Argatroban promotes recovery of spinal cord injury by inhibiting the PAR1/JAK2/STAT3 signaling pathway

Argatroban is a synthetic thrombin inhibitor approved by U.S.Food and Drug Administration for the treatment of thrombosis.However,whether it plays a role in the repair of spinal cord injury is unknown.In this study,we established a rat model of T10 moderate spinal cord injury using an NYU Impactor ModerⅢand performed intraperitoneal injection of argatroban for 3 consecutive days.Our results showed that argatroban effectively promoted neurological function recovery after spinal cord injury and decreased thrombin expression and activity in the local injured spinal cord.RNA sequencing transcriptomic analysis revealed that the differentially expressed genes in the argatroban-treated group were enriched in the JAK2/STAT3 pathway,which is involved in astrogliosis and glial scar formation.Western blotting and immunofluorescence results showed that argatroban downregulated the expression of the thrombin receptor PAR1 in the injured spinal cord and the JAK2/STAT3 signal pathway.Argatroban also inhibited the activation and proliferation of astrocytes and reduced glial scar formation in the spinal cord.Taken together,these findings suggest that argatroban may inhibit astrogliosis by inhibiting the thrombin-mediated PAR1/JAK2/STAT3 signal pathway,thereby promoting the recovery of neurological function after spinal cord injury.

Expression of Vascular Endothelial Growth Factor-C and Vascular Endothelial Growth Factor Receptor-3 in Ovarian Epithelial Tumors

Objective： To explore the role of vascular endothelial growth factor-C （VEGF-C） in the process of angiogenesis, lymphangiogenesis and lymphatic metastasis in epithelial ovarian tumors. Methods： In situ hybridization and immunohistochemical staining for VEGF-C were performed in 30 epithelial ovarian carcinomas, 9 borderline tumors and 26 benign tumors. Endothelial cells were immunostained with anti-VEGFR-3 pAb and anti-CD31 mAb, and VEGFR-3 positive vessels and microvessel density （MVD） were assessed by image analysis. Results： VEGF-C mRNA and protein expression were detected in cytoplasm of carcinoma cells. VEGF-C mRNA and protein expression in ovarian epithelial carcinomas were significantly higher than those in borderline tumors and benign tumors （P〈0.05 or P〈0.01）. In ovarian epithelial carcinomas, VEGF-C protein expression, VEGFR-3 positive vessels and MVD were significantly higher in the cases of clinical stage Ⅲ-Ⅳ and with lymph node metastasis than those of clinical stage Ⅰ-Ⅱ and without lymph node metastasis respectively （P〈0.05 or P〈0.01）. VEGFR-3 positive vessels and MVD were significantly higher in VEGF-C protein positive tumors than negative tumors （P〈0.05）. VEGFR-3 positive vessels was significantly correlated with MVD（P〈0.01）. Conclusion： VEGF-C might play a role in lymphatic metastasis via lymphangiogenesis and angiogenesis in epithelial ovarian tumors, and VBEGF-C could be used as a biologic marker of metastasis in ovarian epithelial tumors.

Study on the Expression of Dopamine Receptor-3(DRD3) in Rats after Sacral Spinal Cord Transection

[Objectives] This study aimed to study the distribution characteristics of DRD3(dopamine receptor-3) and the changes in its expression before and after spinal cord injury(SCI), in order to lay a morphological basis for later research. [Methods] Adult male Wistar rats were randomly divided into sham operation group and SCI group. The rat spinal cord transection model at the sacral 2(S_2) segment was established. Rat tail spasticity score was performed 60 d after SCI, and the rats with 4-5 points were screened for perfusion. The expression of DRD3 in the sacral spinal cord(S+C segment) was detected by immunofluorescence. [Results] In normal rats, DRD3 was mainly distributed in the dorsal horn(DH), intermediate zone(IMZ) and ventral horn(VH) of the gray matter. It was also expressed in the white matter of the spinal cord. After SCI, the distribution of DRD3 in the segment below the injury section was similar to that of normal rats. However, the expression was different(P<0.05). [Conclusions] There was no significant change in the distribution of DRD3 in spinal cord after SCI. After the spinal cord S_2 was completed transected, the expression of DRD3 was significantly reduced in the DH, IMZ and VH regions of the gray matter of the spinal cord.

Expressions of Connexin and Par-3 in the Distal Margin of Rectal Cancer after Ultra-low Anterior Resection

This study examined the expression of connexin and protease-activated receptor 3 （par-3） in the distal resection margin of rectal cancer and the correlation of the expression of the two proteins with tumor relapse. A total of 40 patients with rectal cancer underwent ultra-low anterior resection with curved cutter stapler. The pathological specimens were divided into 3 groups in terms of sampling sites： tumor group, 2.0-cm group （in which the tissues were harvested 2.0 cm distal to the tumor tissues）, 3.0-cm group （in which the tissues were taken 3.0 cm away from the tumor tissues）. All the samples were pathologically observed and then measured for the expression of connexin and par-3 by employing immunohistochemistry and Western blotting. The operations in this series went uneventfully. No anastomotic stoma bleeding, stenosis and death occurred postoperatively. Histopathologically, in the tumor group, epithelial cells lost normal pattern of arrangement and polarity, and were loosely connected and even detached. In the 3.0-cm group, the epithelia had normal appearance, obvious cell polarity and essentially intact cell junction. Immunohistochemistry and Western blotting indicated that the 3.0-cm group had the strongest expression of connexin and par-3, and the expression in the 2.0-cm group and the tumor group was relatively weak. There existed significant difference in the expression of the two proteins among the three groups （P〈0.05 for all）. It was concluded that the down-regulated connexin and par-3 in the distal margin of rectal cancer tissues may indicate the progression of the disease and high likelihood of recurrence and metastasis. Although no tumor cells were found in the sections of the 2.0cm group, the decreased expression of connexin and par-3 may suggest the development of anaplasia and the increased odds of tumor relapse. Therefore, we are led to speculate that tumor resection only including 2.0 cm of unaffected rectum could not completely avoid the distant metastasis and local relapse.

Pioglitazone ameliorates retinal ischemia/reperfusion injury via suppressing NLRP3 inflammasome activities

AIM：To explore the role of Pioglitazone（Pio） on a mouse model of retinal ischemia/reperfusion（I/R） injury and to elucidate the potential mechanism.METHODS：Retinal ischemia was induced in mice by increasing the intraocular pressure,and Pio was administered 4 h though periocular injection before I/R.The number of cells in the ganglion cell layer（GCL） was counted 7 d after retinal I/R injury.Glial fibrillary acidic protein（GFAP）,nuclear factor-kappa B（NF-κB）,p38,phosphorylated-p38,PPAR-γ,interleukin-1β（IL-1β）,Toll-like receptor 4（TLR4）,NLRP3,cleaved caspase-1,caspase-1 were determined by real-time polymerase chain reaction and Western blotting.RESULTS：Pio promoted the survival of retinal cells in GCL following retinal I/R injury（P〈0.05）.Besides,retinal I/R injury stimulated the expression of GFAP and TLR4,which were partially reversed by Pio treatment（P0.05）.Retinal I/R injury-upregulated expression of NLRP3,cleaved caspase-1,IL-1β was attenuated after Pio treatment（P〈0.05）.Moreover,I/R injury induced activation of NF-κB and p38 were inhibited by Pio treatment（P〈0.05）.CONCLUSION：Pio promotes retinal ganglion cells survival by suppressing I/R-induced activation of TLR4/NLRP3 inflammasomes via inhibiting NF-κB and p38 phosphorylation.

Serum VEGFR-3 and survival of advanced gastric cancer patients treated with FOLFOX

AIM: To explore if vascular endothelial growth factor receptor-3 (VEGFR-3) and carcinoembryonic antigen (CEA) can predict overall survival in advanced gastric cancer.METHODS: VEGFR-3 level was assessed by enzymelinked immunosorbent assay,and CEA was assessed by chemiluminescence immunoassay in the sera of 81 advanced gastric cancer patients before treatment with oxaliplatin plus 5-fluorouracil and folinic acid.RESULTS: Median survival time in patients with a low serum VEGFR-3 level was significantly longer than in those with a higher VEGFR-3 level (15.4 mo vs 7.7 mo,P < 0.001).Patients with a low CEA level had a longer survival than those with a higher CEA level (15.8 mo vs 8.6 mo,P < 0.001).Thirty-nine patients with low VEGFR-3 and low CEA levels had a median survival of 19.7 mo (P = 0.0006).The hazard ratio for patients with a high VEGFR-3 level was 2.443 (P = 0.002).CONCLUSION: High serum VEGFR-3 level is correlated significantly with poor survival.In patients with a high serum level of VEGFR-3,alternative chemotherapy regimens should be considered.

Baicalin Attenuates Focal Cerebral Ischemic Reperfusion Injury by Inhibition of Protease-Activated Receptor-1 and Apoptosis

Objective： To investigate the neuro-protective effects of baicaiin in Wistar rats with focal cerebral ischemic reperfusion injury. Methods： Ninety adult male Wistar rats weighing 320-350 g were randomly divided into the following groups （n=5）： （a） sham control group; （b） vehicle group, subjected to middle cerebral artery occlusion and received vehicle intraperitoneally; （c-e） baicalin groups, which were subjected to the middle cerebral artery occlusion and treated with baicalin 25, 50 and 100 mg/kg, respectively. The neurological scores were determined at postoperative 1, 3 and 7 d after the treatment. The expression of protease-activated receptor-1 （PAR-1）, PAR-1 mRNA and Caspase-3 were determined using Western blot, reverse transcription polymerase chain reaction （RT- PCR） analysis and immunohistochemistry, respectively. Results： Significant decrease was noted in the neurological score in the baicalin group compared with that of the vehicle group （P〈0.01）. Additionally, down-regulation of PAR-1 mRNA, PAR-1 and Caspase-3 was observed in the baicalin groups compared with those obtained from the vehicle group （P〈0.01）. Compared with the low-dose baicalin group （25 mg/kg）, remarkable decrease was noted in neurological score, and the expression of PAR-1 mRNA, PAR-1 as well as Caspase-3 in the high-dose group （P〈0.05）. Conclusion： Baicalin showed neuro-protective effects in focal cerebral ischemic reperfusion injury through inhibiting the expression of PAR-1 and apoptosis.

Genome editing of PAR2 through targeted delivery of CRISPR-Cas9 system for alleviating acute lung inflammation via ERK/NLRP3/IL-1β and NO/iNOS signalling

Excessive and uncontrollable inflammatory responses in alveoli can dramatically exacerbate pulmonary disease progressions through vigorous cytokine releases,immune cell infiltration and protease-driven tissue damages.It is an urgent need to explore potential drug strategies for mitigating lung inflammation.Protease-activated receptor 2(PAR2)as a vital molecular target principally participates in various inflammatory diseases via intracellular signal transduction.However,it has been rarely reported about the role of PAR2 in lung inflammation.This study applied CRISPR-Cas9 system encoding Cas9 and sg RNA(p Cas9-PAR2)for PAR2 knockout and fabricated an anionic human serum albuminbased nanoparticles to deliver p Cas9-PAR2 with superior inflammation-targeting efficiency and stability(TAP/p Cas9-PAR2).TAP/p Cas9-PAR2 robustly facilitated p Cas9-PAR2 to enter and transfect inflammatory cells,eliciting precise gene editing of PAR2 in vitro and in vivo.Importantly,PAR2 deficiency by TAP/p Cas9-PAR2 effectively and safely promoted macrophage polarization,suppressed proinflammatory cytokine releases and alleviated acute lung inflammation,uncovering a novel value of PAR2.It also revealed that PAR2-mediated pulmonary inflammation prevented by TAP/p Cas9-PAR2was mainly dependent on ERK-mediated NLRP3/IL-1β and NO/i NOS signalling.Therefore,this work indicated PAR2 as a novel target for lung inflammation and provided a potential nanodrug strategy for PAR2 deficiency in treating inflammatory diseases.

Effect of Thrombin on the Apoptosis of Hippocampal Neurons in vitro

Hippocampal neurons were treated by thrombin and thrombin receptor activatingpeptides (TRAP). Cell survival rate was decreased in a dose-dependent manner by MTT assay. Thenumbers of apoptotic cell and apoptotic rate of hippocampal neurons treated bydifferentconcentrations of thrombin were increased in a dose-dependent manner by terminal deoxynucleotidyltransferase (TdT) mediated dUTP-biotin nick end-labeling (TUNED method and Flow Cytometry. When theconcentration of thrombin is 40 U/mL, TUNEL positive cells and apoptotic rate of hippocampal neuronsreached peak value, were 27. 3 +- 4. 0 and (29. 333 +- 4. 633 ) % , respectively.Immunocytochemistry assay show that Bcl-2 protein expression was down- regulated and Bax proteinexpression was up-regulated with the concentration of thrombin increased. TRAP can mimic the effectof thrombin to induce apoptosis on hippocampal neurons. These data demonstrated that thrombininduced hippocampal neuron apoptosis in a dose-dependent manner through activatingprotease-acti-vated protein-1 (PAR-1). The change in expression of Bcl-2 and Bax was related withthe effect of high concentration thrombin induced apoptosis on hippocampal neurons.

Distribution and localization of fibroblast growth factor-8 in rat brain and nerve cells during neural stem/progenitor cell differentiation

The present study explored the distribution and localization of fibroblast growth factor-8 and its potential receptor, fibroblast growth factor receptor-3, in adult rat brain in vivo and in nerve cells during differentiation of neural stem/progenitor cells in vitro. Immunohistochemistry was used to examine the distribution of fibroblast growth factor-8 in adult rat brain in vivo. Localization of fibroblast growth factor-8 and fibroblast growth factor receptor-3 in cells during neural stem/progenitor cell differentiation in vitro was detected by immunofluorescence. Flow cytometry and immunofluorescence were used to evaluate the effect of an anti-fibroblast growth factor-8 antibody on neural stem/progenitor cell differentiation and expansion in vitro. Results from this study confirmed that fibroblast growth factor-8 was mainly distributed in adult midbrain, namely the substantia nigra, compact part, dorsal tier, substantia nigra and reticular part, but was not detected in the forebrain comprising the caudate putamen and striatum. Unusual results were obtained in retrosplenial locations of adult rat brain. We found that fibroblast growth factor-8 and fibroblast growth factor receptor-3 were distributed on the cell membrane and in the cytoplasm of nerve cells using immunohistochemistry and immunofluorescence analyses. We considered that the distribution of fibroblast growth factor-8 and fibroblast growth factor receptor-3 in neural cells corresponded to the characteristics of fibroblast growth factor-8, a secretory factor. Addition of an anti-fibroblast growth factor-8 antibody to cultures significantly affected the rate of expansion and differentiation of neural stem/progenitor cells. In contrast, addition of recombinant fibroblast growth factor-8 to differentiation medium promoted neural stem/progenitor cell differentiation and increased the final yields of dopaminergic neurons and total neurons. Our study may help delineate the important roles of fibroblast growth factor-8 in brain activities and neural stem/progenitor cell differentiation.

Ischemic preconditioning protects against ischemic brain injury

In this study, we hypothesized that an increase in integrin αβand its co-activator vascular endothelial growth factor play important neuroprotective roles in ischemic injury. We performed ischemic preconditioning with bilateral common carotid artery occlusion for 5 minutes in C57BL/6J mice. This was followed by ischemic injury with bilateral common carotid artery occlusion for 30 minutes. The time interval between ischemic preconditioning and lethal ischemia was 48 hours. Histopathological analysis showed that ischemic preconditioning substantially diminished damage to neurons in the hippocampus 7 days after ischemia. Evans Blue dye assay showed that ischemic preconditioning reduced damage to the blood-brain barrier 24 hours after ischemia. This demonstrates the neuroprotective effect of ischemic preconditioning. Western blot assay revealed a significant reduction in protein levels of integrin αβ, vascular endothelial growth factor and its receptor in mice given ischemic preconditioning compared with mice not given ischemic preconditioning 24 hours after ischemia. These findings suggest that the neuroprotective effect of ischemic preconditioning is associated with lower integrin αβand vascular endothelial growth factor levels in the brain following ischemia.

Blood cellular mutant LXR-α protein stability governs initiation of coronary heart disease

AIM: To investigate the role of [breast and ovarian cancer susceptibility 1(BRCA1)-associated RING domain 1(BARD1)]/BRCA1 E3-ubiquitin ligase complex in governing the stability of mutant liver X receptor-(LXR-α) protein in coronary heart disease(CHD) subjects.METHODS: The expression analysis of various genes was carried out by quantitative real time polymerase chain reaction and western blotting within blood mononuclear cells of human CHD subjects at various stages of coronary occlusion and their corresponding normal healthy counterparts.Immunoprecipitation experiments were performed to establish protein interactions between LXR-αand BARD1.Peripheral blood mononuclear cells were cultured and exposed to Vitamin D_3 and Cisplatin to validate the degradation of mutant LXR-αprotein in CHD subjects by BARD1/BRCA1 complex.RESULTS: The expression of mutant LXR-αprotein in CHD subjects was found to decrease gradually with the severity of coronary occlusion exhibiting a strong negative correlation,r =-0.975 at P < 0.001.Further,the expression of BARD1 and BRCA1 also increased with the disease severity,r = 0.895 and 0.873 respectively(P < 0.001).Immunoprecipitation studies established that BARD1/BRCA1 complex degrades mutant LXR-αvia ubiquitination.The absence of functional LXR-αprotein resulted in increased expression of inflammatory cytokines such as interleukin(IL)-6,IL-8 and interferon-and decreased expression of ABCA1(ATP-binding cassette A1)(r = 0.932,0.949,0.918 and-0.902 with respect to Gensini score;P < 0.001).Additionally,cell culture experiments proved that Vitamin D_3 could prevent the degradation of mutant LXR-αand restore its functional activity to some extent.CONCLUSION: Mutant LXR-αprotein in CHD subjects is degraded by BARD1/BRCA1 complex and Vitamin D_3 can rescue and restore its function.

Response letter regarding the interpretation of gene expression data

This is a response letter to Verna E's comments regarding our previous manuscript published last year in the World Journal of Gastroenterology entitled "Relationship between LYVE-1,VEGFR-3 and CD44 gene expressions and lymphatic metastasis in gastric cancer",which evaluated the relationship between these expression levels and clinicopathological parameters(Ozmen F et al,World J Gastroenterology 2011;17:3220-3228).The mean values for lymphatic vessel endothelial hyaluronan receptor-1,CD44 and vascular endothelial growth factor receptor-3 expression(represented as 2-Ct) were 1.13,1.24 and 1.17,respectively,suggesting an increase in gene expression in tumor tissue compared to normal tissue.Despite the increase in gene expression in the cancer tissues(2-Ct > 1),only some of the results reached statistical significance,which was thoroughly discussed in our paper.In the present letter,we report that his comments are flawed and result in confusion.Therefore,we herein provide more explanation regarding gene expression in gastric cancer.We hope that this letter will address Verna E's misunderstandings.

Immunomagnetic Indirect Positive Sorting of Precartilaginous Stem Cells from Neonatal Rat

To investigate the technique of sorting high-purity precartilaginous stem cells from rat＇s perichondrium, neonatal rat＇s perichondrium cells suspensions were incubated with monoclone antibody of anti-fibroblast growth factor receptor-3 （anti-FGFR-3）, and the labeled cells were separated from the suspension in the magnetic field by immuno-beads coated with the second antibody, Purity of the sorted neural stem cells was found to be 93.0 %-99.0 %, with living cells amounting to 80 % -85 %. The magnetic cell sorting system could effectively separate precartilaginous stem cells from perichondrium cell suspensions.

表皮生长因子受体、人类表皮生长因子受体2、人类表皮生长因子受体3表达与非小细胞肺癌预后的关系

目的：探讨非小细胞肺癌（NSCLC）患者癌组织中表皮生长因子受体（EGFR）、人类表皮生长因子受体（HER）2、HER3蛋白的表达及其与临床病理因素的关系。方法采用MaxVision免疫组织化学两步法检测156例NSCLC患者癌组织中EGFR、HER2、HER3的表达，研究其与患者临床、病理特征间的关系，并回顾性分析蛋白表达与患者生存的关系。结果 EGFR、HER2、HER3蛋白在NSCLC中均存在过表达，过表达率分别为45.9%（73／156）、30.8%（49／156）和21.4%（37／156）。EGFR过表达多见于鳞状细胞癌、低分化、有淋巴结转移的患者，HER2过表达及三者共同过表达多见于晚期NSCLC患者，三者共同过表达多见于有淋巴结转移的患者。 EGFR、HER2、HER3过表达及三者共同过表达的患者生存率均低于低表达组。结论 EGFR、HER2、HER3过表达及三者共同过表达提示NSCLC患者预后不良。三项联合检测可能比单独检测能更好地预测NSCLC患者预后，指导临床治疗。

Pretreatment with Baicalin Attenuates Hypoxia and Glucose Deprivation-Induced Injury in SH-SY5Y Cells

Objective： To explore the neuroprotective effects of baicalin against hypoxia and glucose deprivation- reperfusion （OGD/RO）-induced injury in SH-SY5Y cells. Methods： SH-SY5Y cells were divided into a control group, a OGD/RO group, which was subject to OGD/RO induction; and 3 baicalin groups subject to baicalin （1, 5, 25 μmol/L） for 2 h before induction of OGD/RO （low-, medium-, and high-dose baicalin groups）. Cell viability was detected by thiazolyl blue tetrazoliurn bromide （MTT） assay and flow cytometric analysis was used to detect cell apoptosis. Real-time polymerase chain reaction was performed to determine the mRNA expression of caspase-3 gene. Westem blot analysis was conducted to determine the expression of nuclear factor （NF）- κB and N-methyl-d- aspartic acid receptor-1 （NMDAR1）. Results： Baicalin could significantly attenuate OGD/RO mediated apoptotic cell death in SH-SY5Y cells; the apoptosis rates in the low-, medium- and high-dose groups were 12.1%, 7.9%, and 5.4%, respectively. Western blot and real-time PCR analysis revealed that significant decrease in caspase-3 expression in the baicalin group compared with the OGD/RO group （P〈0.01）. Additionally, down-regulation of NF-K B and NMDAR1 was observed in the baicalin group compared with those obtained from the OGD/RO group. Compared with the low-dose baicalin group, remarkable decrease was noted in the medium- and high-dose groups （P〈0.01）. Conclusion： Baicalin pre-treatment attenuates brain ischemia reperfusion injury by suppressing cellular apoptosis.