Effect of deoxycholic acid intervention on growth of ileum organoids derived from C57BL/6 mice / 南方医科大学学报

<p><b>OBJECTIVE</b>To establish a culture system for mouse intestinal organoids and investigate the effect of deoxycholic acid (DCA) on organoids growth.</p><p><b>METHODS</b>The terminal ileum was collected from 8-month-old C57BL<6 mice. The tissue blocks were treated with EDTA and the crypts were collected and embedded in Matrigel Matrix. Orgnoids growth and buddings were observed in the control group, anhydrous alcohol group, short-term (2 days) 100 µmol<L DCA treatment group, and long-term (10 days) 10 µmol<L DCA treatment group; the orgnoids were further cultured for 10 days after removal of DCA from the medium and observed for orgnoids growth and buddings.</p><p><b>RESULTS</b>Short-term treatment with high-concentration DCA resulted in significantly reduced enterosphere formation, enteroids formation, progression from enterospheres to enteroids and number of crypt buds per enteroid (P<0.05), which remained unchanged even after removal of DCA for a short time (P<0.05); long after DCA removal, the enteroids formation rate and number of the crypt buds still remained lower than those in normal organoids (P<0.05). Short-term treatment with low-concentration DCA only resulted in reduced enteroids formation rate and number of crypt buds (P<0.05), and prolonged treatment caused reduced enterospheres formation rate, enteroids formation rate and number of crypt buds (P<0.05). After DCA removal, enteroids formation rate and the number of crypt buds still remained lower than those in the normal group (P<0.05).</p><p><b>CONCLUSION</b>We successfully established an organoids culture system. The presence of DCA in the culture system affects the growth of the organoids, which can partly recover following a prolonged period after the removal of DCA.</p>

High-throughput sequencing for analysis of structural change of intestinal microbiota in patients with colorectal adenoma / 南方医科大学学报

<p><b>OBJECTIVE</b>To investigate the taxonomic richness and diversity of gut microbiota in patients with colorectal adenoma and elucidate the role of gut microorganisms in precancerous lesions in the colon and rectum.</p><p><b>METHOD</b>Adenomatous tissues from 31 patients with colorectal adenoma and normal intestinal mucosal tissues from 20 healthy control subjects were collected through colonoscopy. The total bacterial genomic DNA was extracted, and the V-Vhypervariable region in bacterial 16S rRNA gene was amplified using polymerase chain reaction and sequenced on an Illumina MiSeq platform.</p><p><b>RESULTS</b>Patients with colorectal adenomas had a higher alpha diversity and richness indices compared to the healthy controls (P<0.01). The mucosal microbiota in colorectal adenoma tissue showed a distinctive structural difference from that in normal intestinal mucosal tissues. At the phylum level, a large decrease in Firmicutes with concomitant relative expansion of Proteobacteria was observed in patients with colorectal adenomas, resulting in a significant decrease in the Firmicutes/Bacteroidetes ratio (P<0.01). At the genus level, Lactococcus and Pseudomonas were enriched whereas Enterococcus, Bacillus, and Solibacillus were reduced obviously in the preneoplastic tissues (P<0.01). We also found a similar gut microbiome composition between low-grade and high-grade intraepithelial neoplasia; the ratio of Escherichia-Shigella tended to increase in high-grade intraepithelial neoplasia, but this change was not statistically significant (P%0.28).</p><p><b>CONCLUSION</b>Significant changes in the structure of the intestinal flora occur in patients with colorectal adenomas, indicating that the association of dysbiosis of the gut microbiota with the occurrence of a pro-oncogenic microenvironment.</p>

Pyrrolidine Dithiocarbamate Inhibits Nuclear Factor kappaB and Toll-Like Receptor 4 Expression in Rats with Acute Necrotizing Pancreatitis

BACKGROUND/AIMS: To investigate the expression of Toll-like receptor 4 (TLR4) in the pancreases of rats with acute necrotizing pancreatitis (ANP) and any changes upon treatment with pyrrolidine dithiocarbamate (PDTC), an inhibitor of nuclear factor kappaB (NF-kappaB), as well as to determine the relationship between TLR4 and NF-kappaB in ANP pathogenesis. METHODS: A total of 72 SD rats were randomly divided into three groups, namely, the control (sham-operation), ANP, and ANP with PDTC pretreatment groups. The PDTC-pretreated group was intraperitoneally injected with PDTC at a dose of 100 mg/kg 1 hour before the induction of ANP. The expressions of TLR4 and NF-kappaB in pancreatic tissue were evaluated by immunohistochemistry and Western blot analysis. The mRNA levels of cytokines tumor necrosis factor alpha, interleukin (IL)-1beta, and IL-6 were measured by reverse transcription polymerase chain reaction. RESULTS: The expressions of TLR4, NF-kappaB, and cytokine (NF-kappaB target) genes in the pancreatic tissue increased more significantly in the ANP groups than in the sham-operation group at 3, 6, and 12 hours. Pretreatment with PDTC alleviated the inflammatory activation in the pancreas with ANP, causing a significant decrease in the expressions of TLR4, NF-kappaB, and cytokine genes in the pancreatic tissue. CONCLUSIONS: The expressions of TLR4 and NF-kappaB were increased in the pancreases of rats with ANP. PDTC not only inhibits NF-kappaB but also suppresses the expression of TLR4 and downregulates the expression of the related cytokine genes.

Pyrrolidine Dithiocarbamate Inhibits Nuclear Factor kappaB and Toll-Like Receptor 4 Expression in Rats with Acute Necrotizing Pancreatitis

BACKGROUND/AIMS: To investigate the expression of Toll-like receptor 4 (TLR4) in the pancreases of rats with acute necrotizing pancreatitis (ANP) and any changes upon treatment with pyrrolidine dithiocarbamate (PDTC), an inhibitor of nuclear factor kappaB (NF-kappaB), as well as to determine the relationship between TLR4 and NF-kappaB in ANP pathogenesis. METHODS: A total of 72 SD rats were randomly divided into three groups, namely, the control (sham-operation), ANP, and ANP with PDTC pretreatment groups. The PDTC-pretreated group was intraperitoneally injected with PDTC at a dose of 100 mg/kg 1 hour before the induction of ANP. The expressions of TLR4 and NF-kappaB in pancreatic tissue were evaluated by immunohistochemistry and Western blot analysis. The mRNA levels of cytokines tumor necrosis factor alpha, interleukin (IL)-1beta, and IL-6 were measured by reverse transcription polymerase chain reaction. RESULTS: The expressions of TLR4, NF-kappaB, and cytokine (NF-kappaB target) genes in the pancreatic tissue increased more significantly in the ANP groups than in the sham-operation group at 3, 6, and 12 hours. Pretreatment with PDTC alleviated the inflammatory activation in the pancreas with ANP, causing a significant decrease in the expressions of TLR4, NF-kappaB, and cytokine genes in the pancreatic tissue. CONCLUSIONS: The expressions of TLR4 and NF-kappaB were increased in the pancreases of rats with ANP. PDTC not only inhibits NF-kappaB but also suppresses the expression of TLR4 and downregulates the expression of the related cytokine genes.

Upregulating expressions of hepatic lipopolysaccharide receptors in nonalcoholic steatohepatitic rats / 中华肝脏病杂志

<p><b>OBJECTIVE</b>The expression of hepatic lipopolysaccharide (LPS) receptors in a rat nonalcoholic steatohepatitis (NASH) model was studied in order to explore the pathogenesis of NASH.</p><p><b>METHODS</b>Forty-five male SD rats were fed with a high fat diet. These rats were sacrificed after high fat feeding at 8, 12, 16, 24 weeks. Hepatic expressions of CD14 were observed by immunohistochemistry and expressions of TLR4 were detected by RT-PCR. Hepatic expressions and serum levels of TNFa were measured by RT-PCR and ELISA. Some rats fed with normal rat food served as controls.</p><p><b>RESULTS</b>At the 8th week fatty livers appeared, and hepatic expressions of CD14 (25.9+/-1.9) and TLR4mRNA (1.75+/-0.81) were upregulated compared to those in the control group (25.9+/-1.9 vs 12.4+/-0.7, 1.75+/-0.81 vs 0.98+/-0.33, P < 0.01, t > 2.756 and P < 0.05, t > 2.045). The hepatic expressions of the two kinds of receptors increased with the appearance of NASH at week 12 (61.8+/-1.9 and 1.88+/-0.72, P < 0.01, t > 2.756 and P < 0.05, t > 2.045), They reached to their peaks at week 16 (71.5+/-1.3 and 5.64+/-0.87, both P < 0.01 and t > 2.756), and decreased slightly at week 24 (67.7+/-6.6 and 4.98+/-0.72, both P < 0.01 and t > 2.756). Hepatic expressions and serum levels of TNFa also increased starting at week 8, and remained at that high level from week 8 to week 24.</p><p><b>CONCLUSION</b>The hepatic expressions of CD14 and TLR4 were up-regulated gradually in the established rat NASH model. It may be one of the factors responsible for the increase of hepatic sensitivity to LPS injury of the NASH rats and may play an important role in the pathogenesis of NASH.</p>

Radiosensitizing effect of celecoxib on human pancreatic carcinoma / 中华消化杂志

Objective To investigate the sensitizing effects and the mechanisms of selective cyclooxygenase-2(COX-2)inhibitor celecoxib on radiotherapy of pancreatic cancer.Methods Radiosen- sitization of celecoxib in pancreatic cancer cell SW1990 in vivo and in vitro were investigated by colony forming assay and xenograft tumor model.Expressions of proliferating cell nuclear antigen(PCNA)and Cyelin D1 were assessed by Western Blot.Effect on apoptosis was studied by TUNEL.Expression of bcl-2 and bax was assayed by RT-PCR.Expression and secretion of matrix metalloproteinases(MMPs) and tissue inhibitors of metalloproteinases(TIMPs)were assessed by RT-PCR and zymography.Results Celecoxib enhanced the effect of radiotherapy on pancreatic cancer in vitro and in vivo.TUNEL demonstrated a significant increase of apoptotic cells in vitro after treatment with celecoxib alone or com bined with radiation,but no change after radiation.Expression of bcl-2 was decreased by celecoxib;radi- ation induced the expression of bcl-2;combination of celecoxib and radiation significantly suppressed the expression of bcl-2.In vitro,angiogenesis and cell invasion potential of pancreatic cancer cells were in- hibited by celecoxib,and celecoxib combined with radiation,but without significant change in radiation group compared with the control group.Expression and secretion of MMP-2 and MMP-9 were closely related to the changes in angiogenesis and cell invasion potential,while the expressions of TIMP-1 and TIMP-2 did not alter significantly in all groups.Conclusions The selective eyclooxygenase 2 inhibitor celecoxib potently enhances the effect of radiation on the treatment of pancreatic cancer. Induction of apoptosis,inhibition of angiogenesis and invasion are involved in the mechanism of cele- coxib treatment.

Influences of angiotensinⅡtype 1 receptor antagonist on pancreatic steilate cells / 中华消化杂志

To investigate the effects of Losartan,an angiotensinⅡ(AngⅡ)receptor(AT_1) antagonist,on pancreatic stellate cells(PSCs)and its possible mechanisms.Methods (1)PSCs were isolated from pancreatic cancerous samples to test the expressions of AT_1 and collagenⅠafter incubated with AngⅡor/and Losartan.(2)Ninety S-D rats were divided into normal group,control group and treatment group,with 30 rats in each.The rats in control and treatment groups were induced pancreatic fibrosis by injection of 2% trinitrobenenze sulfonic acid(TNBS)into biliopancreatic duct.Rats in treat- ment group were then treated with Losartan by garage daily and rats in control group were only given distilled water.The rats were sacrificed on day 3,7,14,21 and 28,respectively,and pancreas were removed.The histological abnormalities were observed by electron microscope.The mRNAs of trans- forming growth factor?_1(TGF?_1)and procollagenⅠwere detected by reverse transcription-polymerase chain reaction(RT-PCR).The expression of TGF?_1 and?-smooth muscle actin(?-SMA)proteins was assessed by immunohistochemistry and the level of?-SMA protein was quantified by Western blot. Results In vitro,there existed AT_1 expression in PSCs,and Losartan reduced expression of collagenⅠ.Losartan treatment reversed the histological abnormalities observed by electron microscope,com- pared to treatment with distill water.The expression of?-SMA,TGF?_1 and procollagenⅠwere signifi- cantly higher in the control group than those in normal group and were reduced by Losartan to different extent in treatment group.Conclusion AT_1 antagonist can inhibit the activation and the profibrogenic action of PSCs by blocking AT_1 receptor-mediated pathways.

A pilot study on establishment of clinical evaluation system for gut barrier dysfunction / 中华消化杂志

Objective To investigate the potential influencing factors which possibly effected the gut barrier function.The effort was made to establish a clinical evaluation system of gut barrier dysfunc- tion.Methods Fifty-three critically ill patients with an APACHEⅡscore of 8 or more and 27 patient which APACHEⅡscore was 6 or less were recruited.Plasma was reserved for measurement of endo- toxin,tumor necrosis factor-?,diamine oxidase,D-lactic acid and high sensitive C reactive protein,uri- nary excretion of lactulose and mannitol and the urinary content of intestinal fatty acid binding protein (IFABP) were determined as well.Analyses was achieved by univariate,multivariate analysis and receiver operating characteristic curve.Results In the logistic regression models,gut barrier was affect- ed by many factors.The ratio of lactulose and mannitol in urine,the urinary content of IFABP of 24 hours and endotoxin level of plasma were identified as the most intimate factors which could associate with gut barrier function.The optimal operating point of plasma endotoxin,ratio of urinary lactulose and mannitol and content of urinary IFABP of 24 hours were 0.145,17 ng and 0,055 EU/ml respectively based on the results of receiver operating characteristic curve,the sensitivity and specificity were 84.5% vs 88%,78% vs 88% and 78% vs 78%.The doubtable value interval of urinary ratio of lactulose and mannitol was limited as 0.178 to 0.082.Conclusion Gut barrier dysfunction should be suspected when critically ill patients presented eertains gastrointestinal symptoms and had the proofs of increasing intesti- nal permeability,hypoperfusion of gut and higher level of plasma endotoxin.

Effect of 5-lipoxygenase and 12-lipoxygenase on proliferation and apoptosis of pancreatic cancer cell / 中华消化杂志

Objective To examine the expression of lipoxygenases (LOXs)on human pancreatic carcinoma and their effects on proliferation and apoptosis of human pancreatic carcinoma in vitro.Methods Expression of 5- LOX and 12-LOX in the tissue of human pancreatic carcinoma and pancreatic cancer cell line (SW1990) was detected by immunohistochemical staining and RT-PCR.To examine the effects of polyunsaturated fatty acids on pancreatic cancer cell line,the proliferation rate of SW1990 cells treated with arachidouic acid (AA),docosahexaenoic acid (DHA) and eicosapentenoic acid (EPA) was analyzed by MTT and the incorporation of bromodeoxyuridine (BrdU) methods.An annexinV/propidium iodide (PI) assay with flow cytometry and in situ terminal deoxynucleotidyl transferase-mediated dUTP-digoxigenin end labeling (TUNEL) assay were used to identify potential induction of apoptosis by the unsaturated fatty acids.The effects of 5-LOX inhibitor (MK-886) and 12-LOX inhibitor (baicalein) on the proliferation of SW1990 cells were determined by above methods.Results Both 5-LOX and 12-LOX were over expressed in the tissue of human pancreatic carcinoma and the pancreatic cancer cell line.AA had a stimulatory effect on the growth of SW1990 cells while EPA and DHA had an inhibitory effect in a dose-dependent manner.The apeptosis of SW1990 cells co-cultured with DHA or EPA lasting for 24 hours was increased markedly.Furthermore, both MK-886 and baicalein in a dose-dependent-manner inhibited the growth of SW1990 cells ard induced a significant increase of apoptotic cells rate.Conclusions Expression of lipoxygenase is up-regulated in human pancreatic cancer. Polyunsaturated fatty acids have regulatory effects on the growth of pancreatic carcinoma in vitro.AA stimulates proliferation of pancreatic cancer cell line,while DHA and EPA suppress proliferation,simultaneously inducing apoptosis of pancreatic cancer cell line.Blocking the functions of lipoxygenase with its inhibitor exerts a negative regulatory effect on the growth of pancreatic carcinoma in vitro.Lipoxygenase might be a novel therapeutic target for the pancreatic cancer.