Intestinal ischemia-reperfusion of macaques triggers a strong innate immune response.

AIM: To investigate inflammatory injury in the intestinal mucosa after intestinal ischemia-reperfusion (IIR) with Toll-like receptor (TLR)-mediated innate immunity. METHODS: Ten macaques were randomized into control and IIR groups. The distribution and expression level of TLR2, TLR4, MD2, nuclear factor (NF)-κB p65 and interferon (IFN)-γ were measured by immunohistochemical stain and western blotting. The mRNA expression of TLR4, TLR2, MD2, interleukin (IL)-1ß and tumor necrosis factor (TNF)-α were measured by reverse transcriptase-polymerase chain reaction. The cytokine levels in blood and intestinal tissues were measured by ELISA. RESULTS: Obvious hemorrhage and erosion of mucosae were seen in the IIR group. Expression of TLR2, TLR4, MD2, NF-κB p65 and IFN-γ was significantly higher in the IIR group than in the control group (0.13 ± 0.04, 0.22 ± 0.04, 0.16 ± 0.06, 0.65 ± 0.12, 0.38 ± 0.10 vs 0.07 ± 0.04, 0.08 ± 0.03, 0.04 ± 0.02, 0.19 ± 0.06, 0.14 ± 0.05, P < 0.05). In addition, the expression of TLR2, TLR4, MD2, IL-1ß and TNF-α mRNA in the IIR group were significantly higher than those of control group(1.52 ± 0.15, 1.39 ± 0.06, 1.94 ± 0.12, 1.48 ± 0.15, 0.66 ± 0.08 vs 0.31 ± 0.05, 0.5 ± 0.04, 0.77 ± 0.05, 0.35 ± 0.08, 0.18 ± 0.04, P < 0.05). Furthermore, IL-1ß, IL-6 and TNF-α levels in the macaques ileum and plasma were significantly higher than in the control group (plasma: 86.3 ± 15.2, 1129 ± 248.3, 77.8 ± 16.2 vs 29.5 ± 7.3, 19.8 ± 8.2, 5.6 ± 1.7; ileum: 273.4. ± 44.7, 1636 ± 168.0, 205.5 ± 30.7 vs 76.8 ± 20.5, 663.4 ± 186.9, 49.0 ± 9.4; P < 0.05). CONCLUSION: After IIR, general inflammatory injury in the intestinal mucosa is correlated with a strong innate immune response, mediated by activation of the TLR-NF-κB-cytokine pathway.

[The study of interfering of endogenous VEGF-C genes and the protein expression gastric cancer cell with siRNA technique].

OBJECTIVE: To explore the interfering effects of siRNA on endogenous VEGF-C genes and the protein expression in gastric cancer cells. METHODS: Cultured gastric cancer cell line SGC7901 cells were prepared in vitro. Five siRNA primers of VEGF-C were designed depending on the sequence of Accession No. BC035212. 1 in Genbank. After homology analysis, the primers were synthesized and transfected into SGC7901 cells. The endogenous VEGF-C mRNA level and its protein expression were observed. RESULTS: VEGF-C-siRNA was inserted into the gastric cancer cell successfully. Five siRNA primers of VEGF-C could inhibit VEGF-C genes and protein expression. CONCLUSION: siRNA could block the endogenous VEGF-C genes and the protein expression in gastric cancer cell.

miR-22 is down-regulated in gastric cancer, and its overexpression inhibits cell migration and invasion via targeting transcription factor Sp1.

Accumulating evidence has shown that microRNAs are involved in multiple processes in cancer development and progression. Recently, miR-22 has been identified as a tumor-suppressing microRNA in many human cancers. However, the specific function of miR-22 in gastric cancer is unclear at this point. In this study, we first measured miR-22 expression level in 30 pairs of gastric cancer and matched normal tissues, two normal and six gastric cancer cell lines by real-time quantitative RT-PCR. We found that the expression of miR-22 in gastric cancer tissues and cell lines was much lower than that in normal control, respectively. Transfection of miR-22 expression plasmid could significantly inhibit the cell migration and invasion in SGC-7901 and NCL-N87 gastric cancer cell lines. Moreover, we also showed that Sp1 was negatively regulated by miR-22 at the posttranscriptional level, via a specific target site within the 3'UTR by luciferase reporter assay. The expression of Sp1 was inversely correlated with miR-22 expression in gastric cancer tissues, and knockdown of Sp1 by siRNA inhibited cell malignant behaviors. Thus, our findings suggest that miR-22 acts as tumor suppressor by targeting the Sp1 gene and inhibiting gastric cancer cell migration and invasion. The findings of this study contribute to current understanding of the functions of miR-22 in gastric cancer.

[A prospective randomized control clinical trial about clopidogrel combined with warfarin versus clopidogrel alone in the prevention of restenosis after femoral-popliteal artery angioplasty].

OBJECTIVE: Using two antithrombotic treatment (clopidogrel vs. clopidogrel combined warfarin) strategies after femoral-popliteal artery angioplasty prospectively, to evaluate which strategy is more effective for the restenosis prevention. METHODS: Totally 50 patients referred for endovascular treatment (including the percutaneous transluminal angioplasty (PTA) and stent implantation) of the superficial femoral artery and popliteal artery from January 2008 to May 2009 were randomly divided into clopidogrel group (group A, 25 cases, 30 limbs) and clopidogrel plus warfarin group (group B, 25 cases, 33 limbs) before operation. Clinical outcomes and restenosis rate of the target lesions were evaluated at 3, 6 and 12 months after operation. RESULTS: Totally 88 patients were screened for participation in the study, 56 patients were included after the follow-up of 12 months. At 3 months, the rates of restenosis were 16.7% in group A and 18.2% in group B (χ² = 0.025, P = 0.874). At 6 months, the accumulated restenosis rates were 36.7% in group A and 36.4% in group B (χ² = 0.001, P = 0.98). At 12 months, the accumulated restenosis rates were 53.3% in group A and 42.4% in group B (χ² = 0.75, P = 0.387). Analysis for the critical limb ischemia sub-group showed that follow-up of 12 months, the accumulated restenosis rate was 8/10 in group A and 6/12 in group B (χ² = 1.023, P = 0.312). CONCLUSION: The clopidogrel alone treatment for PTA or PTA plus stent implantation of femoral popliteal artery has no statistically significant difference in comparison with the clopidogrel combined warfarin treatment in terms of the cumulative vascular restenosis rate at 3, 6, 12 months postoperatively.

[Somatostatin promotes spontaneous contractions of small intestinal circular muscle via activation of subtype 2 somatostatin receptors in myenteric nerve plexus of macaque intestine].

OBJECTIVE: To investigate the mechanism behind the inhibitive effect of somatostatin (SST) on the intestinal migrating contraction in vivo by observing the effect of SST on spontaneous contractions of the circular muscle of the isolated small intestine and visualizing SST and SST receptor 2 (SSTR2) in the intestinal muscle. METHODS: Transverse segments of macaque intestine from laparotomy, were mounted in a perfusion system. The effects of SST on their phasic contractions were recorded. SST and SSTR2 in the intestinal muscles of macaque were visualized by immunohistochemical staining and hybridization in situ. RESULTS: SST (1 x 10(-7) mol/L to 1 x 10(-4) mol/L) significantly increased the frequency of phasic contractions, in a dose-dependent manner, r = 0.984, P < 0.05; (8.33 +/- 1.53) times/min (SST 1 x 10(-7) mol/L) vs (2.67 +/- 0.58) times/min (blank control), P < 0.05. The contractive amplitudes of the isolated circular muscle were also enhanced with the increasing concentration of SST range from 1 x 10(-7) mol/L to 1 x 10(-5) mol/L, r = 0.991, P < 0.05. Both SST and SSTR2 were localized in myenteric nerve plexus between the longitudinal and the circular muscle of the macaque intestine. CONCLUSIONS: SST directly promoted spontaneous contractions of the circular muscle of macaque intestine via mediation of SSTR2 in myenteric nerve plexus between the longitudinal and the circular muscle. It is contributed to the inhibitive effect of SST on the intestinal migrating contractions.

[Correlation of somatostatin receptor expression in human hepatocellular carcinoma tissue to serum alpha-fetoprotein concentration].

BACKGROUND & OBJECTIVE: Somatostatin analogue (SSTA) has inhibitory effect on the growth of hepatocellular carcinoma (HCC). However, the expression of somatostatin receptor (SSTR) in HCC is still unclear and its correlation to serum alpha-fetoprotein (AFP) has not yet been explored. This study was to investigate the expression of SSTR1, SSTR2, SSTR3, SSTR4, and SSTR5 subtypes in HCC, and explore their correlations to serum AFP concentration. METHODS: The mRNA and protein expression of SSTR1, SSTR2, SSTR3, SSTR4, and SSTR5 subtypes in 40 specimens of HCC and 40 specimens of liver cirrhosis were detected by reverse transcription-polymerase chain reaction (RT-PCR) and immunohistochemistry (IHC), respectively. AFP levels in liver tissues and peripheral blood of the 40 HCC patients were measured with enzyme-linked immunosorbent assay (ELISA). RESULTS: The positive rates of SSTR1, SSTR2, SSTR3, SSTR4, and SSTR5 proteins were 47.5%, 70.0%, 50.0%, 65.0%, and 67.5% in HCC, and 55.0%, 67.5%, 52.5%, 60.0%, and 47.5% in liver cirrhosis tissues, respectively. The protein levels of SSTR1, SSTR2, SSTR3, SSTR4, and SSTR5 were markedly higher in HCC than in liver cirrhosis tissues. The expression of SSTR1, SSTR2, SSTR3, SSTR4, and SSTR5 in HCC had curilinear correlations with serum AFP levels (r = 0.882, 0.901, 0.877, 0.854, 0.903, respectively, P < 0.05). Serum AFP levels ranging between 200-800 ng/ml were associated with high expression of SSTRs in HCC; serum AFP levels below or over that range were associated with low expression of SSTRs. CONCLUSIONS: About 60% of the HCC tissues express SSTRs; the protein levels of SSTRs are much higher in HCC than in liver cirrhosis tissues. HCC patients with serum AFP level ranging between 200-800 ng/ml may be good candidates for SSTA therapy.

[Changes of vasoactive intestinal polypeptide and vasoactive intestinal polypeptide receptor 1 in small intestine and liver during macaque development].

The present study was aimed to investigate the changes of vasoactive intestinal polypeptide (VIP) and VIP receptor 1 (VIPR1) in small intestinal and hepatic tissues during macaque development. The tissue samples of small intestine, liver and blood samples from peripheral and portal vein of 4 macaques of 6-month fetus, 2-day neonate, 45-day neonate and adult were obtained after anesthetization. The concentration of VIP in blood or tissues of macaques was measured by radioimmunoassay. The distribution of VIP in small intestinal or hepatic tissues was visualized by immunohistochemical staining. The expression of VIPR1 was detected by in situ hybridization. The results showed that: (1) VIP concentration in intestinal tissue of 6-month fetus was (20.7+/-14.3) ng/mg protein, and a few VIP-positive nerve fibers first appeared in intestinal villus root and submucosal layer but not in muscle layer. The intestinal concentration of VIP increased gradually with macaque development and reached (514.8+/- 49.2) ng/mg protein in adult, significantly higher than that in 6-month fetus (P<0.01). (2) In adult animal, VIP-positive nerve fibers became thicker and gradually extended into the mucosal crypt, submucosal layer nerve, myenteric nerve plexus of annular muscle and indulge muscle, and annular muscle. Correspondingly, the expression of VIPR1 in intestine was up-regulated during development. (3) On the contrary, the levels of VIP and VIPR1 in liver were gradually decreased during development. (4) VIP concentration in small intestinal tissue was higher than that in hepatic tissue during development. The VIP level in portal vein was also significantly higher than that in peripheral blood during development. In conclusion, the levels of VIP and VIPR1 in mucosal crypt, submucosal layer nerve, myenteric nerve plexus of annular muscle and indulge muscle increase rapidly after birth. Most of VIP from intestinal tract is degraded in portal vein before entering liver, suggesting that VIP does not metabolize and decompose in liver, and that VIPR1 is only present in embryo hepatic blood vessels.

[Changes of somatostatin and expression of somatostatin receptor in small intestine and liver tissues during macaque development].

Intestinal tract, which produces more than fifty kinds of gut peptides, is regarded as the largest endocrine organ. With regard to the gut peptides, a number of studies were focused on their structure, function and the roles in some diseases. The changes in output or distribution of gut peptides in the intestinal tract during development have been largely unknown. This study was aimed to investigate the changes of somatostatin (SST) and somatostatin receptor 2 (SSTR2) in small intestinal and hepatic tissues during the development of macaque. The tissue samples of small intestine, liver or blood samples from peripheral and portal vein of 4 macaques in 6-month fetus, 2-day neonate, 45-day neonate and adult were obtained after anesthetization. The concentrations of SST in blood or tissues of macaques were measured by radioimmunoassay. The distributions of SST in small intestinal or hepatic tissues were visualized by immunohistochemical staining. The expression of SSTR2 was detected by in situ hybridization. SST concentration of intestinal tissue in 6-month-old macaque was (27.3+/-16.6) ng /mg protein and light positive staining of SST was localized in mucosal crypts but negative in muscle layer. The intestinal concentration of SST increased gradually with macaque development and reached to the peak [(120.1+/-35.3) ng /mg protein] in adult. It was significantly higher than that in fetus (P<0.01). Strong positive staining of SST was found in both mucosal crypts and myenteric nerve plexus of adult animal. SSTR2 was obviously expressed in intestinal epithelium of fetus but its expression was greatly reduced in epithelium and was shifted to mucosal crypts when grown to adult. Negative staining of SSTR2 in muscle layer of fetal or neonatal macaque turned to be positive in myenteric nerve plexus of adult. The levels of SST or SSTR2 in liver decreased gradually during development. SST concentrations of small intestinal tissue kept significantly higher than those of hepatic tissues in the macaque developing stages. SST levels of portal vein were also maintained significantly higher than those of peripheral blood in the macaque developing stages. In conclusion, the level of SST and expression of SSTR2 in mucosal crypt increased gradually with macaque development. SST from intestinal tract was quickly degraded in portal vein before entering into liver. SST positive myenteric nerve plexus was visualized only in mature macaque.