Synergetic effect of yihuo qingyi decoction (see text) and recombinant staphylokinase in treatment of severe acute pancreatitis of rats.

OBJECTIVE: To investigate the effect of recombinant staphylokinase (r-Sak) and the Chinese medicine Yihuo Qingyi Decoction ((see test) Herbal decoction for severe acute pancreatitis) in the treatment of the severe acute pancreatitis (SAP) in rats, and to observe the synergistic effect of the two. METHODS: One hundred and sixty-two adult male SD rats with the body mass of 250-280 g were randomly divided into the following 5 groups: sham operation group (n = 18), control group (n = 36), Yihuo Qingyi Decoction treatment group (n = 36), r-Sak treatment group (n = 36), and Yihuo Qingyi Decoction plus r-Sak treatment group (n = 36). The SAP ratmodel was prepared by retrograde injection of 5% sodium taurocholate into the cholangiopancreatic duct. Two days before modeling, Yihuo Qingyi Decoction was intragastrically administrated, and r-Sak was intraperitoneally injected. The survival rate within 18 h after modeling was determined. The pancreatic blood flow, the weight of ascites, and the serum amylase and lipase were investigated at 6 h, 12 h, and 18kh after modeling, and the pancreatic tissue was examined under light microscopy to see its pathological change. RESULTS: The 18 h survival count of group A, B, C, D and E rats was 9, 2, 6, 7 and 8 respectively. After r-Sak and Yihuo Qingyi Decoction intervention, the serum amylase and lipase and the weight of ascites were significantly decreased, especially in group E.18 h after modeling, the level of the serum amylase and lipase and the weight of ascites in group E was 1 100 +/- 118 U x L(-1), 1 000 +/- 150 U x L(-1) and 13.40 +/- 1.80 g respectively, obviously lower than that of group B (P < 0.05). After SAP was induced, the pancreatic blood flow showed a tendency to decrease, but the decrease extent in the treatment groups was smaller than that in the control group. 18h after modeling, the pancreatic blood flow in group B and group E was 30.16 +/- 8.96 mL x 100 g(-1) x min(-1), and 129.10 +/- 42.58 mL x 100 g(-1) x min respectively, there was significant difference (P < 0.05). The pathological change of the pancreatic tissue was alleviated in the treatment groups. CONCLUSION: Both r-Sak and Yihuo Qingyi Decoction play a beneficial role in the treatment of rat SAP and there is a synergistic effect between the two.

[High glucose induces INS-1 cell apoptosis by activating nuclear factor-κB].

OBJECTIVE: To study of the role of nuclear transcription factor-κB (NF-κB) in high glucose-induced apoptosis in INS-1 cells. METHODS: Rat insulinoma (INS-1) cells cultured in RPMI 1640 medium were treated with 11.1 mmol/L glucose, 33.3 mmol/L glucose, or 33.3 mmol/L glucose plus 5 µmol/L NF-κB inhibitors for 48 h. The expression of NF-κB subunit P65 protein in the cell nuclei was detected by Western blotting, IKK belta mRNA level by quantitative RT-PCR, and cell apoptosis by Annexin V-PI double staining. RESULTS: Compared with the control levels, IKK belta mRNA levels of the cells significantly increased in response to 33.3 mmol/L glucose exposure (P<0.01), which also resulted in significantly increased P65 protein expression in the cell nuclei (P<0.01) and cell apoptosis rate (P<0.05). Compared with those in the high glucose group, the expression of IKK belta mRNA and P65 protein and cell apoptosis rate decreased significantly after treatment with 33.3 mmol/L glucose plus 5 µmol/L NF-κB inhibitors (P<0.05). CONCLUSION: High glucose induces NF-κB activation in INS-1 cells, and inhibition of NF-κB activation may protect INS-1 cells from high glucose-induced cell apoptosis.

[Effects of heme oxygenase-1 on proteins related to apoptosis in INS-1 cells exposed to intermittent high glucose].

OBJECTIVE: To study the effect of heme oxygenase-1(HO-1) on proteins related to apoptosis in INS-1 cells with exposure to intermittent high glucose. METHODS: INS-1 cells cultured in vitro were divided into control group, persistent high glucose group (PHG), intermittent high glucose group (IHG), CoPP + intermittent high glucose group (CoPP+IHG), and ZnPP+ intermittent high glucose group (ZnPP+IHG). After 72 h of treatment with the corresponding protocols, the cells were examined for expressions of HO-1 protein by Western blotting and for expressions of Bax and Bcl-2 by immunocytochemistry. RESULTS: In comparison with the control group, the cells in both PHG group and IHG group showed significantly increased expressions of HO-1 (P<0.01) and decreased Bcl-2/Bax ratios (P<0.05). The cells in CoPP+ IHG group exhibited a greater HO-1 protein expression but a lower Bcl-2/Bax ratio than those in IHG group (P<0.05) The ZnPP+IHG group demonstrated opposite changes in terms of HO-1, Bax and Bcl-2 expressions compared with the CoPP+IHG group. CONCLUSION: Intermittent high glucose can lower Bcl-2/Bax ratio in INS-1 cells, and HO-1 may protect INS-1 cells against apoptosis possibly by up-regulating the Bcl-2/Bax ratio.

[Effects of metformin on proliferation of human colon carcinoma cell line SW-480].

OBJECTIVE: To observe the effect of metformin on the proliferation of SW-480 cells and study the possible mechanism. METHODS: The proliferation of SW-480 cells treated with different concentrations of metformin was assessed by MTT assay, and the cell cycle changes were analyzed by flow cytometry. The expression of cyclin D1 in the treated cells was detected by Western blotting, and telomerase activity examined by telomeric repeat amplification protocol (TRAP) silver staining. RESULTS: Metformin decreased the proliferation of SW-480 cells in a dose- and time-dependent manner. The proportion of the cells at G0/G1 stage in the control and metformin-treated (5 mmol/L, 72 h) cells was (55.81-/+0.63)% and (63.38-/+0.99)%, the cell proportion at S stage was (31.11-/+3.05)% and (25.29-/+1.64)%, and that at G2/M stage was (13.09-/+3.00)% and (11.33-/+2.60)%, respectively. The expression of cyclin D1 in metformin-treated cells were lowered significantly as compared with that in the control cells. Telomerase activity was also decreased significantly in the cells after treatment with 5 mmol/L metformin for 72 h. CONCLUSION: Metformin can inhibit the growth of SW-480 cells mainly by blocking the cell cycle at G0/G1, down-regulating the expression of cyclin D1 and decreasing telomerase activity.

[Small interfering RNA-mediated islet neogenesis associated protein gene silencing inhibits the proliferation of INS-1 islet cells].

OBJECTIVE: To investigate the effect of small interfering RNA (siRNA)-mediated islet neogenesis associated protein (INGAP) gene silencing on the proliferation of islet cells. METHODS: Different siRNAs targeting INGAP gene were designed and transfected into INS-1 islet cells, and the expression levels of INGAP mRNA and protein following the transfection were detected using RT-PCR, flow cytometry and Western blotting. The proliferation of the transfected INS-1 cells was evaluated using MTT assay. RESULTS: Compared with those in the irrelevant siRNA, empty vector control, and un-transfected groups, the expression levels of INGAP mRNA and protein in the cells transfected with siRNA6 were reduced significantly. The cell proliferation rate significantly increased after transfection with siRNA6 (P<0.05). CONCLUSION: siRNA targeting INGAP can effectively down-regulate INGAP expression and inhibit the proliferation of INS-1 cells.

[High glucose impairs mitochondrial respiratory chain function in pancreatic beta cells].

OBJECTIVE: To investigate the effect of high glucose on mitochondrial respiratory chain function in INS-1 cells. METHODS: The pancreatic beta cell line INS-1 was divided into the normal control (NC), high glucose (HG), and N-acetyl-L-cysteine (NAC) pretreatment groups, which were cultured for 72 h in the presence of 5.5 mmol/L glucose, 16.7 mmol/L glucose, and 16.7 mmol/L glucose with 1.0 mmol/L NAC, respectively. The activities of the enzyme complexes I and III of the respiratory chain in the cells were assessed with spectrophotometry, the ATP levels were examined using a luciferinluciferase kit, and insulin levels detected by radioimmunoassay. RESULTS: The activities of the respiratory chain enzyme complexes I and III were 1.53-/+0.24 and 1.08-/+0.22 micromol.mg(-1).min(-1) in high glucose group, respectively, significantly lower than those in the normal control group (2.31-/+0.33 and 1.92-/+0.39 micromol.mg(-1).min(-1), P<0.01). ATP and insulin levels also decreased significantly in high glucose group as compared with those in the normal control group (P<0.01). The addition of NAC partially inhibited high glucose-induced decreases in the enzyme complex activities, ATP levels and insulin secretion (P<0.05). CONCLUSION: The respiratory chain function is positively correlated to insulin secretion in INS-1 cells, and exposure to high glucose causes impairment of the two enzyme complexes activities through oxidative stress, resulting in the mitochondrial respiratory chain dysfunction. High glucose-induced damages of the mitochondrial respiratory chain function can be partially inhibited by NAC.

[Relationship between expressions of serum amyloid A and insulin resistance in 3T3-L1 adipocytes].

OBJECTIVE: To study the relationship between the expression of serum amyloid A (SAA) and insulin resistance in 3T3-L1 adipocytes. METHODS: 3T3-L1 adipocytes were incubated with different concentrations of dexamethasone (10, 100, and 1000 nmol/L) for 48 h to establish cell models of insulin resistance at different resistant levels (models 1, 2, and 3, respectively). The degree of insulin resistance of 3T3-L1 adipocytes was assayed by 2-deoxy-[(3)H]-D-glucose uptake. Semi- quantitative RT-PCR was performed for quantification of SAA mRNA expression. SAA concentrations in the culture medium were determined by ELISA. RESULT: Dexamethasone did not affect the basal glucose transport (P>0.05). Insulin-stimulated glucose uptake was significantly decreased by 15% (P<0.05), 40% (P<0.01), and 55% (P<0.01) in models 1, 2, and 3 in comparison with the untreated group, respectively; the expressions of SAA mRNA were upregulated by 2.5 (P<0.01), 3.33 (P<0.01), and 4.08 folds (P<0.01) and SAA concentrations increased by 2.05, 3.13, and 4.23 folds, respectively. The expressions of SAA mRNA were positively correlated to the degree of insulin resistance (r=0.773, P<0.01) and SAA concentration (r=0.832, P<0.01). CONCLUSION: A cell model of insulin resistance has been established in 3T3-L1 adipocytes by dexamethasone exposure. SAA is closely associated with insulin resistance and may serve as a marker of insulin resistance.

[Cloning and secretory expression of islet neogenesis-associated protein in Pichia pastoris].

OBJECTIVE: To clone the recombinant human islet neogenesis-associated protein (rhINGAP) gene for its secretory expression in Pichia pastoris. METHODS: INGAP gene was amplified with PCR and inserted between Xho I and EcoR I downstream sites of the alpha factor of the recombinant plasmid alpha/pUC18. The fusion gene of alpha factor and INGAP was subsequently inserted between BamH I and EcoR I sites of the plasmid pPIC9K of P. pastoris. After confirmation with restriction enzyme digestion and sequencing, the positive recombinant plasmid that integrated INGAP gene was linearized with Sal I digestion and transformed into the yeast host strain GS115 through electroporation. The yeast transformants that harbored the INGAP gene with high copies were selected with the auxotroph medium and G418, followed then by PCR verification of the positive transformants, from which the expression of recombinant human INGAP was induced with methanol as the only carbone source. The antigenic activity of the desired protein was then detected using Western blotting and enzyme-linked immunosorbent assay (ELISA). RESULTS AND CONCLUSION: The recombinant expression plasmid INGAP/pPIC9K was successfully constructed, and 3 positive transformants were obtained. The expressed protein showed good antigenic activity as confirmed by Western blotting and ELISA.

[Effect of the recombinant staphylokinase on pancreatic ischemia in severe acute pancreatitis of rats].

OBJECTIVE: To investigate the changes in plasma endothelin-1 (ET-1) , von Willebrand factor (vWF), serum 6-keto-prostaglandin(1alpha) (PGF(1alpha)) , thromboxane B2 (TXB2), platelet aggregation rate maximum (PAGm) and pancreatic blood flow after reproduction of severe acute pancreatitis (SAP) in rat, and the effect of recombinant staphylokinase (r-Sak) on SAP. METHODS: Eighty-one SD rats were divided randomly into the sham-operated group (n=27), the SAP model group (n=27), and the r-Sak treatment group (n=27). SAP was produced by administration of 5% sodium taurocholate into the pancreatic duct. The abdomen of rats was opened at 6, 12 and 18 hours after reproduction of SAP for determining the pancreatic blood flow. Blood was obtained at 6, 12 and 18 hours after reproduction of SAP for determining the concentration of plasma vWF with enzyme-labeled immunosorbent assay (ELISA). The concentration of plasma ET-1 and serum 6-keto-PGF(1alpha), and TXB2 were detected by radioimmunoassay. The PAGm induced by collagen and eicosanoids was assessed. RESULTS: Pancreatic blood flow in the SAP group appeared to have a decreasing trend at 6,12 and 18 hours after operation and were significantly decreased at all time points after reproduction of the model, compared with those of the sham-operated group (all P<0.05). The PAGm, content of plasma ET-1, vWF, and TXB2 were significantly increased at all time points after reproduction of the model, while 6-keto-PGF(1alpha) was significantly decreased, compared with those of the sham-operated group (all P < 0.05). Compared with SAP model group, PAGm, the content of plasma ET-1, vWF, and serum TXB2 in the r-Sak group were decreased at all time points, however, the content of serum 6-keto-PGF(1alpha) was increased (all P<0.05). CONCLUSION: The r-Sak can improve pancreatic microcirculation and enhance pancreatic blood flow in rats with SAP, and may be beneficial in the treatment of SAP.

[Effects of Radix Salviae Miltiorrhizae on the PMN-EC adhesion in vitro at the early stage of endotoxemia].

OBJECTIVE: To evaluate the effects of Radix Salviae Miltiorrhizae (RSM) on the PMN-EC adhesion in vitro at the early stage of endotoxemia, and to probe into the mechanism there in involved. METHODS: The rabbit endotoxemia model was made, and one group was treated with RSM injection (2 ml/kg) instantly and 24 h after the modeling of endotoxemia. PMN-EC adhesion rate, adhesion molecule (CD11a/CD18 and CD11b/CD18) and TNF-alpha level were measured instantly, 2 h, 4 h, 8 h, 16 h, 24 h and 48 h after endotoxemia modeling. RESULTS: It was found that after the modeling of endotoxemia, the levels of CD11a/CD18, CD11b/CD18 and PMN-EC adhesion rate instantly increased in the endotoxemia group and RSM group, they went up to the peak at 4 h in the endotoxemia group, and at 2 h, 4 h, 8 h, 16 h, 24 h and 48 h, they were markedly lower in the RSM group than in the endotoxemia group (P < 0.05). The TNF-alpha level in the endotoxemia group began to increase 2 h after the modeling and reached to the peak at 8 h, with its level still higher than normal at 48 h, while the RSM group had lower level of TNF-alpha at all time-points(P < 0.05). CONCLUSION: The data from this animal experiment indicate that Radix Salviae Miltiorrhizae can decrease PMN-EC adhesion rate and the levels of CD11a/CD18, CD11b/CD18 and TNF-alpha in case of endotoxemia so as to improve the microcirculation.