetic and pharmacological inhibition of N-glycosylation of CD24 enhances the anticancer activity of paclitaxel in vitro and tumor xenograft models. We revealed that the molecular mechanism of N-glycosylation of CD24 in paclitaxel resistance involved inhibition of ferroptosis, a new form that regulates cell death. Inhibition of N-glycosylation of CD24 increased glutathione consumption, iron content, and lipid peroxidation, resulting in paclitaxel-induced ferroptosis. We demonstrated that endoplasmic reticulum (ER)-associated glycosyltransferase STT3 isoforms (including both STT3A and STT3B isoforms) enable N-glycosylation of the L-asparagine (N) site. Knockout of the endogenous STT3 isoform in TNBC cells partially reduced the glycosylation status of CD24. Our results demonstrate the critical role of N-glycosylation of CD24 in weakening drug sensitivity by inhibiting ferroptosis, highlighting new insights that targeting N-glycosylation of CD24 has great potential to promote chemotherapy sensitivity and efficacy.

Itch causes a strong urge to scratch and induces negative emotions, such as aversion and anxiety. Antihistamine medications are key in the clinical management of pruritus, but their therapeutic efficacy in controlling moderate and severe itching remains limited. The neural circuits in the brain that process itching and itch-induced aversion and anxiety remain unclear so far. Human brain imaging suggests that the medial prefrontal cortex (mPFC) is involved in processing the emotional and motivational components of itching. In this study, we investigated the mechanisms by which glutamatergic and GABAergic neurons in mPFC differentially regulated pruritic sensation and emotion through cannabinoid type 1 receptors (CB1Rs). Chloroquinoline (CQ)-induced acute and calcipotriol (MC903)-induced chronic itch models were established. Fiberoptic calcium imaging was used to detect the activity of the two types of neurons in response to itching. The CB1R antagonist AM251 (0.5 mg in 200 nL) was microinjected into the mPFC through the implanted cannula. We showed that chemogenetic activation of glutamatergic neurons and inhibition of GABAergic neurons in the mPFC reduced scratching and chronic itch-induced anxiety. GABAergic, but not glutamatergic, neurons were involved in acute itch-induced aversion. CB1Rs on glutamatergic and GABAergic neurons modulated chronic itch-induced scratching and anxiety in divergent manners. However, CB1Rs did not affect acute itch-induced scratching. CB1Rs on GABAergic, but not glutamatergic, neurons regulated acute itch-induced aversion. These results may guide the development of therapeutic strategies targeting CB1Rs to treat itch-induced sensory and emotional responses.

Despite the vital role of astrocytes in preserving blood-brain barrier (BBB) integrity, their therapeutic potential as targets in ischemic stroke-induced barrier disruption remains underexplored. We previously reported externalization of phosphatidylserine (PS) on astrocytic membranes concurrent with the emergence of PS externalization in neurons. PS externalization of astrocytes induced microglial phagocytosis of astrocytes, resulting in reduced astrocyte-vascular coupling and subsequent BBB breakdown. Annexin A5 (ANXA5) belongs to the superfamily of calcium (Ca²⁺)- and phospholipid-binding proteins. Here, we report two X-ray structures of human ANXA5, including monomeric ANXA5 (1.42 Å) and dimeric ANXA5 (1.80 Å). Through the combination of molecular docking and functional analysis, we explored the mechanism of action of ANXA5 in stroke treatment. In addition, we observed a clear increase in therapeutic efficacy corresponding to the increased affinity of ANXA5 for PS. In summary, the phagocytosis of PS-externalized astrocytes by microglia has emerged as a critical mechanism driving BBB breakdown after ischemia. Our findings offer valuable structural insight into ANXA5 as an innovative pharmacological target for safeguarding blood-brain barrier integrity after cerebral ischemia. These insights may facilitate the development of novel PS-targeting medications aimed at achieving enhanced efficacy with minimal side effects.