Journal of advanced research最新文献

Introduction: Autoimmune uveitis (AU) is a prevalent ocular autoimmune disease leading to significant visual impairment. However, underlying pathogenesis of AU required to develop more efficient therapy remain unclear.

Methods: We isolated peripheral blood mononuclear cells (PBMCs) from AU patients and performed single-cell RNA sequencing (scRNA-seq). Besides, experimental autoimmune uveitis (EAU) model was established and treated with histone deacetylase inhibitor (HDACi) Belinostat or vehicle. We extracted immune cells from Blank, EAU, and HDACi-treated EAU mice and used scRNA-seq, flow cytometry, siRNA, specific inhibitors, and adoptive transfer experiments to explore the role of HDACs and its downstream potential molecular mechanisms in the immune response of EAU and AU.

Results: We found highly expressed histone deacetylases (HDACs) family in AU patients and identified it as a key factor related to CD4⁺ effector T cell differentiation in the pathogenesis of AU. Our further studies showed that targeted inhibition of HDACs effectively alleviated EAU, restored its Th17/Treg balance, and reduced inflammatory gene expression, especially in CD4⁺ T cells. Post-HDACs inhibition, Treg proportions increased with enhanced immunomodulatory effects. Importantly, HDACs exhibited a positive promoting role on Th17 cells. Based on scRNA-seq screening and application of knock-down siRNAs and specific inhibitors in vitro and vivo, we identified CDK6 as a key downstream molecule regulated by HDAC1/3/6 through acetyl-histone H3/p53/p21 axis, which is involved in Th17 pathogenicity and EAU development. Additionally, HDACs-regulated CDK6 formed a positive loop with ID2, inducing PIM1 upregulation, promoting Th17 cell differentiation and pathogenicity, and correlates with AU progression.

Conclusion: Based on the screening of clinical samples and downstream molecular functional validation experiments, we revealed a driving role for HDACs and the HDACs-regulated CDK6/ID2 axis in Th17 cell differentiation and pathogenicity in AU, proposing a promising therapeutic strategy.

Background: Traditional Chinese Medicine (TCM) has gained global attention, particularly after Professor Youyou Tu was awarded the Nobel Prize for her discovery of artemisinin as a treatment for malaria. However, the theory behind TCM is often perceived as a "black-box" with complex components and an unclear structure and mechanism of action. This had hindered the development of TCM within the framework of modern medicine.

Aim of review: The molecular compatibility theory proposed by Professor Tian Xie's team integrates TCM with Western medicine in clinical practice, and provide a feasible direction for TCM modernization. It is necessary to summarize and popularize this theory. This review aims to systematically introduce this theory to provide some new insight for development of TCM.

Key scientific concepts of review: According to the molecular compatibility theory, the desired effects can be achieved by organically combining multiple active molecules from TCM. These TCM molecular compounds have specific ingredients, precise mechanisms, and controllable quality that meet the standards of modern medicine. The molecular compatibility theory has guided the development of antitumor new drug elemene emulsions, and has also revealed extensive compatibility between TCM-derived active molecules and other TCM, Western medicine, or biomaterials. This discovery opens up potential TCM-based treatment options. In conclusion, the molecular compatibility theory holds promise as a strategy for modernizing TCM.

Introduction: Embryo implantation requires synergistic interaction between the embryo and the receptive endometrium. Glycoproteins and glycan-binding proteins are involved in endometrium-embryo attachment. Sialyl Tn (sTn), a truncated O-glycan, is catalyzed by ST6 N-Acetylgalactosaminide Alpha-2,6-Sialyltransferase 1 (ST6GALNAC1) and can be detected by specific Sialic-acid-binding immunoglobulin-like lectins (Siglecs). Whether the sTn-Siglecs axis supports embryo implantation remains unknown.

Objectives: This paper aims to study the role of ST6GALNAC1/sTn-Siglecs axis in embryo implantation.

Methods: ST6GALNAC1 and sTn in human endometrium were analyzed by immunohistochemistry. An in vitro implantation model was conducted to evaluate the effects of ST6GALNAC1/sTn on the receptivity of human endometrial AN3CA cells to JAR spheroids. Immunoprecipitation combined with mass spectrometry analysis was carried out to identify the key proteins modified by sTn in endometrial cells. Siglec-6 in human embryos was analyzed by published single-cell RNA sequencing (scRNA-seq) datasets. Protein interaction assay was applied to verify the bond between the Siglec-6 with sTn-modified CD44. St6galnac1 siRNAs and anti-sTn antibodies were injected into the uterine horn of the mouse at the pre-implantation stage to evaluate the role of endometrial St6galnac1/sTn in embryo implantation. Siglec-G in murine embryos was analyzed by immunofluorescence staining. The function of Siglec-G is evidenced by uterine horn injection and protein interaction assay.

Results: Both human and murine endometrium at the receptive stage exhibit higher ST6GALNAC1 and sTn levels compared to the non-receptive stage. Overexpression of ST6GALNAC1 significantly enhanced the receptivity of AN3CA cells to JAR spheroids. Inhibition of endometrial ST6GALNAC1/sTn substantially impaired embryo implantation in vivo. CD44 was identified as a carrier for sTn in the endometrial cells of both species. Siglec-6 and Siglec-G, expressed in the embryonic trophectoderm, were found to promote embryo attachment, which may be achieved through binding with sTn-modified CD44.

Conclusion: ST6GALNAC1-regulated sTn in the endometrium aids in embryo attachment through interaction with trophoblastic Siglecs.

Introduction: The Septin family of cytoskeletal proteins is abundant in platelets. When these proteins are functionally blocked using the compound forchlorfenuron (FCF), it hampers the normal activation processes of purified human platelets.

Objectives: To evaluate the in vivo effects of FCF on physiological haemostasis and pathological thrombosis in mice and to investigate possible molecular mechanisms.

Methods: The impact of FCF on haemorrhage risk in the brain, liver, and tail of mice was investigated. Using several experimental models, thrombus development in the lung, mesenteric arteries, and postcava was studied. Functional assays were performed on mice and human platelets, both with and without FCF pretreatment. These tests included aggregation, granule release, ROS production, integrin αIIbβ3 activation, cytoskeletal remodeling imaging, and clot retraction.

Results: Neither oral nor intravenous administration of FCF showed any apparent impairment of haemostasis in the tissues studied, but only later administration resulted in a significant reduction in thrombus formation in different mice vessel types. FCF generally inhibited agonist-induced platelet aggregation, degranulation, ROS burst, morphological expansion on the fibrinogen matrix with completely disordered dynamic organizations of the cytoskeleton for septin, tubulin and actin. In addition, FCF was found to antagonise agonist-induced dephosphorylation of VASP (Ser239) and PI3K/AKT and ERK1/2 phosphorylation.

Conclusion: FCF showed preferences in attenuating pathological thrombus formation, apart from physiological haemostasis, with possible mechanisms to prevent cytoskeletal remodelling and signal transduction of AKT, ERK1/2 and VASP signalling pathways, suggesting that Septin may serve as a promising target for the prevention and treatment of thrombotic diseases.

Introduction: Interspecific introgression between Gossypium hirsutum and G. barbadense allows breeding cotton with outstanding fiber length (FL). However, the dynamic gene regulatory network of FL-related genes has not been characterized, and the functional mechanism through which the hub gene GhTUB5 mediates fiber elongation has yet to be determined.

Methods: Coexpression analyses of 277 developing fiber transcriptomes integrated with QTL mapping using 250 introgression lines of different FL phenotypes were conducted to identify genes related to fiber elongation. The function of GhTUB5 was determined by ectopic expression of two TUB5 alleles in Arabidopsis and knockout of GhTUB5 in upland cotton. Yeast two-hybrid, split-luciferase and pull-down assays were conducted to screen for interacting proteins, and upstream genes were identified by yeast one-hybrid, dual-LUC and electrophoretic mobility shift assays.

Results: The 32,612, 30,837 and 30,277 genes expressed at 5, 10 and 15 days postanthesis (dpa) were grouped into 19 distinct coexpression modules, and 988 genes in the MEblack module were enriched in the cell wall process and exhibited significant associations with FL. A total of 20 FL-QTLs were identified, each explaining 3.34-16.04 % of the phenotypic variance in the FL. Furthermore, several FL-QTLs contained 15 genes that were differentially expressed in the MEblack module including the tubulin beta gene (TUB5). Compared with the wild type, the overexpression of GhTUB5 and GbTUB5 in Arabidopsis suppressed root cell length but promoted cellulose synthesis. Knockout of GhTUB5 resulted in longer fiber lines. Protein-based experiments revealed that GhTUB5 interacts with GhZFP6. Additionally, GhTUB5 was directly activated by GhHD-ZIP7, a homeobox-leucine zipper transcription factor, and its paralogous gene was previously reported to mediate fiber elongation.

Conclusion: This study opens a new avenue to dissect functional mechanism of cotton fiber elongation. Our findings provide some molecular details on how GhTUB5 mediates the FL phenotype in cotton.

Introduction: Osteoarthritis (OA) is a highly prevalent degenerative disease worldwide, and tumor necrosis factor (TNF-α) is closely associated with its development. Growth differentiation factor 11 (GDF11) has demonstrated anti-injury and anti-aging abilities in certain tissues; however, its regulatory role in OA remains unclear and requires further investigation.

Objectives: To identify whether GDF11 can attenuate osteoarthritis. To exploring the the potential mechanism of GDF11 in alleviating osteoarthritis.

Methods: In this study, we cultured and stimulated mouse primary chondrocytes with or without TNF-α, analyzing the resulting damage phenotype through microarray analysis. Additionally, we employed GDF11 conditional knockout mice OA model to examine the relationship between GDF11 and OA. To investigate the target of GDF11's function, we utilized NLRP3 knockout mice and its inhibitor to verify the potential involvement of the NLRP3 inflammasome.

Results: Our in vitro experiments demonstrated that endogenous overexpression of GDF11 significantly inhibited TNF-α-induced cartilage matrix degradation and inflammatory expression in chondrocytes. Furthermore, loss of GDF11 led to NLRP3 inflammasome activation, inflammation, and metabolic dysfunction. In an in vivo surgically induced mouse model, intraarticular administration of recombinant human GDF11 alleviated OA pathogenesis, whereas GDF11 conditional knockout reversed this effect. Additionally, findings from the NLRP3-knockout DMM mouse model revealed that GDF11 exerted its protective effect by inhibiting NLRP3.

Conclusion: These findings demonstrate the ability of GDF11 to suppress TNF-α-induced inflammation and cartilage degeneration by preventing mitochondrial dysfunction and inhibiting NLRP3 inflammasome activation, suggesting its potential as a promising therapeutic drug for osteoarthritis.

Introduction: Mitophagy, a selective form of autophagy responsible for maintaining mitochondrial homeostasis, regulates the antiviral immune response and acts as viral replication platforms to facilitate infection with various viruses. However, its precise role in herpes simplex virus 1 (HSV-1) infection and herpes simplex encephalitis (HSE) remains largely unknown.

Objectives: We aimed to investigate the regulation of mitophagy by HSV-1 neurotropic infection and its role in viral encephalitis, and to identify small compounds that regulate mitophagy to affect HSV-1 infection.

Methods: The antiviral effects of compounds were investigated by Western blot, RT-PCR and plaque assay. The changes of Parkin (PRKN)-mediated mitophagy and Nuclear Factor kappa B (NFKB)-mediated neuroinflammation were examined by TEM, RT-qPCR, Western blot and ELISA. The therapeutic effect of taurine or PRKN-overexpression was confirmed in the HSE mouse model by evaluating survival rate, eye damage, neurodegenerative symptoms, immunohistochemistry analysis and histopathology.

Results: HSV-1 infection caused the accumulation of damaged mitochondria in neuronal cells and in the brain tissue of HSE mice. Early HSV-1 infection led to mitophagy activation, followed by inhibition in the later viral infection. The HSV-1 proteins ICP34.5 or US11 deregulated the EIF2S1-ATF4 axis to suppress PRKN/Parkin mRNA expression, thereby impeding PRKN-dependent mitophagy. Consequently, inhibition of mitophagy by specific inhibitor midiv-1 promoted HSV-1 infection, whereas mitophagy activation by PRKN overexpression or agonists (CCCP and rotenone) attenuated HSV-1 infection and reduced the NF-κB-mediated neuroinflammation. Moreover, PRKN-overexpressing mice showed enhanced resistance to HSV-1 infection and ameliorated HSE pathogenesis. Furthermore, taurine, a differentially regulated gut microbial metabolite upon HSV-1 infection, acted as a mitophagy activator that transcriptionally promotes PRKN expression to stimulate mitophagy and to limit HSV-1 infection both in vitro and in vivo.

Conclusion: These results reveal the protective function of mitophagy in HSE pathogenesis and highlight mitophagy activation as a potential antiviral therapeutic strategy for HSV-1-related diseases.

Introduction: A disintegrin and metalloproteinase 8 (ADAM8), a crucial regulator in macrophages, is closely associated with cardiovascular disease progression.

Objectives: This study aimed to explore how ADAM8 regulates macrophage function to inhibit cardiac repair after myocardial infarction (MI).

Methods: Macrophage-specific ADAM8 knockout mice (ADAM8^{flox/flox, Lyz2-Cre}, KO) and corresponding control mice (ADAM8^flox/flox, Flox) were established using the CRISPR/Cas9 system. Bone marrow transplantation was performed, and macrophage-specific ADAM8-overexpressing adeno-associated virus (AAV6-CD68-Adam8) was produced. Finally, proteomics, RNA sequencing, and co-immunoprecipitation/mass spectrometry (COIP/MS) were used to explore the underlying mechanisms involved.

Results: ADAM8 was highly expressed in the plasma of patients with acute myocardial infarction (AMI) and in cardiac macrophages derived from AMI mice. ADAM8 KO mice exhibited enhanced angiogenesis, suppressed inflammation, reduced cardiac fibrosis, and improved cardiac function during AMI, which were reversed by overexpressing macrophage-specific ADAM8 and intervention with the clinical anti-angiogenic biologic bevacizumab. Bone marrow transplantation experiments produced ADAM8 KO phenotypes. RNA sequencing showed that autophagy was activated in bone marrow-derived macrophages (BMDMs) with ADAM8 KO, which was confirmed via p-mTOR Ser2448/mTOR, p62, and LC3II/I detection. Autophagy inactivation suppressed angiogenic factor release and promoted inflammation in BMDMs with ADAM8 KO. Mechanistically, ADAM8 could bind to ANXA2 and promote phosphorylation of the ANXA2 Ser26 site. ADAM8 KO impeded ANXA2 phosphorylation, inhibited mTOR Ser2448 site phosphorylation, and activated autophagy, which were demonstrated using the activation or inactivation of ANXA2 phosphorylation.

Conclusions: ADAM8 was increased in cardiac macrophages after AMI. The ADAM8-ANXA2-mTOR-autophagy axis in macrophages is responsible for regulating angiogenesis and inflammation following MI. Thus, ADAM8 may be a new target in MI treatment.

Introduction: CRISPR/Cas9 gene editing technology has significantly advanced gene therapy, with gene vectors being one of the key factors for its success. Poly (beta-amino ester) (PBAE), a distinguished non-viral cationic gene vector, is known to elevate intracellular reactive oxygen species (ROS) levels, which may cause cytotoxicity and, consequently, impact gene transfection efficacy (T.E.).

Objectives: To develop a simple but efficient strategy to improve the gene delivery ability and biosafety of PBAE both in vivo and in vitro.

Methods: We used glutathione (GSH), a clinically utilized drug with capability to modulating intracellular ROS level, to prepare a hybrid system with PBAE-plasmid nanoparticles (NPs). This system was characterized by flow cytometry, RNA-seq, Polymerase Chain Reaction (PCR) and Sanger sequencing in vitro, and its safety and efficacy in vivo was evaluated by imaging, PCR, Sanger sequencing and histology analysis.

Results: The particle size of GSH-PBAE-plasmid NPs were 168.31 nm with a ζ-potential of 15.21 mV. An enhancement in T.E. and gene editing efficiency, ranging from 10 % to 100 %, was observed compared to GSH-free PBAE-plasmid NPs in various cell lines. In vitro results proved that GSH-PBAE-plasmid NPs reduced intracellular ROS levels by 25 %-40 %, decreased the total number of upregulated/downregulated genes from 4,952 to 789, and significantly avoided the disturbance in gene expression related to cellular oxidative stress-response and cell growth regulation signaling pathway compared to PBAE-plasmid NPs. They also demonstrated lower impact on the cell cycle, slighter hemolysis, and higher cell viability after gene transfection. Furthermore, GSH hybrid PBAE-plasmid NPs exhibited superior safety and improved tumor suppression ability in an Epstein-Barr virus (EBV)-infected murine tumor model, via targeting cleavage the EBV related oncogene by delivering CRISPR/Cas9 gene editing system and down-regulating the expression levels. This simple but effective strategy is expected to promote clinical applications of non-viral vector gene delivery.

Background: The nuclear factor kappa B (NF-κB) is a critical pathway that regulates various cellular functions, including immune response, proliferation, growth, and apoptosis. Furthermore, this pathway is tightly regulated to ensure stability in the presence of immunogenic triggers or genotoxic stimuli. The lack of control of the NF-κB pathway can lead to the initiation of different diseases, mainly autoimmune diseases and cancer, including Renal cell carcinoma (RCC). RCC is the most common type of kidney cancer and is characterized by complex genetic composition and elusive molecular mechanisms.

Aim of review: The current review summarizes the mechanism of NF-κB dysregulation in different subtypes of RCC and its impact on pathogenesis.

Key scientific concept of review: This review highlights the prominent role of NF-κB in RCC development and progression by driving the expression of multiple genes and interplaying with different pathways, including the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt) pathway. In silico analysis of RCC cohorts and molecular studies have revealed that multiple NF-κB members and target genes are dysregulated. The dysregulation includes receptors such as TLR2, signal-transmitting members including RelA, and target genes, for instance, HIF-1α. The lack of effective regulatory mechanisms results in a constitutively active NF-κB pathway, which promotes cancer growth, migration, and survival. In this review, we comprehensively summarize the role of dysregulated NF-κB-related genes in the most common subtypes of RCC, including clear cell RCC (ccRCC), chromophobe RCC (chRCC), and papillary RCC (PRCC).