Macrophages are innate immune cells in the organism and can be found in almost tissues and organs. They are highly plastic and heterogeneous cells and can participate in the immune response, thereby playing a crucial role in maintaining the immune homeostasis of the body. It is well known that undifferentiated macrophages can polarize into classically activated macrophages (M1 macrophages) and alternatively activated macrophages (M2 macrophages) under different microenvironmental conditions. The directions of macrophage polarization can be regulated by a series of factors, including interferon, lipopolysaccharide, interleukin, and noncoding RNAs. To elucidate the role of macrophages in various autoimmune diseases, we searched the literature on macrophages with the PubMed database. Search terms are as follows: macrophages, polarization, signaling pathways, noncoding RNA, inflammation, autoimmune diseases, systemic lupus erythematosus, rheumatoid arthritis, lupus nephritis, Sjogren's syndrome, Guillain-Barré syndrome, and multiple sclerosis. In the present study, we summarize the role of macrophage polarization in common autoimmune diseases. In addition, we also summarize the features and recent advances with a particular focus on the immunotherapeutic potential of macrophage polarization in autoimmune diseases and the potentially effective therapeutic targets. Autoimmune diseases are chronic, refractory clinical common diseases that are mainly stimulated by immune, hormonal, environmental, and genetic factors . To dateγ; can secrete inflammatory factors such as tumor necrosis factor-α (TNF-α), interleukin(IL)-1β, and IL-6; and can generate a large number of reactive oxygen species (ROS) and reactive nitrogen species (RNS), killing invading pathogens, phagocytose, and clear senescent, damaged, and degenerated cells [β (TGF-β); can also produce vascular endothelial growth factor (VEGF) and platelet-derived growth factor (PDGF); can inhibit T cell proliferation and activation; and can participate in T helper (Th) 2-type immunity response, contributing to tissue repair and angiogenesis [Clear and convincing evidence has shown that macrophages are highly plastic and can polarize into different types of macrophages under different microenvironment conditions: classically activated macrophages (M1) and alternatively activated macrophages (M2), a traditional classification, and a simplified, old classification method. Among them, we prefer to interpret M2-type macrophages as activated macrophages other than M1. M1 can be stimulated and activated by lipopolysaccharide (LPS) and interferon- (IFN-) ed cells , 8. M2 cogenesis , 10. Howogenesis . Fibrosiogenesis . ECM canogenesis . Thus, mogenesis , of whicogenesis .Macrophage polarization is a dynamic and reversible process involved in the occurrence and development of many autoimmune diseases, such as uveitis, systemic lupus erythematosus, rheumatoid arthritis, and Sjogren's syndrome –19. StudA search strategy was performed to extract the available literature using the PubMed database. The search terms “macrophages,” “polarization,” “signaling pathways,” “noncoding RNA,” and “inflammation” combined with terms like autoimmune diseases such as systemic lupus erythematosus, rheumatoid arthritis, lupus nephritis, Sjogren's syndrome, Guillain-Barre syndrome, and multiple sclerosis were searched. Original researches, including prospective and retrospective studies and review papers, were included and cross-referenced.In 1908, Nobel Prize winner Ilya Metchnikov discovered cells with phagocytosis from single-celled organisms to vertebrates and called them macrophages . ThroughAccording to the distribution of different physiological locations, macrophages are mainly found in lymph nodes, alveolar walls, bone marrow, liver, spleen, and other organs. They can be divided into subgroups, including microglia in the central nervous system, osteoclasts, alveolar macrophages in the lung, histiocytes in the spleen and interstitial connective tissue, and Kupffer cells in the liver . Macrophα (HIF1-α) expression, and decrease mitochondrial complexes I and IV of OXPHOS, which in turn affect the reprogramming of macrophages in glucose metabolism [Shifts in cellular metabolism are closely related to phenotypic and functional changes in macrophages. M1-type macrophage metabolism mainly depends on glycolysis, followed by the pentose phosphate pathway (PPP), truncated tricarboxylic acid cycle (TCA cycle), and dysfunctional mitochondrial oxidative phosphorylation (OXPHOS) . In conttabolism . Therefotabolism . Itaconatabolism –38. Accotabolism .γ, TNF-α, and granulocyte-macrophage colony-stimulating factor (GM-CSF) stimulation activate M1 macrophages  [β, IL-23, IFN-γ, and TNF-α, producing a large number of ROS and inducible nitric oxide synthase (iNOS) [β (TGF-β), peroxisome proliferator-activated receptor γ (PPARγ), arginase 1 (Arg1), Fizz1, and Ym1 [Mature macrophages undergo phenotypic and morphological differentiation under various factors  and termed macrophage polarization. In the early stage, macrophages are mainly divided into classically/inflammatory activated macrophages (M1 macrophages) and alternatively/wound healing-activated macrophages (M2 macrophages), which are the two terminal states of macrophage polarization depending on their polarization status and function. Later, some scholars referred to the basic principle of the three primary colors in the color wheel and divided macrophages into classically activated macrophages, wound healing macrophages, regulated macrophages, and hybrid macrophage groups with the characteristics of the above two macrophages at the same time . But in nd CD38) , and M1 e (iNOS) –45. There (iNOS) , 47. Proe (iNOS) , 49. In  and Ym1 –52. M2 mβ, and IL-10 can induce the production of M2c, which is characterized by CD163 on the surface and secretes IL-10 and TGF-β1 and participates in inhibiting the immune response and tissue remodeling [β1 positively correlate with M1 polarization in vivo but do not increase in vitro classically activated macrophages [M2a induced by IL-4 and IL-13 is called wound healing macrophage, which highly expresses CD206, IL-1R, and CCL17 and secretes fibronectin and other fibrogenic molecules, promoting tissue repair and remodeling , 53–56. modeling , 59. TLRmodeling –64. Howerophages . Those cγ, LPS, IL-4, TNF-α, GM-CSF-1, and M-CSF, noncoding RNAs can also regulate macrophage polarization. Noncoding RNA refers to RNA that participates in the transcription process of genes but does not translate proteins, mainly including microRNA (miRNA), long noncoding RNA (lncRNA), and circular RNA (circRNA) [In addition to IFN-circRNA) . NoncodimiRNAs are evolutionarily highly conserved noncoding RNAs with a length of about 22 nucleotides involved in immune responses and tumor growth regulation. Currently, a large number of miRNAs have been identified, of which some can regulate macrophage polarization, such as miR-156a, miR-33, miR-let-7a, miR-223, miR-155, miR-21, miR-125a, miR-19b-3p, miR-100-5p, miR-654, and miR-221-3p –79, as slncRNAs are non-protein-coding RNAs with a length of more than 200 nucleotides, which are involved in cell differentiation and proliferation, cycle regulation, tumor development, and other pathophysiological processes. Macrophage polarization is also affected by some lncRNAs, such as ANCR, Mirt2, HITT, GAS5, and RN7S –86, as sIn addition, circRNAs are a new hotspot in the field of RNA research and are a special noncoding RNA. They have also been confirmed in the latest study to promote macrophage polarization to M2 and thus participate in the regulation of disease. circSAFB2 mediates M2 macrophage polarization through the miR-620/JAK1/STAT3 axis , 88, whiAn in vitro study confirmed that lncRNA-GAS5 expression is downregulated in SLE ; meanwhiα (SIRP-α) to promote M2-type macrophages, and sustained M2 macrophage infiltration promotes renal fibrosis. However, microRNA-382 knockout or M2 macrophage depletion can inhibit the expression of α-SMA, fibronectin, and collagen I and exhibit a certain mitigating effect on kidney fibers [It is found that lncRNA NEAT1 is up-regulated in SLE patients and can enhance the expression of cytokines or chemokines such as IL-6, CCL2, and CXCL10 by phosphorylating JNK and ERK. These cytokines can attract Th1 cells and participate in the pathogenesis of LN . Meanwhiy fibers , indicatIn the synovial chamber of RA patients, the expression of miR-221-3p is abnormally increased, and M2 macrophages are transferred to proinflammatory M1 through the JAK3/STAT3 pathway, promoting the development of joint inflammation . In an ilncRNA GAS5 inhibits M2 polarization and intensifies demyelinating by inhibiting transcription of TRF2, a critical factor in M2 macrophage polarization, and interfering with microglial GAS5 in vitro experiments can attenuate the progression of experimental autoimmune encephalomyelitis (EAE) . At the κB pathway in macrophage polarization [κB pathway can recruit MyD88 and IRAK after LPS stimulation, and IRAK phosphorylation activates NF-κB after interacting with TRAF6 to promote M1 production. In an animal model of endotoxin-induced uveitis (EIU), miR-93 was found to bind to IRAK4 in the NF-κB pathway, thereby inhibiting NF-κB activation and thus negatively regulating the generation of M1-type macrophage-related proinflammatory cytokines [In addition, microRNAs are closely associated with autoimmune uveitis. miRNAs can participate in the inflammatory or inflammatory mitigation process of uveitis by acting on signaling molecules of the NF-rization . The tolytokines . miR-30bytokines . Meanwhiytokines , 96. HowInterestingly, studies have shown that different lncRNAs and miRNAs are differentially expressed in primary SS and participate in the pathogenic process of the disease , 98. A rCollectively, a deep understanding of the mechanism of noncoding RNAs regulating macrophage polarization under different conditions can more effectively help us manipulate the expression and silence these noncoding RNAs through drug targeting to control the direction of macrophage polarization, opening up a new horizon for the treatment of inflammatory diseases.Autoimmune diseases are clinical diseases caused by the destruction of autoimmune tolerance or abnormal regulation of autoimmune cells, the continuous immune response of the immune system to autoantigens, and the damage or dysfunction of self-organizing cells induced by some genetic and environmental factors .κB as a cofactor, possessing a certain pathogenic effect on SLE and multiple sclerosis (MS) [Notably, age and gender are the related factors that induce autoimmune diseases. Most autoimmune diseases can occur at any age; however, some autoimmune disorders mainly occur in childhood and adolescence (such as type I diabetes), middle adulthood (such as myasthenia gravis and multiple sclerosis), or the elderly (such as rheumatoid arthritis and primary systemic vasculitis) . The incsis (MS) . Currentsis (MS) , prolongSLE is an autoimmune connective tissue disease that often involves multiple systems, with a wide range of clinical manifestations. It usually occurs in young women aged between 20 and 40. At present, the potential incidence rate and mortality are significant, and the pathogenesis and etiology are still unclear , 107. ClκB2 activation, leading to M1 macrophage polarization and further deterioration of SLE [γ of M2b expressions can be detected in serum samples of SLE patients, which is associated with the deposition of immune complexes in SLE as a good inducer of M2b activation [The pathogenesis of SLE is not only related to the abnormality of B cells and T cells but also related to inflammation-promoting M1 and immunosuppressive M2. It is confirmed that biological factors that promote the polarization of M1 macrophages will exacerbate the inflammation of SLE, and M2 is also involved in the pathogenesis of SLE. Human cytomegalovirus (HCMV) has been shown to be one of the major factors that can trigger SLE. In a clinical study involving SLE patients, HCMV infection-associated human cytomegalovirus protein (US31) was elevated in SLE patients and promoted NF-n of SLE . In addin of SLE . Interesn of SLE . In the n of SLE . Each sun of SLE , 116. M2tivation . A recentivation . Therefotivation .A large number of studies have found that promoting the activation of M2 macrophages and returning the M1/M2 macrophage ratio to normal level play a specific role in alleviating SLE. JAK/STAT signaling pathway is an important pathway that regulates the polarization direction of macrophages. JAK (Janus kinase) kinase family plays a crucial role in the immune system and is a series of pathological therapeutic targets, including autoimmune diseases, COVID-19-related cytokine storms, and blood cancer , 120. Inγ receptors on Fc receptor-carrying cells (monocytes and macrophages) can lead to the release of inflammatory cytokines, thereby causing kidney inflammation [Lupus nephritis (LN) is a common SLE complication that can lead to severe tissue damage and organ failure. The pathogenesis of LN is related to immune complex deposition, macrophage activation, and excessive release of proinflammatory cytokines. Activation of the immune complex of Fcammation . Macrophammation .Evidence has shown that M1 macrophages from the injured kidney have proinflammatory effects and clear apoptotic and injured cells. M2-type macrophages play a role in inhibiting inflammation and promoting tissue repair, while M2a-like macrophages are involved in fibrosis repair and progression , and M2cβ, and IL-60) [The lack of Bruton's tyrosine kinase (BTK) could enhance STAT6 phosphorylation through the STAT signaling pathway, resulting in decreased M1 polarization and increased M2 polarization. In addition, the BTK inhibitor BI-BTK-1 can prevent macrophage activation by inhibiting Fc receptors and certain TLRs, decrease immune complex (IC) deposition, reduce autoantibody IgG levels, and downregulate inflammatory mediators  . These td IL-60) .β and macrophage polarization to proinflammatory M1 metastasis [γ receptor I (Fcγ RI) by inhibiting nuclear factors-κB (NF-κB) could reduce the activation of NLRP3 inflammasome, inhibit renal inflammation, and reduce the toxic effect of LN [The NLRP3 inflammasome is a cytosolic protein composed of the innate immune receptor protein NLRP3, adapter protein ASC, and inflammatory protease caspase-1, playing a vital role in regulating autoimmune diseases . The NLRtastasis . Therefoct of LN . An expect of LN . Interesct of LN .Rheumatoid arthritis (RA), a commonly autoimmune disease in clinical practice, is a chronic synovial proliferative inflammation. Inflammatory changes are mainly seen in the synovial tissue of joints, cartilage, and bones, rarely in extra-articular areas such as skin and blood vessels. The prevalence rate of RA is between 0.4% and 1.3% of the population, and the prevalence rate of women is 2-3 times higher than that of men , 137. ToκB pathway and gasdermin family-driven phosphorylation is all related to the inflammatory process of RA [κB pathway [κB pathway and inhibit LPS/IFN-γ-induced upregulation of TLR4, which in turn inhibits NF-κB activation and reduces M1 polarization [κB-mediated inflammatory response by interacting with the RelA subunit of NF-κB, so when Sirt6 is deficient, it will promote NF-κB activation and endogenous production of IL-6, thereby enhancing macrophage infiltration and M1 macrophage activation in the joint, aggravating inflammation and leading to the development and deterioration of RA [Macrophages can polarize to M1 or M2 when stimulated by different environmental factors, and the dynamic polarization process from M1 to M2 includes the presence of intermediate polarity stages. M1/M2 polarization imbalance contributes to acute or chronic RA , 143. Inss of RA . In the ss of RA . MERTK mss of RA , 147. M2ss of RA . In addi pathway . Wilforlrization . As we arization . Thus, trization . Moreoveon of RA , 154. Maon of RA .Multiple sclerosis (MS) is a progressive demyelinating disease of the central nervous system (CNS). It is also a tremendously challenging autoimmune disease in the clinic. At present, the etiology of MS is still unclear. Given multiple factors, MS is related to genetic and environmental factors, such as virus infection, smoking, and decreased vitamin D levels –158. In β/AMPK pathway, thereby aggravating the continued progression and deterioration of MS [MS is an immune-mediated chronic inflammatory disease, and the homeostasis of M1/M2 macrophages plays a prominent role in developing MS. In multiple sclerosis, M1 and M2 macrophages can coexist and play a dual role, playing a neuroprotective role by producing inflammatory mediators that cause nerve tissue damage and can promote growth support repair. Vogel et al. found that most foam macrophages in active MS lesions can express both M1 and M2 markers, confirming the existence of an intermediate state of macrophage activation . Macrophon of MS .α, IL-1β, and IL-6 synthesized by reducing ERK phosphorylation to promote M2-like macrophages [In the late stage or recovery period of MS, microglia/macrophages polarize to M2, promote tissue repair, and reduce the severity of MS. Therefore, the treatment of MS mainly regulates macrophage polarization and cytokine levels and cytokine levels to improve the immune microenvironment. In recent years, dimethyl fumarate (DMF) has exhibited an excellent therapeutic effect on recurrent remitting MS. DMF can effectively improve the clinical score of MS patients, activate the antioxidant product of Nrf2, and reduce the tissue damage caused by ROS in MS and EAE animal models . In addirophages , 166. Morophages .κB and the expression of proinflammatory genes, which may exert a particular inhibitory effect on macrophage polarization to M1 [κB p65 by forming dopamine quinones in microglia, thereby attenuating proinflammatory cytokine expression, a process that may be associated with reduced polarization of M1 macrophages [At present, biogenic amines' role in treating MS has powerfully attracted attention. Biogenic amines mainly include serotonin (5-HT), dopamine, and norepinephrine. Among them, 5-HT may regulate M2 macrophage polarization . Regardion to M1 . Meanwhirophages . These fβ-mediated NF-κB activation to promote M2 polarization [In addition, studies have also found that mitochondrial fission inhibitor (MDivi-1) can improve the inflammation of EAE mice, mainly by inhibiting TLR2/4 and GSK3rization .Guillain-Barre syndrome (GBS) is a peripheral nerve disease characterized by demyelinating lesions of peripheral nerves, nerve roots, and infiltration of small vascular inflammatory cells . It is aM1 and M2 macrophages can guide T cell polarization in different ways. At different stages of GBS, macrophages play either a proinflammatory or anti-inflammatory role. In the early stage of GBS, M1 macrophages promote cytotoxicity and Th1 cytokine production, leading to inflammatory damage of myelin sheath and disease development ; in the κB signaling is also an effective target for the treatment of EAN. Thus, reducing the polarization of M1 macrophages and promoting the polarization of M2 by inhibiting p65 phosphorylation in the NF-κB pathway can alleviate EAN [The Notch signaling pathway is an important pathway for macrophage polarization. The Notch receptor family consists of 4 members (Notch1-4), the ligand family consists of 5 members , and NICD and RBP-J as the downstream molecules of the Notch signaling pathway are also actively involved in the regulation of M1 macrophage polarization . Oridoniiate EAN .Uveitis is an inflammatory disease of the iris, ciliary body, and choroid tissue in the eye. Clinically, approximately 35% of uveitis patients have a severe visual impairment or even blindness. The etiology of uveitis is complex and can be divided into infectious or noninfectious uveitis. Many studies have confirmed that noninfectious uveitis is mainly related to autoimmunity, that is, the deposition of antigen-antibody complexes in the capillary-rich uvea. It is reported that uveitis primarily occurs in young people. Currently, the main treatment methods include the local or systemic application of glucocorticoids and ciliary muscle paralysis .γt, and Foxp3 is elevated in the pathogenesis of EAU, and increased polarization of M1 macrophages and an imbalance in the ratio of Th17/Treg occur [Experimental autoimmune uveitis (EAU) is an ideal animal model of human autoimmune uveitis. EAU model induction is the immunization of susceptible rodents using proteins or peptides extracted from the retina, iris, or ciliary body. This process is combined with complete Freund's adjuvant and tuberculin. Mice are more accessible to transgene and propagation than other animals, and the eye structure of mice is similar to that of human beings, so most of the research in recent years has used EAU mouse animal models , 182. Maeg occur . Similareg occur , 187. ThκB signaling pathway also plays an essential role in the pathogenic mechanism of EAU. In mammals, the NF-κB family consists of five members, including RelA (p65), RelB, c-Rel, NF-κB1 (p50), and NF-κB2 (p52), which form various dimer complexes that regulate gene transcription by binding to 10bp-specific sequences (-κB sites) on target genes [κB P65 by downregulating the expression of TLR4/MyD88, thereby reducing M1 polarization and contributing to the treatment of EAU [β, can also minimize Th1/Th17-mediated inflammation by inhibiting NF-κB p65 in an animal model of EAU [The NF-et genes . Recent t of EAU . Similarl of EAU .κB signaling pathways described above, PI3K/AKT/FOXO1 phosphorylation is also considered novel pathogenesis of EAU, and phosphodiesterase-4 inhibitors  can reduce the Th1 and Th17 frequencies by inhibiting the downstream transcription factor FOXO1 expression in the PI3K/AKT pathway and enhancing the Treg cell response to alleviate EAU [In addition to the Notch and NF-iate EAU . Interesiate EAU .Sjogren's syndrome (SS) is a chronic autoimmune disease commonly occurring in middle-aged women. Clinically, SS can be divided into primary and secondary SS. Primary SS refers to the separate onset of SS, and secondary SS is mainly induced by SLE and RA. The clinical manifestations of SS are diverse and can involve the whole-body system and specific target organs . It is mThe etiology of SS is still unclear, but sustained B-cell activation and proliferation of Th1 and Th17 cells contribute to disease progression . IFN-indα, IL-6, IL-1β, and IL-12 to play a proinflammatory role and further activate CD4+ T cells to differentiate into Th1 cell lineage, leading to the occurrence and development of submandibular gland inflammation [β and other anti-inflammatory mediators, which contribute to the regression of inflammation and tissue regeneration, and alleviate autoimmune lacrimal gland inflammation, thereby playing an anti-inflammatory role [β signal transduction pathways that induce fibrosis are divided into SMAD regulation and non-SMAD regulation. TGF-β can promote M2 macrophage polarization by activating SMAD2/3/4 trimer complexes, and this pathway can also promote fibroblast-to-myofibroblast transformation [β pathways, can also induce development and fibrosis [κB are activated by MyD88, which induces M1 macrophage polarization to secrete inflammatory factors. Therefore, blocking this pathway has a particular therapeutic effect on SS [After macrophage polarization, both macrophage subtypes exist in patients with primary Sjogren's syndrome. M1 macrophages exist in the early stage of PSS, which generate inflammatory factors such as TNF-ammation . In an iory role . When SSormation . Non-SMAfibrosis –203. It fibrosis . In addifibrosis . Other sfibrosis . Neverthfibrosis . In addict on SS , 207.The role of macrophage polarization balance in the pathogenesis of SS cannot be ignored. Still, in recent years, the research on SS has mainly focused on the pathogenesis of epithelial cells. Hence, the relationship between macrophage polarization and the molecular mechanism of SS remains to be further explored.α-SMA, including FN1 (the gene encoding fibronectin) expression and TGF-β signaling pathway [β activation, macrophages and fibroblasts can activate each other, further increasing tissue thickness and hardness and mediating fibrosis [β) by regulating the NF-κB signaling pathway, so ADAR1 deficiency in macrophages can significantly improve skin and lung sclerosis [Systemic sclerosis (SSc) is an autoimmune disease in which chronic progressive inflammation and fibrosis of tissues and organs are the main lesions after excessive extracellular matrix production . The pat pathway . After Tfibrosis . Variousclerosis . In addiclerosis . Therefoβ/SMAD, Notch, and PI3K-AKT pathways  to prohealing (M2) phenotypes during tissue repair. Moreover, autoimmune diseases can share M1 and M2 phenotypes and have an intermediate polarization state. The imbalance of M1/M2 polarization plays a vital role in autoimmune diseases. Therefore, regulating macrophage polarization's direction can improve autoimmune disease pathogenesis. Presently, the therapeutic strategy of finding therapeutic targets for autoimmune diseases concentrated on regulating macrophage polarization has indeed made significant progress. However, most of the results are based on the data obtained from animal models, and there may be some species differences between animal models and human beings. Therefore, a more comprehensive understanding of the relationship between macrophage polarization and the occurrence and development of autoimmune diseases can find more effective therapeutic targets for autoimmune diseases. It is also the focus of people's efforts in the future.


The safety results of different recommended doses of sodium-glucose cotransporter 2 inhibitors (SGLT-2i) for patients with type 2 diabetes mellitus (T2DM) remain uncertain. This study aims to comprehensively estimate and rank the relative safety outcomes with different doses of SGLT-2i for T2DM.PubMed, Embase, the Cochrane Central Register of Controlled Trials, ClinicalTrials.gov, Chinese National Knowledge Infrastructure, WanFang database, and SinoMed database were searched from the inception to 31 May 2023. We included double-blind randomized controlled trials (RCTs) comparing SGLT-2i with placebo or another antihyperglycemic as oral monotherapy in the adults with a diagnosis of T2DM.Twenty-five RCTs with 12,990 patients randomly assigned to 10 pharmacological interventions and placebo were included. Regarding genital infections (GI), all SGLT-2i, except for ertugliflozin and ipragliflozin, were associated with a higher risk of GI compared to placebo. Empagliflozin 10mg/d  may be the riskiest, followed by empagliflozin 25mg/d ) and canagliflozin 300mg/d  based on probability rankings. Additionally, dapagliflozin 10mg/d ranked highest for urinary tract infections , renal impairment (80.7%), and nasopharyngitis (81.6%) when compared to placebo and other treatments. No increased risk of harm was observed with different doses of SGLT-2i regarding hypoglycemia, acute kidney injury, diabetic ketoacidosis, or fracture. Further subgroup analysis by gender revealed no significantly increased risk of UTI. Dapagliflozin 10mg/d (91.9%) and canagliflozin 300mg/d (88.8%) ranked first in the female and male subgroups, respectively, according to the probability rankings for GI.Current evidence indicated that SGLT-2i did not significantly increase the risk of harm when comparing different doses, except for dapagliflozin 10mg/d, which showed an increased risk of UTI and may be associated with a higher risk of renal impairment and nasopharyngitis. Additionally, compared with placebo and metformin, the risk of GI was notably elevated for empagliflozin 10mg/d, canagliflozin 300mg/d, and dapagliflozin 10mg/d. However, it is important to note that further well-designed RCTs with larger sample sizes are necessary to verify and optimize the current body of evidence.https://www.crd.york.ac.uk/PROSPERO/, identifier CRD42023396023. It is associated with both microvascular and macrovascular complications, causing significant psychological and physical distress for patients and carers while placing a substantial burden on healthcare systems . AccordiThe National Institute for Health and Care Excellence (NICE) clinical practice guidelines recommend metformin, along with lifestyle modifications, as the first-line treatment for T2DM . HoweverThe aim of this study is to conduct a Bayesian network meta-analysis (NMA) to estimate and rank the relative safety outcomes associated with different recommended doses of each approved SGLT-2i for T2DM. The results of this analysis are expected to provide valuable insights for clinical decision-making, enabling the development of optimal treatment strategies for patients with T2DM in the future.2The NMA was performed in accordance with Preferred Reporting Items for Systematic Review and Meta-Analysis extension statement for NMA Table S12.1PubMed, Embase, the Cochrane Central Register of Controlled Trials, ClinicalTrials.gov, China National Knowledge Infrastructure, WanFang database, and SinoMed database were searched from inception to 31 May 2023, using the term “SGLT-2”, “T2DM”, “randomized controlled trials”, and their synonyms shown in 2), pregnancy or lactation, insulin therapy, or allergies or contraindication to the study drugs. Additionally, trials that combined treatment with other antihyperglycemic drugs during the study period were also excluded.We excluded trials that patients with severe hepatic impairment (Child-Pugh class C), severe renal dysfunction or end-stage renal failure (estimated glomerular filtration rate/eGFR < 30mL/min/1.73 mThe outcomes of this study included adverse events (AEs) of special interest, such as confirmed hypoglycemic events (plasma glucose ≤ 3.9 mmol/l and/or requiring assistance), as well as AEs related to urinary tract infections (UTI), genital infections (GI), renal-related AEs, bone fractures, amputations, DKA, and nasopharyngitis.Four reviewers  independently screened the reports against pre-designed eligibility criteria, and any disagreements were resolved through discussion, consulting another reviewer (Z.B. or Z.Q.).2.2Three reviewers  independently screened each trial by reviewing titles, abstracts, and full text using standardized and piloted forms. The baseline information was extracted, including the first author, the publication year, the clinical trials number, participants characteristics , intervention and comparison groups with dosage and usage, as well as the outcomes. Discrepancies were resolved through discussion or by a third reviewer (Z.B. or Z.Q.).2.3Two reviewers (C.L. and X.Q.X.) independently assessed the risk of bias using the Cochrane Collaboration’s Risk of Bias 2 (RoB V.2.0) tool, encompassing domains such as randomization process, deviations from intended interventions, missing outcome data, outcome measurement, and selection of the reported result . Each st2.4We estimated intervention effects by calculating odds ratios (OR) with 95% credible interval (CrI). We performed NMA using Bayesian random effect models with the Markov chain Monte Carlo simulation method for interventions that connected to an evidence network by data available from ≥ 2 studies. For outcomes with inadequate network structure, pairwise meta-analyses were conducted instead. Heterogeneity among studies was assessed using the Q test and I² statistic, with significance defined as p < 0.10 and I² ≥ 50% . ConsistModel convergence was evaluated via visual inspection of four chains, considering the Brooks-Gelman-Rubin diagnostic, as well as trace and density plots . Within Subgroup analyses were performed by gender when sufficient information was available. Additionally, Sensitivity analysis was performed by excluding high risk of bias of studies. A comparison-adjusted funnel plot and Egger test were used to evaluate small-study effects for individual outcomes when at least 10 eligible studies were available . Statist33.1A total of 7,486 studies were identified after removing duplications, 164 potentially eligible studies underwent full-text review. Following the application of eligibility criteria, 25 RCTs involving 12,990 patients were included in NMA. The study selection process is illustrated in The results of the risk of bias assessment are presented in 3.23.2.1Twenty RCTs involving 12,076 patients provided data on GI –38, 40, 3.2.2Twenty-one RCTs involving 12,399 patients reported UTI –41, 43. 3.2.3Seventeen RCTs involving 11,464 patients reported hypoglycemia , 37–39. 3.2.4Thirteen RCTs involving 4,315 patients reported nasopharyngitis , 42–44. 3.2.5Four RCTs involving 5,393 patients reported bone fracture , 32, 43.3.2.6Two RCTs involving 4,705 patients reported DKA , 25. The3.2.7Eight RCTs involving 6,883 patients reported renal-related AEs, with four trials specifically examining renal impairment in the context of dapagliflozin , 42, 43.3.3The pairwise comparisons of heterogeneity in outcome estimates were presented in 4This NMA of 10 pharmacological interventions from 25 double-blind trials, enrolling a total of 12,990 patients, and provides the comprehensive evidence with respect to the key safety outcomes associated with different recommended doses of SGLT-2i. Our study demonstrated that SGLT-2i do not appear to increased risk of DKA, nasopharyngitis, or bone fracture. Through direct comparison and the mixed treatment comparison, empagliflozin 10mg/d and canagliflozin 100mg/d were associated with the higher risk of GI and hypoglycemia, respectively. Moreover, dapagliflozin 10mg/d may be the riskiest according to the probability rankings for both UTI and renal impairment, however, the association for renal impairment with large uncertainty in the estimates owing to the small sample sizes of trials. Notably, this NMA and rankings have the potential to serve as a valuable decision-making tool for clinicians, facilitating informed treatment selection based on the safety profiles of different SGLT-2 inhibitors and their respective doses.SGLT-2i are known for their ability to reduce hyperglycemia in patients by inhibiting renal glucose reabsorption in the proximal tubule of the kidney, leading to increased glucose excretion in the urine. However, this mechanism of action is also associated with an elevated risk of genital and urinary tract infections . RegardiSGLT-2i have shown significant renal protective effects, including a 30% to 50% reduction in proteinuria and favorable outcomes in renal composite hard endpoints. The EMPA-REG OUTCOME trial demonstrated lower composite renal outcomes in patients treated with empagliflozin compared to placebo (HR = 0.68) . SimilarIn 2016 and 2017, the FDA issued warnings regarding a potential increased risk of fractures and leg amputations with the use of canagliflozin , 59. How5This study has several limitations that should be taken into account. First, in order to directly observe the safety of SGLT-2i in patients with T2DM, we excluded the effects of combined with other drugs, and for that reason, the number of original trials included in this study was relatively small, and further confirmation of the findings are necessary. Second, the exclusion of all non-English and non-Chinese language literature may introduce potential publication bias. Third, certain outcomes may have been inadequately characterized within study or with wide 95% CrIs for OR values and imprecise estimates, primarily due to the limited number of studies available. Fourth, the specific types of infection between genital and urinary tract infections were not distinguished in this study, as very few of the included trials reported the specific types of infection in detail, this issue need more detailed clinical trials to address the data gap in the future.6In this NMA, current evidence from RCTs indicated that SGLT-2i were not significantly increased the risk of harm among comparison of different doses, except for dapagliflozin 10mg/d, which showed an increased risk of UTI and may be associated with a higher risk of renal impairment and nasopharyngitis. In terms of GI, empagliflozin 10mg/d, canagliflozin 300mg/d, and dapagliflozin 10mg/d were associated with a higher risk compared to placebo and metformin. Additionally, the evidence does not suggest a significantly increased risk of DKA, nasopharyngitis, and bone fracture with SGLT-2i, over placebo or active comparators. Further well-designed RCTs with larger sample sizes and more detailed information are required to verify and optimize the current body of evidence.The original contributions presented in the study are included in the article/LC: Conceptualization, Data curation, Formal Analysis, Investigation, Methodology, Software, Writing – original draft. QX: Conceptualization, Data curation, Formal Analysis, Methodology, Software, Writing – original draft. CY: Data curation, Formal Analysis, Software, Writing – review & editing. BT: Data curation, Formal Analysis, Writing – review & editing. LW: Data curation, Methodology, Writing – review & editing. BZ: Conceptualization, Supervision, Writing – review & editing. QZ: Conceptualization, Supervision, Writing – review & editing.


These mechanisms expand the fundamental role of dopamine in schizophrenia therapy and may have relevance to considering putatively new strategies for treatment-resistant schizophrenia (TRS), an extremely severe condition epidemiologically relevant and affecting almost 30% of schizophrenia patients. Here, we performed a critical evaluation of the role of antipsychotics in synaptic plasticity, focusing on their canonical and non-canonical mechanisms of action relevant to the treatment of schizophrenia and their subsequent implication for the pathophysiology and potential therapy of TRS.Schizophrenia is a severe psychiatric illness affecting almost 25 million people worldwide and is conceptualized as a disorder of synaptic plasticity and brain connectivity. Antipsychotics are the primary pharmacological treatment after more than sixty years after their introduction in therapy. Two findings hold true for all presently available antipsychotics. First, all antipsychotics occupy the dopamine D2 receptor (D2R) as an antagonist or partial agonist, even if with different affinity; second, D2R occupancy is the necessary and probably the sufficient mechanism for antipsychotic effect despite the complexity of antipsychotics’ receptor profile. D2R occupancy is followed by coincident or divergent intracellular mechanisms, implying the contribution of cAMP regulation, β-arrestin recruitment, and phospholipase A activation, to quote some of the mechanisms considered canonical. However, in recent years, novel mechanisms related to dopamine function beyond or together with D2R occupancy have emerged. Among these potentially non-canonical mechanisms, the role of Na Dopamine cortical–subcortical dysregulation epitomizes the main, albeit not the only, neurotransmitter landmark of schizophrenia pathophysiology and antipsychotics remain, more than sixty years after their introduction in therapy, the cornerstone of schizophrenia treatment.Emerging findings from novel methodologies of receptor–ligand computational and Fluorescence Resonance Energy Transfer (FRET) microscopy modeling at the dopamine D2 receptor (D2R) , analysiAntipsychotics are the mainstay of the pharmacological treatment of schizophrenia and are also prescribed for mania in bipolar disorder as well as, to a lesser extent, augmentation therapy in treatment-resistant depression. Preclinical, clinical, and human functional studies strongly support the occupancy of D2R as a common, necessary, and perhaps sufficient mechanism of action (MOA) for all molecules available at present in clinics, even if they are characterized by different receptor profiles. This mechanism is coherent with a “dopaminocentric hypothesis” of psychosis that was conceptualized almost 50 years ago and is still considered fundamental ,7, even Novel strategies are even more needed with reference to those patients who do not respond to treatment with antipsychotics using at least two antipsychotics  at a dose of 600 mg/equivalent of chlorpromazine for at least 6 weeks of treatment, these patients are defined as treatment-resistant . The onl(1)Is there space for more dopaminergic strategy beyond dopamine D2R occupancy in psychosis treatment?(2)If antipsychotics tune the dopaminergic system by mechanisms other than the non-direct D2R-mediated one, what are the other non-canonical dopaminergic targets?(3)How do antipsychotics acting at D2R impact trans-synaptically other neurotransmitter systems, as well as synaptic and meta-synaptic plasticity?(4)Finally, how the next generation of antipsychotics now appearing on the landscape of schizophrenia treatment may regulate the dopaminergic system?Considering that (i) at present, the dopaminergic strategy to tackle psychosis has been demonstrated to be effective, at least for positive symptoms, (ii) D2R occupancy could be limited by the potential onset of motor and other adverse events, and (iii) multiple domains of the disorder are indirectly related to dopamine through its modulation by other neurotransmitters systems, we addressed the following questions:With these questions in mind, we have addressed the background and the potential relevance for the therapy of non-canonical molecular effects of antipsychotics considering the complexity of antipsychotics’ multiple receptors profile in the framework of a dopaminergic MOA within and beyond dopamine D2R occupancy.The bibliographic search and selection process was conducted according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines . The seaThe PRISMA flow chart has been reported in Dopamine dichotomic cortical–subcortical involvement in schizophrenia pathophysiology, may suggest the coexistence of cortical “hypodopaminergia” and subcortical “hyperdopaminergia” associated with the dysfunction of other neurotransmitter systems, mainly serotonergic and glutamatergic ones ,17. Trea2+-permeable α-ammino-3-idrossi-5-metil-4-isossazol-propionic acid receptors (AMPARs) and the loss of inhibitory postsynaptic currents (IPSCs) dependent on D2R in the middle spiny neurons of the nucleus accumbens (NAc) [substantia nigra are sensitive to reactive oxygen species (ROS) and to the dysregulation of cellular redox homeostasis generated by catecholamine metabolism [2 receptors (5-HT2Rs) [2ARs in the prelimbic cortex and NAc while increasing their density in the caudate-putamen. Therefore, 5-HT2AR activation is necessary for the full expression of DSP induced by antipsychotics, modulating dopamine-dependent behavior. These effects are potentially related to changes in 5-HT2AR density in the prefrontal cortex (PFC) and striatum, suggesting the role of the 5-HT2AR blockade in the regulation of antipsychotic-induced DSP. Moreover, a mechanistic link has been proposed between the development of TD and serotonin 5-HT6R in patients with Parkinson’s disease and transplanted with dopaminergic neurons. The 5-HT6R could be implicated in the dopamine release responsible for the “transplant-induced dyskinesia” side effect induced by the inhibition of autoreceptor feedback [DRD2 gene polymorphism -141 C Ins/Del and poor response to antipsychotics [Iyo and coworkers hypothesized that there is an optimal range in the number of D2Rs available for dopamine binding to achieve adequate treatment in patients with schizophrenia . The autns (NAc) . These fns (NAc) . Furthertabolism , which itabolism . Preclin5-HT2Rs) . Antipsyfeedback . For selfeedback , particufeedback . D2R actfeedback , suggestfeedback . Despitefeedback . Pharmacychotics ,33, suggychotics ,34.ARRB2-knock out (KO) mice [At the molecular level, the mechanisms responsible for D2R trafficking could contribute to DSP . β-arresKO) mice , indicatKO) mice . PCP is KO) mice ,40,41,42KO) mice ,44. It hKO) mice , whereasKO) mice ,35.There is growing evidence that antipsychotics could act as chaperones in the trafficking of dopamine receptors from the intracellular site to the membrane surface; however, not all antipsychotics are equal regarding this feature. Aripiprazole and clozapine have less propensity to act as chaperones of D2R, preventing their translocation and possible upregulation to the cell surface, therefore, this differential mechanism may represent a putative non-canonical MOA to reduce the onset of DSP ,47.i = 0.085 nM, higher than the one for the D2R (Ki = 0.49 nM) [DTNBP1 gene), one of the top candidate genes associated with schizophrenia, affects the intracellular trafficking of D3R. Notably, in both schizophrenia and genetic animal model of the disorder, the concomitant reduction in D3R and DTNBP1 gene expression was associated with pro-cognitive effects. The D3R/dysbindin interaction has been shown to promote D2R/D3R imbalance by favoring an increase in D2R signaling in the PFC but not in the striatum [Even though all antipsychotics occupy both D2R and D3R, the role of the latter one has received less attention in the past, probably for the reported low density and more restricted distribution in a few brain regions. However, more recently, a growing interest at both clinical and preclinical levels has considerably changed the appraisal of the D3R in the framework of antipsychotic therapy. At the clinical level, the introduction of cariprazine has significantly marked the role of dopamine D3R as a relevant addition to the schizophrenia pharmacological armamentarium. Cariprazine is the first available antipsychotic that preferentially acts as a partial agonist at D3R, with K0.49 nM) , demonst0.49 nM) . At the 0.49 nM) ,52,53 by0.49 nM) . Of intestriatum , underlistriatum  and havestriatum ). γ-oscistriatum .globus pallidus, and substantia nigra [in situ hybridization and immunofluorescence histochemistry, confirming the crucial role of D4R in cognitive deficits [D4Rs are predominately located in the prefrontal and temporal areas, sparing the basal ganglia, whereby compounds with a higher affinity for D4Rs than D2Rs could selectively reduce dopaminergic tone in the mesolimbic and mesocortical systems without affecting the nigrostriatal pathway and not producing motor side effects . Furtheria nigra , it has ia nigra . D4R seeia nigra . This efdeficits .2A produces discriminative stimulus effects of “atypicality” similar to clozapine, but the additional antagonism at D2R makes it similar to typical antipsychotics [DRD4 gene is significantly associated with clozapine-induced sialorrhea, suggesting that this duplication may increase the risk of sialorrhea in TRS patients treated with clozapine [A potential difference between atypical and typical antipsychotics could concern binding to the D4R, considering that many atypical antipsychotics show a slightly higher affinity for D4R than D2R, whereas the majority of typical antipsychotics have a higher affinity for D2R than D4R . Despiteychotics . It is nlozapine .v2.2 currents by occluding D1R-like constitutive activity [Dopamine D1R and related intracellular signaling have been longly considered a complex puzzle in the dynamic of dopamine under antipsychotic treatment . One majactivity ,68. Moreactivity , suggestactivity . Accordiactivity . Indeed,activity , acting activity .Preclinical studies reported that clozapine suppressed the acute increase in medial PFC glutamate levels by enhancing NMDAR-mediated neurotransmission that was not induced by D1R-dependent dopaminergic neurotransmission, partially explaining the clozapine-induced attenuation of hyperlocomotion in PCP-treated rats .globus pallidus and the lowest incidence in the caudate putamen [globus pallidus are increased in schizophrenia [However, the results of clinical trials with selective D1R antagonists do not show antipsychotic responses in patients . A diffe putamen . It has  putamen . D1R-D2Rophrenia ,77 and aophrenia  , a complex receptor profile even if dopamine D2R occupancy or blockade is considered to be the necessary and possible sufficient mechanism of antipsychotic action. Exploring which and to what extent receptors that are part of an antipsychotic’s profile are different from dopamine receptors could cooperate with the latter ones to help to unveil potential new antipsychotic mechanisms of non-canonical type.The possibility that non-canonical mechanisms in dopaminergic signaling could intercept serotoninergic signaling has been recently challenged by the discovery of receptor dimers belonging to the different classes of neurotransmitters ,83,84.2A antagonism; specifically, a better affinity for 5-HT2A over D2R, and thus a high 5-HT2A/D2R ratio is considered a possible molecular predictor of atypicality, whose clinically and original definition is the low liability to elicit EPS [2A/D2R dimers. Several preclinical in vitro and in vivo studies have identified dimerization of 5-HT2 with D2R in rat striatum [2A/D2R dimers with D2R agonists is antagonized by co-administration of 5-HT2A-agonists, suggesting a 5-HT2A-mediated trans-inhibition of D2Rs, resulting in increased Gq compared with Gi signaling [2A can form dimers with metabotropic glutamate receptor 2 (mGluR2) [2A/mGluR2 heterodimerization could potentiate mGluR2-Gi signaling and inhibit 5-HT2A-Gq one [2A/mGluR2 heterocomplex, balancing Gi- and Gq-dependent signaling and eliciting antipsychotic action [2A and lower expression of mGluR2, which might reflect a predisposing pattern to psychosis, indicating that the 5-HT2A/mGluR2 complex might be involved in the altered cortical processes of schizophrenia resulting in a promising unconventional target for the treatment of psychosis [3 is involved in the modulation of dopaminergic activity in mesolimbic and nigrostriatal pathways [3 antagonists in mimicking the effects of antipsychotic drugs. Clinical evidence on single-marker and haplotype analyses among different mutations of serotonin receptor subtypes  in patients with TRS found that the daily dosage of neuroleptics received during maintenance therapy was significantly higher in patients with the T/T genotype of the HTR3A rs1062613 polymorphism, supporting the relationship between the HTR3A polymorphism and the potential development of TRS [3 antagonism is yet to be clarified [6 antagonism was shown to attenuate the pro-psychotic effects of both MK-801  [7 receptor antagonist SB-269970 improves ketamine-induced attention and cognitive inflexibility, although its role has not yet been elucidated [1A agonists are known to have effects shared in part by 5-HT2A antagonists in different biological systems [1A agonism contributes to the atypical profile of antipsychotics [HTR1A, as well as solute carrier family 6, member 4 (SLC6A4) genes and clinical outcomes in schizophrenia, have shown that rs6295 and 5-HTTLPR polymorphisms significantly influence clinical symptoms in schizophrenia [1A in schizophrenia pathophysiology is also confirmed by an in vitro preclinical study in PC12 cells treated with clozapine: it is demonstrated a decrease in tyrosine hydroxylase levels, with subsequent reduction in dopamine levels, probably mediated by D2R and 5HT1A stimulation [A trait that has been considered possibly critical for the atypicality of almost all new-generation antipsychotics is 5-HTicit EPS . In the striatum ,83,84. Signaling . Further(mGluR2) ,87. In tA-Gq one . In thisc action . In addisychosis . Severalpathways ,90,91, st of TRS . 5-HT3 alarified ; also 5-t NMDAR)  and PCP t NMDAR) ,96. Furt systems  with anx systems . Moreoveychotics , reducinychotics ,103 and ychotics ,105. In mulation .B receptors expression in the post-mortem PFC and hippocampus [GAD67, GABBR1, and GABBR2 genes, which encode for the GABAB receptor [B receptor firing has been associated with schizophrenia [B-mediated inhibitory transmission [B similarly to baclofen, a GABAB receptor agonist proposed as a non-canonical antipsychotic [1H-MRS) study measured GABA levels in the midcingulate cortex (MCC) of TRS patients, stratifying them into clozapine-resistant schizophrenia (CRS) and TRS patients. The results showed higher GABA levels in CRS compared to TRS patients suggesting the potential implication of GABA to clozapine resistance [A receptors in ventral tegmental area (VTA). As a consequence, GABA neurons are activated, and the GABAergic projection from VTA to the NAc is involved in such antipsychotic effects [The main inhibitory neurotransmitter in the human central nervous system (CNS) is GABA. In postnatal development, GABAergic neurons are instrumental to the formation of brain circuits and are also involved in the pathophysiology of schizophrenia . Animal pocampus ,115, andreceptor . An impaophrenia ,118. Speophrenia ,120, sugophrenia , potentismission ,122. Thrsistance . Converssistance . Abnormasistance ,126. Glusistance . Then, gsistance ,129. Intsistance . α5-GABAsistance . Beyond  effects .1) and alpha-adrenoceptor 2 (α2) antagonism are both considered to be involved in modulating dopamine release in the PFC through firing dopaminergic neurons in the VTA [1 antagonist action of prazosin, which is responsible for enhancing the cognitive performance of MK-801 pretreated rats [1 by antipsychotics may help to suppress positive symptoms, particularly in acute schizophrenia, whereas blocking α2, a prominent effect of clozapine and, to a lesser extent, risperidone, may instead help to partially relieve negative and cognitive symptoms. While α1 blockade may inhibit striatal hyperdopaminergia at the presynaptic level, α2 blockade may enhance and improve dopaminergic function in PFC [2 blockage has been linked to the effects of atypical antipsychotics such as clozapine and antidepressants such as mirtazapine. It has been suggested that α2C and α2A heteroreceptors can regulate non-noradrenergic transmission, such as serotoninergic and dopaminergic, by sharing autoreceptor functions to inhibit noradrenaline release via negative feedback. In addition, while α2A seems to be highly distributed across the CNS, α2C expression is less abundant, suggesting the differential modulation of regional neurotransmission. α2C antagonism could be helpful during states of low endogenous noradrenergic activity, whereas α2A antagonism could be significant during states of high noradrenergic tone. Specifically, preclinical studies have shown that the antagonism at the α2C represents a non-canonical MOA for the regulation of GABA release in the striatum, inducing inhibition of GABA neurons projecting to cortical pyramidal neurons correlating with antidepressant/antipsychotic and procognitive-like effects [2C antagonists could unconventionally mitigate schizophrenia-associated symptoms [1 and α2 receptors could globally stabilize dopaminergic transmission [1 antagonism could affect positive symptoms by attenuating limbic hyperdopaminergia, whereas α2 blockade could act on negative symptoms by increasing prefrontal dopaminergic activity, both contributing to improving secondary negative symptoms [Alpha-adrenoceptor 1  gene are associated with susceptibility to schizophrenia [M5 gene have reported affected sensorimotor gating and PPI mechanisms [Post-mortem studies in schizophrenia patients have reported low levels of M1, M2, and M4 in the striatum affecting motivation and motor control [1 and 5-HT2 receptor subtypes [1A and 5-HT1B receptors in native tissues and/or cloned cell lines and an antagonist of 5-HT2 receptor subtypes [The role of acetylcholine is crucial for hippocampal function, including learning and memory processes impaired in schizophrenia, supporting disturbances of cholinergic systems in such disease ,150. Theignaling  and indiignaling . Of inteophrenia . In addichanisms . Preclinchanisms  and the chanisms . Post-mo control . Indeed, control ,165,166. control . Thus, t control  but acti control , proving control ,168,169. control , as wellsubtypes . Watson subtypes , speculasubtypes ,171. Thesubtypes . A growisubtypes . Clinicasubtypes ; thus xasubtypes  -KO mice that exhibited decreased spontaneous locomotion and poorly respond to methamphetamine [Histamine has a remarkable role as a regulator of several neurotransmitters and critical brain functions . Approxihetamine . In thishetamine . While thetamine ,193,194,hetamine . In addihetamine ,197. In hetamine . Furtherhetamine . Howeverhetamine ,200, is hetamine ,204,205.hetamine . H1 blochetamine . A compaFurthermore, a double-blind, parallel-group RCT study evaluated the potential beneficial effect of the H2 antagonist famotidine on negative symptoms in TRS patients, suggesting the novel pharmacological potential of H2 antagonism as a non-canonical strategy to treat negative symptoms in TRS .Among non-canonical mechanisms intercepting dopaminergic signaling in treating schizophrenia, trace amines and their receptor have emerged strongly. Endogenous trace amines, which structurally resemble monoamines, such as serotonin and dopamine, activate the trace amine-associated receptor 1 (TAAR1) . Since tFurthermore, it should be noted that this molecule acts primarily at the presynaptic level, which is believed to be the pivotal site of dopaminergic dysregulation .Consistent with these results and TAAR1 agonists’ unique MOA, the beneficial effect of these compounds as non-canonical therapy for TRS can be assumed .There is an emerging interest in dissecting non-canonical mechanisms that point to glutamate involvement as an antipsychotic augmentation strategy. The NMDAR hypofunction hypothesis predicts a reduced function of parvalbumin-positive fast-spiking GABA interneurons considered a major determinant of schizophrenia molecular pathophysiology with a possible role in poor response to antipsychotics acting at D2R . NMDAR fEncouraging clinical studies have shown that the strategy of clozapine augmentation with sodium benzoate, which is expected to promote the activation of NMDAR, exhibits beneficial effects overall in patients with CRS  and imprModulators of glutamatergic neurotransmission have been tested in several studies in combination with clozapine. However, no cumulative evidence of significant clinically beneficial differences was shown, except for improvement in negative symptoms with memantine and minocycline . It is pAn increase in glycine availability may also be obtained by administering GlyT1 inhibitors, which have proven to modulate both glutamatergic and dopaminergic neurotransmission in an animal model of schizophrenia . BitoperExperimental strategies to add D-amino acids to antipsychotic drugs to enhance response to treatment in schizophrenia have been investigated for several years, but despite a sufficiently strong rationale and translational background , it is sFluctuations in synaptic glutamate homeostasis led to aberrant activity of NMDARs, which are involved in the pathogenesis of neuropsychiatric disorders, suggesting the clinical relevance of allosteric modulators at this site. A recent preclinical study characterized an allosteric modulator, CNS4, which is responsible for enhancing the currents of NMDARs dependent on the concentration of endogenous glutamate . CNS4 shA further non-canonical treatment approach for schizophrenia is represented by riluzole, a drug used for amyotrophic lateral sclerosis that reduces the synaptic release of glutamate by inhibiting VGSCs and calcium currents ,146. PreCorbett and co-workers proposed that the antipsychotics clozapine and olanzapine may antagonize dopamine-induced and non-competitive NMDAR-associated behaviors . Preclin2 and a thickness of 30–40 nm [1H-MRS or fMRI [The postsynaptic density (PSD) is a macromolecular complex located at the postsynaptic terminals of glutamatergic synapses, detectable with electron microscopy as a disc with a surface area of approximately 0.07 μm30–40 nm ,238,239.30–40 nm  organize30–40 nm ,242. NMD30–40 nm . Patholo30–40 nm  but also30–40 nm ,246. Alt30–40 nm , and sev30–40 nm . These f or fMRI ,249.This complex molecular arrangement may act as a spatio-temporal organizer with multiple converging signaling possibly exiting in the structural changes of synaptic plasticity processes ,251. ConConsistent with the position of PSD in the mechanisms involved in synaptic plasticity, several studies have demonstrated the centrality of this structure in the effect of antipsychotic drugs and the role of dopamine occupancy ,273,274.Homer1 gene [Homer1a is an immediate early gene (IEG), and it can be induced by dopaminergic and glutamatergic manipulations [Homer is a family of proteins located at the PSD associated with schizophrenia, as demonstrated by genetic clinical and preclinical studies investigating the er1 gene ,275,276.er1 gene ,278. Homulations ,280. It ulations ,282.c-Fos [2A and α2 blockade caused by asenapine [Several preclinical studies have investigated the effects of antipsychotics and other drugs in the modulation of Homer1a and Homer1b/c by evaluating the involvement of different receptor profiles, particularly based on D2R occupancy and the timing of administration in the variable induction of these molecules, and thus in the different modes of synapse restructuring . Considec-Fos ,302. On c-Fos ,296,300 c-Fos . Chronicc-Fos . This efsenapine   scaffolding protein interacting with NMDAR through its NR2 subunit, AMPAR through stargazing, shaker-type potassium channels, cell adhesion molecules neuroligins (NLGNs), transmembrane protein Disintegrin, and metalloprotease 22 (ADAM22) ,317,318.2A and α2 adrenoreceptors blockade [2+ release, an effect blocked by a PKA inhibitor. This finding may suggest that D1R promotes NMDAR-Ca2+ signaling via a PKA-dependent mechanism [PSD-95 seems to have the same distribution of other constitutive genes, such as Homer1b/c, when modulated by asenapine or olanzapine, which share a receptor profile that also involves other monoamines stimulating serotonergic and adrenergic receptors. An increase was found in the signal intensity of PSD-95 throughout all subregions of the cortex and striatum, as well as in the NAc, by asenapine at different doses  as well as by olanzapine at 2.5 mg/kg  probablyblockade . Conversblockade . A preclblockade . In ratsblockade . Finallyblockade . Chronicblockade  accordinblockade , favorinblockade . Given tblockade , the incblockade . In ratsblockade . NMDAR iechanism . On the echanism ,340,341.echanism . The stiechanism . On the echanism .De novo mutation (R1117X) in the Shank isoform ProSAP2/Shank3 identified in a patient affected by schizophrenia is responsible for an accumulation of mutated ProSAP2/Shank3 in hippocampal neurons within the nucleus, resulting in an alteration in the transcription of several genes, such as synaptotagmin 1 and leucine-rich repeat transmembrane neuronal protein 1 (LRRTM1) and a reduction in synaptic density [Shank is a PSD protein whose mutations were found to be strongly associated with ASD, ID, and schizophrenia , and tha density . A precl density .There is robust evidence, embedded in almost 30 pieces of research, on the role of dopaminergic receptors in mediating the induction/activation of several IEG programs of immediate and long-lasting type ,349,350.Arc-KO mice exhibit a decrease in the proportion of thin and filopodia-like protrusions as well as in the density of spines in hippocampal neurons [2A [Arc gene compared to both asenapine and olanzapine, leading to the hypothesis that an upregulation of the IEG in the striatum could be related to a perturbation of dopamine neurotransmission through D2R occupancy [The activity-regulated cytoskeleton-associated protein (Arc) is an IEG encoding for a protein characterized by a C-terminus involved in the regulation of cytoskeleton structure through the interaction with F-actin , microtu neurons , probabl neurons , suggest neurons . Arc is  neurons , probablrons [2A ,357,358.ccupancy ,300.C-fos is a transcription factor that, when induced, forms the heterodimer activator protein-1 (AP-1), which binds to the promoter region of numerous target genes . GeneralSome genes involved in neuroprotection appear to be relevant for the pathophysiology of schizophrenia and other neurodevelopmental disorders and may represent novel non-canonical strategies for the treatment of these diseases. Among them, Notch Receptor 4 (NOTCH4) regulates neuronal and glial signaling and maturation ,362,363,Brain-derived neurotrophic factor (BDNF) and tropomyosin receptor kinase B (TrkB) modulate synaptic plasticity ,370,371 Haloperidol and aripiprazole can decrease the formation of D2R-DISC1 complexes by blocking D2R, although haloperidol does not seem to significantly affect neuroprotection . Ray andBeaulieu and colleagues showed that the increased dopaminergic stimulation of D2R results in decreased Akt activity and serine phosphorylation of glycogen synthase kinase 3 (GSK3) . In coheRegarding antiproliferative effect, lumateperone could improve NMDAR- and AMPAR-mediated D1R signaling and increase dopamine and glutamate release in the medial PFC in rats via mTORC ,420,421,During inflammatory processes, quinolinic acid and kynurenic acid are mainly synthesized in microglia and in astrocytes, respectively. The first is an NMDAR agonist with potential excitotoxicity , while tPreclinical studies in mice showed that maternal immune activation affects dopamine receptor expression, increasing dopamine levels in the striatum and decreasing D2R levels in adult offspring ,435. AltDopamine may modulate the activity, migration, differentiation, and proliferation of immune cells ,463,464.Multiple first- and second-generation antipsychotics may decrease oxidative stress by blocking the release of proinflammatory cytokines from activated microglia .+ T cells in the blood [+ T cells of patients treated with clozapine, exhibit an increase in DRD3 expression [2+/CaM/Akt-mediated nuclear factor kappa-light-chain-enhancer of activated B cell (NF-κB) [Maes and colleagues demonstrated that patients with TRS had dysregulation cell-mediated immunity with increased monocytic activity . Fernandhe blood . Typicalhe blood , while ahe blood ,472,473.he blood  and inhihe blood . Here, the blood ; (3) attpression ; (4) actpression ; (5) inh (NF-κB)  and prev (NF-κB) ; (6) reg (NF-κB) ,483,484; (NF-κB) . Akt, in (NF-κB) ,423. Fin (NF-κB) .A post-mortem study showed that in the PFC of schizophrenia patients, there was an increase in NRG1-induced ErbB4 activation and increased ErbB4/PSD-95 interaction, responsible for the suppression of NMDAR signaling activation . The NRG2+ channels [2+ influx from N-type Ca2+ channels [D2R in the striatum may activate the phospholipase C (PLC)/inositol trisphosphate (IP3)/calcineurin signaling pathway and modulate the excitability of GABAergic neurons through the regulation of presynaptic L-type Cachannels . Alteratchannels . Calcinechannels , which ichannels ,492. DARchannels . Specifichannels . In addichannels , whereaschannels ,496. In channels ,498,499,channels , which bchannels . These fchannels .i/o-PKA or serotonin 5HT2A-Gq-PLC signaling pathways [s/Golf, whereas D2Rs are bound to Gi/o and release Gαi/o and Gβγ subunits. Canonically, the function of D2R is implicated in antagonizing cAMP-dependent signaling by inhibiting the activation of PKA [2A-Gq-PLC, the other canonical target of choice for antipsychotics, for second-generation antipsychotics [2A blocking dopamine efflux in all regions of the brain with an excitatory function was facilitated, potentiating dopamine neurotransmission in cortical regions and leading to reduction of dopamine release in the mesolimbic areas that contributes to antipsychotics canonical activity [Multiple lines of evidence have found a link between antipsychotic drugs and epidermal growth factor (EGF) through the signaling of the mitogen-activated protein kinase extracellular signal-regulated kinase (MAPK)-ERK cascade ,503. Clopathways . Dopaminn of PKA ,507. In n of PKA . In thisn of PKA ,390. Bean of PKA . From a n of PKA . Togethen of PKA . On the ychotics . Throughactivity .A preclinical study in the rat cortex and striatum showed that the acute administration of clozapine increased c-Fos levels and induced biphasic phosphorylation of 90 kDa ribosomal s6 kinases (p90RSKs) by MEK/ERK independently of EGF receptor blockade . In contphosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit delta (PIK3CD), a gene encoding for the delta catalytic subunit of PI3K by going on to alter the ErbB4-PI3K pathway [The upregulation of transcripts of the NRG-ErbB signaling pathway in schizophrenia is reported , and it  pathway . This in pathway . Liu and pathway .Finally, antipsychotics drugs may regulate the AKT/mTOR pathway by directly affecting dopaminergic signaling: changes in striatal dopamine neurotransmission are caused, at least in part, by elevated D2R expression and upregulated ERK1/2 activation suggesting the implication of mTOR complex 2 (mTORC2) signaling as a non-canonical pathway in regulating striatal dopamine tone and D2R signaling .An ongoing search exploits the possibility of repurposing antipsychotics for the treatment of medical conditions other than psychiatric diseases, such as cancer ,516,517.According to this possibility, here we consider non-canonical MOAs of first- and second-generation antipsychotics under evaluation for cancer and other diseases.O6-methylguanine-DNA-methyltransferase (MGMT) gene [glioma-associated oncogene 1 [2+ and calmodulin typical of these cancers’ cell types, which are associated with increased DNA synthesis and cell proliferation. This drug is found to bind the IP3  receptor, promoting Ca2+ release from the endoplasmic reticulum and subsequent cell death [Chlorpromazine antagonism at the D2R  could plMT) gene . It was MT) gene ,526,527.MT) gene , relevanMT) gene , and NMDMT) gene  involvedMT) gene . A preclMT) gene . FurtherMT) gene . ConsideMT) gene ,533. PenMT) gene . The decMT) gene . These acogene 1 . Trifluoll death  and actill death . Finallyll death . Anotherll death . The epill death . Insteadll death . Finallyll death ,541. Olall death  and disrll death . Risperill death . Clomiprll death . Clozapill death ,547,548,ll death  through ll death .The potential efficacy of dopamine receptor antagonists, especially at D2R, in cancer therapy, is still in its infancy, and the role of dopamine in cancer biology is worth further exploration . The effs and D2R-Gi signaling complexes) has also open new avenues to understanding the “dopaminergic side” of antipsychotic action allowing [Although all antipsychotics share the common feature represented by D2R occupancy, which is considered the major mechanism of the “anti-psychotic” effect, in recent years, other cellular mechanisms based on dopaminergic function, its modulation, and non-canonical type have emerged ,561,562.allowing ,564. Theallowing . One of allowing ,79. It hallowing , whereasallowing . It has allowing . Concernallowing ,570,571.allowing . A critiallowing ,573, GABallowing , and serallowing  ones; doallowing ,576. Furallowing  and physallowing , possiblallowing . Among tallowing  and the allowing . NPEG4(IETDV)2 (Tat-N-dimer), which binds the tandem PDZ1-2 domain of PSD-95 with a high affinity of 4.6 nM [Second, it could be questioned if and how non-canonical mechanisms can be pragmatically tackled for the development of novel therapeutic strategies that are needed for those conditions, such as TRS, that do not respond or respond poorly to the available antipsychotics. In this regard and concerning presynaptic dopamine regulation, at least two compounds are in an advanced phase of clinical development represented by TAAR1 agonist ,584,585 f 4.6 nM ,588 in tf 4.6 nM .max) and/or D2R high affinity increased number.Third, an open question of clinical relevance is to what extent novel mechanisms based on non-canonical dopamine-related MOA can be “game-changing” in those difficult-to-treat conditions such as TRS. At present, the xanomelin + trospium combination is under scrutiny in a clinical trial specifically designed for subjects with schizophrenia with an inadequate response to their current antipsychotic treatment , and anyAntipsychotics’ dopamine-based non-canonical mechanisms should be exploited (i) for a better understanding of the complex cellular dynamics of antipsychotics beyond D2R occupancy-driven signaling ; (ii) to overcome the limitations, in terms of both efficacy and adverse events of the available compounds. This could allow the exploration of new molecular avenues within the dopamine system, which remains, directly or indirectly, a fundamental pharmacological target in schizophrenia treatment.


Dactylis glomerata L.). This study identified 22 DgTCP transcription factors in orchardgrass and determined their structure, phylogeny, and expression in different tissues and developmental stages. The phylogenetic tree classified the DgTCP gene family into two main subfamilies, including class I and II supported by the exon–intron structure and conserved motifs. The DgTCP promoter regions contained various cis-elements associated with hormones, growth and development, and stress responses, including MBS (drought inducibility), circadian (circadian rhythms), and TCA-element . Moreover, DgTCP9 possibly regulates tillering and flowering time. Additionally, several stress treatments upregulated DgTCP1, DgTCP2, DgTCP6, DgTCP12, and DgTCP17, indicting their potential effects regarding regulating responses to the respective stress. This research offers a valuable basis for further studies of the TCP gene family in other Gramineae and reveals new ideas for increasing gene utilization.Plant-specific TCP transcription factors regulate several plant growth and development processes. Nevertheless, little information is available about the TCP family in orchardgrass ( TB1 from maize [Zea mays]) [C (CYC from snapdragon [Antirrhinum majus]) [PCFs from rice [Oryza sativa]) [The plant-specific TEOSINTE BRANCHED 1/CYCLOIDEA/PROLIFERATING CELL FACTOR (TCP) gene family was first discovered in 1999 [a mays]) , C (CYC  majus]) , and P (sativa]) . Memberssativa]) . Moreovesativa]) . The clesativa]) ,6. ArginAtTCP20 acts upstream of AtTCP9 to regulate leaf senescence via the jasmonic acid (JA) signal pathway [AtTCP16 is necessary for pollen development in developing microspores [Arabidopsis thaliana, five miR319a targets in CIN class II gene subgroups  were confirmed important in leaf growth and morphogenesis [AtTCP4 is essential for the natural life activity of petals, the multifaceted regulation of JA and auxin (IAA) synthesis, the senescence of ripe leaves, and the age-dependent decomposition of leaf photosynthetic complexes [LjCYC1 and LjCYC3 (in Lotus japonicus) and AmCYC (in snapdragon) produce a marked effect in floral development [A thaliana AtTCP1 and GhCYC2 from Gerbera hybrida control symmetrical petal growth in flowers [ZmTB1 in maize, AtBRC1 and AtBRC2 in A thaliana, and OsTB1 in rice modulate branching by negatively regulating axillary bud growth [TCPs regulate the circadian clock and plant morphogenetics. TCP2, 3, 11, and 15 combine with the TGGGC (C/T) element to interact with various compositions of the core circadian rhythm, thus mediating the Arabidopsis circadian clock. Additionally, TCP20 and 22 improve the circadian clock [Many TCP proteins are critical in several plant biological processes ,7, inclu pathway . AtTCP16rospores . In Arabogenesis . AtTCP4 omplexes ,24,25,26elopment . Moreove flowers ,29. ZmTBd growth ,30,31. Ban clock .OsTCP19 induces the typical genes from abscisic acid (ABA), methyl jasmonate (Me-JA), ethylene (ET), IAA, cytokinins (CK), and other signaling pathways in rice. These genes reduce reactive oxygen species, the accumulation of fat droplets, and water loss in transgenic plants, thus improving their tolerance to high salt and mannitol treatments [OsTCP21 and OsPCF6 enhances tolerance to cold stress by altering the scavenging of reactive oxygen species. Similarly, OsPCF5 and OsPCF8 improve tolerance to cold stress [OsPCF2 to the OsNHX1 promoter improves salt and drought tolerance [A thaliana, TCP20 associates with NLP6/7 to modulate signal transduction and nitrate assimilation [TCP genes have also been reported in other species. For example, four miR319 target genes, including AsPCF5, AsPCF6, AsPCF8, and AsTCP14, were downregulated in drought and salt tolerance creeping bentgrass (Agrostis stolonifera) [TCP genes in common bean (Phaseolus vulgaris) and PeTCP10 in moso bamboo (Phyllostachys edulis) enhance tolerance to salt stress [GhTCP genes in cotton (Gossypium hirsutum) were upregulated under drought salt and heat stress [TCP gene family plays a major role in regulating plant life processes, there are no reports of their functioning in orchardgrass (D glomerata).Environmental stresses can affect plant growth and development , and someatments . In riced stress ,36. Bindolerance . In A thmilation . Severalonifera) ,40. Somet stress ,42. Besit stress . AlthougD glomerata has a high nutrient content and is among the four major global economic perennial grasses. It establishes fast, recovers quickly after mowing, and has high shade, drought, and barren tolerance [TCPs in orchardgrass through synthetic analysis of the D. glomerata genome . Preliminary predictions of DgTCP gene evolution involved the analysis of phylogenetic and gene duplication events and collinearity with other plants. Additionally, TCP expression was assessed in different tissues, developmental stages, and abiotic stresses. The results of this study will be helpful in elucidating orchardgrass adaptation to different environments and may reveal the functions of DgTCPs.Orchardgrass is a widely cultivated perennial forage grass that is native to central and western Europe, the temperate regions of Asia, and North Africa . D glomeolerance . Many vahttp://pfam.xfam.org/, accessed on 1 March 2022) [http://orchardgrassgenome.sicau.edu.cn/, accessed on 20 January 2022) using HMMER 3.0 software  [TCPs were aligned with Clustal X2.0 [http://web.expasy.org/protparam/, accessed on 3 March 2022) [Firstly, the TCP domain (PF03634) HMM profile, which was obtained from the Pfam database (ch 2022) , was theof 0.01) . Secondltal X2.0 , and redch 2022) .A thaliana TCP genes were obtained from TAIR  [Brachypodium distachyon TCP protein sequences were obtained from Plant TFDB  [B distachyonwere, and A thaliana were constructed using MEGA7.0 [https://itol.embl.de/itol.cgi/, accessed on 14 April 2022).The sequences of ry 2022) . Next, 2ch 2022) . Multiplch 2022) . Based o MEGA7.0  and thenDgTCPs were generated based on available genomic information and coding sequences from the Gene Structure Display Server  [http://meme-suite.org/, accessed on 11 May 2022) was used to identify the conserved DgTCP proteins motifs (considering ten maximum motifs and default settings) [The exon–intron structures of ay 2022) . The onlettings) .DgTCPs were acquired from the orchardgrass genome database . The 2000 bp region upstream of all DgTCPs was considered the promoter sequence, and the cis-acting promoter elements were appraised via PlantCARE  [The nucleotide sequences of ne 2022) . The putTCP gene was retrieved from orchardgrass genome annotations. MapGene2Chrome  was used to describe the location of the TCPs on the chromosomes. Next, DgTCP gene duplication was analyzed using MCScanX and default parameters [TCP gene collinearity between D glomerata, A thaliana, O sativa, Sorghum bicolor, Hordeum vulgare, and B distachyon  [O sativa, S bicolor, B distachyon, and H vulgare were downloaded from the JGI Genome Portal , and A thaliana was downloaded from TAIR .The chromosomal position information of each rameters . Using til 2022) . The gen3), 100μM abscisic acid (ABA), and 100 μM salicylic acid (SA). The leaves for each treatment were collected separately at 0, 1, 3, 6, 12, and 24 h after treatment, immediately frozen in liquid nitrogen, and then stored at −80 °C for qRT-PCR.Seeds of “Baoxing” orchardgrass were grown in a growth chamber at a 22 °C/14 h (day) and 20 °C/10 h (night) cycle. One week after germination, the seedings were irrigated with 1/2 Hogland solution. When the seedlings reached the third to fourth leaf, they were treated with 1/2 Hoagland solution containing 20% polyethylene glycol (PEG6000), 250 mM sodium chloride (NaCl), 100 μM methyl jasmonate (Me-JA), 200 mM sodium bicarbonate  and AKZ-NRGR66 7 (high-tillering) were obtained from Xu et al.  following the manufacturer’s instructions. Primers for the 14 DgTCPs were designed using Primer 5.0 software (® green qPCR Mix (Monad) on the Bio-Rad CFX96 instrument. GAPDH was the internal reference gene for normalization [−ΔΔCt method [The total RNA of the samples under different treatments was extracted using the Hipure HP plant RNA mini kit . First-strand cDNA was synthesized using the MonScriptsoftware , and qRTlization , and thet method . All qRTDgTCP1–DgTCP22 based on their chromosomal positioning. The protein sequence length, CDS length, molecular weight, isoelectric point (pI), and gene location of the 22 DgTCPs are captured in DgTCP1), and the biggest was 47,382.56 kDa (DgTCP16). The pI ranged from 5.09 (DgTCP7) to 9.92 (DgTCP11), and the protein lengths were 165 (DgTCP17) to 454 (DgTCP6) aa.Twenty-two genes were retrieved from the orchardgrass genome and designated as B distachyon, and 24 A thaliana, TCP proteins were constructed using the neighbor-joining (NJ) method to clarify the phylogenetic relationships and evolutionary history of the TCP gene family , and the other 11 belong to class II (TCP-C) (DgTCPs) and CIN subclasses (7 DgTCPs)  and class II (CIN and CYC/TB1) groups  . The cla DgTCPs) . Thus, w) groups . Class ITCP gene family in orchardgrasss , O sativa, S bicolor, B distachyon, and H vulgare (D glomerata and the 5 species were 33 (O), 31 (S bicolor), 30 (B distachyon), 23 (H vulgare), and 5   . These rDgTCP genes, 93 cis-elements possibly involved in phytohormone response, plant growth and development, and stress response were identified to unravel the functions and regulatory mechanisms of DgTCP genes  that participate in endosperm expression. Besides, NON-box and CAT-box were associated with meristem expression plant growth and development. The seed-specific regulatory element (RY element) and zein metabolism regulatory element (O2 site) were identified in six and nine DgTCPs, respectively. In addition, a circadian control element (circadian), a flavonoid biosynthetic regulation element (MBSI), a cell cycle regulation element (MSA-like), and a palisade mesophyll cells regulatory element (HD-Zip 1) were also discovered in the promoter regions of DgTCPs  (The promoter regions of one and three f DgTCPs . In seveectively . In addierature) .DgTCP genes were the highest in the leaf, spike, and stem, respectively , and after vernalization (upregulated). TCP18 was significantly upregulated during vernalization in “Baoxing” but showed no change in “Donata”. However, it was upregulated in “Baoxing” and “Donata” during the late vernalization stage and similar in “Baoxing” and “Donata”. From after vernalization to the heading stage, DgTCP2, DgTCP4, DgTCP16, DgTCP17, and DgTCP21 were significantly upregulated during the before heading stage in “Baoxing” and the heading stage in “Donata”.The expression patterns of the early- (Baoxing) and late-flowering (Donata) cultivars were analyzed at five flower bud development stages to identify their potential physiological functions in flowering. In most developmental stages, the expression of “Donata” b. TCP15 DgTCPs in regulating growth, and development. All DgTCPs were differentially expressed in the four tissues, contrary to their expression under normal conditions (DgTCPs were highly expressed in the low- (D20170203) and high-tillering (AKZ-NRGR667) varieties. The expression of TCP1 was significantly higher in D20170203 than AKZ-NRGR667 in the four tissues. However, the expression of TCP20 was higher in the leaves of AKZ-NRGR667 than in D20170203 but similar in other tissues. The expression of TCP10 was higher in the tiller bud of D20170203 than in AKZ-NRGR667 but similar in other tissues. Interestingly, the expression of TCP9 was higher in the tiller buds of D20170203 than those of AKZ-NRGR667 but lower in the leaves of D20170203. Thus, DgTCP1 and DgTCP10 probably inhibit tiller bud development in the two varieties in a differential pattern, ultimately resulting in different phenotypes.Gene expression data were retrieved from four tissues of low- (D20170203) and high-tillering (AKZ-NRGR667) orchardgrass to determine the roles of nditions . In tillDgCTPs in “Baoxing” under six abiotic stresses . Salt stress suppressed the expression of two genes (DgTCP8 and DgTCP18) throughout the salt time points and downregulated eleven genes in the early stages (1–3 h) of salt stress. Drought stress upregulated 13 DgTCPs, and the highest values ranged from 1.13-fold (DgTCP3) to 16.89-fold (DgTCP1). Additionally, six genes  showed significantly higher expression 1 h after treatment with an alkali solution. In contrast, other DgTCPs were upregulated at 6 h of alkali treatment, while DgTCP8 was suppressed at all time points. Nine DgTCPs were highly induced under ABA treatment, and DgTCP12 displayed the highest expression. Nevertheless, ABA treatment inhibited DgTCP3, DgTCP8, DgTCP9, DgTCP16, and DgTCP19 at all time points. Me-JA treatment upregulated five , one (DgTCP8), and two genes , which peaked at 3, 6, and 12 h, respectively. These genes were upregulated ranging from 1.26-fold (DgTCP18) to 20.16-fold (DgTCP12). In contrast, Me-JA showed no observable regulation in four genes  but downregulated DgTCP9 and DgTCP19. Furthermore, SA treatment suppressed the expression of DgTCP3, DgTCP9, and DgTCP19 across the time points, with six  and three genes  peaking at 6 and 24 h, respectively. Six stress treatments upregulated DgTCP1, DgTCP2, DgTCP6, DgTCP12, and DgTCP17, but DgTCP levels varied under different stresses and time points when combined with the stress expression pattern data.TCP) gene family are crucial plant-specific transcription factors with various functions in many processes, including hormone biosynthesis, flower development, leaf development, lateral branching, and defence response. To date, the TCP gene family has been identified in many plants, such as switchgrass (Panicum virgatum) [Arabidopsis, and rice [TCP gene family in high-quality forages, such orchardgrass, is lacking.The plant-specific TEOSINTE BRANCHED 1/CYCLOIDEA/PROLIFERATING CELL FACTOR (irgatum) , maize [irgatum) , Arabidoand rice . HoweverTCP genes in the orchardgrass genome [DgTCP proteins have a highly conserved TCP domain (motifs 1 and 2). Thus, DgTCPs possibly have similar DNA-binding and protein–protein interaction patterns [DgTCPs are divided into two major subclasses  [Vitis vinifera) (17) [Fragaria vesca) (19) [Sorghum (20) [DgTCPs was lower than the number in A thaliana (24) [Glycine max) (54) [Nictiana tabacum) (61) [D glomerata experienced whole-genome replication events [DgTCP gene family and no tandem duplication  , grapevira) (17) , strawbeca) (19) , and Sorhum (20) , In contana (24) , maize (ana (24) , soybeanax) (54) , and tobum) (61) . Tandem,um) (61) . Previoun events . This stlication . Segmentechanism ,70.TCP genes are broadly involved in the regulatory processes of plant life [TCP genes in orchardgrass under different abiotic stresses is necessary. In this study, salt and drought treatments upregulated more than half of the identified DgTCPs, similar to the results from rapeseed (Brassica napus) [DgTCPs at all time points. The ABA signal transduction pathway is significant for stress response [Abiotic stresses affect plant growth and development, quality, and yield , and TCPant life . Therefoa napus) , cotton a napus) , and swia napus) . Nonetheresponse .TCPs interact with other genes in JA biosynthesis to influence growth, development, and abiotic stress responses. For instance, TCP4 encodes the enzyme that catalyzes a crucial step in JA synthesis by positively regulating the LOX2 gene [TCP4 in plants downregulates LOX2, thus reducing JA synthesis and increasing plant sensitivity to stress [TCP genes were diverse after Me-JA treatment  and tissues suggested that many DgTCP genes regulate stress tolerance and development in orchardgrass. Specifically, TCP9 probably regulates flowering time, tiller number, and drought stress in D glomerata. This genome-wide analysis of orchardgrass is significant for identifying new DgTCP genes with novel functions and provides a foundation for breeding high-quality orchardgrass varieties and the functional validation of DgTCP genes in the future.Furthermore, the expression of the