Homeobox B9 Promotes the Progression of Hepatocellular Carcinoma via TGF-ß1/Smad and ERK1/2 Signaling Pathways.

Objectives: Homeobox B9 (HOXB9), a homeodomain-containing transcription factor, may play a role in hepatocellular carcinoma (HCC) progression. However, the exact mechanisms underlying its action remain unclear. Materials and methods. Immunohistochemistry was used to investigate the expression of HOBX9 and its prognostic values in HCC patients. HCC cells were transfected with pBabe-HOXB9 and shHOXB9 plasmids, and MTT assay, Transwell assays, and xenograft mouse models were employed to determine the effects of HOXB9 on HCC cell proliferation, migration, and invasion in vitro and in vivo. The biological mechanisms involved in the role of HOXB9 were determined with Western blot and RT-qPCR methods. Results: HOXB9 expression was significantly increased in HCC tissues and cell lines. Patients with higher HOXB9 levels were associated with poor prognosis. Overexpression of HOXB9 in BEL-7405 cells promoted proliferation, migration, and invasion, whereas knockdown of HOXB9 in HepG2 cells significantly reduced cell proliferation, migration, and invasion abilities. Mechanically, a positive correlation was found between HOXB9 expression and transforming growth factor-ß1 (TGF-ß1) and extracellular signal-regulated kinase (ERK)1/2 pathway in HCC tissues. HOXB9 overexpression stimulated TGF-ß1/Smads signaling pathway in BEL-7405 cells. In contrast, HOXB9 knockdown inhibited the TGF-ß1/Smads signaling pathway in HepG2 cells. In addition, the treatment with TGF-ß1 inhibitor, LY364947, significantly reserved HOXB9 overexpression-induced cell proliferation, migration, and invasion abilities. Conclusions: These findings validated that HOXB9 promoted proliferation, migration, and invasion in HCC cells by stimulating the TGF-ß1/Smads and ERK1/2 signaling pathway. HOXB9 could be a promising prognostic biomarker and a potential therapeutic target in HCC.

Circular RNA FOXO3 accelerates glycolysis and improves cisplatin sensitivity in lung cancer cells via the miR-543/Foxo3 axis.

Non-small cell lung cancer (NSCLC) is the most common cause of cancer-associated mortality worldwide. Our previous study revealed that circular RNA (circRNA)-FOXO3 is highly expressed in lung cancer and inhibits cell proliferation. However, to the best of our knowledge, at present, no study has focused on the specific mechanism of circRNA-FOXO3 in drug resistance. Therefore, the present study aimed to provide novel perspectives on the role of circRNA-FOXO3 in cisplatin (DDP) resistance in NSCLC. A Cell Counting Kit-8 assay was used to determine the viability of cells overexpressed with circRNA-FOXO3 and under DDP treatment. Glycolysis was analyzed by measuring glucose consumption and lactate production. The interaction of circRNA-FOXO3, microRNA 543 (miR-543) and Foxo3 was confirmed using a dual-luciferase reporter assay. It was revealed that circRNA-FOXO3 improved cell sensitivity to DDP and repressed glycolysis in DDP-sensitive and DDP-resistant NSCLC cells. Bioinformatics analysis, luciferase reporter assays, quantitative PCR and RNA pull-down assays were employed to verify the binding of circRNA-FOXO3 to miR-543. Functionally, inhibition of miR-543 could sensitize NSCLC cells to DDP, and overexpression of miR-543 at least partially abolished the circRNA-FOXO3-induced decrease in chemoresistance. Furthermore, it was revealed that Foxo3 was a direct target of miR-543. Notably, the inhibitory action of miR-543 silencing on DDP resistance and glycolysis was reversed by overexpression of Foxo3 in DDP-sensitive and DDP-resistant NSCLC cells. In conclusion, the present study demonstrated that circRNA-FOXO3 promoted DDP sensitivity in NSCLC cells by regulating the miR-543/Foxo3 axis-mediated glycolysis balance. The present findings may provide novel perspectives for the treatment of patients with NSCLC resistant to DDP.

Circulating miRNAs as biomarkers for severe acute pancreatitis associated with acute lung injury.

AIM: To identify circulating micro (mi)RNAs as biological markers for prediction of severe acute pancreatitis (SAP) with acute lung injury (ALI). METHODS: Twenty-four serum samples were respectively collected and classified as SAP associated with ALI and SAP without ALI, and the miRNA expression profiles were determined by microarray analysis. These miRNAs were validated by quantitative reverse transcription-polymerase chain reaction, and their putative targets were predicted by the online software TargetScan, miRanda and PicTar database. Gene ontology (GO) and Kyoto encyclopedia of genes and genomes (commonly known as KEGG) were used to predict their possible functions and pathways involved. RESULTS: We investigated 287 miRNAs based on microarray data analysis. Twelve miRNAs were differentially expressed in the patients with SAP with ALI and those with SAP without ALI. Hsa-miR-1260b, 762, 22-3p, 23b and 23a were differently up-regulated and hsa-miR-550a*, 324-5p, 484, 331-3p, 140-3p, 342-3p and 150 were differently down-regulated in patients with SAP with ALI compared to those with SAP without ALI. In addition, 85 putative target genes of the significantly dysregulated miRNAs were found by TargetScan, miRanda and PicTar. Finally, GO and pathway network analysis showed that they were mainly enriched in signal transduction, metabolic processes, cytoplasm and cell membranes. CONCLUSION: This is the first study to identify 12 circulating miRNAs in patients with SAP with ALI, which may be biomarkers for prediction of ALI after SAP.

Dai-Huang-Fu-Zi-Tang alleviates pulmonary and intestinal injury with severe acute pancreatitis via regulating aquaporins in rats.

BACKGROUND: Dai-Huang-Fu-Zi-Tang (DHFZT) is a famous traditional Chinese prescription with intestinal obstruction, acute pancreatitis and cholecystalgia for thousands of years. Our previous work found that DHFZT could act against pulmonary and intestinal pathological injury in rats with severe acute pancreatitis (SAP). But the underlying mechanism has not been fully elucidated. The aim of present study was to investigate whether DHFZT could relieve pulmonary and intestinal injury by regulating aquaporins after SAP induced by sodium taurocholate in rats. METHODS: Forty of SD rats were used for dose dependant experiments of DHFZT.Accurate-mass Time-of-flight liquid chromatography-mass spectrometry was used for qualitative screening of chemical compositions of DHFZT. Twenty-four rats were randomly divided into 3 groups: sham group (n = 8), model group (SAP, n = 8), DHFZT group (SAP with DHFZT treatment, n = 8). SAP models were established by retrograde injections of 5% sodium taurocholate solutions into rat pancreaticobiliary ducts. Blood samples were taken at 0, 12, 24, 48 h post-operation for detecting serum amylase, lipase, endotoxin, TNF-α, IL-6 and IL-10. Protein expression and location of aquaporin (AQP)1, 5, 8 and 9 were assessed by immunohistochemistry, western blot and immunofluorescence respectively. RESULTS: The study showed that 27 kinds of chemical composition were identified, including 10 kinds in positive ion mode and 17 kinds in negative ion mode. The results showed that AQP1, AQP5 of lung, and AQP1, AQP5, AQP8 of intestine in model group were significantly lower than that of sham group (P < 0.05), and which were obviously reversed by treatment with DHFZT. In addition, protein levels of pro-inflammatory cytokines such as TNF-α, IL-6 and endotoxin in peripheral blood were significantly suppressed by DHFZT, and that anti-inflammatory cytokine like IL-10 was just opposite. Finally, we also noted that DHFZT reduced serum levels of amylase, lipase and endotoxin, and also improved edema and pathological scores of lung and intestine after SAP. CONCLUSIONS: DHFZT ameliorated the pulmonary and intestinal edema and injury induced by SAP via the upregulation of different AQPs in lung and intestine, and suppressed TNF-α, IL-6 expression and enhanced IL-10 expression.

Dai-Huang-Fu-Zi-Tang Alleviates Intestinal Injury Associated with Severe Acute Pancreatitis by Regulating Mitochondrial Permeability Transition Pore of Intestinal Mucosa Epithelial Cells.

OBJECTIVE: The aim of the present study was to examine whether Dai-Huang-Fu-Zi-Tang (DHFZT) could regulate mitochondrial permeability transition pore (MPTP) of intestinal mucosa epithelial cells for alleviating intestinal injury associated with severe acute pancreatitis (SAP). METHODS: A total of 72 Sprague-Dawley rats were randomly divided into 3 groups (sham group, SAP group, and DHFZT group, n = 24 per group). The rats in each group were divided into 4 subgroups (n = 6 per subgroup) accordingly at 1, 3, 6, and 12 h after the operation. The contents of serum amylase, D-lactic acid, diamine oxidase activity, and degree of MPTP were measured by dry chemical method and enzyme-linked immunosorbent assay. The change of mitochondria of intestinal epithelial cells was observed by transmission electron microscopy. RESULTS: The present study showed that DHFZT inhibited the openness of MPTP at 3, 6, and 12 h after the operation. Meanwhile, it reduced the contents of serum D-lactic acid and activity of diamine oxidase activity and also drastically relieved histopathological manifestations and epithelial cells injury of intestine. CONCLUSION: DHFZT alleviates intestinal injury associated SAP via reducing the openness of MPTP. In addition, DHFZT could also decrease the content of serum diamine oxidase activity and D-lactic acid after SAP.

UHPLC-MS/MS determination and pharmacokinetic study of plantamajoside in rat plasma after oral administration of single plantamajoside and Plantago asiatica extract.

A sensitive and reliable ultra-high performance liquid chromatography coupled with tandem quadrupole mass spectrometry (UHPLC-MS/MS) method was developed for quantitation of plantamajoside in rat plasma. First, this study compared the pharmacokinetic properties of plantamajoside after oral administration of Plantago asiatica extract and pure plantamajoside in rat plasma with approximately the same dosage of 8.98 mg/kg. Second, chromatographic separation was performed on an Acquity HSS C18 column (50 × 2.1 mm, p.d.1.7 µm) with isocratic elution using methanol-water (80:20, v/v) as mobile phase at a flow rate of 0.25 mL/min. The calibration curves were linear over the range of 0.1-100 ng/mL for plantamajoside. At different time points (0, 0.083, 0.167, 0.25, 0.5, 0.75, 1, 2, 3, 4, 5, 6 and 8 h) after administration, the concentrations of plantamajoside in plasma were measured and the main pharmacokinetic parameters were estimated. The study indicates that the pharmacokinetics of plantamajoside in rat plasma have significant differences between two groups.

Efficacy of Chinese Herbal Medicine as an Adjunctive Therapy on in-Hospital Mortality in Patients with Acute Kidney Injury: A Systematic Review and Meta-Analysis.

Objective. We aimed to systematically assess the efficacy of Chinese herbal medicine (CHM) as an adjunctive therapy on in-hospital mortality in patients with acute kidney injury (AKI). Methods. We did a systematic review of articles published in any language up until Jun 23, 2015, by searching PubMed, Embase, the Cochrane Library, CBM, and CNKI. We included all RCTs that compared outcomes of patients with AKI taking CHM plus Western treatment (WT) with those taking WT alone. We applied Cochrane risk-of-bias tool to assess the methodological quality of the included trials. Results. Of 832 citations, 15 studies involving 966 patients met inclusion criteria. The methodological quality was assessed with unclear risk of bias. In the primary outcome of meta-analysis, pooled outcome of in-hospital mortality showed that patients randomly assigned to CHM treatment group were associated with low risk of in-hospital mortality compared with those randomly assigned to WT alone (RR = 0.41; 95% CI = 0.24 to 0.71; P = 0.001). Conclusions. CHM as an adjunctive therapy is associated with a decreased risk of in-hospital mortality compared with WT in patients with AKI. Further studies with high quality and large sample size are needed to verify our conclusions.

Formaldehyde exposure inhibits the expression of mammalian target of rapamycin in rat testis.

Formaldehyde (FA), a ubiquitous environmental pollutant, has long been suspected of causing adverse male reproductive effects. However, the molecular and cellular mechanisms underlying this phenomenon remain elusive. The overall aim of this study is to clarify the role of mammalian target of rapamycin (mTOR) in male reproductive injuries induced by FA exposure, by which we can further understand the molecular mechanism of FA male reproductive toxicity. In this study, immunohistochemistry, Western blotting, and reverse transcription polymerase chain reaction analysis were used to detect the expression of mTOR molecule in testicular tissues. We found that FA exposure inhibits the expression of mTOR in a dose-dependent manner. Combined with our earlier finding, we found the decreasing expression of mTOR in testicular tissue were consistent with the changes of testicular structure and autophagy levels. In summary, our data suggested that mTOR molecule might be involved in male reproductive injuries induced by FA exposure.

Associations between occupation exposure to Formaldehyde and semen quality, a primary study.

Formaldehyde (FA), a ubiquitous environmental pollutant, has long been suspected of having male reproductive toxicity. However, FA male reproductive toxicity was inconclusive due to dearth of human studies. Therefore, we sought to investigate whether occupational exposure to FA affects semen quality. Semen quality including five conventional parameters and seven kinematics parameters were compared between 114 male workers occupationally exposed to FA and 76 referents. FA exposure index (FEI) was measured and calculated. Our results showed that sperm progressive motility, total sperm motility, VCL, VSL and VAP were statistically significant decreased in FA exposure workers compared with the referents. Moreover, FEI was significantly negative associated with sperm progressive motility (ß = -0.19, P = 0.01) and total sperm motility (ß = -0.23, P = 0.004). In addition, a significant elevated risk of abnormal sperm progressive motility were observed in both low- (OR = 2.58; 95% CI: 1.11-5.97) and high-FA-exposed group (OR = 3.41; 95% CI: 1.45-7.92) respectively. Furthermore, a significant increased risk was also estimated for abnormal total sperm motility in both low- (OR = 3.21; 95% CI: 1.24-8.28) and high-FA-exposed group (OR = 4.84; 95% CI: 1.83-12.81) respectively. In conclusion, our study revealed the adverse effects of FA occupation exposure on semen quality, especially on sperm motion parameters.

HOXB9 promotes epithelial-to-mesenchymal transition via transforming growth factor-ß1 pathway in hepatocellular carcinoma cells.

The function of the epithelial-to-mesenchymal transition (EMT) during hepatocellular carcinoma (HCC) progression is well established. However, the regulatory mechanisms modulating this phenomenon remain unclear. Homeobox B9 (HOXB9) has been proposed as an oncogene in many cancer developments, but its function and underlying mechanisms in HCC metastasis remain unknown. HOXB9 modulates EMT through the transforming growth factor-ß1 (TGF-ß1) pathway, which is a recognized regulator of EMT in HCC cells. The knockdown of HOXB9 decreased the migration and invasion of HCC cells. Conversely, the HOXB9 overexpression led to an increase in the above-mentioned phenotypes in HCC cells. Further analysis of HOXB9-regulated cellular functions showed the ability of this transcription factor to induce EMT. Moreover, we demonstrated that the TGF-ß1 pathway is important in HOXB9-induced EMT in HCC cells. These findings define a novel cellular mechanism regulated by HOXB9, which controls EMT phenotype in HCC. This study is the first to illustrate the pivotal function of HOXB9 in regulating the metastatic behavior of HCC cells.

Homeobox B9 is overexpressed in hepatocellular carcinomas and promotes tumor cell proliferation both in vitro and in vivo.

HomeoboxB9 (HOXB9), a nontransforming transcription factor that is overexpressed in multiple tumor types, alters tumor cell fate and promotes tumor progression. However, the role of HOXB9 in hepatocellular carcinoma (HCC) development has not been well studied. In this paper, we found that HOXB9 is overexpressed in human HCC samples. We investigated HOXB9 expression and its prognostic value for HCC. HCC surgical tissue samples were taken from 89 HCC patients. HOXB9 overexpression was observed in 65.2% of the cases, and the survival analysis showed that the HOXB9 overexpression group had significantly shorter overall survival time than the HOXB9 downexpression group. The ectopic expression of HOXB9 stimulated the proliferation of HCC cells; whereas the knockdown of HOXB9 produced an opposite effect. HOXB9 also modulated the tumorigenicity of HCC cells in vivo. Moreover, we found that the activation of TGF-ß1 contributes to HOXB9-induced proliferation activities. The results provide the first evidence that HOXB9 is a critical regulator of tumor growth factor in HCC.

[Clinical research of Dahuang Fuzi decoction in auxiliary treatment of severe acute pancreatitis: a multi-center observation in 206 patients].

OBJECTIVE: To observe the efficacy of Dahuang Fuzi decoction in patients with severe acute pancreatitis (SAP), and to provide valuable medical evidences for a treatment of SAP with combined traditional Chinese and western medicine. METHODS: A prospective, multi-center, randomized, controlled clinical trial was designed. Two hundred and six adult patients with SAP admitted to intensive care unit (ICU) in three tertiary university teaching hospitals in Dalian from January 2007 to February 2010 were randomly divided into two groups: soapsuds enema control group (control group, n=101) and Dahuang Fuzi decoction enema study group (study group, n=105). The levels of serum amylase, albumin (Alb), D-lactic acid, endotoxin and diamine oxidase (DAO), high-sensitive C-reactive protein (hs-CRP), immunoglobulin (IgG, IgA, IgM), complements (C3, C4), tumour necrosis factor-α (TNF-α), interleukins (IL-6, IL-8) were determined before and after treatment for 2, 4, 7 days. The bowel sound, gastrointestinal function score, the acute physiology and chronic health evaluation II (APACHEII) score and the length of mechanical ventilation (MV), the length of stay in ICU, the mortality rate and average hospital expenses within 28 days were compared. RESULTS: Compared with control group, in the study group the levels of serum amylase, DAO, D-lactic acid and endotoxin were lowered, the Alb was increased, the levels of TNF-α, IL-6, IL-8, hs-CRP were decreased, the function of body immunity was enhanced, intestinal peristalsis was enhanced, gastrointestinal function score and APACHEII score were improved, the length of MV was reduced, the length of stay in ICU was diminished, the 28-day mortality and average hospital expenses were lowered [4 days amylase (U/L): 357.35±137.54 vs. 492.95±189.42, 2 days DAO (kU/L) : 5.20±0.59 vs. 5.45±0.72, 4 days D-lactic acid (mmol/L): 3.31±0.48 vs. 4.15±0.55, 2 days endotoxin (kEU/L): 0.29±0.11 vs. 0.34±0.14, 4 days Alb (g/L): 34.75±3.56 vs. 32.53±3.44, 2 days TNF-α (ng/L): 3.08±0.45 vs. 3.36±1.11, 2 days IL-6 (ng/L): 298.54±67.82 vs. 313.56±73.91, 4 days IL-8 (ng/L): 30.48±8.56 vs. 45.16±10.81, 2 days hs-CRP (mg/L): 32.56±11.83 vs. 40.42±15.10, 4 days IgG (g/L): 7.05±2.56 vs. 9.53±2.94, 2 days IgA (mg/L): 1 600±170 vs. 1 400±140, 4 days IgM (mg/L): 1 310±280 vs. 1 650±290, 4 days C3 (g/L): 1.11±0.09 vs. 1.50±0.15, 4 days C4 (g/L) : 0.32±0.11 vs. 0.41±0.10, 2 days bowel sound (times/min): 1.26±0.45 vs. 1.15±0.41, 2 days gastrointestinal function score: 2.24±0.98 vs. 2.42±1.05, 4 days APACHEII score: 16.4±6.8 vs. 20.1±7.1, the length of MV (days): 6.5±3.1 vs. 10.1±4.6, the length of stay in ICU (days): 11.3±6.3 vs. 13.8±7.5, mortality: 8.6% vs. 16.8%, average hospital expenses (yuan): 72 thousands vs. 86 thousands, P<0.05 or P<0.01]. CONCLUSION: Dahuang Fuzi decoction may enhance the intestinal peristalsis, protect the gastrointestinal barrier function, reduce the bacteria and endotoxin translocation and the releasing of inflammation mediators, protect the function of body immunity, reduce the length of MV, the length of stay in ICU, and lower the 28-day mortality and average hospital expenses, and it can improve the prognosis of patients with SAP.

[Expression and significance of phosphatidylinositol 3-kinase/protein kinase B signal transduction pathway in severe acute pancreatitis-associated lung injury].

OBJECTIVE: To investigate the expression and significance of phosphatidylinositol 3-kinase/protein kinase B (PI3K/PKB) signal transduction pathway in severe acute pancreatitis-associated lung injury. METHODS: Twenty-four healthy male Changbai pigs were randomized into four groups: sham operation group (n = 6), ALI group (n = 6), ALI plus LPS (lipopolysaccharide) group (n = 6) and ALI plus Wortmannin group (n = 6). The expression levels of PI3K and PKB were determined by both RT-PCR and Western blot. Activity changes of NF-kappaB were detected by electrophoretic mobility shift assay (EMSA). Contents of TNF-alpha and IL-1beta in bronchoalveolar lavage fluid (BALF) were examined by ELISA. The histopathological changes of lung were observed. RESULTS: The expressions of mRNA and protein PI3K and PKB, NF-kappaB activity and TNF-alpha, IL-1beta in BALF in ALI group were higher than those in sham operation group (P < 0.05 or P < 0.01). In ALI plus LPS group, they increased significantly in comparison with ALI group and sham operation group after injection of LPS (P < 0.05 or P < 0.01). In ALI plus Wortmannin group, these parameters were significantly inhibited by Wortmannin in comparisons with ALI group and ALI plus LPS group (P < 0.05 or P < 0.01). CONCLUSION: The phosphatidylinositol 3-kinase/protein kinase B signal transduction pathway is found to participate in the pathological process of severe acute pancreatitis-associated lung injury through the up-regulations of NF-kappaB activity, TNF-alpha and IL-1beta.