CXCR4 promotes gefitinib resistance of Huh7 cells by activating the c-Met signaling pathway.

C-X-C chemokine receptor type 4 (CXCR4) expression is associated with poor prognosis of hepatocellular carcinoma (HCC). The aim of this study was to explore the biological role of CXCR4 in gefitinib resistance of HCC. Compared with a normal, non-gefitinib-resistant, human HCC cell line (Huh7), CXCR4 mRNA and protein were highly expressed in gefitinib-resistant Huh7 cells (Huh7-R). Cell proliferation was decreased, and apoptosis was enhanced in Huh7 cells in the presence of gefitinib. These influences conferred by gefitinib treatment on proliferation and apoptosis of Huh7 cells were abolished by CXCR4 overexpression. CXCR4 knockdown reduced the proliferation ability of HuH-7R cells after gefitinib treatment. Importantly, CXCR4 overexpression had no influence on caveolin 1 (Cav-1) expression; similarly, Cav-1 silencing did not cause a substantive change in CXCR4 expression. However, CXCR4 activated Cav-1, c-Met, and Raf-1 in Huh7 cells, whereas Cav-1 silencing repressed the expression of Raf-1 and phosphorylated c-Met in Huh7 cells. CXCR4 overexpression promoted proliferation and repressed apoptosis in gefitinib-treated Huh7 cells, which was partly rescued by PHA-665752 (a c-Met inhibitor) treatment or c-Met deficiency. Finally, we constructed a tumor xenograft model to determine the influence of CXCR4 overexpression on tumor growth of HCC. CXCR4 overexpression accelerated tumor growth of HCC, which was abrogated by c-Met deficiency. These findings demonstrate that CXCR4 overexpression activates c-Met via the Cav-1 signaling pathway, thereby promoting gefitinib resistance of Huh7 cells. Thus, this study highlights novel insights into the mechanism of gefitinib resistance of HCC and CXCR4 may become a potential target for HCC treatment.

Study on the Mechanism of Targeted Poly(lactic-coglycolic acid) Nano-Delivery Carriers in the Treatment of Hemangiomas.

Hemangiomas, also called infantile hemangiomas (IH), are the most common congenital benign vascular tumors in infants and young children. At present, there are many treatment methods for proliferative hemangiomas, which have different effects and lack predictability. Propranolol has gradually replaced glucocorticoids as the first-line treatment for infants and young children with hemangiomas. However, premature discontinuation is prone to relapse, and the efficacy and safety of medication need to be further studied and determined. The exact pathogenesis of hemangiomas is still unclear. Therefore, in this study, poly(lactic-co-glycolic acid) (PLGA) nanoparticles were used as drug delivery carriers, propranolol was encapsulated, and PLGA-propranolol (PLGA-PP) nanodelivery preparations were prepared and targeted. Anisotropy and pharmacokinetics were preliminary studied. At the same time, after the treatment of HemECs cells with PLGA-PP in gradient concentration in vitro, CCK-8 method was used to detect the cell proliferation, and Anyixin-V/PI double staining method was used to detect the apoptosis rate of cells. The effect of PLGA-PP nano-delivery vector on hemangioma was studied by western blot method to detect the expression level of Id-1 protein in HemECs. The results showed that after PLGA-PP treated HemECs for 24 h, PLGA-PP significantly inhibited HeECs proliferation and promoted their apoptosis, and the intracellular Id-1 protein expression was also reduced. Therefore, this study believes that the mechanism of PLGA-PP nano-targeted delivery preparations in the treatment of hemangiomas is achieved by down-regulating the Id-1 gene, thereby inhibiting the colonization of HemECs and promoting its apoptosis effect.

MiR-155 aggravates impaired autophagy of pancreatic acinar cells through targeting Rictor.

The aim of this study was to investigate the role and mechanism of miR-155 in regulating autophagy in a caerulein-induced acute pancreatitis (AP) cellular model. GFP-LC3 immunofluorescence assay was performed to detect autophagy vesicle formation in pancreatic acinar cell line AR42J. AR42J cells were transfected with miR-155 mimic, inhibitor, and corresponding controls to explore the effect of miR-155 on autophagy. The protein levels of LC3-I, LC3-II, Beclin-1, and p62 were analyzed by western blot analysis. Dual-luciferase reporter assay was performed to verify the interaction between miR-155 and Rictor (RPTOR independent companion of MTOR complex 2). The results showed that caerulein treatment induced impaired autophagy as evidenced by an increase in the accumulation of p62 together with LC3-II in AR42J cells, accompanied by miR-155 upregulation. Furthermore, miR-155 overexpression aggravated, whereas miR-155 silencing reduced the caerulein-induced impairment of autophagy. Mechanistically, Rictor was confirmed to be a direct target of miR-155, which could rescue the miR-155 overexpression-mediated aggravation of impaired autophagy. Collectively, these findings indicate that miR-155 aggravates impaired autophagy in caerulein-treated pancreatic acinar cells by targeting Rictor.

miR­92a­3p regulates trypsinogen activation via Egr1 in AR42J cells.

Acute pancreatitis (AP) exhibits high morbidity and mortality rates. The onset of AP is characterized by early trypsinogen activation.The present study aimed to investigate the expression of microRNA (miR)­92a­3p and early growth response protein 1 (Egr1), and the effect of miR­92a­3p on trypsinogen activation in the pancreatic exocrine cell line AR42J. mRNA and miRNA microarrays were used to identify differentially expressed mRNAs and miRNAs in AR42J cells. A miRNA­mRNA network was constructed using bioinformatics software, and Egr1 and its regulated miRNA subnetworks were identified by reviewing previous literature. The results suggested that miR­92a­3p could bind to Egr1 3'untranslated region sequence. Subsequently, miR­92a­3p mimic and inhibitor were used to transfect AR42J cells. Following transfection, reverse transcription­quantitative PCR and western blotting were performed to detect Egr1 expression. Furthermore, AR42J cells were cotransfected with miR­92a­3p inhibitor and small interfering (si)­Egr1. The trypsinogen activation rate of AR42J cells was measured by flow cytometry. Microarrays and bioinformatics results indicated that Egr1 may be a target gene of miR­92a­3p. In addition, the present study suggested that miR­92a­3p downregulated Egr1 in vitro and that miR­92a­3p and Egr1 expression was associated with trypsinogen activation. Furthermore, miR­92a­3p inhibitor reversed the effect of si­Egr1 on trypsinogen activation. In conclusion, miR­92a­3p may negatively regulate the activation of trypsinogen in AR42J cells via Egr1.

The interaction between ANXA2 and lncRNA Fendrr promotes cell apoptosis in caerulein-induced acute pancreatitis.

BACKGROUND: Annexin A2 (ANXA2) plays a crucial role in acute pancreatitis (AP). However, its potential mechanism remains unclear. METHODS: In the present study, we used caerulein-treated AR42J rat pancreatic acinar cells as cell model of AP to investigate the potential functions of ANXA2 and its predicted long noncoding RNA (lncRNA) FOXF1 adjacent noncoding developmental regulatory RNA (lncRNA Fendrr). Cell apoptosis was evaluated by flow cytometry using annexinV-fluorescein isothiocyanate/propidium iodide staining. The expressions of ANAX2 and lncRNA Fendrr were detected by quantitative real-time polymerase chain reaction (qRT-PCR). Furthermore, Western blot analysis was performed to determine the protein levels of ANXA2, Bcl-2, and Bax. The association between lncRNA Fendrr and ANXA2 was disclosed by RNA pull-down, RNA immunoprecipitation, and electrophoretic mobility shift assays. RESULTS: ANXA2 was elevated in caerulein-induced AP model and promoted apoptosis of AR42J cells. LncRNA Fendrr was also upregulated in AP cell model and directly bound ANXA2 protein. Further studies indicated that the interaction between ANXA2 and lncRNA Fendrr contributed to the apoptosis of AR42J cells in AP cell model. CONCLUSION: Our study demonstrated that ANXA2 promoted AP progression via interacting with lncRNA Fendrr in vitro, which will provide a novel insight into the therapeutic target for AP.

Modular bioinformatics analysis demonstrates that a Toll­like receptor signaling pathway is involved in the regulation of macrophage polarization.

In recent years, an increasing number of studies on the roles of macrophages in tumors, immune responses and metabolism have been published, in which macrophage polarization has been an extensively discussed topic. In the present study, differentially expressed genes in various types of macrophages were analyzed using the Gene Expression Omnibus database. Cluster analysis of differentially expressed genes was conducted, and a protein­protein interaction (PPI) network was constructed. Finally, modular analysis and functional enrichment analysis revealed that a Toll­like receptor (TLR) signaling pathway is involved in the regulation of macrophage polarization. Furthermore, the high­degree proteins in the PPI network that are involved in the molecular regulation of macrophage polarization are closely associated with proteins of the TLR signaling pathway. These results suggested that the TLR signaling pathways may be a principal direction of future research on the regulation of macrophage polarization.

miR-352 participates in the regulation of trypsinogen activation in pancreatic acinar cells by influencing the function of autophagic lysosomes.

This study was performed to screen miRNAs and mRNAs that are differentially expressed during trypsinogen activation in acute pancreatitis and to verify their role in the process of trypsinogen activation. The function enrichment analysis showed that the functions of miR-352 and its regulatory targets lysosome-associated membrane protein 2 (LAMP2) and cathepsin L1 (CTSL1) were lysosome related. The results of the verification experiment showed that in the TLC-S-treated AR42J (pancreatic cell line) cells, miR-352 expression increased, expression levels of LAMP2 and CTSL1 were significantly reduced, trypsinogen activation was increased, and the autophagy pathway was blocked. In the miR-352 mimic-transfected cells, miR-352 expression increased, expression levels of LAMP2 and CTSL1 were significantly reduced, trypsinogen activation was increased, intracellular lysosomal pH increased, cathepsins L activity decreased and the amount of autophagolysosomes increased. In the miR-352 inhibitor-transfected cells, miR-352 expression was reduced, expression levels of LAMP2 and CTSL1 were significantly increased, trypsinogen activation was decreased, intracellular lysosomal pH decreased, cathepsins L activity increased and the amount of autophagolysosomes decreased. In the process of taurolithocholic acid 3-sulfate (TLC-S) induced trypsinogen activation, overexpression of miR-352 could down-regulate LAMP2 and CTSL1, resulting in the dysfunction of autophagic lysosome. Thus, the autophagy pathway was blocked, and trypsinogen activation was enhanced.

Role of the c-Jun N-terminal kinase signaling pathway in the activation of trypsinogen in rat pancreatic acinar cells.

Bile acid causes trypsinogen activation in pancreatic acinar cells through a complex process. Additional research is required to further elucidate which signaling pathways affect trypsinogen activation when activated. the changes in the whole­genome expression profile of AR42J cells under the effect of taurolithocholic acid 3­sulfate (TLC­S) were investigated. Furthermore, gene groups that may play a regulatory role were analyzed using the modular approach of biological networks. The aim of the present study was to improve our understanding of the changes in TLC­S­stimulated AR42J cells through a genetic functional modular analysis. whole­genome expression profile chip arrays were applied to detect genes that were differentially expressed in pancreatic acinar AR42J cells treated with TLC­S for 20 min. Based on the human protein reference database, a protein­protein interaction network was obtained, which was then processed by CFinder software to derive 14 modules. Among these 14 modules, the gene ontology biological processes enrichment analysis identified two as modules of interest. Kyoto encyclopedia of genes and genomes map analysis revealed that MAP2K4, MAPK8 and FLNA are part of the c-Jun N-terminal kinase (JNK) pathway. The JNK signaling pathway is involved in regulating trypsinogen activation in rat pancreatic AR42J cells. Next, a regulatory network of seven kinase inhibitors was constructed. SP600125 is an ATP­competitive, efficient, selective and reversible inhibitor of JNK. the results were verified by four sets of experiments and demonstrated that trypsinogen activation is mediated by the JNK signaling pathway in the pathogenesis of acute pancreatitis (AP). The present study provided a useful reference for better understanding the pathogenesis of AP and identifying new targets to regulate trypsinogen activation, in addition to providing valuable information for the treatment of AP.

Association between high mobility group box­1 protein expression and cell death in acute pancreatitis.

The present study used caerulein stimulation of AR42J rat pancreatic cells as an in vitro acute pancreatitis (AP) model to investigate proteins differentially expressed in apoptosis and necrosis. AR42J cells were stimulated with 10­8mol/l caerulein and incubated for 24 h. Apoptosis and necrosis were detected using flow cytometry. The sorted Annexin V­positive cells (apoptotic) and the Annexin V/propidium iodide double­positive cells (necrotic) were analysed using proteomics. Results showed that numerous proteins were differentially expressed in these 2 groups. Functional enrichment analysis was performed on the differentially expressed genes using the Database for Annotation, Visualization and Integrated Discovery. High mobility group box­1 protein (HMGB1) was specifically expressed in the necrosis group. Models of varying degrees of AP were established using caerulein at concentrations of 10­9, 10­8, 10­7, 10­6 and 10­5 mol/l. The percentage of apoptotic and necrotic cells in each group was determined using flow cytometry. Protein expression levels of HMGB1 were detected by western blot analysis. The present study showed that as the concentration of caerulein increased, the percentage of necrotic cells and the protein expression levels of HMGB1 increased. HMGB1 is involved in many biological processes, including the chromosomal protein glycyl lysine isopeptide cross­link. HMGB1 may be involved in the early stage of pancreatitis, potentially by inducing the development of cell death by necrosis. These results provide an experimental basis for clinical intervention in AP.

Is preoperative MRCP necessary for patients with gallstones? An analysis of the factors related to missed diagnosis of choledocholithiasis by preoperative ultrasound.

BACKGROUND: The diagnosis of associated choledocholithiasis prior to cholecystectomy for patients with gallstones is important for the surgical decision and treatment efficacy. However, whether ultrasound is sufficient for preoperative diagnosis of choledocholithiasis remains controversial, with different opinions on whether routine magnetic resonance cholangiopancreatography (MRCP) is needed to detect the possible presence of common bile duct (CBD) stones. METHODS: In this study, a total of 413 patients with gallstones who were admitted to the Department of General Surgery of the First Affiliated Hospital of Harbin Medical University in China for a period of 3 years and underwent both ultrasound and MRCP examinations were retrospectively analysed. After reviewing and screening these cases according to the literature, 11 indicators including gender, age, alanine aminotransferase, aspartate aminotransferase, total bilirubin, direct bilirubin, indirect bilirubin, alkaline phosphatase, γ-aminotransferase, CBD diameter, and concurrent acute cholecystitis were selected and comparatively analysed. RESULTS: Among the 413 patients, a total of 109 cases showed concurrent gallstones and choledocholithiasis, accounting for 26.39 % of all cases. Among them, 60 cases of choledocholithiasis were revealed by ultrasound examination, accounting for 55.05 %, while 49 cases of choledocholithiasis were not detected by ultrasound examination but were confirmed by MRCP instead (the missed diagnosis rate of ultrasound was 44.95 %). The results of statistical analysis suggested that alanine aminotransferase, acute cholecystitis, and CBD diameter were the three most relevant factors for missed diagnosis by ultrasound. CONCLUSION: The accuracy of preoperative ultrasonography for the diagnosis of associated CBD stones for patients with gallstones is not high. However, elevated alanine aminotransferase, concurrent acute cholecystitis, and CBD diameter were identified as key factors that may affect the accuracy of the diagnosis. Thus, routine preoperative MRCP examination is suggested for patients with gallstones to rule out possible concomitant CBD stones.

A novel mechanism of abnormal hematological indices in liver cirrhosis: bone marrow endothelial cell dysfunction caused by humoral inhibitor affects the hematopoietic function of bone marrow.

Abnormal hematological indices (HIs), a complication of liver cirrhosis (LC), present difficulties in the treatment of LC and pose a serious threat to the survival of patients. LC is a dynamic wound-healing process that occurs in response to repeated liver injury and is a chronic disorder associated with changes in various organs and tissues. It has been reported that humoral inhibitor in the formation of LC could affect the hematogenic functions of bone marrow (BM) by acting on erythroid differentiation. This indicates that the BM microenvironment is affected by humoral inhibitor in LC. Bone marrow endothelial cells (BMECs) are very important components of the BM microenvironment that function as the cytoskeleton to support the adhesion of hematopoietic stem cells (HSCs). In addition, they can secrete cytokines, which have important functions in regulating positioning, homing, proliferation, differentiation and other functions of HSCs on the BM microenvironment. These functions of BMECs may be affected due to direct contact with blood and long-term exposure to an environment with humoral inhibitor in the presence of LC. Multiple studies have shown that during the formation of LC, hepatic sinusoid endothelial cells were damaged and secreted cytokines and matrix proteins. Moreover, these cytokines and matrix proteins were involved in the formation and development of LC. Similar in function to mature-stage BM, liver at the embryonic stage also functions as a type of hematogenic organ. With similar anatomical position and functions to that of hepatic sinusoid endothelial cells, BMECs may undergo similar changes and impair hematogenic function of BM. More importantly, we found even more convincing evidence in that the humoral inhibitor in LC could lead to the ultrastructural damage of BMECs that were positively related to the degree of severity of LC. Therefore, we hypothesise the existence of a novel mechanism for abnormal HIs in LC: the continuous humoral inhibitor may lead to abnormal cytokine secretion of BMECs and attenuate their supporting functions, and such alterations of BMECs may lead to BM microenvironment disorder and dysfunction of HSCs, finally causing abnormal HIs.

Proteomic analysis of apoptotic and oncotic pancreatic acinar AR42J cells treated with caerulein.

This study aims to determine the differentially expressed proteins in the pancreatic acinar cells undergoing apoptosis and oncosis stimulated with caerulein to explore different cell death process of the acinar cell. AR42J cells were treated with caerulein to induce cell model of acute pancreatitis. Cells that were undergoing apoptosis and oncosis were separated by flow cytometry. Then differentially expressed proteins in the two groups of separated cells were detected by shotgun liquid chromatography-tandem mass spectrometry. The results showed that 11 proteins were detected in both apoptosis group and oncosis group, 17 proteins were detected only in apoptosis group and 29 proteins were detected only in oncosis group. KEGG analysis showed that proteins detected only in apoptosis group were significantly enriched in 10 pathways, including ECM-receptor interaction, cell adhesion molecules, and proteins detected only in oncosis group were significantly enriched in three pathways, including endocytosis, base excision repair, and RNA degradation. These proteins we detected are helpful for us to understand the process of cell death in acute pancreatitis and may be useful for changing the death mode of pancreatic acinar cells, thus attenuating the severity of pancreatitis.

Construction of network for protein kinases that play a role in acute pancreatitis.

OBJECTIVES: This study aimed to search for protein kinases that play a role in acute pancreatitis and analyze their potential connection with each other. METHODS: Information of human protein kinases were collected in protein kinase database, and then a systematic search was performed using PubMed for studies addressing the association between these kinases and acute pancreatitis. Gene Ontology Annotations were used to build interactions network for acute pancreatitis-associated protein kinases. RESULTS: A total of 570 human protein kinases were found, in which 28 kinases play a role in acute pancreatitis. Among the 28 kinases, RIPK1, JAK2, SRC, EGFR, FYN, MET, JAK1, TYK2, and MTOR were annotated in Gene Ontology database. A gene ontology interactions network was built to visualize the common biological process these kinases participated in. CONCLUSIONS: This study provides observations that protein kinases participate in all the sequential events in the exocrine pancreas in acute pancreatitis and that protein kinases are potential therapeutical target for acute pancreatitis.

Early proteome analysis of rat pancreatic acinar AR42J cells treated with taurolithocholic acid 3-sulfate.

BACKGROUND: Bile acids are the initiating factors of biliary acute pancreatitis. Bile acids can induce the activation of intracellular zymogen, thus leading injury in pancreatic acinar cells. Pathological zymogen activation in pancreatic acinar cells is a common feature of all types of acute pancreatitis. The proteins expressed in pancreatic acinar cells during the activation of zymogen may determine the severity of acute pancreatitis. The present study aims to determine the differentially expressed proteins in taurolithocholic acid 3-sulfate-stimulated pancreatic acinar cells as an in vitro model for acute pancreatitis. METHODS: Rat pancreatic acinar AR42J cells were treated with taurolithocholic acid 3-sulfate for 20 min. Laser confocal scanning microscopy and flow cytometry were used to detect activated trypsinogen in pancreatic acinar AR42J cells. After the determination of trypsinogen activation, proteome analysis was performed to identify the proteins differentially expressed in taurolithocholic acid 3-sulfate-treated cells and non-treated cells. RESULTS: After treatment with taurolithocholic acid 3-sulfate for 20 min, the activation of trypsinogen in AR42J cells was concurrent with changes in the protein expression profile. Thirty-nine differentially expressed proteins were detected; among these, 23 proteins were up-regulated and 16 proteins were down-regulated. KEGG analysis indicated that these proteins are involved in cellular metabolic pathways, cellular defensive mechanisms, intracellular calcium regulation and cytoskeletal changes. CONCLUSION: The expression of proteins in the pancreatic acinar cell changes at the early stage of biliary acute pancreatitis. These differentially expressed proteins will provide valuable information to understand the pathophysiologic mechanism biliary acute pancreatitis and may be useful for prognostic indices of acute pancreatitis.

Metabolomic study of serum from rabbits with acute acalculous cholecystitis.

OBJECTIVES: (1)H-NMR is a powerful approach of metabolomics. This study aimed to apply it to detect the serum metabolites in rabbits with acute acalculous cholecystitis (AAC), and to analyze their potential roles in AAC. METHODS: Fourteen rabbits were randomly divided into two groups, the AAC group and the CON group. In the AAC rabbit model, Escherichia coli solution was injected into the gallbladder, while same volume of saline, instead of E. coli solution, was injected into the gallbladder of the CON rabbit. General morphological, light microscopic and transmission electron microscopic observations were used to evaluate the model. Metabolic profiles of serum from rabbits with AAC were investigated through (1)H-NMR spectroscopy coupled with multivariate statistical analysis, such as principal components analysis and orthogonal partial least-squares discriminant analysis. RESULTS: The pathohistology of gallbladders showed a significant difference between the two groups, proving the successful induction of inflammation in the gallbladders of the AAC group. The serum concentration of lipids (LDL and VLDL) increased during AAC, while the concentrations of phospholipids, lactic acid, 3-hydroxybutyric acid, lysine, citric acid, asparagine, histidine, glucose and some other small molecular metabolites decreased. CONCLUSION: The profiling of serum metabolites in rabbits with acute acalculous cholecystitis changed significantly. These changes referred to the metabolic disturbance of carbohydrate, amino acids and lipids, inhibition of immunological functions and inflammation reaction.

The distribution and expression of claudin-5 and occludin at the rat blood-optic nerve barrier after borneol treatment.

The purpose of this study is to investigate the distribution and expression of the tight junction membrane proteins, claudin-5 and occludin, in rat blood-optic nerve barrier after borneol treatment. Seventy-two female Wistar rats were randomly divided into the borneol gastric lavage group and the equal volume solvent gastric lavage control group. The bilateral optic nerve from the retrobulbar region to the optic chiasma was collected from the rats in the two groups before gastric lavage and at 30 min, 1, 2, 4, and 8 h after gastric lavage. The distribution and expression of claudin-5 and occludin were detected using immunofluorescence staining, Western blotting, and reverse transcription polymerase chain reaction (RT-PCR). Results showed that claudin-5 translocated from the cell membrane to the cytoplasm at 30 min following initiation of borneol treatment, and this translocation peaked at 1 h. During this period of time, a small amount of occludin also translocated from the cell membrane to the cytoplasm. Four hours after initiation of treatment, claudin-5 and occludin levels in the cytoplasm began to decrease and were restored to their normal pattern 8 h after initiation of treatment. There were no significant differences in the levels of claudin-5 or occludin before or after treatment in either group. It was concluded that claudin-5 and occludin translocate within cells of the rat blood-optic nerve barrier after borneol treatment, and this translocation was reversible. Claudin-5 may play a potential role in permeability of the blood-optic nerve barrier following borneol treatment.