P-21-activated kinase 1 contributes to tumor angiogenesis upon photodynamic therapy via the HIF-1α/VEGF pathway.

Photodynamic therapy (PDT) is an effective oncotherapy and has been approved for clinical application. Unfortunately, its therapeutic efficacy is usually overshadowed by tumor angiogenesis. Thus, a detailed understanding of the tumor angiogenesis upon PDT is imperative. This study aimed to investigate the potential contribution and mechanism of P-21-activated kinase 1 (PAK1) in PDT-induced tumor angiogenesis. Firstly, we found that PAK1 was upregulated upon PDT and associated with tumor angiogenesis. Then, we elucidated the underlying molecular mechanism. Activation of PAK1 prevents hypoxia-inducible factor 1 alpha (HIF-1α) protein from ubiquitin-mediated degradation. Thereafter, HIF-1α accumulation results in the upregulation of vascular endothelial growth factor (VEGF), thus promoting tumor angiogenesis. More importantly, we determined that PAK1 knockdown effectually repressed tumor angiogenesis, which contributes to enhance the therapeutic effect of PDT. Together, PAK1 is a potential novel pharmaceutical target for inhibiting PDT-induced tumor angiogenesis, and PAK1 suppression in combination with PDT may be a potentially effective strategy for anti-tumor therapy.

Effect of Huaier granule on recurrence after curative resection of HCC: a multicentre, randomised clinical trial.

OBJECTIVE: There is little evidence that adjuvant therapy after radical surgical resection of hepatocellular carcinoma (HCC) improves recurrence-free survival (RFS) or overall survival (OS). We conducted a multicentre, randomised, controlled, phase IV trial evaluating the benefit of an aqueous extract of Trametes robinophila Murr (Huaier granule) to address this unmet need. DESIGN AND RESULTS: A total of 1044 patients were randomised in 2:1 ratio to receive either Huaier or no further treatment (controls) for a maximum of 96 weeks. The primary endpoint was RFS. Secondary endpoints included OS and tumour extrahepatic recurrence rate (ERR). The Huaier (n=686) and control groups (n=316) had a mean RFS of 75.5 weeks and 68.5 weeks, respectively (HR 0.67; 95% CI 0.55 to 0.81). The difference in the RFS rate between Huaier and control groups was 62.39% and 49.05% (95% CI 6.74 to 19.94; p=0.0001); this led to an OS rate in the Huaier and control groups of 95.19% and 91.46%, respectively (95% CI 0.26 to 7.21; p=0.0207). The tumour ERR between Huaier and control groups was 8.60% and 13.61% (95% CI -12.59 to -2.50; p=0.0018), respectively. CONCLUSIONS: This is the first nationwide multicentre study, involving 39 centres and 1044 patients, to prove the effectiveness of Huaier granule as adjuvant therapy for HCC after curative liver resection. It demonstrated a significant prolongation of RFS and reduced extrahepatic recurrence in Huaier group. TRIAL REGISTRATION: NCT01770431; Post-results.

Nodulisporipyrones A-D, new bioactive α-pyrone derivatives from Nodulisporium sp.

Four new α-pyrone derivatives, nodulisporipyrones A-D (1-4), were isolated from the extract of an endolichenic fungal strain Nodulisporium sp. (65-12-7-1) that was fermented with rice. The structures of 1-4 were elucidated by extensive spectroscopic analysis, and the absolute configurations were determined by modified Mosher's method and electronic circular dichroism experiments. Their antimicrobial activities against Staphylococcus aureus 209P, Escherichia coli ATCC0111, Aspergillus niger R330, and Candida albicans FIM709 were evaluated using a paper disk diffusion method. Nodulisporipyrones A-D (1-4) are the first α-pyrone derivatives from Nodulisporium fungi.

A new alkaloid from Nodulisporium sp.

The genus Nodulisporium, is known to produce secondary metabolites with structural diversity. A new alkaloid, 2-hy- droxy-1,1-dimethyl-1,2,3,9-tetrahydro-4H-carbazol-4-one(1), was isolated from the extract of a fungal strain Nodulisporium sp. fermented with rice, together with three known phenols, tyrosol(2), hydroxytyrosol(3), and hydroxytyrosol acetate(4). Their structures were identified by detailed spectroscopic analyses.

Antiviral therapy after curative treatment of hepatitis B/C virus-related hepatocellular carcinoma: A systematic review of randomized trials.

AIM: Available published work on the benefit of adjuvant antiviral therapy after curative treatment of hepatocellular carcinoma (HCC) reports controversial results. The objective of this systematic review was to evaluate the effect of adjuvant antiviral therapy on recurrence and survival after curative treatment of HCC. METHODS: We conducted an extensive search strategy. All randomized controlled trials comparing adjuvant antiviral therapy versus placebo or no treatment were considered for this review. Results were expressed as hazard ratio for time-to-event outcomes with 95% confidence intervals using RevMan 5. RESULTS: We included nine trials (three of low risk of bias and six of unclear risk of bias) with 954 patients. All the included studies used conventional interferon (IFN) as adjuvant antiviral therapy; none of them used pegylated IFN or nucleoside analogs. There were significant improvements for recurrence-free survival and overall survival in the adjuvant IFN group compared with the control group. Subgroup analysis also showed a significant difference favoring IFN therapy in hepatitis C virus (HCV)-related HCC patients, but for hepatitis B virus (HBV)-related patients, the difference failed to reach statistical significance. A dose reduction was needed in 28.3% of patients and discontinuation of IFN therapy happened in 8.2% of patients due to moderate to severe side-effects. CONCLUSION: Our study suggested potential benefits of adjuvant IFN therapy following curative treatment of HCC, especially for HCV-related HCC. Further high-quality randomized controlled trials of more effective adjuvant antiviral regimens, either used alone or in combination, for virus-related HCC, especially HBV-related HCC, are needed.

Mouse A6-positive hepatic oval cells derived from embryonic stem cells.

Oval cells have a potential to differentiate into a variety of cell lineages including hepatocytes and biliary epithelia. Several models have been established to activate the oval cells by incorporating a variety of toxins and carcinogens, alone or combined with surgical treatment. Those models are obviously not suitable for the study on human hepatic oval cells. It is necessary to establish a new and efficient model to study the human hepatic oval cells. In this study, the hepatocyte growth factor (HGF) and epidermal growth factor (EGF) were used to induce differentiation of mouse embryonic stem (ES) cells into hepatic oval cells. We first confirmed that hepatic oval cells derived from ES cells, which are bipotential, do exist during the course of mouse ES cells' differentiation into hepatic parenchymal cells. RT-PCR and transmission electron microscopy were applied in this study. The ratio of Sca-1+/CD34+ cells sorted by FACS in the induction group was increased from day 4 and reached the maximum on the day 8, whereas that in the control group remained at a low level. The differentiation ratio of Sca-1+/CD34+ cells in the induction group was significantly higher than that in the control group. About 92.48% of the sorted Sca-1+/CD34+ cells on the day 8 were A6 positive. Highly purified A6+/Sca-1+/CD34+ hepatic oval cells derived from ES cells could be obtained by FACS. The differentiation ratio of hepatic oval cells in the induction group (up to 4.46%) was significantly higher than that in the control group. The number of hepatic oval cells could be increased significantly by HGF and EGF. The study also examined the ultrastructures of ES-derived hepatic oval cells' membrane surface by atomic force microscopy. The ES-derived hepatic oval cells cultured and sorted by our protocols may be available for the future clinical application.

Corrigendum: Dual-Targeting Nanoparticle-Mediated Gene Therapy Strategy for Hepatocellular Carcinoma by Delivering Small Interfering RNA.

[This corrects the article DOI: 10.3389/fbioe.2020.00512.].

Dual-Targeting Nanoparticle-Mediated Gene Therapy Strategy for Hepatocellular Carcinoma by Delivering Small Interfering RNA.

As a gene therapy strategy, RNA interference (RNAi) offers tremendous tumor therapy potential. However, its therapeutic efficacy is restricted by its inferior ability for targeted delivery and cellular uptake of small interfering RNA (siRNA). This study sought to develop a dual-ligand nanoparticle (NP) system loaded with siRNA to promote targeted delivery and therapeutic efficacy. We synthesized a dual receptor-targeted chitosan nanosystem (GCGA), whose target function was controlled by the ligands of galactose of lactobionic acid (LA) and glycyrrhetinic acid (GA). By loading siPAK1, an siRNA targeting P21-activated kinase 1 (PAK1), a molecular-targeted therapeutic dual-ligand NP (GCGA-siPAK1) was established. We investigated the synergistic effect of these two targeting units in hepatocellular carcinoma (HCC). In particular, GCGA-siPAK1 enhanced the NP targeting ability and promoted siPAK1 cell uptake. Subsequently, dramatic decreases in cell proliferation, invasion, and migration, with an apparent increase in cell apoptosis, were observed in treated cells. Furthermore, this dual-ligand NP gene delivery system demonstrated significant anti-tumor effects in tumor-bearing mice. Finally, we illuminated the molecular mechanism, whereby GCGA-siPAK1 promotes endogenous cell apoptosis through the PAK1/MEK/ERK pathway. Thus, the dual-target property effectively promotes the HCC therapeutic effect and provides a promising gene therapy strategy for clinical applications.

Biodistribution and biocompatibility of glycyrrhetinic acid and galactose-modified chitosan nanoparticles as a novel targeting vehicle for hepatocellular carcinoma.

Aim: The dual-ligand glycyrrhetinic acid and galactose-modified chitosan nanoparticles were designed to further improve the targeting capability to hepatocellular carcinoma (HCC). Materials & methods: The dual-ligand glycyrrhetinic acid and galactose-modified chitosan nanoparticles were fabricated by using ionic gelation method and their characteristics have been measured. Furthermore, the biodistribution and biocompatibility of this targeting vehicle were investigated in vitro and in vivo, respectively. Results: The targeting vehicle was specifically internalized into hepatoma cells in vitro and accumulated into tumor tissue in vivo with high efficacy. Moreover, the vehicle did not induce inflammation reaction and affect morphologies and organ functions. Conclusion: The targeting accumulation in HCC tissue and great biocompatibility of the dual-ligand modified chitosan nanoparticles highlight the potential of delivering anticancer agents into HCC cells.

Higd1a Protects Cells from Lipotoxicity under High-Fat Exposure.

Hypoxia-inducible gene domain family member 1A (Higd1a) has recently been reported to protect cells from hypoxia by helping to maintain normal mitochondrial function. The potential induction of Higd1a under high-fat exposure and whether it could protect cells from oxidative stress attracted our attention. Initially, 0.4 mM oleic acid and 0.2 mM palmitate were added to the growth media of HepG2 and LO2 cells for 72 hours. We discovered increased Higd1a expression, and knocking down Higd1a impaired mitochondrial transmembrane potential and induced cell apoptosis. We then identified that elevated reactive oxygen species (ROS) is responsible for increased Higd1a expression. Furthermore, we found that ROS promoted Higd1a expression by upregulating HIF-1a and PGC-1a expressions, and these two proteins could exert synergistic effects in inducing Higd1a expression. Taken together, these data suggest that Higd1a plays positive roles in protecting cells from oxidative stress, and ROS could induce Higd1a expression by upregulating PGC-1a and HIF-1a expressions.

Theranostic Nanodots with Aggregation-Induced Emission Characteristic for Targeted and Image-Guided Photodynamic Therapy of Hepatocellular Carcinoma.

Photosensitizer (PS) serves as the central element of photodynamic therapy (PDT). The use of common nanoparticles (NPs) for PDT has typically been rendered less effective by the undesirable aggregation-caused quenching (ACQ) effect, resulting in quenched fluorescence and reduced reactive oxygen species (ROS) generation that diminish the imaging quality and PDT efficacy. To overcome the ACQ effect and to enhance the overall efficacy of PDT, herein, integrin ανß3-targeted organic nanodots for image-guided PDT were designed and synthesized based on a red emissive aggregation-induced emission (AIE) PS. Methods: The TPETS nanodots were prepared by nano-precipitation method and further conjugated with thiolated cRGD (cRGD-SH) through a click reaction to yield the targeted TPETS nanodots (T-TPETS nanodots). Nanodots were characterized for encapsulation efficiency, conjugation rate, particle size, absorption and emission spectra and ROS production. The targeted fluorescence imaging and antitumor efficacy of T-TPETS nanodot were evaluated both in vitro and in vivo. The mechanism of cell apoptosis induced by T-TPETS nanodot mediated-PDT was explored. The biocompatibility and toxicity of the nanodots was examined using cytotoxicity test, hemolysis assay, blood biochemistry test and histological staining. Results: The obtained nanodots show bright red fluorescence and highly effective 1O2 generation in aggregate state. Both in vitro and in vivo experiments demonstrate that the nanodots exhibit excellent tumor-targeted imaging performance, which facilitates image-guided PDT for tumor ablation in a hepatocellular carcinoma model. Detailed analysis reveals that the nanodot-mediated PDT is able to induce time- and concentration-dependent cell death. The use of PDT at a high PDT intensity leads to direct cell necrosis, while cell apoptosis via the mitochondria-mediated pathway is achieved under low PDT intensity. Conclusion: Our results suggest that well-designed AIE nanodots are promising for image-guided PDT applications.

Endogenous danger signals trigger hepatic ischemia/reperfusion injury through toll-like receptor 4/nuclear factor-kappa B pathway.

BACKGROUND: Restoration of blood flow to the ischemic liver lobes may paradoxically exacerbate tissue injury, which is called hepatic ischemia/reperfusion injury (IRI). Toll-like receptor 4 (TLR4), expressed on several liver cell types, and the nuclear factor-kappa B (NF-kappaB) signaling pathway are crucial to mediating hepatic inflammatory response. Because IRI is essentially a kind of profound acute inflammatory reaction evoked by many kinds of danger signals, we investigated TLR4/NF-kappaB signaling pathway activation in a murine model of partial hepatic IRI. METHODS: Wild-type mice (WT, C3H/HeN) or TLR4 mutant mice (C3H/HeJ) were subjected to 45 minutes of partial hepatic ischemia followed by 1 hour, 3 hours of reperfusion. Sham group accepted the same procedure without the obstruction of blood supply. At the end of reperfusion, the compromise of liver function and the histological change of liver sections were measured as the severity of liver injury. The level of endotoxin in the portal vein was measured by limulus assay. NF-kappaB activation was determined by electrophoretic mobility shift assay (EMSA). The levels of tumor necrosis factor-alpha (TNF-alpha) and interleukin-1beta (IL-1beta) in systemic blood after hepatic IRI were assessed by enzyme-linked immunosorbent assay (ELISA). RESULTS: The compromise of liver function and the morphological injuries in mutant mice were relieved more markedly than those in WT mice after partial hepatic IRI. NF-kappaB activation in WT mice was stronger than that in TLR4 mutant mice, and both were stronger than those in the sham operated mice (P < 0.01). Endotoxin in each group was undetectable. The levels of TNF-alpha and IL-1beta in systemic blood were elevated in both strains, but lower in the sham operated group. These mediators were significantly decreased in TLR4 mutant mice compared with those in WT mice (P < 0.01). CONCLUSIONS: The TLR4/NF-kappaB signaling pathway may mediate hepatic IRI triggered by endogenous danger signals. Inhibition of the TLR4/NF-kappaB pathway may be a potential therapeutic target for attenuating ischemia/reperfusion-induced tissue damage in some clinical settings.

[Expression and biological activities of arresten in CHO cells].

OBJECTIVE: To explore the eukaryotic expression of arresten in CHO cells and to investigate its basic biological activities. METHODS: CHO cells were divided into three groups: transfected pSecTag-arresten group, transfected pSecTag group and control group without transfection. PSecTag-arresten was transfected into CHO cells by Lipofectamine 2000 method. The arresten mRNA in CHO cells was assayed by RT-PCR. The protein expression of arresten gene was examined by Western-Blot. The cells expressing arresten were screened out by Zeocin. The effect of arresten on huvec cell migration and anchoring to three-dimensional vascular structures was measured. RESULTS: The result of RT-PCR and Western-blot showed that arresten gene has been successfully transfected into CHO cells and expressed in those cells. Arrssten inhibited huvec cell migration and anchoring to three-dimensional vascular structures. CONCLUSION: CHO cells expressing arresten have been obtained successfully. Arresten can inhibit huvec cell migration and anchoring to three-dimensional vascular structures, indicating that it might be one of its anti-angiogenetic approaches.

[Effect of mitofusin-2 gene in apoptosis of human breast carcinoma cell line in vitro].

OBJECTIVE: To investigate the role of mitofusin-2 gene (mfn2) in apoptosis in human breast carcinoma cell line MCF-7 cells after in vitro transfection. METHODS: pEGFP mfn2 was transfected by sofast in vitro. Expression of GFP was observed by Western blot, and the MCF-7 cell proliferation was measured by MTT and cell counting. Apoptosis in MCF-7 cells was observed in annexin-V/PI and chondrosome transmembrane potential of MCF-7 marked in JC-1 by FCM. The Ultrastructure of cells was observed by transmission electron microscopy. RESULTS: The stable expression of GFP in MCF-7 cells was confirmed by Western blot. Mfn2 significantly inhibited cell proliferation, revealed by MTT, and decrease chondrosome transmembrane potential. Exogenous mfn2 gene significantly induced apoptosis. The apoptotic rate was increased from 3.6% to 16.0% (P < 0.05). Mfn2 gene induced break down and loss of mitochondrial cristae, and rarefaction of mitochondrial ground substance. Swollen mitochondria intensely aggregated around the cell nuclei. CONCLUSION: Mfn2 can strongly induce apoptosis in MCF-7 cells, which may be associated with decrease of mitochondrial transmembrane potential.

Clinical application of carbon nanoparticles in curative resection for colorectal carcinoma.

PURPOSE: To explore the potential of carbon nanoparticles (CNs) for the intraoperative detection of positive and negative lymph nodes in the treatment of colorectal cancer. PATIENTS AND METHODS: The clinical data of 470 patients undergoing surgical procedures for colorectal cancer from June 2010 to February 2013 were analyzed retrospectively. The patients were divided into the CN group (183 males and 161 females; mean age, 58.6±12.4 years), who were given a CN suspension, and the control group (78 males and 48 females; mean age, 59.1±12.2 years), who were not given a CN suspension. The operative time, blood loss, number of lymph nodes detected/positive lymph nodes, and prevalence of postoperative complications were compared between the two groups. Three years after surgery, 444 cases (327 cases in the CN group and 117 cases in the control group) were interviewed, with the remaining 26 cases lost to follow-up. With regard to tumor, node, metastasis staging, the survival and prevalence of recurrence in each group at 3 years were analyzed. RESULTS: The number of positive lymph nodes was higher and the prevalence of blood loss was lower in the CN group than in the control group (p<0.05). There were no significant differences in the operative time, number of lymph nodes detected, or the prevalence of postoperative complications, survival, metastasis, or recurrence between the two groups at 3 years (p>0.05). CONCLUSION: The application of CNs is convenient for the detection of lymph nodes to reduce blood loss and increase the probability of detecting positive lymph nodes accurately and rapidly.

One-Step Formulation of Targeted Aggregation-Induced Emission Dots for Image-Guided Photodynamic Therapy of Cholangiocarcinoma.

Photodynamic therapy (PDT) is a palliative technique that can improve median survival with minimal invasion for cholangiocarcinoma (CC) patients. An ideal photosensitizer (PS) is critical to guarantee the efficacy of PDT. However, conventional PSs have some obvious drawbacks, such as lack of specificity and easy aggregation in aqueous media that limit their further application in the clinic. We herein fully take advantage of a red emissive aggregation-induced emission (AIE) PS to fabricate integrin ανß3 targeted organic AIE dots for image-guided PDT via a simple and straightforward one-step strategy. The obtained AIE dots exhibit high specificity to CC as well as excellent antitumor effect both in vitro and in vivo. Different from conventional PSs and previously reported PS-loaded nanostructures, the AIE dots do not suffer from aggregation-caused fluorescence quenching and reduction in reactive oxygen species production when the AIE PS molecules are in an aggregated state. The significant antitumor effect, as well as good biocompatibility and negligible toxicity, makes the AIE dots promising for future translational research in CC diagnosis and therapy.

Mechanism of dynamic near-infrared fluorescence cholangiography of extrahepatic bile ducts and applications in detecting bile duct injuries using indocyanine green in animal models.

Fluorescence intraoperative cholangiography (IOC) is a potential alternative for identifying anatomical variation and preventing iatrogenic bile duct injuries by using the near-infrared probe indocyanine green (ICG). However, the dynamic process and mechanism of fluorescence IOC have not been elucidated in previous publications. Herein, the optical properties of the complex of ICG and bile, dynamic fluorescence cholangiography and iatrogenic bile duct injuries were investigated. The emission spectrum of ICG in bile peaked at 844 nm and ICG had higher tissue penetration. Extrahepatic bile ducts could fluoresce 2 min after intravenous injection, and the fluorescence intensity reached a peak at 8 min. In addition, biliary dynamics were observed owing to ICG excretion from the bile ducts into the duodenum. Quantitative analysis indicated that ICG-guided fluorescence IOC possessed a high signal to noise ratio compared to the surrounding peripheral tissue and the portal vein. Fluorescence IOC was based on rapid uptake of circulating ICG in plasma by hepatic cells, excretion of ICG into the bile and then its interaction with protein molecules in the bile. Moreover, fluorescence IOC was sensitive to detect bile duct ligation and acute bile duct perforation using ICG in rat models. All of the results indicated that fluorescence IOC using ICG is a valid alternative for the cholangiography of extrahepatic bile ducts and has potential for measurement of biliary dynamics.

[Curative effects of transplantation of hepatocytes differentiated from embryonic stem cells on treatment of fulminant hepatic failure: experiment with mice].

OBJECTIVE: To investigate the curative effects of transplantation of hepatocytes differentiated from embryonic stem cells (ESCs) on treatment of fulminant hepatic failure (FHF). METHODS: Mouse ESCs transfected with green fluorescent protein were cultured. RT-PCR was used to detect the mRNA expression of alpha-fetoprotein (AFP), transthyretin (TTR), hepatic nuclear factor (HNF), albumin (ALB), glucose-6-phosphate (G6P), and tyrosine aminotransferase (TAT) at different time points. Immunocytochemistry (ICC) was used to detect the expression of AFP, ALB, cytokeratin 8 (CK8), and CK11 in the cells. The morphology and distribution of the cells were observed with microscope. Forty mice were randomly divided into 2 equal groups: ESC transplantation group, undergoing transplantation of ICC positive ESCs (hepatocytes) into the hepatic capsule, and control group, undergoing injection of normal saline in the hepatic capsule. Twenty-four hours later carbon tetrachloride was injected intra-peritoneally to induce FHF. The survival status of the mice was observed. Twenty-four hours later venous blood samples were collected to examine the levels of total bilirubin (TB), alanine aminotransferase (ALT), ALB, blood sugar (BS), and prothrombin time (PT). After the death of the mice, their livers were taken out to undergo microscopy and immunohistochemistry to observe the structure of liver tissue, growth of tumor, and expression of ALB. RESULTS: RT-PCR showed that the mRNA expression of AFP, TTR, HNF, ALB, G6P, and TAT emerged since days 3, 3, 5, 9, and 11 respectively and then gradually increased till day 19. ICC showed that the EB cells began to express AFP since day 7, to express CK8 and CK11 since day 9, and to express ALB since day 11. The ICC-positive cells were consistent with the mouse hepatocytes morphologically and were distributed only in the central and marginal areas of the EB cell community. Injected with carbon tetrachloride, the mice showed manifestations of FHF. However, the symptoms of central nervous system emerged later in the mice implanted with the hepatocytes in comparison with the control mice; 8 of the 20 mice in the transplantation group showed ascites in comparison with 14 in the control group. The mean survival time of the transplantation group was 62 hours, significantly longer than that of the control group (23 hours, P < 0.05). Compare with the normal mice, the FHF mice in both groups showed higher ALT and TB and lower ALB and BS (all P < 0.01), however, the levels of ALT and TB were lower, the level of BS was higher, and PT was shorter significantly in the transplantation group than in the control group (all P < 0.05). Pathology showed that no tumor formation was found in both groups and that the transplanted hepatocytes were incorporated well into the liver parenchymal structure and expressed ALB. CONCLUSION: Hepatocytes originating from ESCs exercise the functions of normal hepatocytes. Transplantation of hepatocytes differentiated from ESCs is able to improve the life quality and lengthen the survival time of FHF mice.

[Inhibition of rat RAW264.7 macrophage inflammatory cytokines release by small hairpin RNAi targeting Toll-like receptor].

OBJECTIVE: To construct a eukaryotic expression vector carrying the small hairpin RNA (shRNA) for Toll-like receptor 4 (TLR4) mRNA and a reporter gene of enhanced green fluorescence protein (EGFP) and study the inhibition of cytokine release by rat RAW264.7 macrophages induced by lipopolysaccharide (LPS) stimulation through transfection and expression of shRNA targeting TLR4 gene via the RNAi mechanism. METHODS: The H1 promotor and double BbsIrestrict endoenzyme site from the plasmid psiRNA-hH1neo were cloned into the reporter gene plasmid pEGFP-C1 at the MluIrestrict endoenzymic site, thus forming the plasmid pEGFP-H1/siRNA containing Bbs site and reporter EGFP gene. Then an oligo nuclear hairpin sequence targeting TLR4 gene was designed by the internet tool siRNA Wizard and then inserted into the plasmid pEGFP-H1/siRNA so as to form the plasmid pEGFP-H1/TLR4-siRNA. Rat macrophages of the line RAW264.7 were cultured and transfected with pEGFP-H1/TLR4-siRNA mediated by lipofectamine 2000. Another RAW264.7 cells were transfected with pEGFP-H1/control sequence-siRNA or blank plasmid. Lipopolysaccharide was added into the 3 kinds of culture fluid for 2 and 68 hours respectively. ELSA was used to detect the levels of tumor necrosis factor-alpha (TNF-alpha) in the supernatants. RESULTS: Restriction endonuclease analysis showed that the construction pEGFP-H1/TLR4-siRNA carrying hairpin RNA for TLR4 gene and reporter EGFP gene was successful. The expression of EGFP gene was 50% +/- 8%. The TNF-alpha level of the TLR4-siRNA transfection group 2 hours and after transfection was 825 pg/ml +/- 136 pg/ml, significantly lower than those of the pEGFP-H1/control sequence-siRNA and blank plasmid groups (2190 pg/ml +/- 359 pg/ml and 1265 pg/ml +/- 283 pg/ml respectively, both P < 0.01). The TNF-alpha level of the TLR4-siRNA transfection group 8 hours and after transfection was 1179 pg/ml +/- 240 pg/ml, significantly lower than those of the pEGFP-H1/control sequence-siRNA and blank plasmid groups (4720 pg/ml +/- 227 pg/ml and 4689 pg/ml +/- 310 pg/ml respectively, both P < 0.01). CONCLUSION: shRNA targeting TLR4 gene can inhibit the TNF-alpha release by RAW264.7 cells evoked by LPS.

[Clinical analysis for iatrogenic injuries in the distal part of common bile duct].

OBJECTIVE: To investigate the early diagnosis on iatrogenic injuries in distal part of common bile duct and the prevention of severe retroperitoneal infection. METHODS: From 1990 to 2004, 17 patients with bile duct injures in the distal part of common biliary tract were admitted. And the clinical data of the 17 cases were retrospectively analyzed. RESULTS: Of the 17 cases, the injuries of 15 cases were caused by the operation, and the injuries of the other 2 cases were caused in the process of removing the stone by endoscopic retrograde cholangiopancreatography (ERCP). The injuries of 14 cases were found during the operation, but the other one was not found in time. Before the operation, 16 cases were examined by B-type ultrasonography, 2 by MRCP and 6 by intraoperative choledocho-endoscope after the biliary tract exploration. Ten cases underwent perforating suture repair and T-tube drainage; 2 with Odd's sphincter incision and shaping; 2 with choledochojejunostomy; 1 with duodenum wall and bile duct repair and drainage. When the bile duct injured, the major findings during operation were bile duct explorer located out of the duodenum wall and bile duct, two or more than cleft in the distal part of common biliary tract found by choledocho-endoscopic examination, retroperitoneal edema and liquid accumulation found by irrigating water through T-tube, and/or retroperitoneal tissues stained blue by irrigating methylthioninium chloride through T-tube. The clinical manifestations after injuries were abdominal distention, abdominal pain, pain in the waist and back, fever and shock, et al. Thirteen cases were cured. And the syndromes included 1 case with intestinal fistula, 1 with incisional infection, 4 dead (3 died from infectious shock; 1 from bleeding in gastrectomy). CONCLUSIONS: The postoperative clinical manifestations for iatrogenic injuries in the distal part of common biliary tract lack specificity, CT examinations are necessary to doubtful patients. Early diagnosis and timely management can obtain better results, and can effectively lower severe retroperitoneal infection. The perfect preoperative imaging examinations and intraoperative choledocho-endoscopic examinations before the biliary tract exploration maybe reduce iatrogenic injuries in the distal part of common biliary tract.