Tanshinone IIA protects intestinal epithelial cells from ferroptosis through the upregulation of <i>GPX4</i> and <i>SLC7A11</i>

Background: Inflammatory bowel disease (IBD) is a chronic inflammatory disease of the gastrointestinal tract. The destruction of the intestinal epithelial barrier is one of the major pathological processes in IBD pathology. Growing evidence indicated that epithelial cell ferroptosis is linked to IBD and is considered a target process. Methods: RAS-selective lethal 3 (RSL3) was used to induce ferroptosis in intestinal epithelial cell line No. 6 (IEC-6) cells, and cell ferroptosis and the effects of tanshinone IIA (Tan IIA) were determined by cell counting kit-8 (CCK-8), reactive oxygen species (ROS) staining, Giemsa staining and transmission electron microscope (TEM). The cell viability of natural product library compounds was determined by CCK-8. The expression of ferroptosis-related genes were detected by real-time quantitative polymerase chain reaction (RT-qPCR) and western blot. Results: Treatment of IEC-6 cells results in the accumulation of ROS and typical morphological characteristics of ferroptosis. RSL3 treatment caused rapid cellular cytotoxicity which could be reversed by ferrostatin-1 (Fer-1) in IEC-6 cells. Natural product library screening revealed that Tan IIA is a potent inhibitor of IEC-6 cell ferroptosis. Tan IIA could significantly protect the RSL3-induced ferroptosis of IEC-6 cells. Furthermore, the ferroptosis suppressors, glutathione peroxidase 4 (GPX4), solute carrier family 7 member 11 (SLC7A11), and miR-17-92 were found to be early response genes in RSL3-treated cells. Treatment of IEC-6 cells with Tan IIA resulted in upregulation of GPX4, SLC7A11, and miR-17-92. Conclusion: Our study demonstrated that Tan IIA protects IEC-6 cells from ferroptosis through the upregulation of GPX4, SLC7A11, and miR-17-92. The findings might provide a theoretical grounding for the future application of Tan IIA to treat or prevent IBD.     

Scanning electron microscopy,autolysis, and irradiation as techniques for studying small intestinal morphology

Examination of autolysed control mouse small intestine using scanning electron microscopy has revealed details of the connective tissue components of the mucosa. The cores of the villi are seen collapsed across the intervillous basin. Crypts of Lieberkuhn are seen as tubular channels stretching down from the intervillous basin. Sometimes the crypts are split in two by a connective tissue septum. The mouths of the crypts of Lieberkuhn are, in general, arranged in double rows between the single rows of villi. The ratio of number of crypts to numbers of villi was calculated as 5.01:1. This is close to the figure of 4.53:1, as quoted by Smith & Jarvis (1980) who used differential interference contrast microscopy to investigate the crypt to villus ratio. After radiation, the severe drop in the number of crypt mouths can be clearly seen by the combination of autolysis and scanning electron microscopy: the rows of crypt mouths between the villi have been lost, and many crypt mouths have been occluded by stromal tissue. The arrangement of the crypt mouths and the observation of mucosal abnormalities after irradiation have led to the postulation that cells leaving the crypt mouths move in a spiral manner towards and then up the villous surface: this postulated movement might imply an asymmetry in some properties of enterocytes. The use of scanning electron microscopy in conjugation with autolysis and irradiation has thus forced a critical re-examination of the relationships between crypts and villi.     

The effect of intestinal distension and of section thickness on counts of epithelial nuclei in the rat small intestine.

(1) A method is described for counting nuclei in wax-embedded histological sections of epithelium. The counts are independent of section thickness over a wide range (4-10 mum). (2) counts of nuclei were made in villi and crypts of rat small intestine fixed either collapsed or distended by a hydrostatic pressure equivalent to 350 mm of water. There was no significant difference between the counts of nuclei in collapsed or distended gut. (3) Distension reduced the absolute height of villi, and increased their absolute width at the base. (4) The implications of these findings for studies of the morphological basis of intestinal adaptation are discussed.     

Pollen viability of <i>Polygala paniculata</i> L. (Polygalaceae) using different staining methods

Polygala paniculata L. is a medicinal plant that grows in the Brazilian Atlantic coast, known as ‘barba-de-São-João’, ‘barba-de-bode’, ‘vassourinha branca’, and ‘mimosa’. In this study, pollen viability was estimated by three different staining methods: 2% acetic orcein, 2% acetic carmine, and Alexander’s stain. The young inflorescences of twenty accessions were collected and fixed in a solution of ethanol: acetic acid (3:1) for 24 hours, then stored in ethanol 70% under refrigeration. Six slides per plant, two for each stain, were prepared by squashing, and 300 pollen grains per slide were analyzed. Pollen viability was high (>70%) for most accessions of P. paniculata using the Alexander’s stain, which proved the most adequate method to estimate pollen viability.     

Adjuvant effects of <i>Lactobacillus casei</i> added to a renutrition diet in a malnourished mouse model

Nutritional deficiencies are associated with impaired immune response, affecting the body’s defence mechanisms. It is also known that Lactic Acid Bacteria (LAB) and fermented products such us yogurt have immunopotentiator activity and nutritional properties, and could thus be used as a valuable supplement in a renutrition diet. The aim of this study was to determine, in a non-severe malnutrition model, the effective dose of Lactobacillus casei (L.casei), which when is used as an adjuvant in a renutrition diet, would modulate the mucosal immune system and induce recovery of the integrity of the intestinal barrier. The experiments were performed on groups of malnourished and renourished BALB/c mice. They received after milk renutrition a supplement of different doses and periods of L. casei feeding. We measured body weight; hematologic values and serum proteins. We also characterized small intestine immunoglobulin secreting cells, intraepithelial leukocytes, mastocytes and goblet cells. Structural and ultrastructural studies were performed. Our results suggest that impaired gut barrier and mucosal immune function produced by malnutrition can be reversed by L. casei and that the dose of 10⁷ cfu/day/mouse administered during 5 consecutive days was the optimal one for recovery of the gut mucosal immune system. The clinical significance of these findings suggests ways for improving mucosal immunity, and generating protection against enteropathogens in hosts immunosuppressed by malnutrition.     

Developmental aspects of lipid and lipoprotein synthesis and secretion in human gut

This review article focuses on the ontogeny and the regulatory mechanisms involved in the modulation of the intracellular events governing the assembly and delivery of lipoproteins in human gut. The human fetal intestine organizes villi covered with well-differentiated enterocytes during the end of the first trimester in utero. One striking event is the formation of villi in the colonic mucosa similar to those of the small intestine. The small intestine exhibits very early (14-20 weeks) the capacity to absorb lipids, to elaborate most of the major lipoprotein classes (chylomicrons, very-low-density lipoproteins, low-density lipoproteins, high-density lipoproteins), and to efficiently export these lipoproteins from the intestinal cells. The ontogenic changes of lipid and lipoprotein synthesis are correlated with specific patterns of regulatory enzymes (HMG-CoA reductase, ACAT, MGAT) that are representative of key patterns such as the cholesterol pathway, cholesterol esterification, and neutral lipid pathway. The human fetal colon also has the capability to synthesize lipids, lipoproteins, and apolipoproteins. However, comapred with the small intestine, it is much less efficient at exporting these lipoproteins. Epidermal growth factor, insulin, and hydrocortisone, which are known modulators of the brush border digestive functions of the human gut, differentially modulate the synthesis and secretion of lipoproteins in the small intestine and colon. The use of human fetal gut represents a unique model to further our understanding of the complex biosynthetic molecular events essential for the formation and secretion of lipoproteins relevant to human intestine, both in normal or pathological conditions.     

<i>In vivo</i> protective effect of late embryogenesis abundant protein (ApSK₃ dehydrin) on <i>Agapanthus praecox</i> to promote post-cryopreservation survival

Dehydrins (DHNs), as members of the late embryogenesis abundant protein family, play critical roles in the protection of seeds from dehydration and plant adaptation to multiple abiotic stresses. Vitrification is a basic method in plant cryopreservation and is characterized by forming a glassy state to prevent lethal ice crystals produced during cryogenic storage. In this study, ApSK₃ type DHN was genetically transformed into embryogenic calluses (EC) of Agapanthus praecox by overexpression (OE) and RNA interference (RNAi) techniques to evaluate the in vivo protective effect of DHNs during cryopreservation. The cell viability showed a completely opposite trend in OE and RNAi cell lines, the cell relative death ratio was decreased by 20.0% in ApSK₃-OE EC and significantly increased by 66.15% in ApSK₃-RNAi cells after cryopreservation. Overexpression of ApSK₃ increased the content of non-enzymatic antioxidants (AsA and GSH) and up-regulated the expression of CAT, SOD, POD, and GPX genes, while ApSK₃-RNAi cells decreased antioxidant enzyme activities and FeSOD, POD, and APX genes expression during cryopreservation. These findings suggest that ApSK₃ affects ROS metabolism through chelating metal ions (Cu²⁺ and Fe³⁺), alleviates H₂O₂ and OH· excessive generation, activates the antioxidant system, and improves cellular REDOX balance and membrane lipid peroxidation damage of plant cells during cryopreservation. DHNs can effectively improve cell stress tolerance and have great potential for in vivo or in vitro applications in plant cryopreservation.     

Agmatine pretreatment protects retinal ganglion cells (RGC-5 cell line) from oxidative stress <i>in vitro</i>

Agmatine, 2-(4-aminobutyl)guanidine, has been reported to have neuroprotective effects against various neuronal damages. In this study it was investigated whether agmatine pretreatment rescues the retinal ganglion cells from oxidative injury in vitro. After differentiation of transformed rat retinal ganglion cells (RGC-5 cell line) with staurosporine, agmatine (0.0 to 100.0 μM) pretreatment was performed for 2 hours. Subsequently, they were exposed to hydrogen peroxide (0.0 to 2.5 mM) as an oxidative stress. Cell viability was monitored for up to 48 hours with the lactate dehydrogenase (LDH) assay and apoptosis was examined by the terminal deoxynucleotide transferase-mediated terminal uridine deoxynucleotidyl transferase nick end-labeling (TUNEL) method. As a result, differentiated RGC-5 cells were found to have decreased viability after addition of hydrogen peroxide in a dose-dependent manner. This hydrogen peroxide induced cytotoxicity caused apoptosis characterized by DNA fragmentation. Agmatine pretreatment not only increased cell viability but also attenuated DNA fragmentation. In conclusion, agmatine pretreatment demonstrated neuroprotective effects against oxidative stress induced by hydrogen peroxide in differentiated RGC-5 cells in vitro. This suggests a novel therapeutic strategy rescuing retinal ganglion cells from death caused by oxidative injury     

Fractions of a chloroform extract of ajenjo leaves (<i>Artemisia mendozana</i> DC. var. <i>mendozana</i>) inhibit the proliferation,viability and clonogenicity of B16-F0 melanoma cells

The ajenjo, Artemisia mendozana DC. var. mendozana (Asteraceae), grows in the Andean foothills of Mendoza and San Juan, Argentina, and is used as a medicinal plant for its antispasmodic and antifungal properties. The aim of this work was to obtain fractions of a chloroform extract of ajenjo leaves and to evaluate the in vitro effects on proliferation, viability and clonogenicity of B16-F0 melanoma cells. Using a silica gel chromatography column, 120 fractions were collected and grouped according to the chromatographic profile in 9 main fractions (F1–F9). Their major compounds identified were: terpenes (F1), terpenes and sesquiterpene lactones (F2–F3), sesquiterpenes (F4–F6) and phenols and sesquiterpenes (F7-9). B16-F0 cells were incubated for 72 h with DMSO (vehicle) or 0.1 mg/ml F1–F9. At 72 h of culture, F1 decreased both the growing index (GI) and cell viability. F2 and F3 both decreased GI and only F3 decreased clonogenic activity. F4 and F5 both decreased GI. Only F5 decreased cell viability and F4 decreased clonogenicity. Consequently, fractions F6–F8 did not affect any of the cell parameters assayed, while F9 decreased cell viability and inhibited clonogenicity.     

Immune strategies of phagoctytic cells stimulated <i>in vitro</i> with live and heat-inactivated <i>Streptococcus pyogenes</i>

Streptococcus pyogenes (group A Streptococcus) is frequently involved in a wide range of human diseases. Here we evaluated polymorphonuclear neutrophils and mononuclear cells from healthy subjects for their bactericidal function after stimulation with live and inactivated Streptococcus pyogenes (Streptococcus Group A). Mononuclear cells and Neutrophils were isolated from heparinized blood samples (n=18) using a Ficoll-Hypaque gradient and cultured in RPMI 1640 for 18 hours with a suspension of either live or inactivated Streptococcus pyogenes. Both the respiratory burst (flow cytometry) and nitrite, TNF and IL17 production (ELISA) were measured in the cell culture supernatants. An increased respiratory burst (expressed as R index) was induced by both live and inactivated bacteria. Also, increased nitrite, TNF and IL17 concentrations were found in cell culture supernatants in both cases. These findings may provide some explanation as to the roles played by neutrophils and mononuclear cells in Streptococcus pyogenes immunopathogenicity.     

Prenatal development and histochemical characteristics of gastrointestinal mucins in sheep fetuses

The object of this study was to describe the prenatal development and histochemical properties of mucins in the sheep gastrointestinal tract. To determine changes in the mucin profile, the sections were stained with specific histochemical stains for carbohydrates. While neutral and mixed mucins were observed in the superficial epithelial cells of the abomasal pyloric region, acidic mucins were detected in the secretory ducts and corpus of the glands. Acidic mucins consisted predominantly of sialomucins. In the duodenal villi, the number of goblet cells containing neutral mucins increased toward the end of gestation, whereas Brunner's glands contained acidic mucins until the 95th day of gestation and both acidic and neutral mucins thereafter. The jejunal goblet cells contained either acidic, neutral, or mixed mucins. Goblet cells containing acidic mucins, which were mainly localized to the ileal crypts and villi, mostly contained sulfated mucins. While villi were observed in the proximal colon until the 115th day of gestation, later the typical crypt structure emerged. During the period in which the villi were found in the proximal colon, the goblet cells containing sulphomucins were predominant, whereas the goblet cells containing sialomucins were predominant after the typical crypt structure was formed. In conclusion, gastrointestinal mucins may be involved in the formation of meconium during the prenatal period, and acidic mucins may contribute to the strength of the intestinal barrier against pathogens and digestive enzymes, as the barrier is not fully functional after birth.     

Ultrastructure of the ovariole sheath in <i>Diatraea saccharalis</i> (Lepidoptera: Pyralidae)

The ultrastructure of the ovariole sheath along the Diatraea saccharalis ovariole was studied by scanning and transmission electron microscopy. Each ovariole is surrounded by an epithelial sheath, a tunica propria and scattered lumen cells. These three components of the ovariole sheath show different ultrastructural features along the ovariole, in the germarium or in the vitellarium; these differences are more evident in the epithelial sheath cells. The epithelial sheath is composed by two layers of cells, the external one running longitudinally and the internal one running circularly in the ovariole. These cells, in vitellarium, present cytoplasmic bundles of myofilaments that are arranged parallel to the long axis of the cells; these myofilaments are apparently related to the contraction movements of the follicles within the ovariole. The acellular tunica propria, composed of finely filamentous material, is attached to the adjacent follicle cells by adhesive dense plates. Between the epithelial sheath and the tunica propria there is a population of lumen cells, with morphological features of secretory activity     

Development beyond the gastrula stage and digestive organogenesis in the apple-snail <i>Pomacea canaliculata</i> (Architaenioglossa,Ampullariidae)

Development of Pomacea canaliculata from the gastrula stage until the first day after hatching is described. Trochophore embryos are developed after gastrulation, showing the prototroch as a crown of ciliated orange-brownish cells. However, no true veliger embryos are formed, since the prototroch does not fully develop into a velum. Afterward, the connection between the fore- and midgut is permeated and the midgut becomes full of the pink-reddish albumen, which is stored into a central archenteron’s lake, from where it is accumulated into the large cells forming the midgut wall (“giant cells”). Electron microscopy of giant cells in late embryos showed that albumen is engulfed by large endocytic vesicles formed between the irregular microvilli at the top of these cells. By the end of intracapsular development, giant cells become gradually replaced by two new epithelial cell types which are similar to those found in the adult midgut gland: the pre-columnar and the pre-pyramidal cells. Pre-columnar cells have inconspicuous basal nuclei and are crowned by stereocilia, between which small endocytic vesicles are formed. Pre-pyramidal cells have large nuclei with 2-3 nucleoli and show a striking development of the rough endoplasmic reticulum. The genesis of the three cell lineages (giant, pre-columnar and pre-pyramidal cells) is hypothetically attributed to epithelial streaks that occur at both sides of the midgut since early stages of development.     

Mechanical and histological properties of an electrospun scaffold with a modified surface by plasma polymerization implanted in an <i>in vivo</i> model

This article presents the construction of scaffolds composed of polylactic acid (PLA) with different concentrations of hydroxyapatite (HA) by electrospinning, which were superficially modified with polypyrrole (PPy/I) by plasma polymerization. A preliminary study was conducted of the biological and mechanical behavior of the scaffolds when they were implanted in the back of rabbits for 30 days; bone cells differentiated from mesenchymal stem cells (MSCs) were used. The bone cell and scaffold structures were characterized by histological, immunohistochemical, and mechanical stress tests. Hematoxylin–eosin staining showed good tissue conformation. The immunohistochemical tests highlighted the presence of the main bone tissue proteins, such as collagen, osteocalcin, and osteopontin. The PLA/HA scaffolds were observed to exhibit cell adhesion and proliferation properties; however, the response was much better in the scaffolds that had a higher concentration of HA and that were coated with PPy/I. The results of the mechanical tests of the scaffolds indicated that the plasma treatment improved the adhesion and cell proliferation properties and contributed to the mechanical support, allowing the formation of neotissues with good viability of cell growth.     

Antiproliferative effect of extracts of <i>Sida rhombifolia</i> L. (Malvaceae) on the <i>Allium cepa</i> cell cycle

Field collected roots of four populations of Sida rhombifolia were used for preparing aqueous decoctions at two concentrations: 4g/L; and 16g/L. Afterwards, we used three groups of six onion (Allium cepa) bulbs for testing each population. Slides were made with all bulbs through the smashing technique. Cells in all phases of the cell cycle of A. cepa were analyzed. The mitotic index (% of cells in mitosis) was calculated, and the statistical analysis through the χ2 test was carried out at 5% probability. The results showed that the aqueous extracts of S. rhombifolia have antiproliferative activity at high concentrations. Practically no chromosomal aberrations were induced by treatments.     

Progresses of mycobacteriophage-based <i>Mycobacterium tuberculosis</i> detection

Tuberculosis (TB) remains a major cause of morbidity and mortality worldwide, particularly in developing countries. A rapid and efficient method for TB diagnosis is indispensable to check the trend of tuberculosis expansion. The emergence of drug-resistant bacteria has increased the challenge of rapid drug resistance tests. Due to its high specificity and sensitivity, bacteriophage-based diagnosis is intensively pursued. In this review, we mainly described mycobacteriophage-based diagnosis in TB detection, especially two prevalent approaches: fluorescent reporter phage and phage amplified biologically assay (PhaB). The rationale of reporter phage is that phage carrying fluorescent genes can infect host bacteria specifically. Phage amplified biological assay based on the principle that phages can infect the live Mycobacterium tuberculosis< in the specimen under suitable conditions and produce plaques. Other phage-based diagnostic methods, such as a combination of the amplified biologically assay and nucleic acid amplification or lateral flow assays, are also actively explored. This review will help us improve the understanding of mycobacteriophages in TB detection and better promote the development of the rapid diagnosis of M. tuberculosis.     

A 66-kDa protein of bovine hypophyseal <i>Pars tuberalis</i> induces luteinizing hormone release from rat <i>Pars distalis</i>

In this study, evidence for a factor secreted by bovine hypophyseal pars tuberalis that stimulates luteinizing hormone (LH) release from rat pars distalis cells is shown. The secretion products of bovine pars tuberalis cells into the culture medium were assayed on dispersed rat pars distalis cells in 30 min incubations and superfusion experiments. The culture medium from pars tuberalis total cell populations, added at a dose of 6 μg per tube, induced the greater LH release from pars distalis cells, without effect on follicle stimulating hormone (FSH) release. After pars tuberalis cells separation on a discontinuos Percoll gradient, only the culture medium of cells from 50 and 60% strength Percoll were able to release LH from rat pars distalis cells. Therefore, cell fractions from 50 and 60% strenght Percoll were cultured together. To elicit maximal LH release (6 times the basal output), with the addition of 2 μg of pars tuberalis protein was required, suggesting that these cells produce the factor or factors which affect pars distalis gonadotrope cells. After applying the pars tuberalis culture medium on 12% SDS-PAGE, the band with biological activity was that of 66-kDal. Fifty ng protein of its eluate released almost 9 times the basal output of LH from pars distalis cells. Results suggest a modulating effect of a protein from the bovine pars tuberalis on rat cultured gonadotrope cells from the pars distalis.     

The effect of natural products combination on MCF-7 cells exceeds tamoxifen therapeutic dose effects <i>in vitro</i>

Cancer remains to be one of the most severe sicknesses globally. Cases have kept rising over the years. Breast cancer (BC), which is among the leading types of cancers and predominantly affects women, is the second leading cause of cancer mortality. Researchers have developed interventions over the years; however, the BC survival rate has not improved since the 1980s. This has created the need for novel drug interventions that would manage and treat BC more effectively. This study focused on using a combination of natural product extracts such as phytoestrogen (Ziziphus jujube) and Tannin nanoparticles (NP99) together, which we have referred to as (Z.NP99) and tamoxifen (Tam) as one of the leading BC drugs since the 70s, in treating BC. The effectiveness of Tam if used alone in the treatment and if combined with Z.NP99 was evaluated using MCF-7 cells in vitro. The findings showed that the combination treatment of Z.NP99 affected the proliferation and viability of MCF-7 cells more than Tam at a 10 μg/mL dose. Moreover, Z.NP99 with Tam stimulated the maximum reduction of MCF-7 proliferation and viability in a time-dependent manner. Furthermore, Tam and Z.NP99 augmented the DNA fragmentation percentage combined with the upregulation of the apoptotic genes. Additionally, the results showed that the apoptotic impact of Z.NP99 and Tam on MCF-7 cells may be intermediated by down-regulating some genes such as Claudin-1 followed by down-regulating mRNA expression of MMP-9, VEGF, and BCL-2 genes of treated cells. Combining Tam with Z.NP99 considerably enhanced the effectiveness of conventional therapy. As a result, this study suggested that the Z.NP99 was ideal for developing effective natural treatments that would improve BC outcomes.     

Interaction of bifidobacteria with the gut and their influence in the immune function

Bifidobacteria are predominant in the lumen of the large intestine and confer various health benefits on the host. They are also used in the preparation of new fermented milks (bioyogurts) or added to conventional yogurt to generate probiotic effects. The colonization of the gut by bacteria tends to be host specific due partly to the way in which bacteria adhere to the intestinal wall. Using a homologous strain of Bifidobacterium animalis in an experimental mouse model, we analyzed by immunofluorescence labelledbacteria and transmission electronic microscopy the importance of this bacterial interaction with epithelial an immune cells associated to the gut, and the effect of feeding of B. animalis in the immune response. It was able to adhere and interact with both small and large intestine. In spite of this interaction with the gut, no modifications in the immune state (secretory or systemic response) were observed.
A heterologous strain of Bifidobacterium adolescentis from human faeces, was neither uncapable of binding to the intestine, nor influence the immune system activation, when it was administered during 2, 5 or 7 consecutive days; we believe that using a homologous strain, oral tolerance is developed even when the microorganism interacts with the immune cells associated with the intestine. However, we cannot ignore the beneficial effect of these microorganisms, especially in the prevention of intestinal infections. We think that this property exerted by bifidobacteria is more related to other mechanisms such as competitive inhibition, acid production or others, than enhancement of the immune state.     

Phytohormonal and metabolism analysis of <i>Brassica rapa</i> L. ssp. <i>pekinensis</i> with different resistance during <i>Plasmodiophora brassicae</i> infection

Clubroot of Chinese cabbage (Brassica rapa L. ssp. pekinensis), caused by the obligate parasite Plasmodiophora brassicae, accounts for serious yield losses. The aim of our study was to explore the phytohormone levels and metabolome changes in the roots of resistant and susceptible B. rapa genotypes at a late stage of infection, i.e., 28 days post-infection. Both genotypes showed decreased auxin levels after P. brassicae infection except for indole-3-acetic acid. Overall, the susceptible genotype had higher auxin and cytokinin levels after infection, with the exception of trans-zeatin and 3- indolebutyric acid as compared to the resistant genotype. Jasmonic acid levels declined after infection regardless of the genotype. Resistance against clubroot was evident with the increased levels of salicylic acid in the resistant genotype. The susceptible genotype had a higher number of differentially accumulated metabolites (DAMs) (262) than the resistant genotype (238) after infection. Interestingly, 132 DAMs were commonly detected in both genotypes when infected with the pathogen, belonging to metabolite classes such as phenolic acids, amino acids, and derivatives, glucosinolates, organic acids, flavonoids, nucleotides and derivatives, and fatty acids. The differential metabolite analysis revealed that metabolites related to amino acid biosynthesis, fatty acid biosynthesis and elongation, glutathione metabolism, and glucosinolate metabolism were highly accumulated in the resistant genotype, suggesting their essential roles in resistance against P. brassicae infection.