[Expression of MicroRNA-3162-3p in Different Clinical Stages of Children with Primary Immune Thrombocytopenia and Its Significance].

OBJECTIVE: To explore the expression of microRNA-3162-3p in different clinical stages of childhood primary immune thrombocytopenia (ITP) and its significance. METHODS: Ninety-six children with ITP were enrolled and divided into new diagnosis group (n=40), persistent group (n=30) and chronic group (n=26) according to the course of disease. 80 healthy children were selected as the control group. Peripheral blood mononuclear cells (PBMNC) of ITP children and healthy children were isolated and cultured, and the expression of microRNA-3162-3p in PBMNC of subjects was detected by real-time fluorescence quantitative PCR. The contents of IL-17, IL-23, IL-10 and TGF-ß in PBMNC of subjects were determined by ELISA. The correlation between microRNA-3162-3p and platelet count, IL-17, IL-23, IL-10 and TGF-ß was analyzed. RESULTS: Compared with the control group, the expression of microRNA-3162-3p and IL-10 in PBMNC and platelet count of ITP children were significantly decreased（P < 0.05）, while IL-17, IL-23 and TGF-ß were significantly increased (P < 0.05). With the prolongation of the disease course, the expressions of microRNA-3162-3p and IL-10 in PBMNC and platelet count were significantly decreased（P < 0.05）, while the expressions of IL-17, IL-23 and TGF-ß were significantly increased (P < 0.05). The expression of microRNA-3162-3p in PBMNC was positively correlated with platelet count and IL-10 (r =0.716, 0.667), and negatively correlated with IL-17, IL-23, and TGF-ß (r =-0.540, -0.641, -0.560). CONCLUSION: MicroRNA-3162-3p expression is significantly reduced in PBMNC of children with ITP, and is involved in the regulation of Th17/Treg imbalance, which can be used as a potential therapeutic target of ITP.

Induction of apoptosis and autophagy by dichloromethane extract from Patrinia scabiosaefolia Fisch on acute myeloid leukemia cells.

Patrinia scabiosaefolia Fisch (PS), a perennial herb belonging to the genus Pinus in the family Pinnacle Sauce, has been previously known for its analgesic, anti-inflammatory, antibacterial, and antitumor properties. However, the specific mechanism behind its antileukemic effect remains unknown. This study focused on the cytotoxicity and potential modes of action of the dichloromethane extract from PS (DEPS) in acute myeloid leukemia (AML) cells. Our results demonstrated that DEPS reduced cell viability, arrested the cell cycle in the G2/M phase, disrupted the mitochondrial membrane potential, increased reactive oxygen species (ROS) production, and upregulated the expression of Bax/Bcl-2 and Cleaved caspase-3. However, the impact of DEPS on cell viability and the expression of apoptosis-associated proteins was reversed upon pretreatment with the caspase-3 inhibitor (Z-DEVD-FMK) in HL-60 cells, which demonstrated that DEPS could induce apoptosis through the mitochondria-associated apoptotic pathway. Interestingly, DEPS also influenced autophagy by upregulating the expression of LC3II/I, P62, and Beclin-1 proteins, and the autophagy inhibition chloroquine(CQ) could attenuate the apoptotic effects of DEPS in HL-60 cells. Furthermore, SMART 2.0 analysis predicted that the main components present in DEPS were likely terpenoids. In conclusion, DEPS possibly exerts antileukemic effects by downregulating the PI3K/AKT and ERK pathways, thereby promoting intracellular ROS production, activating the mitochondrial apoptotic pathway, and affecting autophagy, providing valuable insights for the potential future application of PS in the treatment of AML.

[Mechanism of Proliferation and Apoptosis of Acute Promyelocytic Leukemia Cell Line NB4 Induced by TPA].

OBJECTIVE: To investigate the effect of phorbol-12-myristate-13-ace-tate (TPA) on the proliferation and apoptosis of acute promyelocytic leukemia cell line NB4 and its molecular mechanism. METHODS: The effect of different concentrations of TPA on the proliferation of NB4 cells at different time points was detected by CCK-8 assay. The morphological changes of NB4 cells were observed by Wright-Giemsa staining. The cell cycle and apoptosis of NB4 cells after TPA treatment were detected by flow cytometry. The mRNA expressions of NB4 cells after TPA treatment were analyzed by high-throughput microarray analysis and real-time quantitative PCR. Western blot was used to detect the protein expression of CDKN1A, CDKN1B, CCND1, MYC, Bax, Bcl-2, c-Caspase 3, c-Caspase 9, PIK3R6, AKT and p-AKT. RESULTS: Compared with the control group, TPA could inhibit the proliferation of NB4 cells, induce the cells to become mature granulocyte-monocyte differentiation, and also induce cell G1 phase arrest and apoptosis. Differentially expressed mRNAs were significantly enriched in PI3K/AKT pathway. TPA treatment could increase the mRNA levels of CCND1, CCNA1, and CDKN1A, while decrease the mRNA level of MYC. It could also up-regulate the protein levels of CDKN1A, CDKN1B, CCND1, Bax, c-Caspase 3, c-Caspase 9, and PIK3R6, while down-regulate MYC, Bcl-2, and p-AKT in NB4 cells. CONCLUSION: TPA induces NB4 cell cycle arrest in G1 phase and promotes its apoptosis by regulating PIK3/AKT signaling pathway.

Diterpenoids with an unusual tricyclo[10.3.0.02,9]pentadecane skeleton from Pedilanthus tithymaloides as multidrug resistance modulators.

Three new diterpenoids with an unusual carbon skeleton, pedilanins A-C (1-3), and nine new jatrophane diterpenoids, pedilanins D-L (4-12), along with five known ones (13-17), were isolated from Pedilanthus tithymaloides. Compounds 1-3 characterize an unprecedented tricyclo[10.3.0.02,9]pentadecane skeleton. Compounds 4-8 are rare examples of the jatrophanes bearing a cyclic hemiketal substructure. Their structures were determined by an extensive analysis of HRESIMS, NMR, quantum-chemical calculation, DP4+ probability, and X-ray crystallographic data. In the bioassay, compounds 1-12 dramatically reversed multidrug resistance in cancer cells with the fold-reversals ranging from 17.9 to 396.8 at the noncytotoxic concentration of 10 µM. The mechanism results indicated that compounds 2 and 3 inhibited the P-glycoprotein (Pgp) transporter function, thus reversing the drug resistance.

[Effect of Dichloromethane Extraction Phase of Patrinia Scabiosaefolia Fisch. Stem on Proliferation and Differentiation of K562 Cells].

OBJECTIVE: To explore the effect of dichloromethane extraction phase of ethanol extract from stem of Patrinia scabiosaefolia Fisch.(DPSS) on proliferation and differentiation of K562 cells and its related mechanism. METHODS: MTT assay was used to detect the effects of DPSS at 0, 25, 50, 100 and 200 µg/ml on the proliferation of K562 cells at 24, 48 and 72 hours. Flow cytometry was used to analyze the changes of cell cycle and apoptosis at 24 and 48 hours. Wright-Giemsa staining was used to observe the morphological changes of K562 cells. The cell surface antigens CD33 and CD11b were detected by flow cytometry. RESULTS: The proliferation of K562 cells treated with different concentrations of DPSS was inhibited in a time-dose dependent manner (r=-0.96). Cell cycle analysis showed that with the increase of DPSS concentration, cells in G2/M phase increased (r=0.88), and cells were blocked in G2/M phase. Flow cytometry results showed that with the apoptosis rate of K562 cells was the highest when treated with 200 µg/ml DPSS for 48 h. Morphological observation showed that the K562 cell body increased, the amount of cytoplasm increased, the ratio of nucleus to cytoplasm decreased, and the nuclear chromatin was rough after DPSS treatment. Cell differentiation antigen, CD33 and CD11b, were positively expressed after treated with DPSS. CONCLUSION: DPSS can induce apoptosis through cell cycle arrest, inhibit the proliferation of K562 cells, and induce K562 cells to differentiate into monocytes, which has a potential anti-leukemia effect.

Network Pharmacology-Based Investigation on the Mechanism of the JinGuanLan Formula in Treating Acne Vulgaris.

Background: JinGuanLan (JGL) formula is a traditional Chinese medicine (TCM) developed by the Department of Pharmacology at the First Hospital of Lanzhou University. The network pharmacology approach was applied to determine the potential active compounds, therapeutic targets, and main pathways of the JGL formula to evaluate its application value in acne vulgaris. Methods: Data on the active compounds and their related targets were obtained from the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (TCMSP). Acne vulgaris-related targets were searched from the Online Mendelian Inheritance in Man (OMIM) database, GeneCards Database, Comparative Toxicogenomics Database (CTD), Therapeutic Target Database (TTD), and DisGeNET Database. Targets intersecting between JGL- and acne vulgaris-related targets were chosen as potential therapeutic targets. The protein-protein interaction (PPI) network of potential therapeutic targets was visualized using Cytoscape software based on the PPI data collected from the STRING database. Three topological features, namely, "Degree," "MCC," and "EPC" of each node in the PPI network were calculated using the cytoHubba plugin of Cytoscape to excavate the core targets. R program was used for the Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis of the potential therapeutic targets. Finally, the compound-target-pathway network was constructed. Result: Among the 148 active compounds that were identified, quercetin and kaempferol showed the highest degree of target interaction and thus may play essential roles in the pharmacological effect of the JGL formula for acne treatment. Among the 97 potential therapeutic targets that were screened out, the 6 core targets were TNF, JUN, IL6, STAT3, MAPK1, and MAPK3. A total of 2260 terms of GO enrichment analysis were obtained, including 2090 for biological processes (BP), 37 for cellular components (CC), and 133 for molecular function (MF). A total of 156 enriched KEGG pathways were identified, including TNF, IL-17, Th17 cell differentiation, MAPK, PI3K-Akt, T cell receptor, and Toll-like receptor signalling pathways. Conclusion: This work showed that the JGL formula might reverse the pathological changes associated with acne vulgaris through its antiinflammatory effect and regulate the excessive lipogenesis in sebaceous glands via different signalling pathways. This new drug has application value and is worthy of further research and development.

A novel SPTB mutation causes hereditary spherocytosis via loss-of-function of ß-spectrin.

Hereditary spherocytosis (HS) is the most frequently observed chronic non-immune hemolytic disorder caused by altered red cell membrane function. SPTB gene mutation is one of the most common causes of HS, but pathogenicity analyses and pathogenesis research on these mutations have not been widely conducted. In this study, a novel heterozygous mutation of the SPTB gene (c.1509_1518del; p.K503Nfs*67) was identified in a Chinese family with HS by whole-exome sequencing (WES) and was then confirmed by Sanger sequencing. Next, the pathogenicity and pathogenesis of this mutation were studied using peripheral blood. We found that this mutation disrupted the synthesis and localization of ß-spectrin and weakened the interaction between ß-spectrin and ankyrin, which may be caused by the nonsense-mediated mRNA degradation pathway. These changes lead to the transformation of discoid erythrocytes into spherocytes, resulting in hemolytic anemia. Therefore, we classified this novel mutation as a pathogenic mutation leading to loss-of-function of ß-spectrin. It would be insightful to perform the same mutation test and to provide genetic counseling to other relatives of the proband. Our study increases the current understanding of the molecular mechanisms related to mutations in SPTB.

Spectrins and human diseases.

Spectrin, as one of the major components of a plasma membrane-associated cytoskeleton, is a cytoskeletal protein composed of the modular structure of α and ß subunits. The spectrin-based skeleton is essential for preserving the integrity and mechanical characteristics of the cell membrane. Moreover, spectrin regulates a variety of cell processes including cell apoptosis, cell adhesion, cell spreading, and cell cycle. Dysfunction of spectrins is implicated in various human diseases including hemolytic anemia, neurodegenerative diseases, ataxia, heart diseases, and cancers. Here, we briefly discuss spectrins function as well as the clinical manifestations and currently known molecular mechanisms of human diseases related to spectrins, highlighting that strategies for targeting regulation of spectrins function may provide new avenues for therapeutic intervention for these diseases.