Demethyleneberberine Alleviates Pulmonary Fibrosis through Disruption of USP11 Deubiquitinating GREM1.

BACKGROUND: Idiopathic pulmonary fibrosis (IPF) is a fatal and chronic interstitial lung disease. Intricate pathogenesis of pulmonary fibrosis and only two approved medications with side effects and high cost bring us the challenge of fully understanding this lethal disease and urgency to find more safe and low-cost therapeutic alternatives. PURPOSE: Demethyleneberberine (DMB) has been demonstrated to have various anti-inflammatory, antioxidant, antifibrosis and anti-cancer bioactivities. The objective of this study was to evaluate the effect of DMB on pulmonary fibrosis and investigate the mechanism. METHODS: Bleomycin (BLM)-induced pulmonary fibrosis was established in mice to evaluate the antifibrotic effect of DMB in vivo. A549 and MRC5 cells were used to evaluate the effect of DMB on epithelial-mesenchymal transition (EMT) and fibroblast-myofibroblast transition (FMT) in vitro. High throughput sequencing, biotin-avidin system and site-directed mutagenesis were applied to explore the mechanism of DMB in alleviating pulmonary fibrosis. RESULTS: DMB alleviated BLM-induced pulmonary fibrosis in vivo by improving the survival state of mice, significantly reducing pulmonary collagen deposition and oxidative stress and improving lung tissue morphology. Meanwhile, DMB was demonstrated to inhibit epithelial-mesenchymal transition (EMT) and fibroblast-myofibroblast transition (FMT) in vitro. High throughput sequencing analysis indicated that GREM1, a highly upregulated profibrotic mediator in IPF and BLM-induced pulmonary fibrosis, was significantly downregulated by DMB. Furthermore, USP11 was revealed to be involved in the deubiquitination of GREM1 in this study and DMB promoted the ubiquitination and degradation of GREM1 by inhibiting USP11. Remarkably, DMB was demonstrated to selectively bind to the Met776 residue of USP11, leading to disruption of USP11 deubiquitinating GREM1. In addition, DMB presented an equivalent antifibrotic effect at a lower dose compared with pirfenidone and showed no obvious toxicity or side effects. CONCLUSIONS: This study revealed that USP11/GREM1 could be a potential target for IPF management and identified that DMB could promote GREM1 degradation by inhibiting USP11, thereby alleviating pulmonary fibrosis.

Comparative analysis of permeability model construction and risk fields evaluation by roof cutting along the gob in a coal mine.

The productivity of coal mines is seriously threatened by the combined disasters of gas and coal spontaneous combustion, which have become a common disaster mode. It is unclear how the gas and coal spontaneously combusted in the roof cutting along gob working face. The goal of this study is to identify the distinctive features of combined disasters in gob from two different types of roof cutting along working faces. In these two different types of roof cutting along gob working faces, the paper constructs the permeability model of the gob. The findings demonstrate that the data from the field experiment and the simulation results agree, which validates the simulation's reliability. In contrast to single sided roof cutting along gob working faces, double sided roof cutting along gob working faces clearly has a thinner oxidation zone. Moreover, the oxidation zone of the double side roof cutting along gob working faces is closer to the working face, which is located in the shallow area of the gob 50 m behind the working face. The gas explosion area and the coal spontaneous combustion area are divided by the double side roof cutting along gob working face, which reduces the risk of compound disasters. Important theoretical direction for the prevention and control of gob disasters in the roof cutting along gob working face is provided by the simulation results.

[In vivo degradation and histocompatibility of modified chitosan based on conductive composite nerve conduit].

OBJECTIVE: To investigate the in vivo degradation and histocompatibility of modified chitosan based on conductive composite nerve conduit, so as to provide a new scaffold material for the construction of tissue engineered nerve. METHODS: The nano polypyrrole (PPy) was synthesized by microemulsion polymerization, blended with chitosan, and then formed conduit by injecting the mixed solution into a customized conduit formation model. After freeze-drying and deacidification, the nano PPy/chitosan composite conduit (CP conduit) was prepared. Then the CP conduits with different acetyl degree were resulted undergoing varying acetylation for 30, 60, and 90 minutes (CAP1, CAP2, CAP3 conduits). Fourier infrared absorption spectrum and scanning electron microscopy (SEM) were used to identify the conduits. And the conductivity was measured by four-probe conductometer. The above conduits were implanted after the subcutaneous fascial tunnels were made symmetrically on both sides of the back of 30 female Sprague Dawley rats. At 2, 4, 6, 8, 10, and 12 weeks after operation, the morphology, the microstructure, and the degradation rate were observed and measured to assess the in vivo degradation of conduits. HE staining and anti-macrophage immunofluorescence staining were performed to observe the histocompatibility in vivo. RESULTS: The characteristic peaks of the amide Ⅱ band around 1 562 cm -1 appeared after being acetylated, indicating that the acetylation modification of chitosan was successful. There was no significant difference in conductivity between conduits ( P>0.05). SEM observation showed that the surfaces of the conduits in all groups were similar with relatively smooth surface and compact structure. After the conduits were implanted into the rats, with the extension of time, all conduits were collapsed, especially on the CAP3 conduit. All conduits had different degrees of mass loss, and the higher the degree of acetylation, the greater the mass change ( P<0.05). SEM observation showed that there were more pores at 12 weeks after implantation, and the pores showed an increasing trend as the degree of acetylation increased. Histological observation showed that there were more macrophages and lymphocytes infiltration in each group at the early stage. With the extension of implantation time, lymphocytes decreased, fibroblasts increased, and collagen fibers proliferated significantly. CONCLUSION: The modified chitosan basedon conductive composite nerve conduit made of nano-PPy/chitosan composite with different acetylation degrees has good biocompatibility, conductivity, and biodegradability correlated with acetylation degree in vivo, which provide a new scaffold material for the construction of tissue engineered nerve.

Enrichment experiment of ventilation air methane (0.5%) by the mechanical tower.

Methane is one of the most important gases leading to the earth's air pollution. Ventilation air methane(VAM) is an important part of the gas discharged into the atmosphere. The volume concentration of methane is generally less than 0.5% in coal mines. Recycling low concentration is facing challenges. To explore the law of low concentration methane enrichment, the enrichment tower for methane was designed and manufactured. The experiment was divided into two types - free diffusion and weak eddy enrichment, and eight kinds of low concentration gas experimental program. Under free diffusion conditions, the maximum methane concentration of the top (middle) tower is 0.64% (0.53%). In the condition of weak eddy field, the maximum methane concentration is 0.67% (0.69%) in the top (middle) tower. The effect of methane enrichment in the weak eddy field is obvious. Methane enrichment method under the eddy current field can greatly increase methane enrichment efficiency and achieve the goal of CMM (coal mine methane) power generation.

FEN1 inhibitor increases sensitivity of radiotherapy in cervical cancer cells.

BACKGROUND: Cervical cancer is one of the most common causes of cancer-associated mortality among affected women in the world. At present, treatment with weekly cisplatin plus ionizing radiation (IR) therapy is the standard regimen for cervical cancer, especially for locally advanced cervical cancer. The purpose of this study is to determine whether FEN1 inhibitors could enhance the therapeutic effect of IR therapy. METHODS: Western blot was applied to determine the expression of FEN1- and apoptosis-related proteins. Cell growth inhibition assay and colony formation assay were used to determine the effects of FEN1 inhibitor and IR exposure for Hela cells in vitro. CRISPR technology was used to knockdown FEN1 expression level of 293T cells, and tumor xenograft in nude mice was employed to determine the effects of FEN1 inhibitor and IR exposure on tumor growth in vivo. RESULTS: Our data revealed that FEN1 is overexpressed in HeLa cell and can be upregulated further by IR. We also demonstrated that FEN1 inhibitor enhances IR sensitivity of cervical cancer in vitro and in vivo. CONCLUSION: FEN1 inhibitor SC13 could sensitize radiotherapy of cervical cancer cell.

[Preparation and biocompatibility of nano polypyrrole/chitin composite membrane].

Objective: To prepare nano polypyrrole (PPy)/chitin composite membrane and observe their biocompatibility. Methods: The nano PPy was synthesized by microemulsion polymerization, blended with chitosan and then formed membranes. The membranes were then modified by acetylation to get the experimental membranes (nano PPy/chitin composite membranes, group A). The chitosan membranes (group B) and chitin ones (group C) modified by acetylation acted as control. Scanning electron microscopy and FT-IR spectra were used to identify the nano PPy and the membranes of each group. And the conductivity of membranes of each group was measured. Schwann cells were co-cultured in vitro with each group membranes to observe the biocompatibility by inverted microscope observing, living cell staining, cell counting, and immunofluorescence staining. The lysozyme solution was used to evaluate the degradation of the membranes in vitro. Results: The FT-IR spectra showed that the characteristic vibrational absorption peaks of C=C from nano PPy appeared at 1 543.4 cm -1 and 1 458.4 cm -1. Scanning electron microscopy observation revealed that the size of nano PPy particles was about 100-200 nm. The nano PPy particles were synthesized. It was successful to turn chitosan to chitin by the acetylation, which was investigated by FT-IR analysis of membranes in groups A and C. The characteristic peaks of the amide Ⅱ band around 1 562 cm -1 appeared after acetylated modification. Conductivity test showed that the conductivity of membranes in group A was about (1.259 2±0.005 7)×10 -3 S/cm, while the conductivity of the membranes in groups B and C was not detected. The nano PPy particles uniformly distributed on the surface of membranes in group A were observed by scanning electron microscope; the membranes in control groups were smooth. As a result, the nano PPy/chitin composite membranes with electrical conductivity were obtained. The cultured Schwann cells were found to survive with good function by fluorescein diacetate live cell staining, soluble protein-100 immunofluorescence staining, and inverted microscope observing. The cell counting showed that the proliferation of Schwann cells after 2 days and 4 days of group A was more than that of the two control groups, and the differences were significant ( P<0.05). It indicated that the nano PPy/chitin composite membranes had better ability of adhesion and proliferation than those of chitosan and chitin membranes. The degradation of membranes in vitro showed that the degradation rates of membranes in groups A and C were significantly higher than those in group B at all time points ( P<0.05). In a word, the degradation performance of the membranes modified by acetylation was better than that of chitosan membranes under the same condition. Conclusion: The nano PPy and chitosan can be blended and modified by acetylation successfully. Nano PPy/chitin composite membranes had electrical conductivity, degradability, and good biocompatibility in vitro.

[Effect of short-term low-frequency electrical stimulation on nerve regeneration of delayed nerve defect during operation].

Objective: To explore the effect of short-term low-frequency electrical stimulation (SLES) during operation on nerve regeneration in delayed peripheral nerve injury with long gap. Methods: Thirty female adult Sprague Dawley rats, weighing 160-180 g, were used to prepare 13-mm defect model by trimming the nerve stumps. Then all rats were randomly divided into 2 groups, 15 rats in each group. After nerve defect was bridged by the contralateral normal sciatic nerve, SLES was applied in the experimental group, but was not in the control group. The spinal cords and dorsal root ganglions (DRGs) were harvested to carry out immunofluorescence histochemistry double staining for growth-associated proteins 43 (GAP-43) and brain-derived neurotrophic factor (BDNF) at 1, 2, and 7 days after repair. Fluorogold (FG) retrograde tracing was performed at 3 months after repair. The mid-portion regenerated segments were harvested to perform Meyer's trichrome staining, immunofluorescence double staining for neurofilament (NF) and soluble protein 100 (S-100) on the transversely or longitudinal sections at 3 months after repair. The segment of the distal sciatic nerve trunk was harvested for electron microscopy and morphometric analyses to measure the diameter of the myelinated axons, thickness of myelin sheaths, the G ratio, and the density of the myelinated nerve fibers. The gastrocnemius muscles of the operated sides were harvested to measure the relative wet weight ratios. Karnovsky-Root cholinesterase staining of the motor endplate was carried out. Results: In the experimental group, the expressions of GAP-43 and BDNF were higher than those in the control group at 1 and 2 days after repair. The number of labeled neurons in the anterior horn of gray matter in the spinal cord and DRGs at the operated side from the experimental group was more than that from the control group. Meyer's trichrome staining, immunofluorescence double staining, and the electron microscopy observation showed that the regenerated nerves were observed to develop better in the experimental group than the control group. The relative wet weight ratio of experimental group was significantly higher than that of the control group ( t=4.633, P=0.000). The size and the shape of the motor endplates in the experimental group were better than those in the control group. Conclusion: SLES can promote the regeneration ability of the short-term (1 month) delayed nerve injury with long gap to a certain extent.

Morphological Proof of nerve regeneration after long-term defects of rat sciatic nerves.

Unsatisfactory efficacy of clinical cure for long-term delayed injuries and other disadvantages such as the low regeneration rate and speed of axotomized neurons and the questionable reinnervation ability of atrophic target organ lead to inaction to the long-term delayed injuries. Here we attempted to use autologous nerve to bridge a long-term delayed 10-mm defect in SD rats based on some previous positive messages of basic and clinical research. In this study, for experimental groups, the rat sciatic nerve had been transected leaving a 10-mm defect, which was maintained for 3 or 6 months before implantation with the autologous graft. The non-grafted animals served as negative control. Measuring with electrophysiological and histological techniques, we find: (1) A number of long-term axotomized neurons survived and sustained certain degree of axonal regenerative capacity; (2) A few denervated Schwann cells survived and retained their ability to provide trophic support and myelinate axons in at least 6 months; (3) the chronically denervated muscle can partially be reinnervated by regenerated axons. But the quantity and the quality of the regenerated nerve fibers and the reinnervated muscle fibers were all poor. Thus these observations provide new positive morphological proof of nerve regeneration after long-term defects and further studies will be needed to increase the survival rate and the regenerative speed of long-term chronic axotomized neurons, enhance the support provided by denervated distal stumps and protect the target muscle.

Maturation induction of human peripheral blood mononuclear cell-derived dendritic cells.

The aim of the present study was to explore an optimal method for maturation induction of dendritic cells (DCs). Human monocyte-derived DCs were induced in the presence of GM-CSF and IL-4. On Day 6, the maturation of DCs was induced with CD40L, LPS, TNF-α and cocktail of cytokines (TNF-α, IL-6, IL-1ß and PGE2), respectively, for 24 h. Then, DCs were harvested and subjected to flow cytometry (FCM) for the detection of CD80, CD83, CD86 and HLA-DR. FITC-dextran endocytic activity was measured by FCM, IL-12 production by ELISA and T lymphocyte proliferation following DC stimulation by MTT assay. CD40L, LPS, TNF-α and a cocktail of cytokines induced DC maturation. Induction with the cocktail of cytokines was the most efficient, and the expression rate of CD83 was 66.91% (P<0.05). The FITC-dextran endocytic activity of mature DCs was significantly reduced, and IL-12 production was dramatically increased in mature DCs, particularly in those following induction using the cocktail of cytokines. The mature DCs had potent ability to stimulate the proliferation of lymphocytes. The cocktail of cytokines is a favorable strategy for the induction of DC maturation.