Ten sixty-week-old hens from each line were sampled for blood, brain, and cecal content. Neurotransmitters, cytokines, corticosterone, and heterophil/lymphocyte ratios were determined. Cecal microbiota compositions were determined by bacterial 16s rRNA sequencing, and functional predictions were performed. Our data showed that the central serotonin and tryptophan levels were higher in line 63 compared to line 72 (p < 0.05). Plasma corticosterone, heterophil/lymphocyte ratios, and central norepinephrine were lower in line 63 (p < 0.05). The level of tumor necrosis factor α tended to be higher in line 63. Faecalibacterium, Oscillibacter, Butyricicoccus, and Bacteriodes were enriched in line 63 birds, while Clostridiales vadin BB60, Alistipes, Mollicutes RF39 were dominated in line 72. From the predicted bacterial functional genes, the kynurenine pathway was upregulated in line 72. These results suggested a functional linkage of the line differences in serotonergic activity, stress response, innate immunity, and gut microbiota populations.The gut microbiota plays an important role in regulating brain function, influencing psychological and emotional stability. The correlations between conspecific aggression, gut microbiota, and physiological homeostasis were further studied in inbred laying chicken lines, 6 The gut microbiota plays an important role in the bidirectional interactions between the gut and brain. The microbiota–gut–brain axis involves multiple pivotal pathways, including the autonomic nervous system, the enteric nervous system, the neuroendocrine system, and the immune system ,2. The dClostridium, Ruminococcus, Blautia, and Lactobacillus have been identified to be able to degrade TRP to tryptamine, a monoamine that is structurally similar to 5-HT [One possible mechanism of gut microbiota influencing psychiatric health is through the tryptophan (TRP) and serotonin (5-HT) metabolism. The function of the central serotonergic system is highly correlated to depression, aggressiveness, impulsivity, and anger-related personality traits in both humans and animals ,9. The r to 5-HT ,25. Stud to 5-HT . Similar to 5-HT . This hy to 5-HT .Inflammation-associated brain dysfunction is another key factor associated with abnormal behaviors, including aggression . A positLactobacillus spp. as a therapy for feather pecking behaviors. The chicken has been used as a model in developmental biology for over a century for its advantages, such as high productivity, short breeding time, simple management, and a complete genome map [Intensive poultry production systems are continuously developed to meet the increasing food demands for an expanding human population ,41. Howenome map . As a re3) or susceptibility (line 72) to Marek’s disease  [3 birds, the line 72 birds have a higher number of CD4+ T cells but lower number of CD8+ T cells [2 expresses higher levels of both cytokines than the line 63 [2, chickens from line 63 showed fewer aggressive behaviors, including aggressive pecks and fights, as well as duration [Numerous studies have found that aggression is heritable ,54,55. DMI, USA) . The linMI, USA) ,58,59. C T cells ,61. In a line 63 . Line diduration ,63. The duration . The obj3 and 72, were used in the current experiment. Chickens from each line were raised in multiple hen colony cages and raised under the same conditions on the USDA, Agricultural Research Service, Avian Disease and Oncology Laboratory (ADOL) specific-pathogen-free farm at East Lansing, Michigan. All these chickens were managed following preapproved standard operating procedures established by the Institutional Animal Care and Use Committee .Two highly inbred lines, 6g for 20 min at 4 °C. The brain tissues were immediately put on dry ice, while the cecal content samples were snap frozen in liquid nitrogen. All the samples were stored at −80 °C until later analysis. Ten sixty-weekk old hens per line were randomly taken for sample collection. A 10-mL blood sample was collected from each chicken through cardiac puncture (n = 10 per group), following sodium pentobarbital sedation . After blood collection, the chickens were euthanized immediately via cervical dislocation. The raphe nucleus and cecal contents were collected following euthanasia. The blood samples were centrifuged at 700× g for 15 min at 4 °C. The supernatant of each sample was transferred to a new tube and mixed with the mobile phase  at a 1:1 ratio. The mixture was vortexed for 1 min and centrifuged at 18,187× g for 10 min at 4 °C. The supernatant was drawn and filtered through a 0.2 µm polyvinylidene fluoride filter into a sample vial. The mobile phase flow rate was 0.8 mL/min, and the cell potentials were set at 0 mV, 225 mV, and 550 mV. Each sample was tested twice at 10 µL per injection. Each monoamine concentration was calculated according to its corresponding standard and reported as ng/g.The monoamines of NEP, EP, TRP, 5-HT and 5-HIAA of the raphe nuclei were analyzed using high performance liquid chromatography , following previously described procedures . BrieflyThe plasma concentrations of 5-HT and TRP were detected in duplicate using commercially available enzyme-linked immunosorbent assay (ELISA) kits  by following the corresponding protocols. Briefly, 50 µL of standards or samples were added to each well of the antibody pre-coated plate, then 100 µL of horseradish peroxidase-conjugate reagent was added, and the plate was incubated for 60 min at 37 °C. After incubation, the plate was washed and 50 µL of chromogen solutions A and B were added. The plate was placed at dark for 15 min at 37 °C, and then the reaction was stopped by adding a stop solution. The optical density of each sample was read at 450 nm . All the samples were measured in duplicates with a CV ≤ 15% . The plasma levels of IL-6, IL-2, IL-10, tumor necrosis factor (TNF)- α , and immunoglobulin G (IgG)  were measured using the commercially available ELISA kits following each manufacturer’s protocol. The optical density of each sample was read at 450 nm . All the samples were measured in duplicates with a CV ≤ 15% [Plasma corticosterone (CORT) concentration was measured using a commercially available radioimmunoassay kit . The total plasma CORT concentration was analyzed in duplicate following the protocol described by . The H/L ratio was determined from 2 blood smears per bird. A thin layer of blood was applied on the surfaces of glass slides and then air dried overnight. The slides were stained with Wright’s staining solution. One-hundred cells, i.e., heterophils and lymphocytes, were differentiated and counted for each slide  using a microscope with 4000× magnification. The H/L ratio was calculated as the number of heterophils/the number of lymphocytes .The total bacterial community DNA from frozen cecal contents were extracted using a QIAamp Fast DNA Stool Mini Kit . Briefly, 200 mg of the cecal content sample was homogenized with 1 mL of InhibitEX buffer and 25 uL of proteinase K. The supernatant was mixed with buffer AL and incubated at 70 °C for 10 min. Then, the mixture was purified by adding buffer AW1 and AW2 and eluted in 200 uL of ATE buffer. The total DNA concentration was determined using NanoDrop .® 200 PRO, Tecan). Once quantified, the volumes of each of the products are equally pooled into a single tube. The pool was cleaned up using AMPure XP Beads (Beckman Coulter), and then quantified using a fluorometer . After quantification, the molarity of the pool was determined and diluted down to 2 nM, denatured, and then diluted to a final concentration of 6.75 pM with a 10% PhiX spike for sequencing on the Illumina MiSeq. The amplicons were then sequenced on a 151 bp × 12 bp × 151 bp MiSeq run, using customized sequencing primers and procedures [Sample 16S rRNA amplicon sequencing was performed by the Environmental Sample Preparation and Sequencing Facility (ESPSF) of Argonne National Laboratory . Specifically, the V4 region of the 16S rRNA gene (515F-806R) was PCR amplified with region-specific primers that included sequencer adapter sequences used in the Illumina MiSeq flowcell ,68. Briep ≤ 0.05, and a trend was reported when 0.05 < p ≤ 0.10. The physiological data were subjected to one-way ANOVA using the MIXED method of SAS 9.4 software , with the genetic line as the major factor. Data transformation (Box-Cox or log transformation) was performed to improve normality . The stap-values and Spearman p-values using the Benjamini–Hochberg procedure to control the false discovery rate (FDR). The output matrices were ordinated using principal coordinate analysis (PCoA) in R software. A differential relative abundance analysis of the microbiota composition was carried out with negative binomial regression, using the DESeq2 function within the Phyloseq package (Padjusted < 0.05), where negative and positive log2 fold changes were defined as corresponding groups, respectively [The 16S rRNA sequencing data  were analyzed using QIIME2 (version 2020.2) and R studio (version 1.3.1073). The fastq files of the sequenced data were de-multiplexed, quality filtered, and closed reference amplicon sequence variants (ASVs) picking was performed using DADA2, according to the QIIME2 pipeline . In briehttps://github.com/picrust/picrust2 (accessed on 1 December 2020)) was used to predict the functional profiles based on 16S rRNA sequences between the two genetic lines. The plugin q2-picrust2 was applied in QIIME2 (version 2019.10). MetaCyc pathway abundances and Kyoto Encyclopedia of Genes and Genomes (KEGG) Orthology (KO) were predicted based on the Kyoto KEGG database between the two strains and analyzed with the DESeq2 method (Padjusted < 0.05). All the sequencing data were visualized using the R package “ggplot2” [PICRUST2  (https:/ggplot2” .p < 0.05) positive and negative correlations are represented in red and blue, respectively.The relationships between the genus level of the microbiome (relative abundance > 1%), brain neurotransmitters, peripheral 5-HT and TRP, and CORT in each line were calculated for the Spearman’s rank correlation coefficients using the “psych” package in R , and the3-hens tended to have higher levels of EP . No difference was found in IL-6, IL-2, IL-10, and IgG (p > 0.05) between the lines  hallii group, Oscillibacter, Butyricicoccus, Anaeroplasma, Bacteriodes, Parasutterella, Peptococcus, Ruminiclostridium 5, Ruminococcaceae, Ruminococcaceae UCG-005, Ruminococcaceae UCG-008, Ruminococcaceae UCG-009 and Tyzzerella were more enriched in line 63, while Alistipes, Anaerofilum, Anaerostipes, Clostridiales vadinBB60 group, Clostridium sensu stricto 1, Dielma, Family XIII AD3011 group, Lachnoclostridium, Lachnospiraceae FCS020 group, Methanomassiliicoccus, Mollicutes RF39, Oscillospira, Romboutsia, Ruminococcaceae UCG-010, Ruminococcus 1 and Ruminococcus 2 were more enriched in line 72.At the genus level, a deeper analysis using DESeq2 identified 119 out of 993 ASVs that significantly differed between the line 6Fournierella (two out of three ASVs), Lachnospiraceae (nine out of thirteen ASVs), Ruminococcaceae UCG-014 (four out of seven ASVs), were enriched in line 63, while more ASVs from the genus Lactobacillus (three out of four ASVs), and Christensenellaceae R-7 group (two out of three ASVs) were more enriched in line 72. More ASVs from the genus 3 and 72 resulted in the identification of 57 metabolic pathways that differed between the lines of hens , tryptophanase , indolepyruvate decarboxylase  and monoamine oxidase  were detected more often, while aldehyde dehydrogenase  was increased in line 63 hens  and EP  were negatively correlated with the Clostridiales vadin BB60 group, which was the most prominent bacteria in line 72; and brain NEP was negatively correlated to Faecalibacterium , Fournierella , but positively correlated to Romboutsia .The correlations within and between the relative abundance of individual bacterial genera and concentrations of physiological relevant molecules  show thaThe gut microbiome plays an important role in regulating physiological and behavioral homeostasis . The dat3 and 72 chickens. The chickens from line 72 exhibited increased aggressive behaviors with lower brain 5-HT concentrations, compared to line 63 chickens [2 (high aggressive) had lower levels of 5-HT and TRP in the raphe nuclei than that of the line 63 hens (low aggressive). A similar genetic variant in brain monoamines was also reported between highly aggressive and non-aggressive mice, in which excessively aggressive mice had low levels of 5-HT in the prefrontal cortex [3 had higher plasma TRP than line 72. Tryptophan, the major precursor of 5-HT, can pass the BBB and be directly involved in brain 5-HT production [3 and line 72 hens. Because 5-HT cannot cross the BBB, whether the level of peripheral 5-HT contributes to aggression is unknown. In addition, the pathophysiological roles of 5-HT in behavioral and motivational regulation are different between the central and peripheral serotonergic systems [The implications of serotonergic functions in behaviors have been the subject of many studies in both humans and animals in the last century. The outcomes from multiple experimental methods, including neurosurgical lesion of the raphe nucleus, and pharmacological or genetic inhibition of 5-HT synthesis, reuptake, and binding capability of 5-HT receptors, indicate that the hypofunction of the central serotonergic system is associated with increased aggressive and impulsive behaviors ,78. Prevchickens ,54. In tl cortex . A similoduction ,80. The oduction ,82 and aoduction . Unlike  systems ,84. Cont systems . However systems  and in h systems . 3 and line 72 hens. Faecalibacterium, the most prominent genera that was enriched in line 63, has been associated with the behavior of the host. Patients suffering from bipolar depression and major depressive disorder (MDD) have reduced levels of Faecalibacterium [Faecalibacterium prausnitzii  was negatively correlated with the severity of depression between depressive and healthy subjects [Lachnospiraceae, another beneficial microbiota, which was also found in line 63 birds, and its function is closely related to 5-HT and TRP metabolism. A mouse model of autism with deficient TRP and 5-HT metabolism in the intestines has defined a significant decrease in Lachnospiraceae taxa [Lachnospiraceae abundance compared with the healthy controls [Lachnospiraceae and Faecalibacterium genera for the treatment of psychological disorders, including anxiety, depression, and autism [3, Eubacterium hallii group was also found to have a lower abundance in psychological disorder patients than healthy controls [Faecalibacterium and Butyricicoccus, are butyrate-producing organisms. SCFA producers can directly stimulate 5-HT secretion [3 hens, together with the fact that line 63 birds were less aggressive than line 72 [3 birds.The difference in TRP and 5-HT synthesis and metabolism between the two genetic lines could be attributable to the commensal microbiota variance. Approximately 90% of 5-HT is produced by the gut enterochromaffin cells (EC), and gut microbiota plays a crucial role in TRP synthesis, degradation and 5-HT production, which consequently impact peripheral and central 5-HT concentrations ,89,90. Aacterium ,92. In asubjects . More ASeae taxa . Other scontrols ,96,97. Md autism ,99. Othecontrols ,101,102.ecretion ,103,104. line 72 ; this co2, the more dominant genera, Anaerostipes, Clostridium and Lachnoclostridium are consistently found to be higher in MDD patients [Romboutsia is highly involved in metabolic capabilities, including carbohydrate utilization, single amino acids fermentation, and metabolic end products [2 birds. However, another dominated genus Clostridiales vadin BB60 group has been poorly classified for its biological effectiveness [Clostridiales vadin BB60 group is only defined to be associated with an increased risk of developing alopecia universalis, an autoimmune disorder [In line 7patients . The genproducts . The enrtiveness ,108. To 2 birds. In the current study, we observed that the line 72 hens had a higher plasma CORT and H/L ratio compared to 63 hens, the two common stress indicators used in avian species, which suggested an elevated stress status in the line 72 hens. The difference in the stress responding pattern is also indicated by the different changes in neurotransmitters, where the NEP level in the raphe nuclei was significantly higher in the line 72 hens, compared to the line 63. The activation of the HPA stimulates the synthesis of CRH and further regulates central NEP and peripheral CORT [Helicobacter and Streptococcus, and a decrease in anti-inflammatory bacteria, such as Roseburia and Lachnospiraceae species, are observed in individuals under stress conditions [Clostridiales VadinBB60 group (enriched in line 72) but a negative correlation with Faecalibacterium and Fournierella (both enriched in line 63).Elevated stress hormones likely also contribute to the lower serotonergic activity in the 7ral CORT ,110. Botral CORT ,112. Morral CORT . An incrnditions . In the Bacteroides fragilis directly influences the immune system, leading to systemic T cell deficiencies and Th1/Th2 imbalances in lymphoid tissues [Lactobacillus cripatus-based probiotic enhances the anti-inflammatory response of both macrophages and epithelial cells by releasing anti-inflammatory cytokine, IL-10, and reducing proinflammatory cytokines, TNF-α and IL-6 [Faecalibacterium prausnitzii administration increases the IL-10 level and reduces IL-6 as well as CORT levels in depression-like and anxiety-like rats [3 Anaeroplasma and Peptococcus were opportunistic pathogens, which elicited various host immune responses in human diseases [3-hens compared to the 72-hens. On the other side, the more abundant bacteria strains in the line 72 could have an important immunomodulation role in the intestinal mucosa, such as some beneficial Lactobacillus species [Lactobacillus rhamnosus and Lactobacillus gasseri have shown promising anti-inflammatory abilities by inhibiting proinflammatory cytokines [Lactobacillus strains. More research is needed to further identify the correlation between cytokine production and gut microbiota in the two genetic lines.The immune system plays an important role in both brain and the gut integrity and is involved in regulating the function of the microbiota–gut–brain axis via the immune-brain connections . The gut tissues . A Lactoand IL-6 . Faecaliike rats . Howeverdiseases ,125. In  species . For exaytokines ,128,129.3 and line 72 chickens. The majority of the pathways related to biosynthesis and energy metabolism were increased in relative abundance in line 72, which could contribute to the growth and production differences between the two genetic lines. Importantly, the pathway of TRP metabolism varied between the two lines. In line 72, the predicted abundance of tryptophanase (tnaA) and indolepyruvate decarboxylase (ipdC) genes increased, by which TRP can be catalyzed into indole, pyruvate, and or ammonium [2 fold increase of over 2 in the line 72 birds. These results indicate that there could be a higher degradation rate of TRP to kynurenines through the activated kynurenine pathway, rather than 5-HT synthesis [2 might be a possible factor that contributes to the higher aggression reported previously [2 birds in the current study. Moreover, monoamine oxidase (MAO) also increased in abundance in line 72; this could be one of the reasons that they have lower central and peripheral 5-HT levels. However, these hypotheses need to be tested in future studies. PICRUSt2 prediction indicates the different metabolic pathways involved in energy and nutrition metabolism between the line 6ammonium . In addiynthesis . The dysynthesis ,133. Theynthesis . Imbeaulynthesis  also repeviously . Additioeviously , which aThe current results discovered that the ubiquity of host-associated microorganisms indicate that microbes play an important role in chicken behavior, and chickens’ aggression could be reduced by modifying their microbial composition via the microbiota–gut–brain axis. However, the most behavioral differences found among chickens are affected by multiple factors, including the rearing systems. The different housing systems with inherently different designs cause chickens to exhibit different behaviors ,136. Thi2, the line 63 has inherently higher central tryptophan-serotonergic activities, lower stress hormones and increased certain major beneficial bacteria, such as Faecalibacterium and Lachnospiraceae. In addition, the metagenomic function predicted certain gut microbes that could be related to the tryptophan-serotonergic metabolic differences between the two lines. The findings of this study have provided new signals for developing novel biotherapy strategies for controlling aggression in laying hens by identifying gut bacteria and their metabolites correlated with behavioral repertoire.In conclusion, this work has identified the unique correlations of gut bacteria and related metabolic pathways with the different pattens of stress response, neurotransmitter expression, and innate immunity between the two diversely selected lines of chickens. Compared with the aggressive line 7


Chemotherapy-induced intestinal mucositis (CIM) is a significant dose-limiting adverse reaction brought on by the cancer treatment. Multiple studies reported that reactive oxygen species (ROS) is rapidly produced during the initial stages of chemotherapy, when the drugs elicit direct damage to intestinal mucosal cells, which, in turn, results in necrosis, mitochondrial dysfunction, and ROS production. However, the mechanism behind the intestinal redox system-based induction of intestinal mucosal injury and necrosis of CIM is still undetermined. In this article, we summarized relevant information regarding the intestinal redox system, including the composition and regulation of redox enzymes, ROS generation, and its regulation in the intestine. We innovatively proposed the intestinal redox “Tai Chi” theory and revealed its significance in the pathogenesis of CIM. We also conducted an extensive review of the English language-based literatures involving oxidative stress (OS) and its involvement in the pathological mechanisms of CIM. From the date of inception till July 31, 2021, 51 related articles were selected. Based on our analysis of these articles, only five chemotherapeutic drugs, namely, MTX, 5-FU, cisplatin, CPT-11, and oxaliplatin were shown to trigger the ROS-based pathological mechanisms of CIM. We also discussed the redox system-mediated modulation of CIM pathogenesis via elaboration of the relationship between chemotherapeutic drugs and the redox system. It is our belief that this overview of the intestinal redox system and its role in CIM pathogenesis will greatly enhance research direction and improve CIM management in the future. Chemotherapy-induced intestinal mucositis (CIM) is defined as an intestinal disorder caused by chemotherapy in ESMO  , 2. BaseThe following describes a typical CIM presentation in a five-phase model: (1) initiation, (2) upregulation and message generation, (3) signaling and amplification, 4) ulceration, and (5) healing  ulcerati. ExistinThis review has two segments, the first segment details intestinal physiological redox pathways, and the second summarizes available information on CIM. To reiterate, our first segment discusses the intestinal redox system, including the composition and regulation of redox enzymes, as well as reactive oxygen species (ROS) generation and its intestinal regulation. This section details the redox system-mediated modulation of the physiological intestinal function. The second segment discusses the redox system-mediated modulation of CIM pathogenesis via elaboration of the relationship between chemotherapeutic drugs and the redox system and the involvement of ROS in CIM development. We also discussed the significance of intestinal redox system in CIM.The digestive system is responsible for digesting and absorbing nutrients and possesses unique gut morphology. The interaction between intestinal flora and intestinal redox system is not discussed in this review. Since the intestine links an organism with its outside environment, it automatically facilitates protection against luminal toxic agents, which is also primarily done by the redox system. Thus, it is necessary to elucidate the composition and regulation of the intestinal redox system to explain the physical mechanism of the digestive system, particularly in terms of absorption and defense function.2) reduction to form superoxide (O2•−)  . Thus fae (O2•−)  8–10]. –10. 2) rα-helices, a flavin adenine dinucleotide- (FAD-) docking pocket, and a C-terminal NADPH-docking pocket. Multiple reports indicated that the NOX1 molecular mass ranges between 55 and 60 kDa . It. It2S2 lransfers , 38. Botneration .κB site, as well as TNF-α, interferon-γ, and interleukin-1 REs . T. T2O2 or2O2 and other soluble hydroperoxides [GPXs serve an antioxidant role at various locations and cellular compartments. They are widely expressed within the cytosol, mitochondria, and colon  122, 12, wherebyeroxides –127. Usieroxides . GPX2 iseroxides –131. GPXeroxides , and it eroxides . Severaleroxides . GPX4, meroxides , faciliteroxides . GPX5 iseroxides . This preroxides . GPX8 iseroxides , 138.Based on the above evidences, the most significant GPXs are GPX1, GPX2, and GPX3. The GPX2 transcript is commonly expressed in all GI epithelial cells , 139. Ho(Ser)Sec [GPX activity modulation, particularly selenoproteins, depends on the availability of selenium . Scienti(Ser)Sec  and a st(Ser)Sec . SECIS b(Ser)Sec . The euk(Ser)Sec . EIF4a3 (Ser)Sec .2O2, lipid peroxide, and peroxinitrite  that serves as the location of peroxide oxidation [P-SH oxidation to cysteine sulfonic acid (CP–SOH), which, in turn, interacts with a different cysteine residue, named “resolving” Cys (CR), to generate a disulfide, which undergoes subsequent reduction via an electron donor to end one catalytic cycle [R, PRDXs are stratified into typical 2-Cys, atypical 2-Cys, and 1-Cys PRDX subfamilies. PRDX1~4 is a member of the typical 2-Cys, PRDX5 aligns with atypical 2-Cys, and PRDX6 is with 1-Cys PRDX [The peroxide active site structure and sequence are highly conserved among all PRDX classes. It contains a conserved cysteine residue, designated as “peroxidatic,” Cys , and, therefore, intermolecular disulfide formation between these residues takes considerable time. As a result, the Cys51–SOH intermediate is sometimes oxidized to Cys–SO2H, prior to formation of a disulfide . Since sulfinic acid does not undergo reduction, Cys–SO2H-based overoxidation of PRDX enzymes remains catalytically inactive. The CDK- (cyclin-dependent kinases-) mediated phosphorylation of PRDX1, PRDX2, PRDX3, and PRDX4 at the conserved Thr89 residue reduces peroxidase activity of peroxiredoxins [89 phosphorylation abrogates enzymatic activity via disruption of the decameric conformation [The overoxidation of PRDX1~2 and phosphorylation of PRDX 1~4 are known to decrease peroxiredoxin activity, which, in turn, regulates enzyme activity . The conredoxins . Furtherormation . Multiplormation –168. Tarormation .2 and reduced NADPH, HO promotes heme degradation to iron, carbon monoxide, and biliverdin. The two reported HO isoforms are HO-1 and HO-2, and they both express in the ER. HO-1 is a 32 kDa heat shock protein, and it is sensitive to a myriad of toxic stimuli, in locations, such as the lung [2O2 [HO is an oxidase enzyme with varied function. In the process of hemoglobin catabolism, with sufficient molecular Othe lung , 171, lithe lung , 173, anthe lung –176. Mosthe lung , hyperoxthe lung , hypoxiathe lung , H2O2 [1ung [2O2 , hyperthung [2O2 , and endung [2O2 . HO-2 isung [2O2 .α-helices, with heme in between the distal and proximal helices [2 activation. HO is very similar to the globins in the O2-heme iron ligation, induced by an axial histidine ligand [This review focuses on HO-1, which is mainly located in the intestine . HO-1 is helices . The cone ligand , 188.κB/Rel protein family, which are two primary OS-inducible TFs in mammalian cells [HO-1 expression is modulated transcriptionally via porphyrins, metals, progesterone, other molecules, and it is expressed under OS, ischemia, hypoxia, and other disease conditions . The human cells .2 often reduces to free radicals . In contrast, H2O2 is the product of two-electron reduction. Since it is not a radical, it is chemically stable. Electronic excitation often includes a single molecular O2 and excited carbonyl compounds . Additional physiologically significant RS include chlorine and bromine species like hypohalous acids, hypochlorite, and chloramines. Free radicals, generated under both normal and pathological conditions, are extremely reactive and have an unsatisfied electron valence pair. These are very damaging to tissues, as is reported in cases of radiation, environmental chemical damage, and aging. Often, “free radicals” and “ROS” are utilized interchangeably. This may be correct in most cases, but, in certain instances, this terminology can be misleading.Of note, the terminology ROS should be appropriately named using the particular chemical species under consideration. O2·— is the most significant ROS, and it can produce other reactive O2 intermediates. The inner mitochondrial membrane (IM) harbors a multitude of enzyme complexes commonly known as the mitochondrial respiratory chain (MRC). The MRC contains complexes I-IV ), coenzyme Q (CoQ), and a peripheral protein cytochrome c located on the surface of the IM. MRC complexes I and III release elections that reduce molecular O2 to form O2·—[2 to two H2O molecules using a four-electron reduction [2·—to O2 [2, CCO is oxidized and uses ·NO. But, under reduced O2 levels, NO accumulates within the cell [Oform O2·—. CCO , LOOHs, and reactive nonradical compounds like singlet O2 (1O2), H2O2,and hypochlorous acid (HOCl) [2), and nonradical compounds like peroxynitrite (ONOO) and dinitrogen trioxide (N2O3).Indeed, not all free radicals induce OS. In fact, OS is intricately linked to ROS. ROS is extremely reactive, and it is constantly generated via cellular respiration and various enzyme reactions. Among common ROS are as follows: Od (HOCl) . RNS incA majority of the above compounds have poor stability due to the unpaired electrons in their outer electron orbit. Upon ROS accumulation, chief cellular antioxidants like GSH and thioredoxin undergo altered redox states, thereby decreasing antioxidant defenses. Therefore, based on the above review of intestinal oxidoreductases, RS are classified in 2O2 concentration of 0.1 μM can be considered as OeS or OS in different cellular states [Oxidative stress is an imbalance between oxidants and antioxidants in favor of oxidants that leads to the disruption of redox signaling and/or molecular damage . OS strar states .Under BOS conditions, OS is so negligible that it cannot be measured using traditional approaches. Augmented ROS dosages can trigger LOS, in which case, both oxidatively modified structures and endpoint parameters like ROS-driven ROS-sensitive parameters can be measured. LOS may be divided into two components, increasing and decreasing, after it passes maximum. Upon further dosage increases, cells enter the IOS and then HOS stage. In the HOS, both measured function plateau, meaning that all available substrates become potentially oxidized, thus achieving a near maximum response. The redox enzyme regulation is critical for the above complex processes. The specific modulation of each enzyme was described in the previous paragraph, and the regulation mechanism of redox enzyme is summarized below.μM), where GSH levels are high, due to elevated levels in the bile (1–2 mM in rat bile) [Under normal conditions, extracellular GSH concentrations remain quite scarce, other than in the intestinal lumen (60–300 at bile)  and dietat bile) . Luminalat bile) , 205. Daat bile) . Luminalat bile) . In ratsat bile) . Increasat bile) . Given tat bile) .μM and 8-10 μM, respectively, and are determined based on the Cys/CySS in the diet [The Cys/CySS redox couple, with partial help from the GSH system, modulates the extracellular/luminal redox environment . The plathe diet , GSH hydthe diet , thiol-dthe diet , and Cysthe diet . An oxidthe diet . Therefothe diet . Luminalthe diet  and lumethe diet . In rat the diet  and mucuthe diet , producethe diet  and inclthe diet , GSSG rethe diet , with suthe diet , intracethe diet , suggest2O2 does not have charge and is, therefore, optimal for redox sensing and signaling [2O2 reacts well with certain residues, for instance, some cysteinyl residues in peroxiredoxins or selenocysteinyl residues in GPXs (107/M/s) [2O2 has a sluggish reaction, it is able to diffuse further from the production site to react with targets some distance away. In contrast, highly reactive oxidants like hydroxyl radical exhibit a more localized reaction.Hignaling , 225. De107/M/s) . Also, s2O2 sensors and transducers. H2O2 is a crucial member of redox networks [2O2 receptor that particularly conveys oxidative equivalents to the redox-modulated TF STAT3 to generate a redox relay for the H2O2 redox network [2O2-mediated hyperoxidation of peroxiredoxin cysteinyl residues to sulfinic acid. This, in turn, inactivates the peroxidase. This event produces a large buildup of H2O2 at target sites, which then facilitates oxidation of targeted proteins [2O2 concentration range  [2O2 acts as an appropriate secondary messenger in redox signaling. Based on the conclusions of two studies, an intact liver H2O2 production rate is around 50 nmol H2O2 per min per gram, which is about 2% of the entire O2 uptake under steady conditions [Multiple studies suggested potential roles of thiol peroxidases as Hnetworks . For ins network . Anotherproteins . Sulfireproteins . Hence, , 10 nM) , 232, H2nditions .2O2, maintaining the redox balance is not simply by downregulating or upregulating its level or activity, otherwise known as “dynamic balance” or “homeostasis” which is more appropriate. Moreover, given the above factors, at cellular, tissue, and whole level, the “balance” alone cannot appropriately explain the mechanism of the intestinal redox system under physiological or pathological status.There are many theories aimed at generalizing the function and regulation of the intestinal redox system, such as the redox species balance regulated by the intestinal oxidoreductases involves in various physiological functions including absorption and defense , 235. BuTherefore, with reference to the concept of ancient Chinese medicinal theory, we proposed the “Tai Chi” theory of the intestinal redox system to explain the intestinal redox system Figures  and 3. TOur article selection process is detailed in supplemental information. Based on the module and search string in EMBASE Table , PubMed The existing literature and mainstream academic view is that ROS is produced rapidly in the initial stage as the chemotherapy drugs cause direct damage to intestinal mucosa cells, thus resulting in necrosis, mitochondrial dysfunction, and further ROS generation. Through our review of the intestinal redox system, we revealed that the intestinal redox enzyme system is very complex and is regulated by multiple factors. Thus, ROS production is not limited to the early stage but is present throughout the entire course of CIM. Therefore, the redox system is involved in all stages of CIM.Given that some chemotherapeutic drugs produce ROS using different redox enzymes, we first elaborated the mechanism of oxidoreductase and its modulation by chemotherapeutic drugs, in this review. In our analysis, we primarily selected articles that examined alterations in the redox enzyme level or activity as indicators of CIM severity. To circumvent limitations based on our literature selection, we also explored the structural and regulation characteristics of intestinal oxidoreductase to further elucidate their significance in CIM pathogenesis.2•−, H2O2, lipid peroxides, nitrate, and protein carbonyls, while diminishing GSH and NO. 5-FU also triggers an excessive NF-κB production, which modulates oxidoreductases to regulate CIM progression [κB axis in a RAGE-reliant fashion. This triggers glial cell to secrete iNOS-based NO and OS [κB levels [κB) [κB/Rel) [κB) [κB-related pathway.(5-) FU is a highly prevalent chemotherapeutic medication for the management of multiple forms of cancer . Unfortugression –243. In O and OS . Other rB levels –243, whiels [κB) , HO (NF-[κB/Rel) , and CATel) [κB) , 245 regAnother significant mechanism underlying 5-FU-based CIM is the Nrf pathway-mediated regulation of HO , 247. 5-5-FU is highly efficacious in boosting CIM-induced apoptosis. This is carried out via downregulation of Bcl-2 expression while upregulating Bax levels , 256–258MTX is a folic acid counterpart that suppresses dihydrofolate reductase enzyme activity. It is a highly efficacious chemotherapeutic drug against acute leukemia, trophoblastic disease, and intraosseous sarcoma, and it was recently employed as an antirheumatic drug . MTX indMTX also induces mitochondrial dysfunction and respiratory chain defects during CIM –267. It α transcript and protein expressions during CIM [α induces XDH activity in renal epithelial cells [κB site, possible TNF-α, interferon-γ, and interleukin-1 RE. Regrettably, even though we can infer that the activity of XDH, ROS, and TNF is increased in MTX-induced CIM, the direct or indirect relationship between them still remains under investigation.MTX strongly upregulates TNF-ring CIM –270. In al cells  and RAW al cells . Moreoveal cells . To idenMTX enhances PARP levels in the duodenum and ileum , with th9 on the histone stick model and the Lys37, and Arg40-associated histones showed the strongest binding between medication and protein. Additionally, the protein complex-based MTX-driven reduction in histone acetylation pattern also reduces HAT activity. Furthermore, acetylation on histone induces higher CAT mRNA expression [MTX also interacts with histone to form an MTX-protein complex that diminishes histone acetyltransferases (HAT) activity . MTX waspression , which, 2O2, and lipid peroxides, nitrate, while decreasing levels of GSH and NO , nature products, synthesis compound, food extract, probiotics, vitamin, amino acid, and others . UnfortuκB (in inflammation) and caspase-3 (in apoptosis) are the key target molecules influenced by the intestinal redox system, and the above treatments relieve CIM mainly via NF-κB and caspase-3 regulation. However, it is worth noting that the experimental evidence of the studies is not sufficient, particularly in terms of the validation of the pathway, and there is a lack of omics research as well. It is encouraging that there are more and more studies involving TCM, which also applies our “Tai Chi” theory to elaborate the intestinal redox system and its role in the short-term treatment strategies involving CIM.Based on our review of relevant literature discussing the mechanism of various CIM treatment strategies, these treatment strategies can be divided into 3 categories, according to the targets: proanthocyanidins, UFMG A-905, rutin, salecan, melatonin, and magnolol target redox and inflammation; lutein and pomegranate juice target redox and apoptosis; and lastly, vitamin E and FITOPROT target redox, inflammation, and apoptosis . As we dBased on the elaboration of the intestinal redox enzyme and its regulation mechanism, considering that the regulation of the OS level is not simply described as “increased” or “decreased,” and it is observed both inside and outside the cell, the intestine, and even the body, we preliminarily proposed the intestinal redox “Tai Chi” theory and attempted to reveal the role of OS events in the pathogenesis of CIM. However, current studies involving CIM employed oxidoreductases and reactive species as evaluation index of disease, which is inappropriate, as per our review. Moreover, the intestinal redox system participates in the pathogenesis and affects the prognosis of CIM, which is still worthy of in depth study.


Ischemic stroke (IS) is one of the most fatal diseases. Neuroimmunity, inflammation, and oxidative stress play important roles in various complex mechanisms of IS. In particular, the early proinflammatory response resulting from the overactivation of resident microglia and the infiltration of circulating monocytes and macrophages in the brain after cerebral ischemia leads to secondary brain injury. Microglia are innate immune cells in the brain that constantly monitor the brain microenvironment under normal conditions. Once ischemia occurs, microglia are activated to produce dual effects of neurotoxicity and neuroprotection, and the balance of the two effects determines the fate of damaged neurons. The activation of microglia is defined as the classical activation (M1 type) or alternative activation (M2 type). M1 type microglia secrete pro-inflammatory cytokines and neurotoxic mediators to exacerbate neuronal damage, while M2 type microglia promote a repairing anti-inflammatory response. Fine regulation of M1/M2 microglial activation to minimize damage and maximize protection has important therapeutic value. This review focuses on the interaction between M1/M2 microglia and other immune cells involved in the regulation of IS phenotypic characteristics, and the mechanism of natural plant components regulating microglia after IS, providing novel candidate drugs for regulating microglial balance and IS drug development. It is c22.1Following an ischemic attack, a series of events involving the central nervous system (CNS) is triggered . The patWhile all of the above processes occur in the vascular and perivascular spaces, ischemia can also affect the brain parenchyma. Following the impact of ischemia, a series of interrelated cytoplasmic and nuclear events, including bioenergetic exhaustion, excitotoxicity, Ca2+ overload, oxidative stress, and inflammatory responses, begin to occur at the damaged site, culminating in neuronal cell death . Excitot2.2After the occurrence of IS, the immune system starts rapidly, participates in all aspects of the occurrence, development and prognosis of stroke, and plays a corresponding role in different stages of stroke . Intrace2.2.1After the occurrence of IS, with the occurrence of intravascular hypoxia and changes in hemodynamics, platelets, coagulation and complement systems are activated, and the inflammatory response first occurs in the blood vessels . The oxi2.2.2After IS, while the immune system is activated, immunosuppression will occur at the same time. The systemic immune function is down-regulated within a few hours after cerebral ischemia, the cellular immunity is suppressed, the number of various immune cells such as monocytes, T lymphocytes, B lymphocytes and natural killer cells is decreased, apoptosis is increased, or cell dysfunction occurs . Meanwhi33.1Microglia account for approximately 10% of the CNS and are traditionally thought to function as immunocompetent cells of the brain and spinal cord, and undertake sensory functions of injury and infection in tissues . Microgl3.23.2.1M2-type microglia mainly regulate the repair of brain injury after IS. It mainly promotes the survival and recovery of injured neurons by secreting brain-derived neurotrophic factors, insulin secretion factors and transforming growth factors, and at the same time enhances the ability of neurons to withstand stimulation and damage , 71. It 3.2.2As the first line of defense of the immune system in the brain, microglia are rapidly activated within minutes of the acute phase of ischemic stroke, peak around day 2/3 of activation, and persist for several weeks after the onset of IS . At the At present, the main signal pathways that contribute to the polarization of M1/M2 microglia are as follows: (1) IFN-γ secreted by helper T cells 1 (Th1) induces the transformation of microglia into M1 phenotype by activating JAK1/JAK2 and STAT . (2) Ano3.2.3After IS, intracerebral microglia are rapidly activated within minutes of injury , while dIn vitro studies have shown that the interaction between RAGE and its ligand high mobility group box 1 (HMGB1) is critical for neuronal death induced by microglia activation (Many surface receptors involved in regulating the activation and function of microglia/macrophages have been found: (1) TLRs: TLRs represent a series of pattern-recognition transmembrane receptors that recognize relevant molecular patterns on the surface of pathogens. It is an essential component of the innate immune response of microglia and induces microglia to produce neurotoxic factors that contribute to the microglia response to neuronal damage , 93. Stutivation .3.2.4Microglia/macrophages are activated and polarized upon an ischemic injury, and the degree of polarization changes with pathophysiological conditions , 121. DiThe signaling molecules associated with M1 phenotype polarization mainly include the following: (1) NF-κB: NF-κB is a traditional transcription factor that is activated by LPS and regulates the expression of most M1 phenotype marker genes. Substantial evidence suggests that the NF-κB signaling cascade adversely affects cerebral ischemia because of its role in regulating pro-inflammatory mediators, including IL-1, IL-2, IL-6, IL-12, TNF-α, iNOS and cyclooxygenase-2 (COX-2) , 128. InThe signaling molecules associated with M2 phenotype polarization mainly include the following: (1) Peroxisome proliferator-activated receptor γ (PPARγ): In the inflammatory response, PPARγ can inhibit the inflammatory response by competitively inhibiting the inflammatory signaling pathway and the generation of inflammatory mediators. Among them, the crosstalk between Notch and NF-κB signaling pathway can inhibit the expression of PPARγ, which will decrease the expression of PPARγ after stroke, thereby aggravating the inflammatory response , 147. 2, while t3.33.3.1After IS, a large number of nerve cells die due to reduced blood flow and insufficient supply of glucose and oxygen. Dying neuronal cells release injury-associated ligands and excitotoxic glutamate to promote microglia/macrophage activation , thereby3.3.2In vitro studies found that CCL2 released by primary astrocytes contributed to the polarization of M1-type microglia. The proinflammatory mediator lysophosphatidylcholine (LPC) produced by neurons and astrocytes after IS stimulates microglia/macrophages to upregulate the mRNA expression of Mcp-1 and Ccr2, which is involved in mediating the inflammatory response after cerebral ischemia  Interaction between M1-type microglia and Th1/Th17: After ischemia, M1-type microglia can produce pro-inflammatory cytokines leading to BBB disruption . Both Th(2) Interaction between M2-type microglia and Th2/Treg: After cerebral ischemia, the expression of inflammatory mediators is up-regulated, which induces the accumulation of microglia to the injured area and breaks the dynamic balance between M1 and M2 types. The anti-inflammatory cytokines IL4 and IL-10 secreted by Th2 can further promote the polarization of microglia to M2 type , 185. Th44.14.1.1Gastrodia elata Bl  Gastrodin: Gastrodin is an organic compound extracted from the dried roots of elata Bl . Gastrod vertigo . Gastrod effects .(2) Malibatol A: Malibatol A is a natural resveratrol oligomer purified from the leaves of serrata with antioxidant activity. Yang et al.  found thIn vitro and animal experiments have shown that resveratrol has anti-oxidation, anti-inflammatory, inhibition of platelet formation, blood clot adhesion to the vascular wall, anti-cancer and cardiovascular protection ((3) Resveratrol: Resveratrol, a non-flavonoid polyphenolic organic compound, is an antitoxin produced by many plants when stimulated, with a chemical formula of C14H12O3. It can be synthesized in grape leaves and grape skins and is a bioactive component in wine and grape juice . In vitrotection –201. Resotection . In addiotection .(4) 6-Shogaol: 6-shogaol, an active substance isolated from ginger, has a variety of biological activities, including anticancer, anti-inflammatory and antioxidant. For example, 6-shogaol reduced diethylnitrosamine (DEN)-mediated elevation of serum aspartate aminotransferase and alanine aminotransferase and DEN-induced hepatic lipid peroxidation. The induction of Nrf2 and HO-1 by 6-shogaol was also confirmed in mice. 6-shogaol also restores the decreased activity of DEN and the protein expression of liver antioxidant enzymes such as superoxide dismutase, glutathione peroxidase and catalase in mice –206. 6-S(5) 6-Paradoll: 6-Paradoll reduces tMCAO-induced cerebral infarction, neurological deficit, and the inflammatory cascade in the ischemic brain, which is mainly mediated by inhibition of microglia/macrophage activation .Magnolia officinalis Rehd. et Wils. or M. officinalis Rehd. et Wils. var. biloba Rehd. et Wils ((6) Honokiol: Honokiol is derived from the bark, root bark and branches of  et Wils . Honokio et Wils , 211. Ho et Wils , which a(7) Indole-3-propionic acid: Indole-3-propionic acid treatment not only inhibited glial (astrocyte and microglia) activation in the ischemic brain, but also reduced lipid peroxidation, neuronal DNA damage. Its neuroprotective efficacy is mainly related to the fight against glial cell activation .(8) Paeonol: Paeonol is a monomer extracted from the dried roots of Paeonia suffruticosa Andr. or Cynanchum paniculatum (Bge.) Kitag., which has various pharmacological effects . Paeonol(9) Epigallocatechin gallate: Epigallocatechin gallate is a component extracted from green tea. It is the main active and water-soluble component of green tea and is a component of catechins . Catechi(10) Theaflavins: Theaflavins generally refers to tea yellow pigment, is a golden yellow pigment in black tea, is the product of tea fermentation. In biochemistry, tea yellow pigment is a class of polyphenols hydroxyl benzophenone structure of the material, with inhibition of inflammation, anti-oxidative stress and the neuroprotective effect –226. The(11) Propofol: Propofol reduces infarct volume and improves neurological function by reducing microglia/macrophage CD68 and Emr1 levels and inhibiting proinflammatory cytokines including TNF-α, IL-6, and IL-1β. Inhibition of microglial proinflammatory cytokine production during propofol-mediated MCAO contributes to neuroprotection against IS .(12) Probucol: The neuroprotective effect of probucol is related to its anti-inflammatory effect in microglia. It downregulates NF-κB, MAPKs and AP-1 signaling pathways in LPS-activated microglia to reduce inflammatory mediators such as NO, PGE2, IL-1β and IL-6. It also reduces iNOS, COX-2, IL-1 and IL-6 in the brains of MCAO mice .4.1.2Rheum palmatum L., has a wide range of pharmacological properties, including anticancer, hepatoprotective, anti-inflammatory, antioxidant and antibacterial activities ((1) Emodin: Emodin, an active ingredient extracted from the tivities . Previoutivities , 232 emotivities .(2) Chrysophanol: Zhang et al. found that Iba-1 positive cells in the cerebral ischemic penumbra of MCAO model rats increased significantly, and were amoeba-shaped or round; the neurological deficit score, the percentage of cerebral infarction and the relative expression of Notch-1, TNF-α and ICAM-1 proteins in the ischemic penumbra were significantly increased . However4.1.3Astragali radix. The main active ingredients in Astragali radix are astragalus polysaccharides, astragalussaponins and isoflavones. Astragaloside IV is mainly used as a quality control standard to evaluate the quality of Astragali radix ((1) Astragaloside IV: Astragaloside IV is an organic compound with a chemical formula of C41H68O14. It is a white crystalline powder and is extracted from the herbal medicine li radix , 237. Stli radix . In addili radix .(2) Cycloastragenol: Cycloastragenol is directly extracted from the dried roots of Astragalus membranaceus (Fisch.) Bge.var.mongholicus (Bge.) Hsiao or Astragalus membranaceus (Fisch.) Bge., or obtained by hydrolysis of Astragaloside IV , 241. It(3) Triptolide: Tripterygium is derived from the root bark of the traditional Chinese medicine Tripterygium wilfordii, which has anti-inflammatory, antioxidant and anti-cancer effects. Triptolide has been used in the treatment of various diseases, such as tumors [colorectal cancer , hepatoc(4) Artesunate: Artesunate is a derivative of artemisinin with high water solubility and can pass through the BBB for the treatment of cerebral and other types of severe malaria . ArtesunGlycyrrhizae radix et rhizoma, among which Neo-Minophagen C has anti-inflammatory effect, immunomodulatory effect, inhibitory effect on experimental liver cell injury, inhibition of virus proliferation and inactivation of virus ((5) Neo-Minophagen C: Glycyrrhizin is derived from the glycosides of of virus , 260. Neof virus .(6) Hyperforin: Hyperforin, as an active ingredient of Hypericum perforatum L, is used to alleviate mild to moderate depression and has a significant antidepressant effect . Further(7) Ilexonin A: Ilexonin A is a water-soluble compound, 3-succinyl-18-dehydroursolic acid disodium salt, prepared by succinylation of 18-dehydroursolic acid isolated from the rhizome or root bark of Ilex pubescens Hook.et Arn . IlexoniHuperzinaserrata (Thumb.) Trev. It is a potent cholinesterase reversible inhibitor. Its characteristics are similar to neostigmine, but the duration of action is longer than the latter ((8) Huperzine A: Huperzine A is an alkaloid extracted from e latter , 271. Hue latter . Huperzie latter . Huperzie latter .(9) Berberine: Berberine, a quaternary ammonium alkaloid isolated from COPTIDIS RHIZOMA, is the main active ingredient in COPTIDIS RHIZOMA for antimicrobial activity. Studies have shown that berberine regulates immune and inflammatory mechanisms , 275. BeSinomenium acutum (Thunb.) Rehd.et Wils  Sinomenine: Sinomenine is the main active ingredient isolated from .et Wils , which i.et Wils , 279. Sit stroke .4.1.4Epimedii folium, which is an 8-prenyl flavonoid glycoside compound ((1) Icariin: Icariin is the main active ingredient of compound . Icariincompound –284. Tancompound .in vitro and in vivo to combat focal cerebral ischemia. In the post-ischemic brain of mice challenged with tMCAO, eupatin significantly improved neurological function and reduced cerebral infarction. It also significantly reduced Iba1-immunopositive cells, microglia/macrophage proliferation, NF-κB signaling, IKKα/β phosphorylation, IκBα phosphorylation, and IκBα degradation in the tMCAO-attacked brain. This suggests its powerful effect on counteracting the inflammatory response of microglia/macrophages in the ischemic brain ((2) Eupatilin: Eupatilin, a lipophilic flavonoid isolated from ARTEMISIAE ARGYI FOLIUM, is a PPARα agonist with anti-apoptotic, anti-oxidant and anti-inflammatory effects –288. Eupic brain .(3) Heptamethoxyflavonoids: Heptamethoxyflavone protects neuronal cells from ischemia-induced injury by increasing BDNF production, CaMK II phosphorylation, and reducing microglia/macrophage activation .Scutellaria baicalensis Georgi ((4) Wogonin: Wogonin is a flavonoid extracted from the root of s Georgi . Wogonins Georgi , 293. Wos Georgi .(5) Puerarin: Puerarin is the main active ingredient extracted from Pueraria lobata (Willd.) Ohwi . It has (6) Quercetin: Quercetin is a naturally occurring phytochemical with good biological activity. It mainly exists in vegetables, fruits, tea and wine in the form of glycosides and has a healthy effect , 301. Th(7) Fisetin: Fisetin is a compound extracted from natural plants with pharmacological effects against different pathological processes . The conErigeron breviscapus (Vant.) Hand.-Mazz. It is composed of flavonoids, lignans, coumarins, terpenoids, phytosterols and other chemical components  Breviscapine: Breviscapine is the extract of mponents . Modern mponents –313. Bremponents . Brevisca/stroke .Oroxylum indicum (L.) Vent. It is mainly used for intervention in the treatment of hyperlipidemia, cardiovascular and cerebrovascular diseases, anxiety, inflammation, gout, cancer, muscle enhancement, etc. ((9) Chrysin: Chrysin is a flavonoid found in plant species such as nt, etc. –318. Chrnt, etc. .(10) Epicatechin: Epicatechin is a natural plant flavanol compound, chemical formula C15H14O6, with epigallocatechin, catechin gallate, epicatechin gallate, epigallocatechin gallate collectively referred to as catechin compounds , 321. Ep4.1.5(1) Salidroside: Salidroside is a phenylpropanol glycoside extracted from Rhodiola rosea L., which has good anti-inflammatory and antioxidant effects . Han et Forsythia suspensa (Thunb.) Vahl (Forsythia suspensa (Thunb.) Vahl and its constituents have significant effects in improving neurodegenerative diseases and other neuroprotection in the elderly ((2) Forsythin: Forsythin is the dried fruit of b.) Vahl , 329. Mob.) Vahl . Studies elderly . Meanwhi elderly .Panax L ((3) Ginsenosides: Ginsenoside is a steroid compound, also known as triterpenoid saponins, which mainly exists in Panax L . The expPanax L , 334. InPanax L , 335. ThPanax L . GinsenoPanax L .Kaempferol galanga L, which is widely found in various fruits, vegetables and beverages ((4) Kaempferol glycosides: Kaempferol is a flavonoid compound mainly derived from the rhizome of everages , 339. Iteverages –342. Kaeeverages .Paeonia albiflora Pall, P. suffersticosa Andr and P. delarayi Franch, and its content is high in P. lactiflora ((5) Paeoniflorin: Paeoniflorin is derived from the roots of ctiflora . Its phactiflora , 346. Pactiflora .4.1.6Ophiocordyceps sinensis (Berk.) G.H. Sung, J.M.Sung, Hywel-Jones & Spatafora as a lead compound; it is approved by the US Food and Drug Administration for the treatment of relapsing-remitting multiple sclerosis ((1) Fingolimod (FTY720): FTY720 is a high affinity agonist for sphingosine 1-phosphate (S1P) receptors and was developed from a sphingosine analogue extracted from clerosis . Among tclerosis . The theclerosis  found thclerosis , 352, thclerosis  found th(2) 3-N-butylphthalide (NBP): NBP is a compound isolated from celery seeds, and there are three types of derivatives: L-NBP, D-NBP and DL-NBP . L-NBP h(3) Danshenol bornanyl ester: Danshenol bornanyl ester is a new compound with anti-cerebral ischemia effect, which is designed and synthesized based on the active ingredient of Salvia miltiorrhiza Bge.and the effective structural fragment of borneol by using the principle of modern drug design , 358. Da(4) Arctigenin: Arctigenin is a lignan compound from Arctium lappa L., which can effectively inhibit the release of inflammatory factors. It inhibits the proliferation, migration and angiogenesis of human umbilical vein endothelial cells (HUVECs) induced by high glucose, and plays a protective and anti-oxidative stress role in HUVECs injury –362. ArcAcanthopanax sessiliflorus (Rupr.et Maxim.) Seem., the seeds or seed oil of Sesamum indicum DC., and the wood of Paulownia tomentosa (Thunb.) Steud. Its main pharmacological effects are inhibition of inflammation and anti-oxidative stress ((5) Sesamin: Sesamin mainly comes from the roots of e stress , 365. Cue stress .(6) Edaravone: Edaravone ameliorates cognitive decline and delays neuronal death after focal cerebral ischemia by inhibiting inflammatory biomarkers including iNOS, NO, ROS, IL-1β and TNF-α production. In addition, inhibition of inflammation, oxidative stress, and astrocyte activation are thought to be relevant mechanisms for the neuroprotective effect of edaravone against ischemic injury , 367.Ligusticum wallichii  kinase and 20K protein in platelets (via Nrf2/HO-1 ((7) Tetramethylpyrazine (Ligustrazine): Ligustrazine is an alkyl pyrazine extracted from allichii . It has allichii , 370. Liallichii . Ligustrlatelets . Ligustrlatelets . In regurf2/HO-1 .Cannabis sativa L ((8) Cannabidiol: Cannabidiol is an ingredient extracted from sativa L , 375. Atsativa L , 377. Casativa L .(9) Ligustolide: Ligustolide is the main active ingredient in the volatile oil of Angelica sinensis . The cur4.24.2.1Salvia miltiorrhiza Bge. Its main function is to improve the viscous state of blood, with blood circulation, blood stasis, and good clinical tolerance  F. H. Chen ex C. H., mainly containing ginsenoside Rb1, ginsenoside Rg1, notoginsenoside R1 and other components  in hippocampus and cortex, thereby reducing the concentration of inflammatory cytokines including VCAM-1, MMP-9 and MMP-2 and ICAM-1 of IS in a dose-dependent manner. In addition, it can inhibit the activation of MAPKs and NF-κB, thereby ameliorating the neuropathological changes induced by CIRI .4.2.5Fructus Gardenia is a TCM with various pharmacological effects, such as anti-inflammatory, antidepressant, improving cognition and ischemic brain injury. GJ-4 is a natural extract from Fructus Gardenia, GJ-4 can significantly improve memory impairment, cerebral infarction and neurological deficit in MCAO/R-induced vascular dementia (VD) rats. Further studies showed that GJ-4 attenuated VD-induced neuronal damage. In addition, GJ-4 can protect the synapses of VD rats by up-regulating the expression of synaptophysin, postsynaptic density 95 protein (PSD95) and down-regulating the expression of N-methyl-D-aspartate receptor 1 (NMDAR1). Subsequent investigations into the underlying mechanism found that GJ-4 could inhibit neuroinflammatory responses, supported by inhibiting microglia activation and reducing the expression of inflammatory proteins, ultimately exerting neuroprotective effects on VD .The structures of those components are shown in 5in vitro experiments, and the potential mechanism still needs further research. Apoptosis after IS involves many complex signaling pathways. Regulating the programmed death of neurons in the ischemic penumbra can save dying neurons to the greatest extent and promote the recovery of neural function. In addition, activation of SIPRs, TLRs, NLRPs, PPARs, and P2X7R may be the potential mechanisms for regulating microglia after IS, however, the mechanisms need to be further studied.Microglia play a biphasic regulatory role in ischemic stroke. After IS, microglia were activated, migrated, and exerted pro-inflammatory and anti-inflammatory effects through M1/M2 phenotype polarization, respectively, and at the same time played a protective role by inhibiting M1 phenotype polarization or promoting M2 phenotype polarization. However, the research on induced cell polarization is limited to animal experiments and Natural plant compounds may have great potential as therapeutic agents to reduce pro-inflammatory responses after cerebral ischemia. As can be seen from the foregoing, most of the current studies describe M1/M2 polarization, but most studies show a mixed signature of M1 or M2 microglial/macrophage phenotypes after cerebral ischemia. An urgent need to address is the need to define the M1 or M2 microglia/macrophage phenotype in experiments with these natural compounds. Furthermore, the markers currently used to differentiate M1 and M2 phenotypes are not microglia or macrophage specific. Therefore, technologies that can separate M1 from M2 microglia/macrophages (such as microfluidics and single-cell transcriptomics to identify novel microglia-specific biomarkers) are needed in the future.The limitation of this review is that this review focuses on the detailed mechanisms of microglia/macrophages in IS and the mechanisms of natural compound intervention, but lacks a summary of other immune cells such as T cells, B cells, and neutrophils in IS. Since immune cells such as T cells and B cells also play an important role in the pathophysiological process of IS, it is recommended to review the mechanism of these immune cells in IS in the future , 172.In summary, it can be seen that not only single-component natural botanical components can exert a therapeutic effect on IS by regulating microglia, but multi-component natural botanical components also show potential synergistic effects in regulating microglia. This suggests that the combination of natural botanical components to treat multiple inflammatory models (including macrophages and microglia) is a promising direction, and we can see that the combination of natural botanical components will be more effective to achieve the desired therapeutic effect.6Intervention of natural botanical components and their derivatives in microglia-mediated neuroinflammation in IS is a promising research direction. In the absence of effective neuroprotective drugs, we should pay more attention to the mechanisms of natural botanical components in regulating microglia-mediated neuroinflammatory diseases such as IS. Future research directions are suggested as follows: (1) Differentiate between microglia and macrophages through new techniques (such as single-cell transcriptomics and its derivatives) to more accurately determine the regulatory effects of natural compounds. (2) A more standardized and stable IS model is needed to determine the effects of drugs. (3) Using spatiotemporal omics to map the dynamic continuity state of microglia in natural botanical components intervening in IS. (4) Validate the long-term brain-protective effects of natural botanical components and the mechanisms regulating microglia in rodent and mammalian IS models. (5) Pay attention to the pharmacokinetics, pharmacodynamics, and toxicological properties of natural botanical components. (6) Explore the synergistic effect of natural botanical components in inhibiting neuroinflammation. (7) If all results are favorable, the next step is to conduct clinical trials of potential phytochemicals to investigate their neuroprotective effects on cerebral ischemia/stroke .TB, and JZ are responsible for the study concept and design. TB, JZ, KY, SW, WX, XZ, AG, LZ, JG are responsible for the data collection, data analysis and interpretation; JZ and KY drafted the paper; JG supervised the study; all authors participated in the analysis and interpretation of data and approved the final paper.


Several studies have been done to identify comorbidities of COVID-19. In this work, we developed an analytical bioinformatics framework to reveal COVID-19 comorbidities, their genomic associations, and molecular mechanisms accomplishing transcriptomic analyses of the RNA-seq datasets provided by the Gene Expression Omnibus (GEO) database, where normal and infected tissues were evaluated. Using the framework, we identified 27 COVID-19 correlated diseases out of 7,092 collected diseases. Analyzing clinical and epidemiological research, we noticed that our identified 27 diseases are associated with COVID-19, where hypertension, diabetes, obesity, and lung cancer are observed several times in COVID-19 patients. Therefore, we selected the above four diseases and performed assorted analyses to demonstrate the association between COVID-19 and hypertension, diabetes, obesity, and lung cancer as comorbidities. We investigated genomic associations with the cross-comparative analysis and Jaccard's similarity index, identifying shared differentially expressed genes (DEGs) and linking DEGs of COVID-19 and the comorbidities, in which we identified hypertension as the most associated illness. We also revealed molecular mechanisms by identifying statistically significant ten pathways and ten ontologies. Moreover, to understand cellular physiology, we did protein-protein interaction (PPI) analyses among the comorbidities and COVID-19. We also used the degree centrality method and identified ten biomarker hub proteins  that associate COVID-19 with the comorbidities. Finally, we validated our findings by searching the published literature. Thus, our analytical approach elicited interconnections between COVID-19 and the aforementioned comorbidities in terms of remarkable DEGs, pathways, ontologies, PPI, and biomarker hub proteins. The coronavirus disease 2019 (COVID-19) is an infectious disease that was caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). This disease was first reported in December 2019 in Wuhan, Hubei Province, China. At that time, this virus was transmitted directly from one person to another through direct contact or the droplets from the infected person's respiratory system . Also, tIn order to identify comorbidities associated with COVID-19, numerous studies have been conducted. Aktar et al. performed a meta-analysis on the published available global literature and used supervised machine learning algorithms on clinical cohort data with existing comorbidity information to identify significant comorbidities of COVID-19 . ChakrabIn this study, we have used the transcriptomic data derived from the blood samples of COVID-19 patients and healthy people. We analyzed the dataset with GREIN  tool andhttps://www.ncbi.nlm.nih.gov/) public Gene Expression Omnibus (GEO) genomics data repository. The dataset is an RNA-seq dataset prepared from human bronchial epithelial cells of COVID-19 patients and healthy people. GREIN is an interactive web platform that provides options to analyze GEO RNA-seq data for uniform processing [Homo sapiens. Considering all of the criteria, we selected the gestational hypertension dataset for hypertension which is derived from placenta cells of affected and control samples employing Illumina HiSeq 4000 technology. The dataset for diabetes is produced from peripheral blood mononuclear cells of healthy and diseased samples using Illumina HiSeq 2500 technology. The dataset for obesity is obtained by gene expression profiling and Illumina HiSeq 2000 technology from omental adipose tissues of control and case samples. And, the dataset for lung cancer is generated using Illumina HiSeq 2000 technology from human lung tissues of infected and uninfected samples. The summary of the datasets is presented in For the identification of comorbidities, we collected the SARS-CoV-2 (COVID-19) dataset (accession number GSE150819) from NCBI's  for both upregulated and downregulated genes in the diseases can be expressed mathematically as follows:Utilizing neighborhood flourished standard and topological approaches, we constructed bipartite graphs or networks for the gene-disease association where the nodes are genes (round shaped) or diseases  and validated the correlation among COVID-19 and its selected comorbidities. To participate in the network as well as the association or connection, the diseases (COVID-19 and the comorbidities) should have shared one or more significant DEGs within them . We consE) for each node pair through the Jaccard similarity index [The common adjacent neighbors and the interaction are recognized by measuring the edge score  database. We used the medium confidence score of 0.4 as a threshold for the PPI network construction and Cytoscape [i and protein j is mathematically expressed as follows:Ni and Nj denote a set of neighbors i and j.Mathematical and graphical illustration for the structural organization of proteins in a cellular unit is known as protein-protein interaction. To understand cellular/molecular physiology formed by complex biochemical functions and also for drug development, PPI is crucial , 34. Theytoscape  for the Hub proteins are proteins that are connected to several other proteins in a considerable number. They are potential biomarkers because during the development of COVID-19 and its comorbidities, hub proteins play a part in signal transmission as well as they may lead to effective remedy targets . We idenv, N is the total number of nodes in the network  G and avi is the direct connection between nodes v and i.where DC is the degree centrality of a node We also examined the interrelationships between the selected comorbidities. For that, we considered the comorbidities' frequent dysregulated genes or DEGs with COVID-19. Using Equations  and 3),,3), we iP value < 0.05 and |logFC| > 1 and downregulated DEGs were considered with a P value < 0.05 and |logFC| < −1. Our analysis detected total 1164 DEGs for COVID-19, where 576 were upregulated and 588 were downregulated. For hypertension, we identified total 890 DEGs, where 745 DEGs were upregulated and 145 DEGs were downregulated. For diabetes, we got total 897 DEGs, where 417 showed upregulation and the rest 480 showed downregulation. In obesity, we found 997 DEGs, of which 510 were upregulated, and 487 were downregulated. Similarly, in lung cancer, total 4759 genes were differentially expressed with 2504 upregulated DEGs, and 2255 downregulated DEGs. The summary of the DEGs is shown in For investigating the identification and genomic association of COVID-19 comorbidities, we used RNA-seq datasets from NCBI. We selected DEGs eliminating duplicate dysregulated genes for COVID-19 and all other comorbidities, where upregulated DEGs were detected with a statistical After doing all the computational and statistical analyses stated in the methods and analyses section, we picked top 50 diseases from the list of 7,092 diseases and removed homogeneous diseases. As a result, we obtained 27 divergent diseases which are breast cancer, liver cancer, colorectal cancer, prostate cancer, stomach cancer, lung cancer, melanoma, rheumatoid arthritis, ovarian cancer, tumor progression, glioma, Alzheimer's disease, obesity, asthma, glioblastoma, pancreatic cancer, atherosclerosis, hypertension, leukemia, diabetes mellitus, schizophrenia, neuroblastoma, degenerative polyarthritis, renal cell cancer, multiple sclerosis, systemic lupus erythematosus, and multiple myeloma, where hypertension, diabetes, obesity, and lung cancer are selected as comorbidities for further analysis. The selection criteria for the comorbidities are discussed in the methodology section. To prove the associativity, there must have at least one common gene between the two maladies. Therefore, we performed cross-comparative analyses for the shared DEGs identification among COVID-19 and its selected comorbidities, where identified shared DEGs are considered to impact COVID-19 severity. Our findings demonstrated that COVID-19 shared total 78 (70 up and 08 down), 48 (27 up and 21 down), 63 (41 up and 22 down), and 144 (71 up and 73 down) DEGs with hypertension, diabetes, obesity, and lung cancer. In the upregulated shared DEGs, 55 adjacent DEGs are shared only between COVID-19 and hypertension, and these are closely located and shown in We also calculated the Jaccard similarity index utilizing the DEGs (upregulated and downregulated) of COVID-19 and hypertension as 0.039, COVID-19 and diabetes as 0.024, COVID-19 and obesity as 0.030, and COVID-19 and lung cancer as 0.025. The interaction between two nodes can be measured using neighborhood similarity (Jaccard's similarity) . The higUtilizing molecular pathway and gene ontology enrichment analyses, we investigated the effective pathways and ontologies that have a direct or indirect association with the progression of COVID-19 comorbidities. The pathway enrichment analysis identified the pathways, “Immune System,” “Interferon Gamma signaling,” “T cell receptor signaling,” “Apoptosis,” “Tryptophan metabolism,” “Glycolysis/Gluconeogenesis,” “Matrix Metalloproteinases,” “Cytokine-cytokine receptor interaction,” “ECM-receptor interaction,” and “Protein digestion and absorption.” Again, “Inflammatory response,” “Response to lipopolysaccharide,” “Cytokine-mediated signaling pathway,” “Neutrophil degranulation,” “Neutrophil activation involved in immune response,” “Extracellular matrix organization,” “Extracellular structure organization,” “Integral component of plasma membrane,” “Collagen-containing extracellular matrix,” and “Synapse organization” are identified from ontology analysis based on the parameters described in the methodology section. Identified pathways and ontologies are shown in P value is < 1.0E-16. P value is 0.0332. P value is 1.5E-08. P value is 68, 78, and 6.85E-08. To understand cell physiology and the diseases resulting from abnormal PPIs, we investigated PPI analyses. We got highly interacting proteins by removing the disconnected proteins from the PPI analyses. P value for the network is < 1.0E-16. Our identified hub proteins are ranked according to their degrees as follows: IL1B (degree: 50), CXCL8 (degree: 38), FN1 (degree: 37), MMP9 (degree: 35), CXCL10 (degree: 32), IL1A (degree: 21), IRF7 (degree: 20), VWF (degree: 19), CXCL9 (degree: 19), and ISG15 (degree: 18). Identified biomarker hub proteins are shown in By analyzing the PPI network derived from the overlapping DEGs, we identified 10 significant biomarker hub proteins based on the degree centrality using the cytoHubba module. In the network, there are 174 proteins and 545 edges, where hub proteins are marked in a different color. The enrichment A further observation is that our final comorbidities are interacted with each other by exchanging the linking DEGs among themselves. Besides, their interaction, evolvement, and development are also documented in international publications. We ascertained that hypertension, diabetes, obesity, and lung cancer shared 1 DEG; hypertension, diabetes, and obesity shared 2 DEGs; hypertension, obesity, and lung cancer shared 1 DEG; diabetes, obesity, and lung cancer shared 2 DEGs. Again, hypertension and diabetes shared 3 DEGs; obesity and diabetes shared 2 DEGs; hypertension and obesity shared 4 DEGs; also, obesity and lung cancer shared 6 DEGs. Finally, hypertension and lung cancer as well as diabetes and lung cancer shared 7 and 9 DEGs. To determine a disease's comorbidities, several methods and analyses have been developed, including clinical tests and meta-analyses. But the procedures are difficult, exhausting, and time consuming. Therefore, our research is aimed at developing a bioinformatics and system biological framework to identify comorbidities and provide genetic and physiological insights related to COVID-19 along with their interrelationship.Based on clinical and etiological evidence, we found that our identified diseases, breast cancer , liver cWe identified significant overlapping DEGs among COVID-19 and selected comorbidities which verify the susceptibility of the comorbidities to COVID-19 progression and severity. Our distinct (CCAN) networks for the up and downregulated genes indicate that COVID-19 is linked to hypertension, diabetes, obesity, and lung cancer in In order to understand molecular mechanisms underlying COVID-19-associated comorbidities, pathway and ontology enrichments were investigated using shared DEGs. In our identified pathways, the “Immune System” is prominent in the development of hypertension . The “inBased on COVID-19 shared DEGs, we identified 10 highly connected biomarker hub proteins that are responsible for the emergence of selected comorbidities. We validated the emergence by literature review and found a strong affiliation between the hub proteins and the continuation of comorbidities. Interleukin 1 beta (IL-1B) is a member of the interleukin 1 cytokine family that increases hypertension and negatively impacts end-organs during high blood pressure . IL-1B iAgain, our identified significant linking DEGs linked the comorbidities together. We found that PBX2 linked hypertension, diabetes, obesity, and lung cancer with each other. Additionally, HSD11B1 and HLA-DRA linked hypertension, diabetes, and obesity; UCHL1 linked hypertension, obesity, and lung cancer; LENG8 and HSPA1L linked diabetes, obesity, and lung cancer. Besides, hypertension and diabetes are linked by CXCL8, HLA-B, and KANSL1; diabetes and obesity are linked by TRIM26 and MMP8; hypertension and obesity are linked by PLA2G7, FSTL3, STC2, and BCL2A1. Furthermore, PIK3C2G, FIBIN, CHRNA1, NPNT, MEOX1, and POU2F2 linked obesity and lung cancer, while PTPRQ, SSUH2, CILP2, CDH2, ABCA12, CPXM1, and L1CAM linked hypertension and lung cancer. Finally, we found ARG2, PTPRH, CBSL, GSTM1, FKBP5, MCEMP1, OSCAR, CHD5, and SPATA13, which linked diabetes and lung cancer. Public evidence also supports our findings and suggests that hypertension and diabetes have pathophysiological links ; diabeteThe above discussion implies that our identified results from the COVID-19 dataset have a significant association and influence on hypertension, diabetes, obesity, and lung cancer progression. Consequently, we conclude that they are COVID-19 comorbidities, and our framework is valid for comorbidity detection and evaluation.COVID-19 comorbidities increase disease severity and mortality as well as affect organ damage. Therefore, it is crucial for healthcare practitioners to develop an approach to identify comorbidities and explore their biomolecular mechanisms in order to come up with effective therapeutics for COVID-19. In this study, we explain how our methodology identifies COVID-19 comorbidities, explores genomic profiles, and highlights molecular insights and checkpoints related to potential biomarkers such as DEGs, pathways, ontologies, PPIs, hub proteins, and prognostic features relevant to COVID-19. However, it is still necessary to conduct in vitro and in vivo experiments. In fine, we suggest that our model can assist in comorbidity and biomolecular process diagnosis early of other diseases, if transcriptomic datasets are available, and thus, this model can minimize the financial burden on healthcare systems.


The deubiquitinase-mediated cleavage of ubiquitin chains from substrate proteins plays a crucial role in regulating protein degradation, activities, interactions, and localization. The dysregulation of multiple deubiquitinases has been implicated in various human diseases, especially cancer. More importantly, many small molecules targeting oncogenic deubiquitinases have been discovered, some of which have exhibited promising anti-cancer effects and entered clinical trials. Gastric cancer remains one of the most common and fatal malignancies. In this review, we aim to summarize the multifaceted roles of deubiquitinases in gastric tumorigenesis. We also present the upstream regulation of specific deubiquitinases and the research progress of several deubiquitinase-associated small molecules for gastric cancer therapy. Together, this review will improve our understanding of the biological role of deubiquitinases as well as the therapeutic potential of targeting deubiquitinases in gastric cancer.Gastric cancers (GCs) are malignant tumors with a high incidence that threaten global public health. Despite advances in GC diagnosis and treatment, the prognosis remains poor. Therefore, the mechanisms underlying GC progression need to be identified to develop prognostic biomarkers and therapeutic targets. Ubiquitination, a post-translational modification that regulates the stability, activity, localization, and interactions of target proteins, can be reversed by deubiquitinases (DUBs), which can remove ubiquitin monomers or polymers from modified proteins. The dysfunction of DUBs has been closely linked to tumorigenesis in various cancer types, and targeting certain DUBs may provide a potential option for cancer therapy. Multiple DUBs have been demonstrated to function as oncogenes or tumor suppressors in GC. In this review, we summarize the DUBs involved in GC and their associated upstream regulation and downstream mechanisms and present the benefits of targeting DUBs for GC treatment, which could provide new insights for GC diagnosis and therapy. Helicobacter pylori (H. pylori) and Epstein–Barr virus (EBV), also play a significant role in the initiation and progression of GC [Gastric cancer (GC) is the fifth most common cancer and the fourth leading cause of cancer-related mortality worldwide. Based on estimates from the GLOBOCAN database, more than 1 million new GC cases and an estimated 769,000 GC deaths occurred worldwide in 2020 . In addion of GC . Despiteon of GC ,5,6. TheDeubiquitinases (DUBs) are isopeptidases that can cleave a single ubiquitin or entire ubiquitin chains from target proteins, thereby counteracting protein ubiquitylation, a post-translational modification fundamental for the regulation of protein stability, activity, subcellular localization, and interactions ,8. DUBs During the past decades, many studies have demonstrated that multiple DUBs are implicated in the development of GC. In this review, we first summarize and discuss the DUBs related to GC, with a summary of their mechanisms of action and regulation . In addiUSPs constitute the largest family of human DUBs, with 56 members. As shown in E-cadherin and p14ARF [Studies have highlighted the role of USP3 in GC progression. USP3 is overexpressed in GC cells and tissues and can serve as a prognostic marker for patients ,21. USP3d p14ARF . HoweverUSP3 expression by sponging miR-224-5p [USP3 mRNA stability, but also bound to miR-1245b-5p to upregulate USP3 expression, thus leading to SNAIL1 stabilization and inducing the malignant transformation of gastric mucosa cells [Additionally, non-coding RNAs, including circular RNAs, long non-coding RNAs (lncRNAs), and microRNAs (miRNAs), play critical roles in modulating GC ,117,118.R-224-5p . In addiUSP7, also called herpes-virus-associated ubiquitin-specific protease, deubiquitinates many substrate proteins involved in the cell cycle, DNA damage responses, and immune responses ,120. AccH. pylori decreases the expression and activity of USP7 in infected GC cells. However, the exact function of H. pylori in regulating USP7 expression remains unclear [To better understand the USP7 binding network in tumor cells, Anna et al. performed affinity purification coupled with mass spectrometry to identify interactions in GC cells overexpressing USP7. In addition to several reported binding proteins (such as USP11 and TRIP12), this study also identified DHX40 and DDX24, two DEAD/DEAH-box RNA helicases, as novel targets of USP7, providing preliminary evidence that USP7 may regulate RNA metabolism . Further unclear .ALOX15 expression and lipid peroxide accumulation, inhibited ferroptosis, and ultimately lead to acquired chemoresistance in GC cells [In addition, Zhang et al. found that USP7 stabilizes hnRNPA1 in cancer-associated fibroblasts (CAFs) to contribute to the packaging and release of exosomes containing miR-522. Paclitaxel and cisplatin also promoted miR-522 secretion by activating the USP7/hnRNPA1 axis. The lipoxygenase ALOX15 is important in mediating the lipid peroxidation that drives ferroptosis. Further, exosomal miR-522 repressed GC cells .USP9X represses the migration, invasion, and colony formation ability of GC cells. Moreover, hsa_circ_0008434, a miRNA sponge for miR-6838-5p, increased USP9X expression and promoted GC progression [USP9X has been shown to function as either an oncogene or tumor suppressor, depending on the type of cancer . Increasgression . Hippo signaling has been implicated in regulating cell growth, metastasis, and chemoresistance in GC . As the USP49 silencing decreases cell proliferation, migration, and invasion and enhances GC cell sensitivity to chemotherapy; however, this effect can be reversed by YAP1 overexpression. Because USP49 is a target gene modulated by YAP1/TEAD4, this result indicates that USP49 and YAP1 form a positive feedback loop to support the malignant progression of GC [Two studies have suggested that DUB1 and USP49 also contribute to GC development by regulating the Hippo signaling pathway ,67. DUB1USP10 plays a dual role in different human cancer types . Zeng etAs a master transcriptional factor that induces EMT, Snail is frequently overexpressed in tumors and drives tumor progression, cell survival, metastasis, and stem cell properties . E3 ligaUSP14 has been reported to be an oncogene in GC [USP14 depletion did not lead to cell death but sensitized GC cells to cisplatin-induced apoptosis [6-methyladenosine (m6A) plays a crucial role in modulating mRNA processing; studies have indicated that dysregulated m6A regulators are involved in cancer progression [6A reader YTH N6-methyladenosine RNA binding protein 1 (YTHDF1) promotes gastric carcinogenesis by modulating the translation of FDZ7, a key Wnt receptor [6A-dependent manner, and USP14 overexpression reversed the tumor-suppressive effects elicited by YTHDF1 silencing [USP14 is a proteasome-associated DUB that plays a dual role in regulating protein degradation. Although it cleaves ubiquitin, it also promotes protein degradation by activating the proteasome ,138. USPne in GC . USP14 lne in GC . In one ne in GC . Howevergression . The m6Areceptor . YTHDF1 ilencing . Additioilencing ,109. TogUSP15 knockdown attenuated the activity of Wnt/β-catenin signaling and inhibited GC progression both in vitro and in vivo, but the mechanism through which USP15 regulates this pathway remains unclear [The role of USP15 in GC is less defined and contradictory. Zheng et al. suggested that the ectopic expression of USP15 could suppress GC cell growth, migration, and invasion through the deubiquitination of IκBα by the COP9 signalosome (CSN) complex, thereby hindering NF-κB activity ,142. In  unclear . As revi unclear . FurtherUSP22 has been identified as producing cancer-stem-cell-like qualities, including aggressive growth, metastasis, and therapy resistance . Unlike USP22 siRNA-loaded nanoliposomes modified with anti-CD44 (USP22-NLs-CD44), which targets a stem-cell-associated marker. USP22-NLs-CD44 impaired tumorsphere formation and reduced the percentage of CD44+ cells in two human GC cell lines, MKN45 and NCI-N87, presenting an approach to eliminating GC stem cells [To target these USP22-regulated stemness properties, Yang et al. constructed em cells . USP22 is a GC oncogene, a meta-analysis noted contradictions between USP22 expression, tumor size, differentiation state, tumor stage, and clinical outcomes in patients with gastric tumors. However, these inconsistencies may be due to the small sample size, suggesting that the role of USP22 should be analyzed in larger randomized controlled clinical trials [Although the aforementioned studies indicate that l trials . Moreovel trials ,50,51,5250 of 1.10 ± 0.02 μM and induced its degradation at higher doses, thereby exhibiting cytotoxic effects against GC cells [Digitalis lanata. They also showed that lanatoside C partially promoted c-Myc degradation by reducing its interactions with USP28 [USP28 has been validated as an oncoprotein and therapeutic target in various cancer types, and a list of oncogenic substrates of USP28 have been reported, such as JUN, NOTCH1, CCNE, and LSD1 ,152. ForGC cells . In addiGC cells . Hu et ath USP28 . These sTre2 (USP6) oncogene [USP32 was reported to be one protein that contributed to the chimeric oncogene . The prooncogene ,156. Addoncogene . HoweverUSP33 expression is reduced in GC tissues and cell lines, which correlates with poor patient survival ,57. AddiUSP39 expression hinders MGC-803 GC cell proliferation and induces G2/M arrest and PARP cleavage [USP39 knockdown also decreased the proliferation of MGC-803 and HGC-27 cells. Moreover, miR-133a can suppress USP39 expression by directly targeting its 3′ UTR, thereby acting as a tumor suppressor in GC [USP39 depletion inhibits GC proliferation require further investigation.Although USP39 was initially identified as a DUB without ubiquitin hydrolysis activity, other studies have suggested that it plays a role in RNA splicing and oncogenesis in multiple malignant tumors ,158. Intcleavage . In lineor in GC . HoweverUSP44 is regulated by the circFOXO3/miR-143-3p axis in GC, in which circFOXO3 functions as a miR-143-3p sponge to promote USP44-directed malignancy in gastric carcinoma [Aneuploidy is a common characteristic of tumor cells . Severalarcinoma .USP47 is inhibited by miR-204-5p, which represses GC cell proliferation [USP47 knockdown sensitized NCI-N87 cells to camptothecin- or etoposide-induced apoptosis [feration . Interacferation ,165,166.H. pylori, EBV, and CMV infections and CYLD hypermethylation and downregulated protein expression, suggesting that infectious-agent-induced CYLD hypermethylation may be a significant mechanism of GC development [CYLD expression by targeting its 3′-UTR, thus promoting GC cell proliferation, migration, invasion, and treatment resistance [OTUD7B, and TAX1BP1 [CYLD mRNA. However, miR-505 can be sponged by CRAL and increase CYLD levels, which decreases AKT signaling and increases treatment susceptibility in GC cells [CYLD was initially described in cylindromatosis and is currently considered a tumor suppressor that negatively regulates multiple signaling pathways, such as NF-κB, AKT, and Wnt ,168,169.elopment . Severalsistance ,72,75,76sistance . NotablyGC cells . FurtherGC cells . Taken tIn addition to the USPs mentioned previously herein, there are several other GC-associated USPs, including USP1, USP2, USP20, USP21, and USP42. USP1 levels were increased in GC cell lines and clinical samples, and its overexpression confers poor patient survival rates. USP1 knockdown inhibits their proliferation, migration, and invasion ,18. MechUSP42 knockdown inhibited GC cell survival and invasion, suggesting that USP42 may be a therapeutic target [USP20 promotes the tumorigenesis of several cancer types, such as breast and cervical cancer; however, it exhibits inhibitory effects on GC . Past rec target .UCHL1 promoter hypermethylation is a common event in several types of primary digestive tumors, including GC [UCHL1 hypermethylation is more frequent in advanced-stage GC [UCHL1 expression in liver metastases from GC and gastric cardiac adenocarcinoma, likely because UCHL1 overexpression increases the proliferation, migration, and invasion capabilities of GC cells [All members of this family have been reported to be associated with GC . UCHL1, uding GC ,80,81,82stage GC ,81. In astage GC . These sGC cells ,84. LastGC cells . Thus, tSimilar to USP14 and PSMD14, UCHL5 is also a proteasome-associated DUB. Higher UCHL5 expression is a predictor of increased survival in a subgroup of patients with early-stage GC . HoweverOne recent study reported that UCHL3 promotes GC cell migration and invasion by upregulating IGF2 . In addiThree members of this family, including OTUB1, OTUB2, and A20, have been linked to GC . Weng etA20 downregulation suppresses the proliferation, migration, and invasion of MGC-803 GC cells [A20 was found to be the target of miR-200a in GC cells (MGC-803 and SGC7901) resistant to TRAIL-induced apoptosis. Overexpressing miR-200a or depleting A20 enhanced apoptosis by reducing RIP1 polyubiquitination and promoting caspase-8 cleavage [A20 upregulation during H. pylori infection, mainly because USP48 stabilizes nuclear RelA and promotes its transcriptional activity [H. pylori infection and suggested an oncogenic role for A20. Conversely, one study reported that H. pylori infection increased miR-29a-3p, which promoted the migration of gastric epithelial cells by reducing A20 expression, indicating that A20 silencing may induce EMT to promote GC progression [A20, also known as TNFAIP3, is a ubiquitin-editing enzyme with both DUB and E3 ligase activities . A20 expGC cells . In addicleavage . These rcleavage . Later scleavage . Moreovecleavage . Consistactivity ,96,97,98gression . Thus, tThree members of this family, PSMD14, CSN5, and BRCC3, have been found to be associated with GC . PSMD14,CSN5 suppresses GC cell growth and induces apoptosis by regulating P53 and BAX expression [CSN5, the catalytic subunit of the COP9 signalosome, also called COPS5 or JAB1, may play a role in GC . CSN5 ovpression . Additiopression . CSN5 alpression . Previoupression ,184. CSNpression . CollectBRCC3 expression, thereby activating the PI3K/Akt/mTOR pathway, which leads to malignancy [Lastly, Hu et al. reported that BRCC3 is upregulated in GC and is regulated by the lncRNA TMPO-AS1/miR-126-5p axis. TMPO-AS1 sponges miR-126-5p to upregulate lignancy .In the MJD family, only Ataxin-3 has been reported to be associated with GC tumorigenesis. Ataxin-3 levels were found to be decreased in GC tissues and cells, which correlated with clinicopathological characteristics, including tumor size, Lauren classification, histologic differentiation, and p53 mutation status . HoweverIn the past few years, DUBs and tumorigenesis have been linked. Here, we summarize the regulatory roles of DUBs in the occurrence and development of GC. As shown in In addition, DUB regulation is complex. Although the regulatory mechanisms of some GC-related DUBs remain to be discovered, non-coding RNAs have been reported to modulate the expression of DUBs, such as USP3 and CYLD, in GC . IntriguFinally, research on the therapeutic potential of DUB inhibitors for GC therapy is limited despite the efforts made in their development. Some inhibitors show promising therapeutic effects in various cancer types ,185. For