Effect of Codonopsis Radix and Polygonati Rhizoma on the regulation of the IRS1/PI3K/AKT signaling pathway in type 2 diabetic mice.

Objective: Codonopsis Radix and Polygonati Rhizoma (CRPR) has a good hypoglycemic effect. The aims of the present study were to investigate the effect of CRPR on high-fat/high-sugar diet (HFHSD)- and streptozotocin (STZ)-induced type 2 diabetes mellitus (T2DM) mice as well as to investigate the involved mechanism. Methods: A T2DM mouse model was generated by combining HFHSD and STZ. After the model was established, normal and model groups received the same volume of normal saline intragastrically, and the negative control group was treated with metformin (200 mg/kg·BW). The low, medium, and high CRPR groups received four consecutive weeks of oral gavage with CRPR doses of 2.5, 5, and 10 g/kg·BW, respectively, during the course of the study. Body weight and fasting blood glucose (FBG) were measured on a weekly basis. Enzyme-linked immunosorbent assay (ELISAs) were used to evaluate the serum and liver samples. Hematoxylin and eosin (H&E) staining was utilized to observe the pathological status of the liver and pancreas. Western blot (WB) analysis was performed to evaluate the protein expression levels of PI3K, p-PI3K, AKT, and p-AKT. Results: Compared to model mice, each treatment group had significantly elevated levels of FBG, total cholesterol (TC), and triacylglycerol (TG) (P<0.01 and P<0.05, respectively). The levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were significantly reduced in the treatment groups compared to the model group (P<0.01). Compared to the model group, fasting insulin (FINS) levels were elevated in all groups of CRPR (P<0.05), and there were significantly higher levels of high-density lipoprotein cholesterol (HDL-C) in both the low-dose and high-dose CRPR groups (P<0.05). H&E staining indicated that CRPR treatment reduced organ enlargement, improved liver lipid accumulation, and repaired islet injury in T2DM mice. Moreover, WB analysis demonstrated that all CRPR groups significantly upregulated the protein expression of IRS1, p-GSK3ß, PI3K, p-Akt and p-FOXO1(P<0.05) as well as significantly downregulated p-IRS1 and FOXO1 protein expression (P<0.05). Conclusion: The present study demonstrated that CRPR effectively improves the metabolic disturbance of lipids, repairs damaged liver tissues, repairs damaged pancreatic tissues, and reduces insulin resistance (IR) in T2DM mice. The mechanism of action may be associated with upregulation of the IRS1/PI3K/AKT signaling pathway and inhibition of IRS1 phosphorylation.

[Transport characteristics of Shuxiong prescription across Caco-2 cell monolayer model].

Shuxiong prescription (Notoginseng Radix et Rhizoma, Chuanxiong Rhizome and Carthami Flos) has the function of activating blood circulation to dissipate blood stasis, activating meridians to stop pain. This paper was mainly aimed to discuss the transport characteristics of Shuxiong prescription across Caco-2 cell monolayer. Safe concentration range of Shuxiong prescription against Caco-2 cell monolayer model was determined by MTT assay. The mechanism of Shuxiong prescription bidirectional transport was investigated by Caco-2 cell monolayer model. The apparent permeability coefficient Papp of digoxin was determined by high performance liquid chromatography (HPLC). The test results showed that the Papp of extract from Notoginseng Radix et Rhizoma, Chuanxiong Rhizome, Carthami Flos, Chuanxiong Rhizome+Carthami Flos and Shuxiong prescription transport from apical (AP) side to basolateral (BL) side was (3.12±0.73)×10⁻6, (2.58±0.41)×10⁻6, (4.97±0.64)×10⁻6, (4.63±0.57)×10⁻6, (5.79±0.68)×10⁻6 cm·s⁻¹, respectively, indicating that the transport of digoxin across Caco-2 cell monolayer model was active absorption, and the P-gp protein took part in the process. Chuanxiong Rhizome could significantly decrease the transport of digoxin from BL→AP(P<0.01) and increase its transport from AP→BL(P<0.05) significantiy. After the addition of Shuxiong prescription, the transport of digoxin from BL→AP was significantly inhibited(P<0.01). The results suggested that the extract of safflower had no effect on P-gp transport, nor on the independence diffusion of digoxin. The transport of digoxin could be degraded by the extract of Chuanxiong Rhizome and the extract of Shuxiong prescription from BL→AP(P<0.01), significantly; pseudo-ginseng had no effect on the independence diffusion of digoxin; the extract of safflower+Chuanxiong Rhizome had the same experimental result as Chuanxiong Rhizome extract.

[Expression of chemokine receptor CXCR4 in colorectal carcinoma and its relationship with clinicopathological parameters].

OBJECTIVE: To investigate the expression of chemokine receptor CXCR4 in colorectal carcinoma and its relationship with the clinicopathological parameters, and to reveal the role of CXCL12/CXCR4 in the invasion and metastasis of colorectal carcinoma. METHODS: CXCR4 expression was studied in 53 colorectal cancer tissues and 27 normal tissues by immunohistochemistry. Its relationship with clinicopathological characteristics of colorectal cancer patients were analyzed. The CXCR4 expression in tumor and normal specimens and its metastatic sites were assessed by RT-PCR and Western blot. RESULTS: Fifty-three colorectal cancer patients,collected from July 2005 to February 2007 in our hospital,were enrolled in this study. CXCR4 was positive in 39 cancer tissue specimens(73.6%) and its high expression rate (in > 50% of cells) was 45.3%. High CXCR4 expression rate was significantly higher in patients with lymph node metastases (N(1)+N(2): 65.4%) than that in those without metastases(N(0) 25.9%). There were also associations between the high CXCR4 expression and the vascular and lymphatic vessel invasions (P<0.01). Meanwhile, there was a rising trend of high expression rate according to American Joint Committee on Cancer (AJCC) stage and pathologic grade,but no significant difference was found(P>0.05). There were no significant correlation of CXCR4 expression with clinicopathological parameters such as tumor location, tumor size, depth of tumor invasion(P>0.05). In addition, the CXCR4 mRNA expression in primary tumor specimens (n=27) from AJCC stage IIII( patients was significantly higher than that in normal tissues. CXCR4 mRNA expression of liver metastasis specimens(n=5) was significantly higher as compared with the primary colorectal cancer specimens(P<0.01). CONCLUSIONS: Chemokine receptor CXCR4 is associated with the progression of colorectal carcinoma. High CXCR4 expression is associated with metastasis. The CXCL12-CXCR4 signaling pathway may be a potential novel target of therapy for patients with colorectal cancer.

[Promoter methylation and expression of Runx3 gene in gastric cancer].

OBJECTIVE: To analyze the relationships among the aberrant methylation of Runx3 gene promoter, the Runx3 protein expression and clinicopathological features in gastric cancer. METHODS: Methylation specific PCR was used to measure the promoter methylation status of Runx3 gene in tumor and the adjacent normal mucosal tissues from 40 patients with gastric cancer. Protein expression of Runx3 was measured by immunohistochemistry. RESULTS: The frequency of promoter methylation of Runx3 gene in gastric cancer tissue(55.0%) was significantly higher compared to the adjacent normal tissues (12.5%)(P<0.01). The positive rate of protein expression of Runx3 in gastric cancer tissue(37.5%) was significantly lower compared to the adjacent normal tissues (100%). There was marked association between hypermethylation and negative protein expression (P<0.05). The frequency of Runx3 promoter methylation was associated with histological type, N grade, and tumor stage. CONCLUSION: The promoter hypermethylation is a main mechanism of reduced or loss expression of Runx3 gene, which may provide molecular diagnosis and stage evaluation of gastric cancer.

Inhibition of CXCR4 activity with AMD3100 decreases invasion of human colorectal cancer cells in vitro.

AIM: To investigate the effect and mechanism of blockade of the CXC chemokine receptor-4 (CXCR4) signaling pathway by AMD3100, a small non-peptide CXCR4 inhibitor, on invasion and metastasis of colorectal cancer cells in vitro. METHODS: Human colorectal cancer cell line SW480 was treated with AMD3100 at different final concentrations. 3-(4,5-dimethylthiazole-2-yl)-2.5-dipheny-ltetrazolium bromide (MTT) assay was used to detect the effect of AMD3100 on cell proliferation. The invasion ability of SW480 cells was determined by cell invasion assay kit. In the presence of AMD3100, the CXCL12-mediated migratory response of SW480 cells was tested by classical chemotaxis assays. RT-PCR analysis and Western blotting were used to detect the expression of vascular endothelial growth factor (VEGF), matrix metalloproteinase-2 (MMP-2) and -9 (MMP-9) in SW480 cells. RESULTS: Cell viability was significantly suppressed by AMD3100 in a dose-dependent manner. AMD3100 (100 and 1000 ng/mL) significantly inhibited the invasion ability of SW480 cells. Treatment with AMD3100 markedly reduced the expression of VEGF and MMP-9 but not MMP-2 in SW480 cells. CONCLUSION: The CXCL12/CXCR4 system is an important mediator of proliferation and invasion of CXCR4-expressing colorectal cancer cells. AMD3100 inhibited invasion and metastasis activity of the colorectal cancer cell line SW480 through down-regulation of VEGF and MMP-9 expression.

[PCR-SSCP polymorphism of FSHbeta gene and its relationship with prolificacy of Jining grey goats].

The follicle-stimulating hormone beta-subunit (FSHbeta) gene was studied as a candidate gene for the prolificacy in Jining Grey goats. According to the sequence of ovine FSHb gene, nine pairs of primers were designed to detect single nucleotide polymorphisms of 5' regulatory region, exon 1 and exon 2 of FSHbeta gene in both high fecundity breed (Jining Grey goat) and low fecundity breeds (Liaoning Cashmere goat, Boer goat and Angora goat) by PCR-SSCP. The results indicated that the homology of nucleotide sequence of this fragment between goat and sheep was 98 percent. Only the products amplified by primer P9 displayed polymorphism. Three genotypes (AA, AB and AC) were detected in Jining Grey goats and Liaoning Cashmere goats. Three genotypes (AA, CC and AC) were detected in Boer goats. Six genotypes (AA, BB, CC, AB, AC and BC) were detected in Angora goats. Sequencing revealed a G-->A mutation at 94 bp of exon 2 of FSHbeta gene in the BB genotype in comparison to the AA genotype and a C-->T mutation at 174 bp of exon 2 of FSHbeta gene in the CC genotype in comparison to the AA genotype. The former mutation resulted in an amino acid change: alanine-->threonine, and the latter mutation did not cause any amino acid change. Genotype frequency of AA, AB and AC was 0.686, 0.137 and 0.177 in Jining Grey goats, respectively. The does with genotype AA had 0.78 (P<0.05) or 0.64 (P<0.05) kids more than those with genotype AB or AC in Jining Grey goats, respectively.

Diethyldithiocarbamate inhibits iNOS expression in human lens epithelial cells stimulated by IFN-gamma and LPS.

AIM: To investigate the biological activity of human lens epithelial cells (HLEC) in producing inducible nitric oxide synthase (iNOS) and nitric oxide (NO), and to assesse the effect of diethyldithiocarbamate (DDC) on iNOS mRNA levels and expression of NOS. METHODS: The human lens epithelial cell line SRA 01/04 was used in this experiment. Semi-quantitative reverse transcription polymerase chain reaction (RT-PCR) and Western blotting were used to detect, respectively, iNOS mRNA expression and protein production. RESULTS: A costimulation by interferon gamma (IFN-gamma) and lipopolysaccharide (LPS) was necessary for iNOS expression in HLEC. The expression of iNOS was significantly reduced in a dose-dependent manner by adding DDC from 10 micromol/L to 1 mmol/L. CONCLUSION: The expression of iNOS in HLEC needs co-stimulation with IFN-gamma and LPS and it is inhibited by DDC.

Peritoneal lavage cytology and carcinoembryonic antigen determination in predicting peritoneal metastasis and prognosis of gastric cancer.

AIM: To evaluate the role of peritoneal lavage cytology (PLC) and carcinoembryonic antigen (CEA) determination of peritoneal washes (pCEA) in predicting the peritoneal metastasis and prognosis after curative resection of gastric cancer. METHODS: PLC and radioimmunoassay of CEA were performed in peritoneal washes from 64 patients with gastric cancer and 8 patients with benign diseases. RESULTS: The positive rate of pCEA (40.6%) was significantly higher than that of PLC (23.4%) (P<0.05). The positive rates of PLC and pCEA correlated with the depth of tumor invasion and lymph node metastasis (P<0.05). pCEA was found to have a higher sensitivity and a lower false-positive rate in predicting peritoneal metastasis after curative resection of gastric cancer as compared to PLC. The 1-, 3-, and 5-year survival rates of patients with positive cytologic findings or positive pCEA results were significantly lower than those of patients with negative cytologic findings or negative pCEA results (P<0.05). Multivariate analysis indicated that pCEA was an independent prognostic factor for the survival of patients with gastric cancer. CONCLUSION: Intraoperative pCEA is a more sensitive and reliable predictor of peritoneal metastasis as well as prognosis in patients with gastric cancer as compared to PLC method.