Melatonin attenuates acute pancreatitis-associated lung injury in rats by modulating interleukin 22

AIM:To investigate whether therapeutic treatment with melatonin could protect rats against acute pancreatitis and its associated lung injury.METHODS:Seventy-two male Sprague-Dawley rats were randomly divided into three groups:the sham operation(SO),severe acute pancreatitis(SAP),and melatonin treatment(MT) groups.Acute pancreatitis was induced by infusion of 1 mL/kg of sodium taurocholate(4% solution) into the biliopancreatic duct.Melatonin(50 mg/kg) was administered 30 min before pancreatitis was induced,and the severity of pancreatic and pulmonary injuries was evaluated 1,4 and 8 h after induction.Serum samples were collected to measure amylase activities,and lung tissues were removed to measure levels of mRNAs encoding interleukin 22(IL-22) and T helper cell 22(Th22),as well as levels of IL-22.RESULTS:At each time point,levels of mRNAs encoding IL-22 and Th22 were significantly higher(P 0.001) in the MT group than in the SAP group(0.526 ± 0.143 vs 0.156 ± 0.027,respectively,here and throughout,after 1 h;0.489 ± 0.150 vs 0.113 ± 0.014 after 4 h;0.524 ± 0.168 vs 0.069 ± 0.013 after 8 h,0.378 ± 0.134 vs 0.122 ± 0.015 after 1 h;0.205 ± 0.041 vs 0.076 ± 0.019 after 4 h;0.302 ± 0.108 vs 0.045 ± 0.013 after 8 h,respectively) and significantly lower(P 0.001) in the SAP group than in the SO group(0.156 ± 0.027 vs 1.000 ± 0.010 after 1 h;0.113 ± 0.014 vs 1.041 ± 0.235 after 4 h;0.069 ± 0.013 vs 1.110 ± 0.213 after 8 h,0.122 ± 0.015 vs 1.000 ± 0.188 after 1 h;0.076 ± 0.019 vs 0.899 ± 0.125 after 4 h;0.045 ± 0.013 vs 0.991 ± 0.222 after 8 h,respectively).The mean pathological scores for pancreatic tissues in the MT group were significantly higher(P 0.01) than those for samples in the SO group(1.088 ± 0.187 vs 0.488 ± 0.183 after 1 h;2.450 ± 0.212 vs 0.469 ± 0.242 after 4 h;4.994 ± 0.184 vs 0.513 ± 0.210 after 8 h),but were significantly lower(P 0.01) than those for samples in the SAP group at each time point(1.088 ± 0.187 vs 1.969 ± 0.290 after 1 h;2.450 ± 0.212 vs 3.344 ± 0.386 after 4 h;4.994 ± 0.184 vs 6.981 ± 0.301 after 8 h).The severity of SAP increased significantly(P 0.01) over time in the SAP group(1.088 ± 0.187 vs 2.450 ± 0.212 between 1 h and 4 h after inducing pancreatitis;and 2.450 ± 0.212 vs 4.994 ± 0.184 between 4 and 8 h after inducing pancreatitis).CONCLUSION:Melatonin protects rats against acute pancreatitis-associated lung injury,probably through the upregulation of IL-22 and Th22,which increases the innate immunity of tissue cells and enhances their regeneration.     

Increased frequency and clinical significance of myeloid-derived suppressor cells in human colorectal carcinoma

AIM: To investigate the frequency and clinical significance of the myeloid-derived suppressor cells (MDSC) in human colorectal carcinoma (CRC).METHODS: Samples of peripheral blood and tumor tissue from 49 CRC patients were analyzed. Mononuclear cells were isolated by Ficoll-Hypaque density gradient centrifugation and were subjected to a flow cytometry-based immunophenotypic analysis.RESULTS: A considerable increase in the percentage of CD33⁺HLA-DR^- MDSCs was observed in the peripheral blood (1.89% ± 0.75%) and tumor tissues (2.99% ± 1.29%) of CRC patients as compared with that in the peripheral blood of healthy controls (0.54% ± 0.35%). This expanded CD33⁺HLA-DR^- subset exhibited immature myeloid cell markers, but not lineage markers, and showed up-regulation of CD18/CD11b expression as compared with the MDSCs from healthy donors. Further studies showed that the MDSC proportion in CRC peripheral blood was correlated with nodal metastasis(P = 0.023), whereas that in tumor tissues was correlated with nodal/distant metastasis (P = 0.016/P = 0.047) and tumor stage (P = 0.028), suggesting the involvement of MDSCs in CRC tumor development.CONCLUSION: Characterization of MDSCs in CRC suggests the clinical significance of circulating and tumor-infiltrating MDSCs and may provide new insights into the CRC immunotherapy targeting MDSCs.     

Immunohistochemical expression of SP-NK-1R-EGFR pathway and VDR in colonic inflammation and neoplasia

AIM:To determine the expression of neurokinin-1receptor(NK-1R),phosphorylated epidermal growth factor receptor(p EGFR),cyclooxygenase-2(Cox-2),and vitamin D receptor(VDR)in normal,inflammatory bowel disease(IBD),and colorectal neoplasia tissues from Puerto Ricans.METHODS:Tissues from patients with IBD,colitisassociated colorectal cancer(CAC),sporadic dysplasia,and sporadic colorectal cancer(CRC),as well as normal controls,were identified at several centers in Puerto Rico.Archival formalin-fixed,paraffin-embedded tissues were de-identified and processed by immunohistochemistry for NK-1R,p EGFR,Cox-2,and VDR.Pictures of representative areas of each tissues diagnosis were taken and scored by three observers using a4-point scale that assessed intensity of staining.Tissues with CAC were further analyzed by photographing representative areas of IBD and the different grades of dysplasia,in addition to the areas of cancer,within each tissue.Differences in the average age between the five patient groups were assessed with one-way analysis of variance and Tukey-Kramer multiple comparisons test.The mean scores for normal tissues and tissues with IBD,dysplasia,CRC,and CAC were calculatedand statistically compared using one-way analysis of variance and Dunnett’s multiple comparisons test.Correlations between protein expression patterns were analyzed with the Pearson’s product-moment correlation coefficient.Data are presented as mean±SE.RESULTS:On average,patients with IBD were younger(34.60±5.81)than normal(63.20±6.13,P0.01),sporadic dysplasia(68.80±4.42,P0.01),sporadic cancer(74.80±4.91,P0.001),and CAC(57.50±5.11,P0.05)patients.NK-1R in cancer tissue(sporadic CRC,1.73±0.34;CAC,1.57±0.53)and sporadic dysplasia(2.00±0.45)were higher than in normal tissues(0.73±0.19).p EGFR was significantly increased in sporadic CRC(1.53±0.43)and CAC(2.25±0.47)when compared to normal tissue(0.07±0.25,P0.05,P0.001,respectively).Cox-2 was significantly increased in sporadic colorectal cancer(2.20±0.23 vs 0.80±0.37 for normal tissues,P0.05).In comparison to normal(2.80±0.13)and CAC(2.50±0.33)tissues,VDR was significantly decreased in sporadic dysplasia(0.00±0.00,P0.001 vs normal,P0.001 vs CAC)and sporadic CRC(0.47±0.23,P0.001 vs normal,P0.001 vs CAC).VDR levels negatively correlated with NK-1R(r=-0.48)and p EGFR(r=-0.56)in normal,IBD,sporadic dysplasia and sporadic CRC tissue,but not in CAC.CONCLUSION:Immunohistochemical NK-1R and p EGFR positivity with VDR negativity can be used to identify areas of sporadic colorectal neoplasia.VDR immunoreactivity can distinguish CAC from sporadic cancer.     

Amplifications of NCOA3 gene in colorectal cancers in a Chinese population

AIM: To investigate the copy number variation of NACO3 gene in colorectal cancer (CRC) and its correlation with tumor progression. METHODS: A total of 142 samples of case-matched CRC tissues and adjacent normal tissues were obtained from patients undergoing bowel resection. Quantitative real-time polymerase chain reaction method was used to investigate the copy number variations of NCOA3 as well as gene expression in the collected tissues. RESULTS: Copy number gains of NCOA3 were detected in 39 CRC samples (27.5%) and were correlatedwith tumor progression (χ2 = 6.42, P = 0.0112). Moreover, there was a positive correlation between copy number gain and mRNA over-expression of NCOA3 in CRCs (P = 0.0023). Expression level of NCOA3 mRNA was also enhanced in the CRC samples with unaltered copy numbers (3.85 ± 1.23 vs 2.71 ± 0.64, P 0.01). CONCLUSION: Sporadic colorectal cancers exhibit different mechanisms of NCOA3 regulation.     

Expression of monocyte chemotactic protein-1/CCL2 in gastric cancer and its relationship with tumor hypoxia

AIM:To investigate the expression and prognostic value of CCL2 in gastric cancer,as well as its relationshipwith tumor hypoxia.METHODS:Tumor tissues from 68 gastric cancer patients(GC)were analyzed,and the expression of CCL2and hypoxia-inducible factor 1 alpha(HIF-1α)in tumortissues was detected by immunohistochemistry.Statistical evaluations that were used included univariate logrank tests of Kaplan-Meier curves and multivariate Coxregression model analysis.RESULTS:CCL2 was highly expressed in 66.2%(45/68)of gastric cancer specimens.The distribution of CCL2expression in tumor tissue was consistent with thatof HIF-1α.Patients with high CCL2 expression in GChad a lower overall survival rate[50.6 mo(95%CI:44.44-56.93)vs 64.6 mo(95%CI:60.27-68.94),P=0.013].CONCLUSION:CCL2 expression correlates closely with HIF-1αexpression in gastric cancer.CCL2 may be an independent prognostic marker for GC.     

miR-132 inhibits colorectal cancer invasion and metastasis via directly targeting ZEB2

AIM:To investigate the biological role and underlying mechanism of miR-132 in colorectal cancer(CRC)progression and invasion.METHODS:Quantitative RT-PCR analysis was used to examine the expression levels of miR-132 in five CRC cell lines(SW480,SW620,HCT116,HT29 and LoVo)and a normal colonic cell line NCM460,as well as in tumor tissues with or without metastases.The KaplanMeier method was used to analyze the prognostic significance of miR-132 in CRC patients.The biological effects of miR-132 were assessed in CRC cell lines using the transwell assay.Quantitative RT-PCR and western blot analyses were employed to evaluate the expression of miR-132 targets.The regulation of ZEB2 by miR-132was confirmed using the luciferase activity assay.RESULTS:miR-132 was significantly down-regulated in the CRC cell lines compared with the normal colonic cell line(P<0.05),as well as in the CRC tissues withdistant metastases compared with the tissues without metastases(10.52±4.69 vs 23.11±7.84)(P<0.001).Down-regulation of miR-132 was associated with tumor size(P=0.016),distant metastasis(P=0.002),and TNM stage(P=0.020)in CRC patients.Kaplan-Meier survival curve analysis indicated that patients with low expression of miR-132 tended to have worse diseasefree survival than patients with high expression of miR-132(P<0.001).Moreover,ectopic expression of miR-132 markedly inhibited cell invasion(P<0.05)and the epithelial-mesenchymal transition(EMT)in CRC cell lines.Further investigation revealed ZEB2,an EMT regulator,was a downstream target of miR-132.CONCLUSION:Our study indicated that miR-132 plays an important role in the invasion and metastasis of CRC.     

Correlation between circulating myeloid-derived suppressor cells and Th17 cells in esophageal cancer

AIM: To perform a comprehensive investigation into the potential correlation between circulating myeloidderived suppressor cells (MDSCs) and Th17 cells in esophageal cancer (ECA). METHODS: A total of 31 patients newly diagnosed with ECA and 26 healthy subjects were included in the current study. The frequencies of MDSCs and Th17 cells in peripheral blood were determined by flow cytometry. The mRNA expression of cytokines, arginase 1 (Arg1) and inducible NO synthase (iNOS) in peripheral blood mononuclear cells (PBMCs) and plasma Arg1 were assessed by real-time polymerase chain reaction and enzyme-linked immunosorbent assay, respectively. RESULTS: There was an increased prevalence of MDSCs in the peripheral blood from ECA patients (15.21% ± 2.25%) when compared with healthy control (HC) (1.10% ± 0.12%, P 0.0001). The plasma levels of Arg1 in ECA patients were significantly higher than those in HC (28.28 ± 4.10 ng/mL vs 9.57 ± 1.51 ng/ mL, P=0.0003). iNOS mRNA levels in the peripheral blood of ECA patients also showed a threefold increase compared with HC (P=0.0162). The frequencies of Th17 cells (CD4 + IL-17A + ) were significantly elevated in ECA patients versus HC (3.50% ± 0.33% vs 1.82% ± 0.19%, P=0.0001). Increased mRNA expression of IL-17 and ROR-γt was also observed in ECA patients compared with HC (P=0.0041 and P=0.0004, respectively), while the mRNA expression of IL-6 and tumor necrosis factor-α (TNF-α) showed significant decreases (P=0.0049 and P 0.0001, respectively). No obvious correlations were found between the frequencies of MDSCs and Th17 cells in the peripheral blood from ECA patients(r=-0.1725, P=0.3534). Arg1 mRNA levels were positively correlated with levels of IL-6 (r=0.6404, P=0.0031) and TNF-α (r=0.7646, P=0.0001). Similarly, iNOS mRNA levels were also positively correlated with levels of IL-6 (r=0.6782, P=0.0007) and TNF-α (r=0.7633, P 0.0001). CONCLUSION: This study reveals the relationship between circulating MDSCs and Th17 cells, which may lead to new immunotherapy approaches for ECA based on the associated metabolites and cytokines.     

围手术期应用乌司他丁治疗对肝细胞癌患者肝切除术后外周血Th22细胞百分比和血浆白细胞介素-22的影响*

目的 观察围手术期应用乌司他丁对肝细胞癌（HCC）肝切除手术患者术后外周血Th22细胞百分比和血浆白细胞介素（IL）-22水平的影响。方法 2015年7月~2017年4月解放军昆明总医院普通外科行肝切除手术的HCC患者88例,术后51例接受常规治疗,37例另加乌司他丁治疗7 d。另选30例健康人作为对照。分离外周血单个核细胞（PBMC）,使用流式细胞术检测CD3⁺CD4⁺IL-22⁺的Th22细胞百分比,采用ELISA法检测血浆IL-22水平。对符合正态分布的计量资料比较采用独立样本t检验,对于非正态分布的计量资料比较采用Mann-Whitney检验。结果 HCC患者外周血Th22细胞百分比为（1.4±0.3） %,显著高于健康人【（0.8±0.1） %,P<0.0001】;HCC患者血浆IL-22水平为（116.9±32.6） pg/ml,亦显著高于健康人【（42.1±18.2） pg/ml,P<0.0001】;在术后3 d,乌司他丁治疗组和常规治疗组血清ALT分别为【219.4（40.1,510.9） IU/L和450.6（56.1,820.7） IU/L,P<0.05】,血清AST分别为【108.8（82.5,439.1） IU/L和257.3（115.3,7265） IU/L,P<0.01】;在术后7 d,乌司他丁治疗组和常规治疗组血清ALT分别为【72.4（25.6,471.5） IU/L和115.4（35.7,625.2） IU/L,P<0.05】,血清AST分别为【61.4（29.4,351.4） IU/L和90.5（45.5,293.8） IU/L,P<0.05】;术后3 d和术后7 d,乌司他丁治疗组外周血Th22细胞百分比分别为（1.2±0.4）%和（1.1±0.3）%,较常规治疗组显著降低【分别为（1.3±0.3）%,P<0.05和（1.3±0.2） %,P<0.05】;血浆IL-22水平分别为（98.1±20.1） pg/ml和（94.1±24.8） pg/ml,亦较常规治疗组显著降低 【分别为（109.7±29.8） pg/ml,P<0.05和（110.7±37.1）%,P<0.05】。结论 肝切除术后应用乌司他丁治疗可降低外周血Th22细胞比例和血浆IL-22水平,对肝功能具有一定的保护作用     

Interleukin-17 plays a critical role in the acute rejection of intestinal transplantation

AIM:To investigate the role of interleukin(IL)-17 in small bowel allograft rejection.METHODS:We detected the expression of helper T cell 17(Th17)cells in biopsy specimens from 3 cases of living small bowel transplantation in our department through immunofluorescence stain.We then established a rat heterotopic small bowel transplantation model.The rats were sacrificed on the 1st,2nd,3rd,5th, and 7th d after small bowel transplantation.The degrees of transplantation rejection in rat intestine graft were examined through hematoxylin eosin(HE)stain, and the expression of Th17 cells in rat intestine graft were detected through immunofluorescence stain. In addition,the recipient rats undergoing intestinal transplantation were administrated with mouse-anti-rat IL-17 monoclonal antibody(mAb),and the survival of rats was analyzed.The recipient rats which received mouse-anti-rat IL-17 mAb treatment were sacrificed on the 1st,2nd,3rd,5th,and 7th d after small bowel transplantation.The degrees of transplantation rejection and the expression of Th17 cells in rat intestine graft were detected through HE and immunofluorescence stain. The expression of IL-17,IL-1β,tumor necroses factor receptor-α(TNF-α),IL-6,and IL-8 in the intestine graft or serum were also detected. RESULTS:The expressions of Th17 cells ran parallel with the degree of acute rejection in human intestine grafts.The intestine graft rejection of rats was aggravated with prolonged duration after intestinal transplantation,and the expressions of Th17 cells were also correlated with the degree of acute rejection in rat intestine grafts.Administration of mouse-anti-rat IL-17 mAb prolonged the survival of rats after small bowel transplantation(P<0.001).Furthermore,we found that the administration of mouse-anti-rat IL-17 mAb significantly decreased the intensity of CD4+IL-17+Th17 cells in intestine grafts on the 2nd,3rd,5th,and the 7th d (97.22±4.05vs 12.45±2.02 on the 7th d,P<0.0001), and suppressed the severity of acute rejection.The expression of IL-17 in     

Hepcidin expression in colon during trinitrobenzene sulfonic acid-induced colitis in rats

AIM:To investigate hepcidin expression,interleukin-6(IL-6)production and iron levels in the rat colon in the presence of trinitrobenzene sulfonic acid(TNBS)-induced colitis.METHODS:In rats,we evaluated the severity of colitis induced by repeated TNBS administration using macroscopic and microscopic scoring systems and myeloperoxidase activity measurements.The colonic levels of hepcidin,tumor necrosis factor alpha(TNF-α),IL-10 and IL-6 were measured by Enzyme-Linked Immunosorbent Assay,and hepcidin-25 expression and iron deposition were analyzed by immunohistochemistry and the Prussian blue reaction,respectively.Stat-3 phosphorylation was assessed by Western blot analysis.Hematological parameters,iron and transferrin levels,and transferrin saturation were also measured.Additionally,the ability of iron,pathogen-derived molecules and IL-6 to induce hepcidin expression in HT-29 cells was evaluated.RESULTS:Repeated TNBS administration to rats resulted in macroscopically and microscopically detectable colon lesions and elevated colonic myeloperoxidase activity.Hepcidin-25 protein levels were increased in colonic surface epithelia in colitic rats(10.2±4.0pg/mg protein vs 71.0±8.4 pg/mg protein,P<0.01).Elevated IL-6 levels(8.2±1.7 pg/mg protein vs 14.7±0.7 pg/mg protein,P<0.05),TNF-αlevels(1.8±1.2pg/mg protein vs 7.4±2.1 pg/mg protein,P<0.05)and Stat-3 phosphorylation were also observed.Systemic alterations in iron homeostasis,hepcidin levels and anemia were not detected in colitic rats.Iron deposition in the colon was only observed during colitis.Hepcidin gene expression was increased in HT-29 cells after IL-6 and lipopolysaccharide[a toll-like receptor 4(TLR-4)ligand]treatment.Deferoxamine,ferric citrate and peptidoglycan(a TLR-2 ligand)were unable to alter the in vitro expression of hepcidin in HT-29 cells.CONCLUSION:Colitis increased local hepcidin-25 expression,which was associated with the IL-6/Stat-3 signaling pathway.An increase in local iron sequestration was also observed,but additional studies are needed to determine whether this sequestration is a defensive or pathological response to intestinal inflammation.     

Proinflammatory effects and molecular mechanisms of interleukin-17 in intestinal epithelial cell line HT-29

AIM: To evaluate the proinflammatory effects and molecular mechanisms of interleukin (IL)-17 in intestinal epithelial cell line HT-29.METHODS: HT-29 cells were cultured with IL-17, tumor necrosis factor (TNF)-α, or the combination of both IL-17 and TNF-α. Real-time PCR and Western blot were used to measure the gene expression levels of neutrophil chemokines CXCL1, CXCL2, CXCL5, CXCL6, IL-8 and TH-17 cell chemokine CCL20, the phosphorylation levels of p38 and TNF-α, and the expression level of IL-8, after using the p38 inhibitor in HT-29 cells. The stable Act1 knockdown HT-29 cell line was established to further test the phosphorylation changes of p38, after using IL-17 and TNF-α.RESULTS: After HT-29 cells were cultured with IL-17 and TNF-α, the expression levels of neutrophil chemokines (CXCL1, CXCL2, CXCL5, CXCL6, IL-8) and Th17 chemokine (CCL20) significantly improved (24.96 ± 2.53, 28.47 ± 2.87, 38.08 ± 2.72, 33.47 ± 2.41, 31.7 ± 2.38, 44.37 ± 2.73, respectively), and the differences were all statistically significant (P < 0.01). Western blot results showed that IL-17 obviously enhanced the phosphorylation level of p38, which was induced by TNF-α. Compared with the control group, the expression level of IL-8 significantly declined (9.47 ± 1.36 vs 3.06 ± 0.67, P < 0.01) when TH-29 cells were cultured with IL-17 and TNF-α. p38 inhibition assay showed that the p38 pathway played an essential role in the inflammatory response induced by IL-17. p38 phosphorylation levels could not be changed after using IL-17 and TNF-α in the stable Act1 knockdown HT-29 cell line.CONCLUSION: IL-17 significantly promoted the gene expression levels of TNF-α-induced neutrophil chemokines and Th17 cell chemokine. It is obvious that IL-17 and TNF-α have synergistic effects on p38.     

Expression of intercellular adhesive molecule1 in liver cancer tissues and liver cancer metastasis

AIM: To study the relationship between intercellular adhesive molecule-1 (ICAM-1) and liver cancer metastasis and to search for factors to predict metastasis of liver cancer.METHODS: ICAM-1 expression in fresh tissues of normal liver and hepatocellular cancer (HCC) was examined by immunoperoxidase staining. The expression of ICAM-1 in human hepatoma, tumor surrounding tissues and normal livers were semiquantitatively analyzed by Dot immuno blot. Tissue ICAM-1 expression at mRNA level was detected by Northern blot.RESULTS: All 6 cases of normal liver samples were negative in anti-ICAM-1 immunohistochemical staining, 80.0% (36/45) of HCC presented various ICAM-1 expression. The number of positive cells was a little higher in large tumors, tumors with intact capsule and metastasis, but there was no significant difference. Two cases with cancer embolus also had high ICAM-1 expression. ICAM-1 concentration in HCC (13.43 ± 0.09) was higher than that in tumor surrounding tissues (5.89 ± 0.17, P < 0.01) and normal livers (4.27 ± 0.21, P < 0.01). It was also higher in metastasis group (20.24 ± 0.30) than in nonmetastasis group (10.23 ± 0.12, P < 0.05). Northern blot analysis revealed that ICAM-1 expression at mRNA level was also higher in HCC and cancer embolus than that in tumor surrounding tissues and normal livers.CONCLUSION: Tissue ICAM-1 could indicate the growth and metastasis of HCC, and may be an index that can predict liver cancer metastasis.     

Interleukin-8, a promising predictor for prognosis of pancreatic cancer

AIM: To investigate the value of interleukin-8 (IL-8), a pro-inflammatory chemokine, in predicting the prognosis of pancreatic cancer.METHODS: Expression of IL-8 and its receptor CXCR1 was assessed by immunohistochemistry in pancreatic cancer and chronic pancreatitis samples. Enzyme-linked immunosorbent assay was used to detect the serum IL-8 levels in pancreatic cancer patients. Human pancreatic cancer tissues were heterotopically transplanted to the immune-deficiency mice to evaluate the effect of serum IL-8 on the tumorigenesis of the cancer samples.RESULTS: IL-8 and CXCR1 proteins were both over-expressed in pancreatic adenocarcinoma samples (55.6% and 65.4%, respectively) compared with the matched para-cancer tissues (25.9% and 12.3%, P < 0.01), or chronic pancreatitis (0% and 25%, P < 0.05). Serum IL-8 levels in pancreatic cancer patients (271.1 ± 187.7 ng/mL) were higher than in other digestive system tumors, such as gastric cancer (41.77 ± 9.11 ng/mL, P = 0.025), colorectal carcinoma (78.72 ± 80.60 ng/mL, P = 0.032) and hepatocellular carcinoma (59.60 ± 19.80 ng/mL, P = 0.016). In vivo tumorigenesis analysis further proved that tumor tissues from patients with higher serum IL-8 levels grew faster than those with lower IL-8 levels.CONCLUSION: IL-8 can be a fine serum marker for predicting the prognosis pancreatic cancer.     

Moxibustion inhibits interleukin-12 and tumor necrosis factor alpha and modulates intestinal flora in rat with ulcerative colitis

AIM: To investigate the effect of moxibustion on intestinal flora and release of interleukin-12 (IL-12) and tumor necrosis factor-α (TNF-α) from the colon in rat with ulcerative colitis (UC).METHODS: A rat model of UC was established by local stimulation of the intestine with supernatant from colonic contents harvested from human UC patients. A total of 40 male Sprague-Dawley rats were randomly divided into the following groups: normal (sham), model (UC), herb-partition moxibustion (HPM-treated), and positive control sulfasalazine (SA-treated). Rats treated with HPM received HPM at acupuncture points ST25 and RN6, once a day for 15 min, for a total of 8 d. Rats in the SA group were perfused with SA twice a day for 8 d. The colonic histopathology was observed by hematoxylin-eosin. The levels of intestinal flora, including Bifidobacterium, Lactobacillus, Escherichia coli (E. coli), and Bacteroides fragilis (B. fragilis), were tested by real-time quantitative polymerase chain reaction to detect bacterial 16S rRNA/DNA in order to determine DNA copy numbers of each specific species. Immunohistochemical assays were used to observe the expression of TNF-α and IL-12 in the rat colons.RESULTS: HPM treatment inhibited immunopathology in colonic tissues of UC rats; the general morphological score and the immunopathological score were significantly decreased in the HPM and SA groups compared with the model group [3.5 (2.0-4.0), 3.0 (1.5-3.5) vs 6.0 (5.5-7.0), P < 0.05 for the general morphological score, and 3.00 (2.00-3.50), 3.00 (2.50-3.50) vs 5.00 (4.50-5.50), P < 0.01 for the immunopathological score]. As measured by DNA copy number, we found that Bifidobacterium and Lactobacillus, which are associated with a healthy colon, were significantly higher in the HPM and SA groups than in the model group (1.395 ± 1.339, 1.461 ± 1.152 vs 0.045 ± 0.036, P < 0.01 for Bifidobacterium, and 0.395 ± 0.325, 0.851 ± 0.651 vs 0.0015 ± 0.0014, P < 0.01 for Lactobacillus). On the other hand, E. coli and B. fragilis, which are associated with an inflamed colon, were significantly lower in the HPM and SA groups than in the model group (0.244 ± 0.107, 0.628 ± 0.257 vs 1.691 ± 0.683, P < 0.01 for E. coli, and 0.351 ± 0.181, 0.416 ± 0.329 vs 1.285 ± 1.039, P < 0.01 for B. fragilis). The expression of TNF-α and IL-12 was decreased after HPM and SA treatment as compared to UC model alone (4970.81 ± 959.78, 6635.45 ± 1135.16 vs 12333.81 ± 680.79, P < 0.01 for TNF-α, and 5528.75 ± 1245.72, 7477.38 ± 1259.16 vs 12550.29 ± 1973.30, P < 0.01 for IL-12).CONCLUSION: HPM treatment can regulate intestinal flora and inhibit the expression of TNF-α and IL-12 in the colon tissues of UC rats, indicating that HPM can improve colonic immune response.     

Suppression of colorectal cancer metastasis by nigericin through inhibition of epithelial-mesenchymal transition

AIM: To evaluate the effect of nigericin on colorectal cancer and to explore its possible mechanism.METHODS: The human colorectal cancer (CRC) cell lines HT29 and SW480 were treated with nigericin or oxaliplatin under the conditions specified. Cell viability assay and invasion and metastasis assay were performed to evaluate the effect of nigericin on CRC cells. Sphere-forming assay and soft agar colony-forming assay were implemented to assess the action of nigericin on the cancer stem cell properties of CRC cells undergone epithelial-mesenchymal transition (EMT).RESULTS: Compared with oxaliplatin, nigericin showed more toxicity for the HT29 cell line (IC50, 12.92 ± 0.25 μmol vs 37.68 ± 0.34 μmol). A similar result was also obtained with the SW116 cell line (IC50, 15.86 ± 0.18 μmol vs 41.02 ± 0.23 μmol). A Boyden chamber assay indicated that a significant decrease in the number of HT29 cells migrating through polyvinylidene fluoride membrane was observed in the nigericin-treated group, relative to the vehicle-treated group [11 ± 2 cells per high-power field (HPF) vs 19.33 ± 1.52 cells per HPF, P < 0.05]. Compared to the control group, the numbers of HT29 cells invading through the Matrigel-coated membrane also decreased in the nigericin-treated group (6.66 ± 1.52 cells per HPF vs 14.66 ± 1.52 cells per HPF, P < 0.05). Nigericin also reduced the proportion of CD133⁺ cells from 83.57% to 63.93%, relative to the control group (P < 0.05). Nigericin decreased the number of spheres relative to the control group (0.14 ± 0.01 vs 0.35 ± 0.01, P < 0.05), while oxaliplatin increased the number of spheres relative to the control group (0.75 ± 0.02 vs 0.35 ± 0.01; P < 0.05). Nigericin also showed a decreased ability to form colonies under anchorage-independent conditions in a standard soft agar assay after 14 d in culture, relative to the control group (1.66 ± 0.57 vs 7 ± 1.15, P < 0.05), whereas the colony numbers were higher in the oxaliplatin group relative to the vehicle-treated controls (14.33 ± 0.57 vs 7 ± 1.15, P < 0.05). We further detected the expression of E-cadherin and vimentin in cells treated with nigericin and oxaliplatin. The results showed that HT29 cells treated with nigericin induced an increase in E-cadherin expression and a decrease in the vimentin expression relative to vehicle controls. In contrast, oxaliplatin downregulated the expression of E-cadherin and upregulated the expression of vimentin in HT29 cells relative to vehicle controls.CONCLUSION: This study demonstrated that nigericin could partly reverse the EMT process during cell invasion and metastasis.