BACKGROUND: Myelin sheath related inhibitors have been found to have great impact on microenvironment of axon regeneration. Traditional Chinese medicine is gradual y becoming a research hotspot on improving microenvironment of nerve regeneration with its advantage on multiple factors and targets.
OBJECTIVE: To clarify the effect of Jisuikang on Nogo-66 receptor NgR expression after spinal cord injury.
METHODS: 144 rats were randomly divided into six groups: sham surgery group, model group, prednisone group, high-, moderate- and low-dose Jisuikang groups (n=24). Animal models of spinal cord injury were established by the modified Allen’s method in the later five groups. Rats in the prednisone group were daily given 0.06 g/kg prednisone acetate by lavage, once a day. Rats in the high-, moderate- and low-dose Jisuikang groups were daily intragastrically given 12.5, 25 and 50 g/kg Jisuikang, once a day. Rats in the sham surgery and model groups were intragastrically daily given 20 mL of saline, once a day. Rats in each group were administered drugs until death.
RESULTS AND CONCLUSION: Compared with the model group, NgR protein and mRNA expression levels were significantly lower in the prednisone, moderate-and low-dose Jisuikang groups. These data suggested that Jisuikang can improve the recovery of neurological function after spinal cord injury and effectively inhibit NgR protein expression at the site of injury so as to suppress the microenvironment factors harmful to nerve regeneration and further improve the microenvironment of nerve regeneration. Subject headings: Drugs, Chinese Herbal; Spinal Cord Injuries; Axons; Tissue Engineering

Objective To evaluate the role of spinal Toll-like receptor 4 (TLR4) signaling pathway in the development of inflammatory pain in rats.Methods Thirty-six adult male Sprague-Dawley rats were divided into 3 groups (n=12 each) using a random number table:control group,inflammatory pain group and TLR4 signaling pathway inhibitor epigallocatechin gallate (EGCG) group (EGCG group).Inflammatory pain was induced by injecting 50 μl of complete Freund's adjuvant (CFA) into the ankle joint cavity of the left hindpaw of rats anesthetized with isoflurane.At 1-3 days after injection of CFA,EGCG 30 μg was intrathecally injected once a day in group EGCG.At 1,3 (30 min after intrathecal injection),5and 7 days after injection of CFA,the mechanical paw withdrawal threshold (MWT) and thermal paw withdrawal latency (TWL) were measured.The ipsilateral L4.5 segments of the spinal cord were removed at 3 days after CFA injcction for determination of TLR4 expression (by Western blot) and contents of tumor necrosis factor-alpha (TNF-oα),interleukin-6 (IL-6) and IL-1β in the spinal dorsal horn (by enzymelinked immunosorbent assay).Results Compared with control group,the MWT was significantly decreased and the TWL was shortened at each time point after injection of CFA,the expression of TLR4 in the spinal dorsal horn was up-regulated,and contents of TNF-α,IL-6 and IL-1β in the spinal dorsal horn were increased in inflammatory pain group (P＜ 0.05),and no significant change was found in the parameters mentioned above in EGCG group (P＞0.05).Compared with inflammatory pain group,the MWT was significantly increased and the TWL was prolonged at each time point after injection of CFA,the expression of TLR4 in the spinal dorsal horn was down-regulated,and contents of TNF-α,IL-6 and IL-1β in the spinal dorsal horn were decreased in EGCG group (P＜0.05).Conclusion Spinal TLR4 signaling pathway is involved in the development of inflammatory pain in rats.

Purpose To analyze the correlations between PD-L1 expression and clinicopathological factors and their prognostic values in esophageal squamous cell carcinoma (ESCC) patients.Methods PD-L1 expression in the primary tumors from 253 patients with ESCC was evaluated using tissue microarray and immunohistochemistry (IHC).PD-L1 positivity was defined as positive staining of 1％ and 5％ tumor cells.Survival curves were constructed by using the Kaplan-Meier method.Univariate and multivariate Cox proportional hazard regression models were performed to identify associations with outcome variables.Results Overall,tumoral PD-L1 expression was potentially associated with favorable DFS and OS.When the patients were stratified into stage Ⅰ + Ⅱ (60.9％,154/253) and stage Ⅲ + Ⅳa (39.1％,99/253),the prognostic role was not consistent.In patients with stage Ⅰ + Ⅱ disease,tumoral PD-L1 expression was associated with better DFS and OS upon multivariate analysis (1％ as the cutoff:P =0.046 and 0.021,5％ as the cutoff:P=0.011 and0.004).However,PD-L1 expression was not correlated with prognosis in patients with stage Ⅲ + Ⅳa disease (1％ as the cutoff:P =0.586 and 0.682,5％ as the cutoff:P =0.807 and 0.620).Conclusion The prognostic role of tumoral PDL expression is variable in different stages of ESCC,and tumoral PDL expression is an independent favorable predictor in ESCC patients with Stage Ⅰ-Ⅱ disease,but not in stage Ⅲ-Ⅳa or lymph node metastasis.

Objective:To standardize the naming of organ at risk (OAR) and target area during cervical cancer radiotherapy based on AAPM TG-263.Methods:After self-programming of Matlab software to implement the reading and resolution of radiotherapy structure files, the naming of each substructure was automatically output, recorded and restored. After naming all substructures, the structure names were classified by keywords. According to TG-263, a standard naming conversion table of OAR and target area was developed, and the classified structure names were standardized through procedures. Finally, the standardized named radiotherapy structure files were output and imported into the treatment planning system (TPS).Results:The radiation structure of 144 patients with cervical cancer was successfully transformed and displayed correctly in TPS. Before the transformation, the naming of OAR and target area lacked of uniform norms and standards, and the naming of the same structure significantly differed. After the transformation, 43 naming methods of OAR and 74 naming methods of the target area were unified into 20 and 8 naming methods, which were more convenient for staff understanding and communication.Conclusion:The standardization of cervical cancer radiotherapy structure naming can reduce the inconsistency of naming and provide reference for the standardized naming of pelvic tumors.

Objective:To explore the effect of D393E mutation on its intracerebral neurovirulence in mice after infectious clone of the chimeric Japanese encephalitis virus (JEV)/Zika virus (ZIKV) containing envelope protein D393E mutant was built and the virus was rescued.Methods:The full-length cDNA plasmid of JEV/ZIKV chimeric virus containing D393E mutation in the envelope protein was built by SOE PCR and gene cloning technology. The linearized fragment of the plasmid was used for in vitro transcription to obtain viral RNA, followed by electrotransfection into BHK-21 cells to rescue the mutant virus JEV/ZIKV(D393E). The plaque size and growth characteristics of JEV/ZIKV (D393E) and JEV/ZIKV were compared after BHK-21 cells were inoculated with the viruses. The intracerebral neurovirulence of the viruses was calculated after the viruses were inoculated in mice intracerebrally (i.c.).Results:Restriction enzyme digestion evaluation confirmed that the infectious clone of mutant virus was correctly built. The plaque assay demonstrated that the plaque diameter of JEV/ZIKV (D393E) was about 0.7±0.2 mm, which was smaller than that of JEV/ZIKV (1.3±0.2 mm). The LD 50 value of the mutant was 31.51 PFU when injected in mice via i. c. route, which was greater than 2.21 PFU of JEV/ZIKV. Conclusions:The D393E mutation of ZIKV envelope protein in JEV/ZIKA chimeric virus decreases the neurovirulence of the virus in mice.

Objective To investigate the impact and mechanism of Liraglutide on autophagy and apoptosis of human podocyte induced by high glucose.Methods Human podocytes were cultured in vitro,and grouped into normal control group(NC group),high glucose group(HG group),25 nmol/L Liraglutide group(HG+Lir 25 group),50 nmol/L Liraglutide group(HG+Lir 50 group),Liraglutide+LY294002 group(HG+Lir+LY294002 group),and Liraglutide+3-MA group(HG+Lir+3-MA group).The podocyte activity was detected by CCK-8.The apoptosis rate and morphology of podocytes were detected by flow cytometry and Hoechst 33342 staining.The expression of autophagic body and autophagic marker LC3 protein in podocyteswas observed by transmission electron microscopy and immunofluorescence staining.Western blot was applied to detect the expression of apoptosis,autophagy and phosphoinositol 3-kinase(PI3K)/protein kinase B(Akt)/mammalian target of rapamycin(mTOR)pathway related proteins in podocytes.Results Compared with NC group,the activity of podocytes and the expression of Bcl-2,Bcl-2/Bax,LC3 Ⅱ/LC3 Ⅰ,p-PI3K/PI3K,p-Akt/Akt proteins in HG group were decreased(P＜0.05),while the apoptosis rate and the expression of Bax,p62,p-mTOR/mTOR proteins were increased in HG group(P＜0.05).There were many podocytes with pyknotic nuclei,the number of autophagic bodies and the number of green fluorescent spots of LC3 protein were decreased in HG group.Compared with HG group,the activity of podocyte increased,and the expression of Bcl-2,Bcl-2/Bax,LC3 Ⅱ/LC3 Ⅰ,p-PI3K/PI3K,p-Akt/Akt protein increased(P＜0.05),while the apoptosis rate and the expression of Bax,p62,p-mTOR/mTOR protein decreased(P＜0.05)in HG+Lir 25 group and HG+Lir 50 group.The number of podocytes with karyopyknosis was reduced,the number of autophagosomes and the number of green fluorescent spots of LC3 protein were increased in HG+Lir 25 group and HG+Lir 50 group,and the above changes indexes were more obvious in the HG+Lir 50 group group.Compared with HG+Lir 50 group,PI3K/Akt/mTOR pathway could be regulated,and reduce the improvement of Liraglutide on podocyte viability,apoptosis and autophagy induced by high glucose in HG+Lir+LY294002 group and HG+Lir+3-MA group.Conclusion Liraglutide may promote the autophagy of human podocyte induced by high glucose and inhibit its apoptosis through PI3K/Akt/mTOR pathway.

Currently, killed-virus and modified-live porcine reproductive and respiratory syndrome virus (PRRSV) vaccines are used to control porcine reproductive and respiratory syndrome. However, both types of vaccines have inherent drawbacks; accordingly, the development of novel PRRSV vaccines is urgently needed. Previous studies have suggested that yeast possesses adjuvant activities, and it has been used as an expression vehicle to elicit immune responses to foreign antigens. In this report, recombinant Kluyveromyces lactis expressing GP5 of HP-PRRSV (Yeast-GP5) was generated and immune responses to this construct were analyzed in mice. Intestinal mucosal PRRSV-specific sIgA antibody and higher levels of IFN-gamma in spleen CD4+ and CD8+ T cells were induced by oral administration of Yeast-GP5. Additionally, Yeast-GP5 administered subcutaneously evoked vigorous cell-mediated immunity, and PRRSV-specific lymphocyte proliferation and IFN-gamma secretion were detected in the splenocytes of mice. These results suggest that Yeast-GP5 has the potential for use as a vaccine for PRRSV in the future.
Administration, Oral ; Animals ; Antibodies, Viral/*immunology ; B-Lymphocytes/immunology/virology ; Enzyme-Linked Immunosorbent Assay ; *Immunity, Cellular ; *Immunity, Mucosal ; Injections, Subcutaneous ; Kluyveromyces/genetics ; Mice ; Mice, Inbred BALB C ; Porcine respiratory and reproductive syndrome virus/*immunology ; Recombinant Proteins/genetics/immunology ; T-Lymphocytes/immunology/virology ; Viral Envelope Proteins/*genetics/*immunology ; Viral Vaccines/administration & dosage/*pharmacology

OBJECTIVETo explore the pathogenetic mechanism of a family affected with Waardenburg syndrome.

METHODSClinical data of the family was collected. Potential mutation of the MITF, SOX10 and SNAI2 genes were screened. Plasmids for wild type (WT) and mutant MITF proteins were constructed to determine their exogenous expression and subcellular distribution by Western blotting and immunofluorescence assay, respectively.

RESULTSA heterozygous c.763C>T (p.R255X) mutation was detected in exon 8 of the MITF gene in the proband and all other patients from the family. No pathological mutation of the SOX10 and SNAI2 genes was detected. The DNA sequences of plasmids of MITFand mutant MITFwere confirmed. Both proteins were detected with the expected size. WT MITF protein only localized in the nucleus, whereas R255X protein showed aberrant localization in the nucleus as well as the cytoplasm.

CONCLUSIONThe c.763C>T mutation of the MITF gene probably underlies the disease in this family. The mutation can affect the subcellular distribution of MITF proteins in vitro, which may shed light on the molecular mechanism of Waardenburg syndrome caused by mutations of the MITF gene.

Adolescent ; Adult ; Case-Control Studies ; Child ; Child, Preschool ; Female ; Humans ; Male ; Middle Aged ; Mutation ; genetics ; Pedigree ; Waardenburg Syndrome ; genetics ; Young Adult

Objective: To investigate the effects of short chain fatty acid (SCFA) on barrier disruption of human intestinal epithelial cell induced by endotoxin/lipopolysaccharide (LPS) and the related mechanism. Methods: The human intestinal epithelial cell line Caco-2 was used to reproduce monolayer-cells. Cells were divided into control group, LPS group, and SCFA+ LPS group according to the random number table. Cells in control group were only routinely cultured with DMEM medium. Cells in LPS group were cultured with DMEM medium and LPS with final mass concentration of 10 μg/mL. Cells in SCFA+ LPS group were cultured with DMEM medium, LPS and SCFA (consisting of 0.5 mmol/L acetate, 0.01 mmol/L propionate, and 0.01 mmol/L butyrate) with final mass concentration of 10 μg/mL. At post culture hour (PCH) 0, 1, 2, 6, 12, and 24, transepithelial electrical resistance (TER) of cells was determined with an ohmmeter, with sample number of 72. Another portion of cells were divided and treated as above, and then Western blotting was employed to detect the protein expressions of zonula occludens 1 (ZO-1), occludin, and claudin-1 at PCH 24, with sample number of 6. Another portion of cells were divided and treated as above and then immunofluorescence was used to observe cellular morphology and distribution of ZO-1. Data were processed with analysis of variance of factorial design, one-way analysis of variance, least-significant difference test, and Bonferroni correction. Results: (1) Compared with that in control group, TER of cells in LPS group was significantly reduced from PCH 1 to 24 (P<0.01), while TER of cells in SCFA+ LPS group showed no obvious change (P>0.05). TER of cells in SCFA+ LPS group was significantly higher than that in LPS group from PCH 1 to 24 (P<0.01). (2) Compared with the protein expressions of ZO-1, occludin, and claudin-1 of cells in control group (1.25±0.10, 1.17±0.04, and 1.24±0.20), those of cells in LPS group (0.74±0.23, 0.76±0.11, and 0.77±0.11) at PCH 24 were significantly decreased (P<0.05), while those of cells in SCFA+ LPS group (1.23±0.46, 1.05±0.09, and 1.01±0.13) showed no significant differences (P>0.05). Protein expressions of occludin and claudin-1 of cells in SCFA+ LPS group were significantly higher than those in LPS group at PCH 24 (P<0.05). Protein expression of ZO-1 of cells in SCFA+ LPS group was higher than that in LPS group at PCH 24 with no significant difference (P>0.05). (3) At PCH 24, cells in control group were compact in arrangement with pebble-like appearance, and ZO-1 was distributed smoothly and continuously along the cell membrane. In LPS group, cells were sparse in arrangement with change in appearance, and ZO-1 was distributed uncontinuously along the cell membrane with curls and breaks. In SCFA+ LPS group, the appearance of cells and distribution of ZO-1 were remarkably ameliorated compared with those in LPS group. Conclusions SCFA can alleviate the barrier disruption of human intestinal epithelial cell induced by LPS through interdicting the abnormal distribution of ZO-1 and decrease of TER and tight junction proteins′ expressions.

Objective:To find the biomarkers that accurately predict the survival of patients with esophageal squamous cell carcinoma (ESCC).Methods:The immune related genes that were significantly related to the overall survival (OS) of patients with ESCC were screened from The Cancer Genome Atlas (TCGA) database to construct a prognostic risk score model. The prognoses of the high-risk and low-risk groups were compared by Kaplan-Meier method. The accuracy of the model was evaluated by the receiver operating characteristic (ROC) curve. Tumor tissue samples of 83 patients with pathological diagnosis of ESCC were collected from Anyang Cancer Hospital for external verification. Cox regression analysis was used to comprehensively evaluate the effects of prognostic risk score and various clinical characteristics on OS of patients with ESCC.Results:Seven immune-related genes that were significantly related to survival prognosis were selected from the TCGA database and included in the prognostic risk score model, which were S100A12, SLC40A1, FABP9, TNFSF10, IGHA2, IL1F10, and STC2. The 1- and 2-year survival rates of the low-risk group (40 cases) were 94.3% and 82.5%, respectively, while those of the high-risk group (40 cases) were 75.9% and 32.9%, respectively.The prognosis of the high-risk group was worse than that of the low-risk group ( P<0.001). The 83 external validation samples obtained consistent results by using the prognostic risk score model. The prognostic risk score was positively correlated with the content of CD4 + T lymphocytes in ESCC ( rs=0.259, P=0.020), but not correlated with the content of B lymphocytes, CD8 + T lymphocytes, neutrophils, macrophages or dendritic cells ( P>0.05). Conclusions:S100A12, SLC40A1, FABP9, TNFSF10, IGHA2, IL1F10, and STC2 were risk genes significantly associated with OS of patients with ESCC. The prognostic risk score was an independent prognostic factor for the OS of patients with ESCC, and it was correlated with the content of CD4 + T lymphocytes in ESCC tissue.