Hypoxia is a condition commonly observed in the core of solid tumors. The hypoxia-inducible factors (HIF) act as hypoxia sensors that orchestrate a coordinated response increasing the pro-survival and pro-invasive phenotype of cancer cells, and determine a broad metabolic rewiring. These events favor tumor progression and chemoresistance. The increase in glucose and amino acid uptake, glycolytic flux, and lactate production; the alterations in glutamine metabolism, tricarboxylic acid cycle, and oxidative phosphorylation; the high levels of mitochondrial reactive oxygen species; the modulation of both fatty acid synthesis and oxidation are hallmarks of the metabolic rewiring induced by hypoxia. This review discusses how metabolic-dependent factors , and metabolic-independent factors  cooperate in determining chemoresistance in hypoxia. Specific metabolic modifiers, however, can reverse the metabolic phenotype of hypoxic tumor areas that are more chemoresistant into the phenotype typical of chemosensitive cells. We propose these metabolic modifiers, able to reverse the hypoxia-induced metabolic rewiring, as potential chemosensitizer agents against hypoxic and refractory tumor cells. Physiological hypoxia implies an adaptive and homeostatic response, such as vasodilation and/or up-regulation of hypoxia response genes, to maintain stable levels of O2. On the contrary, in pathological hypoxia, the homeostatic mechanisms do not compensate adequately the falling in O2 levels [Depending on the tissue type, there is a wide variability in the oxygen  O2 pressure [2 levels, producing repeated cycles of hypoxia and normoxia within specific areas of tumor bulk [2 are heterogeneously distributed within the tumor bulk, with a prevalence of better oxygenated areas, characterized by a high rate of cell division and tumor growth around the capillaries. The newly generated cells often migrate towards the regions far from vessels [2, hypoxic tumors maintain high the expression of hypoxia-sensitive genes inducing metastasis and resistance to oxidative stress [The fast rate of growth in solid tumors makes them susceptible to O regions ,9. Neo-a regions . Althougpressure ,11,12. Tmor bulk . Moreovemor bulk . Chemoth vessels . Indeed, vessels ,17,18, e vessels ,20. Notae stress , conserve stress .Hypoxia not only affects neoplastic cells, but also implies changes in metabolism and functions of infiltrating cells, such as cancer-associated fibroblasts (CAFs) and tumor-associated macrophages (TAMs). These changes may impair or favor the neoplastic growth, producing cellular quiescence, differentiation, apoptosis, or necrosis, depending on the degree, persistence, and severity of hypoxia. The tolerance to hypoxia, i.e., the ability to enter a quiescent but viable status, determines the persistence of hypoxia-tolerant cells that are aggressive and hard to be eradicated pharmacologically .As a consequence of the different oxygenation, solid tumors are metabolically heterogeneous: better oxygenated regions rely on mitochondrial oxidative phosphorylation (OXPHOS), while hypoxic areas are more dependent on anaerobic metabolism . This me2-regulated HIF-α subunit, and the O2-independent, constitutively expressed aryl hydrocarbon receptor nuclear translocator (ARNT), also called HIF-β [HIF is a heterodimer formed by two subunits, the Oed HIF-β ,26,27,28ed HIF-β . HIF-3α ed HIF-β , but hased HIF-β ,32.2 levels [2 is less than 5 min, but it increases to 60 min as the O2 concentration decreases to 1%. Under normoxia, HIF-α subunit is hydroxylated by prolyl hydroxylase domain (PHD)-containing proteins [2 availability, hypoxia inhibits all of the hydroxylation processes, restoring its transcriptional efficiency [The regulation of stability and activity of HIF-1α and HIF-2α is dependent on O2 levels . Indeed,proteins . This isproteins ,35. Furtproteins . By limificiency ,38.Moreover, microRNAs (miRNAs) play an important role in HIF-1α stabilization and/or in its down-stream effects . Indeed,Besides hypoxia and miRNAs, the stability and activation of HIF-1α and HIF-2α in tumors is also favored by growth factors, cytokine, and oxidative stress . SeveralThe main target genes of HIF-1α are erythropoietin (EPO), the pro-angiogenic vascular endothelial growth factor (VEGF), the pro-invasive factors metalloproteinase 9 (MMP9) and urokinase-type plasminogen activator (uPA) ,47, the The majority of the transcriptional programs activated by hypoxia in tumors are due to the activity of HIF-1α and HIF-2α. The function of HIF-3α remains to be fully elucidated, although it is known to suppress HIF-1α- and HIF-2α-mediated gene expression and to upregulate a different set of genes , thus, f2, ensuring survival and proliferation [+. These metabolic changes are initiated by oncogenes and perpetuated by the homeostatic alterations of the tumor microenvironment (TME) [One of the hallmarks driving aggressiveness in cancer is the reprogramming of energy metabolism . Glycolyferation . This feferation . In parant (TME) ,59. Hypo2 [Cells of solid tumors are often glucose-addicted, although the glycolytic pathway followed by lactic fermentation has a lower efficiency in terms of glucose/ATP ratio, compared to the glycolysis followed by tricarboxylic acid (TCA) cycle and oxidative phosphorylation (OXPHOS). Indeed, although cancer cells take up glucose at higher rates than normal tissues, they oxidize less glucose in TCA and OXPHOS, regardless of the presence of O2 . Despite2 . Indeed,2 .+-coupled glucose transporters (SGLTs) contribute to glucose uptake in non-transformed cells, but they are not modified by hypoxia. The main transporters involved in glucose uptake in cancer cells belong to the GLUTs/SLC2A family [NaA family . An incrA family ,61, appeA family ,62, provA family , determi2 levels.Besides glucose uptake, HIF-1α accelerates the transformation of glucose into lactate, being a transcriptional inducer of the glycolytic enzymes . In para+, promoting the acidosis of TME [2, producing the so-called “reverse Warburg effect” [+ ratio in cancer cells. This event promotes the sirtuin 1 (SIRT1)-induced deacetylation and activation of the peroxisome proliferator-activated receptor gamma coactivator-1α (PGC-1α), an inducer of mitochondrial biogenesis [2 is adequate. Since tumors undergo multiple cycles of hypoxia and re-oxygenation [2 levels, continuing their growth notwithstanding mutable environmental conditions.As a result of the increased anaerobic glycolysis, hypoxic areas of tumors produce huge amounts of lactic acid, which easily dissociates into lactate and Hs of TME . Originas of TME ,66. Lacts of TME ,68, likes of TME . By conts of TME ,71. Thiss of TME ,72 or prs of TME ,73. Indes of TME ,73. The s of TME  that pres of TME . The lac effect” ,75. Notaogenesis . As a regenation , this swBesides its role as energetic substrate, lactate also promotes tumor progression and invasion. For instance, it stimulates the synthesis of VEGF by endothelial cells, promoting neo-angiogenesis ,77. By i+ with lactate by MCT symporters induces a strong acidification of TME, which reaches values between 6 and 6.5. Such acidosis determines metabolic reprogramming [The co-secretion of Hgramming  and invagramming ,81 as weSince the glycolytic pacemaker enzyme PFK1 is inhibited by low pH, it is not surprising that cells growing in acidosis reduce the glycolytic flux, increasing the glucose metabolism in the pentose phosphate pathway (PPP) . This efAn increased fatty acids (FAs) oxidation (FAO) is also observed in acidosis . Such inBesides inducing a strong metabolic reprogramming, hypoxia and lactic acidosis favor the self-maintenance of cancer stem cells (CSCs) ,87, and + T-lymphocytes [+ T-lymphocytes clones with strong anti-tumor activities, characterized by high expansion and production of interferon-γ (IFN-γ) [+ T-lymphocytes: the intracellular acidosis of lymphocytes suppresses the cytotoxic potential and the production of anti-tumor cytokines [+ from cancer cells may affect the efficiency of immune cells in hypoxic areas. For instance, highly glycolytic tumors producing H+ impair the activation of NK and CD8+ T-lymphocytes because the low pH inhibits the transcriptional activity of nuclear factor of activated T-cells (NFAT) [+ T-lymphocytes compete for glucose: in hypoxic and highly glycolytic tumors, the scarcity of glucose left to lymphocytes impair their proliferation and production of IFN-γ [Finally, hypoxia and lactic acidosis alter the TME, promoting an immune-suppressive environment . Hypoxiaphocytes . Activatphocytes . This pr (IFN-γ) . Howeverytokines . As alres (NFAT) , a masteof IFN-γ . Since tof IFN-γ , restoriOverall the higher export of lactate and the consequent acidosis characterizing hypoxic tumor areas, the metabolic cross-talks between cancer cells and non-transformed cells within TME  favor proliferation, invasiveness and immune-escape, reducing the efficacy of anti-tumor pharmacological treatments.+-independent exchanger of cystine and glutamate [As already mentioned, HIF-1α and HIF-2α upregulate the AA transporters xCT (SLC7A11) and LAT1 (SLC7A5) ,50. Whillutamate , the latlutamate . Both AAlutamate .By importing cystine necessary for the biosynthesis of glutathione (GSH), the main intracellular anti-oxidant metabolite, xCT plays a key defensive role against oxidative stress and chemotherapy . AlthougSeveral tumor types also overexpress LAT1 ,103, whi2 levels [Another important reprogramming of cellular energy metabolism in solid tumors is related to the increased glutaminolysis . Glutami2 levels ,113. Alt2 levels . Indeed,2 levels , which nProtein and DNA synthesis, commonly accelerated in growing tumors, are energy-consuming processes . One of On the other hand, AcCoA can be also generated by FAO. Since cancer cells have increased lipogenesis in hypoxia, one should expect a reduced lipid catabolism to sustain rapid cell proliferation. Nevertheless, the scenario is variegate and sometimes contradictory. Indeed, it is a common observation that the acidosis induced by hypoxia up-regulates both FA synthesis and FAO . In someHIF-1α decreases the expression of carnitine palmitoyltransferase 1A (CPT1A), the rate limiting-enzyme of FAO . CPT1A mOver the last three decades, an attenuated mitochondria metabolism associated to hypoxia has been reported in several pathological conditions, including cancer .2 levels and mitochondria are the major O2 consumers, the physiological consequence of hypoxia is a reduced mitochondrial OXPHOS. Indeed, in solid tumors hypoxia reduces the activity of the TCA cycle by up-regulating PDK1 [2 or during re-oxygenation phases. Lactate produced by CAFs increases the mitochondrial mass by upregulating PGC-1α [2 levels.Since hypoxia triggers several mechanisms to adapt tumor cells to low Oing PDK1 ,124, inhing PDK1 , repressing PDK1 , alters ing PDK1 , increasing PDK1 . The suming PDK1 ,73,74, sg PGC-1α , and milg PGC-1α . Consequ2 also leads to an incomplete reduction at complex IV of ETC, increasing the production of partially reduced O2-derived species, i.e., ROS. Mitochondrial ROS (mtROS) can increase the rate of mutations in mitochondrial DNA (mtDNA) that encodes for 13 subunits of complexes I to V [2 uptake and ETC: if these oncogenes are active in hypoxic tumors, the increased ETC coupled with an uncompleted O2 reduction increase mtROS [The scarcity of Os I to V . The pres I to V , are oths I to V . The pres I to V . This sts I to V . Given ts I to V ,136, ROSs I to V ,138. Exps I to V , complexs I to V , complexs I to V . In addise mtROS . Insteadse mtROS . Hence, 2 oscillations, but also pushed towards progression, resistance to chemotherapy and radiotherapy [Within the same tumor, there are normoxic and hypoxic areas: such heterogeneity determines metabolic niches that are associated with more chemosensitive or more chemoresistant cells. The latter are usually characterized by higher stemness and higher tendency to relapse . There aotherapy . SeveralMDR1, encoding for Pgp [Among the metabolism-independent factors , one sim for Pgp . Pgp bel for Pgp . The HIF for Pgp , larynx  for Pgp , lung [1 for Pgp , and bre for Pgp ,152,153  for Pgp  and B-ce for Pgp . The bro for Pgp , lung re for Pgp  and brea for Pgp ,159, areThe high number of CSCs in hypoxic tumor areas ,87,88 isAt the same time, HIF-1α reduces the efficacy of chemotherapeutic drugs damaging DNA by upregulating topoisomerase 2A, which reseals DNA double strand breaks , by actiAs already mentioned, ROS are increased in hypoxic tumor areas because of the OXPHOS dysfunction. The increase in ROS below sub-cytotoxic levels triggers an adaptive response that limits oxidative damages. One ROS-sensitive factor coordinating this response is the Nuclear factor erythroid 2-related factor 2 (Nrf2), which upregulates anti-oxidant enzymes and MRP1 . These tThe metabolic reprogramming induced by hypoxia must also be taken into account as a key player in inducing cancer chemoresistance.The metabolic rewiring in both glycolysis and mitochondria induced by hypoxia cause chemoresistance by cooperating with enhanced pro-survival pathways and reduced apoptosis, EMT activation, increased DNA repair, alterations in drug metabolism, changes in drug targets ,181,182.+/H+ exchanger (NHE) is a typical example of transporter that is up-regulated in response to the acidification: the increased intracellular pH (pHi) produced by its activity creates the optimal conditions for Pgp efflux that is maximally efficient at 7.6-7.8 pH [The increased acidification of TME produced by the upregulation of glycolytic enzymes and MCT4 is a first reason of chemoresistance. On the one hand, the low extracellular pH (pHe) favors the protonation of weak bases, such as anthracyclines, followed by their inactivation and sequestrations within lysosomes once entered within the cancer cell, a mechanism known as “ion trapping” ,184. Sec6-7.8 pH . Indeed,6-7.8 pH . Other a6-7.8 pH ,188. CAX6-7.8 pH  and in p6-7.8 pH .Chemoresistance in hypoxic tumor areas has also been associated with altered mitochondrial metabolism, fusion, fission, and mitophagy . On the 2 shortage, or chemotherapy. We suggest that the pleiotropic effects of hypoxia on metabolic reprogramming all contribute to chemoresistance by different but cooperating mechanisms.Moreover, cancer cells often have the ability to exploit both glycolysis and OXPHOS, fueled by glutaminolysis and FAO. In this way, cells shift from one energetic pathway to the other one, according to the glucose availability . This meOver the last two decades, an enormous effort has been done to look for efficient treatments counteracting chemoresistance. Since the deregulated cell metabolism could cause chemoresistance, specific metabolic modifiers may be potentially effective as chemosensitizer agents.https://clinicaltrials.gov/ct2/results/details?cond=Cancer&term=HIF).The most direct approach consists in targeting HIF-1α, given its central role in controlling metabolic reprogramming, proliferation, invasion, and chemoresistance. Inhibitors of HIF-1α translation , compounds decreasing HIF-1α stability  and transcriptional activity , are the most efficient inhibitors of HIF-1α ,198,199.Targeting the metabolic pathways modulated by HIF-1α and involved in chemoresistance could represent a second chemosensitizing approach .Cancer cells highly dependent on glycolysis display a dramatic decrease in ATP upon treatment with the synthetic glucose analog 2-deoxy-D-glucose (2-DG), which blocks the glycolytic flux . BesidesA chemosensitizing effect of 2-DG in combination with 5-fluorouracil was reported in vitro and in hepatocellular carcinoma xenografts . HoweverThe 3-bromo-pyruvate (3BrPyr) is a potent inhibitor of HK isoform II, one of the pacemaker enzyme of glycolysis, often overexpressed in tumors; therefore, it induces an ATP crisis. Being an alkylating agent on HKII and other enzymes, including anti-oxidant enzymes, it has been proposed as a potential enhancer of alkylating chemotherapeutic drugs. Indeed, 3BrPyr successfully increases the sensitivity to carmustine in hepatocellular carcinoma cells, by inhibiting glycolysis and inactivating several targeting proteins involved in redox homeostasis . Both HKPFK binding protein 3 (PFKBP3) is another critical regulator of glycolysis, because it can stimulate or inhibit PFK-2: recently, the selective PFKBP3 inhibitor PFK158 has shown strong anti-cancer properties, as well as chemosensitizing effects in ovarian cancers resistant to carboplatin and docetaxel . PFKBP3 Moreover, LDH-A, the last pace-maker glycolytic enzyme and another HIF-1α target gene, has been correlated with chemoresistance: indeed, it is over-expressed in taxol-resistant TNBC, but its inhibition with oxamate restores chemosensitivity by inducing apoptosis consequent to the reduced ATP supply . Notably+/lactate symporters. MCT1 inhibitors, such as α-cyano-4-hydroxycinnamate (CHC), AR-C155858 and AZD3965, have been shown to reduce tumor growth and progression [+ T-lymphocytes, which need an active MCT1 to preserve their cytotoxic functions [2 consumption by mitochondria, relieving the intratumoral hypoxia, allowing radiosensitization [Since the TME acidification typical of hypoxic areas up-regulates alkalinizing proteins that contribute to chemoresistance ,189, targression ,213, witunctions . Besidestization  and like2. In turns, the increase in ROS may induce cell death and synergize with chemotherapeutic agents producing ROS, such as cisplatin. On the other hand, the increased amount of ATP derived from OXPHOS, although not sufficient to protect from apoptosis, reduces the glycolytic flux with a feedback inhibition on PFK-2, completely counteracting the metabolic reprogramming induced by hypoxia. Moreover, a direct inhibition of DCA on HIF-1α activity has been documented: this inhibition further contributes to chemosensitization via the direct downregulation of Pgp [Notwithstanding possible toxicities, the shift from a glycolytic to an OXPHOS-based metabolism strongly kill hypoxic cancer cells that rely on glycolysis for their energy production. The glycolysis to OXPHOS shift is achieved for instance by the PDK1 inhibitor dichloroacetate (DCA) that relieves the inhibition on PDH, allowing the generation of AcCoA and fueling TCA cycle . In thesn of Pgp . The supn of Pgp , reversiDual targeting compounds, i.e., agents reprogramming at least two metabolic pathways as DCA and pyruvate, are the most effective chemosensitizing strategies among the metabolic modifiers. In etoposide-resistant neuroblastoma, a constitutively high level of anti-apoptotic survivins induce mitochondria fission, downregulate OXPHOS and increase glycolysis: this signature, similar to that produced by HIF-1α, is associated to a metastatic and chemoresistant phenotype. By relying mainly on glycolysis to obtain ATP, resistant neuroblastoma cells are highly susceptible to glycolytic inhibitors: indeed, the treatment with 2-DG reprograms the metabolic phenotype of resistant neuroblastoma and at the same time sensitizes cells to etoposide . The comSince hypoxia increases FA synthesis, agents targeting this pathway may contribute to chemosensitization. For instance, the FASN inhibitor Orlistat induces chemosensitization to cisplatin in T-cell lymphoma, by pleiotropic mechanisms, including increased ROS levels, decreased MCT1 expression and lactate efflux, downregulated Pgp and MRP1 . Althoug+) to accumulate preferentially within mitochondria [+-derivatives have recently shown to lower mitochondrial biogenesis, mitochondrial mass and energetic metabolism, increasing at the same time the mtROS levels, opening the membrane permeability transition pore, and triggering a caspase-9-mediated apoptosis [+-derivatives may worsen the energetic mitochondrial metabolism of hypoxic cancer cells and increase at the same time the possible oxidative damages. They could be exploited as chemosensitizers in hypoxic areas of tumors, in combination with chemotherapeutic drugs that generate ROS, such as cisplatin, gemcitabine, or doxorubicin [+-conjugate of DCA (mito-DCA) is a synthetic derivative of DCA, conceived as a more potent version of parental compound: the vectorization to mitochondria amplifies the inhibition of PDK1 and the consequent shift from glycolysis to OXPHOS [•O2− and H2O2, and are radio-sensitizers in pancreatic cancer cells [Several small molecules altering mitochondria metabolism, termed “mitocans”, have shown promising anti-cancer efficacy , in partchondria . Phenol poptosis . By reduorubicin ,178,179.o OXPHOS . This pro OXPHOS . Variouso OXPHOS . As a coer cells . Thanks er cells , fully rer cells . IACS-01er cells , showinger cells . For thier cells . Moreoveer cells , forcingOverall, these pharmacological evidences demonstrate that shifting the glycolysis/OXPHOS balance may be an effective chemosensitizing strategy against hypoxic tumor cells.2 supply and forced to rewire their metabolism. This training to survive under unfavorable conditions inevitably make cells more resistant to exogenous stresses such as chemotherapy. HIF-1α-dependent chemoresistance relies on the transcriptional activation of genes determining cell proliferation, metastasis, EMT, maintenance of stem cell-like properties, drug efflux, and metabolic reprogramming.This review provides a deep analysis of the linkages between hypoxia, metabolic rewiring, and chemoresistance in cancers. Drug resistance is well known as the primary cause of therapeutic failure in cancer treatment. The mechanisms of chemoresistance involve a combination of cell-intrinsic factors such as oncogenic drivers or mutations, TME-associated factors, pharmacokinetic factors. Hypoxia is a common feature of TME. It plays an important role in selecting cells challenged by a low OThe typical metabolic signature of hypoxic tumors is characterized by increased glucose uptake and fermentation into lactate, decreased pHe/increased pHi, reduced TCA cycle and OXPHOS, increased production of mtROS, increased uptake of AAs, and increased synthesis of anti-oxidant metabolites as GSH. All of these features contribute to chemoresistance. The accelerated glycolysis supplies cells with sufficient ATP to promote cell survival and with glycolysis intermediates for biosynthetic purposes. The increased mitophagy characterizing hypoxic cells sustains the possibility of recovering ATP. The altered pH inactivates several drugs that are active as weak bases, or sequesters them into lysosomes after protonation. The increased production of sub-cytotoxic mtROS, coupled with the higher levels of anti-oxidant metabolites, train cells to be less susceptible to the oxidative damage induced by chemotherapy.If targeting HIF-1α could represent an effective approach because it blocks a great number of processes determining chemoresistance, HIF-1α inhibitors have the disadvantage of huge toxicity, due to the inhibition of physiologically important HIF-1α-dependent processes, such as ischemia-reperfusion response in non-transformed tissues. Targeting the metabolic pathways controlled by HIF-1α and re-programming them as in normoxic cells may improve the efficacy of chemotherapeutic drugs and/or attenuate chemoresistance. Although metabolic modifiers have already been tested in clinical trials, side effects deriving from the inhibition of metabolic pathways in non-transformed tissues cannot be excluded. However, the metabolic signature of hypoxic cancer cells—based on high anaerobic glycolysis and low mitochondrial metabolism—is quantitatively different from the normal tissues. This quantitative difference may open a therapeutic window at which metabolic modifiers could be safely used against hypoxic cancer cells, without damaging non-transformed tissues. Nanotechnology-based drug delivery, employing tumor-targeting liposomes or nanoparticles, could aid to increase the delivery and the vectorization of the metabolic modifiers towards the tumor, increasing the therapeutic benefits and reducing the side effects. Finally, some endogenous metabolites, such as pyruvate or melatonin, differentially produced by hypoxic cells and non-tumor tissues, have revealed significant chemosensitizing properties, coupled with lower risks of toxicity.Thanks to the deep knowledge of the metabolic rewiring induced by hypoxia, and causing chemoresistance, a precision medicine based on specific metabolic modifiers can be proposed as a novel chemosensitizing strategy against aggressive and refractory tumors.


There is a need to disentangle the etiological puzzle of age-related neurodegenerative diseases, whose clinical phenotypes arise from known, and as yet unknown, pathways that can act distinctly or in concert. Enhanced sub-phenotyping and the identification of in vivo biomarker-driven signature profiles could improve the stratification of patients into clinical trials and, potentially, help to drive the treatment landscape towards the precision medicine paradigm. The rapidly growing field of neuroimaging offers valuable tools to investigate disease pathophysiology and molecular pathways in humans, with the potential to capture the whole disease course starting from preclinical stages. Positron emission tomography (PET) combines the advantages of a versatile imaging technique with the ability to quantify, to nanomolar sensitivity, molecular targets in vivo. This review will discuss current research and available imaging biomarkers evaluating dysregulation of the main molecular pathways across age-related neurodegenerative diseases. The molecular pathways focused on in this review involve mitochondrial dysfunction and energy dysregulation; neuroinflammation; protein misfolding; aggregation and the concepts of pathobiology, synaptic dysfunction, neurotransmitter dysregulation and dysfunction of the glymphatic system. The use of PET imaging to dissect these molecular pathways and the potential to aid sub-phenotyping will be discussed, with a focus on novel PET biomarkers. It is widely accepted that age-related neurodegenerative diseases are increasingly becoming a global public health concern—in particular, Alzheimer’s disease (AD) and other late-onset dementias (LOD), with widespread socioeconomic and healthcare impacts worldwide. The increasing burden of age-related diseases is mainly due to the ageing world population and the unprecedented shift in aging demographics of individuals over 60 years of age, which is predicted to rise to two billion in 2050 . Age-relThe majority of research across age-related neurodegenerative diseases is built upon the clinicopathological nosology model , wherebyAlmost all age-related neurodegenerative diseases can be classified into sporadic or familial forms. The discovery of fully penetrant genetic mutations in several familial neurodegenerative diseases has allowed for the investigation of the early disease pathology prior to the clinical manifestations, in these familial forms, prior to the manifestation of clinical symptoms that could help to unlock causal pathways. In the nonfamilial sporadic forms, in addition to the genetic variants that are noncausative but can confer susceptibility to disease, there is a wide range of risk factors that may affect disease onset and development, including environmental factors and exposures across the life course, cardiovascular status and hypertension, obesity, diabetes, sleep disorders and a variety of factors related to brain biological aging, such as protein misfolding and aggregation, epigenetics and perturbations in DNA damage and repair. However, not all patients with all, or some, of these risk factors will develop symptoms and signs amounting to a clinical diagnosis within their lifetime. While the interaction between genetics and disease mechanisms is indeed complex and has not been fully elucidated, it has been postulated that unraveling the genetics of age-related neurodegenerative diseases might form the basis for sub-phenotyping and/or reclassification based on genotypic divergence aiming to drive forward the application of precision medicine .There is a need to disentangle the etiological puzzle of age-related neurodegenerative diseases, whose clinical phenotypes arise from known, and as yet unknown, pathways that can act distinctly or in concert . Over thWhile preclinical and post-mortem studies have, and will likely continue to, play a key role in the drug discovery process, as well as in understanding the underlying molecular mechanisms, considerations have to be given to their direct translation into humans ,17. For This review will discuss the dysregulation of the main molecular pathways, pathology and biological networks, highlighting where these are distinct and overlapping, across the spectrum of age-related neurodegenerative diseases. The use of molecular PET imaging to disentangle molecular pathways will be highlighted, focusing on novel biomarkers. Finally, the potential for biomarker-driven epigenomic, biological and clinical signatures to improve disease sub-phenotyping for the stratification of patients into future clinical trials towards the precision medicine icon will be discussed.C9orf72, MAPT and GRN, are associated with a similar behavioral variant FTD (bvFTD) presentation, the underlying protein pathology varies such that MAPT mutations are associated with tau pathology and C9orf72 and GRN mutations are associated with Tar-DNA-binding protein (TDP)-43 pathology [While the temporal onset and the rate of progression can vary, clinical phenotypes, such as behavioral, cognitive, metabolic, nonmotor, primary motor and extrapyramidal, often overlap across different age-related neurodegenerative diseases . For exaathology . The comathology , similarathology .It remains to be elucidated why, and how, pathologies diverge towards different clinical phenotypes and if there is a common causal mechanism that links the spectrum of age-related neurodegenerative diseases. The molecular nexopathies paradigm, introduced by Warren and colleagues, proposes that specific pathogenic proteins result in the disintegration of specific neural networks and multiple functional networks, which could give rise to phenotypic variations, as well as overlap between neurodegenerative diseases . A deepePINK-1, Parkin, TREM2, APOE and TOMM40 [There is an increasing body of literature implicating dysfunction of mitochondria and endoplasmic reticulum (ER) dynamics, energy metabolism and oxidative stress within the molecular paradigm of age-related neurodegenerative diseases ,27,28,29d TOMM40 ,33,34,35d TOMM40 ,37,38. Td TOMM40 ,40,41. Cd TOMM40 ,43. 18F)BCPP-EF, for mitochondrial complex 1 (MC1) and (11C)SA-4503 for sigma 1 receptor (σ1R) enables the in vivo investigation of mitochondrial and ER dysfunction SA-4503 and (18F)BCPP-EF PET, respectively [18F)BCPP-EF and (11C)PE2I PET preclinical study demonstrated that the striatal loss of MC1 correlated with the loss of presynaptic nigrostriatal dopaminergic neurons, supporting the interplay and colocalization of mitochondrial and synaptic dysfunction in a PD model [11C)SA-4503 and (18F)BCPP-EF PET, respectively, as part of the MIND-MAPS program (https://lp.invicro.com/mind-maps), which could help to provide a more comprehensive understanding of the mitochondrial-ER-synaptic complex, across the spectrum of age-related neurodegenerative diseases. Preliminary work suggests decreased MC1 density in AD [18F)BCPP-EF uptake has also been shown to correlate with increase tau deposition, using (11C)PPB3 PET, but not with amyloid-β, using (11C)PiB PET, or glucose metabolism, using (18F)FDG PET [Recently, σ1R and MC1 levels were investigated in a cohort of early de novo PD patients using FDG PET . These pTREM2 and CD33, in AD, FTD and PD [Neuroinflammation and alterations in the immune response have been linked with multiple pathological processes associated with age-related neurodegenerative diseases . While tD and PD ,80,81 fu11C)PK1195, (11C)PBR28, (18F)DPA-714, (11C)DAA1106 and (11C)ER176, have been employed [11C)PK1195 uptake has also been associated with higher amyloid-β burden in AD [11C)ER176 [The majority of in vivo PET imaging studies investigating the role of neuroinflammation have focused on the use radioligands targeting the 18-kDa translocator protein (TSPO), expressed in the outer mitochondrial membrane and elevated in activated microglia B. A wideemployed ,83,84. Iemployed ,86,87,88employed , MSA [90employed , CBS [92employed , AD [94,employed ,97 and Aemployed ,99. Incren in AD  and withen in AD , suggesten in AD ,102. Thi1C)ER176 ,83, whic11C)PS13 and (11C)MC1 for cyclooxygenase (COX)-1 and COX-2, respectively, and (11C)JN717 and (11C)SMW139 for the purinergic receptor P2X7 [18F)DPA714 PET in the absence of increased (11C)JN717 PET has been reported in ALS [11C)SMW139 PET (EudraCT Number: 2018-000405-23). The development of the PET radioligand (11C)BU99008 targeting imidazoline 2-binding sites (I2BS) expressed on activated astrocytes [11C)BU99008 binding, has been reported in early PD patients with decreased (11C)BU99008 binding, possibly reflecting that loss of astroglia function occurs in moderate-to-advanced PD disease stages and is associated with longer disease duration and higher global disease burden [11C)BU99008 PET in AD, with the highest levels in amyloid-β-positive AD patients [The identification of reliable molecular targets for microglia activation beyond TSPO has been the focus for the development of the next generation of PET radioligands assessing neuroinflammatory biomarkers. Emerging PET radioligands that could provide key insights into inflammatory pathways include PiB, (18F)Florbetaben, (18F)Flutemetamol and (18F)Florbetapir, has been extensively employed to investigate disease specific patterns across age-related neurodegenerative diseases. Amyloid PET imaging can detect the presence of amyloid-β plaques prior to the onset of AD [Amyloid PET imaging, using Flortaucipir [18F)Flortaucipir PET visual reads for predicting the presence of AD-like tau pathology at autopsy, suggesting the potential clinical use of (18F)Flortaucipir in the diagnosis of AD [18F)JNJ64349311 [18F)APN-1607 ((18F)PM-PBB3) [18F)MK-6240 [18F)GTPI [18F)PI-2620 [18F)RO-948 [18F)PI-2620 PET in AD and PSP illustrate tracer uptake in regions of known tau pathology, in line with post-mortem autoradiography findings [18F)PI-2620 uptake within neocortical regions was associated with cognitive impairment in AD [18F)MK-6240 PET could differentiate between AD and FTD, with patterns of (18F)MK-6240 uptake in line with Braak’s histopathological staging of tau pathology in AD and negligible (18F)MK-6240 uptake in FTD supporting the specificity of this PET tracer for tau tangle conformations [PET studies employing tau ligands, such as APPM-PBB3) , (18F)MK)MK-6240 ,147,148,18F)GTPI , (18F)PI)PI-2620  and (18FF)RO-948 ,152,153,findings ,154. Incnt in AD  and coulMultifaceted neuroimaging approaches, combining tau PET, amyloid-β PET or FDG PET (for glucose metabolism) with functional and structural MRI techniques, could potentially help to unlock network-based connectivity disease signatures. For example, spatial covariance mapping has been employed to derive network topography underlying clinical phenotypes such as cognitive impairments . DiseaseLRRK2 mutations have been linked to mitochondria-dependent intrinsic apoptosis pathways [PINK1 and Parkin mutations show protective effects against stress-induced cytochrome c release [The dysfunction of cellular degradation systems, including the autophagy-lysosome and ubiquitin-protease systems ,159, haspathways , while P release ,170. Neu release ,171,172.Synaptic dysfunction and the loss of synaptic density has been suggested as a common key feature across age-related neurodegenerative diseases ,177,178.2+ dependent exocytosis [11C)UCB-J PET has been employed to study SV2A levels in aging [11C)UCB-J binding was observed in the hippocampus, which correlated with episodic memory [11C)PIB PET scans [11C)UCB-J PET as part of the MIND-MAPS program (https://lp.invicro.com/mind-maps), which could help to provide a comprehensive understanding of the role of SV2A across the spectrum of age-related neurodegenerative diseases. Together, these recent findings provide in vivo evidence to implicate synaptic loss in the pathophysiology of age-related neurodegenerative diseases with potential relevance for clinical phenotypes. Ongoing longitudinal (11C)UCB-J PET studies, such as the TRAID study [Until recently, it was only possible to study synaptic density in post-mortem brain tissue . Howeverocytosis , paved tocytosis . More rein aging , idiopatin aging ,191,192,in aging , as wellin aging ,194. In c memory ,188,195.c memory ,197. In c memory , with SVc memory . In PSP ET scans . The gloET scans . PrelimiET scans , as wellET scans . Work isID study  and the LRRK2 mutation in PD, have also been shown to impair the dynamics of synaptic vesicle endocytosis, recycling, mobilization and storage [Prior to synaptic loss, synaptic function and plasticity is likely to be significantly disrupted. The accumulation of protein aggregates has been linked with dysfunction of synaptic signaling pathways, such as impairing long-term potentiation (LTP) ,201 and  storage ,207.PET imaging studies have collectively demonstrated disruption of neurotransmitter systems E, illust11C)PMP PET, targeting presynaptic acetylcholinesterase (AChE [18F)2FA PET has revealed hippocampal and cortical loss of the postsynaptic α4β2 nicotinic AChE (nACh) receptor in AD [18F)MP4A PET in FTD patients [18F)FEOBV [18F)ASEM [The cholinergic system is predominately associated with the development of cognitive impairment in AD, PD, parkinsonian plus and FTD ,216. (11se (AChE ), has bese FEOBV , and of 18F)ASEM ,233, cou11C)DASB PET has most widely been employed in PD, showing serotonergic pathology starting in premotor stages before the development of overt motor symptoms [18F)MPPF PET studies showing reduced levels of serotonin type 1A (5-HT1A) receptors in the hippocampus of AD patients [18F)2FNQ1P [2A receptors, measured using (11C)MDL100907 PET, has been reported to underlie behavior diseases [11C)WAY100635 PET study also reported decreased 5-HT1A receptor binding in ALS patients within the frontotemporal and cingulate regions [11C)Cimbi-36 PET, a 5-HT2A receptor agonist, could offer a valuable tool to investigate changes in serotonin synaptic levels and, indirectly, to measure serotonin release [Looking at the serotonergic system, MPpatients . The devF)2FNQ1P ,249, coudiseases . A (11C) regions ,252. The release ,254. The release . Given t11C)Raclopride, (11C)PHNO and (18F)Fallypride, as well as presynaptic dopamine integrity assessing DAT with (11C)PE2I, dopamine storage with (18F)DOPA and vesicle monoamine transporter type 1 with (11C)DTBZ [18F)DOPA and (11C)MP4A PET study illustrated that PDD and DLB share similar dopaminergic and cholinergic deficit profiles [The dopaminergic system underpins the development of primary motor and extrapyramidal phenotypes in PD, parkinsonian plus, HD and FTD but has also been linked with behavioral and cognitive phenotypes in PD, parkinsonian plus, FTD ,256 and,11C)DTBZ . While p11C)DTBZ ,261. Acr11C)DTBZ ,263. D2 11C)DTBZ . A combiprofiles . Presynaprofiles ,270,271.profiles , whilst profiles . Decreasprofiles , althoug11C)MRB PET, a selective ligand for NET, has been employed to study noradrenergic synaptic terminals in PD [11C)RTI-32, a marker of both DAT and NET, has also been employed in PD, with evidence suggesting that the loss of dopaminergic and noradrenergic innervation within the limbic system may play a role in the development of depression and anxiety in the PD disease course [The noradrenergic system has been linked with the regulation of several autonomic functions and behavioral and cognitive phenotypes, such as attention, wakefulness, decision making, memory and depression ,275,276.ls in PD ,279,280.e course . Future 18F)FPEB and (11C)ABP688 PET studies demonstrated a reduction of hippocampal mGluR5 in early AD, which was associated with lower episodic memory scores and reduced global cognitive function [11C)UCB-J binding in AD [11C)ABP688 PET uptake have also been reported in patients with the behavior variant FTD (bvFTD) [11C)ITMM and (18F)FIMX radioligands, which are specific for mGluR1 [Glutamatergic neurotransmission is also known to play a central role in supporting higher cognitive function. Excessive glutamate neurotransmission can promote excitotoxic neuronal death contributing to the glutamate hypothesis in AD and FTD ,283,284.function ,286. Whing in AD , the losng in AD  and coulng in AD . Reducti (bvFTD) . PET trar mGluR1 ,291. Givr mGluR1 , it coul11C)Flumazenil PET binding, a marker of GABA-A receptor, has been reported in AD [11C)Flumazenil PET-binding correlates with memory performance in early AD are in accordance with post-mortem evidence of reduced hippocampal GABA-A messenger RNA expression [The GABAergic system represents the main inhibitory neurotransmitter system within the brain and plays an important role in regulating oscillatory dynamics for cognitive control and work memory function ,294. Imped in AD , PSP [29ed in AD  and manied in AD ,298 and ed in AD ,300. Thepression .The glymphatic system is proposed to remove waste products through the cerebrospinal fluid (CSF) to interstitial fluid (ISF) exchange, along the perivascular pathway, with the help of aquaporin-4 water channels, which are polarized in astrocytic end feet towards the capillary vessel walls ,301,302.11C)TGN-020 has been developed for the quantification of aquaporin-4 [11C)TGN-020 within the brain is consistent with known distributions of aquaporin-4—namely, subpial and perivascular end-feet of astrocytes and choroid plexus [11C)TGN-020 PET could have useful applications to help unravel the molecular mechanism by which aquaporin-4 interacts with the glymphatic system and the relationship with disease pathology in age-related neurodegenerative diseases. Dysfunction of the glymphatic system, in late life, has been shown to contribute to the accumulation of amyloid-β ,305. Aquaporin-4 . In humad plexus . TherefoGlymphatic MRI, with intrathecal injections of MRI-based contrasts acting as a CSF tracer, has been developed to quantify CSF-ISF exchange in vivo ,313. RecThe development of novel glymphatic system MRI methodologies, such as 3D phase-contrast MRI ,325,326,18F)FDG PET study revealed a reorganization of regional cerebral metabolic activity following sleep deprivation in healthy individuals, which was associated with the decline in cognitive performance observed after sleep deprivation [11C)PiB to measure the brain amyloid-β load have demonstrated that adults who report less adequate sleep, more sleep problems and greater somnolence have a greater amyloid-β burden in AD-sensitive brain regions, independently of the APOE-ε4 genotype [2A receptors in frontal and parietal cortices [Preclinical animal models have shown that the clearance of amyloid-β is dysregulated under conditions of sleep deprivation and with advanced aging ,346,347.rivation . PET stugenotype ,352. Basgenotype . PET stugenotype ,355,356,genotype  and an ucortices . Togethe11C)Martinostat PET radioligand to measure class I histone deacetylase (HDAC) enzyme density [Challenging the traditional clinicopathological nosology of individual age-related neurodegenerative diseases may open avenues for dissecting disease entities, such as AD or PD, and unraveling their potential heterogeneity into subtypes through in vivo multifaceted biomarker studies. Each with a distinct biomarker-based signature profile, reflecting its distinct pathogenic pathway(s). This approach is not dissimilar to the stratification paradigm, which has, recently, proven its value in several cancer types. Significant recent advances in cancer biology and a biomarker-based subclassification allowed for targeted precision pharmacological interventions, showing significant therapeutic benefit in evidence-based stratified patients, whereas they had previously failed to meet the desired effects . Genetic density . IQ and TauIQ may add future value to the recent AT(N) classification of AD [While PET imaging can be complex, expensive and its wider application may be limited due to infrastructural and logistical constraints, PET imaging remains the only reliable noninvasive in vivo solution to quantify molecular targets and pathologies in patients across the disease life course. The continued development and wider application could help to transfer promising PET techniques from a research to a clinical setting. There is a need for more longitudinal studies to answer a number of key neurobiological questions and to determine the use of PET radioligands as reliable biomarkers to track disease progression. Furthermore, future work should focus on understanding the relationship between PET biomarkers and early signs and symptoms of age-related neurodegenerative diseases. Capitalizing on fully penetrant genetic mutations could allow for the investigation and characterization of early disease pathology in preclinical stages and, accompanied with longitudinal follow-ups, could help to shed further light on the relationship between these PET markers and early disease symptomatology. Recent methodological advances to measure amyloid-β and tau loads from PET imaging, with higher sensitivity and lower variability, could further improve the accuracy of imaging phenotypic stratification and increased power to detect meaningful outcomes and biological effects in future clinical trials ,373. Novon of AD ,11. The The scientific community is slowly moving away from the traditional clinicopathological disease models towards a precision medicine paradigm, which is still lacking in age-related neurodegenerative diseases. Based on the available understanding of interlinked genomic, biological and clinical pathways across diseases, it is likely that a dynamic multifaceted biomarker approach is required to disentangle mechanisms of disease pathogenesis and clinical trajectories. This might include markers of pathology spanning from genetic, epigenetic, proteomic, metabolomic and transcriptomic, clinical and digital and molecular PET and MRI imaging to biological CSF and blood-based biomarkers. Unraveling disease-specific network changes underpinned by genetic and pathological patterns could aid the advancement of personalized precision medicine by providing novel diagnostic tools and stratification approaches, as well as in identifying new therapeutic targets. Molecular PET imaging will likely continue to offer an invaluable tool, reflecting molecular pathology, contributing towards unlocking disease mechanisms and identifying biomarkers throughout the disease course. It is hoped that advances in the development of novel PET radioligands  and thei


Gardenia jasminoides Ellis (G. jasminoides). However, few studies have investigated the biosynthesis and regulation of CQAs in G. jasminoides. In this study, methyl jasmonate (MeJA) was used to enhance CQAs accumulation in cultured G. jasminoides cells. Moreover, the possible molecular mechanism of MeJA-mediated accumulation of CQAs is also explored. To this end, time-course transcriptional profiles of G. jasminoides cells responding to MeJA were used to investigate the mechanism from different aspects, including jasmonate (JAs) biosynthesis, signal transduction, biosynthesis of precursor, CQAs biosynthesis, transporters, and transcription factors (TFs). A total of 57,069 unigenes were assembled from the clean reads, in which 80.7% unigenes were successfully annotated. Furthermore, comparative transcriptomic results indicated that differentially expressed genes (DEGs) were mainly involved in JAs biosynthesis and signal transduction (25 DEGs), biosynthesis of precursor for CQAs (18 DEGs), CQAs biosynthesis (19 DEGs), and transporters (29 DEGs). Most of these DEGs showed continuously upregulated expressions over time, which might activate the jasmonic acid (JA) signal transduction network, boost precursor supply, and ultimately stimulate CQAs biosynthesis. Additionally, various TFs from different TF families also responded to MeJA elicitation. Interestingly, 38 DEGs from different subgroups of the MYB family might display positive or negative regulations on phenylpropanoids, especially on CQAs biosynthesis. Conclusively, our results provide insight into the possible molecular mechanism of regulation on CQAs biosynthesis, which led to a high CQAs yield in the G. jasminoides cells under MeJA treatment.Chlorogenic acid and its derivatives (CQAs) are considered as important bioactive secondary metabolites in  Gardenia jasminoides Ellis (G. jasminoides) is an evergreen shrub of family Rubiaceae, which is widely distributed in Southern China. Its fruits are commonly used in foods and herbal medicines in China , namely, 3-caffeoylquinic acid (3-CQA), 4-caffeoylquinic acid (4-CQA), 5-caffeoylquinic acid (5-CQA), 3,5-dicaffeoylquinic acid , 4,5-dicaffeoylquinic acid , 3,5-dicaffeoyl-4-O-(3-hydroxyl-3-methyl)-glutaroylquinic acid , and malonyl-4,5-O-dicaffeoylquinic acid   . In thesG. jasminoides cells (Jasmonic acid (JA) and methyl jasmonate  are regarded as the most important signaling molecules in plants. Above all, they not only regulate a diverse set of physiological and developmental processes but also trigger the biosynthesis of various plant secondary metabolites such as polyphenolic compounds, alkaloid, and terpenoids through extensive transcriptional reprogramming . Althouges cells , but theL. japonica and identified potential candidate genes that may participate in the biosynthesis of CQAs. Comparative transcriptome analysis was performed between Eucommia ulmoides with high and low CQAs contents, which looked into potential structural genes and transcription factors (TFs) involved in CQAs biosynthesis , 8 h (G8h), 20 h (G20h), and 40 h (G40h) were selected for RNA-seq to investigate the time-course transcriptional profiles in response to MeJA elicitation. Moreover, the abundant transcriptional information was analyzed in detail to gain insight into the transcriptional changes of cells treated by MeJA. In short, this work would provide valuable information to understand the molecular mechanisms of MeJA elicitation for high CQAs production in the cultured G. jasminoides cells.In the current study, MeJA (200 μM) was utilized to increase the yield of CQAs in the cultured G. jasminoides callus used in this study was obtained from Jiangxi Key Laboratory of Natural Products and Functional Food . G. jasminoides cell suspension culture was established according to the method we reported previously  for the suspension cell culture. Six grams (fresh weight) of callus was inoculated into a 100-ml culture flask containing 40 ml MS medium, subsequently cultivated in a rotating shaker with 115 r min–1 at 28 ± 1°C under continuous light , and routinely subcultured every 10 days. After 10 times of subculture, a homogeneous and rapidly growing suspension culture was obtained and used for further experiments.The embryogenic eviously . The detG. jasminoides cells were cultivated in the same medium and conditions as mentioned above. On the fifth day after inoculation, the aforesaid MeJA solution was added into the culture medium at a final concentration of 200 μM, and then the cells were cultured until the scheduled harvest time (0–80 h). The harvested cells were frozen immediately in liquid nitrogen and stored at −80°C prior to use. Each treatment was carried out in triplicate.Methyl jasmonate was dissolved in 75% ethanol and sterilized by filtration prior to elicitation. The collected sample cells were dried to a constant weight. A powder sample (200 mg) was extracted with 10 ml of methanol/water  in an ultrasound extractor for 50 min. The extracts were determined by high-performance liquid chromatography (HPLC) according to our previously reported method . Seven CFour samples, in which cells were treated by MeJA for 0, 8, 20, and 40 h, were prepared by mixing three replicate samples of the harvested cells before RNA extraction. Total RNA was extracted using a Total RNA Extractor  according to the manufacturer’s instructions. mRNA was enriched by Oligo(dT) and used to construct a non-stranded RNA-seq transcriptome library. After fragmentation, reverse transcription, 3′ add A, adaptor ligation, and PCR amplification, the cDNA library was obtained and then subjected to the Illumina HiSeq X Ten (PE150) platform with an average insert length of 350 bp for paired-end sequencing with one technical replicate. In addition, total cDNA libraries of cell samples exposed to MeJA for 0, 8, 20, and 40 h were designated as “G0h,” “G8h,” “G20h,” and “G40h”, respectively, in the current study.de novo assembly was conducted by software Trinity (version number: 2.4.0) with a min_kmer_cov parameter of 2. High-quality reads were subjected to module Chrysalis to obtain unique contigs, which were clustered to generate Bruijn. Module Butterfly was used to process Bruijn to obtain full-length transcripts with alternative splicing and to distinguish paralogs. After transcript de-redundancy, the longest ones of each transcript cluster were regarded as isoforms representing unigenes , and the reads with a length less than 35 bp. Prior to assembly, each library mentioned in section “RNA Extraction, Library Construction, and Illumina Sequencing (RNA-seq)” was pooled. Then, unigenes .E-value of 10–5  non-redundant protein sequences (NR) and Swiss-Prot, for homology comparison using NCBI Blast+ program (version number: 2.28) with identity set at >30% and a cutoff  of 10–5 . By usin of 10–5 . The KEG of 10–5 . Annotat2[fold change (FC)] > 1 (FDR < 0.001) was set as the threshold to identify upregulated differentially expressed genes (DEGs), and Log2(FC) < −1 (FDR < 0.001) was set as the threshold to identify downregulated DEGs , and then significantly overrepresented metabolic and signal transduction pathways were identified and ranked  method was adopted on the RSEM software (version number: 1.0) . The difted DEGs . To furtd ranked . ComparaG. jasminoides cells elicited by MeJA for different periods  using TRIzol® reagent (Invitrogen) following the manufacturer’s recommendations. After treatment with DNase I , 1 μg of RNA was used in reverse transcription with Revert Aid Premium Reverse Transcriptase kits . qRT-PCR was performed using SG Fast qPCR Master Mix  and ran on Step One Plus Real-Time PCR Detection System . The cycling conditions were initial denaturation at 95°C for 3 min, followed by 45 cycles of 95°C for 3 s, 60°C for 30 s, and 72°C for 15 s. Fifteen selected unigenes were validated by qRT-PCR experiments, and qRT-PCR reactions were carried out in triplicate. Gene-specific primer pairs  more than that of control, whereas total CQAs content varied in a little range (0.71–1.21 mg g–1) without MeJA elicitation (CK). The selected cell samples were exposed to MeJA for 0, 8, 20, and 40 h, and both total CQAs content and its fold changes were highest in G40h , followed by G20h , G8h , and G0h . Based on the results, it was observed that MeJA made a differential contribution to individual CQAs production , a plant in Rubiaceae family, followed by Nelumbo nucifera (8.0%), Zea mays (6.6%), and Vitis vinifera (2.9%).A total of 41,201 unigenes were annotated in NR databases  and compGene Ontology is an international classification system for standardized gene functions, aiming to comprehensively describe the properties of genes and their products in any organism. Based on the NR annotation, 30,119 unigenes were classified into three major categories such as “Biological process,” “Cellular component,” and “Molecular function,” which were further grouped into 58 sub-categories . Among tG. jasminoides transcriptome, 22,319 unigenes were distributed into 26 functional groups based on the KOG database  signaling and key biochemical pathways. To identify DEGs and analyze their time-course expression profile during the MeJA-treated process, we analyzed the following comparisons: G0h vs G8h, G0h vs G20h, and G0h vs G40h, in which 3,611 , 3,405 , and 7,926  DEGs were identified, respectively .2(fold change) values revealed a total of 8,264 DEGs in G0h vs G8h, G0h vs G20h, and G0h vs G40h , allene oxide synthase , allene oxide cyclase , 12-oxophytodienoic acid reductase , acyl-CoA oxidase , and acetyl-CoA acyltransferase 1 , were all continuously upregulated, but five DEGs corresponding to OPR (three DEGs), ACCA1 (one DEG), and enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase  were slightly downregulated in G8h, G20h, and G40h. Moreover, the ascending order of transcript abundance for upregulated DEGs was generally as follows: G0h, G40h, G20h, and G8h  and JAZ (three DEGs) showed a significant increase of transcript abundance from G0h and remained in extremely high expressions in G8h, G20h, and G40h. For instance, a MYC2 DEG (c10788_g1) and a JAZ DEG (c1385_g1) were most highly expressed , 3-dehydroquinate dehydratase I , 3-phosphoshikimate 1-carboxyvinyltransferase , arogenate/prephenate dehydratase , and tyrosine aminotransferase . Another eight DEGs showed downregulated expressions, in which seven DEGs participated in tryptophan biosynthesis, namely, anthranilate synthase component II , anthranilate phosphoribosyltransferase , tryptophan synthase beta chain , and tryptophan synthase alpha chain . The above-mentioned results indicated that MeJA could stimulate the expression of genes for the phenylalanine and tyrosine biosynthesis and, meanwhile, downregulated the expression of genes in the branching pathway (tryptophan biosynthesis), which enhanced the supply of precursors for phenylpropanoid biosynthesis.Aromatic amino acids, including phenylalanine, tyrosine, and tryptophan, were generated via the shikimate pathway in plants and microbes , and phetrans-cinnamic acids and quinic acids , trans-cinnamate 4-monooxygenase , 4-coumarate-CoA ligase , shikimate O-hydroxycinnamoyltransferase , and coumaroyl quinate/coumaroyl shikimate 3′-monooxygenase . In particular, the expression of the PAL family showed the most significant upregulation, and PAL (c19588_g1) was upregulated over 28.89-fold in G8h compared with that in G0h. However, none of the hydroxycinnamoyl CoA quinate hydroxycinnamoyl transferase (HQT) gene was even annotated by the BLAST program in our transcriptome data. These results implicated a correlation between the increasing metabolic flux and CQAs accumulation owing to MeJA elicitation.Chlorogenic acids derived from phenylpropanoid biosynthesis pathway are a class of depsides of certain ic acids . A totalic acids  were idecaffeic acid 3-O-methyltransferase (COMT), caffeoyl-CoA O-methyltransferase (CCoAMT), ferulate-5-hydroxylase (F5H), cinnamoyl-CoA reductase (CCR), cinnamyl-alcohol dehydrogenase (CAD), and peroxidase (POD)] were identified, and 18 of these DEGs were upregulated in G8h, G20h, and G40h compared with that in G0h. Our result was in good agreement with the study of IiCAD and IiCCR) and accumulation of several lignans in Isatis indigotica hairy roots.Interestingly, a total of 40 DEGs  related ATP-binding cassette (ABC) transporter and multidrug and toxin extrusion (MATE) protein were reported to be the main transporters for phenylpropanoid. In our transcriptome data  and classified to 55 known TF families . The mosMYB85 family contained the maximum number of DEGs (17 unigenes), and nine of those DEGs displayed an upregulated expression in G8h, G20h, and G40h compared with those in G0h. Besides, MYB111 (one DEG), MYB20 (two DEGs), and MYB15 (one DEG) were found to be upregulated as well. In terms of the negative regulators (MYB60 (one DEG), MYB52 (five DEGs), and MYB4 (five DEGs) families. To this end, these results revealed that MeJA could significantly modulate the expression of MYB TFs, which might participate in phenylpropanoid biosynthetic regulation.Some TF families , especially MYB, usually participate in the regulation on the secondary metabolism. Thus, DEGs in the above-mentioned TF families were classified for further analysis . Among tgulators , 14 DEGsAOS (c18070_g1), JAZ (c1385_g1), MYC2 and (c10788_g1)], CQAs biosynthesis , and TF that modulates the CQAs biosynthesis  were selected for qRT-PCR to validate the relative expression level in RNA-seq with Pearson correlation coefficient (r). Correlation analysis results  related to phenylpropanoid biosynthesis in rice. Meanwhile, higher accumulation of phenolic acids, e.g., cinnamic acid, ferulic acid, and benzoic acid, was also found in the AOC-overexpressed transgenic line  proteins in the absence of a JA signal. Upon sensing of the external stimuli , the SCFCOI1-ubiquitin-proteasome pathway is activated and then leads to the degradation of the JAZ repressor, which paves the way for MYC2 to regulate the JA-responsive gene expression (MYC2 (three DEGs) and JAZ (three DEGs) were significantly upregulated by MeJA treatment (Col 1 (c20119_g3) showed a downward trend in the three comparisons. It was reported that there were positive and negative feedback regulatory loops to regulate JA signaling. On the one hand, the external stimuli promoted the degradation of the JAZ repressor to activate transcription of genes encoding MYC2. Moreover, the external stimuli subsequently facilitate the transcription of genes encoding JAZ, together with the downregulation of Col 1, resulting in resetting the signal transduction pathway and avoiding a runaway response  were upregulated in the overexpression lines  generating CQAs via an activated intermediate (caffeoyl-glycoside); (2) synthesis of CQAs from caffeoyl-CoA and quinic acid by HQT; and (3) hydroxylation of p-coumaroyl-quinic acid by C3H to form CQAs  mentioned above . Subsequently, di-CQAs were generated by HCT from mono-CQA and ultimately converted to di-CQA derivatives in the presence of several unknown structural genes.In our RNA-seq data, a number of genes encoding PAL (nine DEGs), C4H (three DEGs), 4CL (three DEGs), HCT (three DEGs), and C3H (one DEG), 17 of which showed significantly upregulated expressions over time, and the increased expressions of structural genes were consistent with the mono-CQA accumulation in MeJA-treated G. jasminoides cells (C3H (C18639_g1 and C4961_g1) further converted mono-CQA into di-CQAs  was responsible for the membrane translocation of p-coumaric acid, which was a key precursor for CQAs’ and other phenylpropanoid component’s biosynthesis, and silencing the expression of MtABCG10 resulted in a lower accumulation of phenylpropanoid-derived medicarpin and its precursors  was assigned as the putative proanthocyanidin transporter expressed during berry development   . A totalfamilies  in our RG. jasminoides cells , chalcone isomerase (CHI), flavanone 3-hydroxylase (F3H), and flavonol synthase (FLS), and its expression could stimulate flavonoid accumulation  and dihydroflavonol-4-reductase (DFR) in MYB60-overexpressed A. thaliana and showed a negative regulation on flavonoid accumulation  and three kinds of negative regulator  on phenylpropanoid biosynthesis. Most of the positive regulators displayed a continuously upregulated expression, but most of the negative regulators showed a downtrend in expression after MeJA treatment  and JAs signal transduction . Then through the signal transduction network, genes related to biosynthesis of aromatic amino acids, namely, precursors for CQAs, showed an increased expression , which might boost supply of the precursors. Meanwhile, TFs, especially for the MYB family, showed significant response to MeJA treatment and might display positive  or negative  regulations on CQAs biosynthesis, resulting in increased expression of key genes for CQAs biosynthesis . Ultimately, the accumulation of CQAs in G. jasminoides cells was significantly increased. Moreover, our data will surely provide a massive genetic resource for further investigation of CQAs biosynthesis and lay the foundations for genetic engineering to boost the yield of these important compounds in G. jasminoides cells.Our work indicated that the application of exogenous MeJA could effectively stimulate the CQAs accumulation in gulation . In MeJAPRJNA672865).The data presented in the study are deposited in the (NCBI Sequence Read Archive) repository, accession number (ZL: methodology, validation, investigation, data curation, visualization, writing—original draft, and writing—review and editing. AM: formal analysis and writing—review and editing. ZW: methodology, validation, and writing—review and editing. XZ: methodology and validation. YZ and LC: supervision and validation. MG: supervision, project administration, and writing—review and editing. ZY: supervision, funding acquisition, project administration, and writing—review and editing. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Neutrophil granulocytes are the vanguard of innate immunity in response to numerous pathogens. Their activity drives the clearance of microbe- and damage-associated molecular patterns, thereby contributing substantially to the resolution of inflammation. However, excessive stimulation during sepsis leads to cellular unresponsiveness, immunological dysfunction, bacterial expansion, and subsequent multiple organ dysfunction. During the short lifespan of neutrophils, they can become significantly activated by complement factors, cytokines, and other inflammatory mediators. Following stimulation, the cells respond with a defined (electro-)physiological pattern, including depolarization, calcium influx, and alkalization as well as with increased metabolic activity and polarization of the actin cytoskeleton. Activity of ion transport proteins and aquaporins is critical for multiple cellular functions of innate immune cells, including chemotaxis, generation of reactive oxygen species, and phagocytosis of both pathogens and tissue debris. In this review, we first describe the ion transport proteins and aquaporins involved in the neutrophil ion–water fluxes in response to chemoattractants. We then relate ion and water flux to cellular functions with a focus on danger sensing, chemotaxis, phagocytosis, and oxidative burst and approach the role of altered ion transport protein expression and activity in impaired cellular functions and cell death during systemic inflammation as in sepsis. Input and output of water and ions, therefore, require tight regulation in organisms. On a cellular level, various ion and water transport proteins ensure the ion–water balance, which is involved in any physiological and immune function.A major function of the innate immune system is to supply the “first line” of the fluid and cellular defense system, comprising mainly the complement system and neutrophil granulocytes (neutrophils). The complement cascade becomes activated by numerous microorganism and microbial-associated molecular patterns (MAMPs) and in turn generates anaphylatoxins, opsonins, and membrane attack complexes, all of which support bacterial marking and clearance ,2. NeutrSepsis is defined as a life-threatening organ dysfunction caused by a dysregulated host response to infectious microorganisms and corresponding MAMPs . With anIn this review, we discuss neutrophil functions, with a focus on ion and water flux with an emphasis on danger sensing, chemotaxis, phagocytosis, and oxidative burst, highlight pyroptosis and NETosis as neutrophil-related forms of cell death, and transfer these findings into the clinically relevant context of sepsis.Danger sensing of harmful MAMPs, but also of damage-associated molecular patterns (DAMPs), is crucial for any organism, if not a “question of life and death”. Neutrophils, as the most frequent representatives of the first cellular line of defense, can recognize molecular danger by pattern recognition receptors (PRPs), including the membrane-bound formyl-peptide receptors (FPRs), toll-like receptors , complement receptors , and receptor for advanced glycation end products (RAGE) ,20. Ther− efflux, promote NLRP3 inflammasome assembly and subsequent IL-1β secretion, and thus mediate a proinflammatory response [Multiple mechanisms exist by which microbes or corresponding MAMPs can enter host cells. For example, lipopolysaccharide (LPS) can enter the cytoplasm by escaping the bacteria/MAMP-containing vacuole, or via endocytic uptake of LPS encapsulated in outer membrane vesicles originated from bacteria . Upon inresponse ,32. Whetresponse .N-formylmethionine-containing oligopeptides (which are released by many invading microorganisms) via FPRs on neutrophils results in an increase in the mitochondrial membrane potential. This rapidly leads to a robust release of adenosine triphosphate (ATP) by the neutrophil. In turn, the excreted ATP subsequently activates purinergic receptors on the cellular surface in an autocrine manner. The resulting Ca2+-influx finally triggers functional changes, including morphological polarization, directed chemotaxis, phagocytosis, and degranulation [Sensing of nulation ,34.N-formyl-methionyl-leucyl-phenylalanine ) and numerous inflammatory mediators, including among others complement factor 5a (C5a), IL-8, lipid mediators, such as PAF and leukotriene B4 (LTB4), and ATP [Cellular migration is a key function of neutrophils, which is induced by MAMPs from the inflammatory/infectious site , a small GTPase of the Rho family, is required in neutrophils ,39. Chem2+ [In general, neutrophils respond to exposure to chemoattractants with a defined reaction pattern consisting of a rise in cytosolic Ca2+ ,45,46,472+ ,45,46,482+ ,42,49,502+ levels [2+ signaling can be initiated by G protein-coupled receptors  or in general by other ligand-receptor interactions, including Fcɣ-receptors (antibodies), and the activation of CD11b/CD18  as well as by cellular crosstalk, such as platelet–neutrophil interaction  [2) into inositol 1,4,5-trisphosphate (IP3), and diacylglycerol (DAG) [3 triggers Ca2+ release from the endoplasmic reticulum (ER), causing an initial rise in intracellular Ca2+. The decrease in Ca2+ concentration in the ER induces store-operated calcium entry (SOCE). Stromal interaction molecule (STIM) 1 and 2 on the ER membrane translocate to defined regions at the plasma membrane, interacting with Ca2+ release-activated Ca2+ (CRAC) channels (predominantly Orai1 in neutrophils) to initiate the influx of extracellular Ca2+ [2+ of fMLF-stimulated neutrophils by the generation of the nicotinamide adenine dinucleotide (NAD+) metabolites ADP-ribose (ADPr) and to a lesser extent cyclic ADP-ribose (cADPr) [+/Ca2+-influx) [2+-influx from the ER, independent of IP3-SOCE Ca2+-stores) [2+ activates several kinases as well as ion channels and cellular functions, as reviewed in [2+ activates various isoforms of protein kinase C (PKC), which phosphorylate among others p47 phagocyte oxidase , an important step in the activation of the multicomponent enzyme NOX [2+ triggers secondary (electro-)physiological changes, including cellular depolarization via NOX activation [Upon stimulation, neutrophils respond with a rapid and pronounced increase in cellular Ca+ levels ,47,51,52ctively) ,47. GPCRol (DAG) ,54. IP3 lar Ca2+ ,56,57. I (cADPr) , which i-influx)  and ryan-stores) ,61, respzyme NOX ,63,64,65tivation ,67,68, itivation , and cel2), thereby generating superoxide anions (O2•−) [2+ signaling [+ efflux via voltage-gated proton channels (voltage sensing domain only protein (VSOP) / Hv1 as its respective human homologue) [v1-deficient mice, which responded to stimulation by the chemoattractant fMIVIL  with cellular acidification as well as a significantly reduced rise in Ca2+ and subsequent impaired actin depolymerization and cellular migration [Neutrophil stimulation results in the activation of NOX, which transfers two electrons from nicotinamide adenine dinucleotide phosphate (NADPH) to molecular oxygen (Os (O2•−) . In thiss (O2•−) ,69,70,71ignaling ,68,72. Tmologue) ,68,73. Tigration .2+ as well as the intracellular accumulation of H+ generated by NOX initiate H+ extrusion, via Hv1 and the sodium-proton exchanger 1 (NHE1) [v1 is a voltage-dependent H+ channel activated by cellular depolarization [2+ activate calmodulin and other kinases, which can phosphorylate and thereby activate NHE1 together with the initial NOX-mediated increase in intracellular H+ concentration [+/K+-ATPase (by Omeprazole), H+-ATPase (by Bafilomycin), or the Hv1 channel (by zinc) did not prevent C5a-induced alkalization significantly [The rise in intracellular Ca1 (NHE1) ,75,76,77rization ,72,78. Tntration ,80,81. Tficantly .+ via monocarboxylate transporters, thereby contributing to extracellular lactate acidosis [3− [−/HCO3− exchanger  appears to be inhibited by fMLF [−/HCO3− exchange mechanism (likely AE1) appears to mediate the recovery from an alkaline load [3− has frequently not been supplemented in culture media, presumably to facilitate the control of the extracellular pH. Regarding ion fluxes, changes in intracellular pH interact with CRAC-mediated Ca2+-influx and the release of O2•−. For example, intracellular alkalosis increases intracellular Ca2+ and thus cellular function, including migration [2•− release [3− and other important blood ions and proteins in the buffer for neutrophil experiments, it is also noteworthy that most of the described mechanisms have been demonstrated mainly for fMLF and/or C5a stimulated neutrophils.Following initial stimulation, this intracellular alkalization continues for up to an hour and modulates various crucial cellular functions, including fMLF-induced IL-8 release and C5a-induced increase in glucose metabolism ,49. Thisacidosis . It is nosis [3− . Additio by fMLF . Accordiine load ,83. In tigration ,85. Addi release ,86. Besi+ as well as water at the protrusive end, active reorganization of the actin cytoskeleton, and an efflux of ions and water at the retractive pole [The discussed ion–water fluxes in neutrophils after stimulation with chemoattractants also interact closely with cellular migration, which has been addressed in other reviews ,88,89. Hive pole ,87,88,89ive pole ,92. Addiive pole . In the ive pole ,95,96,97ive pole .In sepsis, neutrophils are exposed to a broad spectrum of inflammatory mediators, including cytokines, chemokines, and complement fragments as well as to bacterial endotoxins, which can possibly impact neutrophil ion and water flux and thereby modulate neutrophilic functions. In accordance with this, numerous pivotal functions of neutrophils, including phagocytosis, oxidative burst production, and chemotactic activity, can be impaired during severe sepsis ,100,101.+ transport proteins) for neutrophil chemotaxis in sepsis [In agreement with the proposed alterations of ion transport proteins, an increase in neutrophil size and cell volume can be observed in murine sepsis ,11 as wen sepsis . In thisn sepsis ,102. In n sepsis  or for pn sepsis . However2+ currents and thereby modulate any micro-movement efforts, including during migration, chemotaxis, phagocytosis, and cytokine release [One consequence of this “metabolic switch” when turned on during sepsis may also feature shape changes of neutrophils reflected by an increase in the cellular length/width ratio . Of note release .2+ levels in neutrophils may also constitute a valuable strategy to modulate neutrophil-drive inflammation and/or to restore neutrophil function [2+ was associated with an impairment on phagocytotic activity, which could be ameliorated with the calcium channel blocker amlodipine [2+ is linked with reduced bacterial clearance of pathogens in neutrophils from individuals with cystic fibrosis, which also was improved by reducing intracellular Ca2+ [2+ levels and to explore the possibility to target Ca2+ homeostasis as mediator of neutrophil dysfunction during sepsis.Controlling Cafunction ,105. Forlodipine . Likewislar Ca2+ . FurtherIn addition to ion transport proteins, AQPs modulate fluid shifts in neutrophils, albeit their specific function in sepsis remains rather speculative. AQP5 protein expression has been reported to be downregulated during inflammation as a response to proinflammatory cytokines ,109. IntChanges in neutrophil ion and water homeostasis, frequently measured by changes in cell size, have been of increasing interest as diagnostic and prognostic markers in sepsis and other systemic inflammation conditions. A meta-analysis of the Area Under the Receiver Operating Characteristic Curve (AUROC) reported that a mean neutrophil volume of 0.87 (0.83–0.89) was an indicator for sepsis . In acco2+ influx, K+ efflux, activation of NF-κB as well as synthesis of IL-1β and IL-8 [Some MAMPs and DAMPs can disrupt the integrity of the leukocyte membrane by pore-forming molecules. In the case of MAMPs, various bacteria-derived toxins can generate differently sized pores in neutrophils which in turn lead to ion and water imbalances and in the worst case to cell death. Some sepsis-relevant microbial toxins are exemplarily summarized: Panton-Valentine leukocidin (PVL) is a toxin produced by some staphylococcal strains which can interact with the C5aR1 and induce lytic pores in neutrophils  as well and IL-8 ,120. Oveand IL-8 .2+ and the release of myeloperoxidase and IL-8 [Important representatives for DAMPs are histones and extracellular ATP. For example, histone H4 causes neutrophil membrane depolarization, a rise in intracellular Caand IL-8 . Interesand IL-8 . ATP, whand IL-8 . At highand IL-8 . In pyroand IL-8 . Howeverand IL-8 ,129,130,and IL-8 ,132,133.and IL-8 .2+ sensors STIM1 and 2, which activate the Ca2+ channel Orai1, guarantees sustained Ca2+-influx when activated  and thereby generates intracellular “Ca2+ hotspots” which augment phagocytosis [SOCE in neutrophils, regulated by the Caocytosis  and promocytosis ,135,136.Contradictory reports have been published regarding the phagocytic and oxidative burst activity of neutrophils during sepsis, which, however, appear mainly dependent on the timing and severity of sepsis, the maturity of neutrophils, and the local environment ,138. Durphox to the membrane, where it assembles with other components, including p40phox, p67phox, Rac1/2, and flavocytochrome b558, to the NOX complex [2•−), which is subjected to dismutation generating hydrogen peroxide (H2O2) and ultimately HOCl via the myeloperoxidase [−-conductance for compensation of the electrical charge out of the vacuole [phox and impaired ROS generation [2+ levels of resting neutrophils [To kill phagocytized bacteria, effective antimicrobial mechanisms are essential. These mainly involve an oxidative burst with reactive oxygen species (ROS) generation that can be oxygen-dependent or -independent . Oxygen- complex . The eleroxidase ,150. The vacuole ,152,153.neration ,154, whineration  as well neration , but nottrophils , which ctrophils ,48, LPS-trophils .Moreover, extracellular acidification, as present in local and systemic inflammation, directly inhibits bacterial killing by neutrophils, while in contrast promoting phagocytosis . In acco+/H+ exchanger [+ channel VSOP and its respective human homologue Hv1 are considered essential for the excretion of protons produced by NOX [v1−/− mice had a substantially reduced superoxide production and impaired bacterial clearance both in-vitro and in-vivo indicates the requirement of Hv1 for sufficient antibacterial activity in sepsis [v1 expression or activity actually do contribute to neutrophil dysfunction in severe sepsis has to date hardly been addressed and requires further research.Regarding the oxygen-independent mechanism of bacterial killing, lysosomal enzymes are released into the phagosomes, where after an initial alkalization phase the now phagolysosomes become acidified by activation of the Naxchanger . In addid by NOX ,69. The n sepsis . Notably2+-homeostasis in neutrophils also appears crucially involved in the mounting of an oxidative burst and other antimicrobial functions. Several Ca2+-conductive ion channels are expressed in neutrophils, including the TRP family and store-operated Ca2+ channels. TRPM2, a Ca2+ permeable, non-selective cation channel, which is activated by ADPr, temperature, oxidative stress, and Ca2+ [2+ mobilization and oxidative stress in neutrophils. For example, during experimental sepsis in mice infected with Listeria monocytogenes, the TRPM2 cation channel modulated membrane depolarization, Ca2+ mobilization, and subsequent ROS generation [2+, and aggravated oxidative burst, all of which might be harmful to the host [2+ permeability could limit the oxidative burst response during LPS-induced murine sepsis [Moreover, Caand Ca2+ , has beeneration . In thisthe host . Anothere sepsis . In thise sepsis ,162.Finally, ion concentrations and pH values of the local inflammatory microenvironment of the neutrophils will self-evidently to some extent alter the intracellular milieu and thereby also influence the phagocytic and oxidative burst activity ,163,164.A multitude of triggers and signaling pathways have been described in initiating and regulating neutrophil death ,165. RapPyroptosis is activated by cytosolic inflammasome perturbations. Inflammasome activation can be induced by intracellular pathogens or by intracellular exposure to LPS that result in activation of inflammatory caspases that cleave gasdermin D (GSDMD) ,165. IntN-terminal fragments [Recent findings documented that GSDMD can also be processed by neutrophil elastase. Cleavage occurs upstream of the canonical caspase cleavage site, but still produces pore-forming ragments ,170. Howragments . By contragments . Thereforagments .2+- and Na+-influx and K+-efflux, which results in NLRP3 inflammasome activation [Extracellular ATP can activate inflammasome signaling in neutrophils via activation of P2X7Rs on neutrophils . P2X7R itivation ,173. Whetivation .Activated neutrophils can neutralize invaders by releasing NETs  in a pro2+ mediates ROS production, essential for lytic NETosis. This is probably a result of NOX activation [2+ chelator, inhibited NETosis [2+ levels originates from Ca2+ entry [2+ stores [2+ chelators over a prolonged period could induce a homeostatic response in which a decrease in extracellular Ca2+ results in increased Ca2+ release by the ER to maintain intracellular Ca2+. Such a phenomenon could explain, in part, such a discrepancy between the studies [3−-mediated alkalization of neutrophils favored spontaneous and LPS-induced NETosis, possibly because of modulation of intracellular Ca2+ [Increases in intracellular Cativation ,179, acttivation . In agre NETosis . However2+ entry  or relea+ stores . Dependi studies . Activat studies . Intereslar Ca2+ .2+ signals that themselves depend on intracellular or plasma membrane Ca2+ channels.In lytic  NETosis, NET extrusion occurs via GSDMD pores ,170 and/+ efflux, and NLRP3 inflammasome assembly, which result in NLRP3-dependent caspase-1 activation and GSDMD cleavage [Apoptosis, pyroptosis, and NETosis were traditionally considered to be independent of one another, but emerging evidence indicates that there is extensive cross-talk between them. All three pathways can converge upon the activation of the same cell death effector—the pore-forming protein GSDMD . Similarcleavage .N-terminal domain (GSDMD-N) connected via a linker to the regulatory C-terminal domain. Cleavage of GSDMD at a conserved site releases GSDMD-N from the auto-inhibitory C-domain. GSDMD-N has the ability to translocate to the inner leaflet of the plasma membrane, where it oligomerizes and induces the opening of membrane pores [+) causes an increase in cell volume due to water influx and can result in osmotic cell lysis. This likely depends on the number of GSDMD pores in the plasma membrane. The number of GSDMD pores can be limited by a recently discovered mechanism that actively removes GSDMD pores from the plasma membrane. Ca2+-influx via GSDMD pores recruits endosomal sorting complexes required for transport (ESCRT) to damaged areas of the plasma membrane to remove GSDMD pores and to maintain membrane integrity [+, Cl−, and organic osmolyte  channels that export these solutes and their accompanying water [GSDMD consists of a pore-forming ne pores ,192,193.ne pores . The resntegrity . This isng water . In partng water ,197. A dIon and water fluxes regulate essential neutrophil functions. Their alterations during sepsis impair vital cellular functions and may thereby contribute to immune and organ dysfunctions. Fields of particular interest include first, a further elucidation of the ion–water disbalance upon excessive neutrophil activation, second, an in-depth evaluation of physiological parameters, including intracellular pH and cellular size of neutrophils as a diagnostic and/or prognostic marker of immune dysfunction, and third, a meaningful translation to the bedside by modulating ion–water based alterations, for example, by pharmacological targeting of perpetrator ion transport proteins, such as NHE1, during sepsis.


We aimed to investigate the effect of bromelain, the extract from stems of pineapples on the high-fat diet (HFD)-induced deregulation of hepatic lipid metabolism and non-alcoholic fatty liver disease (NAFLD), and its underlying mechanism in mice. Mice were daily administrated with HFD with or without bromelain (20 mg/kg) for 12 weeks, and we found that bromelain decreased the HFD-induced increase in body weight by ~30%, organ weight by ~20% in liver weight and ~40% in white adipose tissue weight. Additionally, bromelain attenuated HFD-induced hyperlipidemia by decreasing the serum level of total cholesterol by ~15% and triglycerides level by ~25% in mice. Moreover, hepatic lipid accumulation, particularly that of total cholesterol, free cholesterol, triglycerides, fatty acids, and glycerol, was decreased by 15–30% with bromelain treatment. Mechanistically, these beneficial effects of bromelain on HFD-induced hyperlipidemia and hepatic lipid accumulation may be attributed to the decreased fatty acid uptake and cholesteryl ester synthesis and the increased lipoprotein internalization, bile acid metabolism, cholesterol clearance, the assembly and secretion of very low-density lipoprotein, and the β-oxidation of fatty acids by regulating the protein expression involved in the above mentioned hepatic metabolic pathways. Collectively, these findings suggest that bromelain has therapeutic value for treating NAFLD and metabolic diseases. Undynthesis ,5,6. Theynthesis . NAFLD iynthesis ,9,10. Itynthesis . The preynthesis ,13. Therynthesis ,13. Despynthesis ,15, howeBromelain, the main active component extracted from the pineapple stems, has several beneficial effects, including anti-inflammation, anti-coagulation and anti-tumor growth in human and experimental animals ,17,18. TIn this study, we aimed to clarify the interaction between bromelain effects and the high-fat diet (HFD)-induced deregulation of hepatic lipid metabolism and NAFLD. We first investigated the effect of bromelain on body weight, organ weight and the lipid profile of plasma in HFD-fed wild-type (WT) mice and then examined whether bromelain affected the HFD-induced deregulation of lipid metabolism and its underlying molecular mechanisms in the liver. Our findings demonstrate that bromelain decreased fatty acid uptake and cholesteryl ester synthesis and promoted lipoprotein internalization, bile acid metabolism, cholesterol clearance, VLDL assembly and secretion, and the β-oxidation of fatty acids in the liver, and led to the prevention of HFD-induced obesity, hyperlipidemia and the progression of fatty liver.Bromelain was from Cayman Chemical . Mouse antibodies for ATP-binding cassette A1 , carnitine palmitoyltransferase 1  and liver X receptor α ; Rabbit antibodies acyl-CoA:cholesterol acyltransferases 1 , acetyl-CoA carboxylaseα , acyl-coenzyme A oxidase 1 , apoB (ab31992), lipoprotein receptor-related protein 1 , peroxisome proliferator-activated receptor gamma coactivator 1-α coactivator-1α  and scavenger receptor class B type I  were from Abcam . Mouse antibodies for sterol regulatory element-binding protein (SREBP)-1 (557036) and SREBP-2 (557037) were from BD Bioscience . The mouse antibody for β-actin (A5441) was from Sigma-Aldrich . The rabbit antibody for apoE (1930-5) was from Epitomics . The rabbit antibody for low-density lipoprotein receptor  was from Novus Biologicals . The rabbit antibody for apoAI (14427-1-AP) was from Proteintech . The mouse antibody for peroxisome proliferator-activated receptor γ ; Rabbit antibodies for ABCG5 (sc-25796), ABCG8 (sc-30111), long-chain fatty acid coenzyme A ligase 1 , cluster of differentiation 36  cytochrome P450 family 7 subfamily A member 1 , diacylglycerol O-acyltransferase 1 , fatty acid synthase , hydroxymethylglutaryl-CoA reductase , liver-type fatty acid binding protein , LXRβ (sc-1001), PPARα (sc-9000) and retinoid X receptor ; goat antibodies for ACAT2 (sc-30279), DGAT2 (sc-32400) and microsomal triglyceride transfer protein  were from Santa Cruz Biotechnology . ELISA kits for cytokines and adipokines were from R&D systems . Total cholesterol and cholesteryl ester, triglyceride, fatty acid and glycerol fluorometric assay kits were obtained from BioVision .2. The liver, white adipose tissue (WAT) and brown adipose tissue (BAT) were isolated and weighed. These samples were then subjected to histological analysis or stored at −80 °C. The frozen liver, WAT, and BAT were homogenized, and then the lysates were subjected to Western Blot analysis.This study conformed to the Guide for the Care and Use of Laboratory Animals , and all animal experiments were approved by the Animal Care and Utilization Committee of National Yang-Ming University (No. 1070314). Male wild-type (WT) C57BL/6 mice at 8 weeks old were obtained from National Laboratory Animal Center  and were randomly divided into two groups and administrated with either vehicle (PBS) or bromelain (20 mg/kg) by oral gavage with high-fat diet  for 12 weeks. Mice were housed in barrier facilities, maintained in a 12-h/12-h light-dark cycle. Temperature (22 °C) and humidity (40–60%) of the vivarium were tightly controlled. At the end of the experiment, mice were euthanized with COThe liver blocks were cut into 8 μm sections and subjected to histological examination. Cryosections were subjected to oil red O staining and deparaffinized sections were stained with hematoxylin and eosin (H&E), and then viewed under a Motic TYPE 102M microscope .The livers were lysed in immunoprecipitation lysis buffer . Aliquots of tissue lysates (50 μg protein) were mixed with 5 μL loading dye , and then boiled at 100 °C for 5 min. Proteins were separated on 8%, 10% or 15% SDS-polyacrylamide gel by electrophoresis. The proteins were separated and transferred onto a polyvinylidene fluoride membrane , blocked with 5% skim milk for 1 h at room temperature, and then incubated with primary antibodies overnight and then with corresponding secondary antibody for 2 h. The protein bands were detected by using an enhanced chemiluminescence kit  and quantified using ImageQuant 5.2 .2, the blood was collected by cardiac puncture. After clotting and centrifugation, serum was isolated and the levels of triglycerides, total cholesterol, HDL-cholesterol (HDL-c), and non-HDL-c were evaluated by using the corresponding test strips of Spotchem EZ SP 4430 .After euthanizing with COv/v). After drying, the extracted lipids were dissolved by using the commercial solutions and the levels of total cholesterol, free cholesterol, cholesteryl ester, triglycerides, fatty acids, and glycerol were measured by using fluorescence assay kits . The fluorescence intensity was detected at 535 nm excitation and 590 nm emission under the multimode microplate reader Infinite 200 .The lipids in the liver were extracted by use of isopropanol/chloroform/NP-40 , monocyte chemoattractant protein-1 (MCP-1) and interleukin-6 (IL-6), and adiponectin, leptin, and resistin were measured by using ELISA kits according to the manufacturer’s instructions.t-test was used to compare two independent groups. SPSS software v20.0  was used for analysis. Differences were considered statistically significant at p < 0.05.The results were presented as mean ± SEM from 10 mice. Data from mice were evaluated by parametric tests. The unpaired Treatment with bromelain did not affect the appetite of mice. There was no difference in food intake daily between the vehicle group and bromelain group (vehicle group: bromelain group = 4.16 ± 0.25 g: 4.23 ± 0.38 g/mouse). We demonstrated that daily treatment of mice with HFD bromelain for 12 weeks altered HFD-induced change in their appearance A, body wThe liver is the most important organ for the homeostasis of whole-body lipid metabolism ,24. We tde novo lipogenesis, lipoprotein metabolism, bile acid metabolism, triglyceride metabolism, the assembly and secretion of VLDL, and the β-oxidation of fatty acids [The lipid regulation in the liver is tightly controlled by several metabolic pathways, including ty acids ,26,27,28ty acids .Additionally, treatment with bromelain increased the protein expression of LRP-1, LDLR and SR-BI A–D, the Moreover, treatment with bromelain decreased the protein expression of L-FABP and CD36 A–C, two The β-oxidation of fatty acids is also the key metabolic process in regulating the intracellular lipid content of hepatocytes . Thus, wBromelain is known to have beneficial effects on inflammatory diseases . Howeverde novo lipogenesis of cholesterol and fatty acid biosynthesis such as SREBP1 and 2, FAS, ACC, HMGCR in the liver. The previous reports have demonstrated that increased de novo lipogenesis contributes to the development of NAFLD and that the genetic overexpression of regulators involved in de novo lipogenesis exacerbates the NAFLD [de novo lipogenesis, the homeostasis of lipid metabolism in the liver is precisely controlled by several aspects of metabolic pathways, including lipoprotein metabolism, cholesterol metabolism, bile acid metabolism and fatty acid metabolism under physiological condition or pathological situations [Intriguingly, treatment with bromelain increased the protein expression related to the he NAFLD ,41,42. Ttuations ,27,28. TOf particular interest is our finding that treatment with bromelain increased the protein levels of lipoprotein receptors LRP1, LDLR and SR-BI, which might contribute to the decreased serum levels of cholesterol and triglycerides by bromelain. These results are in agreement with the previous reports that the genetic overexpression of these receptors inhibits the development of hyperlipidemia and NAFLD in mice ,44,45. TRegarding with the fatty acid metabolism in the liver, we found that treatment with bromelain decreased the protein expression of L-FABP and CD36, two important regulators for fatty acid uptake from circulation into hepatocytes; however, it increased the protein expression of DGAT1 and DGAT2, the two key enzymes for the biosynthesis of triglycerides . InteresIn particular, we demonstrated that bromelain upregulated the expression of proteins related to ACSL1, ACOX1, CPT1α, RXR, PPARα, PPARγ, and PGC-1α, all involved in the β-oxidation pathway for bioenergetics in mitochondria ,49,50,51Specifically, autophagy is a conserved self-eating process that is important for lipid homeostasis under various stress conditions ,57. It aThe crosstalk between the liver and adipose tissues is crucial for maintaining the homeostasis of whole-body lipid metabolism ,60. It iIn spite of the unique pathways discovered in this study, the detailed mechanisms of bromelain to affect lipid metabolism merit further study. Overall, congruous data have been reported, suggesting the alteration in protein expression of these key molecules regulating lipid metabolism, which again demonstrates the beneficial effects of bromelain on lipid metabolism. In conclusion, our study provides new evidence that bromelain has beneficial effects against the HFD-induced deregulation of hepatic lipid metabolism by decreasing fatty acid internalization and promoting VLDL secretion, lipoprotein metabolism, HDL assembly, cholesterol clearance and fatty acid β-oxidation in male C57BL/6 mice. These key events might work in concert to decrease the HFD-induced lipid accumulation in the liver .Moreover, we discovered a link between bromelain and hepatic lipid metabolism, which broadens its biomedical impact in the treatment of metabolic diseases.