[Expression of Semaphorin 3A after spinal cord injury].

OBJECTIVE: To investigate expression of Semaphorin 3A in rats after spinal cord injury and explore possible mechanism of inhibiting of axonal regeneration after SCI. METHODS: Forty healthy female SD rats, 8 weeks old, weighing (210.00±9.88) g, were randomly divided into control group(20 rats in group A) and model group(20 rats in group B). In control group, removal of T10 lamina and partial removal of T9 and T11 lamina were performed, and no further operation was performed on spinal cord (pseudo operation). In model group, the total T10 and partial T9, T11 partial lamina were incised and the spinal cord transection was performed to create animal models of spinal cord injury. The rats were perfused and spinal cord tissue obtained at 3, 7, 14, 28 and 42 days after surgery (4 rats in each group at each time point), respectively, and then HE staining was performed. Meanwhile, the expression of Semaphoring 3A was detected in accordance with the protocol of SP kit. RESULTS: After a simple spinal cord transection injury, hemorrhagic necrosis, localized edema, neurodegeneration, necrosis, and cyst formation occurred in the injured area, and glial scar formation occurred in glial cells. Semaphorin 3A expression levels in control group was low in the gray matter area. There was no expression of Semaphorin 3A in the injured area of spinal cord injury in model group 3 days after operation. On the 14th day, the expression of Semaphorin 3A in the injured area of spinal cord injury increased significantly and was at a high level. On the 28th day, the expression of Semaphorin 3A was moderate. On the 42th day, the positive expression of Semaphorin 3A returned to normal level. CONCLUSION: The increased expression of Semaphorin 3A after spinal cord injury may be one of the mechanisms that inhibit axonal regeneration.

Remarkable inhibition effects of afatinib alone or combining with paclitaxel in esophageal squamous cell carcinoma.

BACKGROUND AND AIM: Chemotherapy drugs do not work well in esophageal squamous cell carcinoma (ESCC), and none of the targeted drugs have been applied in clinic. This study aims to identify effective targeted drugs and related biomarkers for the treatment of ESCC. METHODS: The effect of 40 Food and Drug Administration-approved small-molecule inhibitors was first tested in five ESCC cell lines. CCK8 assays and xenografts derived from ESCC cell lines were performed to evaluate the anti-ESCC effects of inhibitors or chemotherapeutic agents in vitro and in vivo, respectively. Immunohistochemistry was utilized to analyze the p-EGFR expression in tissues. Western blot combining with gray analysis was conducted to detect the expression of interest protein. Flow cytometry and immunofluorescence assay were used to analyze apoptosis, cell cycle, and mitotic changes after drug treatment. RESULTS: Afatinib showed remarkable effects on inhibiting ESCC cells with higher expression of p-EGFR. Results from combinatorial screening in ESCC cells expressing lower phosphorylation level of EGFR showed that paclitaxel and afatinib presented a significant synergistic inhibitory effect (P < 0.001). Molecular analysis revealed that paclitaxel sensitized afatinib by activating EGFR, and afatinib in combination with paclitaxel effectively blocked MAPK pathway and induced G2/M cell arrest and apoptosis that is an indicator of mitotic catastrophe. CONCLUSIONS: Our data demonstrate that afatinib is an effective drug for patients with ESCC expressing higher phosphorylation level of EGFR. And for patients with lower p-EGFR in tumors, paclitaxel in combination with afatinib might be a promising therapeutic strategy in ESCC.

FKBP10 promotes proliferation of glioma cells via activating AKT-CREB-PCNA axis.

BACKGROUND: Although the availability of therapeutic options including temozolomide, radiotherapy and some target agents following neurosurgery, the prognosis of glioma patients remains poor. Thus, there is an urgent need to explore possible targets for clinical treatment of this disease. METHODS: Tissue microarrays and immunohistochemistry were used to detect FKBP10, Hsp47, p-AKT (Ser473), p-CREB (Ser133) and PCNA expression in glioma tissues and xenografts. CCK-8 tests, colony formation assays and xenograft model were performed to test proliferation ability of FKBP10 in glioma cells in vitro and in vivo. Quantitative reverse transcriptase-PCR, western-blotting, GST-pull down, co-immunoprecipitation and confocal-immunofluorescence staining assay were used to explore the molecular mechanism underlying the functions of overexpressed FKBP10 in glioma cells. RESULTS: FKBP10 was highly expressed in glioma tissues and its expression was positively correlates with grade, poor prognosis. FKBP10-knockdown suppressed glioma cell proliferation in vitro and subcutaneous/orthotopic xenograft tumor growth in vivo. Silencing of FKBP10 reduced p-AKT (Ser473), p-CREB (Ser133), PCNA mRNA and PCNA protein expression in glioma cells. FKBP10 interacting with Hsp47 enhanced the proliferation ability of glioma cells via AKT-CREB-PCNA cascade. In addition, correlation between these molecules were also found in xenograft tumor and glioma tissues. CONCLUSIONS: We showed for the first time that FKBP10 is overexpressed in glioma and involved in proliferation of glioma cells by interacting with Hsp47 and activating AKT-CREB-PCNA signaling pathways. Our findings suggest that inhibition of FKBP10 related signaling might offer a potential therapeutic option for glioma patients.

Olfactory ensheathing cell transplantation alters the expression of chondroitin sulfate proteoglycans and promotes axonal regeneration after spinal cord injury.

Cell transplantation is a potential treatment for spinal cord injury. Olfactory ensheathing cells (OECs) play an active role in the repair of spinal cord injury as a result of the dual characteristics of astrocytes and Schwann cells. However, the specific mechanisms of repair remain poorly understood. In the present study, a rat model of spinal cord injury was established by transection of T10. OECs were injected into the site, 1 mm from the spinal cord stump. To a certain extent, OEC transplantation restored locomotor function in the hindlimbs of rats with spinal cord injury, but had no effect on the formation or volume of glial scars. In addition, OEC transplantation reduced the immunopositivity of chondroitin sulfate proteoglycans (neural/glial antigen 2 and neurocan) and glial fibrillary acidic protein at the injury site, and increased the immunopositivity of growth-associated protein 43 and neurofilament. These findings suggest that OEC transplantation can regulate the expression of chondroitin sulfate proteoglycans in the spinal cord, inhibit scar formation caused by the excessive proliferation of glial cells, and increase the numbers of regenerated nerve fibers, thus promoting axonal regeneration after spinal cord injury. The study was approved by the Animal Ethics Committee of the Medical College of Xi'an Jiaotong University, China (approval No. 2018-2048) on September 9, 2018.

High S phase kinase-associated protein 2 expression is a potential prognostic biomarker for glioma.

S phase kinase-associated protein 2 (SKP2), a substrate recognizing protein, serves an important role in promoting cell cycle progression through ubiquitination and degradation of cell cycle inhibitors. In the present study, the clinical significance of SKP2 in gliomas was studied; 395 glioma specimens and 20 non-neoplastic tissues were collected and immunohistochemical analysis was performed. χ2 test was used to assess the associations between SKP2 expression and clinical parameters. Overall survival (OS) curves were plotted according to the Kaplan-Meier method. In the tested glioma samples, SKP2 expression was mainly observed in glioblastomas (GBMs). Survival analysis demonstrated that the overall survival time of the high SKP2 expression group was lower compared with the low SKP2 expression group (median OS, 10.04 months vs. 16.50 months; P=0.003). Moreover, SKP2 was independently associated with an unfavorable prognosis in GBMs. In addition, the expression of SKP2 was associated with the expression of phosphorylated retinoblastoma protein and the epidermal growth factor receptor. A combination of SKP2 expression along with isocitrate dehydrogenase 1 (IDH1) mutations and telomerase reverse transcriptase (TERT) promoter mutations was used to classify glioma patients for survival analysis. Patients with low SKP2 expression, IDH1 mutation and wild-type TERT promoter demonstrated the longest survival time. The findings of the present study, indicate that SKP2 is a potential prognostic biomarker in patients with GBMs.

Overexpression of IGFBP2 mRNA predicts poor survival in patients with glioblastoma.

The prognosis of patients with glioblastoma (GBM) is dismal. It has been reported that Insulin-like growth factor (IGF) binding protein 2 (IGFBP2) is associated with the mobility and invasion of tumor cells. We investigated the expression of IGFBP2 mRNA in GBMs and its clinical relevance, using tissue microarrays and RNAscope in situ hybridization in 180 GBMs and 13 normal or edematous tissues. The correlations between the expression and clinical pathological parameters as well as some other biomarkers were analyzed. Overexpression of IGFBP2 mRNA was observed in 23.9% of tumors tested. No expression of IGFBP2 mRNA was detected in normal or edematous tissues. Kaplan-Meier survival analysis showed that the survival time of all the patients with high IGFBP2 tumors had shorter survival than those with low IGFBP2 (P<0.01). Univariate regression and multivariate regression both indicated that the expression of IGFBP2 transcript level was an independent prognostic factor (P=0.008 and 0.007, respectively). Furthermore, expression of IGFBP2 mRNA was related to the occurrence of isocitrate dehydrogenase 1 (IDH1) mutation, high heat shock protein 27 (Hsp27) expression and telomerase reverse transcriptase (TERT) promoter mutation (TERTp+) (P=0.013, 0.015 and 0.016, respectively), and patients with TERTp+/IGFBP2high showed the shortest survival. In conclusion, IGFBP2 mRNA expression status is an independent prognostic biomarker in GBMs, and the combination of IGFBP2 mRNA and TERTp status might serve as a prognostic indicator in patients with GBM.

Overexpression of MCM6 predicts poor survival in patients with glioma.

Minichromosome maintenance proteins (MCMs) play an essential role in DNA replication and other cellular activities. However, their expression levels and clinical value in glioma are unclear. In the present study, we analyzed the relationship between MCM mRNA expression and clinical parameters in 325 gliomas and found that MCM6 presented high expression and was associated with poor survival. Immunohistochemistry analysis of an independent data set of 423 glioma tissues confirmed the overexpression of MCM6 protein, especially in glioblastomas with shorter overall survival. Importantly, a combination of MCM6 overexpression with IDH1 mutation further improved the prediction of the prognosis of glioblastomas. Patients with IDH1 mutation and low MCM6 expression exhibited the longest survival, whereas those with high MCM6 expression and wild-type IDH1 showed the shortest. Collectively, our observation indicates that MCM6 is a novel potential biomarker for predicting poor prognosis of the patients with glioma.

Phosphorylated Hsp27 is mutually exclusive with ATRX loss and the IDH1R132H mutation and may predict better prognosis among glioblastomas without the IDH1 mutation and ATRX loss.

AIM: To identify biomarkers for accurate classification of glioma. PATIENTS AND METHODS: We evaluated the heat shock protein 27 (Hsp27), phosphorylated Hsp27 (p-Hsp27), ATRX and IDH1R132Hproteins using immunohistochemistry in 421 glioma tissues. The χ2 test was used to assess the relationship between molecular alterations and clinico-pathological parameters. Kaplan-Meier survival curves were constructed, and differences were detected by the log-rank test. RESULTS: We found that Hsp27 and p-Hsp27 were mainly expressed in aggressive astrocytic gliomas. However, neither Hsp27 nor p-Hsp27 expression was related to survival time for any grade of glioma. Interestingly, p-Hsp27 was mutually exclusive with ATRX loss (ATRX-) and the IDH1R132H mutation, except for one case of anaplastic astrocytoma. We classified glioblastomas (GBMs) into three subtypes: ATRX-/IDH1R132H, high p-Hsp27 expression (p-Hsp27+) and none of these three markers. ATRX-/IDH1R132Hshowed the longest median survival (19.6 months). The prognostic difference between p-Hsp27+ and none of these three markers was significant (15.0 vs 13.1 months, P=0.045). Moreover, p-Hsp27+ predicted better sensitivity for standard therapy among GBMs without the IDH1 mutation and ATRX loss (26.3 vs 15.5 months, P=0.008). CONCLUSION: p-Hsp27 is a novel biomarker of glioma and might have important clinical value for further classification of patients with wild-type IDH1 and normal ATRX expression, for evaluating prognosis and for guidance for adjuvant therapy.

[Effect of lipid accumulation after spinal cord injury in autofluorescence intensity of injury site].

OBJECTIVE: To investigate the relationship between lipid accumulation and autofluorescence intensity of injury site after spinal cord injury (SCI), and explore whether CuSO4 can eliminate autofluorescence in the injury site after SCI. METHODS: Thirty six Wild Type mice at age of 8 to 12 weeks (weight 18 to 24 g) were randomly divided into normal control group (n=4) and SCI group (n=32). Respectively, 8 mice of SCI group were sacrificed randomly at 1 week, 2 weeks, 4 weeks and 8 weeks after injury. Frozen sections of spinal cord tissue with injury site at the center were made to observe autofluorescence under green channel of fluorescence microscope (Specimens of normal control group were taken from the same segment of the spinal cord, and fixed with 4% paraformaldehyde solution). Oil Red O staining was applied to visualize the lipid accumulation in the injury site, and correlation between lipid accumulation and autofluorescence intensity was analyzed. Furthermore, sections were incubated with CuSO4 buffer to eliminate autofluorescence, and CuSO4 concentration and incubation time was optimized. RESULTS: No obvious autofluorescence or lipid staining was found in normal spinal cord tissue sections. By contrast, autofluorescence appeared in the injury site of spinal cord sections, and the intensity increased with passing time after injury. Oil Red O staining showed that lipid accumulated in the injury site with passing time after injury as well, and the correlation between lipid accumulation and autofluorescence intensity was positive. After incubation with CuSO4 buffer, the autofluorescence in the injury site was significantly reduced, especially after optimizing CuSO4 concentration and incubation time. CONCLUSIONS: Lipid accumulation may play an important role to determine the autofluorescence intensity of injury site after SCI, and the autofluorescence intensity can be used as a simple index for evaluating lipid peroxidation damage. Optimized method of using CuSO4 can significantly reduce the autofluorescence in the injury site after SCI, which will be beneficial to the application of immunofluorescence staining technique in the study of SCI.

Glucagon-like peptide-2 protects impaired intestinal mucosal barriers in obstructive jaundice rats.

AIM: To observe the protective effect of glucagon-like peptide-2 (GLP-2) on the intestinal barrier of rats with obstructive jaundice and determine the possible mechanisms of action involved in the protective effect. METHODS: Thirty-six Sprague-Dawley rats were randomly divided into a sham operation group, an obstructive jaundice group, and a GLP-2 group; each group consisted of 12 rats. The GLP-2 group was treated with GLP-2 after the day of surgery, whereas the other two groups were treated with the same concentration of normal saline. Alanine aminotransferase (ALT), total bilirubin, and endotoxin levels were recorded at 1, 3, 7, 10 and 14 d. Furthermore, on the 14(th) day, body weight, the wet weight of the small intestine, pathological changes of the small intestine and the immunoglobulin A (IgA) expressed by plasma cells located in the small intestinal lamina propria were recorded for each group. RESULTS: In the rat model, jaundice was obvious, and the rats' activity decreased 4-6 d post bile duct ligation. Compared with the sham operation group, the obstructive jaundice group displayed increased yellow staining of abdominal visceral serosa, decreased small intestine wet weight, thinning of the intestinal muscle layer and villi, villous atrophy, uneven height, fusion, partial villous epithelial cell shedding, substantial inflammatory cell infiltration and significantly reduced IgA expression. However, no significant gross changes were noted between the GLP-2 and sham groups. With time, the levels of ALT, endotoxin and bilirubin in the GLP-2 group were significantly increased compared with the sham group (P < 0.01). The increasing levels of the aforementioned markers were more significant in the obstructive jaundice group than in the GLP-2 group (P < 0.01). CONCLUSION: GLP-2 reduces intestinal mucosal injuries in obstructive jaundice rats, which might be attributed to increased intestinal IgA and reduced bilirubin and endotoxin.

[Construction of recombinant plasmid pIRES-GM-CSF-IL-21 and its therapeutic effect on orthotopically transplanted liver cancer in mice].

OBJECTIVE: To construct a recombinant plasmid pIRES-GM-CSF-IL-21, and to investigate its antitumor effect on tumors in the mice. METHODS: Fifty Bal b/c mice were included in this study. Cultured hepatoma H22 cells were inoculated in the left lobe of the liver to develop orthotopically transplanted liver tumor models. The mice with orthotopically transplanted liver tumor were randomly divided into 5 groups (n = 10): (1) Each mouse received injection of recombinant plasmid pIRES-GM-CSF-IL-21; (2) Received injection of plasmid pIRES-GM-CSF; (3) pIRES-IL-21; (4) Received injection of ampty plasmid pIRES (H22/neo group); (5) Received injection of PBS (H22 group) via the tail vein, respectively. Therefore, the anti-tumor effect was induced by both GM-CSF and IL-21, or by either of them alone. The serum levels of IFN-γ and IL-2 were detected by ELISA, and the cytotoxicity of spleen NK and CTL cells were tested by MTT colorimetry. RESULTS: Comparing with the H22 and H22/Neo groups, the tumor weight in the mice of H22/GM-CSF group was (0.603 ± 0.223) g, H22/IL21-treated group (0.583 ± 0.290) g and H22/GM-CSF-IL21-treated group (0.303 ± 0.323) g, significantly lower than that in the H22 group [(1.591 ± 0.280) g] and H22/Neo group [(1.489 ± 0.155) g]. Among them the tumor growth was most significantly inhibited in the H22/GM-CSF-IL-21 group (0.303 ± 0.323) g, compared with that of H22 and H22/neo groups (P < 0.01). But there was no significant difference between the tumor weights of the H22/GM-CSF group and H22/IL-21 group, and between the tumor weights of the H22 and H22/Neo groups (P > 0.05). The levels of IFN-γ and IL-2 in peripheral blood of mouse models treated with H22/GM-CSF-IL-21 were significantly increased than that in the H22/GM-CSF group and H22/IL-21 group (all P < 0.01), but significantly decreased in the H22group and H22/Neo group (P < 0.01). The anti-tumor activity of splenic NK cells and CTLs in the H22/GM-CSF-IL21 group was significantly enhanced (P < 0.01), compared with the significantly decreased in the H22 and H22/Neo groups. CONCLUSIONS: Our results demonstrate apparent antitumor effect of the plasmid pIRES-GM-CSF-IL-21 on the orthotopically transplanted liver tumor in mice. The combination of both pIRES-GM-CSF and IL-21 is more effective than that of pIRES/IL21 or pIRES/GM-CSF treatment alone. In addition, the plasmid pIRES-GM-CSF-IL-21 can also promote the secretion of IFN-γ and IL-2 in vivo, and enhance the cytotoxic activity of splenic NK and CTLs against the transplanted liver tumor.