OBJECTIVE To explore the material basis of the anti-inflammatory effect of the petroleum ether extract from Melastoma dodecandrum by establishing its fingerprint and combining it with cellular pharmacodynamics experiments. METHODS HPLC method was adopted； the fingerprints of 20 batches of petroleum ether extract from M. dodecandrum were drawn using The Similarity Evaluation System of TCM Chromatographic Fingerprint （2012 edition）； similarity evaluation and common peak identification were carried out. The lipopolysaccharide-induced inflammation model of mice mononuclear macrophages （RAW264.7） was established； the inhibitory rates of nitric oxide （NO） and tumor necrosis factor α （TNF-α） were used as indexes to investigate the anti-inflammatory activity of the petroleum ether extract from M. dodecandrum； grey correlation degree method and partial least square regression analysis were adopted to study the spectrum-effect relationship. Molecular docking was used to validate the binding activity of the anti-inflammatory active ingredients with TNF-α and iNOS protein receptor. RESULTS There were 19 common peaks in the fingerprint of the petroleum ether extract from M. dodecandrum， the similarity of 20 batches of samples ranged from 0.603-0.990， and five components were identified， such as vitexin （peak 5）， isovitexin （peak 6）， ellagic acid （peak 7）， quercetin （peak 9） and luteolin （peak 10）. The grey correlation degree between 19 common peaks of the petroleum ether extract from M. dodecandrum and the inhibition rates of NO and TNF-α were all greater than 0.7； peaks 19， 13， 9 （quercetin）， 12， 5 （vitexin）， 6 （isovitexin）， 8， 7 （ellagic acid）， 18， 1 were positively correlated with NO inhibition rate， and peaks 8， 10 （luteolin）， 13， 15， 3， 19， 17， 7 （ellagic acid）， 18， 1 were positively correlated with inhibition rate of TNF-α. The binding energies of vitexin， isovitexin and quercetin with iNOS protein receptor were less than －5.0 kcal/mol. CONCLUSIONS Vitexin， isovitexin and quercetin may be the basis of the anti-inflammatory effect of the petroleum ether extract from M.dodecandrum.

Objective: To analyze the safety of endoscopic stapes surgery, and to compare the results with stapes surgery under microscopic approach. Methods: This was a retrospective study. One hundred and thirty seven patients from Eye Ear Nose and Throat Hospital of Fudan University and other seven hospitals were enrolled in this study. Eighty eight patients, in whom 29 were male, and 59 were female, aged from 29 to 66 years old, with an average of 40.1±10.7, underwent endoscopic stapedotomy and 49 patients, in whom 17 were male, and 33 were female, aged from 32 to 64 yeas old, with an arerage of 38.7±9.2, underwent microscopic stapedotomy for otosclerosis. Interventions included endoscopic and microscopic stapes surgeries. Main outcome measures consisted of operating time, preoperative and postoperative hearing, intraoperative findings, and postoperative complications. SPSS 16.0 software was used to analyzed the date (t test and χ² test) . Results: Patients in the group who underwent endoscopic stapes surgery showed a mean operative time of (74.1±26.0) min. Patients in the group treated by microscopic approach had a mean operative time (66.5±15.9) min. Statistical difference was evident (t=1.279, P<0.05) . The average operative time of endoscopic surgery became shorter as the cases increased. The average duration of the last 10 cases was shorter than that of the first 10 cases in both groups. The differences were significant (t value was 3.028, 3.610, both P<0.05). No statistical difference was found in air conduction threshold improvement (t=1.074, P=0.289) , air-bone gap closure (t=-0.135, P=0.893) and bone conduction improvement (t=1.222, P=0.228) between the two groups. No difference regarding the incidence of the postoperative complications (chorda tympanum damage: 6 cases vs 2 cases, χ²=0.08,P>0.05; vertigo:18 cases vs 9 cases,χ²=0.09, P>0.05; facial paralysis: 0 case vs 0 case) between the two groups was found. Conclusion Audiological outcomes achieved by endoscopic surgery are similar to the results obtained through a microscopic approach. Endoscopic stapes surgery is safe.

Objective To evaluate the curative effect of trans-anal surgery vs.conventional laparoscopic surgery in treating sigmoid and high-rectum tumor.Methods From Jan 2014 to Mar 2017,100 patients in Beijing Friendship Hospital participated in this clinical study.45 patients underwent transanal surgery and 55 patients underwent conventional laparoscopic surgery.Results No significant difference was found between trans-anal surgery group and the traditional laparoscopic group in terms of operation time,blood loss,the use of analgesic drugs and the radical evaluation of tumor.While the postoperative pain scores in trans-anal surgery group (2.0 ± 1.0 vs.2.6 ± 1.0,t =2.9,P =0.005) were lower than those in the conventional group.The follow-up data showed one case of local tumor recurrence and one case of multiple peritoneal metastasis in the trans-anal surgery group,compared to two cases of liver metastasis and one case dying of pneumonia in the conventional laparoscopic group.The remaining cases were of no local recurrence,nor distant metastases or any critical complications.Conclusions Trans-anal surgery in the treatment of sigmoid and high-rectum tumor is safe,reliable and having the same clinical efficacy with conventional laparoscopic surgery.

Objective: To assess the incidence and independent risk factors for clinical anastomotic leakage (AL) in patients undergoing anterior resection (AR) or low anterior resection, (LAR) for rectal cancer. Methods: This was a retrospective case-control study of 550 patients with rectal cancer who underwent AR or LAR from April 2007 to March 2017 in Beijing Friendship Hospital, Capital Medical University. The relationship between the incidence of AL and clinicopathological manifestations was analyzed by Chi-squared test and Fisher exact test, and the independent risk factors of AL were analyzed using logistic regression analysis. AL is defined as a defect (including necrosis or abscess formation) of the intestinal wall at the anastomotic site, leading to a communication between the intra- and extra-luminal compartments. AL can be divided into three grades. Grade A anastomotic leakage results in no change in the management of patients, whereas grade B leakage requires active therapeutic intervention but is manageable without re-laparotomy. Grade C anastomotic leakage requires re-laparotomy. Results: AL was noted in 32 (5.8%) of 550 patients with rectal cancer who underwent AR or LAR, including 15 (46.9%) , 4 (12.5%) , and 13 patients (40.6%) with Grades A, B, and C, respectively. Five patients (0.9%, 5/550) died peri-operatively. AL- and non-AL-related deaths occurred in 3 (9.4%, 3/32, all cases were Grade C) and 2 patients (0.4%, 2/518) , respectively, with the two mortality rates being significant difference (P = 0.002) . Chi-squared test or Fisher exact test showed that the incidence of AL was associated with neoadjuvant chemoradiotherapy (P = 0.011) , intraoperative bleeding (≥100 ml) (χ² = 11.980, P = 0.001) , and tension-reducing suture of anastomosis (P = 0.015) . The results of logistic regression analysis showed that the independent risk factors of AL were neoadjuvant chemoradiotherapy (OR = 2.402, 95%CI: 1.004 - 5.749, P = 0.049) , intraoperative bleeding (≥100 ml) (OR = 2.971, 95%CI: 1.269 - 6.957, P = 0.012) and tension-reducing suture of anastomosis (OR = 2.304, 95%CI: 1.008 - 5.263, P = 0.048) . Conclusion The incidence of AL in patients undergoing AR for rectal cancer is 5.8%. The high-risk factors for AL are neoadjuvant chemoradiotherapy, intraoperative bleeding (≥100 ml) , and tension-reducing suture of anastomosis. Patients with these three risk factors have a high risk of AL rate, and a defunctioning stoma should be performed.

Objective To study the effect of paeonol on the proliferation and apoptosis of A375 human melanoma cells and its mechanism.Methods Cell counting kit-8 (CCK8) was used to evaluate the proliferative activity of A375 cells treated with paeonol of 0.5,1,2,4,8 mmol/L for 24,48 and 72 hours respectively.Subsequently,A375 cells were treated with paeonol of 1.25,2.5 and 5 mmol/L for 24 hours followed by double staining with annexin V and propidium iodide for the detection of cell apoptosis,fluorometric assay for the estimation of caspase 3,caspase 8 and caspase 9 activity,and Western blot for the determination of the levels of p53,nuclear factor-κB proteins and some of their target proteins.The A375 cells receiving no treatment served as the blank control group.Statistical analysis was carried out by t test.Results Within the investigated concentration and time ranges,paeonol significantly inhibited the proliferative activity of A375 cells in a concentration-and timedependent manner.Compared with the blank control group,a significant increase was observed in the early apoptosis rate in A375 cells treated with paeonol of 1.25,2.5 and 5 mmol/L for 24 hours (13.74％-± 1.73％,25.95％ ± 0.57％ and 46.44％ ± 0.81％ vs.3.11％ ± 0.53％,P ＜ 0.05 or 0.01),as well as in the activity of caspase 3,8 and 9 in A375 cells treated with paeonol of 2.5 and 5 mmol/L for 24 hours (P ＜ 0.05 or 0.01).After 24-hour treatment,the protein levels of p53 and Bax were elevated,but those of nuclear factor-κB,Bcl-2 and Bcl-XL were decreased in A375 cells with the increase of paeonol concentration.Conclusions Paeonol can inhibit the proliferation but induce the apoptosis of A375 cells,and the apoptosis-inducing effect may be realized through intrinsic and extrinsic pathways by modulating nuclear factor-κB and p53 genes.

Objective To optimize the concentration of a microRNA-21 (miR-21) inhibitor and a miR-494 mimic for the transfection of A375 human melanoma cells,and to estimate the effect of the miR-21 inbihitor and miR-494 mimic on the proliferation of A375 cells.Methods A miR-21 inbihitor and a miR-494 mimic were designed and constructed.To optimize the concentration of the miR-21 inbihitor and miR-494 mimic for transfection,six concentrations (70-250 nmol/L) of the inbihitor and mimic were transfected into A375 cells separately by using LipofectamineTM2000.Then,quantitative fluorescence-based PCR was performed to determine the expression of miR-21 and miR-494 in A375 cells.Some A375 cells were classified into five groups:Mock blank control group remaining untransfected,miR-21 inhibitor group transfected with the miR-21 inhibitor,miR-21 control group transfected with the miR-21 inhibitor negative control,miR-494 mimic group transfected with the miR-494 mimic,and miR-494 control group transfected with the miR-494 mimic negative control.Mter another 48-hour culture,the cells were collected for the analysis of cell apoptosis and cycle by using flow cytometry.Meanwhile,Cy5-labelled miR-494 mimic negative control was transfected into A375 cells for the evaluation of the transfection efficiency by using an inverted fluorescence microscope.Results miRNAs were successfully extracted from A375 cells.As quantitative PCR revealed,the A375 cells transfected with the miR-21 inhibitor at 120 nmol/L showed the lowest expression level (2-△△Ct) of miR-21 (average:0.80; range:0.65-0.92),and those transfected with the miR494 mimic at 250 nmol/L displayed the highest expression level of miR-494 (average:126.82; range:111.52-144.22).The transfection efficiency in A375 cells was higher than 90％.Compared with the corresponding negative control groups,the miR-21 inhibitor group and miR-494 mimic group showed increased apoptosis rate ((27.74 ± 1.39)％ vs.(12.93 ± 0.65)％,(34.30 ± 2.35)％ vs.(15.54 ± 1.02)％,both P ＜ 0.01),percentage of G1-phase cells ((61.61 ± 3.25)％ vs.(50.34 ± 5.62)％,(61.05 ± 3.17)％ vs.(49.95 ± 2.58)％,both P＜ 0.05),but decreased proliferation index ((38.39 ± 3.25)％ vs.(49.66 ± 5.62) ％,(38.95 ± 3.17)％ vs.(50.05 ± 2.58)％,both P ＜ 0.05).Conclusions Both the miR-21 inhibitor and miR-494 mimic can promote the G1-phase arrest and apoptosis in A375 cells,and miR-21 may act as a protooncogene accelerating the proliferation of A375 cells,while miR-494 may founction as a tumor suppressor inhibiting the proliferation of A375 cells.

Objective To study the effects of triptolide on the apoptosis in and proliferation of a human melanoma cell line M14.Methods M14 cells were cultured with the presence of 5 concentrations (12.5,25,50,100,200 nmol/L) of triptolide for 24,48 and 72 hours respectively,and cell counting kit-8 (CCK-8) was used for the detection of cell proliferation.Some M14 cells were treated with triptolide at 10 nmol/L,20 nmol/L and 30 nmol/L for 48 hours followed by the analysis of cell cycle by flow cytometry and detection of cell apoptosis by flow cytometry following annexin V-fluorescein isothiocyanate (FITC)/propidium iodide double staining.The morphological changes of M14 cells treated by triptolide at 30 nmol/L for 48 hours were observed by Hoechest 33258 staining.Results Compared with untreated M14 cells,an increase of cell population in S phase was observed in triptolide-treated cells,along with a decline in cell population in G2/M phase.The apoptosis rate was (2.92 ± 0.17)％,(20.99 ± 0.40)％,(34.28 ± 2.04)％ and (63.38 ± 0.71) ％ respectively in M14 cells treated with triptolide at 0,10,20 and 30 nmol/L for 48 hours,suggesting that triptolide enhanced the proliferation of M14 cells in a dose-dependent manner.After treatment with triptolide of 30 nmol/L,M14 cells showed morphological changes characteristic of apoptosis.Conclusion Triptolide could inhibit the proliferation of and induce the apoptosis in M14 human melanoma cells.

Objective To assess the relationship of methylation status of CpG islands in the promoter region of insulin-like growth factor binding protein 7 (IGFBP7) gene with the expression of IGFBP7 gene in human melanoma cell lines and primary melanocytes.Methods Primary melanocytes from human forcskin tissue as well as 4 human melanoma cell lines,including A375,M14,SK-MEL-1 and MV3,were used in this study.Bisulfite sequencing PCR (BSP) was applied to detect the methylation status of 54 CpG sites in the 5'-flanking promoter region of IGFBP7 gene in all of the melanoma cell lines and primary melanocytes.Results As hierarchical cluster analysis showed,IGFBP7-positive cells (including A375,M14 and SK-MEL-1 ) differed significantly from IGFBP7-negative cells (including MV3 cells and primary melanocytes) in the methylation pattern of IGFBP7 gene promoter region.Conclusion The methylation status of CpG island in the promoter region of IGFBP7 gene may be associated with its expression in melanoma cell lines.

Objective To investigate the expression of CXCR7 in several cutaneous malignant tumors including cutaneous squamous cell carcinoma (SCC),basal cell carcinoma (BCC) and invasive cutaneous malignant melanoma and their cell lines,as well as its significance.Methods Tissue specimens were obtained from the lesions of 30 patients with cutaneous squamous cell carcinoma,25 patients with basal cell carcinoma and 30 patients with cutaneous malignant melanoma.Immunohistochemistry was performed to detect the expression of CXCR7 protein in these tissue specimens and several cell lines (A375 human melanoma cells,M14 human melanoma cells,A431 human epidermoid carcinoma cells,HaCaT human keratinocytes).The mRNA expression of CXCR7 in these cell lines was measured by reverse transcription PCR.Results CXCR7 protein was apparently expressed in invasive cutaneous malignant melanoma.The high expression rate of CXCR7 protein was significantly elevated in cutaneous malignant melanoma tissue specimens compared with SCC and BCC tissue specimens [80％ (24/30) vs.26.67％ (8/30) and 8％ (2/25),x2 =17.16,28.36,both P ＜ 0.05].CXCR7 mRNA was expressed in A375,M14 and A431 cells,but not in HaCaT cells,with the strongest expression observed in A375 cells.Immunohistochemistry revealed the expression of CXCR7 protein only in A375 cells.Conclusions CXCR7 is highly expressed in cutaneous malignant melanoma and A375 cells,which may be involved in the malignant invasion and metastasis of melanoma.

Objective To study the effects of 5-aza-dc on the expression of IGFBP7 in and proliferation of melanoma cell lines A375 and M14.Methods Reverse transcription-PCR and immunocytochemistry were performed to detect the mRNA and protein expression of IGFBP7 in A375 cells and M14 cells after treatment with 5-aza-dc of 10μmol/L for 48 hours,and MTT assay to measure the proliferation of both cell lines treated with 4 different concentrations (2.5,5,10,20μmol/L) of 5-aza-dc for various durations.Results The treatment with 5-aza-dc restored IGFBP7 expression at both mRNA and protein levels.The four concentrations of 5-aza-dc inhibited the proliferation of A375 and M14 cells in a dose-dependent (F=561.12,271.43,respectively,both P<0.01) and time-dependent (F=141.35,549.33,respectively,both P<0.01) manner.Conclusions DNA methylation may be involved in the modulation of aberrant IGFBP7 gene expression in melanoma,and 5-aza-dc could inhibit the proliferation of A375 and M14 cells.