Objective To reveal the molecular mechanism by which nuclear epidermal growth factor receptor(nEGFR)synergistically regulates the expression of cell migration-inducing protein(CEMIP)by forming a complex with the transcription factor Yin Yang 1(YY1),and to investigate the biological functions of the nEGFR-YY1-CEMIP signaling axis in invasion of hepatocellular carcinoma(HCC).Methods After HCC cells were serum-starved for 24 h,the cells were treated with 100 ng/mL EGF.Thus,the cells were divided into a control group and EGF-treated groups at different time points.Nuclear expression and localization changes of EGFR were detected by Western blotting and immunofluorescence(IF).To investigate the interaction between nEGFR and YY1,their nuclear colocalization and interaction were examined by IF and co-immunoprecipitation(Co-IP),respectively.Transcriptional profiling was performed using RNA sequencing(RNA-seq)to identify differentially expressed genes at the genome-wide level.Combined with Gene Ontology(GO)functional enrichment analysis and transcription factor binding profiles via using the JASPAR database,CEMIP was identified as a candidate target gene.To validate the regulatory mechanism,the following experimental groups were established,Control,EGF,siYY1,and siYY1+EGF.The expression of CEMIP at protein and mRNA levels was detected by Western blotting and RT-qPCR.To elucidate the molecular mechanism of nEGFR/YY1 binding to the CEMIP promoter,the control and EGF-treated groups were established.Chromatin immunoprecipitation followed by quantitative PCR(ChIP-qPCR)was performed to assess the enrichment of nEGFR/YY1 at the CEMIP promoter region.Luciferase reporter assay was conducted following transfection with either wild-type EGFR(EGFR-WT),nuclear localization-deficient mutant(EGFR-dNLS),YY1 overexpression plasmid(YY1-OE),or dominant-negative YY1 mutant(YY1-DN)to evaluate changes in promoter activity.Subsequently,cell migration and invasion capabilities were evaluated using scratch wound healing assay and Transwell assay,while hyaluronic acid(HA)level was quantified by ELISA.The expression of matrix metalloproteinases(MMP2/9)was analyzed via Western blotting to assess the regulatory role of the nEGFR/YY1-CEMIP axis in the migration and invasion of HCC cells.By analyzing the CEMIP expression profiles in HCC patients from National Center for Biotechnology Information(NCBI)public databases,its potential association with tumor metastasis risk was validated.Results Western blotting and IF demonstrated that EGF treatment significantly induced nuclear translocation of EGFR,peaking at 30 min(P<0.001).Co-IP and IF assays indicated both physical interaction and nuclear co-localization between nEGFR and YY1.RNA-seq analysis identified CEMIP as a significantly differentially expressed gene.GO enrichment analysis revealed that CEMIP was significantly enriched in biological processes related to cell invasion promotion.JASPAR prediction identified conserved YY1 potential binding region within the CEMIP promoter region.Western blot and RT-qPCR analyses confirmed that EGF treatment up-regulated CEMIP at both protein and mRNA levels(P<0.05).Notably,YY1 knockdown significantly suppressed CEMIP expression,while exogenous EGF supplementation restored CEMIP level in YY1-deficient cells(P<0.05).ChIP-qPCR analysis demonstrated specific enrichment of the nEGFR/YY1 complex at the CEMIP promoter region,with EGF stimulation significantly enhancing its binding affinity(P<0.001).Luciferase reporter assay confirmed that nEGFR/YY1 robustly enhanced CEMIP promoter activity(P<0.01),while either the EGFR-dNLS or the YY1-DN substantially attenuated this transcriptional activation.Functional phenotyping showed that the nEGFR/YY1-CEMIP axis significantly enhanced the migration and invasion of HCC cells by promoting HA catabolism and up-regulating MMP2/9 expression(P<0.05).Analysis of NCBI datasets revealed that CEMIP expression was significantly up-regulated in HCC tumor tissues than adjacent normal tissues(P<0.001).Moreover,HCC patients with elevated CEMIP expression exhibited higher risk of metastasis(P<0.001).Conclusion nEGFR promotes HCC invasion by forming a transcriptional complex with YY1 to cooperatively activate CEMIP expression.

Objective To reveal the mechanism by which the programmed death-ligand 1(PD-L1)-protein tyrosine phosphatase 1B(PTP1B)-focal adhesion kinase(FAK)signaling axis promotes the progression of hepatocellular carcinoma(HCC)and elucidate its effector functions in HCC.Methods GEPIA database was used to plot a 10-year survival curve for PD-L1 and FAK expression levels in HCC patients.Immunohistochemical(IHC)staining was utilized to analyze the relative expression levels of PD-L1 and FAK phosphorylated at the Y397 site[p-FAK(Y397)]in HCC tissues,and the results were compared to those in the adjacent non-tumor tissues.Subsequently,endogenous PD-L1 expression was detected with Western blotting in HCC cell lines with low(SNU-387)and high(Hep3B)PD-L1 expression levels.After lentivirus-transduced SNU-387PDL1+and Hep3BPDL1-cells were constructed,the effect of high and low expression of PD-L1 on the expression of p-FAK(Y397)with Western blotting.To elucidate the functional mechanism of FAK in HCC,functional rescue experiments were performed by administering a FAK inhibitor to SNU-387PDL1+cells and a FAK activator to Hep3BPDL1-cells,combined with wound healing scratch assay,Transwell invasion assay,EdU proliferation assay,and colony formation assay to evaluate tumor malignant effects.The GENEMANIA database predicted functional interactions between protein tyrosine phosphatase 1B(PTP1B),PD-L1,and FAK.IHC staining was performed to analyze the correlation among PD-L1,PTP1B,and p-FAK(Y397)expression.Co-immunoprecipitation(Co-IP)and indirect immunofluorescence(IF)were applied to validate the interaction between PD-L1 and PTP1B.Western blotting was utilized to confirm the regulatory relationship between PD-L1 and PTP1B.In vitro PTP1B phosphatase activity assay measured the changes in PTP1B activity.Subsequently,Western blotting was used to screen cell lines with high endogenous PTP1B expression(SNU-387)and low endogenous PTP1B expression(Hep3B).Furthermore,Hep3BPTP1B+and SNU-387PTP1B-cell lines were generated,and then p-FAK(Y397)levels were then detected in these modified cell lines,and the aforementioned functional effect assays(migration,invasion,proliferation and colony formation)and rescue experiments were repeated.Furthermore,Western blotting was employed to detect changes in downstream signaling pathways following enhancement or attenuation of p-FAK(Y397)in SNU-387 and Hep3B cells.Results IHC staining revealed a positive correlation between PD-L1 and p-FAK(Y397)expression in HCC tissues(95%CI:1.065～3.801,P<0.01).In SNU-387PDL1+cells,PD-L1 overexpression significantly enhanced phosphorylation at the FAK Y397 site(P<0.01)and increased cell migration,invasion,proliferation,and colony formation capabilities(P<0.01),and these effects could be reversed by FAK inhibitor treatment(P<0.05).Conversely,in Hep3BPDL1-cells,PD-L1 knockdown significantly reduced FAK Y397 phosphorylation(P<0.01)and decreased cell migration,invasion,proliferation,and colony formation abilities(P<0.01),and these effects were restored by FAK activator treatment(P<0.05).IHC staining further showed a negative correlation between PTP1B expression and both PD-L1 and p-FAK(Y397)in HCC tissues(95%CI:1.886～3.514,P<0.05).Co-IP and IF assays confirmed a direct interaction between PD-L1 and PTP1B,with PD-L1 suppressing PTP1B expression level and reducing its activity(P<0.01).In SNU-387PTP1B-cells,PTP1B knockdown significantly increased FAK Y397 phosphorylation(P<0.01)and enhanced cell migration,invasion,proliferation,and colony formation(P<0.01),and these effects were reversed by FAK inhibitor(P<0.05).While in Hep3BPTP1B+cells,PTP1B overexpression significantly decreased FAK Y397 phosphorylation(P<0.01)and reduced cell migration,invasion,proliferation,and colony formation(P<0.01),and those effects were restored by FAK activator treatment(P<0.05).Furthermore,enhanced phosphorylation at the FAK Y397 site in SNU-387 cells activated downstream PI3K/AKT and MEK/ERK signaling pathways(P<0.01),whereas inhibition of FAK(Y397)phosphorylation in Hep3B cells attenuated the activation of these signaling pathways(P<0.01).Conclusion PD-L1 activates FAK by suppressing PTP1B,thereby promoting migration,invasion,and proliferation in HCC.

Objective:To explore the therapeutic mechanism of fractional laser combined with pulsed dye laser and multi-point microinjection of triamcinolone in the treatment of hypertrophic scars in rabbit ears.Methods:In this study, 80 hypertrophic scar models of rabbit ears were made and randomly divided into 4 groups: control group, laser combined group, laser combined multi-point microinjection group, simple traditional injection group, 20 scar models in each group.The control group was not given treatment; the laser combined group was treated with pulsed dye laser first, followed by fractional laser at the same time; the laser combined with multi-point microinjection group was treated with pulsed dye laser and fractional laser first, followed by multi-point microinjection of triamcinolone in the scar immediately after laser treatment; the traditional injection group was injected with triamcinolone in the scar.The gross appearance of hypertrophic scars was observed and recorded in 4 groups. At 7 days and 2 months after treatment, the hypertrophic scars were cut and the histological study were performed by HE staining; the expression of collagen was observed by Masson staining and the volume fraction of collagen was measured; the number of micro-vessels was observed by CD31 staining and the micro-vessel density was measured.One way ANOVA was used to show the difference between groups, LSD posthoc test was used to compare between groups, and Pearson correlation coefficient was used to analyze collagen volume fraction and micro-vessel density.Results:Two months after the treatment, the histological results showed that the scar thickness of the laser combined group and the laser combined with multi-point microinjection group was significantly thinner than that of the control group, the texture of the scar was softened, the red color gradually became lighter, and approached to the color of the surrounding skin. Compared with the control group, the scar in the traditional injection group was thinner, softer and lighter. The scar thickness of laser combined with multi-point microinjection group was thinner than that of laser combined group and simple traditional injection group.Masson staining was used to calculate the collagen volume fraction (CVF): the expression of CVF in hypertrophic scar tissue of each group was reduced. The CVF values of each group at the 7th day and the 2nd month were compared. There was statistically significant difference between the laser combined group, the laser combined multi-point microinjection group and the simple traditional injection group ( P<0.05), but there was no statistical difference between the control group ( P>0.05). The CVF value of each group was further compared at 2 months after treatment, the laser combined multi-point microinjection group 0.05). 2 months after treatment, the MVD values of each group were compared with the result as the laser combined with multi-point microinjection group < laser combined group < simple traditional injection group, but there was no statistical difference between the laser combined with multi-point microinjection group and the laser combined group ( P>0.05). There was statistical difference between the laser combined with multi-point microinjection group and the laser combined group and the simple traditional injection group ( P< 0.05). The Pearson correlation coefficient was r=0.972, which indicated that there was a positive correlation between the collagen content and the number of micro-vessels. Conclusions:The use of fractional laser combined with pulsed dye laser and multi-point microinjection of triamcinolone in the treatment of hypertrophic scars in rabbit ears can evenly reduce the number of fibroblasts, promote the normalization of collagen density and arrangement, and reduce collagen volume fraction and micro-vessel density. The curative effect is better than fractional laser combined with pulse dye laser therapy and simple traditional triamcinolone injection therapy. There is a positive correlation between collagen content and micro-vessel density in hypertrophic scars. Simultaneously inhibiting the proliferating blood vessels can improve the therapeutic effectiveness.

Objective:To explore the therapeutic mechanism of fractional laser combined with pulsed dye laser and multi-point microinjection of triamcinolone in the treatment of hypertrophic scars in rabbit ears.Methods:In this study, 80 hypertrophic scar models of rabbit ears were made and randomly divided into 4 groups: control group, laser combined group, laser combined multi-point microinjection group, simple traditional injection group, 20 scar models in each group.The control group was not given treatment; the laser combined group was treated with pulsed dye laser first, followed by fractional laser at the same time; the laser combined with multi-point microinjection group was treated with pulsed dye laser and fractional laser first, followed by multi-point microinjection of triamcinolone in the scar immediately after laser treatment; the traditional injection group was injected with triamcinolone in the scar.The gross appearance of hypertrophic scars was observed and recorded in 4 groups. At 7 days and 2 months after treatment, the hypertrophic scars were cut and the histological study were performed by HE staining; the expression of collagen was observed by Masson staining and the volume fraction of collagen was measured; the number of micro-vessels was observed by CD31 staining and the micro-vessel density was measured.One way ANOVA was used to show the difference between groups, LSD posthoc test was used to compare between groups, and Pearson correlation coefficient was used to analyze collagen volume fraction and micro-vessel density.Results:Two months after the treatment, the histological results showed that the scar thickness of the laser combined group and the laser combined with multi-point microinjection group was significantly thinner than that of the control group, the texture of the scar was softened, the red color gradually became lighter, and approached to the color of the surrounding skin. Compared with the control group, the scar in the traditional injection group was thinner, softer and lighter. The scar thickness of laser combined with multi-point microinjection group was thinner than that of laser combined group and simple traditional injection group.Masson staining was used to calculate the collagen volume fraction (CVF): the expression of CVF in hypertrophic scar tissue of each group was reduced. The CVF values of each group at the 7th day and the 2nd month were compared. There was statistically significant difference between the laser combined group, the laser combined multi-point microinjection group and the simple traditional injection group ( P<0.05), but there was no statistical difference between the control group ( P>0.05). The CVF value of each group was further compared at 2 months after treatment, the laser combined multi-point microinjection group 0.05). 2 months after treatment, the MVD values of each group were compared with the result as the laser combined with multi-point microinjection group < laser combined group < simple traditional injection group, but there was no statistical difference between the laser combined with multi-point microinjection group and the laser combined group ( P>0.05). There was statistical difference between the laser combined with multi-point microinjection group and the laser combined group and the simple traditional injection group ( P< 0.05). The Pearson correlation coefficient was r=0.972, which indicated that there was a positive correlation between the collagen content and the number of micro-vessels. Conclusions:The use of fractional laser combined with pulsed dye laser and multi-point microinjection of triamcinolone in the treatment of hypertrophic scars in rabbit ears can evenly reduce the number of fibroblasts, promote the normalization of collagen density and arrangement, and reduce collagen volume fraction and micro-vessel density. The curative effect is better than fractional laser combined with pulse dye laser therapy and simple traditional triamcinolone injection therapy. There is a positive correlation between collagen content and micro-vessel density in hypertrophic scars. Simultaneously inhibiting the proliferating blood vessels can improve the therapeutic effectiveness.

Angiostrongylus cantonensis invades the central nervous system (CNS) of humans to induce eosinophilic meningitis and meningoencephalitis and leads to persistent headache, cognitive dysfunction, and ataxic gait. Infected mice (nonpermissive host), admittedly, suffer more serious pathological injuries than rats (permissive host). However, the pathological basis of these manifestations is incompletely elucidated. In this study, the behavioral test, histological and immunohistochemical techniques, and analysis of apoptotic gene expression, especially caspase-3, were conducted. The movement and motor coordination were investigated at week 2 post infection (PI) and week 3 PI in mice and rats, respectively. The cognitive impairs could be found in mice at week 2 PI but not in rats. The plaque-like lesion, perivascular cuffing of inflammatory cells, and dilated vessels within the cerebral cortex and hippocampus were more serious in mice than in rats at week 3 PI. Transcriptomic analysis showed activated extrinsic apoptotic pathway through increased expression of TNFR1 and caspase-8 in mice CNS. Immunohistochemical and double-labeling for NeuN and caspase-3 indicated the dramatically increased expression of caspase-3 in neuron of the cerebral cortex and hippocampus in mice but not in rats. Furthermore, western-blotting results showed high expression of cleaved caspase-3 proteins in mice but relatively low expression in rats. Thus, extrinsic apoptotic pathway participated in neuronal apoptosis might be the pathological basis of distinct behavioral dysfunctions in rodents with A. cantonensis infection. It provides the evidences of a primary molecular mechanism for the behavioral dysfunction and paves the ways to clinical diagnosis and therapy for A. cantonensis infection.
Angiostrongylus cantonensis* ; Angiostrongylus* ; Animals ; Apoptosis* ; Behavior Rating Scale ; Caspase 3 ; Caspase 8 ; Central Nervous System ; Cerebral Cortex ; Diagnosis ; Eosinophils ; Gait ; Gene Expression ; Headache ; Hippocampus ; Humans ; Meningitis ; Meningoencephalitis ; Mice ; Neurons* ; Rats ; Receptors, Tumor Necrosis Factor, Type I ; Rodentia*

Objective To discuss the effects of different drying methods on composition and antioxidative activities of the volatile oil fromCymbopogon citrates; To optimize the best drying method for Cymbopogon citrates. MethodsCymbopogon citrates was dried by drying in the sun, drying in the shade and oven drying at 40℃. Volatile oil was extracted by steam distillation. Chemical constituents in the volatile oil were analyzed by GC-MS and the antioxidative activities were determined by ferric reducing antioxidant power (FRAP method).Results Extraction rate of the volatile oil fromCymbopogon citratesunder the environment of freshness, sun drying, shade drying and oven drying at 40℃ were 0.25%, 1.21%, 1.19% and 1.17%, respectively; after dried by different methods, main constituents and antioxidative activities of the volatile oil fromCymbopogon citrates were basically same. Conclusion Different drying methods have little influence on composition and antioxidative activities of the volatile oil fromCymbopogon citrates. Oven drying at 40℃ was the best way to dryCymbopogon citrates.