Systematic Chemical Analysis of Crude Glycan Isolates from the Seven-Herb Decoction Quanzhenyiqitang with Anti-COPD Activity.

The classical Chinese Medicine prescription, Quanzhenyiqitang (QZYQT), containing seven tonic herbs (Shudi, Dangshen, Maidong, Baizhu, Niuxi, Fuzi, and Wuweizi) is clinically used to treat chronic obstructive pulmonary disease (COPD). Although there are studies on the pharmacological effects of QZYQT, little attention has been paid to its active carbohydrate ingredients. We performed a systematic chemical analysis of the crude glycan isolates from the seven-herb decoction (GI-QZYQT) after confirming its anti-COPD activity. GI-QZYQT could enhance lung function, reduce lung damage, and alleviate inflammatory response in mice with COPD. Moreover, two monosaccharides (fructose and glucose) and six oligosaccharides (sucrose, melibiose, 1-kestose, raffinose, mannotriose, and stachyose), accounting for 40.23 % of GI-QZYQT, were discovered using hydrophilic interaction liquid chromatography-evaporative light-scattering detection. Inulin-type fructan with an average molecular weight of 2112 Da was identified using high-performance gel-permeation chromatography in combination with monosaccharide mapping analysis, accounting for 20.10 % of GI-QZYQT in mass. The comparison study showed that the identified monosaccharides, oligosaccharides, and the inulin-type fructan of GI-QZYQT were mainly derived from herbs of Shudi, Dangshen, Maidong, Baizhu, and Niuxi. These findings provide crucial information on the chemical composition of GI-QZYQT, which is vital for the in-depth understanding of its bioactivity, mechanism, and product development.

Understanding the effects of A-site Ag-doping on LaCoO3 perovskite for NO oxidation: Structural and magnetic properties.

The partial substitution of A-site in perovskites is a major strategy to enhance the catalytic oxidation activity. This study explores the use of silver (Ag) to partially replace the lanthanum (La) ion at the A-site in LaCoO3 perovskite, investigating the role of Ag in the ABO3 perovskite structure, elucidating the nitric oxide (NO) oxidation mechanism over La1-xAgxCoO3 (x = 0.1-0.5) perovskites. La0.7Ag0.3CoO3 with an Ag-doping amount of 0.3, exhibited the highest NO oxidation activity of 88.5% at 275 °C. Characterization results indicated that Ag substitution enhanced the perovskite, maintaining its original phase structure, existing in the form of a mixture of Ag0 and Ag+ in the La1-xAgxCoO3 (x = 0.1-0.5) perovskites. Notably, Ag substitution improved the specific surface area, reduction performance, Co3+, and surface adsorption oxygen content. Additionally, the study investigated the relationship between magnetism and NO oxidation from a magnetism perspective. Ag-doping strengthened the magnetism of La-Ag perovskite, resulting in stronger adsorption of paramagnetic NO. This study elucidated the NO oxidation mechanism over La-Ag perovskite, considering structural and magnetic properties, providing valuable insights for the subsequent development and industrial application of high oxidation ability perovskite catalysts.

AQP4-A25Q Point Mutation in Mice Depolymerizes Orthogonal Arrays of Particles and Decreases Polarized Expression of AQP4 Protein in Astrocytic Endfeet at the Blood-Brain Barrier.

Aquaporin-4 (AQP4) is characterized by the formation of orthogonal arrays of particles (OAPs) comprising its M1 and M23 isoforms in the plasma membrane. However, the biological importance of OAP formation is obscure. Here, we developed an OAP depolymerization male mouse model by transgenic knock-in of an AQP4-A25Q mutation. Analyses of the mutant brain tissue using blue native polyacrylamide gel electrophoresis, super-resolution imaging, and immunogold electron microscopy revealed remarkably reduced OAP structures and glial endfeet localization of the AQP4-A25Q mutant protein without effects on its overall mRNA and protein expression. AQP4A25Q/A25Q mice showed better survival and neurologic deficit scores when cerebral edema was induced by water intoxication or middle cerebral artery occlusion/reperfusion. The brain water content and swelling of pericapillary astrocytic endfeet processes in AQP4A25Q/A25Q mice were significantly reduced, functionally supporting decreased AQP4 protein expression at the blood-brain barrier. The infarct volume and neuronal damage were also reduced in AQP4A25Q/A25Q mice in the middle cerebral artery occlusion/reperfusion model. Astrocyte activation in the brain was alleviated in AQP4A25Q/A25Q mice, which may be associated with decreased cell swelling. We conclude that the OAP structure of AQP4 plays a key role in its polarized expression in astrocytic endfeet processes at the blood-brain barrier. Therefore, our study provided new insights into intervention of cerebral cellular edema caused by stroke and traumatic brain injury through regulating AQP4 OAP formation.SIGNIFICANCE STATEMENT Aquaporin-4 (AQP4) is characterized by orthogonal arrays of particles (OAPs) comprising the M1 and M23 isoforms in the membrane. Here, an OAP depolymerization male mouse model induced by AQP4-A25Q mutation was first established, and the functions of OAP depolymerization in cerebral edema have been studied. The results revealed that AQP4 lost its OAP structure without affecting AQP4 mRNA and protein levels in AQP4-A25Q mice. AQP4-A25Q mutation mice has neuroprotective effects on cerebral edema induced by water intoxication and middle cerebral artery occlusion/reperfusion through relieving the activation of astrocytes and suppressed microglia-mediated neuroinflammation. We concluded that the OAP structure of AQP4 plays a key role in its polarized expression in astrocytic endfeet processes at the blood-brain barrier. Therefore, our study provided new insights into intervention of cerebral cellular edema caused by stroke and traumatic brain injury through regulating AQP4 OAP formation.

Association of mitochondrial homeostasis and dynamic balance with malignant biological behaviors of gastrointestinal cancer.

Mitochondria determine the physiological status of most eukaryotes. Mitochondrial dynamics plays an important role in maintaining mitochondrial homeostasis, and the disorder in mitochondrial dynamics could affect cellular energy metabolism leading to tumorigenesis. In recent years, disrupted mitochondrial dynamics has been found to influence the biological behaviors of gastrointestinal cancer with the potential to be a novel target for its individualized therapy. This review systematically introduced the role of mitochondrial dynamics in maintaining mitochondrial homeostasis, and further elaborated the effects of disrupted mitochondrial dynamics on the cellular biological behaviors of gastrointestinal cancer as well as its association with cancer progression. We aim to provide clues for elucidating the etiology and pathogenesis of gastrointestinal cancer from the perspective of mitochondrial homeostasis and disorder.

Behavior of heavy metals in municipal sludge during dewatering: The role of conditioners and extracellular polymeric substances.

Heavy metals, the main harmful substances in the sludge, are easily enriched, have adverse effects on the treatment and disposal of the sludge. In this study, two conditioners (modified corn-core powder, MCCP, and sludge-based biochar, SBB) were separately added and jointly added into municipal sludge to enhance sludge dewaterability. Meanwhile, diverse organics, such as extracellular polymeric substances (EPS), were released under pretreatment. The different organics had different effects on each heavy metal fraction and changed the toxicity and bioavailability of the treated sludge. The exchangeable fraction (F4) and carbonate fraction (F5) of heavy metal were nontoxic and nonbioavailable. When MCCP/SBB was used to pretreat the sludge, the ratio of metal-F4 and -F5 decreased, indicating that MCCP/SBB reduced the biological availability and ecological toxicity of the heavy metals in the sludge. These results were consistent with the calculation of the modified potential ecological risk index (MRI). To understand the detailed function of organics in the sludge network, the relationship between EPS, the secondary structure of the protein, and heavy metals was analyzed. The analyses revealed that the increasing proportion of ß-sheet in soluble EPS (S-EPS) generated more active sites in the sludge system, which enhanced the chelate or complex function among organics and heavy metals, thus reducing the migration risks.

[Medication rules of Chinese herbal compound prescriptions for treating angina in national patent database based on multiple data mining].

This study explored the medication rules of Chinese herbal compound prescriptions for the treatment of angina based on the Chinese herbal compound patents in the patent database of the China National Intellectual Property Administration. The data of eligible Chinese herbal compound patents for the treatment of angina were collected from the patent database of the China National Intellectual Property Administration from database inception to November 10, 2022, and subjected to data modeling, analysis of main syndromes, medication frequency analysis, cluster analysis, association rule analysis, and data visualization by using Excel 2021, IBM SPSS Statistics 26.0, IBM SPSS Modeler 18.0, Cytoscape 3.9.1, and Rstudio R 4.2.2.2 to explore the medication rules for angina. The study included 636 pieces of patent data for angina that met the inclusion criteria, involving 815 drugs, with a total frequency of 6 586. The most common main syndromes were blood stasis obstructing the heart syndrome(222, 34.91%) and Qi deficiency and blood stasis syndrome(112, 17.61%). The top 10 most frequently used drugs were Salviae Miltiorrhizae Radix et Rhizoma, Chuanxiong Rhizoma, Notoginseng Radix et Rhizoma, Astragali Radix, Angelicae Sinensis Radix, Carthami Flos, Glycyrrhizae Radix et Rhizoma, Ginseng Radix et Rhizoma, Borneolum Syntheticum, and Corydalis Rhizoma. High-frequency drugs included blood-activating and stasis-resolving drugs(1 197, 18.17%) and deficiency-tonifying drugs(809, 12.28%). Cluster analysis identified eight drug combinations, including five new prescriptions suitable for clinical use and new drug development, and three drug pairs. The core drug combination of Salviae Miltiorrhizae Radix et Rhizoma-Chuanxiong Rhizoma-Carthami Flos was identified through the complex co-occurrence network analysis of Chinese medicines. Association rule analysis yielded a total of 17 rules, including 13 drug pairs and 4 tripartite combinations. Common drug pairs included Salviae Miltiorrhizae Radix et Rhizoma-Chuanxiong Rhizoma(support degree 25.79%, confidence coefficient 69.49%, lift 1.30) and Salviae Miltiorrhizae Radix et Rhizoma-Notoginseng Radix et Rhizoma(support degree 22.01%, confidence coefficient 61.95%, lift 1.16). Common tripartite combinations included Salviae Miltiorrhizae Radix et Rhizoma-Chuanxiong Rhizoma-Astragali Radix(support degree 10.85%, confidence coefficient 73.40%, lift 1.37) and Salviae Miltiorrhizae Radix et Rhizoma-Chuanxiong Rhizoma-Notoginseng Radix et Rhizoma(support degree 10.69%, confidence coefficient 79.07%, lift 1.48). The results showed that the underlying pathogenesis of angina involved blood stasis obstructing the heart and Qi deficiency and blood stasis. The overall nature of the disease was characterized as asthenia in origin and sthenia in superficiality. In the prescription formulation, blood-activating and stasis-resolving drugs, such as Salviae Miltiorrhizae Radix et Rhizoma, Chuanxiong Rhizoma, and Carthami Flos were often used to resolve the excess manifestation, which were combined with tonifying drugs such as Astragali Radix, Angelicae Sinensis Radix, Glycyrrhizae Radix et Rhizoma, and Ginseng Radix et Rhizoma to reinforce the deficiency. The syndrome, pathogenesis, disease nature, and medication were consistent with clinical practice. Additionally, the new compound prescriptions and drug combinations derived from the multiple data mining in this study could provide references and insights for the clinical diagnosis and treatment of angina and the development of new drugs.

Incidence and Prognosis Nomogram of Small Solitary Lung Cancer (≤2 cm) With Extra-Thoracic Metastasis at Initial Diagnosis:A Population-Based Study.

BACKGROUND: Small solitary lung cancer (≤2 cm) with extra-thoracic metastasis and no nodal metastasis or intra-thoracic metastasis is a rare situation in clinic. METHODS: Lung cancer patients with stage T1aN0M0 and T1aN0M1b from 2010 to 2015 were identified from the Surveillance, Epidemiology, and End Results database. The identified significant parameters were utilized to develop 2 nomogram to predict the extra-thoracic metastasis rates and the overall survival for the group of patients with stage T1aN0M1b. RESULTS: Small solitary lung cancers which occur in the males, younger patients, or locate in the main bronchus or left lung, or with histologic type as small cell lung cancer, or with undifferentiated type, tend to have extra-thoracic metastasis. Application of the nomogram in the intra-group still gave good discrimination and good calibration. Univariable and multivariable analysis identified several clinical data as the prognostic factors for lung cancer patients with stage T1aN0M1b, all the factors above were incorporated into the nomogram. ROC curve analysis showed that the nomogram had good discrimination, with AUC of .779, .786 and .77 for 1-, 3- and 5-year survival in the development group and validation group, respectively. Moreover, decision curve analysis has been implemented to evaluate and compare prediction and prognostic nomogram. CONCLUSIONS: Younger male patients whose lung cancer locates in main bronchus or left lung, or with undifferentiated type, or with histologic type as small cell lung cancer are more likely to have extra-thoracic metastasis. The proposed nomogram reliably predicted OS for lung cancer patients with stage T1aN0M1b, though further validation is needed, it may be a useful tool in clinical practice. These models can be wildly used for easy facilitate the lung cancer individualized prediction of extra-thoracic metastasis and OS.

Cortical mechanisms in migraine.

Migraine is the second most prevalent disorder in the world; yet, its underlying mechanisms are still poorly understood. Cumulative studies have revealed pivotal roles of cerebral cortex in the initiation, propagation, and termination of migraine attacks as well as the interictal phase. Investigation of basic mechanisms of the cortex in migraine not only brings insight into the underlying pathophysiology but also provides the basis for designing novel treatments. We aim to summarize the current research literatures and give a brief overview of the cortex and its role in migraine, including the basic structure and function; structural, functional, and biochemical neuroimaging; migraine-related genes; and theories related to cortex in migraine pathophysiology. We propose that long-term plasticity of synaptic transmission in the cortex encodes migraine.

GRHL2 induces liver fibrosis and intestinal mucosal barrier dysfunction in non-alcoholic fatty liver disease via microRNA-200 and the MAPK pathway.

Non-alcoholic fatty liver disease (NAFLD) serves as the most common subtype of liver diseases and cause of liver dysfunction, which is closely related to obesity and insulin resistance. In our study, we sought to investigate effect of transcription factor grainyhead-like 2 (GRHL2) on NAFLD and the relevant mechanism. NAFLD mouse model was established with a high-fat feed. Then, serum was extracted from NAFLD patients and mice, followed by ectopic expression and depletion experiments in NAFLD mice and L02 cells. Next, the correlation between GRHL2 and microRNA (miR)-200 and between miR-200 and sirtuin-1 (SIRT1) was evaluated. The observations demonstrated that miR-200 and GRHL2 were overexpressed in the serum of NAFLD patients and mice, while SIRT1 was poorly expressed. GRHL2 positively regulated miR-200 by binding to miR-200 promoter region, which negatively targeted SIRT1. The inhibition of miR-200 and GRHL2 or SIRT1 overexpression lowered HA and LN in mouse liver tissue, occludin and ZO-1 in mouse small intestine tissue, TNF-α and IL-6 in mouse serum, glucose, total cholesterol (TC), triglyceride (TG), aspartate aminotransferase (AST) and alanine aminotransferase (ALT) in mouse serum, and also inhibited liver fibrosis and intestinal mucosal barrier dysfunction. Meanwhile, GRHL2 induced activation of MAPK signalling pathway in NAFLD mice. Collectively, GRHL2 played a contributory role in NAFLD by exacerbating liver fibrosis and intestinal mucosal barrier dysfunction with the involvement of miR-200-dependent SIRT1 and the MAPK signalling pathway.

Sodium tanshinone IIA sulfonate protects against Aß-induced cell toxicity through regulating Aß process.

Sodium tanshinone IIA sulfonate (STS) has been reported to prevent Alzheimer's disease (AD). However, the mechanism is still unknown. In this study, two in vitro models, Aß-treated SH-SY5Y cells and SH-SY5Y human neuroblastoma cells transfected with APPsw (SH-SY5Y-APPsw cells), were employed to investigate the neuroprotective of STS. The results revealed that pretreatment with STS (1, 10 and 100 µmol/L) for 24 hours could protect against Aß (10 µmol/L)-induced cell toxicity in a dose-dependent manner in the SH-SY5Y cells. Sodium tanshinone IIA sulfonate decreased the concentrations of reactive oxygen species, malondialdehyde, NO and iNOS, while increased the activities of superoxide dismutase and glutathione peroxidase in the SH-SY5Y cells. Sodium tanshinone IIA sulfonate decreased the levels of inflammatory factors (IL-1ß, IL-6 and TNF-α) in the SH-SY5Y cells. In addition, Western blot results revealed that the expressions of neprilysin and insulin-degrading enzyme were up-regulated in the SH-SY5Y cells after STS treatment. Furthermore, ELISA and Western blot results showed that STS could decrease the levels of Aß. ELISA and qPCR results indicated that STS could increase α-secretase (ADAM10) activity and decrease ß-secretase (BACE1) activity. In conclusion, STS could protect against Aß-induced cell damage by modulating Aß degration and generation. Sodium tanshinone IIA sulfonate could be a promising candidate for AD treatment.

Role of extracellular polymeric substances in sludge dewatering under modified corn-core powder and sludge-based biochar pretreatments.

Extracellular polymeric substances (EPS) which wrapped on sludge particles were deemed to hinder the outflowing of combined water in sludge system. The complex composition of EPS was the bottleneck for revealing its relationship with sludge dewaterability. In this study, a combined modified corn-core powder (MCCP) and sludge-based biochar (SBB) condition was executed to treat sludge for enhancing dehydration performance, and the concentration and the form distribution of organics in EPS, the variances of protein secondary structures were investigated. Correlation between the sludge dewaterability and EPS components were performed, found strong correlations among the net sludge solids yield (YN) and the specific resistance of filtration (SRF) (R = -0.923), Zeta potential (R = -0.971). Furthermore, the relationship between the secondary structures of protein and dehydration performance were strong related. With the optimal dosage of SBB and MCCP, aggregated strands and α -helix were released, indicated that the unfolding and despiralization in soluble EPS (S-EPS) were improved, disordered the sludge network, reduced the flowing resistance of bound water, finally enhancing sludge dewaterability.

Unnecessary Neuroimaging for Patients With Primary Headaches.

Background - Headache may be due to either a primary or secondary disorder, and neuroimaging assessments can play an important role when differentiating between these types of headache. Although many studies have reported no significant differences between primary headache patients and the general population in terms of abnormal neuroimaging findings, others have shown that neuroimaging may be employed to rule out secondary causes of headache that could impact morbidity and mortality. This issue remains under debate. Thus, the present study compared the neuroimaging findings of headache patients and healthy controls. Methods - This study recruited 1070 healthy controls and 1070 primary headache patients from the Chinese People's Liberation Army General Hospital. The primary headache patients were diagnosed by computerized clinical decision support systems, and re-diagnosed by a specialist. All participants were assessed with either computed tomography or magnetic resonance imaging (MRI) scans. The neuroimaging findings were classified as significant abnormalities, non-significant abnormalities, or normal. Results - All the significant abnormalities were found using MRI scans. Significant abnormalities were identified in 4 primary headache patients (0.58%) and 5 healthy controls (0.73%); the rate of significant abnormalities was not significant different between both groups (P > .05). Conclusions - The present study found that neuroimaging was unnecessary for the primary headache patients.

Silencing of COL1A2, COL6A3, and THBS2 inhibits gastric cancer cell proliferation, migration, and invasion while promoting apoptosis through the PI3k-Akt signaling pathway.

This study explores the effect of COL1A2, COL6A3, and THBS2 gene silencing on proliferation, migration, invasion, and apoptosis of gastric cancer cells through the PI3K-Akt signaling pathway. The gastric cancer microarray expression data (GSE19826, GSE79973, and GSE65801) was analyzed. Gastric cancer tissues and corresponding adjacent normal tissues were extracted from patients. Positive expression rate of PI3K, Akt, and p-Akt was measured with immunohistochemistry. Two cell lines, BGC-823 and SGC-7901, were transfected and cells were grouped into blank, negative control, COL1A2-shRNA, COL6A3-shRNA, and THBS2-shRNA groups. Expressions of COL1A2, COL6A3, and THBS2 in gastric cancer cells transfected with corresponding silencing sequences were evaluated by RT-qPCR and Western blot. MTT assay, Transwell, and cell scratch tests were conducted to evaluate cell proliferation, invasion, and migration capacity, respectively. Flow cytometry was used to evaluate cell cycle distribution and apoptosis. The positive expression of PI3K, Akt, and p-Akt was higher in gastric cancer tissues compared with adjacent normal tissues, and the mRNA expression of COL1A2, COL6A3, and THBS2 was increased in gastric cancer tissues. Akt, p-Akt, and PI3K expression drastically decreased in cells transfected with COL1A2, COL6A3, and THBS2 silencing sequences. Cells transfected with COL1A2, COL6A3, and THBS2 silencing sequences exhibited promoted apoptosis but inhibited proliferation, migration, and invasion. This study demonstrates that COL1A2, COL6A3, and THBS2 gene silencing inhibits gastric cancer cell proliferation, migration, and invasion while promoting apoptosis through the PI3K-Akt signaling pathway.

Altered Retinal Nerve Fiber Layer Thickness and Choroid Thickness in Patients with Migraine.

BACKGROUND: Migraine is a chronic neurological disorder. However, its pathogenesis is still unclear. This study aimed to measure the posterior ocular structure in patients with migraine using enhanced depth imaging (EDI)-optical coherence tomography (OCT) and explore the probable pathogenesis of migraine. METHODS: A total of 115 patients diagnosed with migraine and 50 healthy volunteers were recruited. These participants underwent an ocular examination to exclude the ocular diseases. Retinal nerve fiber layer thickness, macular thickness, and choroid thickness were assessed using EDI-OCT. RESULTS: The nasal peripapillary retinal nerve fiber layer (pRNFL) was significantly thinner in the migraine with aura group (p = 0.001) than that in the control group. The inferior inner macular layer was thinner in the migraine with aura group (p = 0.005). The 3 subfields of choroid were significantly thinner in the migraine with aura group (p = 0.044, 0.008, and 0.029). However, there was no difference between the migraine without aura group and the control group. The nasal pRNFL in migraine with aura was negatively correlated with the product of duration (months) and number of attacks/month (p = 0.039). CONCLUSION: The changes in the ocular posterior structure may serve as evidence of the trigeminovascular system mechanism underlying migraine and transneuronal retrograde degeneration of the primary visual cortex, which reflects the cortical spreading depression.

Genetic association of HCRTR2, ADH4 and CLOCK genes with cluster headache: a Chinese population-based case-control study.

BACKGROUND: Cluster headache (CH), a rare primary headache disorder, is currently thought to be a genetic susceptibility which play a role in CH susceptibility. A large numbers of genetic association studies have confirmed that the HCRTR2 (Hypocretin Receptor 2) SNP rs2653349, and the ADH4 (Alcohol Dehydrogenase 4) SNP rs1126671 and rs1800759 polymorphisms are linked to CH. In addition, the CLOCK (Circadian Locomotor Output Cycles Kaput) gene is becoming a research hotspot for CH due to encoding a transcription factor that serves as a basic driving force for circadian rhythm in humans. The purpose of this study was to evaluate the association between CH and the HCRTR2, ADH4 and CLOCK genes in a Chinese CH case-control sample. METHODS: We genotyped polymorphisms of nine single nucleotide polymorphisms (SNPs) in the HCRTR2, ADH4 and CLOCK genes to perform an association study on a Chinese Han CH case-control sample (112 patients and 192 controls),using Sequenom MALDI-TOF mass spectrometry iPLEX platform. The frequencies and distributions of genotypes and haplotypes were statistically compared between the case and control groups to identify associations with CH. The effects of SNPs on CH were further investigated by multiple logistic regression. RESULTS: The frequency of the HCRTR2 SNP rs3800539 GA genotype was significantly higher in cases than in controls (48.2% vs.37.0%). The GA genotypes was associated with a higher CH risk (OR = 1.483, 95% CI: 0.564-3.387, p = 0.038), however, after Bonferroni correction, the association lost statistical significance. Haplotype analysis of the HCRTR2 SNPs showed that among eight haplotypes, only H1-GTGGGG was linked to a reduced CH risk (44.7% vs. 53.1%, OR = 0.689, 95% CI =0.491~0.966, p = 0.030). No significant association of ADH4, CLOCK SNPs with CH was statistically detected in the present study. CONCLUSIONS: Association between HCRTR2, ADH4,CLOCK gene polymorphisms and CH was not significant in the present study, however, haplotype analysis indicated H1-GTGGGG was linked to a reduced CH risk.

Effects of shRNA-Mediated Silencing of PKM2 Gene on Aerobic Glycolysis, Cell Migration, Cell Invasion, and Apoptosis in Colorectal Cancer Cells.

This study aims to explore the effects of shRNA-mediated silencing on Pyruvate kinase type M2 (PKM2) gene during aerobic glycolysis in colorectal cancer (CRC) cells. CRC tissues and adjacent normal tissues were obtained from 136 patients diagnosed with qRT-PCR, Western blotting, and immunohistochemistry (IHC) were performed to detect mRNA and protein expressions of PKM2. CRC cells were divided into a blank, vector, and PKM2-shRNA groups. Hexokinase (HK) and PKM2 activity were both determined by glucose-6-phosphate dehydrogenase (G-6-PD) coupled colorimetric assay and enzyme coupling rate method. The extracellular lactate concentration was measured by ultraviolet spectrophotometer and caspase activity was measured using spectrophotometry. The proliferation, cell cycle, apoptosis, invasion, and migration of CRC cells were detected by cell counting kit-8 (CCK-8) assay, flow cytometry, transwell assay, and scratch test. Three groups of nude mice were injected with 0.2 mL single-cell suspension from the blank, vector, and PKM2-shRNA groups, respectively. PKM2 protein content in CRC tissues was higher than that in adjacent normal tissues. Results showed that the PKM2-shRNA group exhibited significantly lower mRNA and protein expressions of PKM2, decreased PKM2 activity, reduced lactate metabolism level, increased cell apoptosis rate, elevated caspase-3 and caspase-9 activity, weakened proliferation, and a reduction in cell invasion and migration ability compared to the vector and blank groups. The optical density (OD) value was lower in the PKM2-shRNA group than in the blank and vector groups. These findings indicate that shRNA-mediated silencing of PKM2 gene promotes apoptosis and inhibits aerobic glycolysis, proliferation, migration, and invasion in CRC cells. J. Cell. Biochem. 118: 4792-4803, 2017. © 2017 Wiley Periodicals, Inc.

Effects of PKM2 Gene Silencing on the Proliferation and Apoptosis of Colorectal Cancer LS-147T and SW620 Cells.

BACKGROUND/AIMS: This paper aims to explore the effects of pyruvate kinase (PK) M2 gene silencing on the proliferation and apoptosis of colorectal cancer (CRC) LS-147T and SW620 cells. METHODS: CRC LS-147T and SW620 cells highly expressing PKM2 were randomly selected by quantitative real-time polymerase chain reaction (qRT-PCR) and then assigned into the blank (no transfection), PKM2-shRNA (transfection with shRNA) and empty plasmid (transfection with empty plasmid) groups. Immunofluorescence was applied to detect PKM2 protein expression. qRT-PCR and Western blotting were conducted to assess mRNA and protein expression of PKM2, p53 and p21. The cell counting kit-8 (CCK-8) assay was used to assess cell proliferation. Flow cytometry was used to assess the cell cycle and apoptosis rate, and a senescence-associated ß-galactosidase staining kit was used to assess cell senescence. RESULTS: PKM2 exhibited high mRNA expression among CRC LS-147T and SW620 cells with remarkable protein expression noted in the cytoplasm and nucleus. The PKM2-shRNA group exhibited reduced PKM2 mRNA and protein expression, whereas p53 and p21 expression was increased compared with the blank and empty plasmid groups. Cell proliferation in PKM2-shRNA cells decreased significantly compared with the blank group and empty plasmid groups. The PKM2-shRNA group exhibited more cells in the G1 phase and fewer cells in the G2/M phase compared with the blank and empty plasmid groups. In addition, the PKM2-shRNA group exhibited significantly increased apoptosis rates and ß-galactosidase activity compared with the blank and empty plasmid groups. CONCLUSION: Our study demonstrates that PKM2 gene silencing suppresses proliferation and promotes apoptosis in LS-147T and SW620 cells.

Altered microRNA-9 Expression Level is Directly Correlated with Pathogenesis of Nonalcoholic Fatty Liver Disease by Targeting Onecut2 and SIRT1.

BACKGROUND MicroRNA-9 (miR-9) was detected in nonalcoholic fatty liver disease (NAFLD) patients to understand the role of miR-9 in NAFLD development. MATERIAL AND METHODS Between February 2014 and February 2015, 105 cases of NAFLD were recruited and confirmed by liver biopsy pathology, including patients with mild NAFLD (n=58) and moderate-severe NAFLD (n=47); nonalcoholic steatohepatitis (NASH) (n=53) and non-NASH (n=52); and 50 healthy participants were regarded as the healthy control group. MiR-9 expression was measured by qRT-PCR. For in vitro experiments, L-02 normal liver cells were divided into normal control group (cultured with original culture medium), dimethyl sulfoxide (DMSO) group (cultured with DMSO) and oleic acid group (cultured with oleic acid to induce fatty change), and MTT assay was used to measure the effect of different oleic acid concentrations on cell proliferation. Nile red staining was used to detect intracellular accumulation of lipid droplets. Further, synthetic miR-9 mimic and its control and miR-9 inhibitors and its control were independently transfected into L-02 cells. RESULTS MiR-9 levels in the mild NAFLD group and moderate-severe NAFLD group were significantly higher than in the healthy control group (both P<0.05). Mean fluorescence intensity of lipid droplets increased with the duration of induction, and were dramatically higher in oleate-treated L-02 cells; intracellular triglyceride (TG) content was also higher. miR-9 levels significantly increased following oleate induction. Importantly, miR-9 levels were significantly elevated upon miR-9 mimic transfection. Conversely, miR-9 level was lowered with miR-9 inhibitors transfection. Additionally, Onecut2 and SIRT1 were identified as miR-9 targets. CONCLUSIONS A positive relationship between miR-9 and steatosis was established with our results that miR-9 mimic transfection decreased intracellular lipid content. Finally, we identified 2 miR-9 targets, Onecut2 and SIRT1, which may be crucial players in NAFLD development.

AdipoQ T45 G and G276 T Polymorphisms and Susceptibility to Nonalcoholic Fatty Liver Disease Among Asian Populations: A Meta-Analysis and Meta-Regression.

BACKGROUND: Adiponectin (AdipoQ) is an adipose-derived plasma protein that plays an important role in hepatic lipoprotein-lipid metabolism. Emerging evidence have shown that two common polymorphisms (T45 G and G276 T) in the AdipoQ gene may contribute to increasing susceptibility to nonalcoholic fatty liver disease (NAFLD); however individually published studies show inconclusive results. This meta-analysis aimed to derive a more precise estimation of the association of AdipoQ T45 G (rs2241766 T>G) and G276 T (rs1501299 G>T) polymorphisms with NAFLD risk. METHOD: Potential relevant studies were identified covering the following databases: PubMed, Embase, Web of Science, the Cochrane Library, Chinese National Knowledge Infrastructure (CNKI), Chinese Bio-medicine Database (CBM), and Chinese Sci-tech Journals databases. Statistical analyses were calculated using the version 12.0 STATA software (Stata Corp, College Station, TX, USA). Odds ratios (ORs) and its corresponding 95% confidence interval (CI) were calculated. RESULT: Ten case-control studies were included with a total of 2,672 subjects, of these 1,117 being NAFLD patients and 1,555 being healthy controls. Our meta-analysis results revealed that the T variant of AdipoQ rs2241766 T>G polymorphism may be associated with an increased risk of NAFLD. There was also a significant association between the G variant of AdipoQ rs1501299 G>T polymorphism and an increased risk of NAFLD. Country-stratified analysis indicated that a higher AdipoQ rs2241766 T>G polymorphism was closely related with an increased risk of NAFLD in Chinese and Indian populations (all Ps < 0.05); a similar result was observed in Chinese populations between AdipoQ rs2241766 T>G polymorphism and an increased risk of NAFLD (P < 0.05). CONCLUSION: In conclusion, the current meta-analysis indicates that AdipoQ rs2241766 T>G and rs1501299 G>T polymorphisms may contribute to an increasing susceptibility to NAFLD. Moreover, this meta-analysis also suggests for future larger studies with stratified case-control population, and greater focus on the gene-environment interactions regarding NAFLD susceptibility for valid studies.

Clinical and structural characteristics of NEU1 variants causing sialidosis type 1.

Purpose: Sialidosis type 2 has variants that are both catalytically inactive (severe), while sialidosis type 1 has at least one catalytically active (mild) variant. This study aimed to discuss the structural changes associated with these variants in a newly reported family carrying NEU1 variants and explore the clinical characteristics of different combinations of variants in sialidosis type 1. Methods: First, whole-exome sequencing and detailed clinical examination were performed on the family. Second, structural analysis, including energy, flexibility and polar contacts, was conducted for several NEU1 variants, and a sialidase activity assay was performed. Third, previous NEU1 variants were systematically reviewed, and the clinical characteristics of patients in the severe-mild and mild-mild groups with sialidosis type 1 were analyzed. Results: We report a novel family with sialidosis type 1 and the compound heterozygous variants S182G and V143E. The newly identified V143E variant was predicted to be a mild variant through structural analysis and was confirmed by sialidase activity assay. The cherry-red spot was more prevalent in the severe-mild group, and ataxia was more common in the mild-mild group. Impaired cognition was found only in the severe-mild group. Moreover, patients with cherry-red spots and abnormal EEGs and VEPs had a relatively early age of onset, whereas patients with myoclonus had a late onset. Conclusion: Changes in flexibility and local polar contacts may be indicators of the NEU1 pathogenicity. Sialidosis type 1 can be divided into two subgroups according to the variant combinations, and patients with these two subtypes have different clinical characteristics.