Measurement of enzyme activity of insoluble substrates based on ordered porous layer interferometry and the application in evaluation of thrombolytic drugs.

The development of innovative methods for real-time surveillance of enzymatic activity determination processes is essential, particularly for insoluble substrate enzymatic assessments. In this work, a novel method for enzymatic activity determination was devised by assembling a 190 nm silica colloidal crystal (SCC) film onto a glass slide, coupled with Ordered Porous Layer Interferometry (OPLI) technology. By fixing the substrate of the enzyme on the surface of the silica sphere, a solid-liquid interface can be formed for monitoring enzymatic activity. The enzymatic activity is gauged by the change in the SCC film's thickness caused by the digestion of the loaded substrate. The procedure of chymotrypsin-mediated casein digestion was documented in real time, facilitating the examination of chymotrypsin's activity and kinetics. The newly-developed enzymatic activity determination method demonstrated exceptional sensitivity towards chymotrypsin activity, with a linear range spanning 0.0505-2.02 units per mg. Additionally, the method was extended to the assessment of fibrinolysis enzyme activity and kinetic analysis, yielding promising results. Therefore, this technique can serve as a real-time, user-friendly, cost-effective novel approach for enzymatic activity determination, providing fresh perspectives for enzymatic activity determination studies.

The Jiangtang Tongmai Prescription Inhibits Inflammation and Fibrosis of Lung Fibroblast Autophagy Induced by Hyperglycemia by Regulating CAV1 Expression.

OBJECTIVE: The lung is one of the target organs of diabetes. This study aimed to probe the protective mechanism of Jiangtang Tongmai Prescription (JTTMP) against diabetic lung injury. METHODS: JTTMP-containing serum was collected, and a high glucose and high-fat diabetic cell model was established. The cells were treated with a drug-containing serum or a CAV1-associated vector. Transfection efficiency was measured by qRT-PCR and western blot, the cell proliferative capacity was tested by CCK-8 assay, and the expression of autophagosome marker LC3B was measured by immunophluorescence assay. Expression levels of the autophagy markers LC3B, p62, and Beclin-1, and the expression levels of the fibrosis markers α-SMA, FN-1, and TGF-ß1 were determined by western blot, and the levels of inflammatory factors TNF-α and IL-1ß in the supernatants were assessed by ELISA. RESULTS: In high glucose and high fat-induced MRC-5 cells, JTTMP-containing serum impeded the abnormal cell proliferation and the expression levels of autophagy markers, fibrosis markers, as well as inflammatory factors. CAV1 expression was decreased in MRC-5 cells treated with JTTMP-containing serum. In MRC-5 cells upon transfection with the CAV1 overexpression vector and treatment with JTTMP-containing serum, increased cell proliferation, increased LC3B, p62, Beclin-1, α-SMA, FN-1, and TGF-ß1, TNF-α, and IL-1ß levels were found compared with cells treated with JTTMP-containing serum alone. CONCLUSION: This study suggests that JTTMP suppresses CAV1 expression to attenuate diabetic lung injury by reducing abnormal proliferation and autophagy, and reducing levels of fibrosis and inflammation.

Liraglutide Accelerates Ischemia-Induced Angiogenesis in a Murine Diabetic Model.

Background Severe hindlimb ischemia is a chronic disease with poor prognosis that can lead to amputation or even death. This study aimed to assess the therapeutic effect of liraglutide on hind-limb ischemia in type 2 diabetic mice and to elucidate the underlying mechanism. Methods and Results Blood flow reperfusion and capillary densities after treatment with liraglutide or vehicle were evaluated in a mouse model of lower-limb ischemia in a normal background or a background of streptozotocin-induced diabetes. The proliferation, migration, and tube formation of human umbilical vein endothelial cells were analyzed in vitro upon treatment with liraglutide under normal-glucose and high-glucose conditions. Levels of phospho-Akt, phospho-endothelial nitric oxide synthase, and phospho-extracellular signal-related kinases 1 and 2 under different conditions in human umbilical vein endothelial cells and in ischemic muscle were determined by western blotting. Liraglutide significantly improved perfusion recovery and capillary density in both nondiabetic and diabetic mice. Liraglutide also promoted, in a concentration-dependent manner, the proliferation, migration, and tube formation of normal glucose- and high glucose-treated human umbilical vein endothelial cells, as well as the phosphorylation of Akt, endothelial nitric oxide synthase, and extracellular signal-related kinases 1 and 2 both in vitro and in vivo. The liraglutide antagonist exendin (9-39) reversed the promoting effects of liraglutide on human umbilical vein endothelial cell functions. Furthermore, exendin (9-39), LY294002, and PD98059 blocked the liraglutide-induced activation of Akt/endothelial nitric oxide synthase and extracellular signal-related kinases 1 and 2 signaling pathways. Conclusions These studies identified a novel role of liraglutide in modulating ischemia-induced angiogenesis, possibly through effects on endothelial cell function and activation of Akt/endothelial nitric oxide synthase and extracellular signal-related kinases 1 and 2 signaling, and suggested the glucagon-like peptide-1 receptor may be an important therapeutic target in diabetic hind-limb ischemia.

Transcriptome Analysis of Traditional Chinese Medicine 'Kechuanning Plaster' in the Treatment of Asthma.

BACKGROUND: Asthma is a severe chronic inflammatory airway disease. Kechuanning plaster has excellent efficacy in the treatment of asthma. OBJECTIVE: The aim of this study was to analyze the molecular mechanisms of Kechuanning plaster in the treatment of asthma. METHODS: An asthma model was constructed using Sprague Dawley rats. Differentially expressed genes (DEGs) were screened in three rat groups: the control (normal rats), model (asthma rats), and treatment (asthma rats treated with Kechuanning) groups. After enrichment analysis of the DEGs, the protein-protein interactions (PPIs) of the DEGs were analyzed, and transcription factors and microRNAs (miRNAs) that regulate DEGs were predicted. Finally, western blotting (WB) and immunohistochemical (IHC) analysis was performed to validate protein expression. RESULTS: A total of 745 DEGs were identified and enriched in 93 Gene Ontology terms and 25 Kyoto Encyclopedia of Genes and Genomes pathways. A PPI network, consisting of 224 protein nodes and 368 edges, was constructed. The nuclear factor of activated T cells 2 (NFATc2) was predicted to have binding sites in 61 DEGs. The miRNA-target interaction network included 24 DEGs and 9 miRNAs. WB and IHC analysis demonstrated that the fatty acid-binding protein 5 (FABP5) and the chemokine (C-X-C motif) ligand 3 (CXCL3) had higher expression in the model group and lower expression in the control and treatment groups. CONCLUSION: We concluded that FABP5, CXCL3, suppressor of cytokine signaling 3 (SOCS3), E1A binding protein P300 (EP300), NFATc2, microRNA 495 (miR-495), and miR-30 may play important roles in treating asthma.

Jiangtang Tongmai Prescription Reduced Diabetic Lung Injury Through SnoN and TGF-ß1/Smads Signaling Pathway.

By establishing a rat diabetes model in rats with intervening treatment by Jiangtang Tongmai Prescription (JTTMP), this study explored the restorative pairing effect of JTTMP on diabetic lung injury. The model of type II diabetes model was used to establish the rat diabetes model, using a high-fat diet and streptozotocin (STZ) induction. Different doses of JTTMP and metformin were administered as a therapeutic to intervene, and blood was collected to assess the blood glucose level of each group of rats. HE (Hematoxylin and eosin (H&E) staining was performed to detect the morphological changes in rat lung tissue and enzyme-linked immunoassay ELISA was used to detect and quantify the expression of interleukin (IL)-6, TNF tumor necrosis factor-ɑa, and IL-1ß in serum and the lung tissue of each group of rats. The level expression of TGF-ß1 [transforming growth factor (TGF)-ß1), SnoN (transcriptional co-repressor Ski-N terminal (SnoN)], Smad2, Smad3, Smad7, and other signaling pathway proteins were assessed by Western blot. In comparison with the normal control (NC) group, rats in the diabetes model (DM) group lost weight and showed significantly increased blood sugar levels. The levels of TGF-ß1 and Smad2/3 were increased in the DM group but Smad7 decreased. After 8 weeks of JTTMP intervention, the level of TGF-ß1 and Smad2/3 decreased but Smad7 increased, blood sugar decreased significantly and the expression of inflammatory factors in lung tissue decreased. Therefore, JTTMP may activate SnoN and the downstream TGF-ß1/Smads signaling pathway to repair diabetic lung injury, which suggests its application has potential for future clinical treatment of diabetes with lung injury.

Secreted frizzled-related protein 5 promotes angiogenesis of human umbilical vein endothelial cells and alleviates myocardial injury in diabetic mice with myocardial infarction by inhibiting Wnt5a/JNK signaling.

The purpose of this study is to investigate whether secreted frizzled-related protein 5 (SFRP5) affects the proliferation, migration and angiogenesis of human umbilical vein endothelial cells (HUVECs) induced by high glucose (HG). HUVECs were treated with different concentrations of glucose. MTT, wound healing, angiogenesis, and ELISA assays were used to detect cell cytotoxicity, migration, tube formation, and VEGF165 and VEGF165b levels, respectively. The mice model of type 2 diabetes mellitus (T2DM) complicated with myocardial infarction (MI) was established. SFRP5 was injected intrabitoneally for 2 weeks. cardiac output (CO), left ventricular ejection fraction (LVEF) and left ventricular shortening fraction (LVSF) were detected by echocardiography. Western blot was used to detect the protein levels of SFRP5, Wnt5a, JNK1/2/3, p-JNK1/2/3, TGF-ß1, Caspase3, Bax, and Bcl-2. The expression of SFRP5 was declined in HG-induced HUVECs and T2DM-MI. Intervention of SFRP5 promoted the migration of HUVECs and angiogenesis, as evidenced by a lower expression of Bax and caspase3, but a higher expression of Bcl-2. Meanwhile, SFRP5 inhibition repress Wnt5a and p-JNK expression. Howerver, The JNK inhibitor (SP600125) enhanced the down-regulation of Wnt5a/JNK pathway proteins by SFRP5. SFRP5 intervention increased the levels of CO, LVSF, and LVEF in T2DM-MI mice. SFRP5 inhibited myocardial pathological injury and fibrosis in T2DM-MI mice and SFRP5 could down-regulate Wnt5a and p-JNK1/2/3 activation. SFRP5 promotes the proliferation, migration and angiogenesis of HUVECs induced by HG, and inhibits cardiac dysfunction, pathological damage, fibrosis, and myocardial angiogenesis in diabetic myocardial ischemia mice, which is achieved by inhibiting Wnt5a/JNK signaling.

The Hippo Transducer YAP/TAZ as a Biomarker of Therapeutic Response and Prognosis in Trastuzumab-Based Neoadjuvant Therapy Treated HER2-Positive Breast Cancer Patients.

BACKGROUND: We explored the therapeutic and prognostic effect of YAP/TAZ intensityinHER2-positive breast cancer patients. We also investigated the relationship between YAP/TAZ expression and Trastuzumab-resistance. METHODS: We collected clinicopathological information from 397 cases. We evaluated therapeutic and prognostic effect of YAP/TAZ and other variables. We also cultivated Trastuzumab-resistance cell lines and explored relationship between YAP/TAZ and Trastuzumab-resistance. RESULTS: Over-expression of YAP/TAZ was remarkable in Trastuzumab-resistant cells, and so did HER3 and HER2/HER3 heterodimer. Inhibition of YAP/TAZ expression reversed Trastuzumab-resistance.YAP/TAZ deficiency contributed to favorable therapeutic response, and so did hormone receptor insufficiency and chemotherapy dosage inferiority. Deficient YAP/TAZ intensity and abundant hormone receptor intensity contributed to better survival. Over-expression of YAP/TAZ was obvious in recurrent cases in comparison with their matching primary lesions. Prognostic superiority of insufficient YAP/TAZ intensity was more outstanding in hormone receptor negative cases. Over-expression of YAP/TAZ and HER3 was generally synchronous. Absence of HER3 expression in residual lesions might correlate with better breast cancer-free survival. CONCLUSIONS: Over-expression of YAP/TAZ as well as HER-3 and HER2/HER3 heterodimer was synchronously remarkable in Trastuzumab-resistant cell lines. Inhibition of YAP/TAZ expression reversed Trastuzumab resistance. Deficient YAP/TAZ intensity as well as insufficient hormone receptor intensity and high chemotherapy dosage contributed to favorable therapeutic response. Deficient YAP/TAZ intensity and abundant hormone receptor intensity contributed to better survival, and so did absence of HER3expression in residual lesions. Prognostic superiority of YAP/TAZ expression depended on hormone receptor status. Cases with synchronous over-expression of YAP/TAZ and HER3 suffered poor survival, which revealed the potential effect of YAP/TAZ-HER2/HER3 crosstalk in prognosis of HER2-positive patients.

Prognostic Factors for Luminal B-like Breast Cancer.

This study aimed to examine the prognostic factors of luminal B-like breast cancer. Clinical data of 695 luminal B-like breast cancer patients who had been treated in our hospital during the period of past 4.5 years were collected and analyzed. Estrogen receptor (ER), progesterone receptor (PgR), antigen identified by monoclonal antibody Ki-67 (Ki67) were immunohistochemically detected. Different cutoffs of ER, PgR, and Ki67 were evaluated. Pearson χ2 test was performed to compare categorical parameters. Univariate and multivariate models were used to evaluate predictors of disease free survival (DFS). The results showed that patients who were younger, and had larger tumors, and more positive lymph nodes were more likely to receive neo-adjuvent chemotherapy (NAC). Patients with ER-positive tumors having <10% positive cells received more anthracycline- and taxane-based chemotherapy and less endocrine therapy than those with ER-positive tumors having ≥10% positive cells (P=0.004 and P=0.007, respectively); however, patients with ER-positive tumors having <10% positive cells experienced more recurrence (P<0.001). PgR expression levels were not associated with therapeutic schedule and DFS. Patients with tumor tissue Ki67 score ≥30% received more anthracycline- and taxane-based chemotherapy and had worse DFS than those with tumor tissue Ki67 score <30%. Univariate and multivariate analysis showed that clinical T stage, lymph nodes, ER, Ki67, and HER2 status were independent prognostic factors. In conclusion, ER-positive rate <10% and Ki67 score ≥30%, similar to higher clinical T stage, more metastatic lymph nodes, and HER2 positive status, may indicate a worse prognosis for luminal B-like breast cancer patients. Multi-center prospective trials with larger sample sizes are necessary for the continued perfection of our work.

NEK4 kinase regulates EMT to promote lung cancer metastasis.

Epithelial-to-mesenchymal transition (EMT) is a dynamic transitional state from the epithelial to mesenchymal phenotypes. Numerous studies have suggested that EMT and its intermediate states play important roles in tumor invasion and metastasis. To identify novel regulatory molecules of EMT, we screened a siRNA library targeting human 720 kinases in A549 lung adenocarcinoma cells harboring E-cadherin promoter-luciferase reporter vectors. NIMA-related kinase-4 (NEK4) was identified and characterized as a positive regulator of EMT in the screening. Suppression of NEK4 resulted in the inhibition of cell migration and invasion, accompanying with an increased expression of cell adhesion-related proteins such as E-cadherin and ZO1. Furthermore, NEK4 knockdown caused the decreased expression of the transcriptional factor Zeb1 and Smads proteins, which are known to play key roles in EMT regulation. Consistently, overexpression of NEK4 resulted in the decreased expression of E-cadherin and increased expression of Smad3. Using a mouse model with tail vein injection of NEK4 knockdown stable cell line, we found a lower rate of tumor formation and metastasis of the NEK4-knockdown cells in vivo. Thus, this study demonstrates NEK4 as a novel kinase involved in regulation of EMT and suggests that NEK4 may be further explored as a potential therapeutic target for lung cancer metastasis.

Molecular mechanisms and treatment of radiation-induced lung fibrosis.

Radiation-induced lung fibrosis (RILF) is a severe side effect of radiotherapy in lung cancer patients that presents as a progressive pulmonary injury combined with chronic inflammation and exaggerated organ repair. RILF is a major barrier to improving the cure rate and well-being of lung cancer patients because it limits the radiation dose that is required to effectively kill tumor cells and diminishes normal lung function. Although the exact mechanism is unclear, accumulating evidence suggests that various cells, cytokines and regulatory molecules are involved in the tissue reorganization and immune response modulation that occur in RILF. In this review, we will summarize the general symptoms, diagnostics, and current understanding of the cells and molecular factors that are linked to the signaling networks implicated in RILF. Potential approaches for the treatment of RILF will also be discussed. Elucidating the key molecular mediators that initiate and control the extent of RILF in response to therapeutic radiation may reveal additional targets for RILF treatment to significantly improve the efficacy of radiotherapy for lung cancer patients.

Fulvic acid degradation using nanoparticle TiO2 in a submerged membrane photocatalysis reactor.

The degradation of fulvic acid (FA) by nanoparticle TiO2 in a submerged membrane photocatalysis (SMPC) reactor was studied. In this reactor, photocatalytic oxidation and membrane separation co-occured. The continuous air supplier provided O2 for the photocatalytical reaction and mixed the solution through an airflow controller. The particle TiO2 could automatically settle due to gravity without particle agglomeration so it could be easily separated by microfiltration (MF) membrane. It was efficient to maintain high flux of membranes. The effects of operational parameters on the photocatalytic oxidation rate of FA were investigated. Results indicated that photocatalyst at 0.5 g/L and airflow at 0.06 m3/h were the optimum condition for the removal of fulvic acid, the removal efficiency was higher in acid media than that in alkaline media. The effects of different filtration duration on permeate flux rate of MF with P25 powder and with nanoparticle TiO2 were compared. Experimental results indicated that the permeate flux rate of MF was improved and the membrane fouling phenomenon was reduced with the addition of nanoparticle TiO2 catalyst compared with conventional P25 powder. Therefore, this submerged membrane photocatalysis reactor can faciliate potential application of photocatalytic oxidation process in drinking water treatment.