NLRP3-dependent pyroptosis exacerbates coxsackievirus A16 and coxsackievirus A10-induced inflammatory response and viral replication in SH-SY5Y cells.

Coxsackievirus A16 (CV-A16) and coxsackievirus A10 (CV-A10), more commonly etiological agents of hand, foot and mouth disease (HFMD), are capable of causing severe neurological syndromes with high fatalities, but their neuropathogenesis has rarely been studied. Mounting evidence indicated that pyroptosis is an inflammatory form of cell death that might be widely involved in the pathogenic mechanisms of neurotropic viruses. Our study was designed to examine the effects of NLRP3-mediated pyroptosis in CV-A16- and CV-A10-induced inflammatory neuropathologic formation. In this work, it was showed that SH-SY5Y cells were susceptible to CV-A16 and CV-A10, and meanwhile their infections could result in a decreasing cell viability and an increasing LDH release as well as Caspase1 activation. Moreover, CV-A16 and CV-A10 infections triggered NLRP3-mediated pyroptosis and promoted the release of inflammatory cytokines. Additionally, activated NLRP3 accelerated the pyroptosis formation and aggravated the inflammatory response, but inhibited NLRP3 had a dampening effect on the above situation. Finally, it was further revealed that NLRP3 agonist enhanced the viral replication, but NLRP3 inhibitor suppressed the viral replication, suggesting that NLRP3-driven pyroptosis might support CV-A16 and CV-A10 production in SH-SY5Y cells. Together, our findings demonstrated a mechanism by which CV-A16 and CV-A10 induce inflammatory responses by evoking NLRP3 inflammasome-regulated pyroptosis, which in turn further stimulated the viral replication, providing novel insights into the pathogenesis of CV-A16 and CV-A10 infections.

Analysis of clinical characteristics and risk factors of community-acquired pneumonia complicated by parapneumonic pleural effusion in elderly patients.

BACKGROUND: Community-acquired pneumonia (CAP) patients usually present with parapneumonic pleural effusion (PPE), which complicates the treatment of pneumonia. This study aims to investigate the clinical characteristics and risk factors of elderly CAP patients hospitalised with PPE. METHODS: The clinical data of 132 elderly patients with CAP were retrospectively analysed. A total of 54 patients with PPE (PPE group) and 78 patients without PPE (NPPE group) were included in this study. Clinical data, laboratory examinations, treatments and other relevant indicators were collected. Univariate analysis and multivariate logistic regression analysis will be used to explore the possible risk factors for PPE. RESULTS: The proportion of PPE in elderly patients with CAP was 40.9%. PPE patients were significantly more likely to be older, have comorbid neurological diseases, experience chest tightness, and have a lasting fever (P < 0.05). In contrast to NPPE patients, the total number of lymphocytes, serum albumin and blood sodium levels in the PPE group were significantly lower (P < 0.05). The blood D-dimer, C-reactive protein and CURB-65 score of PPE patients were significantly higher (P < 0.05) than those of NPPE patients. Multivariate logistic regression identified chest tightness (OR = 3.964, 95% CI: 1.254-12.537, P = 0.019), long duration of fever (OR = 1.108, 95%CI: 1.009-1.217, P = 0.03), low serum albumin (OR = 0.876, 95%CI: 0.790- 0.971, P = 0.012) or low blood sodium (OR = 0.896, 95%CI: 0.828-0.969, P = 0.006) as independently associated with the development of parapneumonic pleural effusion in the elderly. CONCLUSION: This study has identified several clinical factors, such as chest tightness, long duration of fever, low serum albumin, and low blood sodium, as risk factors for the development of pleural effusion in elderly patients with CAP. Early identification and prompt management of these patients can prevent inappropriate treatment and reduce morbidity and mortality.

HMGB1 is involved in viral replication and the inflammatory response in coxsackievirus A16-infected 16HBE cells via proteomic analysis and identification.

Coxsackievirus A16 (CV-A16) is still an important pathogen that causes hand, foot and mouth disease (HFMD) in young children and infants worldwide. Previous studies indicated that CV-A16 infection is usually mild or self-limiting, but it was also found that CV-A16 infection can trigger severe neurological complications and even death. However, there are currently no vaccines or antiviral compounds available to either prevent or treat CV-A16 infection. Therefore, investigation of the virus‒host interaction and identification of host proteins that play a crucial regulatory role in the pathogenesis of CV-A16 infection may provide a novel strategy to develop antiviral drugs. Here, to increase our understanding of the interaction of CV-A16 with the host cell, we analyzed changes in the proteome of 16HBE cells in response to CV-A16 using tandem mass tag (TMT) in combination with LC‒MS/MS. There were 6615 proteins quantified, and 172 proteins showed a significant alteration during CV-A16 infection. These differentially regulated proteins were involved in fundamental biological processes and signaling pathways, including metabolic processes, cytokine‒cytokine receptor interactions, B-cell receptor signaling pathways, and neuroactive ligand‒receptor interactions. Further bioinformatics analysis revealed the characteristics of the protein domains and subcellular localization of these differentially expressed proteins. Then, to validate the proteomics data, 3 randomly selected proteins exhibited consistent changes in protein expression with the TMT results using Western blotting and immunofluorescence methods. Finally, among these differentially regulated proteins, we primarily focused on HMGB1 based on its potential effects on viral replication and virus infection-induced inflammatory responses. It was demonstrated that overexpression of HMGB1 could decrease viral replication and upregulate the release of inflammatory cytokines, but deletion of HMGB1 increased viral replication and downregulated the release of inflammatory cytokines. In conclusion, the results from this study have helped further elucidate the potential molecular pathogenesis of CV-A16 based on numerous protein changes and the functions of HMGB1 Found to be involved in the processes of viral replication and inflammatory response, which may facilitate the development of new antiviral therapies as well as innovative diagnostic methods.

Comprehensive profiling and characterization of cellular microRNAs in response to coxsackievirus A10 infection in bronchial epithelial cells.

Coxsackievirus A10 (CV-A10), the causative agent of hand, foot, and mouth disease (HFMD), caused a series of outbreaks in recent years and often leads to neurological impairment, but a clear understanding of the disease pathogenesis and host response remains elusive. Cellular microRNAs (miRNAs), a large family of non-coding RNA molecules, have been reported to be key regulators in viral pathogenesis and virus-host interactions. However, the role of host cellular miRNAs defensing against CV-A10 infection is still obscure. To address this issue, we systematically analyzed miRNA expression profiles in CV-A10-infected 16HBE cells by high-throughput sequencing methods in this study. It allowed us to successfully identify 312 and 278 miRNAs with differential expression at 12 h and 24 h post-CV-A10 infection, respectively. Among these, 4 miRNAs and their target genes were analyzed by RT-qPCR, which confirmed the sequencing data. Gene target prediction and enrichment analysis revealed that the predicted targets of these miRNAs were significantly enriched in numerous cellular processes, especially in regulation of basic physical process, host immune response and neurological impairment. And the integrated network was built to further indicate the regulatory roles of miRNAs in host-CV-A10 interactions. Consequently, our findings could provide a beneficial basis for further studies on the regulatory roles of miRNAs relevant to the host immune responses and neuropathogenesis caused by CV-A10 infection.

Comprehensive analysis of differential expression profiles via transcriptome sequencing in SH-SY5Y cells infected with CV-A16.

Coxsackievirus A16 (CV-A16) is one of the viruses that is most frequently associated with hand-foot-and-mouth disease (HFMD). Previous studies have shown that CV-A16 infections are mostly self-limiting, but in recent years, it has been gradually found that CV-A16 infections can also induce neurological complications and eventually cause death in children with HFMD. Moreover, no curative drugs or preventative vaccines have been developed for CV-A16 infection. Therefore, it is particularly important to investigate the mechanism of CV-A16 infection-induced neuropathy. In the current study, transcriptome sequencing technology was used to identify changes in the transcriptome of SH-SY5Y cells infected with CV-A16, which might hide the mechanism of CV-A16-induced neuropathology. The transcriptome profiling showed that 82,406,974, 108,652,260 and 97,753,565 clean reads were obtained in the Control, CV-A16-12 h and CV-A16-24 h groups, respectively. And it was further detected that a total of 136 and 161 differentially expressed genes in CV-A16-12 h and CV-A16-24 h groups, respectively, when compared with Control group. Then, to explore the mechanism of CV-A16 infection, we focused on the common differentially expressed genes at different time points of CV-A16 infection and found that there were 34 differentially expressed genes based on which clustering analysis and functional category enrichment analysis were performed. The results indicated that changes in oxidation levels were particularly evident in the GO term analysis, while only the "Gonadotropin-releasing hormone receptor pathway" was enriched in the KEGG pathway analysis, which might be closely related to the neurotoxicity caused by CV-A16 infection. Meanwhile, the ID2 closely related to nervous system has been demonstrated to be increased during CV-A16 infection. Additionally, the data on differentially expressed non-protein-coding genes of different types within the transcriptome sequencing results were analyzed, and it was speculated that these dysregulated non-protein-coding genes played a pivotal role in CV-A16 infection. Ultimately, qRT-PCR was utilized to validate the transcriptome sequencing findings, and the results of qRT-PCR were in agreement with the transcriptome sequencing data. In conclusion, transcriptome profiling was carried out to analyze response of SH-SY5Y cells to CV-A16 infection. And our findings provide important information to elucidate the possible molecular mechanisms which were linked to the neuropathogenesis of CV-A16 infection.

A Novel Role of VEGFC in Cerebral Ischemia With Lung Injury.

Cerebral ischemia (CI) is a severe brain injury resulting in a variety of motor impairments combined with secondary injury in remote organs, especially the lung. This condition occurs due to insufficient blood supply to the brain during infancy. However, it has a molecular linkage that needs to be thoroughly covered. Here, we report on the role of vascular endothelial growth factor C (VEGFC) in lung injury induced by CI. The middle cerebral artery occlusion (MCAO) was depended to establish the animal model of CI. Rats were used and brain ischemia was confirmed through TTC staining. Serum was used for protein chip analysis to study the proteomic interaction. Immunohistochemistry analyses were used to quantify and locate the VEGFC in the lung and brain. The role of VEGFC was detected by siVEGFC technology in SY5Y, HUCEV, and A549 cell lines, under normal and oxygen glucose deprivation (OGD) conditions in vitro. As a result, the TTC staining demonstrated that the model of brain ischemia was successfully established, and MPO experiments reported that lung damage was induced in MCAO rats. VEGFC levels were up-regulated in serum. On the other hand, immunohistochemistry showed that VEGFC increased significantly in the cytoplasm of neurons, the endothelium of small trachea and the lung cells of CI animals. On a functional level, siVEGFC effectively inhibited the proliferation of SY5Y cells and decreased the viability of HUVEC cells in normal cell lines. But under OGD conditions, siVEGFC decreased the growth of HUVEC and increased the viability of A549 cells, while no effect was noticed on SYSY cells. Therefore, we confirmed the different role of VEGFC played in neurons and lung cells in cerebral ischemia-reperfusion injury. These findings may contribute to the understanding the molecular linkage of brain ischemia and lung injury, which therefore provides a new idea for the therapeutic approach to cerebral ischemia-reperfusion.

Differential roles of p38 MAPK and ERK1/2 in angiopoietin-2-mediated rat pulmonary microvascular endothelial cell apoptosis induced by lipopolysaccharide.

Angiopoietin-2 (Ang-2) is a Tie-2 ligand that destabilizes vascular structures, enhances vascular permeability and induces vascular regression and endothelial cell apoptosis. Although there is evidence for the involvement of the Ang/Tie2 axis in acute lung injury (ALI), the underlying mechanisms involved in Ang-2-induced cell apoptosis are not well understood. In this study, whether Ang-2 contributes to microvascular endothelial cell injury and mediates lipopolysaccharide (LPS)-induced endothelial cell apoptosis and its associated signaling pathways was investigated. Exposure of rat pulmonary microvascular endothelial cells (RPMVECs) to LPS, Ang-2 and related inhibitors was performed to measure the expression levels of Ang-2, the activation of mitogen-activated protein kinases (MAPKs), the phosphorylation of extracellular signal-regulated kinase (ERK)1/2, and expression of the apoptosis-related proteins Bax and Bcl-2 using western blotting, reverse transcription-quantitative polymerase chain reaction, flow cytometry and fluorescence microscopy. The expression of Ang-2 in the RPMVECs was increased by LPS independent of time. The phosphorylation of p38 MAPK and ERK1/2 was significantly upregulated and the activation of apoptosis-related proteins Bax and Bcl was mediated by Ang-2. In addition, inhibition of the p38 pathway by SB203580 attenuated the Ang-2-mediated cell apoptosis, but inhibition of the ERK1/2 pathway by PD98059 exerted an anti-apoptotic effect against Ang-2. In conclusion, LPS-induced apoptosis is partly mediated via stimulation of p38 and ERK1/2 signaling pathways, where Ang-2 acts an inflammation-related factor to participate in the course of cell apoptosis in RPMVECs.

Cooperative Activation of Cobalt-Salen Complexes for Epoxide Hydration Promoted on Flexible Porous Organic Frameworks.

Developing solid catalysts with multiple active sites working cooperatively is desirable for efficient chemical transformations. However, most solid catalysts are rigid and impede the cooperation between their spatially isolated active sites. Two flexible porous organic frameworks (POFs) with integrated Co(salen) as active sites have been successfully synthesized for mimicking the cooperative modes of enzymes. The POFs exhibit second-order rate dependence on Co(salen) concentration in the network and afford much higher TOF (3300 versus 2670 h-1 ) than the homogeneous counterpart in the hydration of propylene epoxide. POFs with a flexible network thus not only facilitate but also enhance the cooperation of nearby Co(salen). Moreover, POFs could catalyze oversized substrates, have a wide substrate scope, and exhibit high stability.

Tuning the Surface Polarity of Microporous Organic Polymers for CO2 Capture.

CO2 capture is very important to reduce the CO2 concentration in atmosphere. Herein, we report the preparation of microporous polymers with tunable surface polarity for CO2 capture. Porous polymers functionalized with -NH2 , -SO3 H, and -SO3 Li have been successfully prepared by using a post-synthesis modification of microporous polymers (P-PhPh3 prepared with 1,3,5-triphenylbenzene as the monomer and AlCl3 as the catalyst) by chemical transformations, such as nitration-reduction, sulfonation, and cationic exchange. The CO2 adsorption selectivity (CO2 /N2 and CO2 /H2 ) and isosteric heats of the microporous polymers increase markedly after modification, P-PhPh3 -NH2 and P-PhPh3 -SO3 Li afford higher CO2 uptake capacity than P-PhPh3 at pressures of less than 0.15 bar due to the enhanced interaction between CO2 and the -NH2 and -SO3 Li functional groups. Moreover, functionalized porous polymers could be stably used for CO2 capture. Surface modification is an efficient approach to tune the CO2 capture properties of porous polymers.

[The levels of angiopoietin-2 in patients with acute respiratory distress syndrome and its value on prognosis].

OBJECTIVE: To approach the correlation between angiopoietin-2 (Ang-2) levels and degree of lung injury and prognosis and its clinical significance in patients with acute respiratory distress syndrome (ARDS). METHODS: A prospective observation was conducted. Fifty-three ARDS patients admitted to Department of Critical Care Medicine of Third Affiliated Hospital of Anhui Medical University from January 2012 to March 2014 were enrolled. According to the criteria of the Berlin Definition of ARDS, the patients were divided into mild group (n=15), moderate group (n=22) and severe group (n=16). Meanwhile, ARDS patients were further divided into survival group(n=29) and non-survival group(n=24) according to 28-day outcomes. Twenty cases of non-ARDS patients were served as control. The acute physiology and chronic health evaluation II(APACHEII) score, sequential organ failure assessment (SOFA) score, oxygenation index (PaO2/FiO2), lung injury score (LIS) were recorded within 24 hours after admission. And the plasma levels of Ang-2, interleukin-6 (IL-6) and C-reaction protein (CRP) were measured. The independent risk factors of ARDS were analyzed by univariate and multivariable logistic regression. Receiver operating characteristic curve (ROC) was plotted to evaluate the value of Ang-2 in predicting ARDS. RESULTS: Compared with non-ARDS group, APACHEII score, SOFA score, LIS score, mortality were significantly increased, PaO2/FiO2 was significantly decreased, and plasma Ang-2, IL-6, CRP were significantly elevated [APACHEII score: 20.7 ± 5.0 vs. 14.1 ± 5.3, SOFA score: 7.7 ± 3.5 vs. 3.5 ± 2.1, LIS score: 1.69 ± 0.71 vs. 0.28 ± 0.27, PaO2/FiO2(mmHg, 1 mmHg = 0.133 kPa): 159.5 ± 61.3 vs. 394.0 ± 3.2, mortality: 45.3% (24/53) vs. 20.0% (4/20), Ang-2(µg/L): 4.73(2.59, 6.99)vs. 1.22 (0.61, 1.52), IL-6(ng/L): 56.50 (27.15, 139.90)vs. 13.05 (4.38, 15.55), CRP (mg/L): 95.75(41.74, 189.72) vs. 10.56 (3.92, 21.36), P<0.05 or P<0.01]. Each index increased or decreased more significantly with the aggravation of the disease. It was shown by correlation analysis that the plasma levels of Ang-2 was significantly positive correlated with IL-6 (r=0.468,P=0.000), CRP(r = 0.492, P = 0.000), APACHEII score (r = 0.560, P = 0.000), SOFA score (r = 0.508, P = 0.000) and LIS score (r = 0.588, P = 0.000), significantly negatively correlated with PaO2/FiO2 (r=-0.685,P=0.000). Factors, APACHEII score, LIS score, PaO2/FiO2, Ang-2 and IL-6 founded statistical significance in univariate analysis were analyzed using multivariable logistic regression. High APACHEII score at admission [odds ratio (OR) = 1.316, 95% confidence interval (95% CI) = 1.040-1.633, P = 0.022] and increased plasma Ang-2 levels (OR = 1.287, 95% CI = 1.041-1.760, P = 0.038) were the independent prognostic factors for the 28-day mortality in ARDS. The area under the ROC curve of Ang-2 was 0.964, the optimal critical value of Ang-2 was 1.79 µg/L, the specificity was 90.0%, and sensitivity was 92.5%. Plasma levels of Ang-2 was better in predicting ARDS than APACHEII score, SOFA score and IL-6. CONCLUSIONS: The plasma level of Ang-2 was significantly increased in patients with ARDS. The plasma level of Ang-2 was correlated with the severity of acute lung injury and had important prognosis evaluation.

Protective effects of total flavonoids of Bidens bipinnata L. against carbon tetrachloride-induced liver fibrosis in rats.

Bidens bipinnata L. is well known in China as a traditional Chinese medicine and has been used to treat hepatitis in clinics for many years. In a previous study we found that total flavonoids of Bidens bipinnata L. (TFB) had a protective effect against carbon tetrachloride (CCl4)-induced acute liver injury in mice. Now this study was designed to investigate its therapeutic effect against CCl4-induced liver fibrosis in rats and to determine, in part, its mechanism of action. The liver fibrosis model was established by subcutaneous injection of 50% CCl4 twice a week for 18 weeks. TFB (40, 80 and 160 mg kg(-1)) was administered by gastrogavage daily from the 9th week. The results showed that TFB (80 and 160 mg kg(-1)) treatment for 10 weeks significantly reduced the elevated liver index (liver weight/body weight) and spleen index (spleen weight/body weight), elevated levels of serum transaminases (alanine aminotransferase and aspartate aminotransferase), hyaluronic acid, type III procollagen and hepatic hydroxyproline. In addition, TFB markedly inhibited CCl4-induced lipid peroxidation and enhanced the activity of the antioxidant enzymes superoxide dismutase and glutathione peroxidase. Moreover, TFB (80 and 160 mg kg(-1)) treatment improved the morphologic changes of hepatic fibrosis induced by CCl4 and suppressed nuclear factor (NF)-kappaB, alpha-smooth muscle actin (SMA) protein expression and transforming growth factor (TGF)-beta1 gene expression in the liver of liver fibrosis of rats. In conclusion, TFB was able to ameliorate liver injury and protect rats from CCl4-induced liver fibrosis by suppressing oxidative stress. This process may be related to inhibiting the induction of NF-kappaB on hepatic stellate cell activation and the expression of TGF-beta1.

Protective effects of total flavonoids of Bidens pilosa L. (TFB) on animal liver injury and liver fibrosis.

The hepatoprotective effects of total flavonoids of Bidens pilosa L. (TFB), a traditional Chinese medicine were evaluated in carbon tetrachloride (CCl(4))-induced liver injury in mice and rats. Total flavonoids of Bidens pilosa L. (25, 50 and 100mg/kg) were administered via gavage daily for 10 days to CCl(4)-treated mice as well as TFB (30, 60 and 90mg/kg) administered for 6 weeks to CCl(4)-treated rats. Liver index (liver weight/body weight), serum levels of transaminases (alanine aminotransferase, ALT and aspartate aminotransferase, AST), hepatic malondialdehyde (MDA) content, superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) activities were evaluated following the 10 days treatment in mice. In addition histopathologic changes and nuclear factor-kappaB (NF-kappaB) expression of the liver were detected with hematoxylin-eosin (HE) and immunohistochemistry methods, respectively. The results showed that TFB (50 and 100mg/kg) effectively reduced the CCl(4)-induced elevated liver index, serum ALT, AST levels, hepatic MDA content, and restored hepatic SOD, GSH-Px activities in acute liver injury mice. TFB (60 and 90mg/kg) treatment significantly inhibited NF-kappaB activation in liver fibrosis of rats. The histopathological analysis suggested that TFB reduced the degree of liver injury in mice and severity of liver fibrosis in rats. These results suggested that TFB had a protective and therapeutic effect on animal liver injury, which might be associated with its antioxidant properties and inhibition of NF-kappaB activation.

Protective effect of total flavonoids from Bidens bipinnata L. against carbon tetrachloride-induced liver injury in mice.

Bidens bipinnata L. is well known in China as a traditional Chinese medicine. This study was designed to evaluate the hepatoprotective activity of the total flavonoids of B. bipinnata L. (TFB) against carbon tetrachloride (CCl4)-induced acute liver injury in mice and to determine its mechanism of action. Oral administration of TFB at doses of 50, 100 and 200 mg kg(-1) for 7 days significantly reduced the elevated relative values of liver weight, serum transaminases (alanine aminotransferase and aspartate aminotransferase) and the hepatic morphologic changes induced by CCl4 in mice. In addition, TFB markedly inhibited CCl4-induced lipid peroxidation and enhanced the activity of the antioxidant enzymes superoxide dismutase and glutathione peroxidase. Moreover, pretreatment with TFB suppressed nitric oxide production and nuclear factor-kappaB activation in CCl4-treated mice. The results suggest that TFB has significant hepatoprotective activity and its mechanism is related, at least in part, to its antioxidant properties. Further research is required to investigate the detailed mechanism of the protective effect of TFB on acute liver injury.

[Study on the property of adsorption and separation of the macroporous resins for total flavonoids of Bidens bipinnata L].

OBJECTIVE: To screen macroporous resins for isolation and purification of total flavonoids of Bidens bipinnata L. (TFB) through investigating the property of adsorption and desorption of 7 kinds of macroporous adsorption resins for TFB. METHODS: The content of total flavonoids was used as the evaluating criteria, and the static and dynamic adsorption-desorption methods were adopted to compare the adsorption quantity, the rate of desorption and adsorption kinetics of different macroporous adsorption resins. RESULTS: Among 7 kinds of macroporous adsorption resins, the HPD 100 resin was the best for isolating and purifying TFB. CONCLUSION: HPD 100 possess a better adsorbability and separating property, and its property is obviously higher than any other resins.