On the mechanism of wogonin against acute monocytic leukemia using network pharmacology and experimental validation.

Wogonin is a natural flavone compound from the plant Scutellaria baicalensis, which has a variety of pharmacological activities such as anti-cancer, anti-virus, anti-inflammatory, and immune regulation. However, the potential mechanism of wogonin remains unknown. This study was to confirm the molecular mechanism of wogonin for acute monocytic leukemia treatment, known as AML-M5. The potential action targets between wogonin and acute monocytic leukemia were predicted from databases. The compound-target-pathway network and protein-protein interaction network (PPI) were constructed. The enrichment analysis of related targets and molecular docking were performed. The network pharmacological results of wogonin for AML-M5 treatment were verified using the THP-1 cell line. 71 target genes of wogonin associated with AML-M5 were found. The key genes TP53, SRC, AKT1, RELA, HSP90AA1, JUN, PIK3R1, and CCND1 were preliminarily found to be the potential central targets of wogonin for AML-M5 treatment. The PPI network analysis, GO analysis and KEGG pathway enrichment analysis demonstrated that the PI3K/AKT signaling pathway was the significant pathway in the wogonin for AML-M5 treatment. The antiproliferative effects of wogonin on THP-1 cells of AML-M5 presented a dose-dependent and time-dependent manner, inducing apoptosis, blocking the cell cycle at the G2/M phase, decreasing the expressions of CCND1, CDK2, and CyclinA2 mRNA, as well as AKT and p-AKT proteins. The mechanisms of wogonin on AML-M5 treatment may be associated with inhibiting cell proliferation and regulating the cell cycle via the PI3K/AKT signaling pathway.

Low frequency of SLC26A4 c.919-2A > G variant among patients with nonsyndromic hearing loss in Yunnan of Southwest China.

BACKGROUND: Gene variants are responsible for more than half of hearing loss, particularly in nonsyndromic hearing loss (NSHL). The most common pathogenic variant in SLC26A4 gene found in East Asian populations is c.919-2A > G followed by c.2168A > G (p.H723R). This study was to evaluate their variant frequencies in patients with NSHL from special education schools in nine different areas of Southwest China's Yunnan. METHODS: We performed molecular characterization by PCR-products directly Sanger sequencing of the SLC26A4 c.919-2AG and c.2168 A > G variants in 1167 patients with NSHL including 533 Han Chinese and 634 ethnic minorities. RESULTS: The SLC26A4 c.919-2A > G variant was discovered in 8 patients with a homozygous state (0.69%) and twenty-five heterozygous (2.14%) in 1167 patients with NSHL. The total carrier rate of the c.919-2A > G variant was found in Han Chinese patients with 4.50% and ethnic minority patients with 1.42%. A significant difference existed between the two groups (P < 0.05). The c.919-2A > G allele variant frequency was ranged from 3.93% in Kunming to zero in Lincang and Nvjiang areas of Yunnan. We further detected the SLC26A4 c.2168 A > G variant in this cohort with one homozygotes (0.09%) and seven heterozygotes (0.60%), which was detected in Baoshan, Honghe, Licang and Pu`er areas. Between Han Chinese group (0.94%) and ethnic minority group (0.47%), there was no statistical significance (P > 0.05). Three Han Chinese patients (0.26%) carried compound heterozygosity for c.919-2A > G and c.2168 A > G. CONCLUSION: These data suggest that the variants in both SLC26A4 c.919-2A > G and c.2168 A > G were relatively less frequencies in this cohort compared to the average levels in most regions of China, as well as significantly lower than that in Han-Chinese patients. These results broadened Chinese population genetic information resources and provided more detailed information for regional genetic counselling for Yunnan.

Therapeutic potential of autophagy in immunity and inflammation: current and future perspectives.

Autophagy is recognized as a lysosomal degradation pathway important for cellular and organismal homeostasis. Accumulating evidence has demonstrated that autophagy is a paradoxical mechanism that regulates homeostasis and prevents stress under physiological and pathological conditions. Nevertheless, how autophagy is implicated in immune responses remains unclear. It is well established that autophagy bridges innate and adaptive immunity, while autophagic dysfunction is closely related to infection, inflammation, neurodegeneration, and tumorigenesis. Therefore, autophagy has attracted great attention from fundamental and translational fields due to its crucial role in inflammation and immunity. Inflammation is involved in the development and progression of various human diseases, and as a result, autophagy might be a potential target to prevent and treat inflammatory diseases. Nevertheless, insufficient autophagy might cause cell death, perpetrate inflammation, and trigger hereditary unsteadiness. Hence, targeting autophagy is a promising disease prevention and treatment strategy. To accomplish this safely, we should thoroughly understand the basic aspects of how autophagy works. Herein, we systematically summarized the correlation between autophagy and inflammation and its implication for human diseases.

Hyperleukocytic Acute Leukemia Circulating Exosomes Regulate HSCs and BM-MSCs.

Hyperleukocytic acute leukemia (HLAL) circulating exosomes are delivered to hematopoietic stem cells (HSCs) and bone marrow mesenchymal stem cells (BM-MSCs), thereby inhibiting the normal hematopoietic process. In this paper, we have evaluated and explored the effects of miR-125b, which is carried by HLAL-derived exosomes, on the hematopoietic function of HSCs and BM-MSCs. For this purpose, we have isolated exosomes from the peripheral blood of HLAL patients and healthy volunteers. Then, we measured the level of miR-125b in exosomes cocultured exosomes with HSCs and BM-MSCs. Moreover, we have used miR-125b inhibitors/mimic for intervention and then measured miR-125b expression and colony forming unit (CFU). Apart from it, HSC and BM-MSC hematopoietic-related factors α-globulin, γ-globulin, CSF2, CRTX4 and CXCL12, SCF, IGF1, and DKK1 expression were measured. Evaluation of the miR-125b and BAK1 targeting relationship, level of miR-125b, and expression of hematopoietic-related genes was performed after patients are treated with miR-125b mimic and si-BAK1. We have observed that miR-125b was upregulated in HLAL-derived exosomes. After HLAL-exosome acts on HSCs, the level of miR-125b is upregulated, reducing CFU and affecting the expression of α-globulin, γ-globulin, CSF2, and CRCX4. For BM-MSCs, after the action of HLAL-exo, the level of miR-125b is upregulated and affected the expression of CXCL12, SCF, IGF1, and DKK1. Exosomes derived from HLAL carry miR-125b to target and regulate BAK1. Further study confirmed that miR-125b and BAK1mimic reduced the expression of miR-125b and reversed the effect of miR-125b mimic on hematopoietic-related genes. These results demonstrated that HLAL-derived exosomes carrying miR-125b inhibit the hematopoietic differentiation of HSC and hematopoietic support function of BM-MSC through BAK1.

Interferon alpha-inducible protein 27 (IFI27) is a prognostic marker for pancreatic cancer based on comprehensive bioinformatics analysis.

Accurate biomarkers to predict the genesis and progression of pancreatic adenocarcinoma (PAAD) are needed in the fight against this deadly disease. Here, we combined multiple datasets (GEO, TCGA and GTEx) to conduct a comprehensive analysis of pancreatic cancer. Through an in-depth analysis, we discovered that the expression of the gene encoding interferon alpha-inducible protein 27 (IFI27) was significantly higher in pancreatic cancer tissues than that in normal tissues, and that higher expression of IFI27 was negatively correlated with the overall survival rate of pancreatic cancer patients. The functional annotation of IFI27 demonstrated relationships to cellular immunity and metabolism, especially glycolysis. Analysis of infiltrating immune cells displayed that higher expression of IFI27 expression correlates with decreased CD8 + T cells and increased M2 macrophages in the tumor immune microenvironment (TIME), then biochemical analyses of a mouse model and immunohistochemical (IHC) staining verified that glycolytic enzymes and M2 macrophages increased significantly in pancreatic cancer tissues. We speculate that IFI27 may affect the tumor microenvironment (TME) of PAAD by regulating cellular immunity and metabolism, thereby promoting the progression of pancreatic carcinoma and worsening the prognosis. These findings of our present study are solid evidence that IFI27 is a potential prognostic biomarker of pancreatic cancer and that it affects the tumor immune microenvironment.

[Relationship between Polymorphism in ALOX5, ALOX5AP and Susceptibility to Myeloid Leukemia].

OBJECTIVE: To investigate the correlation of single nucleotide polymorphisms (SNP) in arachidonate 5-lipoxygenase gene (ALOX5) rs2029253, rs2228064 and rs2228065 sites, 5-lipoxygenase activating protein gene (ALOX5AP) rs10507391, rs4769874 sites with the risk for genesis of adult myeloid leukemia. METHODS: By the approval from the hospital ethics committee and the informed consent of participants. 150 patients with myeloid leukemia (ML) as ML group and 134 healthy people as the control group were selected. The genomic DNA was extracted from the samples. Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) combined with directly sequencing, PCR-amplified products were applied to test the polymorphism of 5 sites in ALOX5 and ALOX5AP gene. RESULTS: A allele frequencies of ALOX5 gene rs2029253 site in the ML group and the control group were 43.0% and 34.3%, respectively. And the G allele frequencies in the ML group and the control group were 57.0% and 65.7%, respectively. The genotype distributions of AA, AG and GG in ALOX5 gene rs2029253 site in the ML group were 32.2%, 21.5% and 46.3% respectively. That in the control group were 15.7%, 37.3% and 47.0% respectively. The genotype AA and A allele frequency of ALOX5 gene rs2029253 site were linked with the increased risk of myeloid leukemia (OR=2.26, 95% CI: 1.43-4.56, P＜0.05; OR=1.44, 95% CI: 1.02-2.03, P＜0.05). And the genotype AG and allele G reduced the susceptibility to myeloid leukemia (OR=0.46, 95% CI: 0.27-0.78, P＜0.01; OR=0.69, 95% CI: 0.50-0.98, P＜0.05), however, the polymorphisms of ALOX5 gene rs2228064 and rs2228065 site not correlated with the risk of myeloid leukemia (P＞0.05). The A allele frequency of ALOX5AP gene rs10507391 site in the ML group and the control group were 30.7% and 36.2% respectirely. The genotype distribution rates of AA, AT and TT in ALOX5AP gene rs10507391 site in the ML group was 1.3%, 58.7% and 40.0% respectively, that in the control group were 9.7%, 53.0% and 37.3% respectively. The genotype AA of ALOX5AP gene rs10507391 site correlated with the decreased risk of myeloid leukemia (OR=0.13, 95% CI: 0.03-0.57, P＜0.05), but the polymorphism of ALOX5AP gene rs4769874 site not correlated with the risk of myeloid leukemia (P＞0.05). CONCLUSION: The genotype AA, AG and allele A, G of ALOX5 rs2029253, as well as ALOX5AP rs10507391 may be correlate with the susceptibility to myeloid leukemia.

Phenotypic characterization of macrophages in the BMB sample of human acute leukemia.

Macrophages within tissues display a strong plastic ability in respond to environmental cues in both physiologic influences and disease. However, the macrophage phenotype and its distribution in the bone marrow biopsies (BMB) samples of human acute leukemia (AL) remain poorly understood. In this study, 97 BMB samples of patients with acute leukemia and 30 iron-deficiency anemias (IDA) as control group were evaluated with immunohistochemistry. In comparison with controls, the counts of CD68+, CD163+, and CD206+macrophages were remarkably increased in BMB samples of acute leukemia (P < 0.01), as well as their infiltration density was roaring up-regulation (P < 0.01). The expression levels of CD68+, CD163+, and CD206+macrophages were decreased in patients with complete remission, but there still existed statistically significant contrast to the control group (P < 0.01). The ratios of the CD163-positive cells or CD206-positive cells to CD68-positive cells were most prevalent in the BMB samples of human acute leukemia compared with the control group (P < 0.01), which support that macrophages were polarized to M2 macrophages.

Assessment of the Number and Phenotype of Macrophages in the Human BMB Samples of CML.

Macrophages have emerged as a key player in tumor biology. However, their number and phenotype in human bone marrow of biopsy (BMB) samples of chronic myeloid leukemia (CML) and their association with disease progression from an initial chronic phase (CP) to accelerated phase (AP) to advanced blast phase (BP) are still unclear. BMB samples from 127 CML patients and 30 patients with iron-deficiency anemia (IDA) as control group were analyzed by immunohistochemistry. The expression levels of CD68, CD163, and CD206 in BMB samples of CML patients were significantly higher than those in the patients of control group (P < 0.01), and we observed that their positive expression was gradually elevated during the transformation of CML-CP to AP to BP (P < 0.01). However, the expressions of CD68, CD163, and CD206 in released group were downregulated and contrasted to these in control group; there exists statistical significance (P < 0.01). The percentage ratio of CD163 and CD206 to CD68 was pronounced to be increasing from CML-CP to AP to BP (P < 0.01). Hence, the higher proportion of CD68+, CD163+ and CD206+ macrophages in BMB samples can be considered a key factor for disease progression of CML patients. Targeting macrophages, especially the M2 phenotype may help in designing therapeutic strategies for CML.

Folate metabolism gene polymorphisms MTHFR C677T and A1298C and risk for preeclampsia: a meta-analysis.

OBJECTIVES: MTHFR C677T and A1298C have been associated with the risk of preeclampsia (PE), but with conflicting results. We performed this meta-analysis to derive a more precise estimation of the association between MTHFR polymorphisms and PE. STUDY DESIGN: An electronic search of PubMed and Chinese Biomedicine database was conducted to select studies for meta-analysis. 54 case controlled studies containing MTHFR C677T and A1298C gene polymorphisms were chosen, and odds ratio (OR) with confidence interval (CI) was used to assess the strength of this association. RESULT: These studies evaluated 7398 cases and 11230 controls for MTHFR C677T. The overall results suggested that MTHFR C677T was associated with the risk of PE. (T vs. C: OR = 1.157, 95% CI: 1.057-1.266, p = 0.002; TT + CT vs. CC: OR = 1.165, 95% CI : 1.049-1.293, P = 0.004; TT vs. CT + CC: OR = 1.371, 95% CI: 1.153-1.63, p < 0.001). We also evaluated 1103 cases and 988 controls for MTHFR A1298C but could not demonstrate an increased risk of PE for this polymorphism (p = 0.667). A symmetric funnel plot, the Egger's test (p = 0.819) suggested a lack of publication bias. CONCLUSION: This meta-analysis supports the idea that MTHFR C677T genotype is associated with increased risk for PE, especially in the case of Asians and Caucasians.

Expression of IARS2 gene in colon cancer and effect of its knockdown on biological behavior of RKO cells.

OBJECTIVE: To investigate the expression level of IARS2 gene in colon cancer tissues and various cell strains of the cancer; to explore cytologically the effect of IARS2 gene knockdown on proliferation, apoptosis and cell cycle of RKO cells in the cancer. METHODS: Real-time, fluorescence-based quantitative PCR (qPCR) was used to detect the expression of IARS2 gene in human colon cancer and surrounding tissues and in various cell strains of the cancer; the RNA interference target of IARS2 gene was designed and the target was detected by Western blot; the IARS2-siRNA lentiviral vector was established and used to infect the RKO cells of colon cancer; qPCR was employed to determine the effect of gene knockdown; changes of the RKO cells in growth, apoptosis, cell cycle and clone formation were observed after IARS2 gene knockdown. RESULTS: The expression of IARS2 gene was higher in human colon cancer tissues than in surrounding tissues; there was expression of IARS2 gene in colon cancer cells, and the expression level of IARS2 gene mRNA was higher in the RKO cells than in the SW480, HCT116, DLD1, HT-29 and SW620 cells. After infection of the RKO cells with IARS2-siRNA lentivirus, the expression of IARS2 gene was inhibited in the level of mRNA; proliferation rate of the RKO cells was significantly inhibited; the G1 phase arrest of the RKO cells was increased with less RKO cells in S phase; the apoptotic RKO cells increased significantly; and the number of colonies of the RKO cells reduced. CONCLUSION: The expression of IARS2 gene is different in human colon cancer and surrounding tissues; after knockdown of IARS2 gene, proliferation of the RKO cells is inhibited; there are more cells in G phase and fewer cells in S phase; apoptosis of cells is increased; and formation of colonies is reduced. IARS2 gene is probably a cancer-promoting gene.

Brain distribution and elimination of recombinant human TIMP-1 after cerebral ischemia and reperfusion in rats.

OBJECTIVE: To investigate recombinant human TIMP-1 ((125)I-rhTIMP-1) half-life in blood and its distribution in rat brain tissue after cerebral ischemia/reperfusion as part of a therapeutic development paradigm. METHOD: A suture model of the middle cerebral artery occlusion was used. (125)I-labeled rhTIMP-1 at 60 µg/kg (11.23 µCi/µg) was administered to rats intravenously at the beginning of reperfusion. Blood and brain tissue were collected. The radioactivity was detected with a gamma counter and analyzed by autoradiography. RESULTS: The blood half-life T(1/2) of (125)I-rhTIMP-1 was 42.2 hours. Thirty minutes after (125)I-rhTIMP-1 administration, an increased accumulation of (125)I-rhTIMP-1 in the ischemic hemisphere was observed. The maximum brain tissue concentration C(max) was 26.1 ng/g at 1.5 hours in the striatum and 13.9 ng/g at 5 hours in the cortex when the uptake percentage of brain tissue to blood was 6.1±0.4 and 6.7±2.1%, respectively. The cortex and striatum elimination half-lives T(1/2) were 45.3 and 39.2 hours, respectively. Electrophoretic analysis of ischemic samples for (125)I-rhTIMP-1 showed a clear 28 kDa band 1.5 hours after (125)I-rhTIMP-1 administration in the cortex and striatum. The intensity of the 28 kDa band decreased after 3.0 hours of the administration. Some (125)I-rhTIMP-1 maintained its molecular integrity for 8.5 hours in ischemic striatum after reperfusion. DISCUSSION: (125)I-labeled rhTIMP-1 was distributed quickly into ischemic brain tissue and had a slow elimination in both blood and brain tissue. These results, along with other studies suggesting therapeutic benefits, will aid in the development of TIMP-1 for protecting ischemic stroke.

[Effects of human bone marrow mesenchymal stem cells on cytokines secretion from allogeneic dendritic cell activated cytokine-induced killer cells].

AIM: to study the effect of human bone marrow derived mesenchymal stem cells (hMSCs) on cytokines secretion (IFN-γ, TNF-α, IL-10, IL-6, IL-4 and IL-2) of allogeneic DC-CIK cells (in co-culture of CIK cells with DC), which investigate the mechanism of immunoregulation induced by hMSCs. METHODS: the hMSCs from bone marrow were isolated, expanded and identified by cell morphology, differentiation into neuron-like cells with NSE, fat-like cells with red-oil stain, and expression of CD29, CD44. The DC and CIK cells from peripheral blood were isolated, expanded and identified by CD1α, HLA-DR or CD3(+);CD56(+);. The hMSCs were co-cultured with DC-CIK cells according to ratio 1:10. The expression of the six cytokines in supernatant was evaluated by flow cytometry after 4 days of DC-activated CIK cells in co-culture with hMSCs. RESULTS: the hMSCs displayed a fibroblast-like morphology and the positive cells of CD29 and CD44 were 96.6%, 94.6%, which have the capacity of differentiation into neuron-like cells with expressed NSE as well as fat-like cells with red-oil stain positive. The expression of CD1α, HLA-DR in DC was (91.9 ± 10.04)% and (88.8 ± 8.92)%. The CD3(+);CD56(+); double positive cells in DC-CIK cells was (29.23 ± 12.23)% compared to CIK cells with (15.98 ± 2.49)%. The cytokines secretion of DC-CIK cells in co-culture with hMSCs was IFN-γ (135.05 ± 48.19) ng/L; TNF-α (11.33 ± 1.42) ng/L; IL-10 (10.15 ± 2.25) ng/L; IL-6 (494.63 ± 235.222) ng/L; IL-4 (7.07 ± 2.30) ng/L and IL-2 (1074.6 3 ± 303.74) ng/L. In control group (DC-CIK cells) the secretion of IFN-γ, TNF-α, IL-10, IL-6, IL-4 and IL-2 was (717.6 ± 248.15) ng/L; (17.78 ± 7.52) ng/L; (29.95 ± 12.76) ng/L; (8.03 ± 0.21) ng/L, (9.08 ± 3.07) ng/L as well as IL-2 1 250 ng/L. CONCLUSION: the secretion of IFN-γ and IL-10 were down-regulated. It probably implied that hMSCs had the effect of immunoregulation on DC-CIK cells.

Peroxynitrite decomposition catalyst prevents matrix metalloproteinase activation and neurovascular injury after prolonged cerebral ischemia in rats.

Matrix metalloproteinases (MMPs) play an important role in reperfusion-induced brain injury following ischemia. To define the effects of peroxynitrite decomposition catalyst on MMP activation and neurovascular reperfusion injury, 5,10,15,20-tetrakis (2,4,6-trimethyl-3,5-disulfonatophenyl)-porphyrin iron (III) (FeTMPyP) was administered intravenously 30 min prior to reperfusion following a middle cerebral artery occlusion. Activation of MMP was assessed by in situ and gel zymography. Neurovascular injury was assessed using endothelial barrier antigen, collagen IV immunohistochemistry and Cresyl violet staining. Results were compared with sham and ischemia alone groups. We found that administration of FeTMPyP just before reperfusion after ischemia inhibited MMP-9 activation and total MMP-2 increases in the cortex and decreased active MMP-9 along with the total amounts of active MMP-9 and active MMP-2 in the striatum. Reperfusion-induced injury to the basal lamina of collagen IV-immunopositive microvasculature and neural cells in cortex and striatum was ameliorated by FeTMPyP. Losses of blood vessel endothelium produced by ischemia or reperfusion were also decreased in the cortex. These results suggest that administration of FeTMPy prior to reperfusion decreases MMP activation and neurovascular injury after prolonged cerebral ischemia. This strategy may be useful for future therapies targeted at preventing breakdown of the blood-brain barrier and hemorrhagic transformation.

[Effect of liver X receptors agonists T0901317 on expression of FAT/CD36 gene mRNA in adult human skeletal muscle cell].

AIM: To investigate the liver X receptors agonists T0901317's effect on expression of FAT/CD36 gene mRNA in adult human skeletal muscle cell. METHODS: Myotubes from humans were exposed to different T0901317 concentrations (0, 0.5, and 1.0 micromol/L) for 24 hours before experiments were performed. Then the expression of FAT/CD36 mRNA in skeletal muscle cell of each experimental group was detected by SYBR Green I real-time quantitative polymerase chain reaction. The relative data were compared among groups by 2-delta delta Ct method. RESULTS: (1) The Ct mean of control group, T0901317 (0.5 micromol/L) group, T0901317 (1 micromol/L) group were analyzed and there was significant difference (P < 0.01). (2) The expression of FAT/CD36 mRNA with liver X receptors agonists T0901317 in human skeletal muscle cell in the T0901317 (0.5 micromol/L) group and T0901317 (1 micromol/L) group were 2.91 times and 3.03 times than the control group. CONCLUSION: The expression of FAT/CD36 mRNA in human skeletal muscle cell afer the treatment of liver X receptors agonists T0901317 is increased, so we may propose that T0901317 may increase the risk of resistance in adult human skeletal muscle.

Effects of activated complements on platelets.

AIM: To observe the effects of activation of complements on platelets and subsequently on vascular endothelial cells (VEC) and vascular smooth muscle cells (VSMC). METHODS: Zymosan A-induced morphological change of platelets was determined with an aggregometer, and prothrombinase expression was quantified using chromogenic substrate. Supernatant of zymosan A-treated platelet rich plasma (PRP) was added to the cultured VEC or VSMC for the observation of cell growth, DNA content, and the membrane microviscosity. RESULTS: Zymosan A induced marked metamorphosis, increased membrane microviscosity, and increased prothrombinase expression of platelets in PRP, but platelet metamorphosis induced by zymosan A was not found in washed platelets and fresh platelet suspended in platelet poor plasma (PPP) which had been pretreated with cobra venom factor (CVF). The effect of zymosan A on platelets was prevented by egtazic acid 5 mmol/L, Mn2+ 10 mmol/L, tetrodotoxin 40 micromol/L or indomethacin 100 micromol/L. The supernatant of zymosan A-treated PRP inhibited the growth of VEC, but accelerated the growth of VSMC and made most VSMC enter G2- and M- phase. The endoplasmic reticulum with rough surface and free ribosome in VSMC and vacuoles appeared in VEC mitochondria. CONCLUSION: Activated complements induced significant shape change of platelets, stimulated platelets to release some factors and subsequently injured VEC, but accelerated the growth of VSMC, which may contribute to the development of blood coagulation and to the chronic inflammation.