Aim To produce poly (lactic-co-glycolic acid) (PLGA) microspheres, containing a staphylokinase variant (K35R, DGR) with reduced immunogenecity and antiplatclet aggregation activities,which allowed the preservation of protein stability during both particle processing and drug release.Methods DGR-loaded microspheres were fabricated using a double emulsion-solvent evaporation technique. The effects of preparative parameters, such as stirring rate, polymer concentration, and the excipients of both internal and external aqueous phase (W2 ), on DGR encapsulation efficiency and microsphere characteristics were investigated. In vitro and in vivo release of DGR were conducted and the cause for instability of DGR during release was also investigated. Results Moderate ultrasonic treatment of aqueous DGR/dichloromethane mixtures caused approximately. Eighty four per cent DGR denaturation.However, the activity recovery of DGR almost amounted to 100% when 2% polyvinyl alcohol (PVA) was added into the aqueous phase. It was found that NaCl in the external water phase significantly increased DGR encapsulation efficiency. Furthermore, NaCl in the external water phase played a role in determining size and surface morphology of microsphere. In vitro release test showed a burst release of DGR from microspheres, followed by sustained release of 50% total activity over 15 days. In vivo experiments showed that DGR released from microspheres sustained 5 days. Denaturation of DGR within microspheres might be resulted from acidic microclimate. Conclusion The stability of DGR was effectively protected during microencapsulation and a relatively high encapsulation efficiency of DGR was obtained. PLGA microspheres could be an effective carrier for DGR.

OBJECTIVETo search novel method for diagnosis and therapy of B-lymphoma, specific small molecular peptide ligands against binding site of tumor cells were screened and its effects on signal transduction and cell apoptosis were tested.

METHODSSpecific peptide ligands were screened by binding with site of human B lymphoma cell (OC1LY8) using peptide-bead libraries. The identified peptides were characterized with responsible cells by rebinding test. The role of tyrosine phosphorylation of peptide ligand was tested by Western blot; and its apoptosispromoting role was observed by confocal fluorescent microscope.

RESULTSSpecific peptide ligand was able to bind specifically to site on cell surface and enter into cytoplasm. Tetrameric peptide ligand was able to strongly trigger signal transduction resulting in tyrosine phosphorylation and cellular apoptosis in OC1LY8 cell line.

CONCLUSIONScreened peptide ligand can effectively bind with OC1LY8 cell, stimulate cellular tyrosine phosphorylation and induce cellular apoptosis.

Amino Acid Sequence ; Apoptosis ; drug effects ; Cell Line, Tumor ; Humans ; Ligands ; Lymphoma, B-Cell ; metabolism ; pathology ; Oligopeptides ; chemistry ; pharmacology ; Peptide Library ; Phosphorylation ; drug effects ; Signal Transduction ; drug effects ; Tyrosine ; metabolism

OBJECTIVETo assess the effects of nuclear translocation of tissue transglutaminase (TTG) and the release of cytochrome C on hepatocyte apoptosis and to reveal the mechanism of signal transduction of early apoptosis in injured hepatocytes.

METHODSHepatocytes isolated from tissue transglutaminase gene knock-out mice and rats were stimulated with ethanol. Proteins from whole cell, cytoplasm and nuclei were extracted for determination of TTG activity by (14)C-putrescine incorporation. Distribution of TTG throughout the entire cell, as well as just nucleus was observed under a confocal scanning microscope. The amount of cytochrome C released from mitochondria was determined by ELISA. Cell apoptosis was observed by fluorescent cytochemistry.

RESULTSTTG activity in whole cells and nuclei was significantly increased after the hepatocytes were treated with ethanol. Cytochrome C release was remarkably increased in the cells isolated from rat and wild-type mouse after treatment with ethanol but not in TTG gene knock-out mice. Cellular apoptosis appeared in hepatocytes isolated from rats and wild-type mice but not in the hepatocytes from TTG gene knock-out mice after stimulation with ethanol.

CONCLUSIONSIncreased TTG in hepatocytes can be translocated into the nucleus and promote release of mitochondrial cytochrome C into the cytoplasm. Passing through a series of signal pathways, hepatocyte apoptosis is induced eventually.

Animals ; Apoptosis ; Cell Nucleus ; metabolism ; Cytochrome c Group ; metabolism ; Hepatocytes ; cytology ; metabolism ; Male ; Mice ; Rats ; Signal Transduction ; Transglutaminases ; metabolism

DNA barcoding has been widely used for herb identification in recent decades,enabling safety and innovation in the field of herbal medicine.In this article,we summarize recent progress in DNA bar-coding for herbal medicine to provide ideas for the further development and application of this tech-nology.Most importantly,the standard DNA barcode has been extended in two ways.First,while conventional DNA barcodes have been widely promoted for their versatility in the identification of fresh or well-preserved samples,super-barcodes based on plastid genomes have rapidly developed and have shown advantages in species identification at low taxonomic levels.Second,mini-barcodes are attractive because they perform better in cases of degraded DNA from herbal materials.In addition,some mo-lecular techniques,such as high-throughput sequencing and isothermal amplification,are combined with DNA barcodes for species identification,which has expanded the applications of herb identification based on DNA barcoding and brought about the post-DNA-barcoding era.Furthermore,standard and high-species coverage DNA barcode reference libraries have been constructed to provide reference se-quences for species identification,which increases the accuracy and credibility of species discrimination based on DNA barcodes.In summary,DNA barcoding should play a key role in the quality control of traditional herbal medicine and in the international herb trade.

ObjectiveTo investigate the different effects of Yunvjian with or without Achyranthis Bidentatae Radix on glucose and lipid metabolism and inflammatory response in diabetic rats with the syndrome of Yin deficiency and internal heat. MethodThe rat model of diabetes due to Yin deficiency and internal heat was established by feeding with a high-sugar and high-fat diet and injection of thyroxine and streptozotocin. The successfully modeled rats were randomized into model control， Yunvjian without Achyranthis Bidentatae Radix （11.8 g·kg^-1）， Yunvjian with Achyranthis Bidentatae Radix （12.8 g·kg^-1）， and Achyranthis Bidentatae Radix （1.0 g·kg^-1） groups （n=10）， and another 10 rats were taken as the normal control group. Each group was administrated with corresponding drugs or saline by gavage for 28 days. The fasting blood glucose （FBG）， fasting insulin （FINS）， total cholesterol （TC）， triglycerides （TG）， low-density lipoprotein cholesterol （LDL-C）， and high-density lipoprotein cholesterol （HDL-C） in rats were measured. Enzyme-linked immunosorbent assay was employed to determine the levels of cyclic adenosine monophosphate （cAMP）， cyclic guanosine monophosphate （cGMP）， triiodothyronine （T₃）， thyroxine （T₄）， interleukin （IL）-1β， IL-6， tumor necrosis factor （TNF）-α， and C-reactive protein （CRP） in the serum. The histopathological changes of the liver were observed. The expression of lipoxygenase-2 （COX-2） was detected by immunofluorescence. The mRNA levels of nuclear transcription factors-κB （NF-κB）， monocyte chemotactic protein-1 （MCP-1）， and intercellular adhesion molecule-1 （ICAM-1） were determined by real-time polymerase chain reaction （Real-time PCR）.Western blot was employed to determine the protein levels of NF-κB in hibitory protein（IκB） kinase β （IKKβ）， IκBα， and phosphorylated IκBα （p-IκBα） in the liver and the protein levels of NF-κB in the cytoplasm and nucleus. ResultCompared with the normal group， the model group showed elevated levels of FBG， FINS， insulin resistance index， TC， TG， LDL-C， cAMP， T₃， T₄， IL-1β， IL-6， TNF-α， and CRP， up-regulated mRNA levels of NF-κB， MCP-1， and ICAM-1， and up-regulated protein levels of COX-2， p-IκBα， and nuclear NF-κB （P<0.01）. Compared with the model group， Yunvjian without Achyranthis Bidentatae Radix lowered the levels of FBG， FINS， insulin resistance index， TC， TG， LDL-C， cAMP， T₃， T₄， IL-1β， IL-6， TNF-α， and CRP， down-regulated the mRNA levels of NF-κB， MCP-1， and ICAM-1， and down-regulated the protein levels of COX-2， p-IκBα and nuclear NF-κB （P<0.05， P<0.01）. Compared with the Yunvjian without Achyranthis Bidentatae Radix， Yunvjian with Achyranthis Bidentatae Radix showed lowered levels of FBG， FINS， insulin resistance index， and inflammatory cytokines， down-regulated mRNA levels of NF-κB， MCP-1， and ICAM-1， and down-regulated protein levels of p-IκBα and nuclear NF-κB （P<0.05， P<0.01）. ConclusionAchyranthis Bidentatae Radix can enhance the performance of Yunvjian in reducing blood glucose and inhibiting inflammation in diabetic rats with the syndrome of yin deficiency and internal heat by down-regulating the IKK/IκB/NF-κB signaling pathway.

Objective:To investigate the diagnostic accuracy of serological indicators and evaluate the diagnostic value of a new established combined serological model on identifying the minimal hepatic encephalopathy (MHE) in patients with compensated cirrhosis.Methods:This prospective multicenter study enrolled 263 compensated cirrhotic patients from 23 hospitals in 15 provinces, autonomous regions and municipalities of China between October 2021 and August 2022. Clinical data and laboratory test results were collected, and the model for end-stage liver disease (MELD) score was calculated. Ammonia level was corrected to the upper limit of normal (AMM-ULN) by the baseline blood ammonia measurements/upper limit of the normal reference value. MHE was diagnosed by combined abnormal number connection test-A and abnormal digit symbol test as suggested by Guidelines on the management of hepatic encephalopathy in cirrhosis. The patients were randomly divided (7∶3) into training set ( n=185) and validation set ( n=78) based on caret package of R language. Logistic regression was used to establish a combined model of MHE diagnosis. The diagnostic performance was evaluated by the area under the curve (AUC) of receiver operating characteristic curve, Hosmer-Lemeshow test and calibration curve. The internal verification was carried out by the Bootstrap method ( n=200). AUC comparisons were achieved using the Delong test. Results:In the training set, prevalence of MHE was 37.8% (70/185). There were statistically significant differences in AMM-ULN, albumin, platelet, alkaline phosphatase, international normalized ratio, MELD score and education between non-MHE group and MHE group (all P<0.05). Multivariate Logistic regression analysis showed that AMM-ULN [odds ratio ( OR)=1.78, 95% confidence interval ( CI) 1.05-3.14, P=0.038] and MELD score ( OR=1.11, 95% CI 1.04-1.20, P=0.002) were independent risk factors for MHE, and the AUC for predicting MHE were 0.663, 0.625, respectively. Compared with the use of blood AMM-ULN and MELD score alone, the AUC of the combined model of AMM-ULN, MELD score and education exhibited better predictive performance in determining the presence of MHE was 0.755, the specificity and sensitivity was 85.2% and 55.7%, respectively. Hosmer-Lemeshow test and calibration curve showed that the model had good calibration ( P=0.733). The AUC for internal validation of the combined model for diagnosing MHE was 0.752. In the validation set, the AUC of the combined model for diagnosing MHE was 0.794, and Hosmer-Lemeshow test showed good calibration ( P=0.841). Conclusion:Use of the combined model including AMM-ULN, MELD score and education could improve the predictive efficiency of MHE among patients with compensated cirrhosis.

Silicosis is a leading cause of occupational disease-related morbidity and mortality worldwide, but the molecular basis underlying its development remains unclear. An accumulating body of evidence supports gasdermin D (GSDMD)-mediated pyroptosis as a key component in the development of various pulmonary diseases. However, there is little experimental evidence connecting silicosis and GSDMD-driven pyroptosis. In this work, we investigated the role of GSDMD-mediated pyroptosis in silicosis. Single-cell RNA sequencing of healthy and silicosis human and murine lung tissues indicated that GSDMD-induced pyroptosis in macrophages was relevant to silicosis progression. Through microscopy we then observed morphological alterations of pyroptosis in macrophages treated with silica. Measurement of interleukin-1β release, lactic dehydrogenase activity, and real-time propidium iodide staining further revealed that silica induced pyroptosis of macrophages. Additionally, we verified that both canonical (caspase-1-mediated) and non-canonical (caspase-4/5/11-mediated) signaling pathways mediated silica-induced pyroptosis activation, in vivo and in vitro. Notably, Gsdmd knockout mice exhibited dramatically alleviated silicosis phenotypes, which highlighted the pivotal role of pyroptosis in this disease. Taken together, our results demonstrated that macrophages underwent GSDMD-dependent pyroptosis in silicosis and inhibition of this process could serve as a viable clinical strategy for mitigating silicosis.