Mechanism of acacetin regulating hepatic stellate cell apoptosis based on network pharmacology and experimental verification.

Background: Hepatic fibrosis is caused by various liver diseases and eventually develops into liver cancer. There is no specific drug approved for the treatment of hepatic fibrosis in the world. Acacetin (AC), a natural flavonoid, is widely present in nature in various plants, such as black locust, Damiana, Silver birch. It has been reported that acacetin can inhibit the proliferation of cancer cells and induce apoptosis. Purpose: In this study, we investigated the effect of acacetin on hepatic stellate cell apoptosis, thereby improving hepatic fibrosis, and combined experimental validation and molecular docking to reveal the underlying mechanism. Result: First, we discovered that acacetin inhibited hepatic stellate cell proliferation as well as the expression of fibrosis-related proteins α-smooth muscle actin (α-SMA) and collagen type I 1 gene (COL1A1) in LX2 cells. Acacetin was then found to promote apoptosis of hepatic stellate cells through the caspase cascade pathway. Network pharmacology screening showed that TP53, CASP3, CASP8, BCL2, PARP1, and BAX were the most important targets related to apoptosis in the PPI network. GO and KEGG analyses of these six important targets were performed, and the top 10 enriched biological processes and related signaling pathways were revealed. Further network pharmacology analysis proved that apoptosis was involved in the biological process of acacetin's action against hepatic stellate cells. Finally, molecular docking revealed that acacetin binds to the active sites of six apoptotic targets. In vitro experiments further confirmed that acacetin could promote the apoptosis of LX2 cells by inducing the activation of P53, thereby improving hepatic fibrosis. Conclusion: acacetin induces P53 activation and promotes apoptosis of hepatic stellate cells thereby ameliorating hepatic fibrosis.

Self-adaptive pyroptosis-responsive nanoliposomes block pyroptosis in autoimmune inflammatory diseases.

Nanoliposomes have a broad range of applications in the treatment of autoimmune inflammatory diseases because of their ability to considerably enhance drug transport. For their clinical application, nanoliposomes must be able to realize on-demand release of drugs at disease sites to maximize drug-delivery efficacy and minimize side effects. Therefore, responsive drug-release strategies for inflammation treatment have been explored; however, no specific design has been realized for a responsive drug-delivery system based on pyroptosis-related inflammation. Herein, we report a pioneering strategy for self-adaptive pyroptosis-responsive liposomes (R8-cardiolipin-containing nanoliposomes encapsulating dimethyl fumarate, RC-NL@DMF) that precisely release encapsulated anti-pyroptotic drugs into pyroptotic cells. The activated key pyroptotic protein, the N-terminal domain of gasdermin E, selectively integrates with the cardiolipin of liposomes, thus forming pores for controlled drug release, pyroptosis, and inflammation inhibition. Therefore, RC-NL@DMF exhibited effective therapeutic efficacies to alleviate autoimmune inflammatory damages in zymosan-induced arthritis mice and dextran sulfate sodium-induced inflammatory bowel disease mice. Our novel approach holds great promise for self-adaptive pyroptosis-responsive on-demand drug delivery, suppressing pyroptosis and treating autoimmune inflammatory diseases.

Single-Cell Microgel Encapsulation Improves the Therapeutic Efficacy of Mesenchymal Stem Cells in Treating Intervertebral Disc Degeneration via Inhibiting Pyroptosis.

While mesenchymal stem cell (MSC) shows great potentials in treating intervertebral disc degeneration, most MSC die soon after intradiscal transplantation, resulting in inferior therapeutic efficacy. Currently, bulk hydrogels are the common solution to improve MSC survival in tissues, although hydrogel encapsulation impairs MSC migration and disrupts extracellular microenvironment. Cell hydrogel encapsulation has been proposed to overcome the limitation of traditional bulk hydrogels, yet this technique has not been used in treating disc degeneration. Using a layer-by-layer self-assembly technique, we fabricated alginate and gelatin microgel to encapsulate individual MSC for treating disc degeneration. The small size of microgel allowed intradiscal injection of coated MSC. We demonstrated that pyroptosis was involved in MSC death under oxidative stress stimulation, and microgel coating suppressed pyroptosis activation by maintaining mitochondria homeostasis. Microgel coating protected MSC in the harsh disc microenvironment, while retaining vital cellular functions such as migration, proliferation, and differentiation. In a rat model of disc degeneration, coated MSC exhibits prolonged retention in the disc and better efficacy of attenuating disc degeneration, as compared with bare MSC treatment alone. Further, microgel-coated MSC exhibited improved therapeutic effects in treating disc degeneration via suppressing the activation of pyroptosis in the disc. For the first time, microgel-encapsulated MSC was used to treat disc degeneration and obtain encouraging outcomes. The developed biocompatible single-cell hydrogel is an effective strategy to protect MSC and maintain cellular functions and may be an efficacious approach to improving the efficacy of MSC therapy in treating disc degeneration. The objective of this study is to improve the efficacy of cell therapy for treating disc degeneration using single-cell hydrogel encapsulation and further to understand related cytoprotective mechanisms.

Synergistically targeting synovium STING pathway for rheumatoid arthritis treatment.

Rheumatoid arthritis (RA) is a common autoimmune disease leading to pain, disability, and even death. Although studies have revealed that aberrant activation of STING was implicated in various autoimmune diseases, the role of STING in RA remains unclear. In the current study, we demonstrated that STING activation was pivotal in RA pathogenesis. As the accumulation of dsDNA, a specific stimulus for STING, is a feature of RA, we developed a spherical polyethyleneimine-coated mesoporous polydopamine nanoparticles loaded with STING antagonist C-176 (PEI-PDA@C-176 NPs) for treating RA. The fabricated NPs with biocompatibility had high DNA adsorption ability and could effectively inhibit the STING pathway and inflammation in macrophages. Intra-articular administration of PEI-PDA@C-176 NPs could effectively reduce joint damage in mice models of dsDNA-induced arthritis and collagen-induced arthritis by inhibiting STING pathway. We concluded that materials with synergistic effects of STING inhibition might be an efficacious strategy to treat RA.

HECTD1-Mediated Ubiquitination and Degradation of Rubicon Regulates Autophagy and Osteoarthritis Pathogenesis.

OBJECTIVE: Osteoarthritis (OA) is one of the most common degenerative joint diseases and is associated with autophagy suppression. However, the molecular mechanism of autophagy regulation in the context of OA is not fully understood. In this study, we sought to determine the role that HECTD1 plays in the pathogenesis of OA. METHODS: We used RNA sequencing analysis to explore the differential expression of E3 ubiquitin ligase genes in healthy human cartilage and human cartilage affected by OA. Using surgery- and aging-induced OA mouse models, we comprehensively analyzed the function of the screened gene Hectd1 in the development of OA; furthermore, we dissected the mechanism by which HECTD1 regulates autophagy and OA progression using a combination of molecular biologic, cell biologic, and biochemical approaches. RESULTS: HECTD1 was significantly down-regulated in human OA cartilage samples compared to healthy cartilage samples. Overexpression of HECTD1 in mouse joints alleviated OA pathogenesis, whereas conditional depletion of Hectd1 in cartilage samples aggravated surgery- and aging-induced OA pathogenesis. Mechanistically, HECTD1 bound to Rubicon and ubiquitinated Rubicon at lysine residue 534, which targets Rubicon for proteasomal degradation. More importantly, HECTD1-mediated Rubicon degradation regulated chondrocyte autophagy, leading to mitigation of stress-induced chondrocyte death and the subsequent progression of OA. CONCLUSION: HECTD1 plays a crucial role in the pathogenesis of OA, in that HECTD1 regulates chondrocyte autophagy by ubiquitinating and targeting Rubicon for proteasomal degradation.

Scavenging of reactive oxygen species can adjust the differentiation of tendon stem cells and progenitor cells and prevent ectopic calcification in tendinopathy.

Tendinopathy is a common disorder that leads to pain and impaired quality of life. Recent studies revealed that osteogenic differentiation of tendon stem/progenitor cells (TSPCs) played an important role in the pathogenesis of tendon calcification and tendinopathy. In this study, we found that the growth hormone-releasing hormone agonist (GA) can prevent matrix degradation and osteogenic differentiation in TSPCs. As oxidative stress is a key factor in the osteogenic differentiation of TSPCs, we used bovine serum albumin/heparin nanoparticles (BHNPs), which have biocompatibility and drug loading capacity, to scavenge reactive oxygen species (ROS) and achieve sustained release of GA at the site of inflammation. The newly developed BHNPs@GA had a synergetic effect on reducing ROS production in TSPCs. In addition, BHNPs@GA effectively inhibited tendon calcification and promoted collagen formation in a rat model of tendinopathy. Focusing on the ROS underlying the differentiation and dedifferentiation of TSPCs, this work demonstrated that sustained release of GA targeting ROS and ectopic ossification is a practical therapeutic strategy for treating tendinopathy. STATEMENT OF SIGNIFICANCE: Osteogenic differentiation of tendon stem/progenitor cells (TSPCs) plays an important role in the pathogenesis of ectopic calcification in tendinopathy. In this study, we found that growth hormone-releasing hormone agonist (GA) can reduce reactive oxygen species (ROS) production and adjust TSPCs differentiation. Bovine serum albumin/heparin nanoparticles (BHNPs) were developed to encapsulate GA and achieve sustained release of GA at the site of inflammation. The developed compound, BHNPs@GA, with a synergistic effect of inhibiting ROS and thus, can effectively adjust TSPCs differentiation, inhibit tendon calcification, and promote collagen formation in tendinopathy. This study highlighted the role of ROS underlying the differentiation and dedifferentiation of TSPCs in tendinopathy, and findings may help to identify new therapeutic targets and develop novel strategy for treating tendinopathy.

Interference Effect of Experimental Parameters on the Mercury Removal Mechanism of Biomass Char under an Oxy-Fuel Atmosphere.

In this paper, the effect of temperature, adsorption bed height, and initial mercury concentration under oxy-fuel combustion on mercury adsorption by 1% NH4Cl-modified biomass char was studied. Modification enriched the pore structure of biomass char and increased the number of surface functional groups. Higher temperature would lead to the destruction of van der Waals and reduce the adsorption efficiency, while the change of adsorption bed height had no obvious effect. Adsorption thermodynamics shows that the mercury removal process is a spontaneous exothermic process. The increase of initial mercury concentration would increase the driving force of mercury diffusion to the surface and improve the adsorption capacity. Meanwhile, three kinetic models including the intraparticle diffusion model, pseudo-first-order model, and pseudo-second-order model were applied to explore the internal mechanism of mercury adsorption by biomass char. The results showed that the pseudo-first-order model and pseudo-second-order model could accurately describe the adsorption process, which meant that the progress of external mass transfer played an important role in the adsorption of mercury while chemical adsorption should not be ignored. The intraparticle diffusion model indicated that internal diffusion was not the only step to control the entire adsorption process and did not have an inhibition on mercury removal. Higher initial mercury concentration would promote the external mass transfer progress and chemical adsorption progress. In addition, higher temperature inhibited the external mass transfer, which was not conducive to the adsorption of mercury.

New MR-based measures for the evaluation of age-related lumbar paraspinal muscle degeneration.

PURPOSE: Although signal intensity on T2W axial images is sensitive in detection of fatty infiltration to assess paraspinal muscle degeneration, it is affected by inhomogeneities of magnetic fields and individual variabilities. The purpose of this study was to propose reference adjusted signal measures on T2W axial images and determine their capacities in reflecting age-related lumbar paraspinal muscle degeneration. METHODS: Lumbar MR images of 421 population-based subjects (177 men and 244 women, mean age 53.1 years, range 19.8-87.9 years) were studied. A custom software Spine Explore (Tulong 2.0) was used to automatically obtain paraspinal measurements of multifidus, erector spinae and psoas major. FCSA/TCSA was defined as functional cross-sectional area relative to total cross-sectional area of paraspinal muscle. Two new signal measures were canal-adjusted and cerebrospinal fluid (CSF)-adjusted signal, defined as the ratio between mean signal measurements and the mean signal of the canal and CSF. RESULTS: The raw signal measurements of the paraspinal muscles were weakly correlated to age (r = 0.28-0.39, P < 0.001). When the signal of canal (r = 0.43-0.59, P < 0.001) or CSF (r = 0.45-0.61, P < 0.001) was used as reference, the correlations substantially increased. Signal measurements of three paraspinal muscles, adjusted or not, were strongly associated with Goutallier score (ρ = 0.60-0.65, P < 0.001) and FCSA/TCSA (r = -0.64 to -0.82, P < 0.001). Greater Goutallier score was associated with greater age (r = 0.38-0.60, P < 0.001), while Lumbar indentation value (LIV) not. CONCLUSION: On routine T2W axial MR images the adjusted signal measurements using an internal reference of CSF or canal can better reflect age-related degenerative changes in the paraspinal muscles.

A thermosensitive, reactive oxygen species-responsive, MR409-encapsulated hydrogel ameliorates disc degeneration in rats by inhibiting the secretory autophagy pathway.

Lumbar disc degeneration is a common cause of chronic low back pain and an important contributor to various degenerative lumbar spinal disorders. However, currently there is currently no effective therapeutic strategy for treating disc degeneration. The pro-inflammatory cytokine interleukin-1ß (IL-1ß) mediates disc degeneration by inducing apoptotic death of nucleus pulposus (NP) cells and degradation of the NP extracellular matrix. Here, we confirmed that extracellular secretion of IL-1ß via secretory autophagy contributes to disc degeneration, and demonstrate that a thermosensitive reactive oxygen species (ROS)-responsive hydrogel loaded with a synthetic growth hormone-releasing hormone analog (MR409) can protect against needle puncture-induced disc degeneration in rats. Methods: The expression levels of proteins related to secretory autophagy such as tripartite motif-containing 16 (TRIM16) and microtubule-associated protein light chain 3B (LC3B) were examined in human and rat disc tissues by histology and immunofluorescence. The effects of TRIM16 expression level on IL-1ß secretion were examined in THP-1 cells transfected with TRIM16 plasmid or siRNA using ELISA, immunofluorescence, and immunoblotting. The in vitro effects of MR409 on IL-1ß were examined in THP-1 cells and primary rat NP cells using ELISA, immunofluorescence, immunoblotting, and qRT-PCR. Further, MR409 was subcutaneously administered to aged mice to test its efficacy against disc degeneration using immunofluorescence, X-ray, micro-CT, and histology. To achieve controllable MR409 release for intradiscal use, MR409 was encapsulated in an injectable ROS-responsive thermosensitive hydrogel. Viscosity, rheological properties, release profile, and biocompatibility were evaluated. Thereafter, therapeutic efficacy was assessed in a needle puncture-induced rat model of disc degeneration at 8 and 12 weeks post-operation using X-ray, magnetic resonance (MR) imaging, histological analysis, and immunofluorescence. Results: Secretory autophagy-related proteins TRIM16 and LC3B were robustly upregulated in degenerated discs of both human and rat. Moreover, while upregulation of TRIM16 facilitated, and knockdown of TRIM16 suppressed, secretory autophagy-mediated IL-1ß secretion from THP-1 cells under oxidative stress, MR409 inhibited ROS-induced secretory autophagy and IL-1ß secretion by THP-1 cells as well as IL-1ß-induced pro-inflammatory and pro-catabolic effects in rat NP cells. Daily subcutaneous injection of MR409 inhibited secretory autophagy and ameliorated age-related disc degeneration in mice. The newly developed ROS-responsive MR409-encapsulated hydrogel provided a reliable delivery system for controlled MR409 release, and intradiscal application effectively suppressed secretory autophagy and needle puncture-induced disc degeneration in rats. Conclusion: Secretory autophagy and associated IL-1ß secretion contribute to the pathogenesis of disc degeneration, and MR409 can effectively inhibit this pathway. The ROS-responsive thermosensitive hydrogel encapsulated with MR409 is a potentially efficacious treatment for disc degeneration.

A Deep-Learning-Based, Fully Automated Program to Segment and Quantify Major Spinal Components on Axial Lumbar Spine Magnetic Resonance Images.

OBJECTIVE: The paraspinal muscles have been extensively studied on axial lumbar magnetic resonance imaging (MRI) for better understanding of back pain; however, the acquisition of measurements mainly relies on manual segmentation, which is time consuming. The study objective was to develop and validate a deep-learning-based program for automated acquisition of quantitative measurements for major lumbar spine components on axial lumbar MRIs, the paraspinal muscles in particular. METHODS: This study used a cross-sectional observational design. From the Hangzhou Lumbar Spine Study, T2-weighted axial MRIs at the L4-5 disk level of 120 participants (aged 54.8 years [SD = 15.0]) were selected to develop the deep-learning-based program Spine Explorer (Tulong). Another 30 axial lumbar MRIs were automatically measured by Spine Explorer and then manually measured using ImageJ to acquire quantitative size and compositional measurements for bilateral multifidus, erector spinae, and psoas muscles; the disk; and the spinal canal. Intersection-over-union and Dice score were used to evaluate the performance of automated segmentation. Intraclass coefficients and Bland-Altman plots were used to examine intersoftware agreements for various measurements. RESULTS: After training, Spine Explorer (Tulong) measures an axial lumbar MRI in 1 second. The intersections-over-union were 83.3% to 88.4% for the paraspinal muscles and 92.2% and 82.1% for the disk and spinal canal, respectively. For various size and compositional measurements of paraspinal muscles, Spine Explorer (Tulong) was in good agreement with ImageJ (intraclass coefficient = 0.85 to approximately 0.99). CONCLUSION: Spine Explorer (Tulong) is automated, efficient, and reliable in acquiring quantitative measurements for the paraspinal muscles, the disk, and the canal, and various size and compositional measurements were simultaneously obtained for the lumbar paraspinal muscles. IMPACT: Such an automated program might encourage further epidemiological studies of the lumbar paraspinal muscle degeneration and enhance paraspinal muscle assessment in clinical practice.

Spine Explorer: a deep learning based fully automated program for efficient and reliable quantifications of the vertebrae and discs on sagittal lumbar spine MR images.

BACKGROUND CONTEXT: Although quantitative measurements improve the assessment of disc degeneration, acquirement of quantitative measurements relies on manual segmentation on lumbar magnetic resonance images (MRIs), which may introduce subjective bias. To date, only a few semiautomatic systems have been developed to quantify important components on MRIs. PURPOSE: To develop a deep learning based program (Spine Explorer) for automated segmentation and quantification of the vertebrae and intervertebral discs on lumbar spine MRIs. STUDY DESIGN: Cross-sectional study. PATIENT SAMPLE: The study was extended on the Hangzhou Lumbar Spine Study, a population-based study of mainland Chinese with focuses on lumbar degenerative changes. From this population-based database, 50 sets lumbar MRIs were randomly selected as training dataset, and another 50 as test dataset. OUTCOME MEASURES: Regions of vertebrae and discs were manually segmented on T2W sagittal MRIs to train a convolutional neural network for automated segmentation. Intersection-over-union was calculated to evaluate segmentation performance. Computational definitions were proposed to acquire quantitative morphometric and signal measurements for lumbar vertebrae and discs. MRIs in the test dataset were automatically measured with Spine Explorer and manually with ImageJ. METHODS: Intraclass correlation coefficient (ICC) were calculated to examine inter-software agreements. Correlations between disc measurements and Pfirrmann score as well as age were examined to assess measurement validity. RESULTS: The trained Spine Explorer automatically segments and measures a lumbar MRI in half a second, with mean Intersection-over-union of 94.7% and 92.6% for the vertebra and disc, respectively. For both vertebra and disc measurements acquired with Spine Explorer and ImageJ, the agreements were excellent (ICC=0.81~1.00). Disc measurements significantly correlated to Pfirrmann score, and greater age was associated with greater anterior disc bulging area (r=0.35~0.44) and fewer signal measurements (r=-0.62~-0.77) as automatically acquired with Spine Explorer. CONCLUSIONS: Spine Explorer is an efficient, accurate, and reliable tool to acquire comprehensive quantitative measurements for lumbar vertebra and disc. Implication of such deep learning based program can facilitate clinical studies of the lumbar spine.

The Landscape of Long Non-Coding RNA Dysregulation and Clinical Relevance in Muscle Invasive Bladder Urothelial Carcinoma.

Bladder cancer is one of the most common cancers in the United States, but few advancements in treatment options have occurred in the past few decades. This study aims to identify the most clinically relevant long non-coding RNAs (lncRNAs) to serve as potential biomarkers and treatment targets for muscle invasive bladder cancer (MIBC). Using RNA-sequencing data from 406 patients in The Cancer Genome Atlas (TCGA) database, we identified differentially expressed lncRNAs in MIBC vs. normal tissues. We then associated lncRNA expression with patient survival, clinical variables, oncogenic signatures, cancer- and immune-associated pathways, and genomic alterations. We identified a panel of 20 key lncRNAs that were most implicated in MIBC prognosis after differential expression analysis and prognostic correlations. Almost all lncRNAs we identified are correlated significantly with oncogenic processes. In conclusion, we discovered previously undescribed lncRNAs strongly implicated in the MIBC disease course that may be leveraged for diagnostic and treatment purposes in the future. Functional analysis of these lncRNAs may also reveal distinct mechanisms of bladder cancer carcinogenesis.

Aerobic α,ß-C(sp3)-H Bond Difunctionalization and C-N Bond Cleavage of Triethylamine: Difunctional Ammonium Iodide Enabling the Regioselective Synthesis of 4-Arylpyrimido[1,2-b]indazoles.

A novel method for the regioselective synthesis of 4-arylpyrimido[1,2-b]indazoles has been developed via the dual C(sp3)-H bond functionalization and C-N bond cleavage of triethylamine. The elusive acyclic enamine intermediates are effectively in situ generated and captured by aromatic aldehydes to form a wide array of tricyclic products from 3-aminoindazoles under the NH4I-mediated aerobic oxidative conditions. This reaction features easily available feedstock, green and economic conditions, and valuable products.

A new method using machine learning for automated image analysis applied to chip-based digital assays.

Chip-based digital assays such as the digital polymerase chain reaction (digital PCR), digital loop-mediated amplification (digital LAMP), digital enzyme-linked immunosorbent assay (digital ELISA) and digital proximity ligation assay (digital PLA) need high-throughput quantification of the captured fluorescence image data. However, traditional methods that are mainly based on image segmentation using either a fixed threshold or an automated hard threshold failed to extract valid signals over a broad range of image characteristics. In this study, we introduce a new method for automated image analysis to extract signals applied to chip-based digital assays. This approach precisely locates each micro-compartment based on the structure design of the chip, thereby eliminating the interference of non-signal noise in the image. Utilizing the principle that the human eyes can distinguish between the positive micro-compartments and the negative micro-compartments, we take the parameters of each micro-compartment together with its surrounding micro-compartments as the training dataset of the Random Forest classifier to classify the micro-compartments and extract valid signals, thus solving the problem caused by the differences among images. Furthermore, we adopted the iteration methodology that adds the output of a model's prediction to the input of the next model's training dataset, until the output of a model's prediction reaches the accuracy we expected, which improves the work efficiency during data training greatly. We demonstrate the method on the dPCR dataset and it performs well without any manual adjustment of settings. The results show that our proposed method can recognize the positive signals from the fluorescence images with an accuracy of 97.78%. With minor modification, bio-instrument companies or researchers can integrate this method into their digital assay devices' software conveniently.