Metabolomic and Proteomic Analysis of ApoE4-Carrying H4 Neuroglioma Cells in Alzheimer's Disease Using OrbiSIMS and LC-MS/MS.

Growing clinical evidence reveals that systematic molecular alterations in the brain occur 20 years before the onset of AD pathological features. Apolipoprotein E4 (ApoE4) is one of the most significant genetic risk factors for Alzheimer's disease (AD), which is not only associated with the AD pathological features such as amyloid-ß deposition, phosphorylation of tau proteins, and neuroinflammation but is also involved in metabolism, neuron growth, and synaptic plasticity. Multiomics, such as metabolomics and proteomics, are applied widely in identifying key disease-related molecular alterations and disease-progression-related changes. Despite recent advances in the development of analytical technologies, screening the entire profile of metabolites remains challenging due to the numerous classes of compounds with diverse chemical properties that require different extraction processes for mass spectrometry. In this study, we utilized Orbitrap Secondary Ion Mass Spectrometry (OrbiSIMS) as a chemical filtering screening tool to examine molecular alterations in ApoE4-carried neuroglioma cells compared to wild-type H4 cells. The findings were compared using liquid chromatography (LC)-MS/MS targeted metabolomics analysis for the confirmation of specific metabolite classes. Detected alterations in peptide fragments by OrbiSIMS provided preliminary indications of protein changes. These were extensively analyzed through proteomics to explore ApoE4's impact on proteins. Our metabolomics approach, combining OrbiSIMS and LC-MS/MS, revealed disruptions in lipid metabolism, including glycerophospholipids and sphingolipids, as well as amino acid metabolism, encompassing alanine, aspartate, and glutamate metabolism; aminoacyl-tRNA biosynthesis; glutamine metabolism; and taurine and hypotaurine metabolism. Further LC-MS/MS proteomics studies confirmed the dysfunction in amino acid and tRNA aminoacylation metabolic processes, and highlighted RNA splicing alterations influenced by ApoE4.

Design, Synthesis, and Biological Evaluation of Novel Tetrahydroacridin Hybrids with Sulfur-Inserted Linkers as Potential Multitarget Agents for Alzheimer's Disease.

Alzheimer's disease (AD) is a complex neurodegenerative disease that can lead to the loss of cognitive function. The progression of AD is regulated by multiple signaling pathways and their associated targets. Therefore, multitarget strategies theoretically have greater potential for treating AD. In this work, a series of new hybrids were designed and synthesized by the hybridization of tacrine (4, AChE: IC50 = 0.223 µM) with pyrimidone compound 5 (GSK-3ß: IC50 = 3 µM) using the cysteamine or cystamine group as the connector. The biological evaluation results demonstrated that most of the compounds exhibited moderate to good inhibitory activities against acetylcholinesterase (AChE) and glycogen synthase kinase 3ß (GSK-3ß). The optimal compound 18a possessed potent dual AChE/GSK-3ß inhibition (AChE: IC50 = 0.047 ± 0.002 µM, GSK-3ß: IC50 = 0.930 ± 0.080 µM). Further molecular docking and enzymatic kinetic studies revealed that this compound could occupy both the catalytic anionic site and the peripheral anionic site of AChE. The results also showed a lack of toxicity to SH-SY5Y neuroblastoma cells at concentrations of up to 25 µM. Collectively, this work explored the structure-activity relationships of novel tetrahydroacridin hybrids with sulfur-inserted linkers, providing a reference for the further research and development of new multitarget anti-AD drugs.

Design, synthesis and biological evaluation of novel 9-N-substituted-13-alkylberberine derivatives from Chinese medicine as anti-hepatocellular carcinoma agents.

A series of novel 9-N-substituted-13-alkylberberine derivatives from Chinese medicine were designed and synthesized with improved anti-hepatocellular carcinoma (HCC) activities. The optimal compound 4d showed strong activities against HepG2, Sk-Hep-1, Huh-7 and Hep3B cells with IC50 values of 0.58-1.15 µM, which were superior to positive reference cisplatin. Interestingly, 4d exhibited over 40-fold more potent activity against cisplatin-resistant HepG2/DPP cells while showing lower cytotoxicity in normal LX-2 cells. The mechanism studies revealed 4d greatly stabilized G-quadruplex DNA leading to intracellular c-MYC expression downregulation, blocked G2/M-phase cell cycle by affecting related p-cdc25c, cdc2 and cyclin B1 expressions, and induced apoptosis by a ROS-promoted PI3K/Akt-mitochondrial pathway. Furthermore, 4d possessed good pharmacokinetic properties and significantly inhibited the tumor growth in the H22 liver cancer xenograft mouse model without obvious toxicity. Altogether, the remarkably biological profiles of 4d both in vitro and in vivo would make it a promising candidate for HCC therapy.

Design, synthesis, and biological evaluation of novel protopanoxadiol derivatives based PROTACs technology for the treatment of lung cancer.

Protopanoxadiol is a key active ingredient derived from Panax ginseng that is well-known to exhibit anti-tumor activity. Previous research focused on the natural protopanaxadiol derivative AD-1 has demonstrated that it possesses broad spectrum anti-tumor activities in vitro and in vivo. However, its limited activity, selectivity, and cell permeability have impeded its therapeutic application. Herein, a series of novel AD-1 derivatives were designed and synthesized based on proteolysis-targeting chimera (PROTAC) technology by linking AD-1 at the C-3 and C-12 positions with pomalidomide through linkers of alkyl chain of differing lengths to achieve the goal of improving the efficacy of the parent compound. Among these synthesized PROTACs, the representative compound A05 exhibited the most potent anti-proliferative activity against A549 cells. Furthermore, mechanistic studies revealed that compound A05 was able to suppress MDM2 expression, disrupt interactions between p53 and MDM2 and readily induce apoptotic death via the mitochondrial apoptosis pathway. Moreover, the in vivo assays revealed that compound A05 exhibited both anti-proliferative and anti-metastatic activities in the zebrafish tumor xenograft model with A549 cells. Together, our findings suggest that AD-1 based PROTACs associated with the degradation of MDM2 may have promising effects for the treatment of lung cancer and this work provide a foundation for future efforts to develop novel anti-tumor agents from natural products.

Asiaticoside Mitigates Alzheimer's Disease Pathology by Attenuating Inflammation and Enhancing Synaptic Function.

Alzheimer's disease (AD) is a prevalent neurodegenerative disorder, hallmarked by the accumulation of amyloid-ß (Aß) plaques and neurofibrillary tangles. Due to the uncertainty of the pathogenesis of AD, strategies aimed at suppressing neuroinflammation and fostering synaptic repair are eagerly sought. Asiaticoside (AS), a natural triterpenoid derivative derived from Centella asiatica, is known for its anti-inflammatory, antioxidant, and wound-healing properties; however, its neuroprotective function in AD remains unclear. Our current study reveals that AS, when administered (40 mg/kg) in vivo, can mitigate cognitive dysfunction and attenuate neuroinflammation by inhibiting the activation of microglia and proinflammatory factors in Aß1-42-induced AD mice. Further mechanistic investigation suggests that AS may ameliorate cognitive impairment by inhibiting the activation of the p38 MAPK pathway and promoting synaptic repair. Our findings propose that AS could be a promising candidate for AD treatment, offering neuroinflammation inhibition and enhancement of synaptic function.

Controlled release of MT-1207 using a novel gastroretentive bilayer system comprised of hydrophilic and hydrophobic polymers.

In the present study, novel gastroretentive bilayer tablets were developed that are promising for the once-a-day oral delivery of the drug candidate MT-1207. The gastroretentive layer consisted of a combination of hydrophilic and hydrophobic polymers, namely polyethylene oxide and Kollidon® SR. A factorial experiment was conducted, and the results revealed a non-effervescent gastroretentive layer that, unlike most gastroretentive layers reported in the literature, was easy to prepare, and provided immediate tablet buoyancy (mean floating lag time of 1.5 s) that lasted over 24 h in fasted state simulated gastric fluid (FaSSGF) pH 1.6, irrespective of the drug layer, thereby allowing a 24-hour sustained release of MT-1207 from the drug layer of the tablets. Furthermore, during in vitro buoyancy testing of the optimised bilayer tablets in media of different pH values (1.0, 3.0, 6.0), the significant difference (one-way ANOVA, p < 0.001) between the respective total floating times indicated that stomach pH effects on tablet buoyancy are important to be considered during the development of non-effervescent gastroretentive formulations and the choice of dosing regimen. To the best of our knowledge, this has not been reported before, and it should probably be factored in when designing dosing regimens. Finally, a pharmacokinetic study in Beagle dogs indicated a successful in vivo 24-hour sustained release of MT-1207 from the optimised gastroretentive bilayer tablet formulations with the drug plasma concentration remaining above the estimated minimum effective concentration of 1 ng/mL at the 24-hour timepoint and also demonstrated the gastroretentive capabilities of the hydrophilic and hydrophobic polymer combination. The optimised formulations will be forwarded to clinical development.

Discovery of Norbornene as a Novel Hydrophobic Tag Applied in Protein Degradation.

Hydrophobic tagging (HyT) is a potential therapeutic strategy for targeted protein degradation (TPD). Norbornene was discovered as an unprecedented hydrophobic tag in this study and was used to degrade the anaplastic lymphoma kinase (ALK) fusion protein by linking it to ALK inhibitors. The most promising degrader, Hyt-9, potently reduced ALK levels through Hsp70 and the ubiquitin-proteasome system (UPS) in vitro without compensatory upregulation of ALK. Furthermore, Hyt-9 exhibited a significant tumor-inhibiting effect in vivo with moderate oral bioavailability. More importantly, norbornene can also be used to degrade the intractable enhancer of zeste homolog 2 (EZH2) when tagged with the EZH2 inhibitor tazemetostat. Thus, the discovery of novel hydrophobic norbornene tags shows promise for the future development of TPD technology.

Development of novel 9-O-substituted-13-octylberberine derivatives as potential anti-hepatocellular carcinoma agents.

A series of novel 9-O-substituted-13-octylberberine derivatives were designed, synthesised and evaluated for their anti-hepatocellular carcinoma (HCC) activities. Compound 6k showed the strongest activity against three human hepatoma cells including HepG2, Sk-Hep-1 and Huh-7 cells with IC50 values from 0.62 to 1.69 µM, which were much superior to berberine (IC50 >50 µM). More importantly, 6k exhibited lower cytotoxicity against normal hepatocytes L-02 with good lipid-water partition properties. The mechanism studies revealed that 6k caused G2/M phase arrest of the cell cycle, stabilised G-quadruplex DNA, and induced apoptosis via a mitochondrial apoptotic pathway. Finally, the in vivo anti-HCC activity of 6k was validated in the H22 liver cancer xenograft mouse model. Collectively, the current study would provide a new insight into the discovery of novel, safe and effective anti-HCC agents.

Development of p-Tau Differentiated Cell Model of Alzheimer's Disease to Screen Novel Acetylcholinesterase Inhibitors.

Alzheimer's disease (AD) is characterized by an initial accumulation of amyloid plaques and neurofibrillary tangles, along with the depletion of cholinergic markers. The currently available therapies for AD do not present any disease-modifying effects, with the available in vitro platforms to study either AD drug candidates or basic biology not fully recapitulating the main features of the disease or being extremely costly, such as iPSC-derived neurons. In the present work, we developed and validated a novel cell-based AD model featuring Tau hyperphosphorylation and degenerative neuronal morphology. Using the model, we evaluated the efficacy of three different groups of newly synthesized acetylcholinesterase (AChE) inhibitors, along with a new dual acetylcholinesterase/glycogen synthase kinase 3 inhibitor, as potential AD treatment on differentiated SH-SY5Y cells treated with glyceraldehyde to induce Tau hyperphosphorylation, and subsequently neurite degeneration and cell death. Testing of such compounds on the newly developed model revealed an overall improvement of the induced defects by inhibition of AChE alone, showing a reduction of S396 aberrant phosphorylation along with a moderate amelioration of the neuron-like morphology. Finally, simultaneous AChE/GSK3 inhibition further enhanced the limited effects observed by AChE inhibition alone, resulting in an improvement of all the key parameters, such as cell viability, morphology, and Tau abnormal phosphorylation.

Novel and Potent Acetylcholinesterase Inhibitors for the Treatment of Alzheimer's Disease from Natural (±)-7,8-Dihydroxy-3-methyl-isochroman-4-one.

Alzheimer's disease (AD) is a neurodegenerative disease that causes memory and cognitive decline as well as behavioral problems. It is a progressive and well recognized complex disease; therefore, it is very urgent to develop novel and effective anti-AD drugs. In this study, a series of novel isochroman-4-one derivatives from natural (±)-7,8-dihydroxy-3-methyl-isochroman-4-one [(±)-XJP] were designed and synthesized, and their anti-AD potential was evaluated. Among them, compound 10a [(Z)-3-acetyl-1-benzyl-4-((6,7-dimethoxy-4-oxoisochroman-3-ylidene)methyl)pyridin-1-ium bromide] possessed potent anti-acetylcholinesterase (AChE) activity as well as modest antioxidant activity. Further molecular modeling and kinetic investigations revealed that compound 10a was a dual-binding inhibitor that binds to both catalytic anionic site (CAS) and peripheral anionic site (PAS) of the enzyme AChE. In addition, compound 10a exhibited low cytotoxicity and moderate anti-Aß aggregation efficacy. Moreover, the in silico screening suggested that these compounds could pass across the blood-brain barrier with high penetration. These findings show that compound 10a was a promising lead from a natural product with potent AChE inhibitory activity and deserves to be further developed for the prevention and treatment of AD.

Magnesium Isoglycyrrhizinate Reduces Hepatic Lipotoxicity through Regulating Metabolic Abnormalities.

The excessive accumulation of lipids in hepatocytes induces a type of cytotoxicity called hepatic lipotoxicity, which is a fundamental contributor to liver metabolic diseases (such as NAFLD). Magnesium isoglycyrrhizinate (MGIG), a magnesium salt of the stereoisomer of natural glycyrrhizic acid, is widely used as a safe and effective liver protectant. However, the mechanism by which MGIG protects against NAFLD remains unknown. Based on the significant correlation between NAFLD and the reprogramming of liver metabolism, we aimed to explore the beneficial effects of MGIG from a metabolic viewpoint in this paper. We treated HepaRG cells with palmitic acid (PA, a saturated fatty acid of C16:0) to induce lipotoxicity and then evaluated the antagonistic effect of MGIG on lipotoxicity by investigating the cell survival rate, DNA proliferation rate, organelle damage, and endoplasmic reticulum stress (ERS). Metabolomics, lipidomics, and isotope tracing were used to investigate changes in the metabolite profile, lipid profile, and lipid flux in HepaRG cells under different intervention conditions. The results showed that MGIG can indeed protect hepatocytes against PA-induced cytotoxicity and ERS. In response to the metabolic abnormality of lipotoxicity, MGIG curtailed the metabolic activation of lipids induced by PA. The content of total lipids and saturated lipids containing C16:0 chains increased significantly after PA stimulation and then decreased significantly or even returned to normal levels after MGIG intervention. Lipidomic data show that glycerides and glycerophospholipids were the two most affected lipids. For excessive lipid accumulation in hepatocytes, MGIG can downregulate the expression of the metabolic enzymes (GPATs and DAGTs) involved in triglyceride biosynthesis. In conclusion, MGIG has a positive regulatory effect on the metabolic disorders that occur in hepatocytes under lipotoxicity, and the main mechanisms of this effect are in lipid metabolism, including reducing the total lipid content, reducing lipid saturation, inhibiting glyceride and glycerophospholipid metabolism, and downregulating the expression of metabolic enzymes in lipid synthesis.

In vitro and in vivo evaluation of a sustained-release once-a-day formulation of the novel antihypertensive drug MT-1207.

Hypertension is one of the most common chronic cardiovascular disorders. Sustained-release formulations are developed to maintain drug therapeutic levels throughout the treatment of hypertension, to promote patient compliance and improve patient outcomes. We have developed and tested in in vivo trials a once-a-day tablet formulation for the novel antihypertensive drug MT-1207. The tablets based upon a hydrophilic polymer matrix underwent post-compression parameter and physicochemical characterisations, along with in vitro drug release testing. The most promising formulation containing 31% w/w HPMC K15M gave a 24-hour release of MT-1207 with an almost constant release rate up to 20 hours. Follow in in vivo studies were carried out in Beagle dogs for the optimised sustained-release tablets in comparison to immediate-release tablets. The results showed that a sustained release of MT-1207 from the new formulation was achieved with a drug t1/2 2-2.5 times longer than the immediate-release tablets. Moreover, the AUC0-24h values of both sustained- and immediate-release tablets were identical at the same dose of 30 mg, indicating that the same amount of drug was absorbed in each case. For treatments based upon MT-1207, this development is significant for future commercial exploitation via scale-up and further trials, and for improved patient outcomes.

Urinary cadmium concentrations and risk of primary ovarian insufficiency in women: a case-control study.

Cadmium, a toxic heavy metal that occurs in the environment in large quantities through human activities, has been shown to have adverse effects on female reproductive health. However, the association between cadmium exposure and primary ovarian insufficiency (POI), one of the most prevalent ovarian diseases in women, has not been examined yet. This case-control study involving 169 POI cases and 209 healthy controls was conducted in Zhejiang Province, China. The urinary concentrations of cadmium were determined by inductively coupled plasma mass spectrometry (ICP-MS). In addition, serum levels of reproductive hormones, including follicle-stimulating hormone (FSH), luteinizing hormone (LH), anti-Mullerian hormone (AMH) and estradiol, were measured. The median concentration of urinary cadmium in POI cases (0.43 µg/L, 0.58 µg/g for creatinine adjustment) was significantly higher than that of controls (0.29 µg/L, 0.43 µg/g for creatinine adjustment). The results of binary logistic regression models showed that the concentrations of urinary cadmium were positively significantly correlated with the odds ratio (ORs) of POI before the adjustment of confounders. After the adjustment, a significantly positive association was still present between the increased concentrations of cadmium and the ORs of POI (2.50, 95% CIs: 1.34-4.65 for the third tertile, p for trend = 0.001). The serum levels of FSH and LH were positively associated with urinary cadmium, while AMH and estradiol levels were inversely correlated. To the best of our knowledge, this is the first reported positive association of cadmium exposure with the risk of POI in women.

Design, synthesis and biological evaluation of isochroman-4-one hybrids bearing piperazine moiety as antihypertensive agent candidates.

7,8-Dihydroxy-3-methyl-isochromanone-4 (XJP), is a polyphenolic natural product with moderate antihypertensive activity. To obtain new agents with stronger potency and safer profile, we employed XJP and naftopidil as the lead compounds to design and synthesize a novel class of hybrids as antihypertensive agent candidates. In the present study, a series of hybrids (6a-r) of XJP bearing arylpiperazine moiety, which is identified as the pharmacophore of naftopidil, were designed and synthesized as novel α1-adrenergic receptor antagonists. The biological evaluation showed that target compounds 6c, 6e, 6f, 6g, 6h, 6m and 6q possessed potent in vitro vasodilation potency and α1-adrenergic receptor antagonistic activity. Furthermore, the most potent compound 6e significantly reduced the systolic and diastolic blood pressure in spontaneously hypertensive rats (SHRs), which was comparable to that of naftopidil, and it had no observable effects on the basal heart rate, suggesting that 6e deserves to be further investigated as a potential clinical candidate for the treatment of hypertension.

Synthesis, molecular properties prediction and biological evaluation of indole-vinyl sulfone derivatives as novel tubulin polymerization inhibitors targeting the colchicine binding site.

Twenty-two novel indole-vinyl sulfone derivatives were designed, synthesized and evaluated as tubulin polymerization inhibitors. The physicochemical and drug-likeness properties of all target compounds were predicted by Osiris calculations. All compounds were evaluated for their antiproliferative activities, among them, compound 7f exhibited the most potent activity against a panel of cancer cell lines, which was 2-7 folds more potent than our previously reported compound 4. Especially, 7f displayed about 8-fold improvement of selective index as compared with compound 4, indicating that 7f might have lower toxicity. Besides, 7f inhibited the microtubule polymerization by binding to the colchicine site of tubulin. Further investigations showed that compound 7f effectively disrupted microtubule network, caused cell cycle arrest at G2/M phase and induced cell apoptosis in K562 cells. Moreover, 7f reduced the cell migration and disrupted capillary-like tube formation in HUVEC cells. Importantly, the in vivo anti-tumor activity of 7f was validated in H22 liver cancer xenograft mouse model without apparent toxicity, suggesting that 7f is a promising anti-tubulin agent for cancer therapy.

Discovery of novel quinazolines as potential anti-tubulin agents occupying three zones of colchicine domain.

A series of novel quinazolines as tubulin inhibitors occupying three zones of colchicine domain have been designed and synthesized inspired by the recently disclosed crystal structure of verubulin analogue 6 with tubulin. Among the newly synthesized compounds, 19c showed noteworthy potency against K562, HepG2, KB, HCT-8 and MDB-MB-231 cancer cells. In vitro microtubule polymerization assays identified 19c as a potent tubulin assembly inhibitor, the binding mode of which with tubulin was confirmed by molecular modeling studies to occupy three zones of tubulin domain. Furthermore, 19c disrupted the intracellular microtubule network, caused G2/M phase arrest, induced cell apoptosis and depolarized mitochondria of K562 cells. 19c also reduced the cell migration and disrupted the capillary-like tube formation of human umbilical vein endothelial cells (HUVECs). Importantly, 19c significantly and dose dependently inhibited tumor growth in H22 liver cancer xenograft mouse model. All these results suggested that 19c deserves further research as a novel and potential anti-tubulin agent for the treatment of cancers.

6,7-Seco-ent-Kauranoids Derived from Oridonin as Potential Anticancer Agents.

Structurally unique 6,7-seco-ent-kaurenes, which are widely distributed in the genus Isodon, have attracted considerable attention because of their antitumor activities. Previously, a convenient conversion of commercially available oridonin (1) to 6,7-seco-ent-kaurenes was developed. Herein, several novel spiro-lactone-type ent-kaurene derivatives bearing various substituents at the C-1 and C-14 positions were further designed and synthesized from the natural product oridonin. Moreover, a number of seven-membered C-ring-expanded 6,7-seco-ent-kaurenes were also identified for the first time. It was observed that most of the spiro-lactone-type ent-kaurenes tested markedly inhibited the proliferation of cancer cells, with an IC50 value as low as 0.55 µM. An investigation on its mechanism of action showed that the representative compound 7b affected the cell cycle and induced apoptosis at a low micromolar level in MCF-7 human breast cancer cells. Furthermore, compound 7b inhibited liver tumor growth in an in vivo mouse model and exhibited no observable toxic effects. Collectively, the results warrant further preclinical investigations of these spiro-lactone-type ent-kaurenes as potential novel anticancer agents.

Targeted intradermal delivery of alpha-arbutin-loaded dissolving polymeric microneedles visualized by three-dimensional Orbitrap secondary ion mass spectrometry (3D OrbiSIMS).

Hyperpigmentation, a prevalent dermatological condition characterized by melanin overproduction, poses treatment challenges due to the hydrophilicity of alpha-arbutin, a widely utilized tyrosinase inhibitor. This study investigates the efficacy of dissolving microneedles (DMNs) in augmenting skin permeation for alpha-arbutin delivery to the targeted epidermal site. Porcine full-thickness skin was employed in a 24-hour Franz cell study, commencing with the assessment of commercial alpha-arbutin-containing products. Solid steel microneedles (CMNs) from Dermapen® were utilized as both pre- and post-treatment modalities to evaluate the influence of different applications on alpha-arbutin delivery. Additionally, alpha-arbutin-loaded polyvinylpyrrolidone-co-vinyl acetate (PVPVA) DMNs, containing 2 % w/w alpha-arbutin, were fabricated and examined for their permeation-enhancing capabilities. HPLC analysis and 3D Orbitrap Secondary Ion Mass Spectrometry (OrbiSIMS) were employed to quantify and visualize alpha-arbutin in various Franz cell components. Results indicate that alpha-arbutin permeation to the skin was restricted (less than 1 %) without microneedle application and significantly increased by 6-fold (4-5 %) with post-treatment CMNs and DMNs, but not with pre-treatment CMNs. Notably, DMNs exhibited a more sustainable and robust capacity than post-treatment CMNs. OrbiSIMS imaging analysis revealed that DMNs visually enhance skin permeation of alpha-arbutin by delivering the compound to the basal layer of the targeted skin location. Overall, this study underscores the potential of DMNs as a promising delivery system for promoting targeted intradermal delivery of alpha-arbutin, providing a comprehensive exploration of various methodologies to identify innovative and improved microneedle approaches for alpha-arbutin permeation.

Transcriptomic profiling revealed immune-related signaling pathways in response to experimental infection of Leishmania donovani in two desert lizards from Northwest China.

Little is known about the immune response of lizards to Leishmania parasties. In this study, we conducted the first liver transcriptome analysis of two lizards (Phrynocephalus przewalskii and Eremias multiocellata) challenged with L. donovani, endemic to the steppe desert region of northwestern China. Our results revealed that multiple biological processes and immune-related signaling pathways are closely associated with the immune response to experimental L. donovani infection in the two lizards, and that both lizards show similar changes to mammals in terms of immunity to Leishmania. However, the interspecific divergence of the two lizards leads to different transcriptomic changes. In particular, in contrast to P. przewalskii, the challenged E. mutltiocellata was characterized by the induction of down-regulation of most DEGs. These findings will contribute to the scarce resources on lizard immunity and provide a reference for further research on immune mechanisms in reptiles.

Unraveling molecular mechanistic disparities in pathogenic visceral Leishmania resistance between reptiles and mammals through comparative transcriptomic analyses.

Leishmaniasis is one of the most important neglected tropical parasitic diseases, manifesting various clinical forms depending on the parasite species and the genetic background of the host. In order to elucidate the underlying mechanisms of reptilian defense against pathogenic Leishmania species and to delineate the global gene expression profile alterations during host-pathogen interaction, we established experimental animal and cell models using both heterothermic lizards (Phrynocephalus przewalskii) and homothermic mammals (BALB/c mice) infected with pathogenic Leishmania infantum (high virulence HCZ strain) and Leishmania donovani (low virulence 801 strain). Overall, the lizards didn't show any obvious clinical symptoms or immune responses in vivo. Using RNA-seq methodology, differentially expressed genes identified in the HCZ and 801-comparison groups of P. przewalskii were primarily associated with arginine biosynthesis, the MAPK signaling pathway and the PI3K-Akt signaling pathway. In contrast, higher parasite loads, exacerbated hepatic inflammatory lesions and enhanced immune responses were observed in BALB/c mice, with DEGs predominantly associated with immunological diseases, innate and adaptive immune responses. By integrating transcriptional data from reptile and mammalian hosts, we elucidated the pivotal role of amino acid metabolism and lipid metabolism in parasite control. In contrast to findings from animal experiments, Leishmania parasites effectively infected peritoneal macrophages of lizards in vitro, demonstrating a high infection rate. Furthermore, we used RT-qPCR to detect changes in cytokine expression in macrophages and found that Th1-type cytokines were significantly upregulated in lizards, facilitating the clearance of the HCZ strain 24 hours post-infection. Conversely, cytokine expression was generally suppressed in BALB/c mice, allowing immune evasion by the parasites.