Synthesis and cytotoxicity of novel 6,8,9-trisubstituted purine analogs against liver cancer cells.

A series of novel 6-(substituted phenyl piperazine)-8-(4-substituted phenyl)-9-cyclopentyl purines, 10-51, were synthesized by a four-step synthesis, achieving an overall yield of about 43 %. The reaction conditions were effectively optimized, and the final products were obtained with high purity and yield in all synthesis steps. The synthesized nucleobases were evaluated for their in vitro cytotoxic activities on selected human cancer cell lines (HUH7 (liver), HCT116 (colon), and MCF7 (breast)) using the Sulforhodamine B (SRB) assay. Among these analogs, compounds bearing 4-trifluoromethyl phenyl (19, 20 and 21), 4-methoxy phenyl (27) and 4-fluoro phenyl (34) substitutions at C-8 of purine were the most potent, and they were also analyzed in drug-resistance and drug-sensitive hepatocellular cancer cell (HCC) panels. Compound 19 displayed remarkable anticancer activities (IC50 = 2.9-9.3 µM) against Huh7, FOCUS, SNU475, SNU182, HepG2, and Hep3B cells compared to the positive control, Fludarabine. Additionally, the pharmacological properties and toxicity profiles of the molecules were investigated computationally by the Swiss-ADME and Pro-Tox II online tools, respectively. Results showed that our compounds have favorable physicochemical characteristics for oral bioavailability and do not reveal any toxicity endpoints such as carcinogenicity, immunotoxicity, mutagenicity, or cytotoxicity.

Discovery of N-(4-((6-(3,5- Dimethoxyphenyl)-9H-purine derivatives as irreversible covalent FGFR inhibitors.

Fibroblast growth factor receptor (FGFR) is an attractive target for cancer therapy, but existing FGFR inhibitors appear to hardly meet the demand for clinical application. Herein, a number of irreversible covalent FGFR inhibitors were designed and synthesized by selecting several five- and six-membered azaheterocycles as parent scaffold with different substituents to take over the hydrophobic region in the active pocket of FGFR proteins. Among the resulting target compounds, III-30 showed the most potent effect on enzyme activity inhibition and anti-proliferative activity against the tested cancer cell lines. Significantly, III-30 could inhibit the enzyme activity by achieving irreversible covalent binding with FGFR1 and FGFR4 proteins. It could also regulate FGFR-mediated signaling pathway and mitochondrial apoptotic pathway to promote cancer cell apoptosis and inhibit cancer cell invasion and metastasis. Moreover, III-30 had a good metabolic stability and showed relatively potent anti-tumor activity in the MDA-MB-231 xenograft tumor mice model.

Purines enrich root-associated Pseudomonas and improve wild soybean growth under salt stress.

The root-associated microbiota plays an important role in the response to environmental stress. However, the underlying mechanisms controlling the interaction between salt-stressed plants and microbiota are poorly understood. Here, by focusing on a salt-tolerant plant wild soybean (Glycine soja), we demonstrate that highly conserved microbes dominated by Pseudomonas are enriched in the root and rhizosphere microbiota of salt-stressed plant. Two corresponding Pseudomonas isolates are confirmed to enhance the salt tolerance of wild soybean. Shotgun metagenomic and metatranscriptomic sequencing reveal that motility-associated genes, mainly chemotaxis and flagellar assembly, are significantly enriched and expressed in salt-treated samples. We further find that roots of salt stressed plants secreted purines, especially xanthine, which induce motility of the Pseudomonas isolates. Moreover, exogenous application for xanthine to non-stressed plants results in Pseudomonas enrichment, reproducing the microbiota shift in salt-stressed root. Finally, Pseudomonas mutant analysis shows that the motility related gene cheW is required for chemotaxis toward xanthine and for enhancing plant salt tolerance. Our study proposes that wild soybean recruits beneficial Pseudomonas species by exudating key metabolites (i.e., purine) against salt stress.

Novel JAK Inhibitors to Reduce Graft-Versus-Host Disease after Allogeneic Hematopoietic Cell Transplantation in a Preclinical Mouse Model.

Allogeneic hematopoietic cell transplantation (allo-HCT) is a highly effective, well-established treatment for patients with various hematologic malignancies and non-malignant diseases. The therapeutic benefits of allo-HCT are mediated by alloreactive T cells in donor grafts. However, there is a significant risk of graft-versus-host disease (GvHD), in which the donor T cells recognize recipient cells as foreign and attack healthy organs in addition to malignancies. We previously demonstrated that targeting JAK1/JAK2, mediators of interferon-gamma receptor (IFNGR) and IL-6 receptor signaling, in donor T cells using baricitinib and ruxolitinib results in a significant reduction in GvHD after allo-HCT. Furthermore, we showed that balanced inhibition of JAK1/JAK2 while sparing JAK3 is important for the optimal prevention of GvHD. Thus, we have generated novel JAK1/JAK2 inhibitors, termed WU derivatives, by modifying baricitinib. Our results show that WU derivatives have the potential to mitigate GvHD by upregulating regulatory T cells and immune reconstitution while reducing the frequencies of antigen-presenting cells (APCs) and CD80 expression on these APCs in our preclinical mouse model of allo-HCT. In addition, WU derivatives effectively downregulated CXCR3 and T-bet in primary murine T cells. In summary, we have generated novel JAK inhibitors that could serve as alternatives to baricitinib or ruxolitinib.

Serum starvation is as efficient as roscovitine on the cycle synchronization in G0/G1 of red-rumped agouti fibroblasts.

Fibroblast cycle synchronization in G0/G1 is an essential step for nuclear reprogramming by cloning or induced cells to pluripotency. Considering the diversity among rodents and the ecological and scientific importance of these animals, we compared the contact inhibition, serum starvation, and 10 µM of roscovitine as methods of synchronization of red-rumped agouti fibroblasts. The effects of each protocol were evaluated on the percentage of cycle phase, morphology, viability, and apoptosis levels. The results showed that culturing the cells to serum starvation for 24 h (75.9%), 48 h (81.6%), 72 h (86.2%), 96 h (84.0%), and 120 h (83.7%) yielded a significantly higher percentage of cells arrested in the G0/G1 (P < 0.05) phase than cells not subjected to any cell cycle synchronization method (31.4%). Also, this effect was not different between the times of 48 and 120 h (P > 0.05). A similar response was observed for cells cultured with roscovitine for 12 h (86.9%), 24 h (74.8%), and 48 h (81.7%), with a higher percentage of synchronized cells in G0/G1 compared to cells not submitted to any synchronization treatment (52.2%). Nevertheless, this effect was best evidenced at 12 h (P < 0.05). Also, the contact inhibition for 24-120 h could not synchronize cells in G0/G1, with values ranging from 70.9 to 77.9% (P > 0.05). Moreover, no difference was observed for morphology, viability, and apoptosis levels in any synchronization method (P > 0.05). Therefore, serum starvation is as efficient as roscovitine on cycle synchronization in G0/G1 of red-rumped agouti fibroblasts.

Differential effects of benzo[a]pyrene exposure on glutathione and purine metabolism in keratinocytes: Dose-dependent and UV co-exposure effects.

Polycyclic aromatic hydrocarbons with the key substance benzo[a]pyrene (B[a]P) are widespread pollutants in the environment and at working places. Nonetheless, the exact underlying mechanisms of toxicological effects caused by B[a]P especially in absence and presence of UV irradiation remain uncertain. This study examines variations in exposure conditions: low B[a]P (4 nM), low B[a]P + UV and high B[a]P (4 µM), selected based on pertinent cytotoxicity assessments. Following cell viability evaluations post-treatment with varied B[a]P concentrations and UV irradiation, the identified concentrations underwent detailed metabolomic analysis via gas chromatography-mass spectrometry. Subsequently, resulting changes in metabolic profiles across these distinct exposure groups are comprehensively compared. Chemometric analyses showed modest regulation of metabolites after low B[a]P exposure compared to control conditions. High B[a]P and low B[a]P + UV exposure significantly increased regulation of metabolic pathways, indicating that additional UV irradiation plus low B[a]P is as demanding for the cells as higher B[a]P treatment alone. Further analysis revealed exposure-dependent regulation of glutathione-important for oxidative defence-and purine metabolism-important for DNA base synthesis. Only after low B[a]P, oxidative defence appeared to be able to compensate for B[a]P-induced perturbations of the oxidative homeostasis. In contrast, purine metabolism already responded towards adversity at low B[a]P. The metabolomic results give an insight into the mechanisms leading to the toxic response and confirm the strong effects of co-exposure on oxidative defence and DNA repair in the model studied.

Spermidine attenuates monocrotaline-induced pulmonary arterial hypertension in rats by inhibiting purine metabolism and polyamine synthesis-associated vascular remodeling.

Ensuring the homeostatic integrity of pulmonary artery endothelial cells (PAECs) is essential for combatting pulmonary arterial hypertension (PAH), as it equips the cells to withstand microenvironmental challenges. Spermidine (SPD), a potent facilitator of autophagy, has been identified as a significant contributor to PAECs function and survival. Despite SPD's observed benefits, a comprehensive understanding of its protective mechanisms has remained elusive. Through an integrated approach combining metabolomics and molecular biology, this study uncovers the molecular pathways employed by SPD in mitigating PAH induced by monocrotaline (MCT) in a Sprague-Dawley rat model. The study demonstrates that SPD administration (5 mg/kg/day) significantly corrects right ventricular impairment and pathological changes in pulmonary tissues following MCT exposure (60 mg/kg). Metabolomic profiling identified a purine metabolism disorder in MCT-treated rats, which SPD effectively normalized, conferring a protective effect against PAH progression. Subsequent in vitro analysis showed that SPD (0.8 mM) reduces oxidative stress and apoptosis in PAECs challenged with Dehydromonocrotaline (MCTP, 50 µM), likely by downregulating purine nucleoside phosphorylase (PNP) and modulating polyamine biosynthesis through alterations in S-adenosylmethionine decarboxylase (AMD1) expression and the subsequent production of decarboxylated S-adenosylmethionine (dcSAM). These findings advocate SPD's dual inhibitory effect on PNP and AMD1 as a novel strategy to conserve cellular ATP and alleviate oxidative injuries, thus providing a foundation for SPD's potential therapeutic application in PAH treatment.

Cananga oil inhibits Salmonella infection by mediating the homeostasis of purine metabolism and the TCA cycle.

ETHNOPHARMACOLOGY RELEVANCE: Cananga oil (CO) is derived from the flowers of the traditional medicinal plant, the ylang-ylang tree. As a traditional antidepressant, CO is commonly utilized in the treatment of various mental disorders including depression, anxiety, and autism. It is also recognized as an efficient antibacterial insecticide, and has been traditionally utilized to combat malaria and acute inflammatory responses resulting from bacterial infections both in vitro and in vivo. AIM OF THE STUDY: The objective of this study is to comprehensively investigate the anti-Salmonella activity and mechanism of CO both in vitro and in vivo, with the expectation of providing feasible strategies for exploring new antimicrobial strategies and developing novel drugs. METHODS: The in vitro antibacterial activity of CO was comprehensively analyzed by measuring MIC, MBC, growth curve, time-killing curve, surface motility, biofilm, and Live/dead bacterial staining. The analysis of the chemistry and active ingredients of CO was conducted using GC-MS. To examine the influence of CO on the membrane homeostasis of Salmonella, we conducted utilizing diverse techniques, including ANS, PI, NPN, ONPG, BCECF-AM, DiSC3(5), and scanning electron microscopy (SEM) analysis. In addition, the antibacterial mechanism of CO was analyzed and validated through metabolomics analysis. Finally, a mouse infection model of Salmonella typhimurium was established to evaluate the toxic side effects and therapeutic effects of CO. RESULTS: The antibacterial effect of CO is the result of the combined action of the main chemical components within its six (palmitic acid, α-linolenic acid, stearic acid, benzyl benzoate, benzyl acetate, and myristic acid). Furthermore, CO disrupts the balance of purine metabolism and the tricarboxylic acid cycle (TCA cycle) in Salmonella, interfering with redox processes. This leads to energy metabolic disorders and oxidative stress damage within the bacteria, resulting in bacterial shock, enhanced membrane damage, and ultimately bacterial death. It is worth emphasizing that CO exerts an effective protective influence on Salmonella infection in vivo within a non-toxic concentration range. CONCLUSION: The outcomes indicate that CO displays remarkable anti-Salmonella activity both in vitro and in vivo. It triggers bacterial death by disrupting the balance of purine metabolism and the TCA cycle, interfering with the redox process, making it a promising anti-Salmonella medication.

Duvelisib: A comprehensive profile.

Duvelisib (DUV) is chemically named as (S)-3-(1-((9H-Purin-6-yl)amino)ethyl)-8-chloro-2-phenylisoquinolin-1(2H)-one. It is a novel drug with a small molecular weight and characterized by dual phosphoinositide-3-kinase (PI3K)- and PI3K-inhibitory activity. The Food and Drug Administration (FDA) recently approved DUV for the management of small lymphocytic lymphoma (SLL) and relapsed or refractory chronic lymphocytic leukemia (CLL) in adult patients. DUV is marketed under the brand name of Copiktra® (Verastem, Inc., Needham, MA, USA). This chapter provides a critical extensive review of the literature, the description of DUV in terms of its names, formulae, elemental composition, appearance, and use in the treatment of CLL, SLL, and follicular lymphoma. The chapter also describes the methods for preparation of DUV, its physical-chemical properties, analytical methods for its determination, pharmacological properties, and dosing information.

Optical Control of Adenosine A2A Receptor Using Istradefylline Photosensitivity.

In recent years, there has been growing interest in the potential therapeutic use of inhibitors of adenosine A2A receptors (A2AR) for the treatment of neurodegenerative diseases and cancer. Nevertheless, the widespread expression of A2AR throughout the body emphasizes the importance of temporally and spatially selective ligands. Photopharmacology is an emerging strategy that utilizes photosensitive ligands to attain high spatiotemporal precision and regulate the function of biomolecules using light. In this study, we combined photochemistry and cellular and in vivo photopharmacology to investigate the light sensitivity of the FDA-approved antagonist istradefylline and its potential use as an A2AR photopharmacological tool. Our findings reveal that istradefylline exhibits rapid trans-to-cis isomerization under near-UV light, and prolonged exposure results in the formation of photocycloaddition products. We demonstrate that exposure to UV light triggers a time-dependent decrease in the antagonistic activity of istradefylline in A2AR-expressing cells and enables real-time optical control of A2AR signaling in living cells and zebrafish. Together, these data demonstrate that istradefylline is a photoinactivatable A2AR antagonist and that this property can be utilized to perform photopharmacological experiments in living cells and animals.

Local and Sustained Baricitinib Delivery to the Skin through Injectable Hydrogels Containing Reversible Thioimidate Adducts.

Janus kinase (JAK) inhibitors are approved for many dermatologic disorders, but their use is limited by systemic toxicities including serious cardiovascular events and malignancy. To overcome these limitations, injectable hydrogels are engineered for the local and sustained delivery of baricitinib, a representative JAK inhibitor. Hydrogels are formed via disulfide crosslinking of thiolated hyaluronic acid macromers. Dynamic thioimidate bonds are introduced between the thiolated hyaluronic acid and nitrile-containing baricitinib for drug tethering, which is confirmed with 1H and 13C nuclear magnetic resonance (NMR). Release of baricitinib is tunable over six weeks in vitro and active in inhibiting JAK signaling in a cell line containing a luciferase reporter reflecting interferon signaling. For in vivo activity, baricitinib hydrogels or controls are injected intradermally into an imiquimod-induced mouse model of psoriasis. Imiquimod increases epidermal thickness in mice, which is unaffected when treated with baricitinib or hydrogel alone. Treatment with baricitinib hydrogels suppresses the increased epidermal thickness in mice treated with imiquimod, suggesting that the sustained and local release of baricitinib is important for a therapeutic outcome. This study is the first to utilize a thioimidate chemistry to deliver JAK inhibitors to the skin through injectable hydrogels, which has translational potential for treating inflammatory disorders.

Synthesis of 6,8-diaminopurines via acid-induced cascade cyclization of 5-aminoimidazole precursors and preliminary anticancer evaluation.

Inspired by the pharmacological interest generated by 6-substituted purine roscovitine for cancer treatment, 5-aminoimidazole-4-carboxamidine precursors containing a cyanamide unit were prepared by condensation of 5-amino-N-cyanoimidazole-4-carbimidoyl cyanides with a wide range of primary amines. When these amidine precursors were combined with acids, a fast cascade cyclization occurred at room temperature, affording new 6,8-diaminopurines with the N-3 and N-6 substituents changed relatively to the original positions they occupied in the amidine and imidazole moieties of precursors. The efficacy and wide scope of this method was well demonstrated by an easy and affordable synthesis of 22 6,8-diaminopurines decorated with a wide diversity of substituents at the N-3 and N-6 positions of the purine ring. Preliminary in silico and in vitro assessments of these 22 compounds were carried out and the results showed that 13 of these tested compounds not only exhibited IC50 values between 1.4 and 7.5 µM against the colorectal cancer cell line HCT116 but also showed better binding energies than known inhibitors in docking studies with different cancer-related target proteins. In addition, good harmonization observed between in silico and in vitro results strengthens and validates this preliminary evaluation, suggesting that these novel entities are good candidates for further studies as new anticancer agents.

L-arginine metabolism inhibits arthritis and inflammatory bone loss.

OBJECTIVES: To investigate the effect of the L-arginine metabolism on arthritis and inflammation-mediated bone loss. METHODS: L-arginine was applied to three arthritis models (collagen-induced arthritis, serum-induced arthritis and human TNF transgenic mice). Inflammation was assessed clinically and histologically, while bone changes were quantified by µCT and histomorphometry. In vitro, effects of L-arginine on osteoclast differentiation were analysed by RNA-seq and mass spectrometry (MS). Seahorse, Single Cell ENergetIc metabolism by profilIng Translation inHibition and transmission electron microscopy were used for detecting metabolic changes in osteoclasts. Moreover, arginine-associated metabolites were measured in the serum of rheumatoid arthritis (RA) and pre-RA patients. RESULTS: L-arginine inhibited arthritis and bone loss in all three models and directly blocked TNFα-induced murine and human osteoclastogenesis. RNA-seq and MS analyses indicated that L-arginine switched glycolysis to oxidative phosphorylation in inflammatory osteoclasts leading to increased ATP production, purine metabolism and elevated inosine and hypoxanthine levels. Adenosine deaminase inhibitors blocking inosine and hypoxanthine production abolished the inhibition of L-arginine on osteoclastogenesis in vitro and in vivo. Altered arginine levels were also found in RA and pre-RA patients. CONCLUSION: Our study demonstrated that L-arginine ameliorates arthritis and bone erosion through metabolic reprogramming and perturbation of purine metabolism in osteoclasts.

Anticancer effect of covalent purine-containing EGFR TKI, ZZC4 and its mechanism of action through network pharmacology.

AIMS: Epidermal growth factor receptor (EGFR) has been documented in many malignancies as participating in the progression of cancer cells. Here, we present a novel EGFR tyrosine kinase inhibitor, ZZC4, and examine its effect on cancer cell proliferation, migration, and tumor-bearing xenograft models. MAIN METHODS: The antiproliferative effect of ZZC4 was assessed in vitro by MTT assay, colony formation, and wound healing assay and in vivo with tumor-bearing xenograft nude mice. Further, Western blotting analysis and computational network pharmacology were used to explore and understand the mechanism of ZZC4. KEY FINDINGS: The results showed that ZZC4 potently inhibited the proliferation of lung, breast, and melanoma cells, and was more sensitive to lung cancer cells HCC827, H1975, and breast cancer cell T47D. In vitro findings were corroborated in vivo as results showed the suppressive effect of ZZC4 on HCC827 and H1975 tumor growth. Western blotting analysis confirmed that ZZC4 is an effective inhibitor of the EGFR pathways as it down-regulated p-EGFR, p-Akt, and p-MAPK. Computational molecular docking confirmed the strong binding affinity between ZZC4 and EGFR. Moreover, network pharmacology suggested that ZZC4 might play a suppressive role in the progression of malignancies with EGFR/PI-3K/Akt axis dysregulation or in cancer-related drug resistance. SIGNIFICANCE: Our study showed that ZZC4 is an anticancer drug candidate.

HDAC specificity and kinase off-targeting by purine-benzohydroxamate anti-hematological tumor agents.

A series of hybrid inhibitors, combining pharmacophores of known kinase inhibitors bearing anilino-purines (ruxolitinib, ibrutinib) and benzohydroxamate HDAC inhibitors (nexturastat A), were generated in the present study. The compounds have been synthesized and tested against solid and hematological tumor cell lines. Compounds 4d-f were the most promising in cytotoxicity assays (IC50 ≤ 50 nM) vs. hematological cells and displayed moderate activity in solid tumor models (EC50 = 9.3-21.7 µM). Compound 4d potently inhibited multiple kinase targets of interest for anticancer effects, including JAK2, JAK3, HDAC1, and HDAC6. Molecular dynamics simulations showed that 4d has stable interactions with HDAC and members of the JAK family, with differences in the hinge binding energy conferring selectivity for JAK3 and JAK2 over JAK1. The kinase inhibition profile of compounds 4d-f allows selective cytotoxicity, with minimal effects on non-tumorigenic cells. Moreover, these compounds have favorable pharmacokinetic profiles, with high stability in human liver microsomes (e.g., see t1/2: >120 min for 4f), low intrinsic clearance, and lack of significant inhibition of four major CYP450 isoforms.

Dual-targeting compounds possessing enhanced anticancer activity via microtubule disruption and histone deacetylase inhibition.

Dual-targeting anticancer agents 4-29 are designed by combining the structural features of purine-type microtubule-disrupting compounds and HDAC inhibitors. A library of the conjugate compounds connected by appropriate linkers was synthesized and found to possess HDACs inhibitory activity and render microtubule fragmentation by activating katanin, a microtubule-severing protein. Among various zinc-binding groups, hydroxamic acid shows the highest inhibitory activity of Class I HDACs, which was also reconfirmed by three-dimensional quantitative structure-activity relationship (3D-QSAR) pharmacophore prediction. The purine-hydroxamate conjugates exhibit enhanced cytotoxicity against MDA-MB231 breast cancer cells, H1975 lung cancer cells, and various clinical isolated non-small-cell lung cancer cells with different epidermal growth factor receptor (EGFR) status. Pyridyl substituents could be used to replace the C2 and N9 phenyl moieties in the purine-type scaffold, which can help to improve the solubility under physiological conditions, thus increasing cytotoxicity. In mice treated with the purine-hydroxamate conjugates, the tumor growth rate was significantly reduced without causing toxic effects. Our study demonstrates the potential of the dual-targeting purine-hydroxamate compounds for cancer monotherapy.

Electrochemistry detection of estrogenic effect: Regulation of de novo purine synthesis and catabolism by gibberellin and fulvestrant.

The estrogenic effect of plant growth regulators has been received little attention, which leads to the lack of relevant toxicity data. In this study, the estrogenic effect induced by gibberellin with ERα-dependent manner was found by E-screen and western blot methods, and the electrochemical signals of MCF-7 cells regulated by gibberellin and fulvestrant were investigated. The results showed that the electrochemical signals of MCF-7 cells were increased by gibberellin, while reduced by fulvestrant significantly, and displayed an extremely sensitive response to the effects of estrogenic effect induced by ERα agonist and antagonist. Western blot results showed that the expressions of phosphoribosyl pyrophosphate amidotransferase and hypoxanthine nucleotide dehydrogenase in de novo purine synthesis and adenine deaminase in catabolism were more effective regulated by gibberellin and fulvestrant, resulting in significant changes of the levels of guanine, hypoxanthine and xanthine in cells, and then electrochemical signals. The results provide a theoretical basis for the establishment of new electrochemical detection method of the estrogenic effect of plant regulators.

Exploring the Multifaceted Potential of Sildenafil in Medicine.

Phosphodiesterase type 5 (PDE5) is pivotal in cellular signalling, regulating cyclic guanosine monophosphate (cGMP) levels crucial for smooth muscle relaxation and vasodilation. By targeting cGMP for degradation, PDE5 inhibits sustained vasodilation. PDE5 operates in diverse anatomical regions, with its upregulation linked to various pathologies, including cancer and neurodegenerative diseases. Sildenafil, a selective PDE5 inhibitor, is prescribed for erectile dysfunction and pulmonary arterial hypertension. However, considering the extensive roles of PDE5, sildenafil might be useful in other pathologies. This review aims to comprehensively explore sildenafil's therapeutic potential across medicine, addressing a gap in the current literature. Recognising sildenafil's broader potential may unveil new treatment avenues, optimising existing approaches and broadening its clinical application.

Activation of Purine Biosynthesis Suppresses the Sensitivity of E. coli gmhA Mutant to Antibiotics.

Inactivation of enzymes responsible for biosynthesis of the cell wall component of ADP-glycero-manno-heptose causes the development of oxidative stress and sensitivity of bacteria to antibiotics of a hydrophobic nature. The metabolic precursor of ADP-heptose is sedoheptulose-7-phosphate (S7P), an intermediate of the non-oxidative branch of the pentose phosphate pathway (PPP), in which ribose-5-phosphate and NADPH are generated. Inactivation of the first stage of ADP-heptose synthesis (ΔgmhA) prevents the outflow of S7P from the PPP, and this mutant is characterized by a reduced biosynthesis of NADPH and of the Glu-Cys-Gly tripeptide, glutathione, molecules known to be involved in the resistance to oxidative stress. We found that the derepression of purine biosynthesis (∆purR) normalizes the metabolic equilibrium in PPP in ΔgmhA mutants, suppressing the negative effects of gmhA mutation likely via the over-expression of the glycine-serine pathway that is under the negative control of PurR and might be responsible for the enhanced synthesis of NADPH and glutathione. Consistently, the activity of the soxRS system, as well as the level of glutathionylation and oxidation of proteins, indicative of oxidative stress, were reduced in the double ΔgmhAΔpurR mutant compared to the ΔgmhA mutant.

Xie Zhuo Tiao Zhi formula modulates intestinal microbiota and liver purine metabolism to suppress hepatic steatosis and pyroptosis in NAFLD therapy.

BACKGROUND: Current evidence indicates a rising global prevalence of Non-Alcoholic Fatty Liver Disease (NAFLD), which is closely associated to conditions such as obesity, dyslipidemia, insulin resistance, and metabolic syndrome. The relationship between the gut microbiome and metabolites in NAFLD is gaining attention understanding the pathogenesis and progression of dysregulated lipid metabolism and inflammation. The Xie Zhuo Tiao Zhi (XZTZ) decoction has been employed in clinical practice for alleviating hyperlipidemia and symptoms related to metabolic disorders. However, the pharmacological mechanisms underlying the effects of XZTZ remain to be elucidated. PURPOSE: The objective of this study was to examine the pharmacological mechanisms underlying the hypolipidemic and anti-inflammatory effects of XZTZ decoction in a mouse model of NAFLD, as well as the effects of supplementing exogenous metabolites on PO induced cell damage and lipid accumulation in cultured hepatocytes. METHODS: A high-fat diet (HFD) mouse model was established to examine the effects of XZTZ through oral gavage. The general condition of mice and the protective effect of XZTZ on liver injury were evaluated using histological and biochemical methods. Hematoxylin and eosin staining (H&E) staining and oil red O staining were performed to assess inflammatory and lipid accumulation detection, and cytokine levels were quantitatively analyzed. Additionally, the study included full-length 16S rRNA sequencing, liver transcriptome analysis, and non-targeted metabolomics analysis to investigate the relationship among intestinal microbiome, liver metabolic function, and XZTZ decoction. RESULTS: XZTZ had a significant impact on the microbial community structure in NAFLD mice. Notably, the abundance of Ileibacterium valens, which was significantly enriched by XZTZ, exhibited a negative correlation with liver injury biomarkers such as, alanine transaminase (ALT) and aspartate transaminase (AST) activity. Moreover, treatment with XZTZ led to a significant enrichment of the purine metabolism pathway in liver tissue metabolites, with inosine, a purine metabolite, showing a significant positive correlation with the abundance of I. valens. XZTZ and inosine also significantly enhanced fatty acid ß-oxidation, which led to a reduction in the expression of pro-inflammatory cytokines and the inhibition of liver pyroptosis. These effects contributed to the mitigation of liver injury and hepatocyte damage, both in vivo and vitro. Furthermore, the utilization of HPLC fingerprints and UPLC-Q-TOF-MS elucidated the principal constituents within the XZTZ decoction, including naringin, neohesperidin, atractylenolide III, 23-o-Acetylalisol B, pachymic acid, and ursolic acid which are likely responsible for its therapeutic efficacy. Further investigations are imperative to fully uncover and validate the pharmacodynamic mechanisms underlying these observations. CONCLUSION: The administration of XZTZ decoction demonstrates a protective effect on the livers of NAFLD mice by inhibiting lipid accumulation and reducing hepatocyte inflammatory damage. This protective effect is mediated by the upregulation of I.valens abundance in the intestine, highlighting the importance of the gut-liver axis. Furthermore, the presesnce of inosine, adenosine, and their derivatives are important in promoting the protective effects of XZTZ. Furthermore, the in vitro approaching, we provide hitherto undocumented evidence indicating that the inosine significantly improves lipid accumulation, inflammatory damage, and pyroptosis in AML12 cells incubated with free fatty acids.