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Patients with Parkinson's disease (PD) often suffer from delayed gastric emptying, but the underlying mechanism remains unclear. We have shown previously that a PD rat model comprising bilateral substantia nigra destruction by 6-hydroxydopamine (6-OHDA rats) exhibits gastroparesis with alteration of neural nitric oxide synthase (nNOS) and acetylcholine in gastric corpus. However, changes in pyloric motility in the 6-OHDA rats have not been characterized. Solid gastric emptying test, immunofluorescence, Western blot, and in vitro pyloric motility recordings were used to assess pyloric motor function in the 6-OHDA rats. The 6-OHDA-treated rats displayed delayed solid gastric emptying and a lower basal pyloric motility index. In the 6-OHDA rats, high K

Liver resection for hepatocellular carcinoma (HCC) patients with portal vein tumor thrombus (PVTT) offers a chance of cure, although survival is often limited. The actual 3-year survival and its associated prognostic factors have not been reported. A nationwide database of HCC patients with PVTT who underwent liver resection with ‘curative’ intent was analyzed. The clinicopathologic characteristics, the perioperative, and survival outcomes for the actual long-term survivors were compared with the non-long-term survivors (patients who died within 3 years of surgery). Univariable and multivariable regression analyses were performed to identify predictive factors associated with long-term survival outcomes. The study included 1590 patients with an actuarial 3-year survival of 16.6%, while the actual 3-year survival rate was 11.7%. There were 171 patients who survived for at least 3 years after surgery and 1290 who died within 3 years of surgery. Multivariable regression analysis revealed that total bilirubin > 17.1 μmol/l, AFP > 400 ng/ml, types of hepatectomy, extent of PVTT, intraoperative blood loss > 400 ml, tumor diameter > 5 cm, tumor encapsulation, R0 resection, liver cirrhosis, adjuvant TACE, postoperative early recurrence (

BACKGROUND AND PURPOSE: Dopamine protects the duodenal mucosa. Here we have investigated the source of dopamine in gastric juice and the mechanism underlying the effects of luminal dopamine on duodenal bicarbonate secretion (DBS) in rodents. EXPERIMENTAL APPROACH: Immunofluorescence, UPLC‐MS/MS, gastric incubation and perfusion were used to detect gastric‐derived dopamine. Immunofluorescence and RT‐PCR were used to examine the expression of dopamine receptors in the duodenal mucosa. Real‐time pH titration and pH(i) measurement were performed to investigate DBS. KEY RESULTS: H(+)‐K(+)‐ATPase was co‐localized with tyrosine hydroxylase and dopamine transporters in gastric parietal cells. Dopamine was increased in in vivo gastric perfusate after intravenous infusion of histamine and in gastric mucosa incubated, in vitro, with bethanechol chloride or tyrosine. D(2) receptors were the most abundant dopamine receptors in rat duodenum, mainly distributed on the apical membrane of epithelial cells. Luminal dopamine increased DBS in a concentration‐dependent manner, an effect mimicked by a D(2) receptor agonist quinpirole and inhibited by the D(2) receptor antagonist L741,626, in vivo D(2) receptor siRNA and in D(2) receptor (−/−) mice. Dopamine and quinpirole raised the duodenal enterocyte pH(i). Quinpirole‐evoked DBS and PI3K/Akt activity were inhibited by calcium chelator BAPTA‐AM or in D(2) receptor(−/−) mice. CONCLUSION AND IMPLICATIONS: Dopamine in the gastric juice is derived from parietal cells and is secreted along with gastric acid. On arrival in the duodenal lumen, dopamine increased DBS via an apical D(2) receptor‐ and calcium‐dependent pathway. Our data provide novel insights into the protective effects of dopamine on the duodenal mucosa.

Background There is currently a lack of consensus regarding the clinical features, diagnosis, treatment indications and options, and risk assessment of hepatic hemangioma patients. Methods This was a multicenter, real-world study that analyzed a large number of hepatic hemangioma cases in China and included patient data on epidemiology, diagnosis, treatment methods, and outcomes. Results A total of 5,143 patients hospitalized for hepatic hemangioma were included, of whom 34.42% were male and 65.58% were female. The age distribution was concentrated between 30 and 60 years old, accounting for 87.41% of the patients. Among the hepatic hemangioma patients, 60.8% had only one tumor, with the most common pathological type being cavernous hemangioma (96.07% of cases). The treatment motivations and indications included anxiety, obvious clinical symptoms, rapid tumor growth, unclear diagnoses and acute emergencies. Overall, 41.4% of the patients were treated for psychological reasons, while 30.59% were treated because they presented obvious (primarily nonspecific) clinical symptoms. Hepatic resection was the main therapeutic method and was based on various indications. There were a small number of patients with Kasabach-Merritt syndrome, according to its generally recognized definition. Conclusions Most patients in this study who were hospitalized for hepatic hemangioma did not meet the indications for requiring treatment. Surveillance is the recommended course of action for definitively diagnosed hepatic hemangioma, and a new classification system is needed to standardize the diagnosis of this condition.

Recent studies have shown that radiofrequency (RF) ablation therapy is a safe, feasible, and effective procedure for hepatic hemangiomas, even huge hepatic hemangiomas. RF ablation has the following advantages in the treatment of hepatic hemangiomas: minimal invasiveness, definite efficacy, high safety, fast recovery, relatively simple operation, and wide applicability. It is necessary to formulate a widely accepted consensus among the experts in China who have extensive expertise and experience in the treatment of hepatic hemangiomas using RF ablation, which is important to standardize the application of RF ablation for the management of hepatic hemangiomas, regarding the selection of patients with suitable indications to receive RF ablation treatment, the technical details of the techniques, therapeutic effect evaluations, management of complications, etc. A final consensus by a Chinese panel of experts who have the expertise of using RF ablation to treat hepatic hemangiomas was reached by means of literature review, comprehensive discussion, and draft approval.

The present study aimed to characterize the function and hemorheology of red blood cells (RBCs) recovered during liver transplantation surgery in patients with hepatitis B and decompensation. A total of 15 hepatitis B patients with decompensation who underwent liver transplantation surgery were included in the present study. Blood samples were recovered during the liver transplantation surgery using an Autologous Blood Recovery System. The morphology and structure of RBCs were characterized and compared between pre-operative and recovered blood samples. In addition, the physiological functions of RBCs were measured and compared between pre-operative and recovered blood samples. No significant differences in the morphological score, 2,3-diphosphoglycerate, Na+K+-ATPase, Ca2+-ATPase, Mg2+-ATPase, malondialdehyde and osmotic fragility were identified between RBCs in the pre-operative and recovered blood samples. The level of free hemoglobin in RBCs of the recovered blood samples was significantly higher than in the pre-operative blood samples (P0.05) in the low-shear blood viscosity, plasma viscosity, relative blood viscosity, erythrocyte aggregation index or Casson yield stress were identified between recovered and pre-operative blood samples. These findings suggested that autologous blood transfusion in liver transplantation surgery in patients with hepatitis B and decompensation had no significant influence on the morphology, structure, function and hemorheology of RBCs.

Celecoxib, a selective cyclooxygenase-2 (COX-2) inhibitor, has been demonstrated to exert antitumor activity in a variety of cancer cells. The underlying mechanism involves inhibition of cell cycle progression and induction of apoptosis. Besides, celecoxib has also been found to induce autophagy in some solid tumor cells. The aim of this study was to investigate the effect of celecoxib on cell proliferation in HL-60 human acute leukemia cells and to explore the potential mechanism. HL-60 cells were exposed to various concentrations of celecoxib and cell viability was evaluated by the MTT assay. Apoptosis was analyzed with flow cytometry and the amount of autophagosome was evaluated by LysoTracker probe labelling. The expression of apoptosis- and autophagy-related proteins was assayed by Western blot and LysoSensor probe labelling was used to detect the effect of celecoxib on the lysosomal functions. The results of this study indicated that celecoxib inhibited cell proliferation in a time- and dose-dependent fashion. The flow cytometry analysis showed that celecoxib induced apoptosis at low concentrations and mainly cell necrosis at high concentrations. The Western blot test confirmed the induction of apoptosis by the upregulation of apoptosis-related proteins cleaved caspase-3 and cleaved PARP. Furthermore, this study demonstrated that celecoxib prevented the autophagic flux by inhibiting lysosome function; the fluorescence intensity of the LysoTracker probe and the level of autophagy-related proteins LC3-II and p62 were increased, but the fluorescence intensity of the LysoSensor probe was weakened. These findings show that celecoxib is an autophagy suppresser and has antitumor effects in HL-60 cells by inducing cell apoptosis and necrosis.

Aim The intestinal barrier is made up of epithelial cells and intercellular junctional complexes to regulate epithelial ion transport and permeability. Dopamine (DA) is able to promote duodenal epithelial ion transport through D1-like receptors, which includes subtypes of D1 (D1R) and D5 (D5R), but whether D1-like receptors influence the duodenal permeability is unclear. Methods FITC–dextran permeability, short-circuit current (ISC), Western blot, immunohistochemistry and ELISA were used in human D5R transgenic mice and hyperendogenous enteric DA (HEnD) rats in this study. Results Dopamine induced a downward deflection in ISC and an increase in FITC–dextran permeability of control rat duodenum, which were inhibited by the D1-like receptor antagonist, SCH-23390. However, DA decreased duodenal transepithelial resistance (TER), an effect also reversed by SCH-23390. A strong immunofluorescence signal for D5R, but not D1R, was observed in the duodenum of control rat. In human D5R knock-in transgenic mice, duodenal mucosa displayed an increased basal ISC with high FITC–dextran permeability and decreased TER with a lowered expression of tight junction proteins, suggesting attenuated duodenal barrier function in these transgenic mice. D5R knock-down transgenic mice manifested a decreased basal ISC with lowered FITC–dextran permeability. Moreover, an increased FITC–dextran permeability combined with decreased TER and tight junction protein expression in duodenal mucosa were also observed in HEnD rats. Conclusion This study demonstrates, for the first time, that DA enhances duodenal permeability of control rat via D5R, which provides new experimental and theoretical evidence for the influence of DA on duodenal epithelial barrier function.

The interaction between bone marrow stromal cells and leukemia cells is critical for the persistence and progression of leukemia, and this interaction may account for residual disease. However, the link between leukemia cells and their environment is still poorly understood. In our study, runt‑related transcription factor 3 (RUNX3) was identified as a novel target gene affected by As2O3 and involved in mesenchymal stem cell (MSC)‑mediated protection of leukemia cells from As2O3‑induced apoptosis. We observed induction of RUNX3 expression and the translocation of RUNX3 into the nucleus after As2O3 treatment in leukemia cells. In K562 chronic myeloid leukemia cells, downregulation of endogenous RUNX3 compromised As2O3‑induced growth inhibition, cell cycle arrest, and apoptosis. In the presence of MSC, As2O3‑induced expression of RUNX3 was reduced significantly and this reduction was modulated by CXCL12/CXCR4 signaling. Furthermore, overexpression of RUNX3 restored, at least in part, the sensitivity of leukemic cells to As2O3. We conclude that RUNX3 plays an important role in As2O3‑induced cellular responses and allows cells to overcome MSC‑mediated drug resistance. Therefore, RUNX3 is a promising target for therapeutic approaches to overcome MSC‑mediated drug resistance.

Constipation is common in Parkinson's disease (PD), in which monoamines (dopamine [DA], norepinephrine [NE], and 5-hydroxytryptamine [5-HT]) play an important role. Rats microinjected with 6-hydroxydopamine (6-OHDA) into the bilateral substantia nigra (SN) exhibit constipation, but the role of monoamines and their receptors is not clear. In the present study, colonic motility, monoamine content, and the expression of monoamine receptors were examined using strain gauge force transducers, ultraperformance liquid chromatography tandem mass spectrometry, immunofluorescence, and Western blot. The 6-OHDA rats displayed a significant reduction in dopaminergic neurons in the SN and a decreased time on rota-rod test and a marked decrease in daily fecal production and fecal water content. The amplitude of colonic spontaneous contraction was obviously decreased in 6-OHDA rats. Blocking D1-like receptor and β3-adrenoceptor (β3-AR) significantly reduced the inhibition of DA and NE on the colonic motility, respectively, whereas the 5-HT and 5-HT4 receptor agonists promoted the colonic motility. Moreover, DA content was increased in the colonic muscularis externa of 6-OHDA rats. The protein expression of β3-ARs was notably upregulated, but 5-HT4 receptors were significantly decreased in the colonic muscularis externa of 6-OHDA rats. We conclude that enhanced DA and β3-ARs and decreased 5-HT4 receptors may be contributed to the colonic dysmotility and constipation observed in 6-OHDA rats.

The development of targeted tyrosine kinase inhibitors (TKIs) has succeeded in altering the course of chronic myeloid leukemia (CML). However, a number of patients have failed to respond or experienced disease relapse following TKI treatment. Proviral integration site for moloney murine leukemia virus-1 (PIM-1) is a serine/threonine kinase that participates in regulating apoptosis, cell cycle, signal transduction and transcriptional pathways, which are associated with tumor progression, and poor prognosis. SMI-4a is a selective PIM-1 kinase inhibitor that inhibits PIM-1 kinase activity in vivo and in vitro. The present study aimed to explore the mechanism underlying the antitumor effect of SMI-4a in K562 and imatinib-resistant K562 (K562/G) cell lines. It was demonstrated that SMI-4a inhibited the proliferation of K562 and K562/G cells using a WST-8 assay. The Annexin V-propidium iodide assay demonstrated that SMI-4a induced apoptosis of K562 and K562/G cells in a dose-, and time-dependent manner. Furthermore, Hoechst 33342 staining was used to verify the apoptosis rate. The clone formation assay revealed that SMI-4a significantly inhibited the colony formation capacity of K562 and K562/G cells. Western blot analysis demonstrated that SMI-4a decreased phosphorylated (p)-Ser9-glycogen synthase kinase (GSK) 3β/pGSK3β and inhibited the translocation of β-catenin. In addition, the downstream gene expression of apoptosis regulator Bax and poly(ADP-ribose) polymerase-1 was upregulated, and apoptosis regulator Bcl-2 and Myc proto-oncogene protein expression levels were downregulated. Immunofluorescence results demonstrated changes in the expression level of β-catenin in the plasma and nucleus. The results of the present study suggest that SMI-4a is an effective drug to use in combination with current chemotherapeutics for the treatment of imatinib-resistant CML.

Aim Gastroparesis is a common non-motor system symptom of Parkinson's disease (PD). However, the mechanism responsible for the gastric motor abnormality is not clear. We previously reported on the impaired gastric motility in 6-hydroxydopamine (6-OHDA) rats, which were treated with a bilateral microinjection of 6-OHDA in the substantia nigra (SN). We hypothesize that the enhanced dopamine system and reduced acetylcholine (Ach) in gastric tissues might contribute to the delayed gastric emptying observed in PD. Methods A strain gauge force transducer, digital X-ray imaging system, Western blot, immunofluorescence and Radio Immunoassay were used in this study. Results Dopaminergic neurones in the SN were greatly reduced following the bilateral microinjection of 6-OHDA. 6-OHDA rats exhibited impaired gastric motility and delayed gastric emptying, accompanied by increased dopamine content and the overexpression of D2 receptors in the stomach. The administration of the D2 receptor antagonist domperidone relieved gastric dysmotility in 6-OHDA rats, but the D1 receptor antagonist SCH23390 failed to do so. Subdiaphragmatic vagotomy prevented the increase in the gastric dopamine content and D2 receptor expression and improved gastric dysmotility in 6-OHDA rats. Conclusion Dopaminergic deficiency in the SN results in impaired gastric motility, possibly as a result of the enhanced activity of dopamine system and reduced Ach in gastric tissue. The vagus nerve plays an important role in peripheral gastric motility disorder.

To study movement disorder in Parkinson's disease (PD), an animal model of PD can be created by injecting lipopolysaccharide (LPS) into the substantia nigra of rats. In addition to body movement disorders, patients with PD often experience gastrointestinal (GI) dysfunction, such as gastroparesis. However, the underlying mechanism of these disorders remains unclear. The dorsal motor nucleus of vagus (DMV) is a well-known visceral nucleus that regulates GI function. The present study investigated alterations in DMV neurons and gastric motility in rats with LPS-induced PD (LPS-PD rats). Gastric motility was recorded using a strain gauge force transducer in vivo. The distributions of tyrosine hydroxylase (TH)- and choline acetyltransferase (ChAT)-positive neurons in the DMV were determined using immunofluorescence and confocal laser microscopy. Our results indicated that in LPS-PD rats, the number of neurons in the substantia nigra, including neurons with TH immunoreactivity, was markedly reduced, although glial cell proliferation was clearly observed. However, enhanced TH immunoreactivity and decreased ChAT immunoreactivity were found in the DMV. Furthermore, weakened gastric motility was recorded in anesthetized LPS-PD rats. In conclusion, rats with LPS-induced PD exhibited gastric dysmotility with an alteration in DMV neurons. This PD model may be used to study autonomic nervous system disorders that are often observed in patients with early-stage PD.

Dopamine (DA) is synthesized in gastrointestinal epithelial cells and performs important regulatory effects on the duodenal mucosa. However, the underlying mechanism remains largely unknown. The present study investigated the effect of DA on the duodenal epithelial ion transport in rats by means of short-circuit current (ISC), real-time pH titration, enzyme-linked immunosorbent assay, and immunohistochemistry. The results indicate that basolateral, but not apical, application of DA induced a concentration-dependent ISC downward deflection with an apparent IC50 of 5.34 μmol/L. Basolateral application of dopaminergic receptor D1 (D1) antagonist, SCH-23390, inhibited DA-induced change in ISC (△ISC) in a dose-dependent manner. D1 agonist, SKF38393, mimicked the effect of DA on the ISC. The clear immunoreactivity of D1 subtype D5 (D1b) was at the both apical and basolatoral sides of Brunner's glands and intestinal crypts. Basolateral pretreatment with adenylate cyclase inhibitor, MDL12330A, significantly inhibited DA- and forskolin-induced △ISC. DA and SKF38393 increased the level of intracellular cyclic adenosine monophosphate (cAMP) from 1.55 ± 0.11 to 2.07 ± 0.11 and 5.91 ± 0.25 pmol/L·mg(-1), respectively. Furthermore, the serosal DA-induced △ISC was remarkably inhibited by apical administration of K(+) channel blockers, Ba(2+) and tetraethylammonium, but not by Cl(-) channel blockers. Serosal DA and D1 agonist did not affect duodenal HCO3(-) secretion. In conclusion, the present results demonstrate that serosal DA is able to promote rat duodenal epithelial K(+) secretion, not HCO3(-) secretion through D1-mediated and cAMP-dependent pathway. The study provides a new insight in the modulation of DA on the ion transport of duodenal epithelia in rats.

Epigenetic therapy has had a significant impact on the management of haematologic malignancies. The aim of this study was to assess whether 5-aza-CdR and TSA inhibit the growth of leukaemia cells and induce caspase-3-dependent apoptosis by upregulating RUNX3 expression.K562 and Reh cells were treated with 5-aza-CdR, TSA or both compounds. RT-PCR and Western blot analyses were used to examine the expression of RUNX3 at the mRNA and protein levels, respectively. Immunofluorescence microscopy was used to detect the cellular location of RUNX3. Additionally, after K562 cells were transfected with RUNX3, apoptosis and proliferation were studied using Annexin V staining and MTT assays.The expression of RUNX3 in leukaemia cell lines was markedly less than that in the controls. Demethylating drug 5-aza-CdR could induce RUNX3 expression, but the combination of TSA and 5-aza-CdR had a greater effect than did treatment with a single compound. The combination of 5-aza-CdR and TSA induced the translocation of RUNX3 from the cytoplasm into the nucleus. TSA enhanced apoptosis induced by 5-aza-CdR, and Annexin V and Hoechst 33258 staining showed that the combination induced apoptosis but not necrosis. Furthermore, apoptosis was dependent on the caspase-3 pathway. RUNX3 overexpression in K562 cells led to growth inhibition and apoptosis and potentiated the effects of 5-aza-CdR induction.RUNX3 plays an important role in leukaemia cellular functions, and the induction of RUNX3-mediated effects may contribute to the therapeutic value of combination TSA and 5-aza-CdR treatment.

Tanshinone I (Tan-I) is a diterpene quinone extracted from the traditional herbal medicine Salvia miltiorrhiza Bunge. Recently, Tan-I has been reported to have anti-tumor effects. In this study, we investigated the growth inhibition and apoptosis inducing effects of Tan-I on three kinds of monocytic leukemia cells (U937, THP-1 and SHI 1). Cell viability was measured by MTT assay. Cell apoptosis was assessed by flow cytometry (FCM) and AnnexinV/PI staining. Reverse transcriptase polymerase chain reaction (RT-PCR) and PCR-enzyme-linked immunosorbent assay (ELISA) were used to detect human telomerase reverse transcriptase (hTERT) expression and telomerase activity before and after apoptosis. The activity of caspase-3 was determined by Caspase colorimetric assay kit and Western blot analysis. Expression of the anti-apoptotic gene Survivin was assayed by Western blot and Real-time RT-PCR using the ABI PRISM 7500 Sequence Detection System. The results revealed that Tan-I could inhibit the growth of these three kinds of leukemia cells and cause apoptosis in a time- and dose-dependent manner. After treatment by Tan-I for 48 h, Western blotting showed cleavage of the caspase-3 zymogen protein with the appearance of its 17-kD subunit, and a 89-kD cleavage product of poly (ADP-ribose) polymerase (PARP), a known substrate of caspase-3, was also found clearly. The expression of hTERT mRNA as well as activity of telomerase were decreased concurrently in a dose-dependent manner. Moreover, Real-time RT-PCR and Western blot revealed a significant down-regulation of Survivin. We therefore conclude that the induction of apoptosis by Tan-I in monocytic leukemia U937 THP-1 and SHI 1 cells is highly correlated with activation of caspase-3 and decreasing of hTERT mRNA expression and telomerase activity as well as down-regulation of Survivin expression. To our knowledge, this is the first report about the effects of Tan-I on monocytic leukemia cells.

Zoledronic acid (ZOL), a nitrogen‑containing bisphosphonate, is widely used in metastatic bone disease. Previous studies indicate that ZOL has marked anti‑leukemia activity, however, the underlying mechanism of action remains to be elucidated. The present study aimed to explore the mechanism of the anti‑leukemia effect of ZOL in leukemia cells. It was observed that ZOL inhibited the proliferation of HL‑60 and adriamycin‑resistant HL‑60 (HL‑60/A) cells using a WST‑8 assay. An Annexin V‑propidium iodide indicated that ZOL induced apoptosis of the two cell types in a dose‑ and time‑dependent manner. Hoechst 33342 staining was also used to verify the levels of apoptosis. The colony formation assay demonstrated that ZOL significantly inhibited colony formation capacity in acute myeloid leukemia (AML) cells. This was achieved by the induction of S‑phase cell cycle arrest, downregulation of B‑cell lymphoma 2 (Bcl‑2) and upregulation of Bcl‑2 associated X protein and cleaved poly (ADP‑ribose) polymerase. The results indicate that ZOL inhibited cell proliferation by inducing apoptosis via the mitochondrial apoptotic pathway and this anti‑leukemic activity appeared notably enhanced in HL‑60/A cells. As ZOL is already available for clinical use, these results indicate that it may be an effective addition to the chemotherapeutic strategies for AML.

Parkinson's disease (PD) is a common neurodegenerative disorder that is often associated with weak tongue motility. However, the link between the degenerated dopaminergic neurons in the substantia nigra (SN) and lingual dysfunction remains unclear. In the present study, we investigated the localization of dopamine receptor 1 (D1) and dopamine receptor 2 (D2) and alternations in their expression in cholinergic motoneurons of the hypoglossal nucleus (HN) using double-label immunofluorescence, Western blotting and semi-quantitative reverse transcription and polymerase chain reaction (SqRT-PCR) in rats that received microinjections of 6-hydroxydopamine bilaterally into the SN (6-OHDA rats). The results revealed that a large population of choline acetyltransferase immunoreactive (ChAT-IR) neurons was distributed throughout HN and that almost all of the ChAT-IR motoneurons were also D1-IR and D2-IR. Several tyrosine hydroxylase (TH)-IR profiles were observed in a nonuniform pattern near the ChAT-IR, D1-IR or D2-IR somas, suggesting potent dopaminergic innervation. In the 6-OHDA rats, TH immunoreactivity in the SN was significantly decreased, but food residue was increased and treadmill occupancy time was shortened. In the HN, protein expression of TH and D2 was increased, whereas that of ChAT and D1 was decreased. A similar pattern was observed in mRNA levels. The present study suggests that dopamine may modulate the activity of cholinergic neurons via binding with D1 and D2 in the HN. Changes in the expression of ChAT, TH, D1 and D2 in the HN of 6-OHDA rats might be associated with the impaired tongue motility in PD. These findings should be further investigated.

Activation of the dopamine (DA) D2 receptor inhibits glucose-stimulated insulin secretion in isolated rodent islets in vitro; however, no information is available regarding the cellular localization of DA receptors (DRs, including D1-D5 receptors) in pancreatic islets in situ. We investigate the protein expression and cellular localization of five types of DRs in pancreatic islets by means of Western blotting and double-labeling immunofluorescence in both normal control and alloxan-induced type 1 diabetes model (T1DM) rats. In control rats, D1 immunoreactivity (-IR) was distributed in the core of the islet and co-localized with insulin-IR, D2-IR was peripherally distributed and found only in somatostatin-immunoreactive cells and D5-IR was co-localized with glucagon-IR and pancreatic polypeptide-IR. No IR for either the D3 or D4 receptor was observed in rat islets. The protein level of the D1 receptor was reduced in T1DM rats (D1/D-glyceraldehyde-3-phosphate dehydrogenase [GAPDH], 0.63 ± 0.05 in control rats compared with 0.16 ± 0.03 in T1DM rats, n = 8, P

Gastrointestinal dysmotility is one of the nonmotor symptoms of Parkinson's disease (PD). Gastroparesis and upregulated β-adrenoceptors (β-ARs) have been reported in rats with bilateral microinjection of 6-hydroxydopamine (6-OHDA) in the substantia nigra, but the underlying mechanism is unclear. The aim of the current study is to investigate the role of β-ARs in gastroparesis in 6-OHDA rats. Gastric motility was studied through strain gauge measurement. Immunofluorescence, real-time reverse transcription-polymerase chain reaction and Western blotting were performed to examine the expression of β-ARs. Norepinephrine (NE) inhibited gastric motility in a dose-dependent fashion in both control and 6-OHDA rats, but much stronger adrenergic reactivity was observed in the 6-OHDA rats. The inhibition of gastric motility by NE in both control and 6-OHDA rats was not affected by tetrodotoxin, a neural sodium channel blocker. Blocking β 1 -AR or β 2 -AR did not affect the inhibition of strip contraction by NE in control rats, but β 1 -AR blockage obviously enhanced the half maximal inhibitory concentration value of NE in 6-OHDA rats. Selective inhibition of β 3 -AR blocked the effect of NE significantly in both control and 6-OHDA rats. The protein expression of β 1 -AR, but not β 2 -AR and β 3 -AR in gastric muscularis externa was increased significantly in 6-OHDA rats. In conclusion, β 3 -AR involves the regulation of gastric motility in control rats, whereas the upregulation of β 1 -AR is responsible for enhanced NE reactivity in 6-OHDA rats and therefore is involved in the formation of gastroparesis. The effect of both β 1 -AR and β 3 -AR on gastric motility is independent of the enteric nervous system.

Vesicular monoamine transporter 2 (VMAT2) has been exploited as a biomarker of β-cell mass in human islets. However, a current report suggested no immunoreactivity of VMAT2 in the β cells of rat islets. To investigate the cellular localization of VMAT2 in islets further, the pancreatic tissues from monkeys and humans were compared with those of rats and mice. The study was performed using among-species comparisons and a type 1 diabetes model (T1DM) for rats by Western blotting, double-label immunofluorescence, and confocal laser scanning microscopy. We found that VMAT2-immunoreactivity (IR) was distributed peripherally in the islets of rodents, but was widely scattered throughout the islets of primates. Consistent with rodent islets, VMAT2-IR did not exist in insulin (INS)-IR cells but was abundantly present in glucagon (GLU)-IR and pancreatic polypeptide (PP)-IR cells in monkey and human islets. VMAT2-IR had no colocalization with INS-IR in any part of the rat pancreas (head, body, and tail). INS-IR cells were reduced dramatically in T1DM rat islets, but no significant alteration in the proportion of VMAT2-IR cells and GLU-IR cells was observed. Furthermore, a strong colocalization of VMAT2-IR with GLU-IR was distributed in the peripheral regions of diabetic islets. For the first time, the current study demonstrates the presence of VMAT2 in α cells and PP cells but not in β cells in the islets of monkeys and humans. This study provides convinced morphologic evidence that VMAT2 is not present in β cells. There needs to be studies for new markers for β cell mass.

The dorsal motor nucleus of vagus (DMV) plays an important role in the regulation of gastrointestinal function. Dopamine (DA) exerts potent neuromodulatory effects on the motoneurons in the DMV via dopamine receptors (DRs). However, the distribution of DRs and their neurochemical phenotypes in the DMV are unclear. In the present study, the distribution of DRs D1- and D2-immunoreactive (IR) neurons and their neurochemical phenotypes in the DMV of rats were investigated using a double-labeling immunofluorescence technique combined with confocal microscopy. The results indicated that a considerable quantity of D1 and D2 was expressed throughout the DMV. A large amount of choline acetyltransferase (ChAT)-IR and a few tyrosine hydroxylase (TH)-IR neurons were observed in the DMV. Nearly all of the neurons were also D1-IR and D2-IR. In conclusion, the present study demonstrates the wide distribution of D1 and D2 in the cholinergic and catecholaminergic neurons in the DMV of rats. The DRs might play an important role in the regulation of DA on the activity of cholinergic and catecholaminergic neurons in the DMV.

The motility of distal colon could be inhibited by dopamine (DA), yet, the involved receptor is controversial according to the published reports. The goal of present study was to investigate DA receptor(s) mediated inhibition of DA on the colonic motility in rat. The contraction of isolated colon strips was assessed through isometric force transducer. The expressions of DA receptors in distal colon were detected through immunofluorescence and Western blot. DA concentration in colonic smooth muscle was measured by liquid chromatography/mass spectrometry. The results showed that DA inhibited the spontaneous motility of distal colon in a dose-dependent manner with EC50 8.3 μM. Tetrodotoxin increased colonic contractive frequency, but failed to affect the inhibition of DA on the colonic motility. Pretreatment with SCH-23390, an antagonist of dopaminergic receptor D1, shifted the dose-response curve to the right with EC50 of DA 37 μM. However, blocking dopaminergic receptor D2 with sulpiride, had no effect. The immunoreactivity of D1 and D2 were detected in the distal colon including myenteric plexus and smooth muscle. Acute cold-restraint stress (CRS) enhanced spontaneous contraction of rat distal colon, which was more sensitive to DA compared with control. Moreover, DA content and D1 expression in smooth muscle layer were increased under CRS condition. In conclusion, D1 in the smooth muscle is mediated DA inhibition on the spontaneous contraction of rat distal colon. The increased DA content and D1 receptor expression in the smooth muscle layer could be a compensatory effect under CRS condition to balance the enhanced colonic motility.

This study was aimed to investigate the effects of methylation of runx3 gene promoter on pathogenesis of acute leukemia (AL) and its clinical significance. The methylation of runx3 gene promoter in cells of bone marrow or peripheral blood from 40 cases of AL and 10 healthy persons as well as in CHRF, U937 and K562 cell lines were assuaged by methylation specific polymerase chain reaction (MS-PCR), the expression of runx3 gene were detected with reverse transcription polymerase chain reaction (RT-PCR). The results indicated that no methylation was detected in all of cell lines and healthy persons while expression of runx3 gene could be deteted, methylation of runx3 gene promoter was found in 35% (14/40) AL patients and its percentage was significant higher than that healthy persons (0%), the difference in methylation for runx3 between two kinds of samples was statistically significant (p0.05), while methylation rate in AML was 30.43% (7/23), ALL was 41.18% (7/17), there was no significant difference between them (p0.25). All of methylated samples had no expression of runx3 gene. Patients without methylation of runx3 gene had a lower percentage of blasts in bone marrow and a higher complete remission rate of first chemotherapy than those with methylation of runx3 gene. It is concluded that methylation of runx3 gene promoter probably plays a role in the pathogenesis of AL and may have clinical significance in predicting prognosis of AL.

AIM: To assess the patency of pancreaticoenterostomy and pancreatic exocrine function after three surgical methods. METHODS: A pig model of pancreatic ductal dilation was made by ligating the main pancreatic duct. After 4 wk ligation, a total of 36 piglets were divided randomly into four groups. The piglets in the control group underwent laparotomy only; the others were treated by three anastomoses: (1) end-to-end pancreaticojejunostomy invagination (EEPJ); (2) end-to-side duct-to-mucosa sutured anastomosis (ESPJ); or (3) binding pancreaticojejunostomy (BPJ). Anastomotic patency was assessed after 8 wk by body weight gain, intrapancreatic ductal pressure, pancreatic exocrine function secretin test, pancreatography, and macroscopic and histologic features of the anastomotic site. RESULTS: The EEPJ group had significantly slower weight gain than the ESPJ and BPJ groups on postoperative weeks 6 and 8 (P < 0.05). The animals in both the ESPJ and BPJ groups had a similar body weight gain. Intrapancreatic ductal pressure was similar in ESPJ and BPJ. However, pressure in EEPJ was significantly higher than that in ESPJ and BPJ (P < 0.05). All three functional parameters, the secretory volume, the flow rate of pancreatic juice, and bicarbonate concentration, were significantly higher in ESPJ and BPJ as compared to EEPJ (P < 0.05). However, the three parameters were similar in ESPJ and BPJ. Pancreatography performed after EEPJ revealed dilation and meandering of the main pancreatic duct, and the anastomotic site exhibited a variable degree of occlusion, and even blockage. Pancreatography of ESPJ and BPJ, however, showed normal ductal patency. Histopathology showed that the intestinal mucosa had fused with that of the pancreatic duct, with a gradual and continuous change from one to the other. For EEPJ, the portion of the pancreatic stump protruding into the jejunal lumen was largely replaced by cicatricial fibrous tissue. CONCLUSION: A mucosa-to-mucosa pancreaticojejunostomy is the best choice for anastomotic patency when compared with EEPJ. BPJ can effectively maintain anastomotic patency and preserve pancreatic exocrine function as well as ESPJ.

To evaluate the feasibility and efficacy of radiofrequency ablation (RFA) therapy combined with suture and ligation surgery for patients with giant hepatic cavernous hemangioma (HCH).Between June 2004 and June 2005, a total of 30 patients were treated by RFA therapy after suture and ligation surgery (SL group, n = 15, with 18 liver lesions) or RFA therapy without suture and ligation surgery (non-SL group, n = 15, with 17 liver lesions) under general anesthesia. All patients had obvious symptoms such as abdominal discomfort, pain and swelling. Preoperative diagnosis of HCH was established by means of ultrasonography, helical computed tomography (CT) scans, and magnetic resonance imaging (MRI). The mean maximum diameter of the lesions was 8.8 cm +/- 1.4 cm. All of the 35 lesions were located on the liver surface, in the caudate lobe of the liver, or adjacent to the gallbladder. Seven patients had chronic calculous cholecystitis, 6 common duct stones, 5 thrombocytopenias, and one posthepatitic cirrhosis. Thirteen of the 30 patients had previous laparotomy. Therapeutic efficacy and clinical data of RFA therapy were compared between these two groups.RFA therapy under ultrasound guidance was performed successfully in all the patients. Cholecystectomy was performed simultaneously for gallstones in 7 patients and for abutting of gallbladder from hemangioma in 2 patients. Choledochotomy with T-tube drainage was performed in 6 patients. The mean blood loss, the mean RFA time per lesion and reduction rate of maximum diameter of the lesions 6 months after RFA in the SL group and non-SL group were 88.0 ml +/- 22.4 ml vs. 255.0 ml +/- 71.7 ml (P0.001), 23.0 min +/- 7.5 min vs. 53.3 min +/- 16.0 min (P0.001), and 61.8% vs. 44.8% (P0.001) respectively. No severe complication related to RFA was observed in all patients. At a median follow-up of 12 months (6 approximately 17 months), a complete lesion necrosis was achieved on the contrast-enhanced helical CT scans in both groups. During the follow-up, all of the 15 patients were free of upper abdominal pain in the SL group, and 12 patients were symptom-free and 3 obtained significant amelioration of symptoms in the non-SL group.RFA therapy combined with suture and ligation surgery is a feasible, safe, and effective treatment modality for patients with giant HCHs. It can reduce blood loss, shorten RFA therapy time, and increase therapeutic efficacy of RFA. Intraoperative ultrasonography is a useful adjunct for increasing the therapeutic efficacy of RFA and reducing the complications related to RFA.

To evaluate the feasibility, safety and efficacy of radiofrequency ablation (RFA) therapy in patients with hepatic cavernous hemangioma (HCH) and investigate its optimal operative approach.Between March 2001 and June 2004, a total of 68 patients, 18 males and 50 females, age 43.1 (30-64), with 104 HCHs 2.5-11 cm in diameter with the mean size of 5.6 cm, were treated by ultrasound-guided RFA, via percutaneous (n = 19), laparoscopic (n = 29), or open surgical (n = 20) approach. In 7 patients with hepatic lesions larger than 7 cm in diameter, Pringle maneuver was used to occlude the hepatic blood flow during the laparoscopic and open RFA therapy. All patients were followed up with helical computed tomographic (CT) scans and ultrasonography for 19 months (6-36 months).Additional intrahepatic lesions not detected preoperatively were found in 2 patients (with 2 new lesions) via laparoscopy and 3 patients (with 4 new lesions) via celiotomy. All patients were treated with RFA successfully. The mean blood loss in the Pringle group (90.0 ml +/- 22.4 ml) was significantly fewer than that in the non-Pringle group (249 ml +/- 56 ml) (P0.01). The mean RFA time per lesion in the Pringle group (29.0 min +/- 7.5 min) was shorter markedly compared to the non-Pringle group (55.4 min +/- 12.4 min) (P0.01). In the laparoscopic RFA group, laparoscopic cholecystectomy was performed simultaneously in 15 patients with chronic calculous cholecystitis and in another 2 patients because of tumors abutting the gallbladders, and laparoscopic fenestration with intraperitoneal drainage was performed in 3 patients with simple hepatic cysts. In the open RFA group, cholecystectomy was performed in 5 patients with gallbladder diseases, partial cystectomy was performed in one patient with a hepatic cyst, and choledochotomy was performed in 3 patients with common bile duct stones. Postoperative fever and abnormal serum transaminase (ALT and AST) levels were observed in 29 patients (42.6%). A transient hematuria occurred in one patient after open RFA. No specific complications developed during or after RFA. The follow-up showed a complete lesion necrosis rate of 99% (103/104). One patient showed an incomplete lesion necrosis in the margin of RFA site 6 months after percutaneous RFA therapy and obtained retreatment with percutaneous RFA.RFA therapy is a safe, feasible and effective treatment options for patients with HCHs. This procedure can be performed via percutaneous, laparoscopic, or open approach. To prevent the RFA-related complications and to increase the therapeutic efficacy of RFA, the choice of optimal operative approach should be based on the lesion size, number, and location and on the patient's clinical status. Hepatic inflow occlusion by Pringle maneuver during laparoscopic or open RFA therapy can reduce the blood loss and increase the therapeutic efficacy significantly.

To study the inhibitory effect of retroviral packaging cells injected intrasplenically encoding mouse interleukin-12 (mIL-12) and human interleukin-2 (hIL-2) fusion gene on the growth of hepatocellular carcinoma.The retroviral vectors encoding mIL-12 gene, hIL-2 gene, and mIL-12 and hIL-2 genes, GCIL12EXPN, GCXEIL2PN, and GCIL12EIL2PN were constructed and then transfected into the retroviral packaging cells PA317 to construct cells PA317-GCIL12EXPN, PA317-GCXEIL2PN, and PA317-GCIL12EIL2PN. Rat hepatocellular carcinoma cells CBRH3 were implanted into the livers of Wistar rats to establish hepatoma animal model. Then the rats were divided into 5 groups to be injected intrasplenically with normal saline one day after the implantation (0.8 ml/rat, group I, n = 10), blank vector PA317-GCXEXPN one day after the implantation (10(7) cells/rat, group II, n = 10), PA317-GCIL12EXPN containing IL-2 gene (1 x 10(7) cells/rat 1, 3, 5, or 7 days after the implantation, group III, n = 40), PA317-GCXEIL2PN containing mIL-12 gene (1 x 10(7) cells/rat 1, 3, 5, or 7 days after the implantation, group IV, n = 40), and PA317-GCIL12EIL2PN containing IL-12-IL-2 fusion gene (1 x 10(7) cells/rat 1, 3, 5, or 7 days after the implantation, group V, n = 40) respectively. The rats surviving longer than 2 months were re-injected with hepatocellular carcinoma cells. The therapeutic effect, immune function and toxic effect were evaluated. CT was conducted on the liver before and after the experiment. Laparotomy was performed 3 and 7 days after treatment to resect some of the carcinoma tissues to undergo pathological examination and OX8 immunohistostaining. Serum mIL-12 and hIL-2 were detected one day before and 3, 7, 30, and 60 days after treatment.The average survival times of the rats treated with IL-12-IL-2 fusion gene at the first, third, fifth and seventh day after tumor implantation were 53.3 +/- 3.7 days, 49.3 +/- 4.2 days, 31.0 +/- 2.1 days, and 24.3 +/- 1.4 days respectively, longer than those treated with IL-2 gene (25.0 +/- 2.5 days, 23.5 +/- 2.0 days, 18.3 +/- 2.4 days, and 12.0 +/- 1.8 days respectively, P0.001), and those treated with IL-12 gene (39.0 +/- 4.8 days, 32.0 +/- 3.9 days, 23.0 +/- 2.5 days, and 19.4 +/- 2.1 days respectively, P0.001). Long survival (or= 60 days) rate in the rats treated with IL-12-IL-2 fusion gene on the first and third day was 30%. The serum mIL-12 and hIL-2 levels in these rats remained high on the 60th day after treatment. The pathological study showed that the number of infiltrating lymphocytes in liver tumor tissues was increased in the IL-12-IL-2 fusion gene treatment group.The retroviral packaging cell line injected intrasplenically encoding mIL-12 and hIL-2 fusion gene inhibits the growth of hepatocellular carcinoma significantly in rats. The therapeutical efficacy of early administration is superior to that of late one.

Abstract 4418 IL-27 acts as a critical regulator in many pathological processes. Although IL-27 has been studied in many solid tumors, its effect on leukemia remains elusive. Here, we investigated expression of IL-27 receptor (WSX-1 and gp130) on bone marrow mononuclear cells (BM-MNCs) of 31 patients with acute myeloid leukemia (AML) and 8 controls (including patients with IDA, ITP and non-hematological diseases) by RT-PCR, exploring IL-27's property in malignant hematological diseases. In addition, we detected the secretion of antitumor cytokines, cell proliferation and apoptosis in human monocytic leukemic cellline (U397 cells) treated with IL-27. We found that expression rate of IL-27 receptor subunits in AML patients was significantly higher than those with benign hematological diseases, especially on the acute monocytic leukemia subtype (AML-M5 subtype). ELISA assay revealed that antitumor cytokines, including TNF-α and IL-12, were produced by IL-27 stimulated-U937 cells, with an optimal concentration of IL-27 at 60ng/mL and an optimal time at 24h. U937 cells' growth inhibition and apparent apoptosis was induced by IL-27 in a dose dependent manner. U937 cells displayed apparent apoptosis when treated by IL-27 over 40ng/ml. The apoptosis rate of U937 tumor cells reached peak level while the stimulation concentration was 100ng/ml. Hoechst 33258 staining also presented typical apoptotic bodies in U937 cells treated with IL-27. Based on the high expression of IL-27 receptor in BM-MNCs of AML patients, our data provided the first evidence that IL-27 functioned as an anti-leukemia cytokine, suppressing cell growth and inducing apoptosis, offering a new anticancer agent in leukemia therapy. Disclosures: No relevant conflicts of interest to declare.

AIM: To test the efficacy of gene therapy in rat liver tumor. METHODS: A retroviral vector GCIL12EIL2PN encoding human IL-2 (hIL-2) and mouse IL-12 (mIL-12) fused gene and its packaging cell were constructed. The packaging cell lines contained of IL-2 and/or IL-12 genes were injected intrasplenically to transfect splenocyte at different time. The therapeutic effect, immune function and toxic effect were evaluated. RESULTS: The average survival times of the 4 groups using IL genes at days 1, 3, 5 and 7 after tumor implantation were 53.3±3.7, 49.3±4.2, 31.0±2.1 and 24.3±1.4 d respectively in IL-2/IL-12 fused gene group, 25.0±2.5, 23.5±2.0, 18.3±2.4 and 12.0±1.8 d respectively in IL-2 gene treatment group, and 39.0±4.8, 32.0±3.9, 23.0±2.5 and 19.4±2.1 d respectively in IL-12 gene treatment group (P