Early administration of Wumei Wan inhibit myeloid-derived suppressor cells via PI3K/Akt pathway and amino acids metabolism to prevent Colitis-associated Colorectal Cancer.

ETHNOPHARMACOLOGICAL RELEVANCE: Wumei Wan (WMW), a traditional Chinese medicine prescription, has been proved to be effective in treating Colitis-associated colorectal cancer (CAC), but it has not been proven to be effective in different stages of CAC. AIM OF THE STUDY: The purpose of our study is to investigate the therapeutic effect and mechanism of WMW on the progression of CAC. MATERIALS AND METHODS: Azioximethane (AOM) and dextran sulfate sodium (DSS) were used to treat mice for the purpose of establishing CAC models. WMW was administered in different stages of CAC. The presentative chemical components in WMW were confirmed by UHPLCQTOF/MS under the optimized conditions. The detection of inflammatory cytokines in the serum and colon of mice were estimated by qRT-PCR and ELISA. The changes of T cells and myeloid-derived suppressor cells (MDSCs) in each group were detected by flow cytometry. The metabolic components in serum of mice were detected by UPLC-MS/MS. Expression of genes and proteins were detected by eukaryotic transcriptomics and western blot to explore the key pathway of WMW in preventing CAC. RESULTS: WMW had significant effect on inhibiting inflammatory responses and tumors during the early development stage of CAC when compared to other times. WMW increased the length of mice's colons, reduced the level of IL-1ß, IL-6, TNF-α in colon tissues, and effectively alleviated colonic inflammation, and improved the pathological damage of colon tissues. WMW could significantly reduce the infiltration of MDSCs in the spleen, increase CD4+ T cells and CD8+ T cells in the spleen of CAC mice, and effectively reform the immune microenvironment in CAC mice. Transcriptomics analysis revealed that 2204 genes had different patterns of overlap in the colon tissues of mice between control group, AOM+DSS group, and early administration of WMW group. And KEGG enrichment analysis showed that PI3K/Akt signaling pathway, ECM-receptor interaction, IL-17 signaling pathway, MAPK signaling pathway, pancreatic secretion, thermogenesis, and Rap1 signaling pathway were all involved. The serum metabolomics results of WMW showed that the metabolic compositions of the control group, AOM+DSS group and the early stage of WMW were different, and 42 differential metabolites with the opposite trends of changes were screened. The metabolic pathways mainly included pyrimidine metabolism, glycine, serine and threonine metabolism, tryptophan metabolism, and purine metabolism. And amino acids and related metabolites may play an important role in WMW prevention of CAC. CONCLUSION: WMW can effectively prevent the occurrence and development of CAC, especially in the initial stage. WMW can reduce the immune infiltration of MDSCs in the early stage. Early intervention of WMW can improve the metabolic disorder caused by AOM+DSS, especially correct the amino acid metabolism. PI3K/Akt signaling pathway was inhabited in early administration of WMW, which can regulate the amplification and function of MDSCs.

MicroRNA­449a is a potential predictor of colitis­associated colorectal cancer progression.

An early diagnosis of colitis­associated colorectal cancer (CAC) is important for its clinical management. However, it is currently difficult to distinguish the different stages of CAC development. MicroRNA dysregulation is common in human colorectal disorders, however little is known regarding whether miRNA affects tumor progression by regulating inflammation. In the present study, we identified a novel miRNA (miR­449a), the expression of which was significantly reduced in CAC tissues than in paired adjacent non­cancerous tissues (ANTs). Notably, the level of miR­449a was in a markedly decreased pattern during the neoplastic transformation of ulcerative colitis (UC)­to­CAC, as demonstrated by both clinical investigations and the experimental mouse model induced by AOM/DSS treatment. In addition, we observed that decreased miR­449a expression was associated with advanced T or N status, later clinical stage and poor histological differentiation of CAC. Mechanistic studies revealed that miR­449a inhibited the growth and metastasis of human colon cancer cells by directly binding to the 3'­UTR of Notch­1 and thereby, suppressed the activation of the Notch signaling pathway. Therefore, these findings provide strong evidence for the translational potential of miR­449a in the discrimination of patients with UC that is likely to progress into CAC, from those unlikely to progress, as well as in the prognosis and diagnosis of CAC.

Prediction of hepatic necroinflammatory activity in patients with chronic hepatitis B by a simple noninvasive model.

BACKGROUND: A model was constructed using clinical and serum variables to discriminate between chronic hepatitis B (CHB) patients with and without significant necroinflammatory activity (score 4-18 vs. score 0-3). METHODS: Consecutive CHB patients who underwent liver biopsy were divided into two sequential groups: a training group (n = 401) and a validation group (n = 401). Multivariate analysis identified alanine aminotransferase, γ-glutamyltransferase, prothrombin time and albumin as independent predictors of necroinflammatory activity. RESULTS: The area under the receiver operating characteristic curve was 0.826 for the training group and 0.847 for the validation group. Using a cut-off score of H ≤ 0.375, significant necroinflammatory activity (score 4-18) was excluded with high accuracy [78.2% negative predictive value (NPV), 72% positive predictive value (PPV), and 90.8% sensitivity] in 238 (59.4%) of 401 patients in the training group and with the same certainty (88.1% NPV, 61.2% PPV, and 95.1% sensitivity) among 204 (50.9%) of 401 patients in the validation group. Similarly, applying a cut-off score of H > 0.720, significant necroinflammatory activity was correctly identified with high accuracy (90.8% PPV, 57.7% NPV, and 92.0% specificity) in 150 (37.4%) of 401 patients in the training group and with the same certainty (91.8% PPV, 64.6% NPV, and 95.4% specificity) in 188 (46.9%) of 401 patients in the validation group. CONCLUSIONS: A predictive model based on easily accessible variables identified CHB patients with and without significant necroinflammatory activity with a high degree of accuracy. This model may decrease the need for liver biopsy for necroinflammatory activity grading in 72.1% of CHB patients.

18α-glycyrrhetinic acid extracted from Glycyrrhiza radix inhibits proliferation and promotes apoptosis of the hepatic stellate cell line.

OBJECTIVE: To evaluate the effect of 18α-glycyrrhetinic acid (18α-GA) on the proliferation and apoptosis of hepatic stellate cells (HSCs) and its underlying mechanisms. METHODS: HSCs (both human and rat HSCs) were pretreated with or without selective peroxisome proliferator-activated receptor-γ (PPAR-γ) antagonist, GW9662, before 18a-GA treatment. Cell cycle and apoptosis of HSCs were analyzed by flow cytometry, and changes in cell cycle and apoptosis-related proteins were analyzed by Western blot. The effect of 18α-GA on nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) DNA-binding activity was measured by ArrayStar transcription factor activity assay. RESULTS: 18α-GA markedly reduced LX-2 cell numbers by 14.8% and 31.2% after 48 h and 72 h of treatment, respectively (P < 0.05). 18α-GA also significantly increased the percentage of LX-2 cells in phase G0/G1 and decreased it in phase S after treated for 48 h and 72 h compared with the control group. 18α-GA increased apoptosis to 6.8% at 48 h, compared with control (2.5%), and at 72 h the percentages of apoptotic cells in control and the treatment groups were 3.1% and 15.6%, respectively, in LX-2 cells (P < 0.01). Similar changes occurred in CCl4-cirrhotic fat-storing cells. Furthermore, 18α-GA induced expression of PPAR-γ and altered some cell cycle and apoptosis-related proteins. 18α-GA also inhibited NF-κB DNA-binding activity. All these effects were abolished by GW9662. CONCLUSIONS: 18α-GA inhibits the proliferation of activated HSCs and induces apoptosis in culture. It also increases PPAR-γ expression and decreases NF-κB DNA-binding activity, which may be involved in these effects.

Prediction of significant fibrosis and cirrhosis in hepatitis B e-antigen negative patients with chronic hepatitis B using routine parameters.

AIM: As liver biopsy has considerable limitations in the assessment of liver fibrosis, non-invasive models have achieved great progress in the past. However, many tests consist of variables that are not readily available, and there are few data about patients with hepatitis B e-antigen (HBeAg) negative chronic hepatitis B (CHB). The aim of this study was to develop a model using routine data to predict liver fibrosis in HBeAg negative CHB patients. METHODS: We randomly divided 349 patients who underwent liver biopsy into training (n = 200) and validation (n = 149) sets. Multivariable logistic regression and receiver-operator curve (ROC) analyses were used to develop a model for predicting both significant fibrosis (stages 2-4) and cirrhosis (stage 4) in the training set. The model was validated in 149 patients in comparison to FIB-4, Forn's, S and aspartate aminotransferase-to-platelet ratio index indices using ROC. RESULTS: Multivariable logistic regression analysis showed that the parameters of the model for predicting both significant fibrosis and cirrhosis included sex, age, prothrombin time, platelet count, cholesterol and γ-glutamyltransferase. In the training set, the areas under the ROC (AUC) for predicting significant fibrosis and cirrhosis were 0.856 and 0.956, respectively. In the validation group, the AUC for predicting significant fibrosis and cirrhosis were 0.889 and 0.937, respectively. Using the best cut-off values, significant fibrosis and cirrhosis can be accurately predicted in 40.9% and 91.3% of patients, respectively. CONCLUSION: Our model can accurately predict both significant fibrosis and cirrhosis and may decrease the need of liver biopsy in a considerable proportion of patients with HBeAg negative CHB.

Suppression of pancreatic carcinoma growth by activating peroxisome proliferator-activated receptor gamma involves angiogenesis inhibition.

AIM: To study the possible actions and mechanisms of peroxisome proliferator-activated receptor gamma (PPARgamma), a ligand-activated transcription factor, in pancreatic carcinogenesis, especially in angiogenesis. METHODS: Expressions of PPARgamma and retinoid acid receptor (RXRalpha) were examined by reverse-transcription polymerase chain reaction (RT-PCR) with immunocytochemical staining. Pancreatic carcinoma cells, PANC-1, were treated either with 9-cis-RA, a ligand of RXRalpha, or with 15-deoxy-Delta(12,14) prostaglandin J(2) (15d-PGJ(2)), a ligand of PPARgamma, or both. Antiproliferative effect was evaluated by cell viability using methyltetrazolium (MTT) assay. A pancreatic carcinoma xenograft tumor model of nude mice was established by inoculating PANC-1 cells subcutaneously. Rosiglitazone, a specific ligand of PPARgamma, was administered via water drinking in experimental group of nude mice. After 75 d, all mice were sacrificed. Expression of proliferating cell nuclear antigen (PCNA) in tumor tissue was examined with immunohistochemical staining. Expression of vascular endothelial growth factor (VEGF) mRNA in PANC-1 cells, which were treated with 15d-PGJ(2) or 9-cis-RA at various concentrations or different duration, was detected by semi-quantitative RT-PCR. Effects of Rosiglitazone on changes of microvascular density (MVD) and VEGF expression were investigated in xenograft tumor tissue. Neovasculature was detected with immunohistochemistry staining labeled with anti-IV collagen antibody, and indicated by MVD. RESULTS: RT-PCR and immunocytochemical staining showed that PPARgamma and RXRalpha were expressed in PANC-1 cells at both transcription level and translation level. MTT assay demonstrated that 15d-PGJ(2), 9-cis-RA and their combination inhibited the growth of PANC-1 cells in a dose-dependent manner. 9-cis-RA had a combined inhibiting action with 15d-PGJ(2) on the growth of pancreatic carcinoma. In vivo studies revealed that Rosiglitazone significantly suppressed the growth of pancreatic carcinoma as compared to control group (0.48+/-0.23 cm(3) vs 2.488+/-0.59 cm(3), P<0.05), and the growth inhibition rate was 80.7%. Immunohistochemistry study showed that PCNA was down regulated in Rosiglitazone-treated group compared to the control group. 15d-PGJ(2), 9-cis-RA and their combination inhibited the expression of VEGF mRNA in PANC-1 cells in a dose- and time-dependent manner. MVD was decreased more significantly in Rosiglitazone-treated mice (10.67+/-3.07) than in the control group (31.44+/-6.06) (P<0.01). VEGF expression in xenograft tumor tissue was also markedly down-regulated in Rosiglitazone-treated mice. CONCLUSION: Activation of PPARgamma inhibits the growth of pancreatic carcinoma both in vitro and in vivo. Suppression of tumor angiogenesis by down-regulating the expression of VEGF may be one of the mechanisms by which PPARgamma activation inhibits the growth of pancreatic carcinoma.

[The role of lipopolysacchride-binding protein in the pathogenesis of animal model of acute necrotizing pancreatitis].

OBJECTIVE: To explore the pathogenic role of lipopolysacchride-binding protein (LBP) in the pathogenesis of acute necrotizing pancreatitis (ANP) by applying anti-LBP antibody to the animal model of ANP in mice. METHODS: Sixty BALB/c mice were randomly divided into four groups, including ANP group (n = 18), ANP treated with anti-LBP antibody group (n = 18), anti-LBP antibody group (n = 18) and normal control (n = 6). ANP model was induced by seven times administration of cerulein (50 micro g/kg.body weight), challenged by lipopolysaccharide (LPS) (5 mg/kg) intravenous injection. Treatment with anti-LBP antibody was started 15 minutes before LPS injection in ANP treated with anti-LBP antibody group. Anti-LBP antibody group only received intravenous injection of anti-LBP antibody, normal saline was administrated intraperitoneally instead of cerulein and LPS. At 9 h, 12 h and 24 h after the first injection of cerulein (or saline), the serum levels of amylase and lactate dehydrogenase (LDH) were measured. The severity of pancreatitis was evaluated by histological scoring system. Intrapancreatic TNF-alpha, IL-1beta, ICAM-1 and E-selectin mRNA expressions were studied by semi-quantitative RT-PCR. The activation of nuclear factor-kappaB (NF-kappaB) in the pancreas was investigated by the methods of immunohistochemistry and Western blot. The activity of PMN myeloperoxidase (MPO) was determined by zymohistochemistry. RESULTS: Compared with the ANP group, a marked elevation of serum amylase was observed 9 h and 12 h after cerulein administration and a marked elevation of serum LDH was observed 24 h after cerulein administration in the ANP treated with anti-LBP antibody group. Histologically, treatment with anti-LBP group increased the severity of pancreatic injury including edema at 9 h and 12 h after, and inflammatory cell infiltration and necrosis 24 h after. Intrapancreatic TNF-alpha, IL-1beta, ICAM-1 and E-selectin mRNA levels were increased. The activity of MPO was increased significantly 12 h and 24 h after in the anti-LBP antibody group. Immunohistochemistry and Western blotting showed up-regulation of NF-kappaB. However, there was no significant difference in serum parameters and pathologic scoring results between LBP antibody group and normal control. CONCLUSION: LBP plays a protective role in the pathogenesis of ANP, and this action may be mediated by inhibiting of NF-kappaB activation and down-regulation of proinflammatory mediators.

[Effect of selective cyclooxygenase-2 inhibitor celebrex on expression of vascular endothelial growth factor (VEGF) in pancreatic carcinoma].

BACKGROUND & OBJECTIVE: The previous study has identified that cyclooxygenase-2 (COX-2) may have a close relation with tumor genesis, particularly with digestive tract tumors, and its inhibitor can exert the chemoprevention role on carcinogenesis. This study was designed to investigate the effect of celebrex, a selective cyclooxygenase-2 inhibitor, on the expression of vascular endothelial growth factor (VEGF) in pancreatic carcinoma of xenografted nude mice induced by pancreatic carcinoma PC-3 cell lines. METHODS: The effect of celebrex on tumor growth was observed.The expression of VEGF in the tumors was determined by reverse transcription polymerase chain reaction (RT-PCR), Western blot analysis, and enzyme-linked immunosorbent assay (ELISA). RESULTS: Average tumor volume and tumor weight from control mice were 0.438+/-0.052 cm(3) and 0.552+/-0.064 g as compared with 0.215+/-0.038 cm(3) and 0.244+/-0.042 g from treated mice (inhibition rate:51.6%,P< 0.05). VEGF expression was significantly down-regulated in the celebrex-treated tumors. ELISA revealed that the expression levels of VEGF were 1.11+/-0.11(microg/g) in control mice and the 0.66+/-0.11(microg/g) in the treated mice. The inhibition rate of VEGF was 40.6% (P< 0.05). CONCLUSION: COX-2 may play an important role in the angiogenesis of pancreatic carcinoma. The selective COX-2 inhibitor, celebrex, can result in the inhibition of angiogenesis and tumor growth.

[Inhibition of angiogenesis in pancreatic carcinoma by cyclooxygenase-2 antisense oligodeoxynucleotides].

OBJECTIVE: To investigate the effect of cyclooxygenase-2 antisense oligodeoxynucleotides (COX-2 AS-ODNs) on the angiogenesis in pancreatic carcinoma and to evaluate the intermediary effect of prostaglandin 2 in this process. METHODS: Specific targeting COX-2 AS-ODNs were designed and synthesized, and transfected into the PC3 human pancreatic carcinoma cells cultured in vitro. Fluorescence microscopy was used to observe the PC3 cells 0.12. 24, 40, and 72 hours after the transfection. the second cultured PC3 cells were divided into 5 groups: control group, Lipo group (transfected with Lipofectin only), C1 group (transfected with 1 micro g COX-2 AS-ODN + Lipo/well), C2 group (transfected with 2 micro g COX-2 AS-ODN + Lipo/well), and C3 group (transfected with 3 micro g COX-2 AS-ODN + Lipo/well). RT-PCR was used to observe the expression of COX-2 mRNA in the PC3 cells. The third batch of PC3 cells were transfected with 3 micro g COX-2 AS-ODN + Lipo/well, and the expression of COX-2 mRNA was observed 0, 12, 24, 48, and 72 hours later by RT-PCR. 3 micro g COX-2 AS-ODN + Lipo/bottle and 9 micro g COX-2 AS-ODN + Lipo/bottle were added into the cultured PC3 cells and Western blotting was used to observe the expression of COX-2 protein 24 hours later. 24 chicken eggs were inoculated with PC3 cells into the chorio-allantoic membrane and then divided equally into 5 groups; control group, Lipo group, COX-2 AS-ODN + Lipo group, and COX-2 AS-ODN + Lipo + PGE2 group. Leica microscopy was used to observe the angiogenesis in the transplanted carcinoma. RESULTS: RT-PCR showed that the downregulation of expression of COX-2 mRNA in the PC3 cells with the increase of the COX-2 AS-ODN concentration, peaking at the concentration of 0.2 micro mol/L. The effect of COX-2 AS-ODN was strongest by the 12th hour after transfection and then began to decrease and basically disappeared 48 hours after. Western blotting showed that COX-2 AS-ODN, especially that of the concentration of the expression of 9 micro g/bottle, inhibited the expression of COX-2 AS-ODN. The angiogenesis of the transplanted carcinoma in the eggs was significantly inhibited in the Lipo + COX-2 AS-ODN group, the density of newly generated vessels in the Lipo + COX-2 AS-ODN + PGE2 group was between those of the other 2 groups. CONCLUSION: COX-2 AS-ODN significantly inhibits the angiogenesis in the pancreatic carcinoma. Endogenous COX-2 AS-ODN may play an important role in such a process and PGE2 may play an intermediate role therein.

[Regulatory effects of peroxisome proliferator-activated receptor gamma on the growth of pancreatic carcinoma].

OBJECTIVE: To examine the effects of peroxisome proliferator-activated receptor (PPAR)gamma activation on the growth of human pancreatic carcinoma both in vitro and in vivo. METHODS: The expression of PPARgamma and RXRalpha were examined by RT-PCR. SW1990 pancreatic cancer cells were treated with 9-cis-RA, ligand of PPARgamma, 15d-PGJ(2), and both. Antiproliferative effect was evaluated with cell viability by using MTT assay. Pancreatic cancer xenograft tumor model was established in nude mice by inoculating SW1990 cells subcutaneously and rosiglitazone, a PPARgamma activator, was administered via water drinking in experimental group. The nude mice were sacrificed after 75 days, the volume and weight of the xenograft tumor were measured. Expression of PCNA was observed by immunohistochemical staining. RESULTS: RT-PCR showed that PPARgamma and RXRalpha mRNA were expressed in SW1990 cell line. MTT assay demonstrated that 15d-PGJ(2), 9-cis-RA and the combination of both had a potent inhibitory effect on the growth of SW1990 cells with a dose-dependent manner. SW1990 cells were suppressed to more than 50% of the control at the concentration of 10 micro mol/L 15d-PGJ(2), 20 micro mol/L 9-cis-RA and 5 micro mol/L 15d-PGJ(2) plus 10 micro mol/L 9-cis-RA, respectively. 9-cis-RA had a synergic action with 15d- PGJ(2) on the growth inhibition of pancreatic carcinoma. In vivo studies, rosiglitazone suppressed the growth of pancreatic carcinoma in a statistically significant manner (P < 0.05). The average tumor volume and tumor weight in the experimental group were less than those in the control group, the growth inhibition rate of rosiglitazone was 80.7%. PCNA was present in both groups, but immunohistochemistry showed a down-regulation trend of PCNA in the experimental group as compared with the control group. CONCLUSIONS: Activation of PPARgamma exerts a negative regulatory effect on the growth of pancreatic carcinoma both in vitro and in vivo. These results suggest that PPARgamma might be a novel therapeutic target for the pancreatic carcinoma. Activation of RXRalpha has a synergic action with PPARgamma agonist on the growth inhibition of pancreatic carcinoma.