Fibroblasts overpressing WNT2b cause impairment of intestinal mucosal barrier / 南方医科大学学报

OBJECTIVE@#To investigate the mechanism by which fibroblasts with high WNT2b expression causes intestinal mucosa barrier disruption and promote the progression of inflammatory bowel disease (IBD).@*METHODS@#Caco-2 cells were treated with 20% fibroblast conditioned medium or co-cultured with fibroblasts highly expressing WNT2b, with the cells without treatment with the conditioned medium and cells co-cultured with wild-type fibroblasts as the control groups. The changes in barrier permeability of Caco-2 cells were assessed by measuring transmembrane resistance and Lucifer Yellow permeability. In Caco-2 cells co-cultured with WNT2b-overexpressing or control intestinal fibroblasts, nuclear entry of β-catenin was detected with immunofluorescence assay, and the expressions of tight junction proteins ZO-1 and E-cadherin were detected with Western blotting. In a C57 mouse model of dextran sulfate sodium (DSS)-induced IBD-like enteritis, the therapeutic effect of intraperitoneal injection of salinomycin (5 mg/kg, an inhibitor of WNT/β-catenin signaling pathway) was evaluated by observing the changes in intestinal inflammation and detecting the expressions of tight junction proteins.@*RESULTS@#In the coculture system, WNT2b overexpression in the fibroblasts significantly promoted nuclear entry of β-catenin (P < 0.01) and decreased the expressions of tight junction proteins in Caco-2 cells; knockdown of FZD4 expression in Caco-2 cells obviously reversed this effect. In DSS-treated mice, salinomycin treatment significantly reduced intestinal inflammation and increased the expressions of tight junction proteins in the intestinal mucosa.@*CONCLUSION@#Intestinal fibroblasts overexpressing WNT2b causes impairment of intestinal mucosal barrier function and can be a potential target for treatment of IBD.

Mechanism of intestinal injury induced by WNT2B high-expressed fibroblasts in Crohn's disease / 中华儿科杂志

Objective: To explore the mechanism of intestinal tissue damage induced by macrophages activated by WNT2B high-expressed fibroblasts. Methods: This study involved biological information analysis, pathological tissue research and cell experimental research. The biological information of the colon tissue from the children with inflammatory bowel disease in previous study was analyzed again with single-cell sequencing. The pathological tissues were collected by colonoscopy from 10 children with Crohn's disease treated in the Department of Gastroenterology of Guangzhou Women and Children's Medical Center from July 2022 to September 2022. According to the findings of colonoscopy, tissues with obvious inflammation or ulceration were classified as the inflammatory group, while tissues with slight inflammation and no ulceration were classified as the non-inflammatory group. HE staining was performed to observe the pathological changes of the colon tissues. Macrophage infiltration and CXCL12 expression were detected by immunofluorescence. In terms of cell experiments, fibroblasts transfected with WNT2B plasmid or empty plasmid were co-cultured with salinomycin treated or non-treated macrophages, respectively; the expression of proteins through Wnt classical pathway were detected by western blotting. Macrophages treated with SKL2001 were used as the experimental group, and those with phosphate buffer as the control group. The expression and secretion of CXCL12 in macrophages were detected by quantitative Real-time PCR and enzyme-linked immunosorbent assay (ELISA). T-test or rank sum test were used for the comparison between groups. Results: Single-cell sequencing analysis suggested that macrophages were the main cells in inflammatory bowel disease colon tissue, and there was interaction between WNT2B high-expressed fibroblasts and macrophages. HE staining of the 10 patients ((9.3±3.8) years old, 7 males and 3 females) showed that the pathological score of colon tissue in the inflammatory group was higher than that in the non-inflammatory group (4 (3, 4) vs. 2 (1, 2) points, Z=3.05, P=0.002). Tissue immunofluorescence indicated that the number of infiltrating macrophages in the inflammatory group was significantly higher than that in the non-inflammatory group under high power field of view (72.8±10.4 vs.8.4±3.5, t=25.10, P<0.001), as well as the number of cells expressing CXCL12 (14.0±3.5 vs. 4.7±1.9, t=14.68, P<0.001). In cell experiments, western blotting suggested an elevated level of glycogen synthase kinase-3β phosphorylation in macrophages co-cultured with fibroblast transfected with WNT2B plasmid, and salinmycin could reverse this change. Real-time PCR suggested that the transcription level of CXCL12 in the experimental group was higher than that in the control group (6.42±0.04 vs. 1.00±0.03, t=183.00, P<0.001), as well as the expression and secretion of CXCL12 by ELISA ((465±34) vs. (77±9) ng/L, t=13.21, P=0.006). Conclusion: WNT2B high-expressed fibroblasts can secrete WNT2B protein and activate the Wnt classical signaling pathway thus enhancing the expression and secretion of CXCL12 in macrophages, inducing the development of intestinal inflammation of Crohn's disease.

Relationship between prognosis and different surgical treatments of zygomatic defects: A retrospective study / 北京大学学报(医学版)

OBJECTIVE@#To evaluate the effect and summarize the characteristics of different treatment methods in repairing zygomatic defect.@*METHODS@#A total of 37 patients with zygomatic defect were reviewed in the Department of Oral and Maxillofacial Surgery, Peking University School and Hospital of Stomatology from August 2012 to August 2019. According to the anatomical scope of defect, the zygomatic defects were divided into four categories: Class 0, the defect did not involve changes in zygomatic structure or continuity, only deficiency in thickness or projection; Class Ⅰ, defect was located in the zygomatic body or involved only one process; Class Ⅱ, a single defect involved two processes; Class Ⅲa, referred to a single defect involving three processes and above; Class Ⅲb, referred to zygomatic defects associated with large maxillary defects. The etiology, defect time, defect size and characteristics of zygomatic defects, the repair and reconstruction methods, and postoperative complications were collected and analyzed. Postoperative computed tomography (CT) data were collected to evaluate the outcome of zygomatic protrusion. Chromatographic analysis was used to assess the postoperative stability.@*RESULTS@#Among the causes of defects, 25 cases (67.57%) were caused by trauma, and 11 cases (29.73%) were of surgical defects following tumor resection. We performed autologous bone grafts in 19 cases, 6 cases underwent vascularized tissue flap, 5 cases underwent external implants alone, and 7 cases underwent vascularized tissue flap combined with external implants. After the recovery of the affected side, the average difference of the zygomatic projection between the navigation group and the non-navigation group was 0.45 mm (0.20-2.50 mm) and 1.60 mm (0.10-2.90 mm), with a significant difference (P=0.045). Two patients repaired with titanium mesh combined with anterolateral thigh flap had obvious deformation or fracture of titanium mesh; 2 patients with customized casting prosthesis had infection after surgery and fetched out the prosthesis finally.@*CONCLUSION@#Autologous free grafts or alloplastic materials may be used in cases without significant structural changes. Pedicle skull flap or vascularized bone tissue flap is recommended for zygomatic bone defects with bone pillar destruction, chronic inflammation, oral and nasal communication or significant soft tissue insufficiency. Titanium mesh can be used to repair a large defect of zygomatic bone, and it is suggested to combine with vascularized bone flap transplantation.

Efficacy and safety analysis of anlotinib in treatment of advanced gastric cancer / 肿瘤研究与临床

Objective:To investigate the efficacy and safety of anlotinib in the treatment of advanced gastric cancer.Methods:Eighty patients with advanced gastric cancer in Longhua District Central Hospital of Shenzhen City from February 2015 to May 2016 were selected. The patients were randomly divided into anlotinib group (anlotinib 12 mg) and placebo group by random number table method. The anlotinib or placebo was given once a day for two weeks and discontinued for one week, and three weeks were a course of treatment. The relief situation, total survival time as well as adverse reactions after treatment of all patients were compared between the two groups.Results:The remission rate in the anlotinib group was higher than that in the placebo group [61.6% (37/60) vs. 5.0% (1/20)], and the difference was statistically significant ( χ2 = 19.315, P < 0.05). The overall survival time of the anlotinib group was longer than that of the placebo group [(22.8±1.0) months vs. (10.3±0.9) months], and the difference was statistically significant ( P < 0.01). The adverse reactions mainly included hypertension, diarrhea, nausea and vomiting, liver damage, etc. The adverse reactions were mild, and no drug-related deaths occurred. There was no statistically significant difference in the incidence of adverse reactions between the two groups (all P > 0.05). Conclusion:Anlotinib is effective and safe in the treatment of advanced gastric cancer.

Efficacy of early cold budesonide atomization inhalation on postoperative sore throat following double-lu-men endotracheal intubation / 实用医学杂志

Objective To evaluate the efficacy of early controlled cold atomization inhalation of budesonide in the treatment of post-operative sore throat (POST) after double-lumen endotracheal intubation. Methods A total of 105 ASAⅠ~Ⅲpatients having POST after thoracic surgery with double-lumen endotracheal intubation were randomly divided into three groups(n = 35 each). The patients in the control group were treated with atomization inhalation of 12 mL 20℃saline(Group C)and those in the observation groups were treated with 10 mL 20℃saline plus 2 mL(1 mg)budesonide(Group R)or 10 mL 0℃saline plus 2 mL(1 mg)budesonide (Group L)for 15 minutes immediately after extubation. The 4-score scale was used to evaluate sore throat,dry throat,hoarseness and swallowing difficulty 1,2,6 and 24 h after the extubation and QoR-40 scale to assess post-operative recovery at 24 h. Results The scores of sore throat and dry throat were significant lower in group L than those in group C(P < 0.05)at 1,2 and 24 h and the score of swallowing difficulty(dysphagia)was also signifi-cant relieved at 1,6 and 24 h after the extubation in group L. Furthermore,the score of sore throat was significant lower in group L than that in group R(P<0.05)at 1 h. There were no significant differences of hoarseness in three groups(P > 0.05). The total score of QoR-40 scale was the significantly highest in group L than that in group C and group R(P<0.05)24 h postoperatively. Conclusion Cold atomization inhalation of budesonide immediately after the extubation of double-lumen endotracheal can alleviate POST and bring more benefits to patients which help to enhance the recovery after throracic surgery.

Gingerol activates noxious cold ion channel TRPA1 in gastrointestinal tract / 中国天然药物

TRPA1 channels are non-selective cation channels that could be activated by plant-derived pungent products, including gingerol, a main active constituent of ginger. Ginger could improve the digestive function; however whether ginger improves the digestive function through activating TRPA1 receptor in gastrointestinal tract has not been investigated. In the present study, gingerol was used to stimulate cell lines (RIN14B or STC-1) while depletion of extracellular calcium. TRPA1 inhibitor (rethenium red) and TRPA1 gene silencing via TRPA1-specific siRNA were also used for mechanistic studies. The intracellular calcium and secretion of serotonin or cholecystokinin were measured by fura-2/AM and ELISA. Stimulation of those cells with gingerol increased intracellular calcium levels and the serotonin or cholecystokinin secretion. The gingerol-induced intracellular calcium increase and secretion (serotonin or cholecystokinin) release were completely blocked by ruthenium red, EGTA, and TRPA1-specific siRNA. In summary, our results suggested that gingerol derived from ginger might improve the digestive function through secretion releasing from endocrine cells of the gut by inducing TRPA1-mediated calcium influx.

System evaluation on efficacy and safety of Kushen Gel for treatment of bacterial and trichomonas vaginitis / 中草药

Related randomly controlled trials about Kushen Gel and Metronidazole in recent 10 years were collected by various kinds of database according to strict inclusion and exclusion criteria. Valid data were extracted to conduct Meta-analysis by RevMan5.0. To evaluate the efficacy and safety of Kushen Gel and Metronidazole for bacterial and trichomonas vaginitis. A total of 63 articles were retrieved, but only four of them were finally included. Meta-analysis showed that the effective proportion of Kushen Gel was significantly higher than that of Metronidazole [RR=1.13, 95% CI (1.06 and 1.20), P < 0.01]. The incidence of adverse effects was significantly lower in Kushen group compared to the Metronidazole group [RR=0.29, 95% CI (0.15 and 0.56), P < 0.01]. Kushen Gel can improve symptoms of vaginitis more effectively than Metronidazole and tend to have lower incidence of adverse effects.

Significance of changes in hepatic histology from patients with HBeAg(+)or HBeAg(-)chronic hepatitis B and normal or mildly elevated alanine aminotransferase / 临床肝胆病杂志

Alanine aminotransferase (ALT)abnormality is an indicator of the degree of liver inflammation caused by immune activation in patients with chronic hepatitis B (CHB).However,approximately half of the CHB patients with normal or mildly elevated ALT levels have concealed significant changes in hepatic histology.CHB patients with normal or mildly elevated ALT levels may have significant histopatho-logical changes in hepatic tissues,and those changes vary between HBeAg(+)and HBeAg(-)CHB patients.Attention and investigation on the clinical management of CHB patients with normal or mildly elevated ALT levels may have great significance in grasping the right treat-ment opportunity and reducing the risk of liver cirrhosis.

Construction of a full-genome HCV replicon with self-cleaving double ribozyme sequences and characterization in vitro and in vivo / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To construct a full-genome hepatitis C virus (HCV) replicon that will allow for direct initiation of replication and generation of infectious viral particles in an in vitro and in vivo cell system.</p><p><b>METHODS</b>Self-cleaving ribozyme sequences were added to each side of the HCV cDNA clone JFH1 and the replication-deficient clone JFH1/GND, then inserted into the pcDNA3.1 vector downstream of the CMV promoter. The resultant recombinant plasmids, pcDNA3.1-RZ-JFH1 and pcDNA3.1-RZ-JFH1/GND, were tested for activity in vitro and in vivo by transiently transfecting into Huh7.5 cells (5 mug/100 mm culture dish) and injecting by high-pressure tail vein injection into Kunming mice (10 - 30 mug/mouse). Quantitative reverse transcription-PCR, immunofluorescence, immunohistochemistry, and serological testing were performed to determine the replication ability and assess the properties of the recombinant plasmids in the two systems.</p><p><b>RESULTS</b>HCV RNA (1 - 3 * 10(6) copies/ml) was detected in the supernatant of transfected Huh7.5 cells up to 16 weeks after transfection. In addition, the viral particles from the supernatant were able to infect nave Huh7.5 cells. However, only transient viremia was achieved upon tail vein injection of the plasmid, and no HCV antigen-positive cells were detected by immunohistochemistry nor HCV-specific antibodies by serological testing.</p><p><b>CONCLUSION</b>The constructed HCV replicon was capable of stable expression in cultured cells and of efficiently generating infectious viral particles in the in vitro system over a long period. However, the HCV replicon did not show infective characteristics in an in vivo mouse system. The full-length HCV replicon may represent a useful tool for in vitro study of HCV pathological mechanisms, possibly including anti-HCV drug screening.</p>

Comprehensive evaluation on the curative effect of TCM treating type 2 diabetes with three type dialectical therapy by Fuzzy Mathematics / 国际中医中药杂志

ObjectiveTo comprehensively evaluate the curative effect ofTCM reating type 2 diabetes with three type dialectical therapy by Fuzzy Mathematics.Methods261 cases of 2 type diabetes were divided into three groups by syndrome differentiation,as follows:type of heat with yin deficiency,type of deficiency of both qi and yin,and type of deficiency of both yin and yang,and received corresponding therapies.The course of treatment was 24 weeks,in the first 12 weeks (period 1),on the basis of western medicine treatment,patients of the three groups were respectively treated by qingrungranules,ziyigranule and shuangtiaogranules.In the next 12 weeks (period 2),all patients were only treated by western medicine.The value of FBG,P2BG,HbAlc,TC,TG,BMI of the two periods were comprehensively evaluated by the methods of fuzzy mathematics.ResultsAt the end of period 1,the total effective rate of the type of heat with yin deficiency was 91.38％,the type of deficiency of both qi and yin was 87.16％,and the type of deficiency of both yin and yang was 83.34％; while at the end of period 2,the total effective rate of the three types was 59.18％,48.65％a nd32.65％ respectively.ConclusionThe effective rate was obviously improved in period 1; while in period 2,the effective rate was decreased.

Variable fragment of heavy chain antibody against the terminal protein region of hepatitis B virus polymerase introduced by Tat protein transduction domain inhibits the replication of hepatitis B virus in vitro / 中华传染病杂志

Objective To study a functional variable fragment of heavy chain(VH)antibody against the terminal protein(TP)region of hepatitis B virus(HBV)polymerase introduced by human immunodeficiency virus Tat protein transduction domain(TAT)and the inhibitive activity of TAT-VH on the replication of HBV in vitro.Methods The gene encoding TAT-VH was cloned into prokaryotic expression vector pET28a(+).Recombinant plasmid was transduced into E coli BL21(DE3)LysS,then the protein was expressed and purified.The purified TAT-VH fusion protein was added into HepG2.2.15 cell culture.The transduction efficiency was evaluated by indirect fluorescence assay(IFA).The cytotoxicity of TAT-VH was detected by Methabenzthiazuron(MTT)assay.HBV DNA level in HepG2.2.15 cell culture was measured using quantitative polymerase chain reaction(PCR).The data were analyzed by one-factor analysis of variance and t test.Results TAT-VH fusion protein was successfully expressed and purified.It was confirmed by IFA and MTT assay that TAT-VH was introduced into HepG2.2.15 cells and the cell growth was not affected.The level of HBV DNA in supernatant of HeDG2.2.15 cell culture with 5 000 nmol/L TAT-VH was(1.211±0.132)lg copy/mL,which was significantly lower than control group[(5.325±0.041)lg copy/mL,t=72.91,P＜0.05].Meanwhile,the level of intracellular HBV DNA was(3.521±0.411)lg copy/mL,which was significantly lower than control group[(8.532±0.132)lg copy/mL.t=28.41,P＜0.05].Conclusion The HBV replication is inhibited by anti-TP TAT-VH antibodies in vitro,which provides valuable experimemal basis for developing therapy of HBV infection with intracellular antibody.

Study of the quasispecies dynamics of serum hepatitis B virus in a patient with acute exacerbations of chronic hepatitis B / 中华肝脏病杂志

<p><b>OBJECTIVES</b>To investigate the quasispecies dynamics of hepatitis B virus (HBV) during the course of exacerbation and resolution of chronic hepatitis B in a patients.</p><p><b>METHODS</b>Five serum samples were collected from a patient with two episodes of exacerbation and resolution of chronic hepatitis B. A method of PCR-TA cloning-conformation sensitive gel electrophoresis (CSGE)-sequencing was performed to study the dynamic changes of HBV quasispecies in basal core promoter (BCP), precore and core regions of HBV genome.</p><p><b>RESULTS</b>Quasispecies complexity was 10 and 12 at the points of exacerbations, and 14 and 17 at the points of resolutions (t = 3.133, P < 0.05). Ratio of dominant quasispecies in HBV population was high (42.4% and 51.5%) during exacerbations and low (30.3% and 21.2%) during resolutions (t = 3.295, P < 0.05). All dominant quasispecies, except the one during the second resolution, carried core P5T, L60V, S155T, and precore G1896A mutations.</p><p><b>CONCLUSION</b>The composition of HBV quasispecies changes due to the change of host immune status, and immune pressure might lead to the selection of immune escape mutants.</p>

Preliminary study on hepatitis B virus quasispecies in a patient with chronic hepatitis B / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To investigate whether the hepatitis B virus (HBV) has quasispecies character by studying nucleotide sequence polymorphism and mutation features of HBV PreC/C gene region, and preliminaryly explore the heterogeneity of HBV quasispecies.</p><p><b>METHODS</b>The serum sample was obtained from a patient with chronic hepatitis B, and the whole HBV PreC/C gene region was amplified by PCR and cloned. Thirty-four clones that contained HBV PreC/C gene fragments were sequenced.</p><p><b>RESULTS</b>There were 28 kinds of different nucleotide sequences in 34 clones, and the nucleotide sequences diversity ranged from 0.2% to 2.1%. The mutation points were almost distributed in the whole region, but there wasn't mutation at PreC region nt.1 896 point in all sequences.</p><p><b>CONCLUSION</b>Hepatitis B virus has complex quasispecies character in the patients with chronic hepatitis B.</p>

Quasispecies and mutation of hepatitis B virus polymerase gene in lamivudine- treated patients / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To study the quasispecies and mutation features of hepatitis B virus polymerase (HBV P) gene in chronic hepatitis B patients before and after lamivudine treatment.</p><p><b>METHODS</b>The HBV P gene was amplificated with PCR and cloned, then single strand conformation polymorphism / heteroduplex analysis (SSCP/HDA) was applied to analyze the quasispecies complexity and mutation characters of HBV P gene.</p><p><b>RESULTS</b>The quasispecies number of HBV P gene before treatment was larger than that after treatment (7 to 14 vs. 4 to 8, t = 3.98, P < 0.05). Six patients had one or two predominant quasispecies before therapy, but the percentages of predominant quasispecies were lower than those after therapy (33.3% to 81.8% vs. 78.8% to 90.9%, t = 3.42, P < 0.05). By sequencing the predominant clones, there were 2 patients with M550V/L528M mutation, 3 patients with M550I mutation and 1 patient with wild type after lamivudine therapy. Additionally, there were individualization mutations which had no obvious tender.</p><p><b>CONCLUSION</b>Quasispecies of hepatitis B virus are changed under the selection of lamivudine, meanwhile, the mutation at YMDD motif emerges.</p>

Isolation of the variable fragments of heavy chain against the terminal protein region of hepatitis B virus polymerase with protein fragment complementation assay / 中国免疫学杂志

Objective:To isolate the variable fragments of heavy chain(VH) against the terminal protein(TP) region of hepatitis B virus(HBV) DNA polymerase(Pol) with protein fragment complementation assay(PCA).Methods:The TP region of HBV secreted by the HepG2.2.15 cells was used as an antigen,and the antibodies were selected with PCA.In this assay,two interacting proteins(target and antibody) are genetically fused to the two halves of the dissected enzyme dihydrofolate reductase.Binding of the two partners reassembles this enzyme and reconstitutes its activity,thus allowing growth on minimal mediem.Results:There were three TP region antigen-specific VHs could be directly in vivo selected with PCA.Sequence analysis showed that each TP-specific VH had a different sequence.Conclusion:Our results show that TP region antigen-specific VH could be directly in vivo selected with PCA.This system were powerful as a routine system for generating antibodies,especially in functional genomics.