Comprehensive transcriptomic analysis of immune-related genes in diabetic foot ulcers: New insights into mechanisms and therapeutic targets.

BACKGROUND: Diabetic foot ulcers (DFU), affecting a quarter of diabetic patients and leading to high rates of amputation and mortality, pose significant health and economic burdens. Wound healing in DFU is often compromised by chronic inflammation, underscoring the critical role of immune cells. However, the systematic investigation of immune-related genes (IRGs) in DFU pathogenesis remains elusive. To address this gap, our study aims to explore the association between IRGs and DFU. METHODS: To explore biological changes in immune related gene expression in DFU, RNA-seq was performed on wound biopsies derived from 10 DFU patients and 11 healthy controls. Differentially expressed genes (DEGs) between DFU and normal samples were obtained by DEseq2. By intersecting the IRG list from the ImmPort database, the immune-related differentially expressed genes were identified. Function enrichment analysis and protein-protein interaction (PPI) analysis were applied by clusterProfiler and STRING database, and the hub genes of the PPI network were calculated by the cytoHubba plug-ins in Cytoscape. CIBERSORT algorithms was applied to analyze immune infiltration in DFU. And the correlation between immune cells infiltration and hub genes was explored by correlation analysis. Finally, to validate our findings, the transcriptional change of hub genes in DFU was confirmed using external scRNA-seq dataset and RT-qPCR. RESULTS: RNA-seq analysis detected 8,800 DEGs in DFUs, with 2,351 upregulated and 6,449 downregulated.526 differential IRGs were obtained from intersection of DEGs and IRGs. 526 differential IRGs were obtained from intersection of DEGs and IRGs. Enrichment function analysis of DEGs showed that they played a significant role in immune response. The PPI network was constructed, and the most significant module containing 4 hub genes was identified. CIBERSORT analysis showing that there was a significant difference between DFU and normal controls in the infiltration of immune cells. Compared with normal tissue, DFU tissue contained a higher proportion of resting NK cell, M0 macrophages, and activated mast cell, while resting dendritic cell, activated mast cell, and activated NK cell contributed to a relatively lower portion. Additionally, the analysis for M1/M2 polarization of macrophage cells shown that DFU tissue contained a higher M1/M2 ratio than normal group. Finally, the expression levels of 4 hub genes were confirmed by external scRNA-seq dataset and RT-qPCR. CONCLUSIONS: The immune related hub genes and the difference in immune infiltration between DFU tissue and normal controls might provide new insight for understanding DFU healing.

Gynura divaricata (L.) DC. promotes diabetic wound healing by activating Nrf2 signaling in diabetic rats.

THE ETHNOPHARMACOLOGICAL SIGNIFICANCE: Diabetic chronic foot ulcers pose a significant therapeutic challenge as a result of the oxidative stress caused by hyperglycemia. Which impairs angiogenesis and delays wound healing, potentially leading to amputation. Gynura divaricata (L.) DC. (GD), a traditional Chinese herbal medicine with hypoglycemic effects, has been proposed as a potential therapeutic agent for diabetic wound healing. However, the underlying mechanisms of its effects remain unclear. AIM OF THE STUDY: In this study, we aimed to reveal the effect and potential mechanisms of GD on accelerating diabetic wound healing in vitro and in vivo. MATERIALS AND METHODS: The effects of GD on cell proliferation, apoptosis, reactive oxygen species (ROS) production, migration, mitochondrial membrane potential (MMP), and potential molecular mechanisms were investigated in high glucose (HG) stimulated human umbilical vein endothelial cells (HUVECs) using CCK-8, flow cytometry assay, wound healing assay, immunofluorescence, DCFH-DA staining, JC-1 staining, and Western blot. Full-thickness skin defects were created in STZ-induced diabetic rats, and wound healing rate was tracked by photographing them every day. HE staining, immunohistochemistry, and Western blot were employed to investigate the effect and molecular mechanism of GD on wound healing in diabetic rats. RESULTS: GD significantly improved HUVEC survival, decreased apoptosis, lowered ROS production, restored MMP, improved migration ability, and raised VEGF expression. The use of Nrf2-siRNA completely abrogated these effects. Topical application of GD promoted angiogenesis and granulation tissue growth, resulting in faster healing of diabetic wounds. The expression of VEGF, CD31, and VEGFR was elevated in the skin tissue of diabetic rats after GD treatment, which upregulated HO-1, NQO-1, and Bcl-2 expression while downregulating Bax expression via activation of the Nrf2 signaling pathway. CONCLUSION: The findings of this study indicate that GD has the potential to serve as a viable alternative treatment for diabetic wounds. This potential arises from its ability to mitigate the negative effects of oxidative stress on angiogenesis, which is regulated by the Nrf2 signaling pathway. The results of our study offer valuable insights into the therapeutic efficacy of GD in the treatment of diabetic wounds, emphasizing the significance of directing interventions towards the Nrf2 signaling pathway to mitigate oxidative stress and facilitate the process of angiogenesis.

Bortezomib inhibits ZIKV/DENV by interfering with viral polyprotein cleavage via the ERAD pathway.

Flaviviruses have posed a serious threat to human health in the past decades, and effective therapeutic drugs are lacking; thus, treatment of flavivirus infection is a great challenge. The flavivirus protease NS2B3 is an attractive target for antiviral drug screening. Here, we developed an intracellular Zika virus (ZIKV) NS2AB3 self-cleavage assay to identify inhibitors that interfere with viral polyprotein cleavage and block ZIKV/dengue virus (DENV) replication. Bortezomib was identified as the most potent inhibitor, with a half-maximal effective concentration (EC50) in the nanomolar range. We found that instead of directly inhibiting NS2B3 protease activity, bortezomib dramatically induced the ubiquitination and aggregation of NS3, leading to the attenuation of its protease activity in cells. Two E3 ligases, HRD1 and RNF126, were found to be responsible for NS3 ubiquitination. Our study identifies bortezomib as a potential drug for the treatment of ZIKV/DENV infection and reveals the central role of the ERAD pathway in the inhibition of flaviviruses by bortezomib.

Electrostatic Interaction Between NS1 and Negatively Charged Lipids Contributes to Flavivirus Replication Organelles Formation.

Flavivirus replication occurs in membranous replication compartments, also known as replication organelles (ROs) derived from the host ER membrane. Our previous study showed that the non-structural (NS) protein 1 (NS1) is the essential factor for RO creation by hydrophobic insertion into the ER membrane. Here, we found that the association of NS1 with the membrane can be facilitated by the electrostatic interaction between NS1 and negatively charged lipids. NS1 binds to a series of negatively charged lipids, including PI4P, and a positively charged residue, R31, located on the membrane-binding face of NS1, plays important roles in this interaction. The NS1 R31E mutation significantly impairs NS1 association with negatively charged membrane and its ER remodeling ability in the cells. To interfere with the electrostatic interaction between NS1 and negatively charged lipids, intracellular phosphatidylinositol phosphates (PIPs) level was downregulated by the overexpression of Sac1 or treatment with PI3K and PI4K inhibitors to attenuate flavivirus replication. Our findings emphasize the importance of electrostatic interaction between NS1 and negatively charged lipids in flavivirus RO formation.

Zika NS1-induced ER remodeling is essential for viral replication.

Zika virus (ZIKV), a recently emerged member of the flavivirus family, forms replication compartments at the ER during its lifecycle. The proteins that are responsible for the biogenesis of replication compartments are not well defined. Here, we show that Zika nonstructural protein 1 (NS1)-induced ER remodeling is essential for viral replication. NS1 expressed in the ER lumen induced ER perinuclear aggregation with an ultrastructure resembling that of the replication compartment. Data from model membrane system indicated that the membrane-binding and membrane-remodeling properties of NS1 depend on its hydrophobic insertion into the membrane. These findings demonstrate that NS1 plays a crucial role in flavivirus replication compartment formation by remodeling the ER structure.

Zika NS2B is a crucial factor recruiting NS3 to the ER and activating its protease activity.

Zika virus (ZIKV) is an emergent flavivirus associated with severe neurological disorders. ZIKV NS3 protein is a viral protease that cleaves the ZIKV polyprotein precursor into individual viral proteins. In this study, we found that ZIKV NS3 by itself exhibited mitochondrial localization, which was quite different from its endoplasmic reticulum (ER) localization in ZIKV-infected cells. We screened viral proteins and identified NS2B as the bona fide recruiter of NS3 to the ER. The NS2B C-terminal tail interacted with NS3 protease domain to retain NS3 on the ER. ß-Sheet motifs that formed between NS2B and the NS3 protease domain played important roles in their interaction, while mutation in the ß-strand of NS2B attenuated NS2B-NS3 interaction and impaired the ability of NS3 protease to cleave the polyprotein precursor into multiple viral proteins. Consequently, NS2B mutations led to severe inhibition of ZIKV replication and production due to insufficient NS3 protease activity. In summary, our study reveals the critical role of NS2B in NS3 recruitment and protease function and provides mechanistic insight into ZIKV replication.

Zika virus NS3 is a canonical RNA helicase stimulated by NS5 RNA polymerase.

Zika virus is a positive single-strand RNA virus whose replication involved RNA unwinding and synthesis. ZIKV NS3 contains a helicase domain, but its enzymatic activity is not fully characterized. Here, we established a dsRNA unwinding assay based on the FRET effect to study the helicase activity of ZIKV NS3, which provided kinetic information in real time. We found that ZIKV NS3 specifically unwound dsRNA/dsDNA with a 3' overhang in the 3' to 5' direction. The RNA unwinding ability of NS3 significantly decreased when the duplex was longer than 18 base pairs. The helicase activity of NS3 depends on ATP hydrolysis and binding to RNA. Mutations in the ATP binding region or the RNA binding region of NS3 impair its helicase activity, thus blocking viral replication in the cell. Furthermore, we showed that ZIKV NS5 interacted with NS3 and stimulated its helicase activity. Disrupting NS3-NS5 interaction resulted in a defect in viral replication, revealing the tight coupling of RNA unwinding and synthesis. We suggest that NS3 helicase activity is stimulated by NS5; thus, viral replication can be carried out efficiently. Our work provides a molecular mechanism of ZIKV NS3 unwinding and novel insights into ZIKV replication.

Maltol, a food flavor enhancer, attenuates diabetic peripheral neuropathy in streptozotocin-induced diabetic rats.

SCOPE: Maltol (3-hydroxy-2-methy-4-pyrone), a potent antioxidative agent, typically is used to enhance flavor and preserve food. This study evaluated its effects on preventing diabetic peripheral neuropathy (DPN) in streptozotocin (STZ)-induced diabetic rats and explored its mechanisms. METHODS AND RESULTS: We intraperitoneally injected Sprague-Dawley (SD) rats with STZ (65 mg kg-1, ip) and treated the rats with different doses of maltol after 4 weeks of injection. During treatment, we evaluated motor nerve conduction velocity (MNCV) and thermal and mechanical hyperalgesia and assayed the oxidative stress, Na+-K+-ATPase activity, and apoptosis. Repeated treatment with maltol for 12 weeks significantly improved thermal and mechanical hyperalgesia, increased the MNCV, elevated the Na+-K+-ATPase activity, and ameliorated oxidative stress and apoptosis in STZ-induced diabetic rats. We coincubated RSC96 cells, a Schwann cell line, with maltol and hydrogen peroxide (H2O2, 0.6 mM). Evidently, maltol increased cell viability and inhibited apoptosis after injury by H2O2. CONCLUSIONS: Maltol was demonstrated to prevent DPN development and may provide a new alternative for the treatment of DPN.

Vesicular stomatitis virus G protein transmembrane region is crucial for the hemi-fusion to full fusion transition.

Viral fusion proteins are essential for enveloped virus infection. These proteins mediate fusion between the virus envelope and host cellular membrane to release the viral genome into cells. Vesicular stomatitis virus G (VSV G) protein is a typical type III viral fusion protein. To study the mechanism of VSV G protein mediated membrane fusion, we set up a cell-cell fusion system in which cells are marked by different fluorescent proteins. Taking advantage of this system, we performed real-time recording and quantitative analysis of the cell fusion mediated by VSV G. We found that the time scale required for VSV G mediated cell-cell fusion was approximately 1-2 minutes. Next, we specifically examined the function of the transmembrane (TM) region of VSV G protein in membrane fusion by replacing the TM region with those of other fusion proteins. The TM region replacements dramatically impaired VSV G protein function in the cell-cell fusion assay and diminished VSV G mediated lentivirus and recombinant VSV infection efficiency. Further experiments implied that the TM region played a role in the transition from hemi-fusion to full fusion. Several residues within the TM region were identified as important for membrane fusion. Overall, our findings unraveled the important function of the TM region in VSV G mediated viral fusion.

Selenite inhibits glutamine metabolism and induces apoptosis by regulating GLS1 protein degradation via APC/C-CDH1 pathway in colorectal cancer cells.

Glutaminolysis is important for metabolism and biosynthesis of cancer cells, and GLS is essential in the process. Selenite is widely regarded as a chemopreventive agent against cancer risk. Emerging evidence suggests that it also has chemotherapeutic potential in various cancer types, but the mechanism remains elusive. We demonstrate for the first time that supranutritional dose of selenite suppresses glutaminolysis by promoting GLS1 protein degradation and apoptosis. Mechanistically, selenite promotes association of APC/C-CDH1 with GLS1 and leads to GLS1 degradation by ubiquitination, this process is related to induction of PTEN expression. In addition, GLS1 expression is increased in human colorectal cancer tissues compared with normal mucosae. Our data provide a novel mechanistic explanation for the anti-cancer effect of selenite from a perspective of cell metabolism. Moreover, our results indicate that glutaminolysis especially GLS1 could be an attractive therapeutic target in colorectal cancer.

Selenite-induced autophagy antagonizes apoptosis in colorectal cancer cells in vitro and in vivo.

In the present study, we aimed to investigate the relationship between autophagy and apoptosis in selenite­treated colorectal cancer (CRC) cells. The effects of selenite on HCT116 and SW480 cell apoptosis were investigated with an Annexin V/propidium iodide (PI) double staining kit by flow cytometry. The punctate of LC3 protein following treatment with selenite was observed by a laser scanning confocal microscope and by transmission electron microscopy. Using western blot assays, we detected the apoptotic and autophagic markers in both CRC cells and mouse xenograft tumor models. We found that sodium selenite induced autophagy in the two CRC cell lines. Consistent with the in vitro results, we observed that the expression of autophagy marker LC3 was increased. Finally, we discovered that modulation of reactive oxygen species by MnTMPyP inhibited autophagy, while H2O2 activated autophagy. These results help to elucidate the anticancer effect of selenium, providing further evidence to exploit novel anticancer drugs targeting selenium.

The switch from ER stress-induced apoptosis to autophagy via ROS-mediated JNK/p62 signals: A survival mechanism in methotrexate-resistant choriocarcinoma cells.

BACKGROUND: Human choriocarcinoma, a highly curable solid tumour, is exceptionally sensitive to methotrexate-based chemotherapy at the metastatic stage. The present study aimed to investigate the molecular basis for this resistance to methotrexate therapy occurs in some cases, and these patients subsequently die from progressive and advanced disease. METHODS: The autophagy and apoptotic activity regulated by PERK/ATF4 axis in methotrexate-resistant JEG-3 and parental cells were evaluated with western blotting and chromatin immunoprecipitation (ChIP). The regulatory relationships among the reactive oxygen species (ROS), JNK/p62 axis, PERK/ATF4-mediated apoptosis and autophagy were assessed with western blotting, RT-PCR, fluorescence-activated cell sorting as well as ChIP. RESULTS: The decreased apoptosis in methotrexate-resistant JEG-3 cells was observed with an up-regulation of protective autophagy, suggesting a switch from apoptosis to autophagy, which was regulated via the PERK/ATF4 pathway under condition of endoplasmic reticulum (ER) stress. Further experiments demonstrated that this cell death switch was regulated by ROS-mediated JNK/p62 pathway and subsequently lead to the resistance of choriocarcinoma cells to methotrexate treatment. CONCLUSIONS: This study provides evidence to explain a survival mechanism of the switch from ER stress-induced apoptosis to autophagy via ROS-mediated JNK/p62 signals in methotrexate-resistant choriocarcinoma cells and may implicate the chemotherapy of methotrexate resistance in choriocarcinoma.

The p38 MAPK-regulated PKD1/CREB/Bcl-2 pathway contributes to selenite-induced colorectal cancer cell apoptosis in vitro and in vivo.

Supranutritional selenite has anti-cancer therapeutic effects in vivo; however, the detailed mechanisms underlying these effects are not clearly understood. Further studies would broaden our understanding of the anti-cancer effects of this compound and provide a theoretical basis for its clinical application. In this study, we primarily found that selenite exposure inhibited phosphorylation of cyclic adenosine monophosphate (cAMP)-response element binding protein (CREB), leading to suppression of Bcl-2 in HCT116 and SW480 colorectal cancer (CRC) cells. Moreover, the selenite-induced inhibitory effect on PKD1 activation was involved in suppression of the CREB signalling pathway. Additionally, we discovered that selenite treatment can upregulate p38 MAPK phosphorylation, which results in inhibition of the PKD1/CREB/Bcl-2 survival pathway and triggers apoptosis. Finally, we established a colorectal cancer xenograft model and found that selenite treatment markedly inhibits tumour growth through the MAPK/PKD1/CREB/Bcl-2 pathway in vivo. Our results demonstrated that a supranutritional dose of selenite induced CRC cell apoptosis through inhibition of the PKD1/CREB/Bcl-2 axis both in vitro and in vivo.

WITHDRAWN: Selenite induces apoptosis in colorectal cancer cells through interaction with thioredoxin reductase.

Ahead of Print article withdrawn by publisher. The chemopreventive and chemotherapeutic effects of selenium compounds has been well validated in colorectal cancer by epidemiologic and pre-clinical studies, but the underlying mechanisms still remains elusive. We provide evidence that interaction between selenium metabolites and selenoproteins participates in colorectal cancer suppression induced by selenite. We found that supranutritional doses of selenite could induces inhibition of thioredoxin system and apoptosis in SW480 cells, and cause direct modifications of thiol groups on TrxR protein. Moreover, we showed that TrxR ablation sensitized apoptosis induced by selenite, and the synergistic effect was related to Bax activation and mitochondrial apoptotic pathway. Our results proposed that TrxR is an important target for selenite in colorectal cancer suppression, and selenite in combination with TrxR inhibition could be an effective strategy to treat colorectal cancer.

ATF4 activation by the p38MAPK-eIF4E axis mediates apoptosis and autophagy induced by selenite in Jurkat cells.

Previous studies have shown that selenite exerts pro-apoptosis and pro-autophagy effects and is associated with the activation of ER stress in T-cell acute lymphoblastic leukemia (T-ALL). Herein we demonstrate the underlying mechanisms by which the activation of p38MAPK plays essential roles in apoptosis and autophagy and the coordination of cellular metabolic processes during leukemia therapy. MKK3/6-dependent activation of p38MAPK is required for the phosphorylation of eIF4E, thus initiating the translation of ER stress-related transcription factor ATF4. Upregulated ATF4 results in the transcriptional initiation of the apoptosis-related chop gene and autophagy-related map1lc3b gene, through which selenite links ER stress to apoptosis and autophagy during leukemia treatment. Moreover, autophagy induction enhances cell apoptosis under this condition.

Sodium selenite alters microtubule assembly and induces apoptosis in vitro and in vivo.

BACKGROUND: Previous studies demonstrated that selenite induced cancer-cell apoptosis through multiple mechanisms; however, effects of selenite on microtubules in leukemic cells have not been demonstrated. METHODS: The toxic effect of selenite on leukemic HL60 cells was performed with cell counting kit 8. Selenite effects on cell cycle distribution and apoptosis induction were determined by flow cytometry. The contents of cyclin B1, Mcl-1, AIF, cytochrome C, insoluble and soluble tubulins were detected with western blotting. Microtubules were visualized with indirect immunofluorescence microscopy. The interaction between CDK1 and Mcl-1 was assessed with immunoprecipitation. Decreasing Mcl-1 and cyclin B1 expression were carried out through siRNA interference. The alterations of Mcl-1 and cyclin B1 in animal model were detected with either immunohistochemical staining or western blotting. In situ detection of apoptotic ratio was performed with TUNEL assay. RESULTS: Our current results showed that selenite inhibited the growth of HL60 cells and induced mitochondrial-related apoptosis. Furthermore, we found that microtubule assembly in HL60 cells was altered, those cells were arrested at G2/M phase, and Cyclin B1 was up-regulated and interacted with CDK1, which led to down-regulation of the anti-apoptotic protein Mcl-1. Finally, in vivo experiments confirmed the in vitro microtubule disruption effect and alterations in Cyclin B1 and Mcl-1 levels by selenite. CONCLUSIONS: Taken together, the results from our study indicate that microtubules are novel targets of selenite in leukemic HL60 cells.

Activation of p53 by sodium selenite switched human leukemia NB4 cells from autophagy to apoptosis.

It was revealed by our previous research that sodium selenite repressed autophagy accompanied by the induction of apoptosis in human leukemia NB4 cells. The inhibition of autophagy exerted a facilitative effect on apoptosis. In the present study, we further explored the mechanisms underlying the switch from autophagy to apoptosis and elucidated p53 played a key role. Selenite induced phosphorylation of p53 at the vital site Ser15 via p38MAPK and ERK. Subsequently p53 dissociated with its inhibitory protein mouse double minute 2 (MDM2). Meanwhile, the nucleolar protein B23 transferred from the nucleolus to the nucleoplasm and associated with MDM2, probably stabilizing p53. The active p53 participated in the decrease of autophagic protein Beclin-1 and LC-3, as well as activation of apoptosis-related caspases. Furthermore, in p53 mutant U937 leukemia cells, selenite could not elicit such a switch from autophagy to apoptosis, laying emphasis on the crucial role p53 played in this process.

Sodium selenite-induced activation of DAPK promotes autophagy in human leukemia HL60 cells.

Autophagy has been suggested as a possible mechanism for non-apoptotic death despite evidence from many species that autophagy represents a survival strategy of cells under stress. From our previous findings that supranutritional doses of sodium selenite induced apoptosis in human leukemia cells, now we show autophagic cell death occurred after selenite exposure in HL60, suggested an alternative mechanism for the potential therapeutic properties of selenite. Additionally, Death-associated Protein Kinase (DAPK) performed a significantly increased expression during this process, concomitantly with gradually decreased phosphorylation at Ser(308). We further reveal that the up-regulation of DAPK which depends on selenite-activated ERK had no effect on autophagy. However, activation of DAPK via PP2A-mediated dephosphorylation at Ser(308) serves as a new strategy for autophagy induction. In conclusion, these results indicate that PP2A-mediated activated DAPK sensitizes HL60 cells to selenite, ultimately triggers autophagic cell death pathway to commit cell demise.

Selenite induces apoptosis in colorectal cancer cells via AKT-mediated inhibition of ß-catenin survival axis.

Mounting evidence reveals that selenium possesses chemotherapeutic potential against cancer cells. However, the molecular mechanisms underlying the anti-cancer effect of selenium remain elusive. In this study, with the aim to explore the detailed mechanisms how selenite induces apoptosis in colorectal cancer cells, we investigated the role of AKT/ß-catenin signaling, a critical regulator of cell proliferation, survival and tumorigenesis, in selenite-induced apoptosis of colorectal cancer cells and xenograft tumors. We showed that selenite exerted a remarkable inhibitory effect on activation of AKT, leading to suppression of ß-catenin activity and expression of its targets: cyclin D1 and survivin. Further experiments by transient expression of AKT and ß-catenin revealed that inhibition of AKT/ß-catenin was closely correlated with selenite-triggered apoptosis. Importantly, MnTMPyP pretreatment implied reactive oxygen species (ROS) was a crucial upstream signal for selenite-triggered inhibition of AKT/ß-catenin. Overall, these observations demonstrate that selenite could induce apoptosis through ROS-dependent inhibition of AKT/ß-catenin signaling in colorectal cancer cells in vitro and in vivo, and our findings yield novel insights into elucidating the mechanisms involved in the anti-cancer effect of selenium.