OBJECTIVETo verify the existence and significance of calcium/calmodulin dependent serine protein kinase/inhibitors of differentiation 1 (CASK/Id1) pathway in fibroblasts of human keloid.

METHODSImmunofluorescence laser was used to confirm CASK and Id1 protein expression and localization in fibroblasts of the keloid and normal skin. RT-PCR and Western-blot were adopted to analysis the CASK and Id1 expression and differences between keloid and normal skin fibroblasts. The natural combination of CASK and Id1 protein of keloid fibroblasts was tested by immunoprecipitation.

RESULTSCASK and Id1 protein expression were both found in fibroblast cells of keloid and normal skin under normal circumstances. Most of CASK and Id1 were distributed in the cytoplasm and nucleus of fibroblasts. The results of RT-PCR showed that the expression of CASK mRNA in the keloid group was 0.658 +/- 0.024, which was lower than that in the normal control group (1.076 +/- 0.008, t = 11.159, P < 0.05). The expression of Id1 mRNA was 0.497 +/- 0.014, which was higher than that in the normal control group (0.307 +/- 0.017, t = 15.148, P < 0.05). The results of Western-blot showed that the expression level for CASK protein in the keloid group was 0.057 +/- 0.006, which was lower than that in the normal control group (0.168 +/- 0.012, t = 13.524, P < 0.05); the expression of Id1 protein was 0.812 +/- 0.035, which was higher than that in the normal control group (0.368 +/- 0.031, t = 16.356, P < 0.05). The results of immunoprecipitation showed that Id1 could be detected in the CASK precipitate, while CASK also could be detected in the Id1 precipitate. There was a natural binding of CASK and Id1 in keloid fibroblasts.

CONCLUSIONCASK/Id1 signal pathway may be existed and involved in the proliferation of keloid fibroblasts, which is related with the occurrence of keloid.

Cell Proliferation ; genetics ; Cyclin-Dependent Kinase Inhibitor Proteins ; genetics ; metabolism ; Fibroblasts ; metabolism ; Humans ; Inhibitor of Differentiation Protein 1 ; genetics ; metabolism ; Keloid ; metabolism ; pathology ; RNA, Messenger ; metabolism ; Signal Transduction

Objective To investigate the expression of gastric and intestinal phenotypic markers in Siewert typeⅡand Ⅲ early gastroesophageal junction(GEJ) cancer, and to explore its correlation with clinic-pathological features.Methods From April 2010 to July 2015, 53 cases diagnosed as early GEJ cancer were enrolled.The gastric and intestinal phenotypic markers such as mucin5AC(MUC5AC),mucin6(MUC6),mucin2(MUC2),caudal related homeodomain transcription 2(CDX2) and cluster of differentiation 10(CD10) were detected, and then the patients were divided into gastric type, gastrointestinal type, intestinal type and non-classified type according to the results of immunohistochemical staining.Combined with Siewert classification the clinicopathological features were analyzed.Chi square test or Fisher′s exact test was performed for statistical analysis.Results In the cancer tissues of 47 patients with Siewert type Ⅱand Ⅲ early GEJ cancer, the case numbers of positive expression of MUC5AC,MUC6,MUC2, CDX2 and CD10 were 21(44.7%),19(40.4%),31(66.0%),27(57.4%) and 17(36.2%),respectively;the case numbers of gastric type, gastrointestinal type, intestinal type and non-classified type were 11(23.4%),14(29.8%),21(44.7%) and one(2.1%), respectively.The positive expression rates of MUC5AC and MUC6 in Siewert typeⅡwere 55.9%(19/34) and 50.0%(17/34),which were higher than those of Siewert typeⅢ(2/13), and the positive expression rate of MUC2 was 55.9%(19/34), which was lower than that of Siewert typeⅢ(12/13), and the differences were statistically significant (x2=6.240,4.679 and 4.053;all P<0.05).In Siewert typeⅡ, the proportion of intestinal type was 32.4%(11/34), which was lower than that of Siewert typeⅢ(10/13), and the differences were statistically significant (x2=7.142,P=0.010).In patients with Siewert typeⅡand Ⅲ early cancer, males predominated in intestinal type which were mostly well differentiated type with less submucosal carcinoma.The maximum diameter of tumor was less than those of gastric type and gastrointestinal type.In paracancerous mucosal tissues, the incidences of intestinal metaplasia in gastrointestinal type and intestinal type were 11/14 and 81.0%(17/21), which were higher than that of gastric type (3/11);the incidences of atrophy in gastrointestinal type and intestinal type were 12/14 and 85.7%(18/21),which were higher than that of gastric type (4/11),and the differences were statistically significant (Fisher′s exact test,all P<0.05).Conclusions Siewert typeⅡand Ⅲ early GEJ cancer can directly originated not only from gastric mucosa, but also from gastrointestinal and intestinal mucosa.Atrophy and intestinal metaplasia could exist before cancer genesis.

Objective To noninvasively predict isocitrate dehydrogenase(IDH)status of glioma via combining imaging and clini-cal features before surgery,so as to provide basis for individualized clinical treatment decision.Methods A total of 47 patients with glioma confirmed by pathological and molecular genetic tests were included,including 20 with IDH mutant type and 27 with IDH wild type.After diffusion tensor imaging(DTI)scanning,fractional anisotropy(FA)and mean diffusivity(MD)values of tumor paren-chyma were calculated.Combining DTI parameters with MRI morphological features of tumor,blood neutrophil/lymphocyte ratio(NLR)and patient's age,binary logistic regression model was established to effectively predict IDH status of glioma patients before surgery.Results There were significant differences in FAmean/FANAWM,MDmin,NLR,tumor location and age between IDH mutant type and IDH wild type groups(P<0.05).The binary logistic regression model concluding,FAmean/FANAWM,MDmin,cystic degeneration,NLR and age,predicted IDH status of glioma with area under the curve(AUC)of 0.961 and 95%confidence interval(CI)of 0.914-1.00.Conclusion The regression model established via combining DTI,MRI morphological features and blood NLR has great performance in classifying IDH status of glioma,and can help predict IDH status noninvasively before surgery,so as to assist clinical individualized treatment.

Objective To investigate the basic clinical features in 371 cases of colorectal polyps and its relationship with fecal occult blood and carcinoembryonic antigen(CEA).Methods The retrospective analysis was performed on 371 inpatients with colo-rectal polyps.The relationship among gender,number of polyps and polyps anatomical site in different ages of patients was investi-gated,and the relationship between fecal occult blood and CEA with polyp canceration was analyzed by 1.5?3.0 years follow-up. Results Among 371 cases of colorectal polyps,the female patients were gradually increased and single polyp was gradually de-creased along with the age increase;due to different ages,there was the statistically significant difference in the polyp locations (χ2 =9.759,P=0.045);the distribution difference of the patients with polyp canceration among three age groups was statistically significant(χ2 =5.138,4.107,13.153,P<0.05).The cases of fecal occult blood positive and CEA abnormal increase were gradual-ly increased with age increasing(χ2 =15.544,11.959,P<0.01);with the number of polyps increasing,the cases of fecal occult blood positive showed the increasing trend(χ2 =14.043,P=0.001);the canceration rate in colorectal polyp cases of fecal occult blood positive and CEA abnormal increase was significantly higher than that in the cases of fecal occult blood negative and CEA normal range(χ2 =40.165,43.249,all of P< 0.001).Conclusion The fecal occult blood test and CEA detection results have a certain significance to the follow up for preventing colorectal polyps canceration.

Objective To investigate the effect of case management in the perioperative period of patients with gastrointestinal tumor. Methods The quasi-experiment study were took to design the research. A total of 100 patients were enrolled in this study. Patients were divided into control group (n=50) and intervention group (n=50) by the admission time. The control group received routine nursing care, while the intervention group received routine perioperative care and case management during the perioperative period. The uncertainty of illness was compared between the two groups at the time point admission, on discharge and 5 weeks after surgery. The satisfaction during the process of treatment services, postoperative complications and return visit rate were compared too. After 5 weeks follow up, 10 cases in the control group and 4 cases in the intervention group were lost to follow-up, 86 cases were included in data analysis. Results Two groups patients all had medium uncertainty of illness at the three-different time points(preoptrative, 1 day before discharge and 5 weeks after surgery). The scores of the control group were 103.38 ± 14.94, 109.58 ± 16.06 and 113.90 ± 15.95 respectively.Those of the intervention patients were 108.02 ± 15.29,109.89 ± 16.52 and 113.41 ± 14.70.The uncertainty of illness elevated on the 5th week after surgery. Significant statistical differences were identified in the uncertainty of illness within each group (F=14.117, P<0.01). Significant statistical difference was identified in the visit rate, 100% (46/46)vs.87.5% (35/40) (χ2=6.105,P<0.05). The score of preoperative satisfaction of intervention patients (14.41 ± 1.13) was obviously higher than that of control patients (13.58 ± 1.55) (t=-1.89, P<0.05). Whereas, incidence of postoperative complications was not statistically significant between two groups. Conclusions The specificity of gastrointestinal tumor and the complexity of treatment bring about great pressure to patients. As the type of entire journey care model, the case management could integrate the fragmented management of conditional management model. Meantime, it also could effectively improve the medical experience and outpatient visit rate of patients with gastrointestinal tumor.

ObjectiveTo decipher the mechanism of modified Sanpiantang in the treatment of nitroglycerin-induced migraine in rats. MethodSeventy-two Wistar rats were randomized into the control， model （nitroglycerin， 10 mg·kg^-1）， positive control （rizatriptan， 0.89 mg·kg^-1）， and high- （12.96 g·kg^-1）， medium- （6.48 g·kg^-1）， and low-dose （3.24 g·kg^-1） modified Sanpiantang groups. The rat model of migraine was established by intraperitoneal injection of 10 mg·kg^-1 nitroglycerin. The behavioral test was carried out to measure the mechanical pain thresholds （MPT） of the periorbital region and hindpaw after successful modeling. The serum levels of nitric oxide （NO）， tumor necrosis factor-α （TNF-α）， interferon-γ （IFN-γ）， and interleukin-1β （IL-1β） in rats were determined by enzyme-linked immunosorbent assay （ELISA）. Immunohistochemistry （IHC） was employed to determine the expression of inducible nitric oxide synthase （iNOS） in the trigeminal nucleus caudalis （TNC）. Western blot was employed to determine the protein levels of iNOS and phospho-p38 mitogen-activated protein kinase （p-p38 MAPK） in the TNC. Real-time fluorescence quantitative polymerase chain reaction（Real-time PCR） was performed to measure the mRNA levels of iNOS， p38 MAPK， and IL-1β in the TNC. ResultCompared with the control group， the model group showed decreased MPT （P<0.01）， elevated serum levels of NO， TNF-α， IFN-γ， and IL-1β （P<0.01）， and up-regulated expression levels of p38 MAPK， iNOS， and IL-1β in the TNC （P<0.05， P<0.01）. Compared with the model group， modified Sanpiantang increased the MPT （P<0.05）， and the medium-dose group showed the most significant effect （P<0.01）. In addition， modified Sanpiantang down-regulated the mRNA levels of iNOS， p38 MAPK， and IL-1β and the protein levels of p-p38 MAPK and iNOS in the TNC of migraine rats （P<0.05， P<0.01） and lowered the serum levels of NO， TNF-α， IFN-γ， and IL-1β （P<0.05， P<0.01）. ConclusionModified Sanpiantang may treat migraine by down-regulating the expression of pro-inflammatory factors such as NO， TNF-α， IFN-γ， and IL-1β in the p38 MAPK/iNOS signaling pathway to reduce the neurogenic inflammation.

OBJECTIVE To prepar e and characterize danazol （DAZ）-sodium caseinate （SC）composite nanoparticles ，and to study the mechanism of preparing nanoparticles in “bottom-up”technology. METHODS SC was used as a stabilizer for regulating nanoparticles，so that DAZ-SC composite nanoparticles were prepared by anti-solvent precipitation method. The particle size ， Zeta-potential，micro-morphology，stability，encapsulation efficiency ，drug loading and in vitro dissolution rate were characterized. Fluorescence spectra ，IR spectra ，FBRM and other methods were used to analyze the interaction mechanism between DAZ and SC. RESULTS The particle size of DAZ-SC composite nanoparticles was （223.7±12.5）nm，and the polydispersity index was 0.274± 0.012. Zeta-potential was －（17.81±1.63）mV（n＝3）. The stability of nano-suspension was good ，the solid properties of DAZ were greatly improved ，and the dissolution rate was significantly increased. SC was statically quenched under the action of DAZ and the secondary structures of SC were changed. The crystallization process of DAZ was controlled under the action of SC ，and the interaction between DAZ and SC was mainly hydrogen bond and van der Waals force. CONCLUSIONS In this study ，DAZ-SC composite nanoparticles are successfully prepared. In the “bottom-up”technology，the interaction between SC and DAZ caused by hydrogen bond and van der Waals force inhibits the growth and agglomeration of drug crystals .

To investigate the effect and mechanism of metformin on intestinal epithelial barrier injury in ulcerative colitis. A cell model of colitis was established by co-culture of human colon cancer cell line Caco-2 and human monocyte cell line THP-1. The colitis model cells were treated with metformin at concentration of for Flow cytometry was used to detect Caco-2 cell apoptosis, and Western blotting was used to detect the protein expression of tight junction proteins and endoplasmic reticulum stress-related proteins. After metformin treatment, the apoptosis rate of Caco-2 cells was decreased from (14.22±2.34)% to 0.61)% (=3.119, <0.05), and the expression levels of tight junction protein-1 and claudin-1 increased (=5.172 and 3.546, both <0.05). In addition, the expression levels of endoplasmic reticulum-related proteins glucose regulated protein (GRP) 78, C/EBP homologous protein (CHOP) and caspase-12, as well as the phosphorylation level of PRKR-like endoplasmic reticulum kinase (PERK) and eukaryotic translation initiation factor 2α (eIF2α) decreased (all <0.05). Metformin may alleviate the intestinal epithelial barrier damage in colitis by reducing intestinal epithelial cell apoptosis and increasing the expression of tight junction proteins, which may be associated with the inhibition of endoplasmic reticulum stress-induced apoptotic pathway.
Apoptosis ; Caco-2 Cells ; Colitis, Ulcerative ; Endoplasmic Reticulum Stress ; Humans ; Metformin/pharmacology*

ObjectiveTo investigate the effect of Yiqi Tongxin formula （YQTM） on liver inflammation in apolipoprotein E^-∕- （ApoE^-∕-） mice by regulating the nuclear transcription factor-κB （NF-κB）/NOD-like receptor protein 3 （NLRP3） signaling pathway. MethodForty ApoE^-∕- mice were randomly divided into a model group， an atorvastatin group （positive drug group）， and low-， medium-， and high-dose YQTM groups （0.39， 0.78， 1.56 g·kg^-1）. Each drug administration group was given the corresponding concentration of the drug by gavage on the basis of high-fat feeding for 12 consecutive weeks. Eight C57BL/6J mice were used as a blank group and fed with normal chow. After 12 weeks， oil red O staining and Masson staining were used to observe the aortic lesions in mice and to determine whether the modeling was successful. Oil red O staining was used to observe the lipidosis in the livers of mice. Hematoxylin-eosin （HE） staining was used to observe the tissue lesions in the livers of mice. Masson staining was used to observe the distribution of collagen fibers in the livers of mice. Enzyme markers were used to detect the total cholesterol （TC）， triglyceride （TG）， low-density lipoprotein （LDL-C）， aspartate aminotransferase （AST）， and alanine aminotransferase （ALT） in mouse serum， as well as total cholesterol （TC） and triglyceride （TG） in the liver. Interleukin-1β （IL-1β） and IL-18 were detected in mouse liver by enzyme-linked immunosorbent assay （ELISA）. Immunohistochemistry （IHC） was utilized to observe the expression regions of NF-κB and NLRP3 in the livers of mice. Western blot was employed to detect the protein expression levels of NF-κB， NF-κB inhibitory protein （IκB）， IκB kinase β （IKKβ）， phosphorylated NF-κB （p-NF-κB）， phosphorylated IκB （p-IκB）， phosphorylated IKK β （p-IKKβ）， NLRP3， and Caspase-1 in the livers of mice. ResultCompared with the blank group， the model group showed severe aortic lipidosis， and the intracellular fat droplets in the livers aggregated in large quantities. The cytoplasm was filled with fat vacuoles（P<0.01）. The serum levels of TG， TC， LDL-C， AST， and ALT were significantly elevated in the mice（P<0.01）. TG and TC levels were elevated in the liver（P<0.01）. The levels of IL-1β and IL-18 in liver tissue， as well as the protein expression levels of NF-κB， IκB， IKKβ， p-NF-κB， p-IκB， p-IKKβ， NLRP3， and Caspase-1 in the liver were significantly elevated（P<0.01）. Compared with the model group， the aortic arch plaques of mice in each YQTM group were attenuated， and the fat aggregation in the liver was reduced. The inflammatory cell infiltration was alleviated（P<0.05，P<0.01）. The serum levels of TG， TC， LDL-C， AST， and ALT were significantly reduced（P<0.05，P<0.01）. TG and TC levels in the liver were reduced. The IL-1β and IL-18 levels in liver tissue， as well as protein expression levels of NF-κB， IκB， IKKβ， p-NF-κB， p-IκB， p-IKKβ， NLRP3， and Caspase-1 in the liver were significantly reduced（P<0.05，P<0.01）. ConclusionThe intervention mechanism of YQTM on liver inflammation in ApoE^-∕- mice may be related to the down-regulation of the NF-κB/NLRP3 signaling pathway.

Objective: To investigate the effects of interferon inducible protein 16 (IFI16), a cytosolic DNA sensor, on the expression of human T-cell leukemia virus type 1 (HTLV-1) proteins and pro-inflammatory cytokines in adult HTLV-1-positive T cells. Methods: IFI16 expression in different HTLV-1-positive T cell lines was detected by immunoblot assay. Specific siRNA targeting the IFI16 gene was constructed and the gene silencing efficiency was detected by immunoblot assay. Expression of HTLV-1 Tax protein at mRNA and protein levels was respectively detected by real-time PCR and immunoblot assay after knocking down the expression of IFI16 in HTLV-1-positive T cells with siRNA. Expression of interferon (IFN)-α, IFN-γ, tumor necrosis factor (TNF)-α, Tax and Env were detected by real-time PCR. Results: Compared with the HTLV-1-negative T cell line Jurkat, IFI16 expression was enhanced in the HTLV-1-positive T cell lines MT2, MT4 and C8166. Tax expression was increased, while that of IFN-α, IFN-γ and TNF-α was decreased in MT2 and MT4 cells after silencing the expression of IFI16 with siRNA. Conclusions IFI16 expression was increased in HTLV-1-positive MT2 and MT4 cells. Meanwhile, IFI16 promoted the production of interferon and pro-inflammatory cytokines and inhibited the expression of HTLV-1 proteins.