Overexpression of CHD1L is associated with poor survival and aggressive tumor biology in esophageal carcinoma.

Esophageal carcinoma (EC) is a malignancy with high metastatic potential. Chromosomal helicase/ATPase DNA binding protein 1-like (CHD1L) gene is a newly identified oncogene located at Chr1q21, and it is amplified in many solid tumors. However, the status of CHD1L protein expression in EC and its clinical significance is uncertain. This study was designed to investigate the significance of CHD1L expression in human EC and its biological function in EC cells. The expression of CHD1L was examined by immunohistochemistry in 191 surgically resected ECs. The associations between CHD1L expression and clinical pathological parameters and the prognostic value of CHD1L were analyzed. Western blot analysis showed that CHD1L was overexpressed in EC cell lines. In addition, positive CHD1L expression was strongly related to advanced clinical stage (P<0.01), and lymph node metastasis (P<0.01) of EC. The Kaplan-Meier curve indicated that high expression of CHD1L may result in poor prognosis of EC patients (P<0.01), and multivariate analysis showed that CHD1L overexpression was an independent predictor of overall survival. Furthermore, suppression of CHD1L in EC cells increased apoptosis and decreased cell proliferation invasion ability. Our results suggest that CHD1L is a target oncogene with the potential to serve as a novel prognostic biomarker in EC pathogenesis.

Pharmacokinetic comparisons of two different combinations of Shaoyao-Gancao Decoction in rats: competing mechanisms between paeoniflorin and glycyrrhetinic acid.

ETHNOPHARMACOLOGICAL RELEVANCE: Shaoyao-Gancao Decoction (SGD), a well-known traditional Chinese medicine prescription, is a combination of Radix Paeoniae Alba (Paeonia lactiflora Pall, root) and Glycyrrhizae uralensis (Glycyrrhiza uralensis Fisch., root and rhizome, honeyed) for spasmolysis and emergency pain relief. Paeoniflorin (PF) and glycyrrhetinic acid (GA) are two typical active components of SGD for pain relief. AIM OF THE STUDY: To study comparative pharmacokinetics of ten bioactive compounds in SGDs with two different combinations of RP and GU, and therefore to investigate the herb-herb interaction mechanisms of Shaoyao-Gancao Decoction for better spasmolysis and emergency pain relief in rats. MATERIALS AND METHODS: Herbal IR macro-fingerprinting was implemented to provide the full chemical fingerprints of RP, GU and SGD decoctions and to investigate the variation rule of the full chemical profile of SGDs with various combinations of RP and GU. A specifically developed HPLC-MS/MS assay coupled with protein precipitation method was employed to determine the plasma concentrations of the ten analytes. Male Wistar rats were orally administered with SGD1 (RP:GU, 1:1 (w/w)) and SGD2 ((RP:GU, 4:1 (w/w)) equivalent to 9.5 g/kg body weight of GU. RESULTS: Full chemical fingerprints of RP, GU and SGDs with various combinations of RP and GU were provided in the form of IR macro-fingerprints. Except for liquiritin, there were statistically significant differences (p<0.05 or p<0.01) of these analytes between SGD1 and SGD2 in in vivo pharmacokinetic study. Compared with the results when oral administrated with SGD1, six glycosides (PF, albiflorin, oxypaeoniflorin, isoliquiritin, ononin, and glycyrrhizin) exhibited higher systematic exposure levels (AUC0-t) and slower elimination rates (CL) whereas two glycones (GA and isoliquiritigenin) were the reverse when administrated with SGD2. CONCLUSIONS: Increasing the amount of RP attenuated the inhibitory effect of GA via competing being consumed by intestinal bacteria (or ß-glucosidase) to reduce the conversion amount of glycyrrhizin to GA and subsequently to afford significantly higher bioavailability and longer efficacy of PF, glycyrrhizin, albiflorin, oxypaeoniflorin, isoliquiritin, and ononin, leading to better spasmolysis and emergency pain relief.

[Study on different proportion formulas of activating blood circulation by FTIR].

Fourier transform spectroscopy (FTIR) and two-dimensional correlation spectroscopy were used to analyze different compatibility forms of sargentgloryvine stem, radix paeoniae rubra and cortex moutan in order to study the FTIR spectra of different proportion formulas. The results indicate that different proportion formulas have distinct change regularity in FTIR spectra, second derivative spectra and synchronous 2D. Key components of sargentgloryvine stem's characteristic absorption band are 1 610, 1 518 and 1 446 cm(-1) which are characteristic absorption band of aromatic material, and in the original formula, that shows stronger peak than others, suggesting that original formula can make the best of composition of drug action. In the different proportion formulas, 1 614 cm(-1) is nearer to 1 610 cm(-1), which is sargentgloryvine stem's characteristic absorption band, than to 1 706 cm(-1), illustrating that sargentgloryvine stem is more influential formula than others. According to identification and ascription of characteristic absorption band, it was initially revealed that different proportion of drug dosage can affect pharmacodynamic action of integral formula.

[Analysis and comparison of daxueteng and their extracts by FTIR].

The present study is to compare and analyze Sargentodora cuneata (oliv) Rehd. etwils herbs, the aqueous, anthraquinone extracts and residue in Jiang Xi and An Hui quickly and undamagedly by Fourier transform infrared spectroscopy(FTIR) and two-dimensional correlation spectroscopy. In the spectra of DaXueTeng, there are two strong peak areas at about 1400-1620 cm(-1) and 1000-1200 cm(-1), and it was concluded that DaXueTeng contains much glycoside and anthraquinone. Anthraquinone extracts only exhibit strong peaks of 1400-1620 cm(-1) which were stronger than that of 1000-1200 cm(-1), and this illustrated that this method was adapted to extracting anthraquinone; then aqueous only show powerful peaks of 1000-1200 cm(-1), so the authors know that the craftwork was suitable for extracting glycoside; finally, the authors also found that the residue of DaXueTeng contained much calcium oxalate. All of these illustrated that FTIR could not only analyze and identify traditional Chinese medicine (TCM) and extract components, but also discriminate contents of different extracts of TCM. The authors developed the new method to analyze and evaluate the DaXueTeng and their pharmacodynamic extracts successfully.

Beneficial effect of HHI-I on cerebral microcirculation, blood-brain barrier in rats and anti-hypoxic activity in mice.

OBJECTIVE: To investigate the effect of HHI-I (I) on the cerebral microcirculation, the blood-brain barrier permeability in rats and anti-hypoxic activity in mice. METHODS: (1) The blood microcirculation of the brain in rats was investigated by laser Doppler flowmetry with the probes laid on the cerebral pia mater or inserted into the brain parenchyma. (2) The protective action of HHI-I against the brain microcirculation disturbance induced by intravenous injection of high-molecular dextran (10%, 9 mL/kg) was observed. (3) The protective effect of HHI-I against lethal hypoxia in mice was observed with a hypoxic chamber containing 5% oxygen. (4) The disruption of the blood-brain barrier (BBB) permeability in rats was caused by phenylephrine-induced hypertension, and the effect of intravenous injection of HHI-I on the BBB permeability was determined using Evans blue as the marker. RESULTS: HHI-I could increase the blood flow of the cerebral microcirculation in rats and possess some protective effects on the brain microcirculatory disturbance. Besides, HHI-I could decrease the brain edema occurring in the process of lethal hypoxia in mice. While increasing the blood flow of brain, HHI-I could lower the BBB permeability in rats. CONCLUSION: HHI-I has several beneficial effects on the cerebral microcirculation, blood-brain barrier in rats and anti-hypoxic activity in mice.

Da-Cheng-Qi-Tang promotes the recovery of gastrointestinal motility after abdominal surgery in humans.

In order to examine the effects of Da-Cheng-Qi-Tang (DCQT) on gastrointestinal motility functions after abdominal surgery in humans, 33 patients with abdominal surgeries and 36 patients with cholecystectomies were divided into the DCQT and the control groups at random. Electrogastrography (EGG) and gastroduodenojejunal manometry was performed and the levels of plasma motilin were measured by radioimmunoassay. The results were as follows: (1) on the day of surgery, the ratio of EGG normal frequency in the DCQT group was higher than in the control group (P=0.0016); (2) the power of EGG in the DCQT group was higher than in the control group on the second and third days after surgeries (P=0.0011 and P=0.0215, respectively); (3) the percentage of normal bowel peristalsis was significantly higher in the DCQT group than in the control group (P<0.01); and (4) in the DCQT group, the plasma motilin level reached its peak earlier than in the control group. Our results suggest that DCQT can increase plasma motilin, enhance gastrointestinal motility, improve gastric dysrythmia, and reduce gastroparesis after abdominal surgery.

Effect of Tetrandrine on LPS-induced NF-kappaB activation in isolated pancreatic acinar cells of rat.

AIM: To investigate the effect of Tetrandrine (Tet) on LPS-induced NF-kappaB activation and cell injury in pancreatic acinar cells and to explore the mechanism of Tetrandrine preventing LPS-induced acinar cell injury. METHODS: Male rat pancreatic acinar cells were isolated by collagenase digestion, then exposed to LPS (10 mg/L), Tet (50 micromol/L, 100 micromol/L) or normal media. At different time point (30 min, 1 h, 4 h, 10 h) after treatment with the agents, cell viability was determined by MTT, the product and nuclear translocation of subunit p65 of NF-kappaB was visualized by immunofluorescence staining and nuclear protein was extracted to perform EMSA which was used to assay the NF-kappaB binding activity. RESULTS: LPS induced cell damage directly in a time dependent manner and Tet attenuated LPS-induced cell damage (50 micromol/L, P < 0.05; 100 micromol/L, P < 0.01). NF-kappaB p65 immunofluorescence staining in cytoplasm increased and began showing its nuclear translocation within 30 min and the peak was shown at 1 h of LPS 10 mg/L treatment. NF-kappaB DNA binding activity showed the same alteration pattern as p65 immunofluorescence staining. In Tet group, the immunofluorescence staining in cytoplasm and nuclear translocation of NF-kappaB were inhibited significantly. CONCLUSION: NF-kappaB activation is an important early event that may contribute to inflammatory responses and cell injury in pancreatic acinar cells. Tet possesses the protective effect on LPS-induced acinar cell injury by inhibiting NF-kappaB activation.

[Study on protective effect of tetrandrine on lipopolysaccharide induced pancreatic acinar cell damage and its mechanism].

OBJECTIVE: To evaluate the protective effect of tetrandrine (Tet) on lipopolysaccharide (LPS)-induced pancreatic acinar cell damage and to explore its mechanism. METHODS: SD male rat's pancreatic acinar cells were isolated by collagenase digestion and they were pre-treated with Tet (50 micromol/L and 100 micromol/L), then exposed to LPS (10 mg/L) or conventional culture medium respectively. At 0, 1, 4, 10 hours after treatment with the agents, cell viability was determined by methyl thiazolyl tetrazolium (MTT), and the supernatant supernate of cells was collected for determination of the content of malondialdehyde (MDA) and the activity of superoxide dismutase (SOD) and phospholipase A(2) (PLA(2)). Some cells were loaded with Fluo-3/AM, and the dynamic change in intracellular Ca(2+) ([Ca(2+)]i) in single pancreatic acinar cell was determined by laser scanning confocal microscopy. RESULTS: Tet attenuated LPS-induced cell damage (P<0.05 and P<0.01) and inhibited the elevation of cytosolic free calcium of rat pancreatic acinar cells. In the supernatant, Tet pretreatment decreased the content of MDA and the activity of PLA(2) and increased the activity of SOD (all P<0.05). CONCLUSION: Tet attenuates LPS-induced cell damage by blocking [Ca(2+)]i overload, inhibiting superoxide response, decreasing activity of pancreatic enzyme, thus it shows a protective effect on pancreatic acinar cells.

Upregulation of human telomerase reverse transcriptase mRNA expression by in vitro transfection of hepatitis B virus X gene into human hepatocarcinoma and cholangiocarcinoma cells.

AIM: To study the changes of human telomerase reverse transcriptase (hTERT) mRNA expression in human hepatocarcinoma cell lines (HepG2) and cholangiocarcinoma cell lines (QBC939) after HBx gene transfection and to illustrate the significance of transcriptional regulation of hTERT gene by HBx gene in the carcinogenesis. METHODS: HepG2 and QBC939 cell lines were cultured and co-transfected with eukaryotic expression vector containing the HBx coding region and cloning vector containing enhanced green fluorescent protein (EGFP) coding sequence using lipid-mediated gene transduction technique. Thirty-six hours after transfection, EGFP expression in cells was used as the indicator of successful transfection. Flow cytometry was performed to determine the transfection efficiency. Cells were harvested and total RNA was extracted using TRIzol reagent. The expression of hTERT mRNA in HepG2 and QBC939 cell lines was assayed by reverse transcription-polymerase chain reaction. The expression of HBx protein in both cell lines was detected by immunocytochemical staining and Western blotting. RESULTS: Flow cytometry showed that the transfection efficiency was 46.4% in HepG2 cells and 29.6% in QBC939 cells for both HBx gene expression vector and blank vector. The expression of hTERT mRNA was meaningfully increased in HepG2 and QBC939 cell lines when transfected with HBx gene expression vector compared to those transfected with OPTI-MEM medium and blank vector. Immunocytochemical staining and Western blotting revealed HBx protein expression in HepG2 and QBC939 cells only when transfected with HBx gene. CONCLUSION: HBx gene transfection can upregulate the transcriptional expression of hTERT mRNA. The transactivation of hTERT gene by HBx gene is a newfound mechanism for pathogenesis of hepatocarcinomas and cholangiocarcinomas after HBV infection.

[A study on the cell differentiation induced by tanshinone IIA and its molecular mechanism in retinoic acid: resistant acute promyelocytic leukemia].

OBJECTIVE: To investigate retinoic acid-resistant acute promyelocytic leukemia (APL) cell differentiation induced by tanshinone IIA (Tan IIA) and its molecular mechanism. METHODS: NB4 cells treated with 0.5 mg/L Tan IIA was regarded as positive control. After in vitro incubation of MR-2 cells with Tan IIA at the concentration of 1.0 mg/L for 4 days, the cell differentiation was observed by growth status, cytomorphology, and nitroblue tetrazolium test. Cell cycle, membrane cluster differentiation (CD) antigens (CD(33), CD(11b)) and expression of some oncogene (c-myc, c-fos, p53 and bcl-2) were analysed by flow cytometry. RESULTS: The growth of MR-2 and NB4 cells was inhibited after Tan IIA treatment, the inhibition rate were 73.5% and 67.7% respectively (P < 0.01, P < 0.01) without significant difference. After Tan IIA treatment, MR-2 and NB4 cells were induced to undergo morphological differentiation, which exhibited small cell bulk decreased nucleus/cytoplasm proportion, rough chromatin, disappearance of nucleolus and formation of azurophil granules and anomalous nucleus. MR-2 cells could be induced to metamyelocyte while NB4 could be induced to band form. NBT reduction of MR-2 and NB4 cells treated with Tan IIA showed that positive cells accounted for (95.30 +/- 0.76)% and (93.20 +/- 1.04)% respectively; but the positive rate of either group of the treated positive cells was significantly higher than that of untreated, being (3.50 +/- 1.32)% and (2.80 +/- 0.29)% respectively (P < 0.01). Flow cytometry showed that the expression of CD(33) was reduced, while that of CD(11b) was increased. The quantity of treated cells in G(0)/G(1) phase increased but that in S phase decreased. The proliferous index was also decreased. After treated with Tan IIA, the expressions of anti-oncogene p53 and c-fos were up-regulated while those of oncogene bcl-2 and c-myc were down-regulated (P < 0.01). CONCLUSIONS: 1.0 mg/L Tan IIA could inhibit proliferation of MR-2 cells and induce differentiation of MR-2 cells into mature granulocyte, the effectivity was the same as 0.5 mg/L Tan IIA treated NB4 cells. Its possible molecular mechanism might be related to modulation of oncogene expressions associated proliferation and differentiation as well as inhibition of DNA synthesis. Tan IIA probably can be applied to treat the patients with APL, particularly to the relapsed and drug resistant patients with broad prospect.

Overexpression of sterol carrier protein-2 mRNA in patients with cholesterol gallstones.

BACKGROUND: Hypersecretion of biliary cholesterol is believed to be one of the important causes of lithogenic bile. Sterol carrier protein-2(SCP2) participates in cholesterol trafficking and metabolism and may play a key role in cholesterol gallstone formation. This study was undertaken to investigate the expression of liver SCP2 mRNA in patients with cholesterol gallstone and those patients with non-cholesterol gallstone. METHODS: The expression of liver SCP2mRNA was studied in 36 patients with cholesterol gallstone and 30 patients with non-cholesterol gallstone by reverse transcription-polymerase chain reaction (RT-PCR). RESULT: The expression of SCP2 mRNA was increased more significantly in patients with cholesterol gallstone than in patients with non-cholesterol gallstone. CONCLUSION: The SCP2 gene was overexpressed in patients with cholesterol gallstone, indicating that SCP2 may be one of the important causes of cholesterol gallstone.

[Intensive effect of traditional Chinese medicines activating blood to resolve stasis on medicines dredging intestines--influence on peristalsis of small intestine in guinea pigs].

OBJECTIVE: To observe the changes of peristalsis of small intestine in guinea pigs after administration of traditional Chinese medicines activating blood to resolve stasis (Compound Danshen Decoction, CDSD) or/and medicines dredging intestines (Dachengqi Decoction, DCQD), and to explore the synergetic or intensive effect of CDSD on DCQD. METHODS: By means of BL-420 Biological Experimental System, peristalsis of small intestine was recorded and analyzed following administration of DCQD, CDSD or Huoxue Chengqi Decoction (HXCQD, compound of CDSD and DCQD) respectively in different experimental periods. RESULTS: The amplitude and frequency of intestinal peristaltic wave obviously increased following administration of the three decoctions, but HXCQD appeared to be most dominantly. CONCLUSION: The effect of DCQD can be further enhanced by combining use of CDSD, suggesting that the traditional Chinese medicines activating blood to resolve stasis have an intensive effect on medicines dredging intestines.

[The expression and significance of human telomerase reverse transcriptase protein and gene in bile duct carcinomas and their adjacent tissues].

OBJECTIVE: To detect the expression of human telomerase reverse transcriptase (hTERT) protein and mRNA in bile duct carcinomas and the adjacent tissues and to elucidate its role in bile duct carcinogenesis. METHODS: The expression of hTERT protein and hTERT mRNA in the formalin-fixed paraffin-embedded specimens of 71 cases of bile duct cancers and 39 cases of adjacent tissues was detected by streptavidin-peroxidase immunostaining and in situ hybridization. The correlation was analysed statistically between the expression of hTERT protein and mRNA and clinicopathological parameters bile duct carcinomas. RESULTS: The positive rate of hTERT protein expression and mRNA expression in malignant specimens was 78.9% (56/71) and 67.6% (48/71), while that in the adjacent tissues was 35.9% (14/39) and 23.1% (9/39), respectively. All the positive signals were found in the hyperplastic biliary epithelia. No significant correlation was established between hTERT expression and clinicopathological parameters. CONCLUSION: hTERT gene transcription and protein expression is most likely involved in the proliferation and malignant transformation of bile epithelia and the malignant progression of bile duct carcinomas. The detection of hTERT expression may serve elucidating the carcinogenesis of bile duct.

[In situ nucleic acid detection of HBV X gene in extrahepatic biliary tract carcinomas and its clinicopathological significance].

OBJECTIVE: To detect the expression of HBV X gene (HBx mRNA) in extrahepatic biliary tract carcinomas and the adjacent non-cancerous tissues, and to analyzed the relationship between HBV infection and incidence of biliary tract carcinomas, thereby to elucidate the possible role of HBx in the carcinogenesis of biliary tract. METHODS: The plasmid pSPX46 was digested by appropriate restriction enzyme. HBx fragment was obtained through gel extraction kit. The digoxigenin-labeled DNA probes for HBx mRNA were prepared by a random prime technique. The expression of HBx mRNA was detected in formalin-fixed- paraffin-embedded specimens from 71 cases of biliary tract carcinomas and 39 specimens of non-cancerous tissues adjacent to cancer by in situ hybridization. The correlations between HBx mRNA expression and clinicopathological parameters were statistically analysed in 71 cases of biliary duct carcinomas. RESULTS: Forty-three of 71 malignant specimens had detectable HBx mRNA expression with a positive rate being 61%. Only 7 of 39 specimens of non-cancerous tissues adjacent to cancer had weak HBx mRNA expression, with a positive rate being 18%, and all these positive signals were found in the hyperplastic biliary epithelium. No significant correlation was found between HBx mRNA expression and clinicopathological parameters, but a strong positive correlation was found between HBx mRNA and protein expression. CONCLUSION: There is a high frequency of HBx mRNA expression in extrahepatic biliary tract carcinomas. HBV infection and its gene integration might play a role to certain extent in the development of biliary tract carcinomas.

Effects of lipopolysaccharides on calcium homeostasis in isolated pancreatic acinar cells of rat.

AIM: To investigate the effects of lipopolysaccharides (LPS, endotoxin) on the calcium content in pancreatic acinar cells and the origin of Ca2+ during calcium overload induced by LPS, further to explore the mechanism of LPS in inducing calcium overload and pancreatic acinar cell injury. METHODS: Male rat pancreatic acinar cells were isolated by collagenase digestion and loaded with Fluo-3/AM, then exposed to varying doses of LPS (from 1 mg/L to 20 mg/L). The dynamic change of [Ca2+]i in single pancreatic acinar cell in the absence and presence of Ca2+ in extracellular fluid was determined by laser scanning confocal microscopy. Cell viability was determined by MTT at different time points after treatment with LPS. RESULTS: Under physiological calcium concentration in extracellular fluid, LPS (10 mg/L) initiated a rapid, concentration-dependent rise in intracellular [Ca2+]i and consequent cell damage (P<0.05). LPS induced a slight rise of [Ca2+]i in the calcium-free extracellular fluid containing egtazic acid 1 mmol/L and addition of extracellular calcium in the presence of LPS resulted in a more immediate and remarkable rise of [Ca2+]i, which reached the peak value within 150 s and maintained the value sustainedly. Egtazic acid attenuated LPS-induced cell damage (P<0.05). The increase in intracellular [Ca2+]i preceded the pathological alteration of pancreatic acinar cells. CONCLUSION: LPS directly induced the injury and the disorder of calcium homeostasis in isolated rat pancreatic acinar cell. Calcium overload is an early event in the pathogenesis of LPS-induced cell damage. Origin of the [Ca2+]i in cytoplasma of pancreatic acinar cells during calcium overload is mainly due to the influx of extracellular Ca2+. Calcium homeostasis disorder may be one of the causes or at least an important mediator of LPS-induced pancreatic acinar cell damage.

[Changes in pro-inflammatory cytokines and media and peptide hormones during multiple organ dysfunction syndrome following acute abdominal diseases].

OBJECTIVE: To inquire into effects of cytokines and other inflammatory media, and peptide hormones during multiple organ dysfunction syndrome (MODS) subsequent to acute abdominal diseases. METHODS: In 19 patients with MODS due to acute abdominal diseases, tumor necrosis factor-alpha (TNF-alpha), interleukin-6 (IL-6), thromboxane B(2) (TXB(2)), 6-keto-prostaglandin F(1alpha) (6-keto-PGF(1alpha)), endotoxin, gene-related peptide(CGRP), endothelin-1 (ET-1) and substance P (SP) in plasma, and lipid peroxide (LPO) and nitric oxide (NO) in serum were determined dynamically. RESULTS: Both TNF-alpha and IL-6 at increased significantly in MODS patients; IL-6 on day 0 in patients without treatment of endoscopic retrograde bile duct drainage (ERBD) were higher than that in patients with correspondent treatment, IL-6 in severe acute cholangitis patients was higher than that in patients with acute necrotic pancreatitis, it approached 24,000 ng/L during toxic shock. TNF-alpha and IL-6 during early stage of MODS were higher than that during systemic inflammatory response syndrome (SIRS) respectively. Endotoxin and LPO levels in MODS patients increased significantly. The levels of NO in emergency patients with MODS was elevated, but lowered in patients with acute necrotic pancreatitis, hepatocarcinoma, advanced age's patients with long time fever due to hepatic abscess. TXB(2) and 6-keto-PGF(1alpha) during early stage rose significantly, both decreased after treatment. ET-1 and CGRP during early stage increased significantly, SP peaked on day 0. CONCLUSION: The level of IL-6 persistently higher than 300 ng/L suggests the diagnosis of MODS. The levels of IL-6 and TNF-alpha could be taken as an indication of the degree of SIRS. NO maybe either increased or decreased, ET-1, CGRP, TXB(2), 6-keto-PGF(1alpha), endotoxin, and LPO are found to be increased MODS.