AIM:To investigate the production and activation of caspase-3 in primary rat renal proximal tubule cells in response to tumor necrosis factor-α(TNF-α) and the implication of nuclear factor-κB (NF-κB) in the process. METHODS:Isolated rat renal proximal tubule cells (PTCs) from male adult Sprague Dawley rats were treated with TNF-α according to the indicated time courses. A specific NF-κB inhibitor,Bay11-7082,was used alone or as a pretreatment for 1 h followed by exposure to TNF-α for 24 h.The protein levels of cleaved caspase-3,caspase-3,I-κBα,phosphorylated I-κBα,and GAPDH were detected by Western blotting using specific antibodies. RESULTS:The protein level of cleaved caspase-3 relative to caspase-3 was significantly increased in the presence of TNF-α for 6 h,12 h,and 24 h. Protein levels of caspase-3 were significantly decreased by 12 h and returned to baseline by 24 h in the presence of TNF-α. Treatment with Bay11-7082 for 25 h alone or pretreatment with Bay11-7082 for 1 h followed by addition of TNF-α for 24 h caused a remarkable reduction in both cleaved caspase-3 and caspase-3 as compared to control and TNF-α treated groups. An increase in phosphorylated I-κBα was observed from 15 min to 60 min after treatment with TNF-α at a dose of 10 μg/L in PTCs. CONCLUSION:NF-κB is not only associated with the activation of caspase-3 but also the production of caspase-3 in primary rat renal proximal tubule cells in response to TNF-α.

Objective To investigate the expression of DNA methyltransferase 3b (DNMT3B) in hepatocellular carcinoma (HCC) and its effect and mechanism on the proliferation,invasion and migration of HCC cells.Methods The expression of DNMT3B gene was detected by qRT-PCR in 46 cases of HCC tissues and corresponding adjacent tissues;the results and clinical pathological parameters were analyzed.SiRNA targeting DNMT3B was transfected into MHCC97-H cells by RNA interference (RNAi) technique.The mRNA and protein expression levels of related genes were detected by qRT-PCR and Western blot.The cell proliferation was measured by MTT assay,and the invasion and migration abilities were measured by Transwell assay.Results In 46 HCC patients,the expression of DNMT3B (73.91％) was significantly higher in HCC than in adjacent normal tissue.The high expression of DNMT3B gene was associated with histological type and tumor size of HCC (all P＜0.05).Inhibition of DNMT3B gene expression decreased proliferation,invasion and migration of MHCC97-H cells.Interference with DNMT3B gene increased the expressions of tumor suppressor genes RASSFA1,APC and MTSS1 at mRNA and protein levels.Conclusion DNMT3B is associated with the progression of HCC.It may inhibit the proliferation,invasion and migration of HCC cells by regulating the methylation of downstream tumor suppressor gene.

ABSTRACT:Objective To observe the influence of saikosaponin-d (SSd)on the proliferation and the function of autophagy of human hepatocellular carcinoma (HCC)cell line SMMC-7721 to explore the possible mechanisms. Methods SMMC-7721 was cultured invitro and then treated with SSd of various concentrations (5.0,7.5,10.0, 12.5,15.0 and 17.5 mg/L)for 24,48 and 72 h.We used MTT to detect cell proliferation,selected the optimal concentration and time,and detected the expressions of BECN1 at mRNA and protein levels by PCR and Western blot.Results The inhibition rate of the proliferation of SMMC-7721 cell line increased with the increase of the concentration of SSd,and the highest inhibition rate (60%)appeared when the concentration reached 12.5 mg/L. The expression of BECN1 in the group with SSd was obviously higher than that in the control group (P<0.05). 3-MA decreased not only the expressions of BECN1 at mRNA and protein levels but also the expression of BECN1 when used in conjunction with SSd.Conclusion The inhibiting function of SSd on SMMC-7721 presents a dependency between drug concentration and function time,basically in line with the drug dose-effect relationship. SSd induces the occurrence of autophagic cell death through up-regulating the expression of BECN1 ,thus inhibiting the proliferation of SMMC-7 7 2 1 .

Objective:To explore the role of matrix metalloproteinase 12 (MMP12) in airway macrophages and pulmonary neu-rogenic substance P ( SP ) in the pathogenesis of asthma by analyzing their relationship in different categories of asthmatic patients.Methods:Twenty patients of asthma remission phase ( remission asthma group ) , twenty ones of mild acute exacerbation asthma (mild asthma group) and twenty healthy adults (normal control group) were included,respectively.After lung function was measured,the numbers of macrophage in induced sputum were counted.The expression levels of MMP12 mRNA and protein in sputum macrophages were detected by quantitative reverse transcription polymerase chain reaction and Western blot.The concentration of sputum SP was assayed by enzyme immunometric assay.Results: ( 1 ) Compared with the subjects in normal control group, forced expiratory volume in 1 second%predicted ( FEV1 ) and forced expiratory flow rates at 50% of the forced vital capacity % predicted ( FEF50 ) were much lower and the numbers of sputum macrophages were much higher in the patients in different asthmatic groups.Compared with the patients in remission asthma group,FEV1 and FEF50 were much lower in the ones in mild asthma group.(2) MMP12 expressions in the macrophages and the concentrations of SP in sputum were significantly increased in the patients in different asthmatic groups compared with those in normal control group;Furthermore,MMP12 and SP in mild asthma group were much higher than in remission asthma.(3) In all patients from different asthmatic groups,mRNA expressions of MMP12 in the macrophages were positively correlated with the levels of sputum SP or the numbers of sputum macrophages,whereas negative correlations between mRNA expressions of MMP 12 and FEV 1 or FEF50 were observed.Conclusion: The regulatory imbalance of macrophages′MMP12 and pulmonary neurogic SP may participate in the pathogenesis of asthma and become the potential targets for asthma therapy.

Objective To detect the expression profile of transcription factor C/EBPβ in human immortalized normal hepatic cell lines and hepatocellular carcinoma cell lines so as to determine the correlation between C/EBP3 with cell death mediated by endoplasmic reticulum stress in hepatocellular cells.Methods We cultured the human immortalized normal hepatic cells lines HHL5 and HL7702 and hepatocellular carcinoma cell lines SMMC7721;Bel7402,HepG2 and Hep3B.Hep3B cells were used as the cell model in tunicamycin-induced endoplasmic reticulum stress.Cellular morphology was observed under an inverted optical microscope.MTT assay was used to assess the inhibition of cell growth.To detect cell apoptosis,the cells were dyed with Hoechst 33258 and observed using a fluorescence microscope.RToPCR and Western blotting were used to detect the expression of at mRNA and protein levels,respectively.Results We found that normally the mRNA and protein isoform of C/EBPβ,C/EBPβ-1,were both expressed in all of the four hepatocellular cell lines and the two immortalized normal hepatic cell lines,while C/EBPβ protein isoform C/EBPβ-3 was only expressed in the two immortalized normal hepatic cell lines.Tunicamycin increased the expressions of both mRNA and protein of C/EBPβ in Hep3B cells and the increase of protein isoform C/EBPβ-3 was the most remarkable.In Hep3B cells,cell death was induced by tunicamycin through endoplasmic reticulum stress activity.Apoptosis as well as paraptosis was observed in tunicamycin-induced cell death.Conclusion C/EBPβ-3,one of the protein isoforms of C/EBPβ,is only expressed in normal hepatic cell lines,but not in hepatocellular cell lines.C/EBPβ is involved in cell death mediated by endoplasmic reticulum stress activity in hepatocellular carcinoma cells.

Objective: To investigate MAML2 gene-translocation in primary pulmonary mucoepidermoid carcinoma (PMEC) and pulmanary adenosquamous carcinoma, and the optimal diagnostic immunohistiochemical (IHC) panel in distinguishing PMEC from adenosqumous carcinoma. Methods: Twenty-four cases of PMEC and 44 adenosqumous carcinoma diagnosed in the Guangdong General Hospital were tested for MAML2 translocation by fluorescent in-situ hybridization (FISH) using tissue array. An IHC panel including TTF1, Napsin A, CK5/6, p63, p40 and Ki-67 was performed on the cohort. The clinical data for all cases were collected and all PMEC patients had follow-up information. Results: The patients′ age ranged form 6 to 73 years, with a median age of 32 years. The male to female ratio was 1.4∶1.0. MAML2 translocation was found in 16/24 (66.7%) cases of PMEC whereas all 44 cases adenosqumous carcinoma were negative for translocation. All the cases of the PMEC were negative for TTF1 and Napsin A but positive for CK5/6, p63 and p40 in the intermediate cells and epidermal-like cells. In most PMEC cases, the Ki-67 expression index was lower than 10%. In contrast, most cases of adenosqumous carcinomas expressed TTF1 and Napsin A in the adenomatous component and CK5/6, p63 and p40 in the squamous component, which expression pattern was different from that of PMEC. Based on IHC staining, 2 cases of highly invasive ALK-positive adenocarcinoma mimicing PMEC were also found in the study. Conclusions MAML2 gene translocation can be detected in about two-third of PMEC. Translocation of MAML2 gene and lower morphology grading are associated with good prognosis. The combined use of IHC antibodies panel is helpful to distinguish PMEC from the adenosqumous carcinoma and adenocarcinoma mimicing PMEC.

Objective: To evaluate the application of FISH testing of bcl-2/IgH gene translocation and IgH/L gene rearrangement in different stages of follicular lymphoma. Methods: In 32 follicular lymphoma cases, which were collected at Guangdong General Hospital from September 2014 to December 2016, the bcl-2/IgH gene ectopic state was detected by FISH while the IgH/L gene rearrangement was tested using PCR-GeneScan to analyze the relationship between bcl-2/IgH gene translocation, different stages of follicular lymphoma and clonal immunoglobulin (IgH/L) gene rearrangements. Results: From the paraffin sections of all 32 follicular lymphomas, 17 cases showed bcl-2/IgH gene translocation, and the percentages of FL1, FL2 and FL3 translocation were 12/13, 3/5 and 2/14, respectively. Among the 24 cases of IgH/L gene arrangements identified from the total sample, the occurrence rates of FL1, FL2 and FL3 gene arrangement were 7/13, 4/5 and 13/14, respectively. Spearman′s rank correlation analysis and χ² analysis showed that bcl-2/IgH gene translocation was negatively correlated with follicular lymphoma stage and the association was statistically significant. In more advanced stages of follicular lymphoma, the occurrence of bcl-2/IgH gene translocation tended to decrease with distinct FL1, FL2 and Fl3 gene expression (P<0.05). As IgH/L gene rearrangement in FL3 was higher than that in FL1 and FL2, its detection may be complimentary to FISH test for bcl-2/IgH gene translocation in diagnosing follicular lymphoma. Conclusions The combined use of FISH and PCR-GeneScan increases the positive rate of follicular lymphoma diagnosis, and this combination is more sensitive than FISH or clonal analysis only to detect the chromosomal abnormality or the gene rearrangement.

Objective: To investigate the clinicopathological features, treatment and prognosis of duodenal-type follicular lymphoma. Methods: Four cases of duodenal-type follicular lymphoma diagnosed at Guangdong General Hospital from 2014 to 2015 with detailed clinical data were included. The histomorphology, immunophenotype, treatment and prognoses were analyzed. Results: The patients′ age ranged from 51 to 57 years (mean 54 years), and there were 2 males and 2 females. The involved sites were gastric fundus in one case, second portion of the duodenum in two cases and terminal ileum in one case. All patients presented with multiple mucosal granules or nodules at endoscopy. Microscopically, there were multiple mucosal neoplastic follicles, constituting grade 1-2 disease based on nodal follicular lymphoma grading system. The tumor cells were positive for CD20, CD10, bcl-6 and bcl-2. CD21 highlighted the follicular dendritic meshwork mainly at the periphery of the follicles. Proliferation index was low. Three patients received rituximab monotherapy for 4 cycles, leading to complete remission. One patient refused therapy and the disease progressed to systemic lymphoma 15 months after the initial diagnosis. Conclusions Duodenal-type follicular lymphoma is a special variant of follicular lymphoma with indolent clinical course. The tumor exhibits morphology of low grade follicular lymphoma with characteristic dendritic meshwork at the periphery of the follicles and a low proliferation index. Prognosis is excellent. Rituximab monotherapy is treatment of choice, but a small minority of patients may progress to systemic disease.

Objective:To evaluate the clinical effect of free anterior lateral thigh flap (ALTF) with cutaneous nerve on repair and reconstruction after radical operation of tongue cancer.Methods:79 patients with tongue cancer who underwent radical resection in the stomatology department of Hunan Provincial People′s Hospital from October 2015 to October 2020 were selected and divided into observation group (38 cases) and control group (41 cases) according to the treatment method. The observation group was reconstructed with free ALTF with cutaneous nerve, and the control group was reconstructed with free ALTF without cutaneous nerve. The survival of the flap was observed and the function and morphology of the reconstructed tongue were evaluated.Results:All flaps survived and had good blood supply; the blood supply was good, the wounds healed in the first stage, and there were no cases of infection and arterial crisis. The appearance of the reconstructed tongue was plump and showed mild atrophy. The observation group was significantly better than the control group in terms of language clarity, masticatory efficiency, sensory excellence rate and University of Washington Quality of Life questionnaire (UW-QOL) score ( P<0.05). Conclusions:The ALTF with cutaneous nerve for repair and reconstruction after radical operation of tongue cancer, can significantly improve the reconstruction of tongue sensory function and the quality of life of patients.

Objective:To evaluate the feasibility and effectiveness of magnetic anchor-guided endoscopic submucosal dissection (MAG-ESD).Methods:A total of 36 patients with gastrointestinal tumors at different sites who underwent MAG-ESD in the First Affiliated Hospital of Xi'an Jiaotong University from March 2020 to October 2022 were enrolled. The anchor success rate, en bloc resection rate, the anchor time, the procedure time, and the complication incidence were observed and analyzed.Results:Among the 36 patients, there were 9 lesions in stomach, 2 in duodenum, 6 in cecum and 19 in colorectum. Thirty-five (97.2%) patients successfully underwent magnetic anchor, and en bloc resection of lesions were completed. No adverse events such as bleeding or perforation occurred. The anchor time and procedure time was 4.0 (2.0-9.5) min and 36 (16-82) min, respectively.Conclusion:MAG-ESD is feasible and effective for gastrointestinal tumors at different sites, with a high anchor success rate and en bloc resection rate, and shorter operation time, especially for difficult submucosal dissection.