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.

[Study on tanshinones regulating root-associated microbiomes of Salvia miltiorrhiza].

The plant root-associated microbiomes include root microbiome and rhizosphere microbiome, which are closely related to plant life activities. Nearly 30% of photosynthesis products of plants are used to synthesize root compounds, there is evidence that root compounds regulate and significantly affect the root microbiome Tanshinones are the main hydrophobic components in Salvia miltiorrhiza. In order to study whether these compounds can regulate the root-associated microbiomes of S. miltiorrhiza, our study first identified a white root S. miltiorrhiza(BG) which contains little tanshinones. Retain of the fifth intron of tanshinones synthesis key enzyme gene SmCPS1 leading to the early termination of the SmCPS1 gene, and a stable white root phenotype. Further, wild type(WT) and BG were planted in greenhouse with nutrient soil(Pindstrup, Denmark) and Shandong soil(collected from the S. miltiorrhiza base in Weifang, Shandong), then high-throughput sequencing was used to analyze the root-associated microbiomes. The results showed that the tanshinones significantly affected the root-associated microbiomes of S. miltiorrhiza, and the impact on root microbiomes was more significant. There are significant differences between WT and BG root microbiomes in species richness, dominant strains and co-occurrence network. Tanshinones have a certain repelling effect on Bacilli which belongs to Gram-positive, while specifically attract some Gram-negative bacteria such as Betaproteobacteria and some specific genus of Alphaproteobacteria. This study determined the important role of tanshinones in regulating the structure of root-associated microbiomes from multiple angles, and shed a light for further improving the quality and yield of S. miltiorrhiza through microenvironment regulation.

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.

Genetic Polymorphisms of SP-A, SP-B, and SP-D and Risk of Respiratory Distress Syndrome in Preterm Neonates.

BACKGROUND We examined selected polymorphisms in 3 pulmonary surfactant-associated proteins (SP) for their influence on serum SP levels and risk of respiratory distress syndrome (RDS) in preterm neonates. MATERIAL AND METHODS Premature infants from a Han population were enrolled, including 100 premature infants with RDS (case group) and 120 premature infants without RDS (control group). SNP genotyping for SP-A (+186A/G and +655C/T), SP-B (-18A/C and 1580C/T), and SP-D (Met11ThrT/C and Ala160ThrG/A) used polymerase chain reaction-restriction fragment length polymorphism. Haplotypes were calculated with Shesis software and serum SP-A/B/D levels were quantified by ELISA. RESULTS Case and control groups exhibited significant differences in genotype and allele frequencies of SP-A (+186A/G, +655C/T) and SP-B (1580C/T). However, no statistically significant differences were observed in the allele and genotype frequencies of SP-B -18A/C, SP-D Met11ThrT/C, and SP-D Ala160ThrG/A. Importantly, serum SP-A and SP-B levels were reduced in RDS patients carrying SP-A (+186A/G, +655C/T) and SP-B (1580C/T) polymorphisms. AA genotype of +186A/G, SP-A level, and CC genotype of 1580C/T were independently correlated with increased RDS risk. CONCLUSIONS SP-A (+186A/G) and SP-B (1580C/T) polymorphisms are strongly associated with the risk of RDS in preterm infants. Notably, reduced serum SP-A levels were correlated with a high risk of RDS and may serve as novel biomarkers for RDS detection and monitoring.

[Effect of the Cultivation Pattern on Photosynthesis and Yield of Salvia miltiorrhiza].

Objective: To investigate the effect of cultivation pattern on photosynthesis and yield of Salvia miltiorrhiza. Methods: The covering plastic mulch, the uncorering plastic mulch, and the traditional cultivation pattern were used to analysed. LI-6400 XT photosynthesis was used to determine the photosynthetic parameter of Salvia miltiorrhiza, and some growth indexes of Salvia militiorrhiza were measured,and the accumulation was measured. Results: The change of stomatal conductance in the plants of different treatments were as follows, the covering plastic mulch > the uncovering plastic mulch > the traditional cultivation pattern; the change of intercellular CO2 concentration in the plants of different treatments was as follows, the uncovering plastic mulch > the covering plastic mulch > the traditional cultivation pattern; the change of transpiration rates in the plants of different treatments was as follows, the covering plastic mulch> the uncovering plastic mulch > the traditional cultivation pattern; the change of net photosynthetic rates in the plants of different treatments was as follows, the covering plastic mulch > the uncovering plastic mulch > the traditional cultivation pattern. The change of fresh and dry weight in root of Salvia miltiorrhiza of different treatments was as follows, the covering plastic mulch > the uncovering plastic mulch > the traditional cultivation pattern. Compared to the uncovering plastic mulch and the traditional cultivation pattern, the fresh and dry weight in the root of Salvia miltiorrhiza of the covering plastic mulch were increased to 16. 62%,18. 20% and 14. 68%,48. 62%. Conclusion: The cultivation pattern of covering plastic mulch reduced water stress by increasing the water content of soil to increase photosynthesis efficiency, thus increase the yield of Salvia miltiorrhiza.

Influence of naturaldecay of fibrousroots on growth and content of active compositions of Salvia miltiorrhiza f. alba.

The change of yield and contents. of active compositions were studied while the fibrous roots were decayed naturally. HPLC method was used to detect the contents of active composition. The results show that fibrousroots could decrease the production of plant by 38.60% (20 g) and 30.99% (40 g), respectively. Treatment 1 could increase the contents of dihydrotanshinone and cryptotanshinone of Salvia miltiorrhiza f. alba by 26.08% and 22.64%, respectively. Compared with the comparison, treatment 2 decreased the contents of ihydrotanshinone, cryptotanshinone, tanshinone I and tanshinone II(A) of S. miltiorrhiza f. alba by 60.87%, 79.24%, 84.61% and 88.99%, respectively. Meanwhile, the total contents of the liposoluble constituents reduced by 86.27%. The different concentration of fibrousroots could increase the content of salvianolic acid B by 4.98% (20 g) and 23.64% (40 g), respectively. Meanwhile, the content of rosemary acid was increased by 4.98% (20 g) and 23.64% (40 g), respectively. The content of water-soluble constituents positively correlated to the mount of added fibrousroots, and the change was significantly. The result indicted that the decay of fibrousroots has a significant impact on the growth and the content of the active composition of S. miltiorrhiza f. alba under the condition of continuous cropping. Fibrousroots could decrease the content of biomass and liposoluble constituents significantly, which maybe one of the main factors to S. miltiorrhiza f. alba continuous cropping obstacle formation.

[Biological and Phenological Properties of Salvia miltiorrhiza f. alba for Anti-continuous Cropping].

OBJECTIVE: Morphological characteristics and phenological phase of Salvia miltiorrhiza f. alba for anti-continuous cropping was studied in the field. METHODS: The data of fixed plants of different stages were collected and analyzed. RESULTS: The resistance of the strains for anti-continuous cropping was obviously higher than the others. The phenological phase of strains for anti-continuous cropping was divided into seedling stage, elongation stage,flower stage, fructification stage,root enlargement stage and dead stage. The whole development period was divided into the vegetative growth and reproduction growth by the flower stage. CONCLUSION: From the appearance characteristics and resistance observation, the growth of Salvia miltiorrhiza f. dlba for anti-continuous cropping was better than the other strains in the next year.

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.

Fabrication of fluorescent magnetic Fe3O4@ZnS nanocomposites.

A novel Fe3O4@ZnS nanomaterial with fluorescent and superparamagnetic properties has been successfully fabricated via TOPO-TOP synthesis with an additional coordinating component (OAm). The adsorption of OAm on the preformed magnetite nanoparticles, which were prepared in phenyl ether with oleic acid and oleyl amine, played an essential role in directing the structure of the Fe3O4@ZnS composites. The obtained materials were characterized by FTIR, TEM, XRD, X-ray photoelectron spectroscopy (XPS), UV-vis, fluorescence spectrophotometer and VSM. The results indicated that the Fe3O4 nanoparticles were successfully combined with ZnS and the coating of ZnS can be controlled by adjusting the molar ratio of Fe3O4 to ZnS. The saturation magnetization values of Fe3O4, Fe3O4@ZnS (1:2) and Fe3O4@ZnS (1:5) nanoparticles are 57.0 emu g(-1), 44.4 emu g(-1) and 34.2 emu g(-1), respectively at 300 K and the nanocomposites exhibit better fluorescence without evident quenching. The combined magnetic and fluorescent properties endow the nanocomposites with great potential applications in "nano-conveyer-belt" platform technology for drug targeting, bioseparation, diagnostic analysis and so on.

[Influence of continuous cropping years on yield and active compounds in Salvia miltiorrhiza f. alba].

Biological characteristic of Salvia miltiorrhiza f. alba in field was studied. HPLC method was used to determine the lipophilic constituents (dihydrotanshinone, cryptotanshinone, tanshinone, tanshinone II (A) and miltione) and hydrophilic constituents (salvianolic acid, rosemarinic acid). The results showed that the fresh weight of S. miltiorrhiza f. alba which cropped for 2 years was decreased by 80.47%, while dry weight decreased by 79.42%. The normal diameter of the root was 0.3-0.5 cm, however, the diameter was 0.2-0.4 cm after 2 years, it was said that the decrease of the root diameter was the main reason for the decrease of the yield. The average contents of dihydrotanshinone, cryptotanshinone, tanshinone, tanshinone II (A), miltione, salvianolic acid and rosemarinic acid were decreased by 35.26%, 32.26%, 19.35%, 3.39%, 64.40%, 66.93% in plant which continuously cropped for 2 years, respectively. The yield and active constituents were mostly effected in the plant of S. miltiorrhiza f. alba, which continuously cropped for 2 years.

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.

[Study on thermal decompositon properties of hexafluoropropane clean gaseous fire-extinguishing agent].

The thermal decomposition properties of hexafluoropropane clean gaseous fire-extinguishing agent were studied in tubular reactor from 500 to 750 degrees C and the decomposed gas was characterized by gas chromatography(GC), Fourier transform infrared spectroscopy (FTIR) and gas chromatography-mass spectrometry (GC-MS). Hydrogen fluoride was detected after the decomposed gas was analyzed by pH testing, while pentafluoropropylene was found by GC-MS. The results showed that hydrogen fluoride eliminated from hexafluoropropane was the main reaction, while pentafluoropropylene was the primary product during hexafluoropropane decomposition under high temperature. GC and FTIR results indicated that the reaction temperatures had significant effects on the thermal decomposition of hexafluoropropane. Haxafluropropane was steady at 500 degrees C, whereas started to decompose weakly at 600 degrees C. The degree of the thermal decomposition of hexafluoropropane was enhanced with the temperature increase. And hexafluoropropane underwent intense decompositon at 750 degrees C. FTIR can be used as a new method to study extinguishing mechanism of fluorine-containing fire extinguishing agent online.

[Primary research of anti-influenza virus activity of "Benovoair Concentrate"].

OBJECTIVE: Anti-influenza virus activity of "Benovoair Concentrate". METHODS: The different dilution of samples were mixed with the same quantity of 100 TCID50 virus at 37 degrees C for 30 minutes. Add suitable quantity mixture in wells containing cells. Every 3 wells were the same mode. Viruses control, cells control and samples control of different dilution were performed and set in the CO2 incubator at 37 degrees C. CPE was observed every day. When CPE appears in viruses control as "++++", stopped testing and performed the hemagglutination titration. RESULTS: "Benovoair Concentrate" with dilution of 1:60, 1:120, 1:240 and 1:480 have 100% anti-influenza A and anti-influenza B activities. "Benovoair Concentrate" with dilution of 1:960 and 1:1920 have 25%-50% anti-influenza A and anti-influenza B activities. CONCLUSION: The test was the proof of anti-influenza virus activities which provided for the development of "Benovoair Concentrate".

[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.