Immune checkpoint inhibitor (ICI)-based treatment beyond progression with prior immunotherapy in patients with driver-gene negative advanced non-small cell lung cancer.

BACKGROUND: No definite conclusion has yet to be reached for immunotherapy beyond progression(IBP) of first-line immunotherapy as the second-line treatment for advanced NSCLC patients with negative driver genes. Therefore a retrospective study was conducted to evaluate the efficacy of IBP in this population and investigated whether the cycles best response and progressive mode of first-line immunotherapy could affect the results. PATIENTS AND METHODS: The clinical data of patients with advanced NSCLC whose response was evaluated as progressive disease (PD) after receiving a PD-1/PD-L1 inhibitors as first-line therapy were retrospectively collected and the patients were assigned to the IBP and non-IBP groups. The overall survival (OS), progression-free survival (PFS) were evaluated between the two groups. The survival effects of cycles best response and progressive mode of first-line immunotherapy were also evaluated. RESULTS: Between January 2019 and January 2022, a total of 121 patients was evaluated as PD after first-line immunotherapy in our institution; 53 (43.8%) patients were included in the IBP group and 68 (56.2%) patients were included in the non-IBP group. The OS and PFS were no significantly different between the two groups in whole population. Further analysis revealed the OS was prolonged with the prolongation of first-line medication cycle. The median OS was 15.4m (15.4 vs 10.8 p=0.047) 16.1m (16.1 vs 10.8 p=0.039), 16.3m (16.3 vs 10.9 p=0.029) for patients with ≥4, ≥6, ≥8 cycles in first-line immunotherapy, respectively. The advantages of OS and PFS were also seen in the subgroup of PR (best response) and oligo progression of first-line immunotherapy. CONCLUSIONS: The clinical outcomes of IBP were similar to those of non-IBP in patients with PD after first-line immnuotherapy in advanced NSCLC. But more cycles, PR as best response and oligo progression in first-line was benefit.

Application of dose-gradient function in reducing radiation induced lung injury in breast cancer radiotherapy.

OBJECTIVE: Try to create a dose gradient function (DGF) and test its effectiveness in reducing radiation induced lung injury in breast cancer radiotherapy. MATERIALS AND METHODS: Radiotherapy plans of 30 patients after breast-conserving surgery were included in the study. The dose gradient function was defined as DGH=VDVp3, then the area under the DGF curve of each plan was calculated in rectangular coordinate system, and the minimum area was used as the trigger factor, and other plans were triggered to optimize for area reduction. The dosimetric parameters of target area and organs at risk in 30 cases before and after re-optimization were compared. RESULTS: On the premise of ensuring that the target dose met the clinical requirements, the trigger factor obtained based on DGF could further reduce the V5, V10, V20, V30 and mean lung dose (MLD) of the ipsilateral lung in breast cancer radiotherapy, P < 0.01. And the D2cc and mean heart dose (MHD) of the heart were also reduced, P < 0.01. Besides, the NTCPs of the ipsilateral lung and the heart were also reduced, P < 0.01. CONCLUSION: The trigger factor obtained based on DGF is efficient in reducing radiation induced lung injury in breast cancer radiotherapy.

Re-evaluating the risk factors for radiation pneumonitis in the era of immunotherapy.

As one of the common complications of radiotherapy, radiation pneumonia (RP) limits the prognosis of patients. Therefore, better identifying the high-risk factors that lead to RP is essential to effectively prevent its occurrence. However, as lung cancer treatment modalities are being replaced and the era of immunotherapy has arrived, literature that reviews the parameters and mode of radiotherapy, chemotherapy drugs, targeted drugs and current hot immune checkpoint inhibitors related to RP is lacking. This paper summarizes the risk factors for radiation pneumonia by retrieving and analysing previously published literature and the results of large clinical trials. The literature primarily included retrospective analyses, including clinical trials in different periods and a part of the literature review. A systematic literature search of Embase, PubMed, Web of Science, and Clinicaltrials.gov was performed for relevant publications up to 6 Dec. 2022. Search keywords include, but are not limited to, "radiation pneumonia", "pneumonia", "risk factors", "immunotherapy", etc. The factors related to RP in this paper include physical parameters of radiotherapy, including V5, V20, and MLD; chemoradiotherapy mode and chemotherapy drugs, including paclitaxel and gemcitabine; EGFR-TKI; ALK inhibitors; antiangiogenic drugs; immune drugs and the underlying disease of the patient. We also introduce the possible mechanism of RP. In the future, we hope that this article not only sounds the alarm for clinicians but also helps to identify a method that can effectively intervene and reduce the occurrence of RP, significantly improve the quality of life and prognosis of patients, and more effectively improve the therapeutic effect of radiation therapy.

Circular RNA CircCDKN2B-AS_006 Promotes the Tumor-like Growth and Metastasis of Rheumatoid Arthritis Synovial Fibroblasts by Targeting the miR-1258/RUNX1 Axis.

Rheumatoid arthritis (RA) is an autoimmune polyarthritis in which synovial fibroblasts (SFs) play a major role in cartilage and bone destruction through tumor-like proliferation, migration, and invasion. Circular RNAs (circRNAs) have emerged as vital regulators for tumor progression. However, the regulatory role, clinical significance, and underlying mechanisms of circRNAs in RASF tumor-like growth and metastasis remain largely unknown. Differentially expressed circRNAs in synovium samples from patients with RA and patients with joint trauma were identified via RNA sequencing. Subsequently, in vitro and in vivo experiments were performed to investigate the functional roles of circCDKN2B-AS_006 in RASF proliferation, migration, and invasion. CircCDKN2B-AS_006 was upregulated in synovium samples from patients with RA and promoted the tumor-like proliferation, migration, and invasion of RASFs. Mechanistically, circCDKN2B-AS_006 was shown to regulate the expression of runt-related transcription factor 1 (RUNX1) by sponging miR-1258, influencing the Wnt/ß-catenin signaling pathway, and promoting the epithelial-to-mesenchymal transition (EMT) in RASFs. Moreover, in the collagen-induced arthritis (CIA) mouse model, intra-articular injection of lentivirus-shcircCDKN2B-AS_006 was capable of alleviating the severity of arthritis and inhibiting the aggressive behaviors of SFs. Furthermore, the correlation analysis results revealed that the circCDKN2B-AS_006/miR-1258/RUNX1 axis in the synovium was correlated with the clinical indicators of RA patients. CircCDKN2B-AS_006 promoted the proliferation, migration, and invasion of RASFs by modulating the miR-1258/RUNX1 axis.

The role of E2A in ATPR-induced cell differentiation and cycle arrest in acute myeloid leukaemia cells.

Acute myeloid leukaemia (AML) is a biologically heterogeneous disease with an overall poor prognosis; thus, novel therapeutic approaches are needed. Our previous studies showed that 4-amino-2-trifluoromethyl-phenyl retinate (ATPR), a new derivative of all-trans retinoic acid (ATRA), could induce AML cell differentiation and cycle arrest. The current study aimed to determine the potential pharmacological mechanisms of ATPR therapies against AML. Our findings showed that E2A was overexpressed in AML specimens and cell lines, and mediate AML development by inactivating the P53 pathway. The findings indicated that E2A expression and activity decreased with ATPR treatment. Furthermore, we determined that E2A inhibition could enhance the effect of ATPR-induced AML cell differentiation and cycle arrest, whereas E2A overexpression could reverse this effect, suggesting that the E2A gene plays a crucial role in AML. We identified P53 and c-Myc were downstream pathways and targets for silencing E2A cells using RNA sequencing, which are involved in the progression of AML. Taken together, these results confirmed that ATPR inhibited the expression of E2A/c-Myc, which led to the activation of the P53 pathway, and induced cell differentiation and cycle arrest in AML.

Nesfatin-1 exerts protective effects on acidosis-stimulated chondrocytes and rats with adjuvant-induced arthritis by inhibiting ASIC1a expression.

Nesfatin-1, a newly identified energy-regulating peptide, has been reported to possess antioxidant, anti-inflammatory, and antiapoptotic properties; however, to date, its effect on rheumatoid arthritis (RA) has not been previously explored in detail. We previously showed that activation of acid-sensing ion channel 1a (ASIC1a) by acidosis plays an important role in RA pathogenesis. Therefore, in this study, we evaluated the effects of nesfatin-1 on acidosis-stimulated chondrocyte injury in vitro and in vivo and examined the involvement of ASIC1a and the mechanism underlying the effects of nesfatin-1 on RA. Acid-stimulated articular chondrocytes were used to examine one of the several possible mechanisms underlying RA pathogenesis in vitro. The mRNA expression profile of acid-induced chondrocytes treated or not treated with nesfatin-1 was investigated by RNA sequencing. The effects of nesfatin-1 on oxidative stress, inflammation, and apoptosis in acid-induced chondrocytes were measured. The mechanistic effect of nesfatin-1 on ASIC1a expression and intracellular Ca2+ in acid-stimulated chondrocytes was studied. Rats with adjuvant-induced arthritis (AA) were used for in vivo analysis of RA pathophysiology. Cartilage degradation and ASIC1a expression in chondrocytes were detected in rats with AA after intraarticular nesfatin-1 injection. The in vitro experiments showed that nesfatin-1 decreased acidosis-induced cytotoxicity and elevation of intracellular Ca2+ levels in chondrocytes. Moreover, it attenuated acid-induced oxidative stress, inflammation, and apoptosis in chondrocytes. Nesfatin-1 decreased ASIC1a protein levels in acid-stimulated chondrocytes via the mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) and nuclear factor kappa-B (NF-κB) signaling pathways. In vivo analysis showed that nesfatin-1 ameliorated cartilage degradation and decreased ASIC1a expression in the chondrocytes of rats with AA. Collectively, nesfatin-1 suppressed acidosis-induced oxidative stress, inflammation, and apoptosis in acid-stimulated chondrocytes and alleviated arthritis symptoms in rats with AA, and its mechanism may be related to its ability to decrease ASIC1a protein levels via the MAPK/ERK and NF-κB pathways.

Estrogen antagonizes ASIC1a-induced chondrocyte mitochondrial stress in rheumatoid arthritis.

BACKGROUND: Destruction of articular cartilage and bone is the main cause of joint dysfunction in rheumatoid arthritis (RA). Acid-sensing ion channel 1a (ASIC1a) is a key molecule that mediates the destruction of RA articular cartilage. Estrogen has been proven to have a protective effect against articular cartilage damage, however, the underlying mechanisms remain unclear. METHODS: We treated rat articular chondrocytes with an acidic environment, analyzed the expression levels of mitochondrial stress protein HSP10, ClpP, LONP1 by q-PCR and immunofluorescence staining. Transmission electron microscopy was used to analyze the mitochondrial morphological changes. Laser confocal microscopy was used to analyze the Ca2+, mitochondrial membrane potential (Δψm) and reactive oxygen species (ROS) level. Moreover, ASIC1a specific inhibitor Psalmotoxin 1 (Pctx-1) and Ethylene Glycol Tetraacetic Acid (EGTA) were used to observe whether acid stimulation damage mitochondrial function through Ca2+ influx mediated by ASIC1a and whether pretreatment with estrogen could counteract these phenomena. Furthermore, the ovariectomized (OVX) adjuvant arthritis (AA) rat model was treated with estrogen to explore the effect of estrogen on disease progression. RESULTS: Our results indicated that HSP10, ClpP, LONP1 protein and mRNA expression and mitochondrial ROS level were elevated in acid-stimulated chondrocytes. Moreover, acid stimulation decreased mitochondrial membrane potential and damaged mitochondrial structure of chondrocytes. Furthermore, ASIC1a specific inhibitor PcTx-1 and EGTA inhibited acid-induced mitochondrial abnormalities. In addition, estrogen could protect acid-stimulated induced mitochondrial stress by regulating the activity of ASIC1a in rat chondrocytes and protects cartilage damage in OVX AA rat. CONCLUSIONS: Extracellular acidification induces mitochondrial stress by activating ASIC1a, leading to the damage of rat articular chondrocytes. Estrogen antagonizes acidosis-induced joint damage by inhibiting ASIC1a activity. Our study provides new insights into the protective effect and mechanism of action of estrogen in RA.

ZEB1 serves as an oncogene in acute myeloid leukaemia via regulating the PTEN/PI3K/AKT signalling pathway by combining with P53.

Acute myeloid leukaemia is a complex, highly aggressive hematopoietic disorder. Currently, in spite of great advances in radiotherapy and chemotherapy, the prognosis for AML patients with initial treatment failure is still poor. Therefore, the need for novel and efficient therapies to improve AML treatment outcome has become desperately urgent. In this study, we identified the expression of ZEB1 (a transcription factor) and focused on its possible role and mechanisms in the progression of AML. According to the data provided by the Gene Expression Profiling Interactive Analysis (GEPIA), high expression of ZEB1 closely correlates with poor prognosis in AML patients. Additionally, the overexpression of ZEB1 was observed in both AML patients and cell lines. Further functional experiments showed that ZEB1 depletion can induce AML differentiation and inhibit AML proliferation in vitro and in vivo. Moreover, ZEB1 expression was negatively correlated with tumour suppressor P53 expression and ZEB1 can directly bind to P53. Our results also revealed that ZEB1 can regulate PTEN/PI3K/AKT signalling pathway. The inhibitory effect of ZEB1 silencing on PTEN/PI3K/AKT signalling pathway could be significantly reversed by P53 small interfering RNA treatment. Overall, the present data indicated that ZEB1 may be a promising therapeutic target for AML treatment or a potential biomarker for diagnosis and prognosis.

Acidosis induces synovial fibroblasts to release vascular endothelial growth factor via acid-sensitive ion channel 1a.

Acid-sensitive ion channel 1a (ASIC1a) is a member of the extracellular H+ activated cation channel family. Studies have shown that tissue acidification contributes to the formation of microvessels in rheumatoid arthritis (RA) synovial tissue, but its underlying mechanisms remain unclear. The purpose of this study was to investigate the role of tissue acidification in microvascular formation of arthritic synovial tissue and the effect of ASIC1a on vascular endothelial growth factor (VEGF) release from arthritic synovial tissue. Our results indicate that ASIC1a expression, VEGF expression, and microvessel density (MVD) are elevated in RA synovial tissue and adjuvant arthritis (AA) rat synovial tissue. When AA rats were treated with ASIC1a-specific blocker psalmotoxin-1 (PcTx-1), the expression of ASIC1a, VEGF expression, and MVD were all reduced. Acidification of RA synovial fibroblasts (RASF) can promote the release of VEGF. PcTx-1 and ASIC1a-short hairpin RNA can inhibit acid-induced release of VEGF. In addition, the ASIC1a overexpression vector can promote acid-induced VEGF release. This indicates that extracellular acidification induces the release of VEGF by RASF via ASIC1a. These findings suggest that blocking ASIC1a mediates the release of VEGF from synoviocytes may provide a potential therapeutic strategy for RA therapy.

ASIC1a promotes the proliferation of synovial fibroblasts via the ERK/MAPK pathway.

Synovial hyperplasia, a profound alteration in the structure of synovial tissue, is the basis for cumulative joint destruction in rheumatoid arthritis (RA). It is generally accepted that controlling synovial hyperplasia can delay the progression of RA. As one of the most intensively studied isoforms of acid-sensing ion channels (ASICs), ASIC1a contributes to various physiopathologic conditions, including RA, due to its unique property of being permeable to Ca2+. However, the role and the regulatory mechanisms of ASIC1a in synovial hyperplasia are poorly understood. Here, rats induced with adjuvant arthritis (AA) and human primary synovial fibroblasts were used in vivo and in vitro to investigate the role of ASIC1a in the proliferation of RA synovial fibroblasts (RASFs). The results show that the expression of ASIC1a was significantly increased in synovial tissues and RASFs obtained from patients with RA as well as in the synovium of rats with AA. Moreover, extracellular acidification improved the ability of RASFs colony formation and increased the expression of proliferation cell nuclear antigen (PCNA) and Ki67, which was abrogated by the specific ASIC1a inhibitor psalmotoxin-1 (PcTX-1) or ASIC1a-short hairpin RNA (ASIC1a-shRNA), suggesting that extracellular acidification promotes the proliferation of RASFs by activating ASIC1a. In addition, the activation of c-Raf and extracellular signal-regulated protein kinases (ERKs) signaling was blocked with PcTX-1 or ASIC1a-shRNA and the proliferation of RASFs was further inhibited by the ERK inhibitor (U0126), indicating that ERK/MAPK signaling contributes to the proliferation process of RASFs promoted by the activation of ASIC1a. These findings gave us an insight into the role of ASIC1a in the proliferation of RASFs, which may provide solid foundation for ASIC1a as a potential target in the treatment of RA.

E2F4 functions as a tumour suppressor in acute myeloid leukaemia via inhibition of the MAPK signalling pathway by binding to EZH2.

Acute myeloid leukaemia (AML) is an aggressive and mostly incurable haematological malignancy with frequent relapse after an initial response to standard chemotherapy. Therefore, novel therapies are urgently required to improve AML clinical outcome. Here, we aim to study the dysregulation of a particular transcription factor, E2F4, and its role in the progression of AML. In this study, human clinical data from the Gene Expression Profiling Interactive Analysis (GEPIA) revealed that increased E2F4 expression was associated with poor prognosis in AML patients. Moreover, the experimental results showed that E2F4 was aberrantly overexpressed in human AML patients and cell lines. Depletion of E2F4 inhibited the proliferation, induced the differentiation and suppressed the growth of AML cells in a nude mouse model. By contrast, overexpression of E2F4 promoted the proliferation and inhibited the differentiation of AML cells in vitro. Additionally, E2F4 expression not only is positively correlated with EZH2 but also can bind to EZH2. RNA microarray results also showed that E2F4 can regulate MAPK signalling pathway. EZH2 can reverse the inhibitory effect of E2F4 silencing on MAPK signaling pathway. In summary, our data suggest that E2F4 may be a potential therapeutic target for AML therapy.

ATPR triggers acute myeloid leukaemia cells differentiation and cycle arrest via the RARα/LDHB/ERK-glycolysis signalling axis.

Acute myeloid leukaemia (AML) remains a therapeutic challenge and improvements in chemotherapy are needed. 4-Amino-2-trifluoromethyl-phenyl retinate (ATPR), a novel all-trans retinoic acid (ATRA) derivative designed and synthesized by our team, has been proven to show superior anticancer effect compared with ATRA on various cancers. However, its potential effect on AML remains largely unknown. Lactate dehydrogenase B (LDHB) is the key glycolytic enzyme that catalyses the interconversion between pyruvate and lactate. Currently, little is known about the role of LDHB in AML. In this study, we found that ATPR showed antileukaemic effects with RARα dependent in AML cells. LDHB was aberrantly overexpressed in human AML peripheral blood mononuclear cell (PBMC) and AML cell lines. A lentiviral vector expressing LDHB-targeting shRNA was constructed to generate a stable AML cells with low expression of LDHB. The effect of LDHB knockdown on differentiation and cycle arrest of AML cells was assessed in vitro and vivo, including involvement of Raf/MEK/ERK signalling. Finally, these data suggested that ATPR showed antileukaemic effects by RARα/LDHB/ ERK-glycolysis signalling axis. Further studies should focus on the underlying leukaemia-promoting mechanisms and investigate LDHB as a therapeutic target.

ATPR induces acute promyelocytic leukemia cells differentiation and growth arrest by blockade of SHP2/Rho/ROCK1 pathway.

Acute promyelocytic leukemia (APL) is a form of acute myeloid leukemia with a unique chromosome translocation t (15;17), commonly complicated by a complex coagulopathy. 4-Amino-2-trifuoromethyl-phenyl retinate (ATPR), a novel all-trans retinoic acid (ATRA) derivative, was synthesized by our group and known to possess obvious biological anti-tumor activities. It has previously been shown that ATPR could induce differentiation and inhibit proliferation of APL cells, although the mechanism responsible for this effect was not well understood. In this study, we demonstrated that ATPR remarkably inhibited the expression and activity of SHP2. Further experiments showed silencing SHP2 or using SHP2 inhibition (SHP099) enhanced the effect of ATPR on cell proliferation and maturation. In addition, we also demonstrated that Rho/ROCK1 might be regulated by SHP2. Using Y-27632, a ROCK inhibitor, further proved that ROCK1 played an important role in ATPR-induced differentiation and proliferation suppression. In conclusion, the results from this study revealed that ATPR induced APL cells terminal differentiation and growth arrest by blockade of SHP2/Rho/ ROCK1 pathway.

Acid-sensing ion channel 1a mediates acid-induced pyroptosis through calpain-2/calcineurin pathway in rat articular chondrocytes.

The pyroptosis is a causative agent of rheumatoid arthritis, a systemic autoimmune disease merged with degenerative articular cartilage. Nevertheless, the precise mechanism of extracellular acidosis on chondrocyte pyroptosis is largely unclear. Acid-sensing ion channels (ASICs) belong to an extracellular H+ -activated cation channel family. Accumulating evidence has highlighted activation of ASICs induced by extracellular acidosis upregulate calpain and calcineurin expression in arthritis. In the present study, to investigate the expression and the role of acid-sensing ion channel 1a (ASIC1a), calpain, calcineurin, and NLRP3 inflammasome proteins in regulating acid-induced articular chondrocyte pyroptosis, primary rat articular chondrocytes were subjected to different pH, different time, and different treatments with or without ASIC1a, calpain-2, and calcineurin, respectively. Initially, the research results showed that extracellular acidosis-induced the protein expression of ASIC1a in a pH- and time-dependent manner, and the messenger RNA and protein expressions of calpain, calcineurin, NLRP3, apoptosis-associated speck-like protein, and caspase-1 were significantly increased in a time-dependent manner. Furthermore, the inhibition of ASIC1a, calpain-2, or calcineurin, respectively, could decrease the cell death accompanied with the decreased interleukin-1ß level, and the decreased expression of ASIC1a, calpain-2, calcineurin, and NLRP3 inflammasome proteins. Taken together, these results indicated the activation of ASIC1a induced by extracellular acidosis could trigger pyroptosis of rat articular chondrocytes, the mechanism of which might partly be involved with the activation of calpain-2/calcineurin pathway.

MicroRNA-155 Participates in the Expression of LSD1 and Proinflammatory Cytokines in Rheumatoid Synovial Cells.

MicroRNA-155 (miRNA-155) is abundant in fibroblast-like synoviocytes (FLS) in rheumatoid arthritis (RA). Lysine-specific demethylase 1 (LSD1) has been found that it can ameliorate the severity of RA. Tumor necrosis factor-alpha, interleukin-1 beta, and interleukin-6 are key proinflammatory cytokines implicated in the pathogenesis of RA. In our study, we investigated whether miRNA-155 participates in the expression of LSD1 and proinflammatory cytokines in rheumatoid synovial cells. First of all, flow cytometry and cell counting kit-8 analysis were employed to explore the apoptosis and proliferation of FLS, respectively. Subsequently, reverse transcription-quantitative polymerase chain reaction (RT-qPCR) was applied to probe into the level of miRNA-155 in FLS when stimulated by miRNA-155 molecules. Moreover, RT-qPCR was used to explore the relative LSD1 miRNA expression in FLS when stimulated by miRNA-155 molecules, and Western blot and immunofluorescence assay were applied to probe into the expression level of LSD1. Finally, enzyme-linked immunosorbent assay was employed to analyze the secreting level of proinflammatory cytokines in FLS when stimulated by miRNA-155 molecules. RA-FLS showed a higher apoptosis rate than normal FLS. The cell proliferation of both HFLS and MH7A cells was promoted by miRNA-155 upregulation. Meanwhile, the expression of LSD1 and proinflammatory cytokines in the FLS of RA was also changed by miRNA-155 regulation. In conclusion, miRNA-155 participates in the expression of LSD1 and proinflammatory cytokines in rheumatoid synovial cells. These findings imply a potential function and interaction of miRNA-155 and LSD1.

Effects of autophagy on apoptosis of articular chondrocytes in adjuvant arthritis rats.

Rheumatoid arthritis (RA) is a chronic, systemic autoimmune disease that eventually leads to joint deformities and loss of joint function. Previous studies have demonstrated a close relationship between autophagy and the development of RA. Although autophagy and apoptosis are two different forms of programmed death, the relationship between them in relation to RA remains unclear. In this study, we explored the effect of autophagy on apoptosis of articular chondrocytes in vivo and in vitro. Adjuvant arthritis (AA) and acid-induced primary articular chondrocyte apoptosis were used as in vivo and in vitro models, respectively. Articular chondrocyte autophagy and apoptosis were both observed dynamically in AA rat articular cartilage at different stages (15 days, 25 days and 35 days). Moreover, chondrocyte apoptosis and articular cartilage injury in AA rats were increased by the autophagy inhibitor 3-methyladenine (3-MA) and decreased by the autophagy activator rapamycin. In addition, pre-treatment with 3-MA increased acid-induced chondrocyte apoptosis, while pre-treatment with rapamycin reduced acid-induced chondrocyte apoptosis in vitro. These results suggest that autophagy might be a potential target for the treatment of RA.

ß-Estradiol Protects Against Acidosis-Mediated and Ischemic Neuronal Injury by Promoting ASIC1a (Acid-Sensing Ion Channel 1a) Protein Degradation.

Background and Purpose- Sex differences in the incidence and outcome of stroke have been well documented. The severity of stroke in women is, in general, significantly lower than that in men, which is mediated, at least in part, by the protective effects of ß-estradiol. However, the detailed mechanisms underlying the neuroprotection by ß-estradiol are still elusive. Recent studies have demonstrated that activation of ASIC1a (acid-sensing ion channel 1a) by tissue acidosis, a common feature of brain ischemia, plays an important role in ischemic brain injury. In the present study, we assessed the effects of ß-estradiol on acidosis-mediated and ischemic neuronal injury both in vitro and in vivo and explored the involvement of ASIC1a and underlying mechanism. Methods- Cultured neurons and NS20Y cells were subjected to acidosis-mediated injury in vitro. Cell viability and cytotoxicity were measured by methylthiazolyldiphenyl-tetrazolium bromide and lactate dehydrogenase assays, respectively. Transient (60 minutes) focal ischemia in mice was induced by suture occlusion of the middle cerebral artery in vivo. ASIC currents were recorded using whole-cell patch-clamp technique while intracellular Ca2+ concentration was measured with fluorescence imaging using Fura-2. ASIC1a expression was detected by Western blotting and quantitative real-time polymerase chain reaction. Results- Treatment of neuronal cells with ß-estradiol decreased acidosis-induced cytotoxicity. ASIC currents and acid-induced elevation of intracellular Ca2+ were all attenuated by ß-estradiol treatment. In addition, we showed that ß-estradiol treatment reduced ASIC1a protein expression, which was mediated by increased protein degradation, and that estrogen receptor α was involved. Finally, we showed that the level of ASIC1a protein expression in brain tissues and the degree of neuroprotection by ASIC1a blockade were lower in female mice, which could be attenuated by ovariectomy. Conclusions- ß-estradiol can protect neurons against acidosis-mediated neurotoxicity and ischemic brain injury by suppressing ASIC1a protein expression and channel function. Visual Overview- An online visual overview is available for this article.

The role of Ca2+ in acid-sensing ion channel 1a-mediated chondrocyte pyroptosis in rat adjuvant arthritis.

Rheumatoid arthritis is an autoimmune disease with a poor prognosis. Pyroptosis is a type of proinflammatory programmed cell death that is characterised by the activation of caspase-1 and secretion of the proinflammatory cytokines interleukin (IL)-1ß/18. Previous reports have shown that pyroptosis is closely related to the development of some autoimmune diseases, such as rheumatoid arthritis. The decrease in the pH of joint fluid is a main pathogenic feature of RA and leads to excessive apoptosis in chondrocytes. Acid-sensitive ion channels (ASICs) are extracellular H+-activated cation channels that mainly influence Na+ and Ca2+ permeability. In this study, we investigated the role of Ca2+ in acid-sensing ion channel 1a-mediated chondrocyte pyroptosis in an adjuvant arthritis rat model. The expression of apoptosis-associated speck-like protein, NLRP3, caspase-1, ASIC 1a, IL-1ß and IL-18 was upregulated in the joints of rats compared with that in normal rats, but the expression of Col2a in cartilage was decreased. However, these changes were reversed by amiloride, which is an inhibitor of ASIC1a. Extracellular acidosis significantly increased the expression of ASIC1a, IL-1ß, IL-18, ASC, NLRP3 and caspase-1 and promoted the release of lactate dehydrogenase. Interestingly, Psalmotoxin-1 (Pctx-1) and BAPTA-AM inhibited these effects. These results indicate that ASIC1a mediates pyroptosis in chondrocytes from AA rats. The underlying mechanism may be associated with the ability of ASIC1a to promote [Ca2+]i and upregulate the expression of the NLRP3 inflammasome.

ATPR-induced differentiation and G0/G1 phase arrest in acute promyelocytic leukemia by repressing EBP50/NCF1 complex to promote the production of ROS.

Our previous study has demonstrated that 4-amino-2-trifluoromethyl-phenyl Retinate (ATPR) can induce human leukemia NB4 cells differentiation and G0/G1 phase arrest, but the underlying mechanism is still unclear. In this study, we used proteomics to screen differentially expressed protein profiles in NB4 cells before and after ATPR treatment in vitro. We analyzed the peptides digested from total cellular proteins by reverse phase LC-MS/MS and then performed label-free quantitative analysis. We found 27 significantly up-regulated proteins in the ATPR group compared to the control group. NCF1 was the most significantly changed protein. Immunoprecipitation and double immunofluorescent staining showed that EBP50 bind to NCF1. We further explored the potential molecular mechanism of EBP50/NCF1 complex in ATPR-induced differentiation and G0/G1 phase arrest. The results showed that ATPR remarkably reduced the expression of EBP50 in vivo and in vitro. Interestingly, the reduction of EBP50 contributed to ROS release by modulating the subcellular localization of NCF1. The reduction of EBP50 also contributed to G0/G1 phase arrest by inhibiting CyclinD1, CyclinA2 and CDK4, as well as promoting the differentiation of NB4 cells by increasing the expression of CD11b. Furthermore, we found that the overexpression of EBP50 restrained the effects of ATPR on differentiation and G0/G1 phase arrest in NB4 cells. These results suggest that ATPR-induced differentiation and G0/G1 phase arrest in acute promyelocytic leukemia (APL) by repressing EBP50/NCF1 complex to promote the production of ROS, and the results from in vivo experiments were consistent with those from in vitro studies. Therefore, our finding results suggest that EBP50 may be a new target for ATPR in the treatment of APL.

ROS play an important role in ATPR inducing differentiation and inhibiting proliferation of leukemia cells by regulating the PTEN/PI3K/AKT signaling pathway.

BACKGROUND: Acute myeloid leukemia (AML) is an aggressive and mostly incurable hematological malignancy with frequent relapses after an initial response to standard chemotherapy. Therefore, novel therapies are urgently required to improve AML clinical outcomes. 4-Amino-2-trifluoromethyl-phenyl retinate (ATPR), a novel all-trans retinoic acid (ATRA) derivative designed and synthesized by our team, has been proven to show biological anti-tumor characteristics in our previous studies. However, its potential effect on leukemia remains unknown. The present research aims to investigate the underlying mechanism of treating leukemia with ATPR in vitro. METHODS: In this study, the AML cell lines NB4 and THP-1 were treated with ATPR. Cell proliferation was analyzed by the CCK-8 assay. Flow cytometry was used to measure the cell cycle distribution and cell differentiation. The expression levels of cell cycle and differentiation-related proteins were detected by western blotting and immunofluorescence staining. The NBT reduction assay was used to detect cell differentiation. RESULTS: ATPR inhibited cell proliferation, induced cell differentiation and arrested the cell cycle at the G0/G1 phase. Moreover, ATPR treatment induced a time-dependent release of reactive oxygen species (ROS). Additionally, the PTEN/PI3K/Akt pathway was downregulated 24 h after ATPR treatment, which might account for the anti-AML effects of ATPR that result from the ROS-mediated regulation of the PTEN/PI3K/AKT signaling pathway. CONCLUSIONS: Our observations could help to develop new drugs targeting the ROS/PTEN/PI3K/Akt pathway for the treatment of AML.