Phosphatases maintain low catalytic activity of SGK1: DNA damage resets the balance in favor of phosphorylation.

The serum- and glucocorticoid-induced kinase 1 (SGK1) promotes cell survival under stress conditions and facilitates the emergence of drug resistance in cancer. The underlying mechanisms of these observations are not fully understood. In this study, we found that SGK1 activity is suppressed by the action of the S/T phosphatases PP5 and PP2A, which constantly dephosphorylate SGK1. Using newly developed anti-phospho SGK1 antibodies and inhibitors of phosphatases, we determined that the high degree of dephosphorylation is caused by two factors: the tendency of SGK1 to unfold, which makes it dependent on Hsp90 chaperone complexes composed of four proteins, Hsp90/CDC37/PP5/SGK1, and where the phosphatase PP5 persistently dephosphorylates SGK1 within the complex. SGK1 binding to PP2A regulatory subunits B55γ and B55δ brings PP2A catalytic subunit close to exposed SGK1 phosphoresidues. A further association of phosphorylated pS37-FAM122A-an endogenous inhibitor of PP2A-to the holoenzyme diminishes dephosphorylation of SGK1 mediated by PP2A. Our study also reveals that genotoxic stress can reverse the dominant impact of phosphatases over kinases by activating the DNA-dependent protein kinase, which enhances mTORC2 activity directed to SGK1. Thus, our results provide insight into a molecular pathway that enables SGK1 to gain phosphorylation and catalytic activity and promote cell survival, potentially diminishing the efficacy of cancer treatments. As the DNA damage response operates in many cancer cells and is further induced by chemotherapies, the findings of this study could have significant implications for the development of novel cancer therapies targeting SGK1.

The HIV-1 Viral Protease Is Activated during Assembly and Budding Prior to Particle Release.

HIV-1 encodes a viral protease that is essential for the maturation of infectious viral particles. While protease inhibitors are effective antiretroviral agents, recent studies have shown that prematurely activating, rather than inhibiting, protease function leads to the pyroptotic death of infected cells, with exciting implications for efforts to eradicate viral reservoirs. Despite 40 years of research into the kinetics of protease activation, it remains unclear exactly when protease becomes activated. Recent reports have estimated that protease activation occurs minutes to hours after viral release, suggesting that premature protease activation is challenging to induce efficiently. Here, monitoring viral protease activity with sensitive techniques, including nanoscale flow cytometry and instant structured illumination microscopy, we demonstrate that the viral protease is activated within cells prior to the release of free virions. Using genetic mutants that lock protease into a precursor conformation, we further show that both the precursor and mature protease have rapid activation kinetics and that the activity of the precursor protease is sufficient for viral fusion with target cells. Our finding that HIV-1 protease is activated within producer cells prior to release of free virions helps resolve a long-standing question of when protease is activated and suggests that only a modest acceleration of protease activation kinetics is required to induce potent and specific elimination of HIV-infected cells. IMPORTANCE HIV-1 protease inhibitors have been a mainstay of antiretroviral therapy for more than 2 decades. Although antiretroviral therapy is effective at controlling HIV-1 replication, persistent reservoirs of latently infected cells quickly reestablish replication if therapy is halted. A promising new strategy to eradicate the latent reservoir involves prematurely activating the viral protease, which leads to the pyroptotic killing of infected cells. Here, we use highly sensitive techniques to examine the kinetics of protease activation during and shortly after particle formation. We found that protease is fully activated before virus is released from the cell membrane, which is hours earlier than recent estimates. Our findings help resolve a long-standing debate as to when the viral protease is initially activated during viral assembly and confirm that prematurely activating HIV-1 protease is a viable strategy to eradicate infected cells following latency reversal.

Structural basis of neuropeptide Y signaling through Y1 receptor.

Neuropeptide Y (NPY) is highly abundant in the brain and involved in various physiological processes related to food intake and anxiety, as well as human diseases such as obesity and cancer. However, the molecular details of the interactions between NPY and its receptors are poorly understood. Here, we report a cryo-electron microscopy structure of the NPY-bound neuropeptide Y1 receptor (Y1R) in complex with Gi1 protein. The NPY C-terminal segment forming the extended conformation binds deep into the Y1R transmembrane core, where the amidated C-terminal residue Y36 of NPY is located at the base of the ligand-binding pocket. Furthermore, the helical region and two N-terminal residues of NPY interact with Y1R extracellular loops, contributing to the high affinity of NPY for Y1R. The structural analysis of NPY-bound Y1R and mutagenesis studies provide molecular insights into the activation mechanism of Y1R upon NPY binding.

Live imaging approach of dynamic multicellular responses in ERK signaling during vertebrate tissue development.

The chemical and mechanical responses of cells via the exchange of information during growth and development result in the formation of biological tissues. Information processing within the cells through the signaling pathways and networks inherent to the constituent cells has been well-studied. However, the cell signaling mechanisms responsible for generating dynamic multicellular responses in developing tissues remain unclear. Here, I review the dynamic multicellular response systems during the development and growth of vertebrate tissues based on the extracellular signal-regulated kinase (ERK) pathway. First, an overview of the function of the ERK signaling network in cells is provided, followed by descriptions of biosensors essential for live imaging of the quantification of ERK activity in tissues. Then adducing four examples, I highlight the contribution of live imaging techniques for studying the involvement of spatio-temporal patterns of ERK activity change in tissue development and growth. In addition, theoretical implications of ERK signaling are also discussed from the viewpoint of dynamic systems. This review might help in understanding ERK-mediated dynamic multicellular responses and tissue morphogenesis.

Regulation of the NLRP3 Inflammasome by Posttranslational Modifications.

Inflammasomes are important in human health and disease, whereby they control the secretion of IL-1ß and IL-18, two potent proinflammatory cytokines that play a key role in inflammatory responses to pathogens and danger signals. Several inflammasomes have been discovered over the past two decades. NLRP3 inflammasome is the best characterized and can be activated by a wide variety of inducers. It is composed of a sensor, NLRP3, an adapter protein, ASC, and an effector enzyme, caspase-1. After activation, caspase-1 mediates the cleavage and secretion of bioactive IL-1ß and IL-18 via gasdermin-D pores in the plasma membrane. Aberrant activation of NLRP3 inflammasomes has been implicated in a multitude of human diseases, including inflammatory, autoimmune, and metabolic diseases. Therefore, several mechanisms have evolved to control their activity. In this review, we describe the posttranslational modifications that regulate NLRP3 inflammasome components, including ubiquitination, phosphorylation, and other forms of posttranslational modifications.

Acetylation-dependent regulation of BRAF oncogenic function.

Aberrant BRAF activation, including the BRAFV600E mutation, is frequently observed in human cancers. However, it remains largely elusive whether other types of post-translational modification(s) in addition to phosphorylation and ubiquitination-dependent regulation also modulate BRAF kinase activity. Here, we report that the acetyltransferase p300 activates the BRAF kinase by promoting BRAF K601 acetylation, a process that is antagonized by the deacetylase SIRT1. Notably, K601 acetylation facilitates BRAF dimerization with RAF proteins and KSR1. Furthermore, K601 acetylation promotes melanoma cell proliferation and contributes to BRAFV600E inhibitor resistance in BRAFV600E harboring melanoma cells. As such, melanoma patient-derived K601E oncogenic mutation mimics K601 acetylation to augment BRAF kinase activity. Our findings, therefore, uncover a layer of BRAF regulation and suggest p300 hyperactivation or SIRT1 deficiency as potential biomarkers to determine ERK activation in melanomas.

GPCR activation mechanisms across classes and macro/microscales.

Two-thirds of human hormones and one-third of clinical drugs activate ~350 G-protein-coupled receptors (GPCR) belonging to four classes: A, B1, C and F. Whereas a model of activation has been described for class A, very little is known about the activation of the other classes, which differ by being activated by endogenous ligands bound mainly or entirely extracellularly. Here we show that, although they use the same structural scaffold and share several 'helix macroswitches', the GPCR classes differ in their 'residue microswitch' positions and contacts. We present molecular mechanistic maps of activation for each GPCR class and methods for contact analysis applicable for any functional determinants. This provides a superfamily residue-level rationale for conformational selection and allosteric communication by ligands and G proteins, laying the foundation for receptor-function studies and drugs with the desired modality.

Structural basis and mechanism of activation of two different families of G proteins by the same GPCR.

The ß1-adrenergic receptor (ß1-AR) can activate two families of G proteins. When coupled to Gs, ß1-AR increases cardiac output, and coupling to Gi leads to decreased responsiveness in myocardial infarction. By comparative structural analysis of turkey ß1-AR complexed with either Gi or Gs, we investigate how a single G-protein-coupled receptor simultaneously signals through two G proteins. We find that, although the critical receptor-interacting C-terminal α5-helices on Gαi and Gαs interact similarly with ß1-AR, the overall interacting modes between ß1-AR and G proteins vary substantially. Functional studies reveal the importance of the differing interactions and provide evidence that the activation efficacy of G proteins by ß1-AR is determined by the entire three-dimensional interaction surface, including intracellular loops 2 and 4 (ICL2 and ICL4).

A Drug-Drug Interaction Study Evaluating the Effect of Givosiran, a Small Interfering Ribonucleic Acid, on Cytochrome P450 Activity in the Liver.

Givosiran (trade name GIVLAARI) is a small interfering ribonucleic acid that targets hepatic delta-aminolevulinic acid synthase 1 (ALAS1) messenger RNA for degradation through RNA interference (RNAi) that has been approved for the treatment of acute hepatic porphyria (AHP). RNAi therapeutics, such as givosiran, have a low liability for drug-drug interactions (DDIs) because they are not metabolized by cytochrome 450 (CYP) enzymes, and do not directly inhibit or induce CYP enzymes in the liver. The pharmacodynamic effect of givosiran (lowering of hepatic ALAS1, the first and rate limiting enzyme in the heme biosynthesis pathway) presents a unique scenario where givosiran could potentially impact heme-dependent activities in the liver, such as CYP enzyme activity. This study assessed the impact of givosiran on the pharmacokinetics of substrates of 5 major CYP450 enzymes in subjects with acute intermittent porphyria (AIP), the most common type of AHP, by using the validated "Inje cocktail," comprised of caffeine (CYP1A2), losartan (CYP2C9), omeprazole (CYP2C19), dextromethorphan (CYP2D6), and midazolam (CYP3A4). We show that givosiran treatment had a differential inhibitory effect on CYP450 enzymes in the liver, resulting in a moderate reduction in activity of CYP1A2 and CYP2D6, a minor effect on CYP3A4 and CYP2C19, and a similar weak effect on CYP2C9. To date, this is the first study evaluating the DDI for an oligonucleotide therapeutic and highlights an atypical drug interaction due to the pharmacological effect of givosiran. The results of this study suggest that givosiran does not have a large effect on heme-dependent CYP enzyme activity in the liver.

Molecular basis for kinin selectivity and activation of the human bradykinin receptors.

Bradykinin and kallidin are endogenous kinin peptide hormones that belong to the kallikrein-kinin system and are essential to the regulation of blood pressure, inflammation, coagulation and pain control. Des-Arg10-kallidin, the carboxy-terminal des-Arg metabolite of kallidin, and bradykinin selectively activate two G protein-coupled receptors, type 1 and type 2 bradykinin receptors (B1R and B2R), respectively. The hyperactivation of bradykinin receptors, termed 'bradykinin storm', is associated with pulmonary edema in COVID-19 patients, suggesting that bradykinin receptors are important targets for COVID-19 intervention. Here we report two G protein-coupled complex structures of human B1R and B2R bound to des-Arg10-kallidin and bradykinin, respectively. Combined with functional analysis, our structures reveal the mechanism of ligand selectivity and specific activation of the bradykinin receptor. These findings also provide a framework for guiding drug design targeting bradykinin receptors for the treatment of inflammation, cardiovascular disorders and COVID-19.

Grazing intensity changed the activities of nitrogen assimilation related enzymes in desert Steppe Plants.

BACKGROUND: Nitrogen, as a limiting factor for net primary productivity in grassland ecosystems, is an important link in material cycles in grassland ecosystems. However, the nitrogen assimilation efficiency and mechanisms of grassland plants under grazing disturbance are still unclear. This study investigated Stipa breviflora desert steppe which had been grazed for 17 years and sampled the root system and leaf of the constructive species Stipa breviflora during the peak growing season under no grazing, light grazing, moderate grazing and heavy grazing treatments. The activities of enzymes related to nitrogen assimilation in roots and leaves were measured. RESULTS: Compared with no grazing, light grazing and moderate grazing significantly increased the activities of nitrate reductase (NR), glutamine synthetase (GS), glutamic oxaloacetic transaminase (GOT) and glutamic pyruvate transaminase (GPT) in leaves, and GS, GOT and GPT in roots of Stipa breviflora, while heavy grazing significantly decreased the activities of GS in leaves and NR in roots of Stipa breviflora. NR, GOT and GPT activities in leaves and roots of Stipa breviflora were positively correlated with nitrogen content, soluble protein, free amino acid and nitrate content. CONCLUSIONS: Grazing disturbance changed the activities of nitrogen assimilation related enzymes of grassland plants, and emphasized that light grazing and moderate grazing were beneficial for nitrogen assimilation by grassland plants. Therefore, establishing appropriate stocking rates is of great significance for material flows in this grassland ecosystem and for the stability and sustainable utilization of grassland resources.

Lack of Cdk5 activity is involved on Dopamine Transporter expression and function: Evidences from an animal model of Attention-Deficit Hyperactivity Disorder.

Attention deficit/Hyperactivity disorder (ADHD) is one of the most diagnosed psychiatric disorders nowadays. The core symptoms of the condition include hyperactivity, impulsiveness and inattention. The main pharmacological treatment consists of psychostimulant drugs affecting Dopamine Transporter (DAT) function. We have previously shown that genetically modified mice lacking p35 protein (p35KO), which have reduced Cdk5 activity, present key hallmarks resembling those described in animal models useful for studying ADHD. The p35KO mouse displays spontaneous hyperactivity and shows a calming effect of methylphenidate or amphetamine treatment. Interestingly, dopaminergic neurotransmission is altered in these mice as they have an increased Dopamine (DA) content together with a low DA turnover. This led us to hypothesize that the lack of Cdk5 activity affects DAT expression and/or function in this animal model. In this study, we performed biochemical assays, cell-based approaches, quantitative fluorescence analysis and functional studies that allowed us to demonstrate that p35KO mice exhibit decreased DA uptake and reduced cell surface DAT expression levels in the striatum (STR). These findings are supported by in vitro observations in which the inhibition of Cdk5 activity in N2a cells induced a significant increase in constitutive DAT endocytosis with a concomitant increase in DAT localization to recycling endosomes. Taken together, these data provide evidences regarding the role of Cdk5/p35 in DAT expression and function, thus contributing to the knowledge of DA neurotransmission physiology and also providing therapeutic options for the treatment of DA pathologies such as ADHD.

Heat-dependent opening of TRPV1 in the presence of capsaicin.

Transient receptor potential vanilloid member 1 (TRPV1) is a Ca2+-permeable cation channel that serves as the primary heat and capsaicin sensor in humans. Using cryo-EM, we have determined the structures of apo and capsaicin-bound full-length rat TRPV1 reconstituted into lipid nanodiscs over a range of temperatures. This has allowed us to visualize the noxious heat-induced opening of TRPV1 in the presence of capsaicin. Notably, noxious heat-dependent TRPV1 opening comprises stepwise conformational transitions. Global conformational changes across multiple subdomains of TRPV1 are followed by the rearrangement of the outer pore, leading to gate opening. Solvent-accessible surface area analyses and functional studies suggest that a subset of residues form an interaction network that is directly involved in heat sensing. Our study provides a glimpse of the molecular principles underlying noxious physical and chemical stimuli sensing by TRPV1, which can be extended to other thermal sensing ion channels.

Dysfunction in Mitochondrial Electron Transport Chain Complex I, Pyruvate Dehydrogenase Activity, and Mutations in ND1 and ND4 Gene in Autism Spectrum Disorder Subjects from Tamil Nadu Population, India.

Autism spectrum disorder (ASD) is a neurodevelopmental disorder characterised by impaired social interaction and behavioural abnormalities. Growing evidence proved that impairment in mitochondrial functions could inhibit energy production and may contribute to the onset of ASD. Genetic variants in the genes of mitochondrial DNA (mtDNA) could interrupt the normal energy metabolism and production in the brain which lead to a wide range of structural and functional changes in the brain resulting in ASD. The present study aims to compare the activities of mitochondrial electron transport chain (ETC) complex I, pyruvate dehydrogenase (PDH) and specific mitochondrial DNA gene (MT-ND1 and MT-ND4) variants associated with ASD subjects in the Tamil Nadu population. Mutational analysis revealed that most mutations in ASD subjects showed synonymous type followed by missense in both the ND1 and ND4 genes. Interestingly, we found that the complex I and PDH dysfunctions may have a role in ASD compared to the controls (p ≤ 0.0001). Hence, the results of the present study suggest that mitochondrial dysfunction, specifically the complex I genes, may play a major role in the onset of ASD, concluding that further research on mitochondrial genes are mandatory to unravel the mechanism behind ASD pathogenesis.

Evaluation of the effects of metformin as adenosine monophosphate-activated protein kinase activator on spatial learning and memory in a rat model of multiple sclerosis disease.

In patients with multiple sclerosis (MS) disease, cognitive deficits have been detected because of destruction of hippocampus. Cognitive impairment is one of the common signs in MS. Recent studies showed that metformin (Met) has wide-ranging effects in the treatment of diseases. Here, we have tried to study the preservative effects of Met as adenosine monophosphate-activated protein kinase (AMPK) activator on the hippocampus dentate gyrus (DG) neuronal firing pattern, motor coordination, and learning & memory loss following MS induction. The MS induction was done by local ethidium bromide (EB) injection into the rat hippocampus. Then, rats were treated with Met (200 mg/kg) for two weeks. Spatial memory and learning status were assessed using Morris water maze. A neuronal single-unit recording was measured from hippocampus DG. After decapitation, the bilateral hippocampi separated to measure malondialdehyde (MDA). Treatment with Met ameliorated latency times and path lengths (P < 0.05, P < 0.01, P < 0.001 in 1th, 2th, 3th and 4th days) in the Met + MS group respectively. The percent of total time spent in goal quarter and the average number of spikes/bin were decreased significantly in MS rats compared with the sham group (p < 0.001) but significantly increased in the metformin-treated MS group (Met + MS), (p < 0.01, p < 0.001). Met treatment in rats with MS significantly reduced the concentration of MDA, which is an indicator of lipid peroxidation compared to untreated groups. These observations show that increase of neuronal activity, sensory-motor coordination, and improvement of spatial memory in MS rats treated with Met appears via an increment of AMPK.

Static and Dynamic Projections of Drug-Drug Interactions Caused by Cytochrome P450 3A Time-Dependent Inhibitors Measured in Human Liver Microsomes and Hepatocytes.

Cytochrome P450 3A (CYP3A) is a frequent target for time-dependent inhibition (TDI) that can give rise to drug-drug interactions (DDI). Yet many drugs that exhibit in vitro TDI for CYP3A do not result in DDI. There were 23 drugs with published clinical DDI evaluated for CYP3A TDI in human liver microsomes (HLM) and hepatocytes (HHEP), and these data were used in static and dynamic models for projecting DDI caused by inactivation of CYP3A in both liver and intestine. TDI parameters measured in HHEP, particularly the maximal rate of enzyme inactivation, were generally lower than those measured in HLM. In static models, the use of estimated average unbound organ exit concentrations offered the most accurate projections of DDI with geometric mean fold errors of 2.0 and 1.7 for HLM and HHEP, respectively. Use of maximum organ entry concentrations yielded marked overestimates of DDI. When evaluated in a binary fashion (i.e., projection of DDI of 1.25-fold or greater), data from HLM offered the greatest sensitivity (100%) and specificity (67%) and yielded no missed DDI when average unbound organ exit concentrations were used. In dynamic physiologically based pharmacokinetic modeling, accurate projections of DDI were obtained with geometric mean fold errors of 1.7 and 1.6 for HLM and HHEP, respectively. Sensitivity and specificity were 100% and 67% when using TDI data generated in HLM and Simcyp modeling. Overall, DDI caused by CYP3A-mediated TDI can be reliably projected using dynamic or static models. For static models, average organ unbound exit concentrations should be used as input values otherwise DDI will be markedly overestimated. SIGNIFICANCE STATEMENT: CYP3A time-dependent inhibitors (TDI) are important in the design and development of new drugs. The prevalence of CYP3A TDI is high among newly synthesized drug candidates, and understanding the potential need for running clinical drug-drug interaction (DDI) studies is essential during drug development. Ability to reliably predict DDI caused by CYP3A TDI has been difficult to achieve. We report a thorough evaluation of CYP3A TDI and demonstrate that DDI can be predicted when using appropriate models and input parameters generated in human liver microsomes or hepatocytes.

AMPK hyperactivation promotes dendrite retraction, synaptic loss, and neuronal dysfunction in glaucoma.

BACKGROUND: The maintenance of complex dendritic arbors and synaptic transmission are processes that require a substantial amount of energy. Bioenergetic decline is a prominent feature of chronic neurodegenerative diseases, yet the signaling mechanisms that link energy stress with neuronal dysfunction are poorly understood. Recent work has implicated energy deficits in glaucoma, and retinal ganglion cell (RGC) dendritic pathology and synapse disassembly are key features of ocular hypertension damage. RESULTS: We show that adenosine monophosphate-activated protein kinase (AMPK), a conserved energy biosensor, is strongly activated in RGC from mice with ocular hypertension and patients with primary open angle glaucoma. Our data demonstrate that AMPK triggers RGC dendrite retraction and synapse elimination. We show that the harmful effect of AMPK is exerted through inhibition of the mammalian target of rapamycin complex 1 (mTORC1). Attenuation of AMPK activity restores mTORC1 function and rescues dendrites and synaptic contacts. Strikingly, AMPK depletion promotes recovery of light-evoked retinal responses, improves axonal transport, and extends RGC survival. CONCLUSIONS: This study identifies AMPK as a critical nexus between bioenergetic decline and RGC dysfunction during pressure-induced stress, and highlights the importance of targeting energy homeostasis in glaucoma and other neurodegenerative diseases.

Differences in Hydrolase Activities in the Liver and Small Intestine between Marmosets and Humans.

For drug development, species differences in drug-metabolism reactions present obstacles for predicting pharmacokinetics in humans. We characterized the species differences in hydrolases among humans and mice, rats, dogs, and cynomolgus monkeys. In this study, to expand the series of such studies, we attempted to characterize marmoset hydrolases. We measured hydrolase activities for 24 compounds using marmoset liver and intestinal microsomes, as well as recombinant marmoset carboxylesterase (CES) 1, CES2, and arylacetamide deacetylase (AADAC). The contributions of CES1, CES2, and AADAC to hydrolysis in marmoset liver microsomes were estimated by correcting the activities by using the ratios of hydrolase protein levels in the liver microsomes and those in recombinant systems. For six out of eight human CES1 substrates, the activities in marmoset liver microsomes were lower than those in human liver microsomes. For two human CES2 substrates and three out of seven human AADAC substrates, the activities in marmoset liver microsomes were higher than those in human liver microsomes. Notably, among the three rifamycins, only rifabutin was hydrolyzed by marmoset tissue microsomes and recombinant AADAC. The activities for all substrates in marmoset intestinal microsomes tended to be lower than those in liver microsomes, which suggests that the first-pass effects of the CES and AADAC substrates are due to hepatic hydrolysis. In most cases, the sums of the values of the contributions of CES1, CES2, and AADAC were below 100%, which indicated the involvement of other hydrolases in marmosets. In conclusion, we clarified the substrate preferences of hydrolases in marmosets. SIGNIFICANCE STATEMENT: This study confirmed that there are large differences in hydrolase activities between humans and marmosets by characterizing marmoset hydrolase activities for compounds that are substrates of human CES1, CES2, or arylacetamide deacetylase. The data obtained in this study may be useful for considering whether marmosets are appropriate for examining the pharmacokinetics and efficacies of new chemical entities in preclinical studies.

MicroRNA-30d-5p ameliorates lipopolysaccharide-induced acute lung injury via activating AMPKα.

OBJECTIVES: Acute lung injury (ALI) is a devastating lung disease characterized by uncontrolled pulmonary inflammation and oxidative stress. Currently, no effective therapeutic strategies are available for ALI and its prognosis remains poor. The present study aims to investigate the role and potential mechanism of microRNA-30d-5p (miR-30d-5p) in the progression of ALI. METHODS: Mice were intravenously treated with miR-30d-5p agomir, antagomir or their respective controls for 3 consecutive days and then were exposed to a single intratracheal injection of lipopolysaccharide (LPS) for 12 h at a dosage of 5 mg/kg to induce ALI. To inhibit adenosine monophosphate-activated protein kinase α (AMPKα) or phosphodiesterase 4 D (PDE4D), compound C (CpC) and rolipram were used. RESULTS: miR-30d-5p expression in the lungs was significantly inhibited by LPS treatment. miR-30d-5p agomir significantly alleviated, while miR-30d-5p antagomir aggravated pulmonary inflammation, oxidative damage, and dysfunction in ALI mice. Besides, we found that miR-30d-5p agomir ameliorated LPS-induced ALI via activating AMPKα and that the inhibition of AMPKα by CpC completely abolished these beneficial effects of miR-30d-5p agomir. Further findings validated that PDE4D downregulation was required for the activation of AMPKα by miR-30d-5p agomir. CONCLUSION: miR-30d-5p ameliorates LPS-induced ALI via activating AMPKα and it is a valuable therapeutic candidate in the treatment of ALI.