Discovering metabolic vulnerability using spatially resolved metabolomics for antitumor small molecule-drug conjugates development as a precise cancer therapy strategy

Against tumor-dependent metabolic vulnerability is an attractive strategy for tumor-targeted therapy.However,metabolic inhibitors are limited by the drug resistance of cancerous cells due to their metabolic plasticity and heterogeneity.Herein,choline metabolism was discovered by spatially resolved metabolomics analysis as metabolic vulnerability which is highly active in different cancer types,and a choline-modified strategy for small molecule-drug conjugates(SMDCs)design was developed to fool tumor cells into indiscriminately taking in choline-modified chemotherapy drugs for targeted cancer therapy,instead of directly inhibiting choline metabolism.As a proof-of-concept,choline-modified SMDCs were designed,screened,and investigated for their druggability in vitro and in vivo.This strategy improved tumor targeting,preserved tumor inhibition and reduced toxicity of paclitaxel,through targeted drug delivery to tumor by highly expressed choline transporters,and site-specific release by carboxylesterase.This study expands the strategy of targeting metabolic vulnerability and provides new ideas of developing SMDCs for precise cancer therapy.

Integrated mass spectrometry imaging reveals spatial-metabolic alteration in diabetic cardiomyopathy and the intervention effects of ferulic acid

Diabetic cardiomyopathy(DCM)is a metabolic disease and a leading cause of heart failure among people with diabetes.Mass spectrometry imaging(MSI)is a versatile technique capable of combining the molecular specificity of mass spectrometry(MS)with the spatial information of imaging.In this study,we used MSI to visualize metabolites in the rat heart with high spatial resolution and sensitivity.We optimized the air flow-assisted desorption electrospray ionization(AFADESI)-MSI platform to detect a wide range of metabolites,and then used matrix-assisted laser desorption ionization(MALDI)-MSI for increasing metabolic coverage and improving localization resolution.AFADESI-MSI detected 214 and 149 metabolites in positive and negative analyses of rat heart sections,respectively,while MALDI-MSI detected 61 metabolites in negative analysis.Our study revealed the heterogenous metabolic profile of the heart in a DCM model,with over 105 region-specific changes in the levels of a wide range of metabolite classes,including carbohydrates,amino acids,nucleotides,and their derivatives,fatty acids,glycerol phospholipids,carnitines,and metal ions.The repeated oral administration of ferulic acid during 20 weeks significantly improved most of the metabolic disorders in the DCM model.Our findings provide novel insights into the molecular mechanisms underlying DCM and the potential of ferulic acid as a therapeutic agent for treating this condition.

Promise of spatially resolved omics for tumor research

Tumors are spatially heterogeneous tissues that comprise numerous cell types with intricate structures.By interacting with the microenvironment,tumor cells undergo dynamic changes in gene expression and metabolism,resulting in spatiotemporal variations in their capacity for proliferation and metastasis.In recent years,the rapid development of histological techniques has enabled efficient and high-throughput biomolecule analysis.By preserving location information while obtaining a large number of gene and molecular data,spatially resolved metabolomics(SRM)and spatially resolved transcriptomics(SRT)approaches can offer new ideas and reliable tools for the in-depth study of tumors.This review provides a comprehensive introduction and summary of the fundamental principles and research methods used for SRM and SRT techniques,as well as a review of their applications in cancer-related fields.

Integration of network pharmacology with experimental verification reveals the hypoglycemic mechanism of coptisine in Jinqi Jiangtang tablets:inhibition of the FoxO1 signaling pathway and hepatic gluconeogenesis

Background:Jinqi Jiangtang tablets(JQJT)have been approved for the treatment of type 2 diabetes mellitus(T2DM)in China for many years.Exploring the effective substances and mechanisms of JQJT is important for its clinical application and further drug research and development.This study aimed to explore the chemical basis and mechanisms of JQJT in the treatment of T2DM.Methods:With network pharmacology,we screened substances in JQJT and their possible targets,then constructed the action network and enriched the biological functions and pathways associated with the active components,and identified the potential targets and mechanisms of JQJT in the treatment of T2DM.Based on the network pharmacology data,we explored the hypoglycemic mechanisms of coptisine in JQJT through western blot and quantitative real-time polymerase chain reaction.Results:Forty-three compounds with good pharmacokinetic properties were identified in JQJT,together with 146 potential biological targets.Among these potential targets,74 were associated with treatment of T2DM.A compound-target network of the 43 compounds against T2DM was constructed.Biological process and signal pathway enrichment analysis of the network highlighted the FoxO signaling pathway.Western blot and quantitative real-time polymerase chain reaction results showed that coptisine,but not epiberberine,significantly inhibited expression of key genes involved in hepatocyte gluconeogenesis by regulating the FoxO1 signaling pathway.Conclusion:Network pharmacology analysis and cell experiments showed that coptisine regulated glucose homeostasis by inhibiting the FoxO1 signaling pathway and hepatic gluconeogenesis,which may be one of the mechanisms of JQJT in the treatment of T2DM.

Expression of Bmi-1 and EZH2 in tissues adjacent to human epithelial ovarian cancer cells of orthotopic implantation in nude mice

Objective This study investigated the feasibility of screening residual normal ovarian tissues based on the expression of Bmi-1 and EZH2 in tissues adjacent to orthotopic ovarian carcinomas in nude mice. Methods The human epithelial ovarian cancer cell line OVCAR3 was grown in subcutaneous tissues and the tumor tissues were orthotopically implanted. The expression levels of Bmi-1 and EZH2 were detected by immunohistochemical staining and RT-PCR in cancer tissues, proximal and remote tissues with respect to the cancer tissues, and normal ovarian tissues of nude mice.Results Thirty-five ovarian tissue samples with normal biopsy results were obtained from 40 cases of human epithelial ovarian cancer in the nude mice in which the tumor tissues were orthotopically implanted. Bmi-1 and EZH2 expression levels were lower in proximal paraneoplastic tissue samples than in cancer tissue samples(P < 0.05) and higher than in remote paraneoplastic tissue samples(P < 0.01). No significant difference was found in the expression levels of Bmi-1 and EZH2 using immunohistochemistry among residual normal ovarian tissues obtained from orthotopically implanted models that differed in severity. The expression of Bmi-1 and EZH2 was negative in 20 normal ovarian tissue samples.Conclusion The expression levels of Bmi-1 and EZH2 were reduced with increasing distance from the cancer tissues. Negative expression of these tumor-associated genes can be used as a standard for the screening of normal ovarian tissues adjacent to tumor tissues. Normal ovarian tissues can be obtained from the tissues adjacent to tumors.

Hepatitis associated with hepatitis B virus in broilers

Infection by hepatitis B virus （HBV） results in acute and chronic liver damages in humans. Liver products of broilers as a primary food consumed in our daily life have a close connection with public health. The prevalence of the virus in livers and serum of broilers is of great significance, owning to the potential transmission between chickens and humans. Liver tissues and serum samples were tested to investigate the prevalence of hepatitis B virus infection in slaughtered broilers, for expression of HBV antigens and antibodies. The distribution and positive rate of hepatitis B surface antigen （HBsAg）, hepatitis B core antigen （HBcAg） and hepatitis B e antigen （HBeAg） in liver samples were examined using immunohistochemistry. HBsAg was mainly located in the cytoplasm of hepatocytes with a positivity of 81.61% whereas HBeAg and HBcAg were primarily located in the nucleus of hepatocytes with a positivity of 40.13 and 49.10%, respectively. Enzyme-linked immunosorbent assay （ELISA） analysis of serum for HBV serological markers demonstrated a high prevalence of hepatiits B surface antibody （HBsAb, 54.91%） and hepatitis B core antibody （HBcAb, 27.68%）, whereas HBeAb, HBsAg and HBeAg were rarely detectable. Classic hepatitis pathological changes, including swollen hepatocytes, focal parenchymal necrosis, lymphocytic infiltration and hyperplasia of fibrous connective tissues were observed using histopathological analysis. Some of the liver samples were found positive for HBV DNA using nested PCR. Sequence comparison confirmed that all sequences shared 97.5-99.3% identity with human HBV strains. These results demonstrated the existence of HBV in livers and serums of broilers. Animals or animal products contaminated with HBV could raise an important public health concern over food safety and zoonotic risk.

Clinical Efficacy on the Treatment of Chronic Colitis with Feng-Liao-Chang-Wei-Kang Granules Combined with Mesalazine

[Objectives]To analyze the clinical effect of Feng-Liao-Chang-Wei-Kang Granule combined with mesalazine on chronic colitis.[Methods]120 patients with chronic enteritis admitted to Qionghai Hospital of Traditional Chinese Medicine from October 2020 to March 2022 were selected as the research objects,and were randomly divided into combined group(62 cases)and conventional group(58 cases).Patients in the conventional group were treated with oral mesalazine on the basis of conventional treatment,while patients in the combined group were treated with Feng-Liao-Chang-Wei-Kang Granule combined with mesalazine.The clinical efficacy,inflammatory factor level,colonic mucosal lesion degree,quality of life and TCM syndrome scores of the two groups were compared.[Results]The total clinical effective rate in the combined group was higher than that in the conventional group(P<0.05).After treatment,the levels of inflammatory factors in the two groups were lower than those before treatment,and the levels of interleukin-6(IL-6),C-reactive protein(CRP)and tumor necrosis factor-α(TNF-α)in the combined group were lower than those in the conventional group(P<0.001).After treatment,the Baron and Geboes scores of the two groups were lower than those before treatment,and the Baron and Geboes scores of the combined group were lower than those of the conventional group(P<0.001).After treatment,the scores of quality of life in the two groups were higher than those before treatment,while the scores of TCM syndromes were lower than those before treatment.The scores of quality of life in the combined group were higher than those in the conventional group(P<0.001).[Conclusions]Feng-Liao-Chang-Wei-Kang Granule combined with mesalazine can obviously improve the curative effect,reduce the level of inflammatory factors,improve the degree of colonic mucosal lesions and improve the quality of life of patients with chronic colitis,which is worthy of popularization and application in clinical practice.

Honokiol alleviated neurodegeneration by reducing oxidative stress and improving mitochondrial function in mutant SOD1 cellular and mouse models of amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis(ALS)is a progressive neurodegenerative disease affecting both upper and lower motor neurons(MNs)with large unmet medical needs.Multiple pathological mechanisms are considered to contribute to the progression of ALS,including neuronal oxidative stress and mitochondrial dysfunction.Honokiol(HNK)has been reported to exert therapeutic effects in several neurologic disease models including ischemia stroke,Alzheimer’s disease and Parkinson’s disease.Here we found that honokiol also exhibited protective effects in ALS disease models both in vitro and in vivo.Honokiol improved the viability of NSC-34 motor neuron-like cells that expressed the mutant G93A SOD1 proteins(SOD1-G93A cells for short).Mechanistical studies revealed that honokiol alleviated cellular oxidative stress by enhancing glutathione(GSH)synthesis and activating the nuclear factor erythroid 2-related factor 2(NRF2)-antioxidant response element(ARE)pathway.Also,honokiol improved both mitochondrial function and morphology via fine-tuning mitochondrial dynamics in SOD1-G93A cells.Importantly,honokiol extended the lifespan of the SOD1-G93A transgenic mice and improved the motor function.The improvement of antioxidant capacity and mitochondrial function was further confirmed in the spinal cord and gastrocnemius muscle in mice.Overall,honokiol showed promising preclinical potential as a multiple target drug for ALS treatment.

Acanthopanax senticosus Protects Structure and Function of Mesencephalic Mitochondria in A Mouse Model of Parkinson's Disease

Objective： To investigate the neuro-protective effects of Acanthopanax senticosus Harms（EAS） on mesencephalic mitochondria and the mechanism of action, using a mouse model of Parkinson＇s disease（PD）. Methods： The chemical fingerprint analysis of the extract of Acanthopanax senticosus Harms（EAS） was performed using the ultra performance liquid chromatograph and time of flight mass spectrometry. Thirty mice were randomly divided into the control group, the MPTP model group, and the EAS treated group with MPTP（MPTP＋EAS group, 10 in each group）. The MPTP model group and the MPTP＋EAS group received MPTP-HCl（30 mg/kg i.p） once a day for 5 days. The control group received an equal volume of saline（20 m L/kg i.p） once a day for 5 days. Induced by 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine hydrochloride daily（MPTP-HCl, 30 mg/kg） for 5 days, the PD mice were treated with EAS at 45.5 mg/kg daily for 20 days. The behavioral testing of mice was carried out using the pole-climbing test. The integrity and functions of neurons were examined in mesencephalic mitochondria in a PD mouse model, including nicotinamide adenine dinucleotide dehydrogenase ubiquinone flavoprotein 2（NDUFV2）, mitochondrially encoded nicotinamide adenine dinucleotide dehydrogenase 1（MT-ND1）, succinate dehydrogenase complex subunit A（SDHA）, and succinate dehydrogenase cytochrome b560 subunit（SDHC）. Results： After treatment with EAS, the behavioral changes induced by MPTP were attenuated significantly（P〈0.05）. EAS protected the mesencephalic mitochondria from swelling and attenuated the decreases in their membrane potential（both P〈0.05）, which was supported by an ultra-structural level analysis. The changes in reactive oxygen species（ROS）, malonic dialdehyde（MDA）, oxidative phosphorylation（OXPHOS） system 4 subunits levels and PD-related proteins expressions（parkin, Pink1, DJ-1, α-synuclein, and Lrrk2） reverted to near normal levels（all P〈0.05）, based on the results of immune-histological and Western blotting observations. Conclusions： The neuro-protective effects of EAS are linked to protecting mice against MPTPinduced mitochondrial dysfunction and structuraldamage.Therefore,EAS is a promising candidate for the prevention or treatment of mitochondrial neurodegenerative disorders,such as PD.

In situ metabolomics in nephrotoxicity of aristolochic acids based on air flow-assisted desorption electrospray ionization mass spectrometry imaging

Understanding of the nephrotoxicity induced by drug candidates is vital to drug discovery and development.Herein,an in situ metabolomics method based on air flow-assisted desorption electrospray ionization mass spectrometry imaging(AFADESI-MSI)was established for direct analysis of metabolites in renal tissue sections.This method was subsequently applied to investigate spatially resolved metabolic profile changes in rat kidney after the administration of aristolochic acid I,a known nephrotoxic drug,aimed to discover metabolites associated with nephrotoxicity.As a result,38 metabolites related to the arginine-creatinine metabolic pathway,the urea cycle,the serine synthesis pathway,metabolism of lipids,choline,histamine,lysine,and adenosine triphosphate were significantly changed in the group treated with aristolochic acid I.These metabolites exhibited a unique distribution in rat kidney and a good spatial match with histopathological renal lesions.This study provides new insights into the mechanisms underlying aristolochic acids nephrotoxicity and demonstrates that AFADESI-MSI-based in situ metabolomics is a promising technique for investigation of the molecular mechanism of drug toxicity.

Effect of Total Flavone of Haw Leaves on Nuclear Factor Erythroid-2 Related Factor and Other Related Factors in Nonalcoholic Steatohepatitis Rats

Objective： To investigate the effect of total flavone of haw leaves（TFHL） on the expression of nuclear factor erythroid-2 related factor（Nrf2） and other related factors in nonalcoholic steatohepatitis（NASH） rats induced by high-fat diet and then to further discuss the mechanism of TFHL＇s prevention against NASH. Methods： High-fat diet was fed to 40 rats to establish the NASH model. Then model rats were intragastrically administrated with 40, 80, 160 mg/（kg·day） TFHL, respectively. The pathological changes of liver tissues in NASH rats were detected by oil red O and hematoxylin-eosin（HE） stainings. The expression of Nrf2 in rat liver was examined through immunohistochemistry. The level of 8-iso-prostaglandin F2α in serum was detected through enzyme linked immunosorbent assay（ELISA）. The mR NA and protein levels of Nrf2 and other related factors in liver tissue were measured by real-time reverse transcriptionpolymerase chain reaction and western blot. Results： Lipid deposition, hepatic steatosis, focal necrosis in lobular inflammation and ballooning degeneration were emerged in livers of NASH rats. The 8-iso-prostaglandin F2α in the serum of NASH rats increased significantly compared with the control group（P〈0.05）. The mR NA of Nrf2, hemeoxyenase1（HO-1） and the mR NA and protein levels of quinine oxidoreductase（NQO1） in NASH rats liver tissue showed a striking increase, while the mR NA levels of Keap1, r-glutamylcysteine synthethase（rG CS） and glutathione S-transferase（GST） were significantly decreased compared with the control group（P〈0.05）. After TFHL treatment, 8-iso-prostaglandin F2α level in serum significantly decreased, and Nrf2 mR NA and protein levels in hepatocytes nucleus enhanced compared with the model group（P〈0.05 or 0.01）. Meanwhile the Keap1 mR NA, the mR NA and protein levels of HO-1, NQO1 antibody, rG CS antibody, GST increased after TFHL treatment（P〈0.05 or 0.01）. Conclusions： Nrf2 and other related factors were involved in development of NASH, and they also served as an important part in its occurrence. By regulating expression of Nrf2 and other related factors, TFHL may play a role in antioxidative stress and prevention of NASH.

Spatial-resolved metabolomics reveals tissue-specific metabolic reprogramming in diabetic nephropathy by using mass spectrometry imaging

Detailed knowledge on tissue-specific metabolic reprogramming in diabetic nephropathy(DN)is vital for more accurate understanding the molecular pathological signature and developing novel therapeutic strategies.In the present study,a spatial-resolved metabolomics approach based on air flowassisted desorption electrospray ionization(AFADESI)and matrix-assisted laser desorption ionization(MALDI)integrated mass spectrometry imaging(MSI)was proposed to investigate tissue-specific metabolic alterations in the kidneys of high-fat diet-fed and streptozotocin(STZ)-treated DN rats and the therapeutic effect of astragalosideⅣ,a potential anti-diabetic drug,against DN.As a result,a wide range of functional metabolites including sugars,amino acids,nucleotides and their derivatives,fatty acids,phospholipids,sphingolipids,glycerides,carnitine and its derivatives,vitamins,peptides,and metal ions associated with DN were identified and their unique distribution patterns in the rat kidney were visualized with high chemical specificity and high spatial resolution.These region-specific metabolic disturbances were ameliorated by repeated oral administration of astragaloside Ⅳ(100 mg/kg)for 12 weeks.This study provided more comprehensive and detailed information about the tissue-specific metabolic reprogramming and molecular pathological signature in the kidney of diabetic rats.These findings highlighted the promising potential of AFADESI and MALDI integrated MSI based metabolomics approach for application in metabolic kidney diseases.

Genotoxicity and Embryotoxicity Study of Bicyclol Methyl Ether, Main Impurity in Bicyclol

Objective: To assess the genotoxicity and embryotoxicity of bicyclol methyl ether(BME), the main impurity in bicyclol. Methods: Five concentrations of BME(0.5, 5, 50, 500 and 5000 μg/plate) were used in the Ames test to detect gene mutation. In the chromosome aberration test, Chinese hamster lung cells were used to detect chromosomal aberration of BME(15, 30, 60, 120 μg/m L) with or without S9 mixture. Embryotoxicity test was also conducted to determine any embryotoxicity of BME(7.5, 22.5, 67.5 μg/L) using zebrafish embryos. Results: No significant differences were observed in the Ames test and the chromosome aberration test in the BME groups compared with the vehicle control group. The zebrafish embryos toxicity test also showed no embryo development toxicity of BME, including hatching rate, body length, pericardial area and yolk sac area. Conclusions: Bicyclol methyl ether has no genotoxicity in vitro and embryotoxicity in zebrafish embryos, and the impurity in bicyclol is qualified.

Rapid and sensitive liquid chromatography–tandem mass spectrometric method for the quantitative determination of potentially harmful substance 5,5′-oxydimethylenebis (2-furfural) in traditional Chinese medicine injections

With the rapid development and wide application of traditional Chinese medicine injection(TCMI), a number of adverse events of some TCMIs have incessantly been reported and have drawn broad attention in recent years. Establishing effective and practical analytical methods for safety evaluation and quality control of TCMI can help to improve the safety of TCMIs in clinical applications. In this study, a sensitive and rapid high-performance liquid chromatography–tandem mass spectrometry(HPLC–MS/MS)method has been developed and validated for the quantitative determination of potentially harmful substance5,5′-oxydimethylenebis(2-furfural, OMBF) in TCMI samples. Chromatographic separation was performed on a C18 reversed-phase column(150 mm × 2.1 mm, 5 μm) by gradient elution, using methanol–water containing 0.1% formic acid as mobile phase at the flow rate of 0.3 m L/min. MS/MS detection was performed on a triple quadrupole mass spectrometer with positive electrospray ionization in the multiple reaction-monitoring mode. The method was sensitive with a limit of quantification of 0.3 ng/m L and linear over the range of 0.3–30 ng/m L(r = 0.9998). Intra-and inter-day precision for analyte was o9.52% RSD withrecoveries in the range 88.0–109.67% at three concentration levels. The validated method was successfully applied to quantitatively determine the compound OMBF in TCMIs and glucose injections. Our study indicates that this method is simple, sensitive, practicable and reliable, and could be applied for safety evaluation and quality control of TCMIs and glucose injections.

Anti-anaphylactic potential of benzoylpaeoniflorin through inhibiting HDC and MAPKs from Paeonia lactiflora

Guided by cell-based anti-anaphylactic assay,eighteen cage-like monoterpenoid glycosides(1−18)were obtained from the bioactive fraction of P.lactiflora extract.Among these,compounds 1,5,6,11,12,15,and 17 significantly reduced the release rate ofβ-HEX and HIS without or with less cytotoxicity.Furthermore,the most potent inhibitor benzoylpaeoniflorin(5)was selected as the prioritized compound for the study of action of mechanism,and its anti-anaphylactic activity was medicated by dual-inhibiting HDC and MAPK signal pathway.Moreover,molecular docking simulation explained that benzoylpaeoniflorin(5)blocked the conversion of L-histidine to HIS by occupying the HDC active site.Finally,in vivo on PCA using BALB/c mice,benzoylpaeoniflorin(5)suppressed the IgE-mediated PCA reaction in antigen-challenged mice.These findings indicated that cage-like monoterpenoid glycosides,especially benzoylpaeoniflorin(5),mainly contribute to the anti-anaphylactic activity of P.lactiflora by dual-inhibiting HDC and MAPK signal pathway.Therefore,benzoylpaeoniflorin(5)may be considered as a novel drug candidate for the treatment of anaphylactic diseases.

A temporo-spatial pharmacometabolomics method to characterize pharmacokinetics and pharmacodynamics in the brain microregions by using ambient mass spectrometry imaging

The brain is the most advanced organ with various complex structural and functional microregions. It is often challenging to understand what and where the molecular events would occur for a given drug treatment in the brain. Herein, a temporo-spatial pharmacometabolomics method was proposed based on ambient mass spectrometry imaging and was applied to evaluate the microregional effect of olanzapine(OLZ) on brain tissue and demonstrate its effectiveness in characterizing the microregional pharmacokinetics and pharmacodynamics of OLZ for improved understanding of the molecular mechanism of drugs acting on the microregions of the brain. It accurately and simultaneously illustrated the levels dynamics and microregional distribution of various substances, including exogenous drugs and its metabolites, as well as endogenous functional metabolites from complicated brain tissue. The targeted imaging analysis of the prototype drug and its metabolites presented the absorption, distribution, metabolism, and excretion characteristics of the drug itself. Moreover, the endogenous functional metabolites were identified along with the associated therapeutic and adverse effects of the drug, which can reflect the pharmacodynamics effect on the microregional brain. Therefore, this method is significant in elucidating and understanding the molecular mechanism of central nervous system drugs at the temporo and spatial metabolic level of system biology.