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Effects of FK866 on migration of A549 cells and related mechanism
XIE Xian,XU Xiaofang,WANG Qi,LU Yunbi,WU Ming,ZHANG Weiping
J Zhejiang Univ (Med Sci), 2018, 47(1): 1-9.   https://doi.org/10.3785/j.issn.1008-9292.2018.02.01
Abstract( 1649 )   HTML( 59 )     PDF(851KB)( 308 )

Objective: To investigate the effect of nicotinamide phosphoribosyltransferase (NAMPT) inhibitor FK866 on the migration of human non-small cell cancer A549 cells and related mechanism. Methods: The inhibition effect of FK866 on A549 cells was tested by MTT assay. A549 cells were treated with 1.0 and 10.0 nmol/L FK866, and the cell migration was evaluated by modified wound scratch assay. The mRNA expression of E-cadherin and vimentin was detected by real-time RT-PCR, and the expression of ERK1/2 and pERK1/2 was determined by Western blotting. Results: FK866 inhibited the proliferation of A549 cells in a time- and concentration-dependent manner; after treatment for 72 h, the IC50 of FK866 was 9.55 nmol/L. When 1.0 nmol/L or 10.0 nmol/L FK866 was continuously applied 48 h before and 48 h after a scratch was made in wound scratch assay, the migration of A549 cells was significantly inhibited. However, when the FK866 was applied only 48 h after the scratch, the migration of A549 cells was inhibited by 10.0 nmol/L but not by 1.0 nmol/L FK866. The mRNA expression of E-cadherin and vimentin, and the activated ERK1/2 were significantly increased after 1.0 nmol/L FK866 treatment for 72 h. The pretreatment with nicotinamide adenine dinucleotide (NAD) precursor nicotinamide mononucleotide(1.0 mmol/L) or ERK1/2 inhibitor U0126 (10.0 μmol/L) reversed the up-regulation of E-cadherin and vimentin expression induced by FK866. Conclusions: Low concentration of FK866 decreases the migration of A549 cells through the inhibition of NAD level, activation of ERK1/2 and up-regulation of E-cadherin expression. However, it also up-regulates the expression of vimentin, indicating that it may have dual effects on the migration of tumor cells.

Effects of cysteinyl leukotriene receptors on phagocytosis of mouse microglial cells
WANG Xiaorong,LU Yunbi,ZHANG Weiping,WEI Erqing,FANG Sanhua
J Zhejiang Univ (Med Sci), 2018, 47(1): 10-18.   https://doi.org/10.3785/j.issn.1008-9292.2018.02.02
Abstract( 1077 )   HTML( 28 )     PDF(1059KB)( 187 )

Objective: To determine the effects of cysteinyl leukotriene receptors (CysLT1R and CysLT2R) on phagocytosis of mouse BV2 microglial cells. Methods: BV2 cells were stimulated with microglial activators lipopolysaccharide (LPS) or CysLT receptor agonists LTD4. The phagocytosis of BV2 cells was observed by immunofluorescence analysis and flow cytometry. The intracellular distributions of CysLT1R and CysLT2R in BV2 cells were examined with immunofluorescence staining. Results: Both LPS and LTD4 could significantly enhance the phagocytosis of BV2 cells, and such effect could be inhibited by CysLT1R selective antagonist Montelukast and CysLT2R selective antagonist HAMI 3379. The activation of BV2 cells induced by LTD4 or LPS resulted in changes in intracellular distributions of CysLT1R and CysLT2R. CysLT1R and CysLT2R was co-localization with a similar distribution. Conclusion: CysLT1R and CysLT2R regulate the phagocytosis of mouse BV2 microglial cells with a synergistic effect.

Effects of cysteinyl leukotrienes receptor antagonists on chronic brain injury after global cerebral ischemia/reperfusion
WANG Hao,GUO Honggang,LOU Qi,SHI Qiaojuan
J Zhejiang Univ (Med Sci), 2018, 47(1): 19-26.   https://doi.org/10.3785/j.issn.1008-9292.2018.02.03
Abstract( 777 )   HTML( 11 )     PDF(1117KB)( 127 )

Objective: To investigate the effects of cysteinyl leukotrienes receptor (CysLTR) antagonists on global cerebral ischemia/reperfusion (CI/R) injury in gerbils, and to explore its mechanism. Methods: Totally 40 gerbils weighting 45-65 g were randomized into sham, saline, Pranlukast and HAMI 3379 groups with 10 animals in each. The CI/R model was established in gerbils by bilateral common carotid occlusion for 10 min followed by reperfusion. After ischemia, the CysLTR antagonists Pranlukast (0.1 mg/kg) and HAMI 3379 (0.1 mg/kg) were injected intraperitoneally for 5 consecutive days in the last two groups, while the former two groups were injected with saline only (10 mL/kg). After 24 h or 14 d reperfusion, neurological deficit score was evaluated and the behavioral dysfunction was assessed, respectively. And 14 d after reperfusion, the neuron morphology of cerebral cortex was observed in brain sections stained with Cresyl violet. In addition, the Iba-1 (microgila) and GFAP (astrocyte) positive cells in cerebral cortex were observed by using immunohistochemitry method. Results: CI/R models were successfully established in 21 out of 30 gerbils with 7 in saline group, 6 in Pranlukast group, and 8 in HAMI 3379 group. Compared with saline group, Pranlukast and HAMI 3379 significantly attenuated neurological deficits, improved the behavioral function 24 h after reperfusion(all P < 0.01); Pranlukast and HAMI 3379 also significantly improved the behavioral function 14 days after reperfusion(P < 0.05 or P < 0.01). Compared with saline group, the neurological symptom scores in Pranlukast and HAMI 3379 groups presented a trend of amelioration 14 d after reperfusion, but it was not significant(P > 0.05). In addition, Pranlukast and HAMI 3379 also inhibited the neuron loss and injury, suppressed microgila and astrocyte activation 14 d after reperfusion(all P < 0.01). Conclusion: CysLTR antagonists Pranlukast and HAMI 3379 have long-term neuroprotective effect on chronic brain injury induced by global cerebral ischemia/reperfusion in gerbils.

Chlorogenic acid inhibits non-enzymatic glycation and oxidation of low density lipoprotein
CAI Rui,CHEN Shuqing,JIANG Shenhua
J Zhejiang Univ (Med Sci), 2018, 47(1): 27-34.   https://doi.org/10.3785/j.issn.1008-9292.2018.02.04
Abstract( 557 )   HTML( 16 )     PDF(1075KB)( 123 )

Objective: To investigate the effect of chlorogenic acid (CGA) on non-enzymatic glycation and oxidation of low density lipoprotein (LDL). Methods: The non-enzymatic glycation incubation system of LDL-glucose was established. The contents of early glycation products (Amodori product) and intermediate products (dicarbonyl compound) were determined by ultraviolet-visible spectrophotometry, and the content of advanced glycation end products (AGEs) was determined by fluorescence spectrophotometry. The LDL oxidation incubation system was established. The contents of thiobarbituric acid reactive substances(TBARS) and conjugated diene were determined by ultraviolet-visible spectrophotometry. The tryptophan fluorescence quenching, and the content of lipofuscin, total fluorescence products, active aldehydes and malondialdehyde were determined by fluorescence spectrophotometry, and further verified by three-dimensional fluorescence spectroscopy. Results: In the LDL glycation experiment, 150 μg/mL and 300 μg/mL CGA inhibited the formation of Amadori product, dicarbonyl compounds and AGEs. In the LDL oxidation experiment, 15 μg/mL and 25 μg/mL CGA inhibited the formation of TBARS effectively; 5 μg/mL and 10 μg/mL CGA inhibited tryptophan fluorescence quenching, and the formation of active aldehydes, malondialdehyde, total fluorescence products, lipofuscin and conjugated diolefine. And the three-dimensional fluorescence spectroscopy showed the same results. Conclusion: CGA can inhibit non-enzymatic glycation and oxidation of LDL.

TLR/NF-κB independent signaling pathway in TNF-α suppression of diabetic MyD88-knockout mice after Lycium barbarum polysaccharides administration
LIU Tingting,WANG Lingxiao,YANG Xiaohui,YAO Zhiqing,CAI Huizhen
J Zhejiang Univ (Med Sci), 2018, 47(1): 35-40.   https://doi.org/10.3785/j.issn.1008-9292.2018.02.05
Abstract( 828 )   HTML( 13 )     PDF(1031KB)( 213 )

Objective: To investigate the effect of Lycium barbarum polysaccharides (LBPs) on TLR/NF-κB independent pathway and serum tumor necrosis factor (TNF-α) level in diabetic MyD88-knockout mice. Methods: Diabetes was induced by feeding high-fat/high-sugar diet and injection of low-dose streptozotocin in MyD88-knockout mice. The diabetic mice were randomly divided into model group, positive control group and LBPs group. The expressions of TRAM, TRIF, TRAF6, RIP1 and TNF-α mRNA and proteins in mouse peritoneal macrophages were detected by real-time RT-PCR and Western blotting after LBPs treatment for 3 month. Serum TNF-α was determined with ELISA kit. Results: Real time RT-PCR showed that compared with model group, the relative expressions of Tram, Trif, Traf6 and Tnf-α mRNA in macrophages of LBPs group were significantly decreased and expression of Rip1 was significantly increased (all P < 0.05). Expression of TRAM, TRIF, TRAF6, RIP1 and TNF-α proteins as well as serum TNF-α level had no significant difference between LBPs group and model group (all P > 0.05). Conclusion: LBPs may not inhibit serum TNF-α level through TLR/NF-κB independent pathway.

Screening of mitochondrial DNA damage repair genes in rats with septic acute kidney injury
YANG Jingjuan,WU Fengfeng,CHEN Jianghua,YANG Yi
J Zhejiang Univ (Med Sci), 2018, 47(1): 41-50.   https://doi.org/10.3785/j.issn.1008-9292.2018.02.06
Abstract( 1026 )   HTML( 13 )     PDF(1272KB)( 146 )

Objective: To screen genes involved in mitochondrial DNA (mtDNA) damage repair in rats with septic acute kidney injury (SAKI). Methods: Forty male C57BL/6J mice were randomly divided into SAKI group (n=28) and sham operation group (n=12). The SAKI mouse model was established by cecal ligation and puncture. Blood and kidney samples were collected at 8, 24, and 48 h after surgery. Serum creatinine and urea nitrogen were measured by a dry biochemical analyzer. Serum inflammatory cytokines were detected by ELISA. Histopathological changes were observed with HE staining. The mtDNA damage repair related genes were screened by RNA sequencing and bioinformatics analysis; the mRNA and protein expression levels of related genes were detected by real-time quantitative RT-PCR and immunohistochemisry, respectively. Results: Symptoms of sepsis were observed in SAKI group, and 16 out of 28 mice were died in the SAKI group; serum TNF-α, IL-6, creatinine and urea nitrogen levels were higher than those in the sham group (P < 0.05 or P < 0.01). Histopathological examination in SAKI group showed that renal tubular epithelial cells were swollen, inflammatory cells infiltrated, and a large number of cell vacuoles were seen, suggesting successful modeling. Mitochondrial DNA damage repair related genes Gadd45α, Bcl2l1, Cdkn1a, Jun, Rela, Nfkbia and Nfkb1 were screened out. The expression of these genes was detected by real-time RT-PCR, and the results were consistent with RNA sequencing trends. Immunohistochemical staining showed that Gadd45α was mainly expressed in the nucleus of renal tubular epithelial cells, and the positive rate of Gadd45α in SAKI group was higher than that in the sham operation group (P < 0.05). Conclusion: Gadd45α, Bcl2l1, Cdkn1a, Jun, Rela, Nfkbia and Nfkb1 genes are involved in mtDNA damage repair in rats with SAKI, indicating that these genes may be used as new targets for prevention and treatment of SAKI.

Application of PeriCam PSI blood flow imaging perfusion system in rat brain ischemia reperfusion injury models
WU Chao,CHEN Guoxian,ZHAO Liuyang,LI Shu,HONG Yun
J Zhejiang Univ (Med Sci), 2018, 47(1): 51-56.   https://doi.org/10.3785/j.issn.1008-9292.2018.02.07
Abstract( 1085 )   HTML( 5 )     PDF(1111KB)( 116 )

Objective: To study the feasibility and effect of PeriCam PSI system guiding the establishment of ischemia/reperfusion injury model in rats. Methods: A total of 70 adult male Sprague-Dawley rats were divided into the control group(n=6), PSI monitoring group(n=34) and traditional operation group(n=30). Ischemia reperfusion model was established with reference to improve Zea-Longa line plug method. After the model established, the blood flow to the brain of control group, PSI monitoring group (ischemic 2 h, 24 h reperfusion) were observed and recorded respectively with PSI. The rats were then executed after 24 h, and the 2, 3, 5-triphenyltetrazolium chloride (TTC) staining and HE staining were used to observe the brain tissue. Results: The survival rate and modeling success rate of PSI monitoring group were higher than those of the traditional operation group(all P < 0.05). The blood perfusion in the brain and the distribution of blood vessels were clearly observed in the control group, and the data were normal. In 2 h ischemic group, the arterial flow was interrupted in the right cerebral artery, and the blood flow in the middle arterial blood supply was significantly decreased than that in the control group(P < 0.05). After the recovery of 24 h, the artery in the right side of the brain was restored to blood flow, but the blood flow in the partial supply area decreased, unable to recover to normal level. The TTC staining results indicated that there were obvious infarcts in the right brain tissue of PSI monitoring group, and the infarct area was more stable than that of the traditional operation group. The results of HE staining showed that the structure of brain tissue in the control group was normal, and the morphological rules of nerve cells were not change. While in brain tissue from PSI monitoring group, cortex and ischemia half dark stripe, nerve cell degeneration, necrosis and glial fiber disintegration, liquefaction, and light color, screen mesh in ischemic central area were observed. Conclusion: PSI system can guide ischemia reperfusion model building and improve the success rate of the model.

Expression of g6pd gene in wild type zebrafish embryos of early development
XIA Haixiong,LI Li,ZHOU Yanhua,REN Pingping,HE Zhixu,SHU Liping
J Zhejiang Univ (Med Sci), 2018, 47(1): 57-63.   https://doi.org/10.3785/j.issn.1008-9292.2018.02.08
Abstract( 1373 )   HTML( 8 )     PDF(1153KB)( 172 )

Objective: To observe the expression of g6pd gene in the early development stage of wild zebrafish embryos. Methods: The collinearity of g6pd gene and the sequence similarity of G6pd protein were analyzed with gene database and BLAST software, respectively. Expression of g6pd gene in different development stages of zebrafish embryos was detected by in situ hybridization. The g6pd-EGFP-pCS2+ recombinant plasmids were microinjected into zebrafish embryos, and fluorescence was observed under a fluorescence microscope. The expression of G6pd protein at 24, 48 and 72 hour post fertilization (hpf) zebrafish embryos was detected by Western blotting; the enzyme activity of G6pd at 24, 48 and 72 hpf zebrafish embryos was detected by modified G6pd quantitative ratio method. Results: The G6pd protein similarity of zebrafish and human was 88%, and that of zebrafish and mouse was 87%. The results of in situ hybridization showed that the g6pd gene was mainly expressed in the hematopoietic tissues of zebrafish; the results observed after microinjection of g6pd-EGFP-pCS2+ recombinant plasmid were consistent with the results of in situ hybridization. At 24, 48 and 72 hpf, the relative expression levels of G6pd protein in zebrafish embryos were 1.44±0.03, 1.47±0.05, and 1.54±0.02, respectively(P > 0.05); the G6pd enzyme activity levels were 1.74±0.17, 1.75±0.12, 1.71±0.22, respectively (P > 0.05). Conclusion: The study has observed the expression of g6pd gene and G6pd protein, and G6pd enzyme activity in zebrafish embryos at different development phases, which provides a reference for the establishment of a zebrafish G6PD deficiency model.

Experimental factors affecting in vitro recovery of puerarin in microdialysis
ZHAN Shuyu,RUAN Yu'er,LIU Guoqiang,DING Baoyue,SHAO Qing
J Zhejiang Univ (Med Sci), 2018, 47(1): 64-70.   https://doi.org/10.3785/j.issn.1008-9292.2018.02.09
Abstract( 754 )   HTML( 2 )     PDF(1089KB)( 82 )

Objective: To analyze experimental factors affecting in vitro recovery of puerarin in microdialysis. Methods: Puerarin concentration in microdialysate samples was determined by high performance liquid chromatography. The methods of direct dialysis, retrodialysis and the zero-net flux were used to calculate in vitro recovery, respectively. The effects of perfusate composition, the analyte concentration, perfusate flow rate, medium temperature and stir rates of the dialysis medium on recovery were investigated. Results: There were significant differences in the recovery values among direct dialysis, retrodialysis and zero-net flux methods. The recovery for 0.9% NaCl solution, Ringer's solution, PBS and anticoagulant dextrose solution as perfusate fluid were (71.25±2.36)%, (73.48±1.41)%, (68.50±2.43)% and (74.98±1.16)%, respectively. The composition of perfusate fluid had significant influence on the recovery(P < 0.01). At the same flow rate, recovery was independent of the analyte concentration. At the same concentration, the recovery was decrease with the increasing flow rate in an exponential relationship. The recovery increased with the raising temperature and stir rate of the dialysis medium, and the recovery remained stable when the stir rate reached above 200 rpm. Conclusion: A study method for in vitro recovery of puerarin in microdialysis has been established, and the recovery of puerarin is affected by calculating methods, perfusate fluids, flow rate, medium temperature and stir rate, but not affected by analyte concentrations.

A case of neuromyelitis optica spectrum disorders complicated with systemic lupus erythematosus and thymoma
ZHOU Haijin,XIA Ping,HU Xingyue
J Zhejiang Univ (Med Sci), 2018, 47(1): 71-74.   https://doi.org/10.3785/j.issn.1008-9292.2018.02.10
Abstract( 947 )   HTML( 8 )     PDF(1006KB)( 137 )

A 53-year-old male patient presented with hypopsia of his right eye for 2 months and lower extremities weakness for 8 days. Thoracic MRI demonstrated a lesion at T3 level appearing as hyperintense on T2-weighted images with non-enhancement by contrast medium and demyelinating lesion was considered. Aquaporin-4-Ab was positive and the antibody titer was 1:320 in serum. The diagnosis of neuromyelitis optica spectrum disorders was made. In addition, systemic lupus erythematosus and thymoma coexisted in this patient. After methylprednisolone impact treatment, plasma exchange and immunosuppressive therapy, the right vision and lower extremities weakness of the patient were improved.

Regulatory effect of Vav1 on T cells and its relation to clinical diseases
ZHANG Yuchuan,CHEN Wei
J Zhejiang Univ (Med Sci), 2018, 47(1): 75-81.   https://doi.org/10.3785/j.issn.1008-9292.2018.02.11
Abstract( 730 )   HTML( 7 )     PDF(1054KB)( 127 )

Vav1, as a key downstream signaling molecule of T cell receptor, includes a catalytic core DH-PH-ZF domain with the function as guanine nucleotide exchange factor (GEF), and a SH3-SH2-SH3 domain with the function as adaptor protein. These two structures of Vav1 play different roles in the development, activation, proliferation and function of T cells, and thereby exert the different regulatory effect on the occurrence and development of autoimmune disease, graft rejection, cancer and other clinical conditions, implicating that Vav1 might be a potential therapeutic target for these diseases. This paper reviews the role of Vav1 in T cells and the occurrence of related diseases.

Protein ubiquitination on the regulation of inflammatory bowel disease
LING Jing,LI Hongrui,CHEN Weilin
J Zhejiang Univ (Med Sci), 2018, 47(1): 82-88.   https://doi.org/10.3785/j.issn.1008-9292.2018.02.12
Abstract( 706 )   HTML( 6 )     PDF(1074KB)( 258 )

Inflammatory bowel disease refers to chronic inflammatory disorders that affect the gastrointestinal tract. Ubiquitination is an important protein post-translational modification. In recent years, the research of ubiquitination-deubiquitination system in the development of inflammatory bowel disease has become a hot spot. Up to now, the E3 ubiquitin ligases such as ring finger protein 183 (RNF183), RNF20, Itch and A20 were well studied in inflammatory bowel disease. RNF183 promotes the activation of the NF-κB pathway by increasing the ubiquitination and degradation of IκBα; RNF20 drives histone H2B monoubiquitylation, downregulates a panel of inflammation-associated genes; Itch inhibits IL-17-mediated colon inflammation by retinoid acid related orphan receptor γt ubiquitination; A20 has ubiquitinating-deubiquitinating activity to regulates colon inflammation. This article reviews the role and regulatory mechanism of RNF183, RNF20, Itch and A20 in the pathogenesis of inflammatory bowel disease.

Research progress on receptor interacting proteins in inflammation
DING Jingjing,LU Yunbi
J Zhejiang Univ (Med Sci), 2018, 47(1): 89-96.   https://doi.org/10.3785/j.issn.1008-9292.2018.02.13
Abstract( 1472 )   HTML( 23 )     PDF(1149KB)( 234 )

Receptor interacting proteins (RIPs) are a group of threonine/serine protein kinases, which have relatively conserved kinase domains and different non-kinase domains, and are involved in physiological and pathological processes including innate immune response and inflammation. In recent years, many studies have shown that RIPs mediate cell necroptosis and triggers inflammatory responses by participating in the formation of necrotic complexes, and RIP1 and RIP3 are particularly closely related to cell necrosis. Cell necroptosis is a well-regulated way of cell death. The death signal that transmit through the TNF signaling pathway and the Toll-like receptor signaling pathway can recruit and phosphorylate mixed lineage kinase domain-like protein (MLKL), and eventually leading to disintegration and death of cells, and the release of cells intercellular material after cell disintegration can trigger an inflammatory reaction. This review mainly focuses on the major signaling pathways and molecular mechanisms that are involved in the mediation of necrosis and inflammation by RIPs. It also highlights the importance of RIPs in the development of inflammatory diseases and their potentials as therapeutic targets for inflammatory diseases.

Research progress on genotyping of Helicobacter pylori
WANG Jiajing,GU Haiying
J Zhejiang Univ (Med Sci), 2018, 47(1): 97-103.   https://doi.org/10.3785/j.issn.1008-9292.2018.02.14
Abstract( 1028 )   HTML( 18 )     PDF(1078KB)( 253 )

Helicobacter pylori (Hp) is widely disseminated in human, and Hp infection causes various gastrointestinal diseases, including gastric cancer. Different genotypes of Hp may cause different diseases, so the genotyping is important for clinical and basic research of Hp. This article introduces the methods for Hp genotyping, including multilocus sequence typing, pulsed-field gel electrophoresis, random amplified polymorphic DNA, amplified fragment length polymorphism, and whole-genome sequencing. By reviewing the application of these techniques in Hp genotyping and comparing their advantages and disadvantages, the article provides a theoretical basis for research into the pathogenesis, antibiotic resistance, and epidemiology of Hp infection.

Methodology for survival assessment of cancer patients using population-based cancer registration data
TANG Huijuan,JIANG Xiyi,LOU Jianlin,CHEN Tianhui
J Zhejiang Univ (Med Sci), 2018, 47(1): 104-109.   https://doi.org/10.3785/j.issn.1008-9292.2018.02.15
Abstract( 651 )   HTML( 8 )     PDF(1288KB)( 162 )

Evaluating and monitoring long-term survival of cancer patients and reporting the survival rate are routinely employed by cancer registries. Long-term survival rate is a necessary indicator in evaluating the effect of cancer therapy and cancer burden. Cohort method is a traditional approach for survival analysis, but it essentially reflects the survival expectations of patients diagnosed many years ago, therefore survival status of cancer patients was often disclosed with delay. Given the limitation of cohort method, period analysis and model-based period analysis are subsequently proposed and gradually applied in assessment of survival rates in recent years. Period analysis includes the patients of interest period, which reflects more up-to-date estimates of long-term survival of cancer patients. While model-based period analysis can use the existing data to calculate survival rates and to assess the trend, and predict survival rates in the future. Compared with cohort approach, period analysis and model-based period analysis are better in timeliness and precision in survival analysis. This article reviews the definition and theory, calculation and application of cohort method, period analysis and model-based period analysis, in order to provide a basis on up-to-date and precise assessment of survival rates of cancer patients.

15 articles