Radioprotective effectiveness of a novel delta-tocotrienol prodrug on mouse hematopoietic system against 60Co gamma-ray irradiation through inducing granulocyte-colony stimulating factor production.

Considering the increasing risk of nuclear attacks worldwide, the development of develop potent and safe radioprotective agents for nuclear emergencies is urgently needed. γ-tocotrienol (GT3) and δ-tocotrienol (DT3) have demonstrated a potent radioprotective effect by inducing the production of granulocyte-colony stimulating factor (G-CSF) in vivo. However, their application is limited because of their low bioavailability. The utilization of ester prodrugs can be an effective strategy for modifying the pharmacokinetic properties of drug molecules. In this study, we initially confirmed that DT3 exhibited the most significant potential for inducing G-CSF effects among eight natural vitamin E homologs. Consequently, we designed and synthesized a series of DT3 ester and ether derivatives, leading to improved radioprotective effects. The metabolic study conducted in vitro and in vivo has identified DT3 succinate 5b as a prodrug of DT3 with an approximately seven-fold higher bioavailability compared to DT3 alone. And DT3 ether derivative 8a were relatively stable and approximately 4 times more bioavailable than DT3 prototype. Furthermore, 5b exhibited superior ability to mitigate radiation-induced pancytopenia, enhance the recovery of bone marrow hematopoietic stem and progenitor cells, and promote splenic extramedullary hematopoiesis in sublethal irradiated mice. Similarly, 8a shown potential radiation protection, but its radiation protection is less than DT3. Based on these findings, we identified 5b as a DT3 prodrug, and providing an attractive candidate for further drug development.

Tubule-specific cyclin-dependent kinase 12 knockdown potentiates kidney injury through transcriptional elongation defects.

Direct tubular injury caused by several medications, especially chemotherapeutic drugs, is a common cause of AKI. Inhibition or loss of cyclin-dependent kinase 12 (CDK12) triggers a transcriptional elongation defect that results in deficiencies in DNA damage repair, producing genomic instability in a variety of cancers. Notably, 10-25% of individuals developed AKI after treatment with a CDK12 inhibitor, and the potential mechanism is not well understood. Here, we found that CDK12 was downregulated in the renal tubular epithelial cells in both patients with AKI and murine AKI models. Moreover, tubular cell-specific knockdown of CDK12 in mice enhanced cisplatin-induced AKI through promotion of genome instability, apoptosis, and proliferative inhibition, whereas CDK12 overexpression protected against AKI. Using the single molecule real-time (SMRT) platform on the kidneys of CDK12RTEC+/- mice, we found that CDK12 knockdown targeted Fgf1 and Cast through transcriptional elongation defects, thereby enhancing genome instability and apoptosis. Overall, these data demonstrated that CDK12 knockdown could potentiate the development of AKI by altering the transcriptional elongation defect of the Fgf1 and Cast genes, and more attention should be given to patients treated with CDK12 inhibitors to prevent AKI.

Sequential administration of delta-tocotrienol ameliorates radiation-induced myelosuppression in mice and non-human primates through inducing G-CSF production.

To date only four recombinant growth factors, including Filgrastim (rhG-CSF), have been approved by FDA as radiomitigators to ameliorate hematopoietic acute radiation syndrome (H-ARS). These approved agents are not stable under room-temperature, needing to be stored at 2-8 °C, and would not be feasible in a mass casualty scenario where rapid and cost-effective intervention is crucial. Delta-tocotrienol (δ-T3H), the most potent G-CSF-inducing agent among vitamin E isoforms, exhibited efficiency and selectivity on G-CSF production in comparison with TLR and STING agonists in mice. Five-dose δ-T3H was utilized as the optimal therapeutic regimen due to long-term G-CSF production and the best peripheral blood (PB) recovery of irradiated mice. Comparable with rhG-CSF, sequential administration of δ-T3H post-irradiation improved hematologic recovery and accelerated the regeneration of hematopoietic stem cells (HSCs) and hematopoietic progenitor cells (HPCs) in the bone marrow (BM) and spleen of 6.5Gy irradiated mice; and consistently enhanced repopulation of BM-HSCs. In 4.0Gy irradiated nonhuman primates, δ-T3H exhibited comparable efficacy as rhG-CSF to promote PB recovery and colony-formation of BM-HPCs. Altogether, we demonstrated that sequential administration of delta-tocotrienol ameliorates radiation-induced myelosuppression in mice and non-human primates through inducing G-CSF production, indicated δ-T3H as a promising radiomitigator for the management of H-ARS, particularly in a mass casualty scenario.

Shenshuaifu Granule Attenuates Acute Kidney Injury by Inhibiting Ferroptosis Mediated by p53/SLC7A11/GPX4 Pathway.

Background: Acute kidney injury (AKI) is a common clinical condition resulting in a rapid decline in renal function, and requires improvement in effective preventive measures. Ferroptosis, a novel form of cell death, is closely related to AKI. Shenshuaifu granule (SSF) has been demonstrated to prevent AKI through suppressing inflammation and apoptosis. Objective: This study aimed to explore whether SSF can inhibit ferroptosis in AKI. Methods: Active ingredients in SSF were detected through HPLC-MS/MS, and their binding abilities with ferroptosis were evaluated by molecular docking. Then, male C57/BL/6J mice were randomly divided into control, cisplatin, and cisplatin+SSF groups. In the latter two groups, mice were intraperitoneally injected with 20 mg/kg of cisplatin. For five consecutive days prior to cisplatin injection, mice in the cisplatin+SSF group were gavaged with 5.2 g/kg of SSF per day.72 h after cisplatin injection, the mice were sacrificed. Serum creatinine (SCr) and blood urea nitrogen (BUN) were measured to evaluate renal function. H&E and PAS staining were used to observe pathological damage of kidney. Cell death was observed by TUNEL staining, and iron accumulation in kidneys of mice was detected by Prussian blue staining. Western blotting, immunohistochemistry, and immunofluorescence were used to investigate the presence of inflammation, oxidative stress, mitochondrial dysfunction, iron deposition, and lipid peroxidation in mouse kidneys. Results: Active ingredients in SSF had strong affinities with ferroptosis. SSF reduced SCr (p<0.01) and BUN (p<0.0001) levels, pathological damage (p<0.0001), dead cells in the tubular epithelium (p<0.0001) and iron deposition (p<0.01) in mice with cisplatin induced AKI. And SSF downregulated macrophage infiltration (p<0.01), the expressions of high mobility group box 1 (HMGB1, p<0.05) and interleukin (IL)-17 (p<0.05), upregulated superoxide dismutase (SOD) 1 and 2 (p<0.01), and catalase (CAT, p<0.05), and alleviated mitochondrial dysfunction (p<0.05). More importantly, SSF regulated iron transport and intracellular iron overload and reduced the expression of ferritin (p<0.05). Moreover, it downregulated the expressions of cyclo-oxygenase-2 (Cox-2, p<0.001), acid CoA ligase 4 (ACSL4, p<0.05), and solute carrier family 7, member 11 (SLC7A11, p<001), upregulated glutathione peroxidase 4 (GPX4, p<0.01) and p53 (p<0.01), and decreased 4-hydroxynonenal (4-HNE) level (p<0.001). Conclusion: SSF attenuates AKI by inhibiting ferroptosis mediated by p53/SLC7A11/GPX4 pathway.

Design, Synthesis, and Antitumor Activity Evaluation of Proteolysis-Targeting Chimeras as Degraders of Extracellular Signal-Regulated Kinases 1/2.

Inhibition of the extracellular signal-regulated kinases 1/2 (ERK1/2) alone or in combination with other targets has emerged as a promising treatment strategy for a variety of human tumors. In addition to the development of inhibitors, the development of ERK1/2 degraders is an alternative approach to decrease its activity. We synthesized proteolysis-targeting chimeras (PROTACs) as effective ERK1/2 degraders, among which B1-10J showed high degradative activity, with DC50 of 102 nM and cytotoxic IC50 of 2.2 µM against HCT116 cells. Moreover, B1-10J dose-dependently inhibited tumor cell migration. Xenograft experiments in nude mice demonstrated that B1-10J inhibited HCT116 tumor cell growth and achieved significant regression of tumors at a daily dose of 25 mg/kg.

Design, Synthesis, and Biological Evaluation of Proteolysis-Targeting Chimeras as Highly Selective and Efficient Degraders of Extracellular Signal-Regulated Kinase 5.

Extracellular signal-regulated kinase 5 (ERK5) is recognized as a key member of the mitogen-activated protein kinase family and is involved in tumor growth, migration, and angiogenesis. However, the results of ERK5 inhibition in multiple studies are controversial, and a highly specific ERK5-targeting agent is required to confirm physiological functions. Using proteolysis-targeting chimera technology, we designed the selective ERK5 degrader PPM-3 and examined its biological effect on cancer cells. Interestingly, the selective degradation of ERK5 with PPM-3 did not influence tumor cell growth directly. Based on proteomics analysis, the ERK5 deletion may be associated with tumor immunity. PPM-3 influences tumor development by affecting the differentiation of macrophages. Therefore, PPM-3 is an effective small-molecule tool for studying ERK5 and a promising immunotherapy drug candidate.

Quercetin alleviates tubulointerstitial inflammation by inhibiting exosomes-mediated crosstalk between tubular epithelial cells and macrophages.

BACKGROUND: Tubulointerstitial inflammation (TII) is a critical pathological feature of kidney disease leading to renal fibrosis, and its treatment remains a major clinical challenge. We sought to explore the role of quercetin, a potential exosomes inhibitor, in exosomes release and TII. METHODS: The effects of quercetin on exosomes release and TII were examined by two TII mouse models: the unilateral ureteral obstruction (UUO) models and the LPS-induced mouse models. In vitro, exosomes-mediated crosstalk between tubular epithelial cells (TECs) and macrophages was performed to investigate the mechanisms by which quercetin inhibited exosomes and TII. RESULTS: In this study, we found that exosomes-mediated crosstalk between TECs and macrophages contributed to the development of TII. In vitro, exosomes released from LPS-stimulated TECs induced increased expression of inflammatory cytokines and fibrotic markers in Raw264·7 cells and vice versa. Interestingly, heat shock protein 70 (Hsp70) or Hsp90 proteins could control exosomes release from TECs and macrophages both in vivo and in vitro. Importantly, quercetin, a previously recognized heat shock protein inhibitor, could significantly reduce exosomes release in TII models by down-regulating Hsp70 or Hsp90. Quercetin abrogated exosomes-mediated intercellular communication, which attenuated TII and renal fibrosis accordingly. CONCLUSION: Quercetin could serve as a novel strategy for treatment of tubulointerstitial inflammation by inhibiting the exosomes-mediated crosstalk between tubules and macrophages.

Network pharmacology, molecular docking, and experimental verification reveal the mechanism of San-Huang decoction in treating acute kidney injury.

Background: Cisplatin is an effective anti-tumor drug. However, its usage is constrained by side effects such as nephron toxicity. Cisplatin-induced acute kidney injury (AKI) appears in approximately 20%-30% of cases. Hence, finding an effective protective strategy is necessary. San-Huang decoction (SHD) is a Chinese herbal decoction with good efficacy in treating chronic kidney disease (CKD). Nevertheless, the mechanism of SHD on AKI remains unclear. Consequently, we proposed to explore the potential mechanism of SHD against cisplatin-induced AKI. Methods: Active compounds, core target proteins, and associated signaling pathways of SHD were predicted through network pharmacology. Then confirmed by molecular docking. In vivo experiment, Cisplatin + SHD group was treated with SHD (6.5 g/kg/day) for 6 days before building the model. An AKI model was established with a single intraperitoneal injection of cisplatin at 20 mg/kg. After 72 h of cisplatin injection, all mice were sacrificed to collect blood and kidney tissues for verification of network pharmacology analysis. Results: We found that calycosin, rhein, and ginsenoside Rh2 may be SHD's primary active compounds in treating cisplatin-induced AKI, and AKT, TNF-α, IL-6, IL-1ß, caspase-3, and MMP9 are the core target proteins. The relationship between the compound and target protein was further confirmed by molecular docking. The Gene Ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses predicted that SHD has an anti-inflammatory role through the TNF and IL-17 signaling pathway. Moreover, Western blot and immunohistochemistry validated the potential molecular mechanisms of SHD, predicted from network pharmacology analysis. The mechanism of cisplatin-induced AKI involves apoptosis and inflammation. In apoptosis, Caspase-3, caspase-8, caspase-9, and Bax proteins were down-regulated, while Bcl-2 was up-regulated by SHD. The differential expression of MMP protein is involved in the pathological process of AKI. MMP9 protects from glomerular tubule damage. MMP9 and PI3K/AKT anti-apoptosis pathway were up-regulated by SHD. In addition, we discovered that SHD alleviated AKI by inhibiting the NF-κB signaling pathway. Conclusion: SHD plays a critical role in anti-inflammation and anti-apoptosis via inhibiting the NF-κB signaling pathway and activating PI3K/AKT anti-apoptosis pathway, indicating that SHD is a candidate herbal drug for further investigation in treating cisplatin-induced AKI.

An herbal formulation "Shenshuaifu Granule" alleviates cisplatin-induced nephrotoxicity by suppressing inflammation and apoptosis through inhibition of the TLR4/MyD88/NF-κB pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Shenshuaifu Granule (SSF) is an in-hospital preparation approved by the Guangdong Food and Drug Administration of China. It has been clinically used against kidney diseases for more than 20 years with a definite curative effect. AIM OF THE STUDY: Cisplatin (CDDP) is a first-line chemotherapeutic drug in clinical practice, primarily excreted by the kidney with nephrotoxicity as a common side effect. Approximately 5-20% of cancer patients develop acute kidney injury (AKI) after chemotherapy; however, prevention and control strategies are currently unavailable. Therefore, it is important to identify safe and effective drugs that can prevent the nephrotoxicity of CDDP. SSF is an herbal formulation with 8 herbs, and has been used to protect the kidney in China. Nonetheless, its mechanism in relieving CDDP nephrotoxicity remains unclear. Therefore, this work attempt to prove that SSF can alleviate CDDP nephrotoxicity. We also explore its mechanism. MATERIALS AND METHODS: First, Thin Layer Chromatography (TLC) of a few herbs in SSF were performed for quality control. Several open-access databases were used to identify the active ingredients of SSF, their corresponding targets, and CDDP-induced nephrotoxicity targets. We performed Protein-Protein Interaction (PPI), Gene Ontology (GO), and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses. Next, the results of network pharmacology were validated using CDDP-induced nephrotoxicity mouse models. Renal function in the mice was assessed by analyzing the levels of serum creatinine (Scr) and blood urea nitrogen (BUN). On the other hand, renal damage was assessed by determining the level of tubular injury and apoptotic cells using Periodic acid-Schiff (PAS) staining and Terminal Dutp Nick End-Labeling (TUNEL) staining, respectively. The expression of inflammatory and apoptotic-related targets including IL-1ß, IL-6, TNF-α, Cox-2, Bax, Bcl-2, Cleaved-caspase 3, and Cleaved-caspase 9 was determined using Western Blot (WB) and Immunohistochemistry (IHC). Furthermore, WB was used to analyze the expression of proteins associated with the TLR4/MyD88/NF-κB pathway in the kidneys of mice with CDDP-induced nephrotoxicity. Finally, molecular docking simulations were performed to evaluate the binding abilities between major active ingredients of SSF and core targets. RESULT: Through network pharmacology, we identified 127 active ingredients of SSF and their corresponding 134 targets. Additional screening identified 14 active ingredients and 17 targets for further analysis. In biological process (BP), the targets were enriched in inflammation and apoptosis, among others. In KEGG terms, they were enriched in apoptosis and NF-κB pathways. Animal experiments revealed that SSF significantly reduced the levels of Scr and BUN and prevented renal tubular damage in mice treated with CDDP. In addition, SSF inhibited inflammation and apoptosis by targeting the TLR4/MyD88/NF-κB pathway. Molecular docking revealed good binding capacities of active ingredients and core targets. CONCLUSION: In summary, the experimental findings were consistent with the network pharmacological predictions. SSF can inhibit inflammation and apoptosis by targeting the TLR4/MyD88/NF-κB pathway. Taken together, our data suggest that SSF is an alternative agent for the treatment of CDDP-induced nephrotoxicity.

Optimization of 4-arylthiophene-3-carboxylic acid derivatives as inhibitors of ANO1: Lead optimization studies toward their analgesic efficacy for inflammatory pain.

Current pain management is largely limited to opioids and non-steroidal anti-inflammatory drugs. Developing new analgesic drugs remains important to address the unmet medical needs of chronic pain patients. Calcium-activated chloride channel anoctamin-1 (ANO1) is a potential analgesic target. ANO1 is activated by noxious stimuli in peripheral sensory neurons and further induced neural depolarization. Downregulation of ANO1 reduced hyperalgesia and allodynia caused by inflammation and nerve injury. Here we developed a series of 4-arylthiophene-3-carboxylic acid derivatives for proof-of-concept studies of ANO1-targeted analgesia. These efforts led to the identification of the compound DFBTA, 4-(4-chlorophenyl)-2-(2,5-difluorobenzamido)thiophene-3-carboxylic acid, which displays dramatic ANO1 inhibition with IC50 of 24 nM. DFBTA displays very weak cytotoxicity, cardiotoxicity, and acute toxicity (HEK293 proliferation IC50 > 30 µM, hERG IC50 > 30 µM, mouse minimum lethal dosage, MLD>1000 mg/kg), as well as excellent pharmacokinetics properties with oral bioavailability >75% and little brain penetration (<1.5% brain/plasma). Finally, the analgesic efficacy of ANO1 inhibitor was evaluated in animal models. DFBTA shown comparable efficacy to clinical drugs in all inflammatory pain models induced by complete Freund's adjuvant, formalin, and capsaicin. These works provide a useful tool compound and promising results for ANO1-targenting analgesic development.

Discovery of novel ataxia telangiectasia mutated (ATM) kinase modulators: Computational simulation, biological evaluation and cancer combinational chemotherapy study.

Ataxia-telangiectasia mutated (ATM) kinase is a serine/threonine protein kinase and plays a key role in DNA double-strand breaks repair. Thus, ATM is considered a promising target for radiotherapy and chemotherapy sensitizing. Herein, we report the discovery of ATM agonist A22 and inhibitor A41 by computational methods and further biological evaluation. Among them, A22 exhibited low cytotoxicity in vitro and might serve as a useful tool for ATM research. Moreover, we firstly proved that ATM inhibitors could sensitize Irinotecan and Etoposide in a time-dependent manner on MCF-7 and SW480 cells, antagonism in a short period treatment while synergy at a long-term treatment and ATM agonist worked in an opposite way of ATM inhibitors. Further mechanism study demonstrated that the antagonism effect of ATM inhibitors with chemotherapeutic agents in a short period was resulting from inhibiting the p53/p21 axis to accelerate G1/S phase cell-cycle transition and promote cell survival. Additionally, A41 displayed antitumor effects combined with a chemotherapeutic drug in the SW480 xenograft model, indicating that A41 is a promising ATM inhibitor, which could increase the antitumor effect of chemotherapeutic drugs in vivo. All in all, these findings will guide the combination of ATM inhibitors with chemotherapeutic agents in further preclinical and clinical studies.

DUSP9 alleviates hepatic ischemia/reperfusion injury by restraining both mitogen-activated protein kinase and IKK in an apoptosis signal-regulating kinase 1-dependent manner.

Hepatic ischemia/reperfusion (I/R) injury occurs frequently in various liver operations and diseases, but its effective treatment remains inadequate because the key switch that leads to hepatic explosive inflammation has not been well disclosed. Dual specificity phosphatase 9 (DUSP9) is widely involved in the innate immune response of solid organs and is sometimes regulated by ubiquitin. In the present study, we find that DUSP9 is reduced in mouse hepatic I/R injury. DUSP9 enrichment attenuates hepatic inflammation both in vivo and in vitro as revealed by western blot analysis and qRT-PCR. In contrast, DUSP9 depletion leads to more severe I/R injury. Mechanistically, DUSP9 inhibits the phosphorylation of apoptosis signal-regulating kinase 1 (ASK1) by directly binding to ASK1, thereby decreasing tumor necrosis factor receptor-associated factor 6 (TRAF6), K63 ubiquitin and the phosphorylation of p38/JNK1 instead of ERK1. The present study documents a novel role of DUSP9 in hepatic I/R injury and implies the potential of targeting the DUSP9/ASK1 axis towards mitogen-activated protein kinase and TRAF6/inhibitor of κB kinase pathways.

Screening and identification of key biomarkers of papillary renal cell carcinoma by bioinformatic analysis.

BACKGROUND: Papillary renal cell carcinoma (PRCC) is the most common type of renal cell carcinoma after clear cell renal cell carcinoma (ccRCC). Its pathological classification is controversial, and its molecular mechanism is poorly understood. Therefore, the identification of key genes and their biological pathways is of great significance to elucidate the molecular mechanisms of PRCC occurrence and progression. METHODS: The PRCC-related datasets GSE7023, GSE48352 and GSE15641 were downloaded from the Gene Expression Omnibus (GEO) database. Differentially expressed genes (DEGs) were identified, and gene ontology (GO) term enrichment analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis were performed. Cytoscape and STRING were used to construct the protein-protein interaction network (PPI) and perform module analysis to identify hub genes and key pathways. A heatmap of hub genes was constructed using the UCSC cancer genomics browser. Overall survival and recurrence-free survival of patients stratified by the expression levels of hub genes were analysed using Kaplan-Meier Plotter. The online database UALCAN was applied to analyse gene expression based on tissue type, stage, subtype and race. RESULTS: A total of 214 DEGs, specifically, 205 downregulated genes and 9 upregulated genes, were identified. The DEGs were mainly enriched in angiogenesis, kidney development, oxidation-reduction process, metabolic pathways, etc. The 17 hub genes identified were mainly enriched in the biological processes of angiogenesis, cell adhesion, platelet degranulation, and leukocyte transendothelial migration. Survival analysis showed that EGF, KDR, CXCL12, REN, PECAM1, CDH5, THY1, WT1, PLAU and DCN might be related to the carcinogenesis, metastasis or recurrence of PRCC. UALCAN analysis showed that low expression of PECAM1 and PLAU in PRCC tissues was related to stage, subtype and race. CONCLUSIONS: The DEGs and hub genes identified in the present study provide insight into the specific molecular mechanisms of PRCC occurrence and development and may be potential molecular markers and therapeutic targets for the accurate classification and efficient diagnosis and treatment of PRCC.

Fisetin mitigates hepatic ischemia-reperfusion injury by regulating GSK3ß/AMPK/NLRP3 inflammasome pathway.

BACKGROUND: Hepatic ischemia-reperfusion (I/R) injury (IRI) represents a crucial challenge in liver transplantation. Fisetin has anti-inflammatory, anti-aging and anti-oxidative properties. This study aimed to examine whether fisetin mitigates hepatic IRI and examine its underlying mechanisms. METHODS: Sham or warm hepatic I/R operated mice were pretreated with fisetin (5, 10 or 20 mg/kg). Hepatic histological assessments, TUNEL assays and serum aminotransferase measurements were performed. An in vitro hypoxia/reoxygenation (H/R) model using RAW264.7 macrophages pretreated with fisetin (2.5, 5 or 10 µmol/L) was also used. Serum and cell supernatant concentrations of interleukin-1ß (IL-1ß), IL-18 and tumor necrosis factor-α (TNF-α) were determined by enzyme-linked immunosorbent assay (ELISA). Protein levels of p-GSK3ß, p-AMPK and NLR family pyrin domain-containing 3 (NLRP3)-associated proteins were detected by Western blotting. RESULTS: Compared with the I/R group, fisetin pretreatment reduced pathological liver damage, serum aminotransferase levels, serum concentrations of IL-1ß, IL-18 and TNF-α in the murine IRI model. Fisetin also reduced the expression of NLRP3 inflammasome-associated proteins (NLRP3, cleaved caspase-1, IL-1ß and IL-18) in I/R-operated liver. The experiments in vitro showed that fisetin decreased the release of IL-1ß, IL-18 and TNF-α, and reduced the expression of NLRP3 inflammasome-associated proteins in H/R-treated RAW264.7 cells. Moreover, fisetin increased the expressions of p-GSK3ß and p-AMPK in both models, indicating that its anti-inflammatory effects were dependent on GSK3ß/AMPK signaling. The anti-inflammatory effects of fisetin were partially inhibited by the AMPK specific inhibitor compound C. CONCLUSIONS: Fisetin showed protective effects against hepatic IRI, countering inflammatory responses through mediating the GSK3ß/AMPK/NLRP3 inflammasome pathway.

SET8 mitigates hepatic ischemia/reperfusion injury in mice by suppressing MARK4/NLRP3 inflammasome pathway.

AIMS: Hepatic ischemia/reperfusion (I/R) injury is a critical factor affecting the prognosis of liver surgery. The aim of this study is to explore the effects of SET8 on hepatic I/R injury and the putative mechanisms. MAIN METHODS: The expression of SET8 and MARK4 in I/R group and sham group were detected both in vivo and in vitro. In addition, mouse and RAW 264.7 cells were transfected with MARK4 siRNA and SET8 siRNA knockdown of MARK4 and SET8, respectively. The expression of SET8, MARK4 and NLRP3-associated proteins were detected after different treatments. The pathology of liver and the serologic detection were detected after different treatments. KEY FINDINGS: Our present study identified SET domain-containing protein 8 (SET8) as an efficient protein, which can negatively regulate hepatic I/R-mediated inflammatory response and ameliorate hepatic I/R injury by suppressing microtubule affinity-regulating kinase 4 (MARK4)/ NLR family pyrin domain containing 3 (NLRP3) inflammasome pathway. The data showed that MARK4 deficiency inhibited hypoxia/reoxygenation (H/R)-induced NLRP3 inflammasome activation, while SET8 deficiency showed the opposite effect. We further demonstrated that SET8 restrained NLRP3 inflammasome activation by inhibiting MARK4. Moreover, we verified SET8 made protective effect on hepatic I/R injury. SIGNIFICANCE: SET8 plays an essential role in hepatic ischemia/reperfusion injury in mice by suppressing MARK4/NLRP3 inflammasome pathway. Our results may offer a new strategy to mitigate hepatic I/R injury.

Synthesis and biological evaluation of organoselenium (NSAIDs-SeCN and SeCF3) derivatives as potential anticancer agents.

A series of organoselenium compounds based on the hybridization of nonsteroidal antiinflammatory drugs (NSAIDs) scaffolds and Se functionalities (-SeCN and -SeCF3) were synthesized and characterized, and evaluated against four types of cancer cell lines, SW480 (human colon adenocarcinoma cells), HeLa (human cervical cancer cells), A549 (human lung carcinoma cells), MCF-7 (human breast adenocarcinoma cells). Interestingly, most of the investigated compounds showed active in reducing the viability of different cancer cell lines. The most active compound 3h showed IC50 values lower than 20 µM against the four cancer cell lines, particularly to SW480 and MCF-7 with IC 50 values of 4.9 and 3.4 µM, respectively. Furthermore, NSAIDs-SeCN derivatives (2h and 2i) and NSAIDs-SeCF3 derivatives (3h and 3i) were selected to investigate their ability to induce apoptosis in MCF-7 cells via modulation the expression of anti-apoptotic Bcl-2 protein, pro-inflammatory cytokines (IL-2) and proapoptotic caspase-3 protein. Moreover, the redox properties of the synthesized organoselenium candidates were conducted by 2, 2-didiphenyl-1-picrylhydrazyl (DPPH), bleomycin dependent DNA damage and glutathione peroxidase (GPx)-like assays. Taken together, these NSAIDs-Se candidates could provide promising new lead derivatives for further potential anticancer drug development.

Inhibition of miR-450b-5p ameliorates hepatic ischemia/reperfusion injury via targeting CRYAB.

Hepatic ischemia/reperfusion injury (IRI) is an unavoidable course in liver transplantation, during which the immune response of inflammation plays a leading part. MicroRNA-450b-5p (miR-450b-5p), which has been reported to participate in several inflammatory diseases, was investigated in this study. The purpose of this study is to identify the potential function of miR-450b-5p toward remission of hepatic IRI and elucidate the specific mechanism. Herein we found that expression of miR-450b-5p, interleukin (IL)-1ß, tumor necrosis factor-α (TNF-α), and IL-6 was stimulated in hepatic IRI. Inhibition of miR-450b-5p could remarkably alleviate mouse hepatic IRI and improve liver function measured by hematoxylin-eosin (HE) staining, terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL), and enzyme-linked immunosorbent assay (ELISA). We further assessed protein expression undergoing Western blot and immunofluorescence, and discovered that miR-450b-5p suppressed alpha B-crystallin (CRYAB), thus restraining the inhibitory κB kinase (IKK) ß-mediated canonical nuclear factor-κB (NF-κB) signaling, instead of the noncanonical path guided by IKKα in hepatic IRI. In addition, we demonstrated CRYAB as an activator of M2 polarization through protein kinase B (Akt) 1/mammalian target of rapamycin (mTOR), thus resulting in relief of liver IRI. Combination treatment containing both paths revealed a better antidamage efficacy than adjusting either path alone, suggesting that the joint therapy might be a promising solution in hepatic IRI.

NLRC3 alleviates hypoxia/reoxygenation induced inflammation in RAW264.7 cells by inhibiting K63-linked ubiquitination of TRAF6.

BACKGROUND: NOD-like receptor family CARD domain containing 3 (NLRC3) plays an important role in both innate and adaptive immunity. This study was to explore the function and related mechanisms of NLRC3 in a hypoxia/reoxygenation (H/R)-induced inflammatory response in RAW264.7 cells. METHODS: Liver ischemia-reperfusion (I/R) model in mice and H/R model in RAW264.7 cells were constructed. Western blotting was used to determine the protein expression level of NLRC3 in liver tissue and NLRC3, TRAF6, p-p65, p65, IκB-α, and the K63-linked ubiquitination level of TRAF6 in cells. The immunofluorescence assay was performed to evaluate the nuclear level of the NF-κB (p65). ELISA was conducted to measure the content of IL-1ß in serum and cell supernatant. The interaction between NLRC3 and TRAF6 in cells was analyzed by the Co-IP assay. RESULTS: The NLRC3 protein level in liver tissue was decreased with the prolongation of reperfusion time (P < 0.05). The expression of NLRC3 and IκB-α protein in RAW264.7 was decreased gradually, while the expression of p-p65 and TRAF6 proteins and K63-linked ubiquitination of TRAF6 were increased gradually with the prolongation of reoxgenation time (P < 0.05). The Co-IP assay revealed that NLRC3 and TRAF6 can bind to each other directly. However, NLRC3 had no effect on the expression of TRAF6 protein. The ubiquitination test results showed that the K63-linked ubiquitination level of TRAF6 in H/R + Lv-NLRC3 group was significantly lower than that in the H/R + negative control (NC) group (P < 0.05). Moreover, the activation of NF-κB in H/R + Lv-NLRC3 group was inhibited compared with that in the H/R + NC group, and the level of the inflammatory factor IL-1ß in the cell culture supernatant was also decreased accordingly (P < 0.05). CONCLUSIONS: NLRC3 might alleviate H/R-induced inflammation in RAW264.7 cells by inhibiting K63-linked ubiquitination of TRAF6.

Designed Artificial Transcription Factors Inhibit Hepatitis B Virus Transcription in HepG2.2.15 Cells.

Hepatitis B virus (HBV) causes serious health issues worldwide. Despite this, current treatment options for HBV have many drawbacks. Strategies to safely and specifically target HBV replication and survival at the transcriptional level within host cells are needed to combat current drawbacks in treatment. In this study, we designed a novel artificial transcription factor (ATF) with suppressive function to target and bind to the HBV core promoter, a component that plays a central role in the viral life cycle. ATF has attached specifically to the intended target site by using electrophoretic mobility shift assays (EMSA). We tested whether targeting this suppressive ATF had any effect on HBV gene expression by transfection factor, western blotting, and real-time PCR. In the presence of ATF, viral mRNA and DNA levels were significantly decreased within HepG2.2.15 cells compared to control cells. The HBV-derived protein expression of HBV-e antigen (HBeAg) and HBV-c antigen (HBcAg) was also significantly inhibited. These results show that ATF treatment targeting the HBV core protein promoter has an antiviral effect and inhibits HBV infection in host cells. These results further suggest that the design of new artificial transcription factors may be valuable antiviral therapies to treat HBV patients.

Direct Gating of the TRPM2 Channel by cADPR via Specific Interactions with the ADPR Binding Pocket.

cADPR is a well-recognized signaling molecule by modulating the RyRs, but considerable debate exists regarding whether cADPR can bind to and gate the TRPM2 channel, which mediates oxidative stress signaling in diverse physiological and pathological processes. Here, we show that purified cADPR evoked TRPM2 channel currents in both whole-cell and cell-free single-channel recordings and specific binding of cADPR to the purified NUDT9-H domain of TRPM2 by surface plasmon resonance. Furthermore, by combining computational modeling with electrophysiological recordings, we show that the TRPM2 channels carrying point mutations at H1346, T1347, L1379, S1391, E1409, and L1484 possess distinct sensitivity profiles for ADPR and cADPR. These results clearly indicate cADPR is a bona fide activator at the TRPM2 channel and clearly delineate the structural basis for cADPR binding, which not only lead to a better understanding in the gating mechanism of TRPM2 channel but also shed light on a cADPR-induced RyRs-independent Ca2+ signaling mechanism.