[Application of precision-cut lung slice technology to study the role of DDR2 in pulmonary fibrosis].

Pulmonary fibrosis is a severe lung interstitial disease characterized by the destruction of lung tissue structure, excessive activation and proliferation of fibroblasts, secretion and accumulation of a large amount of extracellular matrix (ECM), and impaired lung function. Due to the complexity of the disease, a suitable animal model to mimic human pulmonary fibrosis has not yet been established. Precision-cut lung slice (PCLS) has been a widely used in vitro method to study lung physiology and pathogenesis in recent years. This method is an in vitro culture technology at the level between organs and cells, because it can preserve the lung tissue structure and various types of airway cells in the lung tissue, simulate the in vivo lung environment, and conduct the observation of various interactions between cells and ECM. Therefore, PCLS can compensate for the limitations of other models such as cell culture. In order to explore the role of discoidin domain receptor 2 (DDR2) in pulmonary fibrosis, Ddr2flox/flox mice were successfully constructed. The Cre-LoxP system and PCLS technology were used to verify the deletion or knockdown of DDR2 in mouse PCLS. Transforming growth factor ß1 (TGF-ß1) can induce fibrosis of mouse PCLS in vitro, which can simulate the in vivo environment of pulmonary fibrosis. In the DDR2 knock down-PCLS in vitro model, the expression of various fibrosis-related factors induced by TGF-ß1 was significantly reduced, suggesting that knocking down DDR2 can inhibit the formation of pulmonary fibrosis. The results provide a new perspective for the clinical study of DDR2 as a therapeutic target in pulmonary fibrosis.

Neurokinin-1 receptor drives PKCɑ-AURKA/N-Myc signaling to facilitate the neuroendocrine progression of prostate cancer.

The widespread application of antiandrogen therapies has aroused a significant increase in the incidence of NEPC, a lethal form of the disease lacking efficient clinical treatments. Here we identified a cell surface receptor neurokinin-1 (NK1R) as a clinically relevant driver of treatment-related NEPC (tNEPC). NK1R expression increased in prostate cancer patients, particularly higher in metastatic prostate cancer and treatment-related NEPC, implying a relation with the progression from primary luminal adenocarcinoma toward NEPC. High NK1R level was clinically correlated with accelerated tumor recurrence and poor survival. Mechanical studies identified a regulatory element in the NK1R gene transcription ending region that was recognized by AR. AR inhibition enhanced the expression of NK1R, which mediated the PKCα-AURKA/N-Myc pathway in prostate cancer cells. Functional assays demonstrated that activation of NK1R promoted the NE transdifferentiation, cell proliferation, invasion, and enzalutamide resistance in prostate cancer cells. Targeting NK1R abrogated the NE transdifferentiation process and tumorigenicity in vitro and in vivo. These findings collectively characterized the role of NK1R in tNEPC progression and suggested NK1R as a potential therapeutic target.

NaClO-Mediated Cross Installation of Indoles and Azoles Benefits Anticancer Hit Discovery.

Innovations in synthetic chemistry have a profound impact on the drug discovery process, and will always be a necessary driver of drug development. As a result, it is of significance to develop novel simple and effective synthetic installation of medicinal modules to promote drug discovery. Herein, we have developed a NaClO-mediated cross installation of indoles and azoles, both of which are frequently encountered in drugs and natural products. This effective toolbox provides a convenient synthetic route to access a library of N-linked 2-(azol-1-yl) indole derivatives, and can be used for late-stage modification of drugs, natural products and peptides. Moreover, biological screening of the library has revealed that several adducts showed promising anticancer activities against A549 and NCI-H1975 cells, which give us a hit for anticancer drug discovery.

Neurokinin-1 receptor promotes non-small cell lung cancer progression through transactivation of EGFR.

Despite the great advances in target therapy, lung cancer remains the top cause of cancer-related death worldwide. G protein-coupled receptor neurokinin-1 (NK1R) is shown to play multiple roles in various cancers; however, the pathological roles and clinical implication in lung cancer are unclarified. Here we identified NK1R as a significantly upregulated GPCR in the transcriptome and tissue array of human lung cancer samples, associated with advanced clinical stages and poor prognosis. Notably, NK1R is co-expressed with epidermal growth factor receptor (EGFR) in NSCLC patients' tissues and co-localized in the tumor cells. NK1R can crosstalk with EGFR by interacting with EGFR, transactivating EGFR phosphorylation and regulating the intracellular signaling of ERK1/2 and Akt. Activation of NK1R promotes the proliferation, colony formation, EMT, MMP2/14 expression, and migration of lung cancer cells. The inhibition of NK1R by selective antagonist aprepitant repressed cell proliferation and migration in vitro. Knockdown of NK1R significantly slowed down the tumor growth in nude mice. The sensitivity of lung cancer cells to gefitinib/osimertinib is highly increased in the presence of the selective NK1R antagonist aprepitant. Our data suggest that NK1R plays an important role in lung cancer development through EGFR signaling and the crosstalk between NK1R and EGFR may provide a potential therapeutic target for lung cancer treatment.

Interrelation Between Fibroblasts and T Cells in Fibrosing Interstitial Lung Diseases.

Interstitial lung diseases (ILDs) are a heterogeneous group of diseases characterized by varying degrees of inflammation and fibrosis of the pulmonary interstitium. The interrelations between multiple immune cells and stromal cells participate in the pathogenesis of ILDs. While fibroblasts contribute to the development of ILDs through secreting extracellular matrix and proinflammatory cytokines upon activation, T cells are major mediators of adaptive immunity, as well as inflammation and autoimmune tissue destruction in the lung of ILDs patients. Fibroblasts play important roles in modulating T cell recruitment, differentiation and function and conversely, T cells can balance fibrotic sequelae with protective immunity in the lung. A more precise understanding of the interrelation between fibroblasts and T cells will enable a better future therapeutic design by targeting this interrelationship. Here we highlight recent work on the interactions between fibroblasts and T cells in ILDs, and consider the implications of these interactions in the future development of therapies for ILDs.

Inhibition of autophagy by YC-1 promotes gefitinib induced apoptosis by targeting FOXO1 in gefitinib-resistant NSCLC cells.

Non-small cell lung cancer (NSCLC) is the most common cancer in the world. Gefitinib, an inhibitor of EGFR tyrosine kinase, is highly effective in treating NSCLC patients with activating EGFR mutations (L858R or Ex19del). However, despite excellent disease control with gefitinib therapy, innate resistance and inevitable acquired resistance represent immense challenges in NSCLC therapy. Gefitinib potently induces cytoprotective autophagy, which has been implied to contribute to both innate and acquired resistance to gefitinib in NSCLC cells. Currently, abrogation of autophagy is considered a promising strategy for NSCLC therapy. In the present study, YC-1, an inhibitor of HIF-1α, was first found to significantly inhibit the autophagy induced by gefitinib by disrupting the fusion of autophagosomes and lysosomes and thereby enhancing the proapoptotic effect of gefitinib in gefitinib-resistant NSCLC cells. Furthermore, the combinational anti-autophagic and pro-apoptotic effect of gefitinib and YC-1 was demonstrated to be associated with an enhanced of forkhead box protein O1 (FOXO1) transcriptional activity which resulted from an increase in the p-FOXO1 protein level in gefitinib-resistant NSCLC cells. Our data suggest that inhibition of autophagy by targeting FOXO1 may be a feasible therapeutic strategy to overcome both innate and acquired resistance to EGFR-TKIs.

YC-1 potentiates the antitumor activity of gefitinib by inhibiting HIF-1α and promoting the endocytic trafficking and degradation of EGFR in gefitinib-resistant non-small-cell lung cancer cells.

The tyrosine kinase inhibitor (TKI) gefitinib exerts good therapeutic effect on NSCLC patients with sensitive EGFR-activating mutations. However, most patients ultimately relapse due to the development of drug resistance after 6-12 months of treatment. Here, we showed that a HIF-1α inhibitor, YC-1, potentiated the antitumor efficacy of gefitinib by promoting EGFR degradation in a panel of human NSCLC cells with wild-type or mutant EGFRs. YC-1 alone had little effect on NSCLC cell survival but significantly enhanced the antigrowth and proapoptotic effects of gefitinib. In insensitive NSCLC cell lines, gefitinib efficiently inhibited the phosphorylation of EGFR but not the downstream signaling of ERK, AKT and STAT3; however, when combined with YC-1 treatment, these signaling pathways were strongly impaired. Gefitinib treatment induced EGFR arrest in the early endosome, and YC-1 treatment promoted delayed EGFR transport into the late endosome as well as receptor degradation. Moreover, the YC-1-induced reduction of HIF-1α protein was associated with the enhancement of EGFR degradation. HIF-1α knockdown promoted EGFR degradation, showing synergistic antigrowth and proapoptotic effects similar to those of the gefitinib and YC-1 combination treatment in NSCLC cells. Our findings provide a novel combination treatment strategy with gefitinib and YC-1 to extend the usage of gefitinib and overcome gefitinib resistance in NSCLC patients.

ß-Arrestin 1 has an essential role in neurokinin-1 receptor-mediated glioblastoma cell proliferation and G2/M phase transition.

Glioblastoma is the most common malignant brain tumor and has a poor prognosis. Tachykinin receptor neurokinin-1 (NK1R) is a promising target in glioblastoma therapy because of its overexpression in human glioblastoma. NK1R agonists promote glioblastoma cell growth, whereas NK1R antagonists efficiently inhibit cell growth both in vitro and in vivo However, the molecular mechanisms involved in these effects are incompletely understood. ß-Arrestins (ARRBs) serve as scaffold proteins and adapters to mediate intracellular signal transduction. Here we show that the ARRB1-mediated signaling pathway is essential for NK1-mediated glioblastoma cell proliferation. ARRB1 knockdown significantly inhibited NK1-mediated glioblastoma cell proliferation and induced G2/M phase cell cycle arrest. ARRB1 knockdown cells showed remarkable down-regulation of CDC25C/CDK1/cyclin B1 activity. We also demonstrated that ARRB1 mediated prolonged phosphorylation of ERK1/2 and Akt in glioblastoma cells induced by NK1R activation. ERK1/2 and Akt phosphorylation are involved in regulating CDC25C/CDK1/cyclin B1 activity. The lack of long-term ERK1/2 and Akt activation in ARRB1 knockdown cells was at least partly responsible for the delayed cell cycle progression and proliferation. Moreover, we found that ARRB1-mediated ERK1/2 and Akt phosphorylation regulated the transcriptional activity of both NF-κB and AP-1, which were involved in cyclin B1 expression. ARRB1 deficiency increased the sensitivity of glioblastoma cells to the treatment of NK1R antagonists. Taken together, our results suggest that ARRB1 plays an essential role in NK1R-mediated cell proliferation and G2/M transition in glioblastoma cells. Interference with ARRB1-mediated signaling via NK1R may have potential significance for therapeutic strategies targeting glioblastoma.

[Anesthesia experiences on laparoscopic nephrectomy with da Vinci S robotics].

OBJECTIVE: To summarize the clinical anesthesia experiences in 20 patients who underwent laparoscopic nephrectomy with da Vinci S robotics.
 METHODS: Anesthesia data of 20 patients from Sichuan Provincial People's Hospital, who underwent laparoscopic nephrectomy with da Vinci S robotics from August 2014 to November 2014, were analyzed and summarized. The anesthesia time, operation time, CO(2) pneumoperitoneum time, PaCO(2) and PETCO(2) were recorded.
 RESULTS: All patients were anesthetized and underwent surgery with da Vinci S robotics. The anesthesia time was (220±14) min, the operation time was (187±11) min, and the CO(2) pneumoperitoneum time was (180±13) min. The PaCO(2) and PETCO(2) were significantly elevated at 1.5 h after operation compared with those at the baseline (before pneumoperitoneum) (P<0.05). The pH value was significantly decreased at 2.5 h after operation compared to that at the baseline (P<0.05). The peak airway pressure of inspiration was significantly elevated at 0.5 h after the beginning of pneumoperitoneum compared to that at the baseline (P<0.05).
 CONCLUSION: The hemodynamics is stable during the laparoscopic nephrectomy with da vinci S robotics. However, the duration of CO(2) pneumoperitoneum is significantly increased compared to that of other surgical procedures, resulting in high airway resistance and acid-base disturbance.

[Clinical research on the application of trans-esophageal echocardiography in monitoring Nuss surgery].

OBJECTIVE: To investigate the validity and safety of trans-esophageal echocardiography (TEE) in monitoring of Nuss surgery. METHODS: A total of 140 patients with pectus excavatum from Sichuan Provincial People's Hospital underwent Nuss surgery from August, 2011 to Aµgust, 2013. Among them, 72 patients received TEE monitoring while 68 patients didn't. The injury of heart and large vessels by the introducer and Nuss steel bar was observed by intraoperative TEE monitoring under middle-esophageal four chamber view and middle-esophageal aortic short axis view. RESULTS: The operation in all patients had been performed successfully without any severe complications. Satisfactory TEE images were obtained in all patients. The procedure of inserting the inducer and Nuss steel bar behind sternum and steel bar overturn could be seen clearly. No injury in heart and large vessels was detected. Local streak-like hemorrhage in 3 patients was observed under intra-operative TEE screen, but no further new bleeding was found in postoperative TEE examination. The blood was absorbed and couldn't see under trans-thoracic echocardiography in 1 month after the operation. CONCLUSION: The TEE is a non-invasive monitoring method. It is sensitive to detect the status of the heart and large vessels and can prevent the severe complications due to Nuss surgery.

Iron-induced oxidative rat liver injury after non-heart-beating warm ischemia is mediated by tumor necrosis factor α and prevented by deferoxamine.

This study investigated iron-induced injury after warm ischemia in a non-heart-beating (NHB) rat liver model and the effects of deferoxamine (DFO). Livers from heart-beating (HB) rats or rats that were NHB for 60 minutes were stored in University of Wisconsin solution for 5 hours at 4°C [cold storage (CS)] and then were subjected to 2 hours of machine reperfusion (MRP) at 37°C. Three NHB groups were compared: (1) no DFO, (2) DFO 30 minutes before cardiac arrest and during CS and MRP, and (3) DFO during CS and MRP. Aspartate aminotransferase (AST) and lactate dehydrogenase (LDH) levels in the NHB perfusate were significantly elevated (P < 0.01) in comparison with levels in HB controls after CS and MRP. After CS, the levels of iron and tumor necrosis factor α (TNF-α) were 0.077 ± 0.007 µmol/g and 151 ± 26 pg/g, respectively, in the NHB group and 0.022 ± 0.004 µmol/g and 17 ± 7 pg/g, respectively, in the HB group (P < 0.01). After MRP, LDH significantly correlated with iron (R(2) = 0.81, P < 0.01). The DFO pretreatment of NHB donors decreased AST (7.3 ± 0.8 versus 4.0 ± 0.5 U/g of liver, P < 0.05) and LDH (42.5 ± 4.1 versus 20.4 ± 2.5 U/g of liver, P < 0.05) with 2 hours of MRP and increased bile flow during MRP (142 ± 34 versus 240 ± 18 µL/g, P < 0.05). It also reduced the levels of iron (0.077 ± 0.007 versus 0.050 ± 0.008 µmol/g, P < 0.05) and TNF-α (151 ± 26 versus 51 ± 13 pg/g, P < 0.05) after CS and the levels of lipid peroxidation products F2-isoprostane (149 ± 11 versus 99 ± 10 ng/g, P < 0.05) and malondialdehyde (1.58 ± 0.1 versus 1.14 ± 0.08 µmol/g, P < 0.05) after MRP. In conclusion, iron-initiated oxidative stress is likely involved in NHB donor liver injury, and importantly, DFO pretreatment reduces liver damage.

Expression of the HPV18E2 gene in cervical cancer and premalignant lesions and its clinical significance.

This study aims to observe the expression of the HPV18E2 gene in cervical cancer and premalignant lesions and to investigate its clinical significance. The expression of the HPV18E2 gene in the cervical tissues obtained from 38 women with cervical lesions was detected using the RT-PCR method. The pathological changes were graded based on cervical intraepithelial neoplasia (CIN) criteria. The HPV18E2 gene was expressed mainly in cervical premalignant lesions, 60 % in Grade I CIN, 33.3 % in Grade II CIN, and 28.6 % in Grade III CIN. No expression was detected in cervical cancer and chronic cervical inflammation. This study suggests that peptides vaccine targeting the HPV18E2 protein may disrupt and prohibit the progress of diseases induced by HPV 18 infection (i.e., CIN and cervical cancer).

The analysis of transesophageal oxygen saturation photoplethysmography from different signal sources.

The photoplethysmography (PPG) signals detected by transesophageal oximetry sensor toward aorta arch (AA), descending aorta (DA), and left ventricle (LV) under the guidance of transesophageal echocardiography (TEE) were investigated, and the effects of filter application on PPG signals were evaluated. Eleven cardiac surgical patients were involved. After anesthesia was induced, the TEE probe with a modified pulse oximetry sensor was inserted. Under the guidance of TEE, the AA PPG, DA PPG and LV PPG were detected respectively when ventilator was on and off. The mean alternating current (AC) amplitudes and direct current (DC) values of original and filtered PPG signals were measured. The ratio of AC and DC value (AC/DC) and ventilation-induced AC variations were calculated. Satisfactory PPG waveforms were obtained in all patients under the guidance of TEE. The AC amplitude in LV PPG was significant larger than in AA and DA PPG, and both AC/DC and ventilation-induced AC variation in LV PPG were significantly higher than in AA PPG or DA PPG either. There were no significant differences of AC amplitude between filtered and ventilation off PPG signals. The AC amplitudes and AC/DC toward LV are significantly higher than transesophageal oximeter toward AA or DA, and the effect of mechanical ventilation on transesophageal PPG can be obviously reduced by filtering techniques.

Neurotrophic artemin promotes motility and invasiveness of MIA PaCa-2 pancreatic cancer cells.

OBJECTIVE: To analyze the capacity of neurotrophic artemin to promote the motility and invasiveness of MIA PaCa-2 pancreatic cancer cells. METHODS: MIA PaCa-2 was cultured in vitro and studied using transwell chambers for motility and invasiveness on treatment with different concentrations of aArtemin or its receptor GFRα3 were also determined. Expression of matrix metalloproteinase-2 (MMP-2) and epithelial cadherin (E-cadherin) was quantified using RT-PCR and Western blotting. RESULTS: MIA PaCa-2 pancreatic cancer cell motility and invasiveness was significantly increased with artemin and its receptor GFRα3 with dose dependence (P<0.01). MMP-2 production was also significantly increased (t=6.35, t=7.32), while E-cadherin was significantly lowered (t=4.27, t=5.61) (P<0.01). CONCLUSION: Artemin and its receptor GFRα3 can promote pancreatic cancer cell motility and invasiveness and contribute to aggressive behavior. The mechanism may be related to increased expression of MMP-2 molecule and down-regulation of E-cadherin expression.

Enhancing DNA vaccine potency against hantavirus by co-administration of interleukin-12 expression vector as a genetic adjuvant.

BACKGROUND: The heavy incidence and mortality of hemorrhagic fever with renal syndrome, as well as no specific drugs in curing the disease, clearly indicate the need for development of the more effective hantavirus vaccine. Refining the DNA vaccination strategy to elicit more clinically efficacious immune responses is now under intensive investigation. In the present study, we examined the effects of using an interleukin-12 expression plasmid as a genetic adjuvant to enhance the immune responses induced by a DNA vaccine based on the S gene encoding nucleocapsid protein against hantavirus. METHODS: BALB/c mice were immunized three times by intramuscular inoculations of DNA vaccine encoding of hantanvirus nucleocapsid protein alone or in combination with a plasmid expressing murine interleukin-12 (pcIL-12). Booster immunizations were employed 2 times at 2-week interval. To evaluate the humoral and cellular immune responses, antigen-specific lymphocyte proliferation and antibody production were assayed by MTT method and ELISA respectively. The level of interleukin-4 and interferon-gamma in the splenic lymphocytic cultured supernatant were detected with ELISA kit at day 5, 10, 17, 35 and 42 after primary immunization. RESULTS: Antigen-specific IgG antibodies was increased markedly at day 17 in the experiment groups and reached a plateau after day 35. As pcIL-12 co-injected, a significant inhibition of antigen-specific IgG levels was displayed over the period and the antibody mean titre was decreased to only about 1:50 at day 42 after primary immunization, significantly lower than the group immunized with pcDNA3.1 + S alone, in which the mean titre was about 1:70. Interferon-gamma was increased remarkably by the co-injection of pcIL-12 compared with the injection of pcDNA3.1 + S alone. However, the production of interleukin-4 was inhibited by pcIL-12 co-injection. Furthermore, pcIL-12 co-injection efficiently enhanced antigen-specific lymphocyte proliferation. CONCLUSION: Humoral and cytokine responses elicited by pcDNA3.1 + S inoculation can be modulated by co-inoculation with pcIL-12 and efficiently induced Th1-dominant immune responses.

Novel TLR4-antagonizing peptides inhibit LPS-induced release of inflammatory mediators by monocytes.

Toll-like receptor 4 (TLR4) has become a new target for combating Gram-negative bacterium-induced sepsis. In this study, we screened peptides that can interact with TLR4 from a random 16-peptide library using yeast two-hybrid system and performed functional identification for the obtained peptides. We got two positive clones out of 1.28x10(7) transformants. The peptides were sequenced and synthesized. Protein sequence comparison confirmed that the two peptides had no homologous proteins. The two peptides were found to significantly inhibit LPS-induced NF-kappaB activation in HEK-293 cells that were transfected with TLR4 cDNA, LPS-induced IkappaBalpha (IkappaB alpha) phosphorylation and NF-kappaB activation in monocytes, and release of IL-1, IL-6, and TNF-alpha by monocytes. We further confirmed that the No. 9 peptide could bind to TLR4 extracellular domain, but the No. 24 peptide could not, suggesting that two novel peptides were identified as the antagonists of TLR4, which significantly inhibited the effects of endotoxin in vitro. The No. 9 peptide may function through binding to TLR4 extracellular domain. Our findings suggest a promising countermeasure against Gram-negative bacterium-induced sepsis.

Focal nodular hyperplasia of the liver: pathological analysis of 11 cases.

BACKGROUND: Focal nodular hyperplasia (FNH) is a benign tumor-like lesion of the liver, predominantly affecting women. Its etiology is obscure and its pathogenesis is poorly understood. FNH should be differentiated from other benign and malignant hepatic lesions. The aim of this study was to explore the pathological characteristics of FNH of the liver. METHODS: Eleven patients with FNH were studied retrospectively by using hematoxylin and eosin, immunohistochemical and histochemical staining. RESULTS: In 8 female and 3 male FNH patients aged 19 to 54 years (mean 32), most of lesions showed central scars macroscopically. Microscopically 8 patients were found of classical type, 2 were of telangiectic type, and 1 was of mixed type. CONCLUSION: FNH is an uncommon benign hyperplastic lesion of the liver. It should be differentiated from hepatocellular adenoma, alpha-fetoprotein negative hepatocellular carcinoma, and fibrolamellar carcinoma.

Clinical experience in liver transplantation from an organ transplantation center in China.

OBJECTIVE: To sum up the experience in liver transplantation in a period of ten years at a single center. METHODS: We retrospectively reviewed the clinical records of 120 patients receiving liver transplantation from April 1993 to October 2002. The patients' clinical characteristics, surgical techniques, complications and survival were compared in the phases of 1993-1997 (phase I), 1999 (phase II), and 2000-2002 (phase III). RESULTS: Malignant liver diseases were major indications for liver transplantation in phase I (100%) and II (53.3%), but decreased markedly in percentage in phase III (34.0%). When compared with recipients in phase I and II, the survival of recipients with benign liver diseases in phase III was significantly improved with the 3-month, 6-month and 1-year survival rates of 85.7%, 84.5% and 83.1%, respectively. For patients with malignant liver diseases, the 3-month, 6-month and 1-year survival rates were 87.4%, 81.1% and 46.0%, respectively. The reinfection rate of hepatitis B virus was 24% 12 months after transplantation. With technical refinements, the incidence of postransplantation vascular complications has significantly decreased from 29.4% in phase I and II to 4.9% in phase III. Biliary complications remained one of the major obstacles to long-term survival. No veno-venous bypass was applied in phase III, providing a promising outcome. CONCLUSION: Strict selection of potential recipients, technical refinement, appropriate management of vascular and biliary complications, and prophylaxis of recurrences of hepatitis B and malignant liver diseases are important to obtain long-term survival of patients receiving liver transplantation in China.