Microglial voltage-dependent anion channel 1 signaling modulates sleep deprivation-induced transition to chronic postsurgical pain.

STUDY OBJECTIVES: This study verified that sleep deprivation before and after skin/muscle incision and retraction (SMIR) surgery increased the risk of chronic pain and investigated the underlying roles of microglial voltage-dependent anion channel 1 (VDAC1) signaling. METHODS: Adult mice received 6 hours of total sleep deprivation from 1 day prior to SMIR until the third day after surgery. Mechanical and heat-evoked pain was assessed before and within 21 days after surgery. Microglial activation and changes in VDAC1 expression and oligomerization were measured. Minocycline was injected to observe the effects of inhibiting microglial activation on pain maintenance. The VDAC1 inhibitor 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) and oligomerization inhibitor VBIT-4 were used to determine the roles of VDAC1 signaling on microglial adenosine 5' triphosphate (ATP) release, inflammation (IL-1ß and CCL2), and chronicity of pain. RESULTS: Sleep deprivation significantly increased the pain duration after SMIR surgery, activated microglia, and enhanced VDAC1 signaling in the spinal cord. Minocycline inhibited microglial activation and alleviated sleep deprivation-induced pain maintenance. Lipopolysaccharide (LPS)-induced microglial activation was accompanied by increased VDAC1 expression and oligomerization, and more VDAC1 was observed on the cell membrane surface compared with control. DIDS and VBIT-4 rescued LPS-induced microglial ATP release and IL-1ß and CCL2 expression. DIDS and VBIT-4 reversed sleep loss-induced microglial activation and pain chronicity in mice, similar to the effects of minocycline. No synergistic effects were found for minocycline plus VBIT-4 or DIDS. CONCLUSIONS: Perioperative sleep deprivation activated spinal microglia and increases the risk of chronic postsurgical pain in mice. VDAC1 signaling regulates microglial activation-related ATP release, inflammation, and chronicity of pain.

Mechanically Activated Calcium Channel PIEZO1 Modulates Radiation-Induced Epithelial-Mesenchymal Transition by Forming a Positive Feedback With TGF-ß1.

TGF-ß-centered epithelial-mesenchymal transition (EMT) is a key process involved in radiation-induced pulmonary injury (RIPI) and pulmonary fibrosis. PIEZO1, a mechanosensitive calcium channel, is expressed in myeloid cell and has been found to play an important role in bleomycin-induced pulmonary fibrosis. Whether PIEZO1 is related with radiation-induced EMT remains elusive. Herein, we found that PIEZO1 is functional in rat primary type II epithelial cells and RLE-6TN cells. After irradiation, PIEZO1 expression was increased in rat lung alveolar type II epithelial cells and RLE-6TN cell line, which was accompanied with EMT changes evidenced by increased TGF-ß1, N-cadherin, Vimentin, Fibronectin, and α-SMA expression and decreased E-cadherin expression. Addition of exogenous TGF-ß1 further enhanced these phenomena in vitro. Knockdown of PIEZO1 partly reverses radiation-induced EMT in vitro. Mechanistically, we found that activation of PIEZO1 could upregulate TGF-ß1 expression and promote EMT through Ca2+/HIF-1α signaling. Knockdown of HIF-1α partly reverses enhanced TGF-ß1 expression caused by radiation. Meanwhile, the expression of PIEZO1 was up-regulated after TGF-ß1 co-culture, and the mechanism could be traced to the inhibition of transcription factor C/EBPß expression by TGF-ß1. Irradiation also caused a decrease in C/EBPß expression in RLE-6TN cells. Dual luciferase reporter assay and chromatin immunoprecipitation assay (ChIP) confirmed that C/EBPß represses PIEZO1 expression by binding to the PIEZO1 promoter. Furthermore, overexpression of C/EBPß by using the synonymous mutation to C/EBPß siRNA could reverse siRNA-induced upregulation of PIEZO1. In summary, our research suggests a critical role of PIEZO1 signaling in radiation-induced EMT by forming positive feedback with TGF-ß1.

PIEZO1 Ion Channel Mediates Ionizing Radiation-Induced Pulmonary Endothelial Cell Ferroptosis via Ca2+/Calpain/VE-Cadherin Signaling.

Pulmonary endothelial cell dysfunction plays an important role in ionizing radiation (IR)-induced lung injury. Whether pulmonary endothelial cell ferroptosis occurs after IR and what are the underlying mechanisms remain elusive. Here, we demonstrate that 15-Gy IR induced ferroptosis characterized by lethal accumulation of reactive oxygen species (ROS), lipid peroxidation, mitochondria shrinkage, and decreased glutathione peroxidase 4 (GPX4) and SLC7A11 expression in pulmonary endothelial cells. The phenomena could be mimicked by Yoda1, a specific activator of mechanosensitive calcium channel PIEZO1. PIEZO1 protein expression was upregulated by IR in vivo and in vitro. The increased PIEZO1 expression after IR was accompanied with increased calcium influx and increased calpain activity. The effects of radiation on lung endothelial cell ferroptosis was partly reversed by inhibition of PIEZO1 activity using the selective inhibitor GsMTx4 or inhibition of downstreaming Ca2+/calpain signaling using PD151746. Both IR and activation of PIEZO1 led to increased degradation of VE-cadherin, while PD151746 blocked these effects. VE-cadherin knockdown by specific siRNA causes ferroptosis-like phenomena with increased ROS and lipid peroxidation in the lung endothelial cells. Overexpression of VE-cadherin partly recused the ferroptosis caused by IR or PIEZO1 activation as supported by decreased ROS production, lipid peroxidation and mitochondria shrinkage compared to IR or PIEZO1 activation alone. In summary, our study reveals a previously unrecognized role of PIEZO1 in modulating ferroptosis, providing a new target for future mitigation of radiation-induced lung injury.

Repeated Whole-Body Exposure to Low-Dose Radiation Combined With Topical Application of Basic Fibroblast Growth Factor and Zinc Accelerates Wound Healing in Diabetic Rats.

We reported the acceleration of skin wound healing in diabetic rats by repeated exposure to low-dose radiation (LDR). Here, we explored whether the wound healing could be further improved when LDR was combined with a topical application of basic fibroblast growth factor (bFGF) or zinc. Wounds were established on the backs of type 1 diabetic rats induced by a single injection of streptozotocin. Rats were treated daily with normal saline (Diabetes), LDR, bFGF, zinc, or combined 3 treatments for 5 consecutive days with a 2-day break between each consecutive 5-day treatment. Changes in wound size, histopathology, and microvessel density were assessed on days 5, 10, and 15, respectively, once treatment is started. All treatment regimens significantly accelerated skin wound healing, tissue remodeling, and new vessel formation compared to diabetes group. However, the combined LDR plus bFGF and zinc provided a better beneficial effect on wound healing than either one of these treatments alone. Further, we found that the effects of LDR and bFGF were similar, whereas zinc alone induced a weaker response. Our results suggest that whole-body LDR plus the topical application of bFGF and zinc can further accelerate wound healing in diabetic rats.

High expression of CD39/ENTPD1 in malignant epithelial cells of human rectal adenocarcinoma.

The ectonucleotidase CD39 is pivotal in the conversion of immunostimulatory adenosine triphosphate (ATP) into immunosuppressive adenosine which potently inhibits host immune responses against cancer. This study investigated the expression level and prognostic significance of CD39 in human rectal adenocarcinoma. Our data demonstrated that CD39 staining strongly marked malignant epithelial cells where the protein and messenger RNA (mRNA) expression levels of CD39 were significantly increased compared with paracancerous controls. In addition to primary tumors, CD39 was also abundantly expressed in liver metastases and tumor-draining lymph nodes from metastatic rectal adenocarcinoma. Although patients with higher CD39 density in tumor cells were more likely to have favorable characteristics (early TNM and N stages) and overall survival, the singular parameter cannot be used as an independent factor for predicting patients' prognosis. Intriguingly, combined analysis of CD39 and CD73 expression was more efficient to foretell patient's outcome where patients with increased CD73 but decreased CD39 levels displayed a worst prognosis. Taken together, the current study revealed that malignant epithelial cells of human rectal adenocarcinoma strongly express CD39 that may play a potential role in the tumor invasion and metastasis. Although high expression of CD39 in tumor cells is correlated with favorable clinical outcome, the combination of CD39 and CD73 expression may have a better prognostic value.

Radiation induces phosphorylation of STAT3 in a dose- and time-dependent manner.

BACKGROUND: We have reported the radiation could activate STAT3, which subsequently promotes the invasion of A549 cells. We here explored the dose- and time-response of STAT3 to radiation and the effect of radiation on upstream signaling molecules. MATERIALS AND METHODS: A549 cells were irradiated with different doses of γ-rays. The expression of and nucleus translocation of p-STAT3 in A549 cells were detected by immunoblotting and immunofluorescence, respectively. The level of phosphorylated EGFR was also assessed by immunoblotting, and IL-6 expression was detected by real time PCR and ELISA. RESULTS: Radiation promoted the phosphorylation of STAT3 at Y705 in a dose- and time-dependent manner and nuclear translocation. The level of phosphorylated EGFR in A549 cells increased after radiation. In additional, the mRNA and protein levels of IL-6 in A549 cells were also up regulated by radiation. CONCLUSIONS: STAT3 is activated by radiation in a dose-and time-dependent manner, probably due to radiation-induced activation of EGFR or secretion of IL-6 in A549 cells.

S100A4 siRNA inhibits human pancreatic cancer cell invasion in vitro.

OBJECTIVE: Pancreatic cancer is one of the most deadly cancers, which is characterized by its high metastatic potential. S100A4 is a major prometastatic protein involved in tumor invasion and metastasis which precise role in pancreatic cancer has not been fully investigated. We knocked down the S100A4 gene in the Bxpc-3 pancreatic cancer cell line via RNA interference to study the changes in cell behavior. METHODS: Real-time polymerase chain reaction and western blotting were used to detect mRNA and protein expression levels of S100A4, matrix metalloproteinase (MMP)-2, E-cadherin and thrombospondin (TSP)-1. Transwell chambers were used to detect the migration and invasion abilities; a cell adhesion assay was used to detect adhesion ability; colony forming efficiency was used to detect cell proliferation; flow cytometry was used to detect apoptosis. RESULTS: S100A4 mRNA expression was reduced to 17% after transfection with S100A4-siRNA, and protein expression had a similar trend. mRNA and protein expression of MMP-2 was reduced and that of E-cadherin and TSP-1 was elevated, indicating that S100A4 affects their expression. S100A4-silenced cells exhibited a marked decrease in migration and invasiveness and increased adhesion, whereas overall proliferation and apoptosis were not overtly altered. CONCLUSION: S100A4 and its downstream factors play important roles in pancreatic cancer invasion, and silencing A100A4 can significantly contain the invasiveness of pancreatic cancer.

Toll-like receptor 5 agonist inhibition of growth of A549 lung cancer cells in vivo in a Myd88 dependent manner.

The purpose of this study was to examine the effect of a Toll-like receptor 5 (TLR5) agonist, CBLB502, on the growth and radiosensitivity of A549 lung cancer cells in vivo. Expression of myeloid differentiation factor 88 (MyD88) or TLR5 was stably knocked down in human lung cancer cells (A549) using lentivirus expressing short hairpin RNA targeting human MyD88 or TLR5. Lack of MyD88 or TLR5 expression enhanced tumor growth in mouse xenografts of A549 lung cancer cells. CBLB502 inhibited the growth of A549 lung cancer cells, not A549-MyD88-KD cells in vivo in the murine xenograft model. Our results showed that the inhibition of A549 by CBLB502 in vivo was realized through regulating the expression of neutrophil recruiting cytokines and neutrophil infiltration. Finally, we found that activation of TLR5 signaling did not affect the radiosensitivity of tumors in vivo.

[Biological effect of gamma-rays and LPS on the murine macrophage cell line RAW264.7 in mice].

AIM: To explore the effect of activating the murine macrophage cell line RAW264.7 by both gamma-rays and lipopolysaccharide (LPS) and to study the expression of calcium-binding protein S100A8 induced by gamma-rays and LPS. METHODS: The RAW264.7 cells were observed by phase contrast microscope. The cell cycle and the level of reactive oxygen intermediates (ROIs) were detected by flow cytometry (FCM). The production of NO was measured by colorimetric Griess reaction. The mRNA expression of S100A8 was recorded by real-time quantitative RT-PCR method. RESULTS: The exposure of RAW264.7 cells to gamma-rays and LPS resulted in the morphological change of cells, the rise of cells number of aneuploid and apoptosis, and the rise of the level of ROI, NO and S100A8 mRNA. The effect of using both gamma-rays and LPS was stronger than that of single gamma-rays or LPS treatment. CONCLUSION: The mechanism of using both gamma-rays and LPS for activating macrophages is owing to the various biological effects including the change of cell cycle, the change of the level of messenger molecules and the expression of inflammation factor such as S100A8. The expression of S100A8 gene is closely correlated with the function and state of macrophages.