Advances in the understanding of circRNAs that influence viral replication in host cells.

Circular RNAs (circRNAs) are non-coding RNAs discovered in recent years, which are produced by back-splicing involving the 3' and 5' ends of RNA molecules. There is increasing evidence that circRNAs have important roles in cancer, neurological diseases, cardiovascular and cerebrovascular diseases, and other diseases. In addition, host circRNAs and virus-encoded circRNAs participate in the body's immune response, with antiviral roles. This review summarizes the mechanisms by which host and viral circRNAs interact during the host immune response. Comprehensive investigations have revealed that host circRNAs function as miRNA sponges in a particular manner, primarily by inhibiting viral replication. Viral circRNAs have more diverse functions, which generally involve promoting viral replication. In addition, in contrast to circRNAs from RNA viruses, circRNAs from DNA viruses can influence host cell migration, proliferation, and apoptosis, along with their effects on viral replication. In summary, circRNAs have potential as diagnostic and therapeutic targets, offering a foundation for the diagnosis and treatment of viral diseases.

Spatial-temporal distribution characteristics of pollutants of heavy-duty diesel vehicles in urban road networks: a case study of Kunming City.

With the continuous promotion of urbanization in China, the economic level of small and medium-sized cities has been further improved. The transportation industry is crucial in promoting urban-rural integration and construction. Still, motor vehicle emissions also bring air pollution problems to cities, with heavy-duty diesel vehicle emissions severely impacting the urban environment. This study used a bottom-up approach to analyze the spatial emission characteristics of heavy-duty diesel vehicles under different road types in Kunming, a typical medium-sized city in China. A high-resolution emission inventory (1 km × 1 km) of heavy-duty diesel vehicles was developed using the vehicle emission inventory model (VEIN) and ArcGIS, and the vehicle emission standards were determined by the Weibull survival rate curve. The VEIN emission model was optimized using a velocity correction curve. The results showed that heavy-duty vehicles had a more significant impact on the emissions during the morning and evening peak hours, with low emission levels during the day and high emission levels at night and early morning. The total daily emissions of carbon monoxide (CO), hydrocarbons (HC), nitrogen oxides (NOx), and particulate matter (PM10 and PM2.5) from heavy-duty diesel vehicles in Motorway, Trunk, Primary, Secondary, and Tertiary were 14.44 tons, 5.26 tons, 4.78 tons, 7.02 tons, and 3.83 tons, respectively. China III heavy-duty diesel vehicles mainly contributed to CO, HC, NOx, and PM emissions. This study can be used as an essential reference for controlling the exhaust emissions of HDDVs in Kunming.

Transcriptional Analysis of lncRNA and Target Genes Induced by Influenza A Virus Infection in MDCK Cells.

BACKGROUND: The MDCK cell line is the primary cell line used for influenza vaccine production. Using genetic engineering technology to change the expression and activity of genes that regulate virus proliferation to obtain high-yield vaccine cell lines has attracted increasing attention. A comprehensive understanding of the key genes, targets, and molecular mechanisms of viral regulation in cells is critical to achieving this goal, yet the post-transcriptional regulation mechanism involved in virus proliferation-particularly the effect of lncRNA on influenza virus proliferation-is still poorly understood. Therefore, this study used high-throughput RNA-seq technology to identify H1N1 infection-induced lncRNA and mRNA expression changes in MDCK cells and explore the regulatory relationship between these crucial lncRNAs and their target genes. RESULTS: In response to H1N1 infection in MDCK cells 16 h post-infection (hpi) relative to uninfected controls, we used multiple gene function annotation databases and initially identified 31,501 significantly differentially expressed (DE) genes and 39,920 DE lncRNAs (|log2FC| > 1, p < 0.05). Among these, 102 lncRNAs and 577 mRNAs exhibited predicted correlations with viral response mechanisms. Based on the magnitude of significant expression differences, related research, and RT-qPCR expression validation at the transcriptional level, we further focused on 18 DE mRNAs and 32 DE lncRNAs. Among these, the differential expression of the genes RSAD2, CLDN1, HCLS1, and IFIT5 in response to influenza virus infection was further verified at the protein level using Western blot technology, which showed results consistent with the RNA-seq and RT-qPCR findings. We then developed a potential molecular regulatory network between these four genes and their six predicted lncRNAs. CONCLUSIONS: The results of this study will contribute to a more comprehensive understanding of the molecular mechanism of host cell non-coding RNA-mediated regulation of influenza virus replication. These results may also identify methods for screening target genes in the development of genetically engineered cell lines capable of high-yield artificial vaccine production.

CDC20 is a potential target gene to inhibit the tumorigenesis of MDCK cells.

MDCK is currently the main cell line used for influenza vaccine production in culture. Previous studies have reported that MDCK cells possess tumorigenic ability in nude mice. Although complete cell lysis can be ensured during vaccine production, host cell DNA released after cell lysis may still pose a risk for tumorigenesis. Greater caution is needed in the production of human vaccines; therefore, the use of gene editing to establish cells incapable of forming tumors may significantly improve the safety of influenza vaccines. Knowledge regarding the genes and molecular mechanisms that affect the tumorigenic ability of MDCK cells is crucial; however, our understanding remains superficial. Through monoclonal cell screening, we previously obtained a cell line, CL23, that possesses significantly reduced cell proliferation, migration, and invasion abilities, and tumor-bearing experiments in nude mice showed the absence of tumorigenic cells. With a view to exploring tumorigenesis-related genes in MDCK cells, DIA proteomics was used to compare the differences in protein expression between wild-type (M60) and non-tumorigenic (CL23) cells. Differentially expressed proteins were verified at the mRNA level by RT-qPCR, and a number of genes involved in cell tumorigenesis were preliminarily screened. Immunoblotting further confirmed that related protein expression was significantly reduced in non-tumorigenic cells. Inhibition of CDC20 expression by RNAi significantly reduced the proliferation and migration of MDCK cells and increased the proliferation of the influenza virus; therefore, CDC20 was preliminarily determined to be an effective target gene for the inhibition of cell tumorigenicity. These results contribute to a more comprehensive understanding of the mechanism underlying cell tumorigenesis and provide a basis for the establishment of target gene screening in genetically engineered non-tumorigenic MDCK cell lines.

Suspended cell lines for inactivated virus vaccine production.

INTRODUCTION: Inactivated virus vaccines are the most widely used tool to prevent disease. To meet vaccine production demands, increasing attention has been placed on identifying methods to improve vaccine production efficiency. The use of suspended cells can greatly increase vaccine production. Suspension acclimation is a traditional method to convert adherent cells to suspension strains. Furthermore, as genetic engineering technology has developed, increasing attention has focused on the development of suspension cell lines using targeted genetic engineering techniques. AREAS COVERED: This review systematically summarizes and analyzes the development and research progress of various inactivated viral vaccine production suspension cell lines and provides protocols and candidate target genes for the engineered establishment of additional suspension cell lines for vaccine production. EXPERT OPINION: The use of suspended cells can significantly improve the production efficiency of inactivated virus vaccines and other biological products. Presently, cell suspension culture is the key component to improve many vaccine production processes.

TGM2 inhibits the proliferation, migration and tumorigenesis of MDCK cells.

Madin-Darby canine kidney (MDCK) cells are one of the main cell lines used for influenza vaccine production due to their high virus yield and low mutation resistance. Due to their high tumorigenicity, the safety of vaccines produced from these cells is controversial. TGM2 is a multifunctional protein that plays an important role in the adhesion and migration of cells and is associated with tumor formation. We found that the expression level of TGM2 was significantly up-regulated in low tumorigenic MDCK cells. We first analyzed TGM2-overexpressed and knockout MDCK cells in vitro. Scratch-wound assay and Transwell chamber experiments showed that TGM2 overexpression significantly inhibited the migration and invasion of MDCK cells and significantly reduced their proliferation. TGM2 knockout significantly enhanced cell migration, invasion, and proliferation. The tumorigenesis results in nude mice were consistent with those in vitro. TGM2 knockout significantly enhanced the tumorigenesis rate of MDCK cells in nude mice. We also investigated the effects of TGM2 gene expression on the replication of the H1N1 influenza A virus in MDCK cells. The results showed that TGM2 induced the negative regulation of H1N1 replication. These findings contribute to a comprehensive understanding of the tumor regulation mechanism and biological functions of TGM2.

An Improved Deep Reinforcement Learning Method for Dispatch Optimization Strategy of Modern Power Systems.

As a promising information theory, reinforcement learning has gained much attention. This paper researches a wind-storage cooperative decision-making strategy based on dueling double deep Q-network (D3QN). Firstly, a new wind-storage cooperative model is proposed. Besides wind farms, energy storage systems, and external power grids, demand response loads are also considered, including residential price response loads and thermostatically controlled loads (TCLs). Then, a novel wind-storage cooperative decision-making mechanism is proposed, which combines the direct control of TCLs with the indirect control of residential price response loads. In addition, a kind of deep reinforcement learning algorithm called D3QN is utilized to solve the wind-storage cooperative decision-making problem. Finally, the numerical results verify the effectiveness of D3QN for optimizing the decision-making strategy of a wind-storage cooperation system.

Inhibition of SMAD3 effectively reduces ADAMTS-5 expression in the early stages of osteoarthritis.

OBJECTIVE: As one of the most important protein-degrading enzymes, ADAMTS-5 plays an important role in the regulation of cartilage homeostasis, while miRNA-140 is specifically expressed in cartilage, which can inhibit the expression of ADAMTS-5 and delay the progression of OA (osteoarthritis). SMAD3 is a key protein in the TGF-ß signaling pathway, inhibiting the expression of miRNA-140 at the transcriptional and post-transcriptional levels, and studies have confirmed the high expression of SMAD3 in knee cartilage degeneration, but whether SMAD3 can mediate the expression of miRNA-140 to regulate ADAMTS-5 remains unknown. METHODS: Sprague-Dawley (SD) rat chondrocytes were extracted in vitro and treated with a SMAD3 inhibitor (SIS3) and miRNA-140 mimics after IL-1 induction. The expression of ADAMTS-5 was detected at the protein and gene levels at 24 h, 48 h, and 72 h after treatment. The OA model of SD rats was created using the traditional Hulth method in vivo, with SIS3 and lentivirus packaged miRNA-140 mimics injected intra-articularly at 2 weeks, 6 weeks and 12 weeks after surgery. The expression of miRNA-140 and ADAMTS-5 in the knee cartilage tissue was observed at the protein and gene levels. Concurrently, knee joint specimens were fixed, decalcified, and embedded in paraffin prior to immunohistochemical, Safranin O/Fast Green staining, and HE staining analyses for ADAMTS-5 and SMAD3. RESULTS: In vitro, the expression of ADAMTS-5 protein and mRNA in the SIS3 group decreased to different degrees at each time point. Meanwhile, the expression of miRNA-140 in the SIS3 group was significantly increased, and the expression of ADAMTS-5 in the miRNA-140 mimics group was also significantly downregulated (P < 0.05). In vivo, it was found that ADAMTS-5 protein and gene were downregulated to varying degrees in the SIS3 and miRNA-140 mimic groups at three time points, with the most significant decrease at the early stage (2 weeks) (P < 0.05), and the expression of miRNA-140 in the SIS3 group was significantly upregulated, similar to the changes detected in vitro. Immunohistochemical results showed that the expression of ADAMTS-5 protein in the SIS3 and miRNA-140 groups was significantly downregulated compared to that in the blank group. The results of hematoxylin and eosin staining showed that in the early stage, there was no obvious change in cartilage structure in the SIS3 and miRNA-140 mock groups. The same was observed in the results of Safranin O/Fast Green staining; the number of chondrocytes was not significantly reduced, and the tide line was complete. CONCLUSION: The results of in vitro and in vivo experiments preliminarily showed that the inhibition of SMAD3 significantly reduced the expression of ADAMTS-5 in early OA cartilage, and this regulation might be accomplished indirectly through miRNA-140.

RSAD2 Is an Effective Target for High-Yield Vaccine Production in MDCK Cells.

Increasingly, attention has focused on improving vaccine production in cells using gene editing technology to specifically modify key virus regulation-related genes to promote virus replication. In this study, we used DIA proteomics analysis technology to compare protein expression differences between two groups of MDCK cells: uninfected and influenza A virus (IAV) H1N1-infected cells 16 h post infection (MOI = 0.01). Initially, 266 differentially expressed proteins were detected after infection, 157 of which were upregulated and 109 were downregulated. We screened these proteins to 23 genes related to antiviral innate immunity regulation based on functional annotation database analysis and verified the mRNA expression of these genes using qPCR. Combining our results with published literature, we focused on the proteins RSAD2, KCNN4, IDO1, and ISG20; we verified their expression using western blot, which was consistent with our proteomics results. Finally, we knocked down RSAD2 using lentiviral shRNA expression vectors and found that RSAD2 inhibition significantly increased IAV NP gene expression, effectively promoting influenza virus replication with no significant effect on cell proliferation. These results indicate that RSAD2 is potentially an effective target for establishing high-yield vaccine MDCK cell lines and will help to fully understand the interaction mechanism between host cells and influenza viruses.

[Transglutaminase 2 inhibits the proliferation of H1 subtype influenza virus in MDCK cells].

Transglutaminase 2 (TGM2) is a ubiquitous multifunctional protein, which is related to the adhesion of different cells and tumor formation. Previous studies found that TGM2 is involved in the interaction between host cells and viruses, but the effect of TGM2 on the proliferation of influenza virus in cells has not been reported. To explore the effect of TGM2 during H1N1 subtype influenza virus infection, a stable MDCK cell line with TGM2 overexpression and a knockout cell line were constructed. The mRNA and protein expression levels of NP and NS1 as well as the virus titer were measured at 48 hours after pot-infection with H1N1 subtype influenza virus. The results showed that overexpression of TGM2 effectively inhibited the expression of NP and NS1 genes of H1N1 subtype influenza virus, while knockout of TGM2 up-regulated the expression of the NP and NS1 genes, and the expression of the NP at protein level was consistent with that at mRNA level. Virus proliferation curve showed that the titer of H1N1 subtype influenza virus decreased significantly upon TGM2 overexpression. On the contrary, the virus titer in TGM2 knockout cells reached the peak at 48 h, which further proved that TGM2 was involved in the inhibition of H1N1 subtype influenza virus proliferation in MDCK cells. By analyzing the expression of genes downstream of influenza virus response signaling pathway, we found that TGM2 may inhibit the proliferation of H1N1 subtype influenza virus by promoting the activation of JAK-STAT molecular pathway and inhibiting RIG-1 signaling pathway. The above findings are of great significance for revealing the mechanism underlying the interactions between host cells and virus and establishing a genetically engineering cell line for high-yield influenza vaccine production of influenza virus.

Host Non-Coding RNA Regulates Influenza A Virus Replication.

Outbreaks of influenza, caused by the influenza A virus (IAV), occur almost every year in various regions worldwide, seriously endangering human health. Studies have shown that host non-coding RNA is an important regulator of host-virus interactions in the process of IAV infection. In this paper, we comprehensively analyzed the research progress on host non-coding RNAs with regard to the regulation of IAV replication. According to the regulation mode of host non-coding RNAs, the signal pathways involved, and the specific target genes, we found that a large number of host non-coding RNAs directly targeted the PB1 and PB2 proteins of IAV. Nonstructural protein 1 and other key genes regulate the replication of IAV and indirectly participate in the regulation of the retinoic acid-induced gene I-like receptor signaling pathway, toll-like receptor signaling pathway, Janus kinase signal transducer and activator of transcription signaling pathway, and other major intracellular viral response signaling pathways to regulate the replication of IAV. Based on the above findings, we mapped the regulatory network of host non-coding RNAs in the innate immune response to the influenza virus. These findings will provide a more comprehensive understanding of the function and mechanism of host non-coding RNAs in the cellular anti-virus response as well as clues to the mechanism of cell-virus interactions and the discovery of antiviral drug targets.

Developing a dual-RCA microfluidic platform for sensitive E. coli O157:H7 whole-cell detections.

Aptamer based microfluidic platforms have been developed rapidly in recent years, and strategies to improve detection sensitivities of such platforms have attracted a significant amount of attention. To achieve whole cell sensitive detections by microfluidic devices, a new dual-rolling circle amplification (RCA) detection approach is presented in this study. This dual-RCA approach includes a capturing RCA (cRCA) reaction that is designed to modify microfluidic channel surfaces with long tandem repeating aptamers (i.e. poly-aptamers) to effectively capture target E. coli O157:H7 cells. We demonstrate that this poly-aptamers modified microchannels capture 3-fold more target cells in comparison with microchannels modified with mono-aptamers against the target cells. In addition, signalling RCA (sRCA) is employed in the dual-RCA design to further enhance detection signals. Our results show that the detection signals are enhanced by up to 50 times by sRCA when compared with those with single fluorescence probes. Furthermore, by combing both the cRCA and the sRCA in one dual-RCA detection system, we demonstrate that the detection signals can be significantly enhanced by ∼250-fold. We also show that E. coli O157:H7 detections with the dual-RCA approach can be used in different food matrices, including orange juice and milk where the limit of detection of 80 cells/mL is achieved. In conclusion, this microfluidic device in combination with a dual-RCA to enhance both target capturing and detection signals is a simple and promising approach to sensitive whole-cell detections for food safety inspections.

Aptamer surface functionalization of microfluidic devices using dendrimers as multi-handled templates and its application in sensitive detections of foodborne pathogenic bacteria.

A microfluidic system that incorporates both dendrimers and aptamers to detect E. coli O157:H7 is developed. To achieve this, generation 7-polyamidoamine dendrimers were immobilized onto the detection surfaces of PDMS microfluidic channels; subsequently aptamers against E. coli O157:H7 were conjugated onto the microchannel surfaces via the immobilized dendrimers as templates. Surface modifications were characterized by FTIR, XPS, water contact angles, fluorescence microscopy and AFM to confirm the success of each surface modification steps. The efficacy of this simple microchannel in detection was investigated using E. coli O157:H7 spiked samples. Our results showed that this interesting approach significantly increased the amount of aptamers available on the microfluidic channel surfaces to capture E. coli O157:H7 cells to allow sensitive detection, which in turn resulted in detections of E. coli O157:H7 cells at a low limit of detection of 102 cells mL-1. The results also demonstrated that in comparison with the generation 4-polyamidoamine dendrimers (G4) modified microchannels, those modified with G7 showed enhanced detection signals, improved target capturing efficiencies, and at higher throughput. This simple whole cell detection design has not been reported in the literature and it is an interesting and effective approach to developing a sensitive and rapid detection platform for foodborne pathogenic bacteria.

Bidirectional effects of moxifloxacin on the pro­inflammatory response in lipopolysaccharide­stimulated mouse peritoneal macrophages.

Sepsis is a systemic inflammatory condition in response to life­threatening infections, and macrophages are a key source of inflammatory cytokines. Moxifloxacin (MXF) has antibacterial activity in Gram­positive and Gram­negative bacteria. The present study investigated the effects of MXF on a lipopolysaccharide (LPS)­stimulated inflammatory response and gene expression in macrophages. Peritoneal macrophages were isolated from male C57BL/6J mice and treated with LPS and/or MXF. The mRNA and protein expression of toll­like receptor 4 (TLR4), sphingosine kinase 1 (SPHK1) and nuclear factor (NF)­κB was determined by quantitative polymerase chain reaction, western blotting and immunofluorescence analysis. The expression of tumor necrosis factor (TNF)­α and interleukin (IL)­6 was determined with ELISAs. The data demonstrated that MXF dose­dependently decreased the viability of macrophages, and 8 and 16 µg/ml MXF prevented the LPS­induced increase in TLR4, SPHK1, NF­κB p65, TNF­α and IL­6 expression. The inhibition was most effective at a concentration of 16 µg/ml MXF, whereas, 64 µg/ml MXF exerted a pro­inflammatory effect. Collectively, the data demonstrated a bidirectional effect of MXF: Lower MXF concentrations (8 and 16 µg/ml) inhibited the inflammatory response; however, a higher MXF concentration (64 µg/ml) had a pro­inflammatory effect on LPS­treated mouse peritoneal macrophages. In conclusion, these results suggested the importance of MXF as an inhibitor of the inflammatory response at an optimal dose. MXF inhibition of the inflammatory response may be mediated by TLR4 signaling.

Multifunctional Theranostic Agent of Cu2(OH)PO4 Quantum Dots for Photoacoustic Image-Guided Photothermal/Photodynamic Combination Cancer Therapy.

Image-guided phototherapy is considered to be a prospective technique for cancer treatment because it can provide both oncotherapy and bioimaging, thus achieving an optimized therapeutic efficacy and higher treatment accuracy. Compared to complicated systems with multiple components, using a single material for this multifunctional purpose is preferable. In this work, we strategically fabricated poly(acrylic acid)- (PAA-) coated Cu2(OH)PO4 quantum dots [denoted as Cu2(OH)PO4@PAA QDs], which exhibit a strong near-infrared photoabsorption ability. As a result, an excellent photothermal conversion ability and the photoactivated formation of reactive oxygen species could be realized upon NIR irradiation, concurrently meeting the basic requirements for photothermal and photodynamic therapies. Moreover, phototherapeutic investigations on both cervical cancer cells in vitro and solid tumors of an in vivo mice model illustrated the effective antitumor effects of Cu2(OH)PO4@PAA upon 1064-nm laser irradiation, with no detectable lesions in major organs during treatment. Meanwhile, Cu2(OH)PO4@PAA is also an exogenous contrast for photoacoustic tomography (PAT) imaging to depict tumors under NIR irradiation. In brief, the Cu2(OH)PO4@PAA QDs prepared in this work are expected to serve as a multifunctional theranostic platform.

Effects of human umbilical cord mesenchymal stem cells on renal ischaemia-reperfusion injury in rats.

OBJECTIVE: This study aims to observe the function of umbilical cord-mesenchymal stem cells (UC-MSCs) labelled with enhanced green fluorescent protein (eGFP) in the repair of renal ischaemia-reperfusion (I/R) injury, to determine the effects on inflammatory cascade in an established rat model and to explore possible pathogenesis. MATERIALS AND METHODS: Sixty rats were randomly divided into three groups: the sham-operated, I/R and UC-MSC treatment groups. All rats underwent right nephrectomy. Ischaemia was induced in the left kidney by occlusion of the renal artery and vein for 1hour, followed by reperfusion for 24 hours or 48 hours. Kidney samples were collected to observe morphological changes. Immunohistochemistry was performed to assess the expression of intercellular adhesion molecule 1 (ICAM-1) in the renal tissue sample, as well as the number of infiltrating polymorphonuclear neutrophils (PMNLs) and UC-MSCs with positive eGFP. RESULTS: Renal histopathological damages and the expression of ICAM-1 and PMNL increased significantly in the I/R group compared with those in the sham-operated group, whereas the damages were less conspicuous in the UC-MSC treatment group. CONCLUSIONS: Renal ICAM-1, which mediated PMNL infiltration and contributed to renal damage, was significantly up-regulated in the I/R group. UC-MSCs were identified to inhibit these pathological processes and protect the kidney from I/R injury.

Effects of Human Umbilical Cord Mesenchymal Stem Cells on Renal Ischaemia-reperfusion Injury in Rats

Objective This study aims to observe the function of umbilical cord-mesenchymal stem cells (UC-MSCs) labelled with enhanced green fluorescent protein (eGFP) in the repair of renal ischaemia-reperfusion (I/R) injury, to determine the effects on inflammatory cascade in an established rat model and to explore possible pathogenesis. Materials and Methods Sixty rats were randomly divided into three groups: the sham-operated, I/R and UC-MSC treatment groups. All rats underwent right nephrectomy. Ischaemia was induced in the left kidney by occlusion of the renal artery and vein for 1hour, followed by reperfusion for 24 hours or 48 hours. Kidney samples were collected to observe morphological changes. Immunohistochemistry was performed to assess the expression of intercellular adhesion molecule 1 (ICAM-1) in the renal tissue sample, as well as the number of infiltrating polymorphonuclear neutrophils (PMNLs) and UC-MSCs with positive eGFP. Results Renal histopathological damages and the expression of ICAM-1 and PMNL increased significantly in the I/R group compared with those in the sham-operated group, whereas the damages were less conspicuous in the UC-MSC treatment group. Conclusions Renal ICAM-1, which mediated PMNL infiltration and contributed to renal damage, was significantly up-regulated in the I/R group. UC-MSCs were identified to inhibit these pathological processes and protect the kidney from I/R injury. .

Knockdown of sphingosine kinase 1 inhibits the migration and invasion of human rheumatoid arthritis fibroblast-like synoviocytes by down-regulating the PI3K/AKT activation and MMP-2/9 production in vitro.

To investigate the potential regulation of sphingosine kinase 1 (SPHK1) on the migration, invasion, and matrix metalloproteinase (MMP) expression in human rheumatoid arthritis fibroblast-like synoviocytes (RA-FLS). RA-FLS were transfected control siRNA or SPHK1 siRNA. The migration and invasion of unmanipulated control, control siRNA or SPHK1 siRNA- transfected RA-FLS in vitro were measured by the transwell system. The relative levels of SPHK1, PI3K, and AKT as well as AKT phosphorylation in RA-FLS were determined by Western blot. The levels of MMP-2/9 secreted by RA-FLS were detected by ELISA. Knockdown of SPHK1 significantly inhibited the spontaneous migration and invasion of RA-FLS, accompanied by significantly reduced levels of PI3K expression and AKT phosphorylation. Similarly, treatment with LY294002, an inhibitor of the PI3K/AKT pathway, inhibited the migration and invasion of RA-FLS. Knockdown of SPHK1 and treatment with the inhibitor synergistically inhibited the migration and invasion of RA-FLS, by further reducing the levels of PI3K expression and AKT phosphorylation. In addition, knockdown of SPHK1 or treatment with LY294002 inhibited the secretion of MMP-2 and MMP-9, and both synergistically reduced the production of MMP-2 and MMP-9 in RA-FLS in vitro. Knockdown of SPHK1 expression inhibits the PI3K/AKT activation, MMP-2 and MMP-9 expression, and human RA-FLS migration and invasion in vitro. Potentially, SPHK1 may be a novel therapeutic target for RA.

Comparison of the Inhibitory Potential of Bavachalcone and Corylin against UDP-Glucuronosyltransferases.

Bavachalcone and corylin are two major bioactive compounds isolated from Psoralea corylifolia L., which has been widely used as traditional Chinese medicine for many years. As two antibiotic or anticancer drugs, bavachalcone and corylin are used in combination with other drugs; thus it is necessary to evaluate potential pharmacokinetic herb-drug interactions (HDI) of the two bioactive compounds. The aim of the present study was to compare the effects of liver UDP-glucuronosyltransferase (UGT) 1A1, UGT1A3, UGT1A7, UGT1A8, UGT 1A10, and UGT2B4 inhibited by bavachalcone and corylin. 4-Methylumbelliferone (4-MU) was used as a nonspecific "probe" substrate. Bavachalcone had stronger inhibition on UGT1A1 and UGT1A7 than corylin which did not inhibit UGT1A1, UGT1A3, UGT1A7, UGT1A8, UGT1A10, and UGT2B4. Data fitting using Dixon and Lineweaver-Burk plots demonstrated the noncompetitive inhibition of bavachalcone against UGT1A1 and UGT1A7-mediated 4-MU glucuronidation reaction. The values of inhibition kinetic parameters (Ki) were 5.41 µ M and 4.51 µ M for UGT1A1 and UGT1A7, respectively. The results of present study suggested that there was a possibility of UGT1A1 and UGT1A7 inhibition-based herb-drug interaction associated with bavachalcone and provided the basis for further in vivo studies to investigate the HDI potential between bavachalcone and UGT substrates.