[Impact and related mechanisms of glucose fluctuations on aortic fibrosis in type 1 diabetic rats].

Objective: To investigate the impact and related mechanisms of glucose fluctuations on aortic fibrosis in rats with type 1 diabetes mellitus. Methods: After injection of streptozotocin (STZ), male Sprague Dawley (SD) (8-12 weeks) rats (n=24) were randomly divided into three groups in accordance with the random number table: controlled STZ-induced diabetes (C-STZ) group (n=8); uncontrolled STZ-induced diabetes (U-STZ) group (n=8); STZ-induced diabetes with glucose fluctuations (STZ-GF) group (n=8). After three weeks, rats were sacrificed and aorta was obtained, aortic fibrosis was detected by Masson trichrome staining. The expression of collagen type 1 (collagen Ⅰ) was tested by immunofluorescence. The expression of runt-related transcription factor 2 (Runx2) was tested by immunohistochemistry. The mRNA levels of collagen Ⅰ and Runx2 were detected by quantitative real-time PCR (qRT-PCR). The protein expressions of collagen Ⅰ, Runx2 and nuclear factor (NF)-κB were determined by Western blot. Primary rat aortic smooth muscle cells (VSMCs) were cultured in three conditions: normal glucose (NG), high glucose (HG) and glucose fluctuations (GF). Cells in GF group were incubated for 72 hours with glucose alternating between 5.5 and 25 mmol/L every 12 hours. TPCA-1, the inhibitor of NF-κB, the expression of collagenⅠin different groups of cells was tested by immunofluorescence. The protein expressions of collagen Ⅰ, Runx2 and NF-κB were also determined by Western blot. Results: (1) The quantitative ratios of the area of fibrosis in the C-STZ group, U-STZ group, STZ-GF group were (8.42±0.10)%, (21.30±0.74)% and (44.39±1.09)% (P<0.05), respectively. The means of integral optical density (IOD) of collagenⅠ in the three groups were 11.92±0.88, 50.04±3.56 and 77.52±2.69, respectively (P<0.05). The mRNA levels of collagenⅠ in the three groups were 1.00±0.10, 2.02±0.28 and 2.83±0.33, respectively (P<0.05). The protein expressions of collagenⅠ in the three groups were 1.05±0.03, 2.06±0.32 and 4.93±0.25, respectively (P<0.05). (2) The average IOD of Runx2 in the three groups were 150.00±7.35, 204.84±2.32 and 391.48±7.13, respectively (P<0.05). The mRNA levels of Runx2 in the three groups were 1.02±0.02, 1.27±0.04 and 2.18±0.12, respectively (P<0.05). The protein expressions of Runx2 in the three groups were 1.03±0.01, 2.34±0.36 and 4.52±0.75, respectively (P<0.05). (3) The protein expressions of NF-κB in the three groups were 1.02±0.01, 1.96±0.13 and 2.64±0.21, respectively (P<0.05). (4) In vitro, application of inhibitor of NF-κB reversed glucose fluctuations-induced upregulation of protein levels of Col Ⅰ and Runx2 (P<0.05). Conclusion: Glucose fluctuations could aggravate aortic fibrosis through activating Runx2 via NF-κB signaling pathways.

[Effects of n-3 polyunsaturated fatty acid on store-operated calcium channels in coronary artery smooth muscle cells derived from diabetic rat].

Objective: To investigate the impact of n-3 polyunsaturated fatty acid (n-3 PUFA) on function and expression of store-operated calcium channels (SOCC) in coronary artery smooth muscle cells (SMC) derived from diabetic rat. Methods: A total of 180 healthy male Sprague-Dawley (SD) rats were randomly divided into normal group (N, n=45), placebo-treated diabetic group (D, n=45), lose dose n-3 PUFA treated diabetic group (DL, n=45) and high dose n-3 PUFAs treated diabetic group (DH, n=45). Streptozotocin-induced diabetic rat animal model was established by two consecutive intraperitoneal injections. After modeling, rats in group DL and DH were treated with 10 mg·kg(-1)·d(-1) and 50 mg·kg(-1)·d(-1) n-3 PUFAs respectively per gavage for eight weeks. After eight weeks, rat coronary artery SMC was isolated by enzyme digestion. Changes of cytosolic calcium concentration in coronary artery SMC were examined by calcium fluorescence imaging technique, coronary artery tension was detected by myograph system, and protein expressions of SOCC on coronary artery SMC were measured by Western blot. Results: SOCC induced ΔF340/F380 of group N, D, DL and DH were 0.425±0.023, 0.838±0.037, 0.342±0.052 and 0.364±0.045 respectively, which was significantly lower in group N, DL, DH than in group D (P<0.05). SOCC induced changes of tensions were 0.94±0.09, 1.95±0.18, 1.35±0.24 and 1.01±0.18 in the group N, D, DL and DH, respectively, which was significantly lower in group N and DH than in group D (P<0.05). Protein expressions of STIM1, Orai1 and TRPC1 were significantly higher in diabetic rat coronary SMC than in group N (P<0.05). STIM1 protein expressions were significantly lower in group DL and DH than in group D, and Orai1 and TRPC1 protein expressions were similar among group. Conclusions: Coronary artery tension, cytosolic calcium concentration and protein expressions of SOCC are higher in diabetic rat coronary artery SMC when compared with normal rats. n-3 PUFA intervention could downregulate the protein expression of SOCC, reduce cytosolic calcium concentration and coronary artery tension, and is protective to the diabetic injury in coronary artery.

[Composition of potassium channels in normal rat coronary smooth muscle cells and activation effects of docosahexaenoic acid].

OBJECTIVE: To investigate the composition of potassium channels in normal rat coronary smooth muscle cells (CASMCs) and the activation effects of docosahexaenoic acid (DHA). METHODS: CASMCs were isolated by enzyme digestion.Effects of different types of potassium channel blockers and/or DHA on potassium channels currents were studied by whole-cell patch clamp technique. RESULTS: Potassium currents were significantly increased with 5 µmol/L DHA perfusion (P<0.05). The current density was increased from (52.80±6.68) pA/pF to (110.09±13.39) pA/pF (P<0.05) after DHA perfusion when the stimulation voltage was 100 mV.Compared with baseline, potassium currents were significantly decreased by various inhibitor perfusion (tetraethylammonium: (49.63±5.75) pA/pF vs. (13.96±2.18) pA/pF; ibritoxin: (50.67±7.89) pA/pF vs. (26.53±4.68) pA/pF; TRAM-34: (52.60±7.02) pA/pF vs. (46.05±7.60) pA/pF; apamin: (51.97±3.83) pA/pF vs. (44.89±5.04) pA/pF; 4-aminopyridine: (51.19±3.44) pA/pF vs. (29.92±2.81) pA/pF; glyburide: (49.67±1.77) pA/pF vs. (49.61±1.87) pA/pF, all P<0.05). In presence of different inhibitors, potassium channel current densities were increased after DHA perfusion except tetraethylammonium (tetraethylammonium: ( 12.79±1.89) pA/pF; ibritoxin: (67.08±5.54) pA/pF; TRAM-34: (117.91±21.79) pA/pF; apamin: (108.33±7.06) pA/pF; 4-aminopyridine: (127.73±20.56) pA/pF; glyburide: (121.53±13.83) pA/pF, all P<0.05 compared with baseline). CONCLUSIONS: Large-conductance calcium-activated potassium channel and voltage-gated potassium channel are the major constituents of potassium channels in CASMCs.DHA can activate potassium channels in CASMCs, mainly the large conductance calcium-activated potassium channel, thus dilate coronary arteries.

[Docosahexaenoic acids activate large conductance calcium-activated potassium channels via phospholipase C- inositol triphosphate- calcium pathway in normal rat coronary smooth muscle cells].

OBJECTIVE: To investigate the mechanisms of docosahexaenoic acids (DHA) on activating large conductance calcium-activated potassium channels (BK channels) in normal rat coronary smooth muscle cells. METHODS: Normal coronary smooth muscle cells were isolated by enzyme digestion from Sprague-Dawley rats. BK currents were recorded by patch clamp in whole cell and single channel configurations, respectively. The effects of DHA on cytosolic calcium concentrations were examined by recording the changes of fluorescence intensity ratios. RESULTS: DHA (1 µmol/L) could activate BK channels. Open probabilities (NP0) of BK channels at test potential 60 mV, and calcium concentrations in external solution at 0, 0.01, 0.1, 1, 3, 10, 50 and 100 µmol/L were 0.002 7±0.000 4, 0.006 0±0.001 4, 0.097 2±0.010 6, 0.137 9±0.032 9, 0.468 7±0.163 7, 2.097 1±0.310 4 and 3.120 4±0.242 7, respectively (P<0.05, n=4). Before DHA perfusion, the fluorescence intensity ratio was 0.51±0.01, and the ratios were 0.53±0.02 and 0.55±0.01 after 0.001 and 0.01 µmol/L DHA perfusion, respectively (P>0.05, n≥5). The ratios were 0.64±0.01, 0.65±0.01, 0.70±0.01, 0.69±0.01, 0.68±0.01 and 0.67±0.02 after 0.1, 0.3, 1, 3, 5 and 10 µmol/L DHA perfusion, respectively, and EC50 was (0.04±0.02) µmol/L(P<0.05, n≥4). They were all higher than that before DHA perfusion. After incubating with phospholipase C (PLC) blocker U73122 and inositol triphosphate (IP3) blocker 2-APB, the ratios were 0.52±0.01 and 0.49±0.02 on the setting of 0.1 µmol/L DHA, respectively. Compared with control group(0.64±0.01), the ratios decreased after incubating with blockers (P<0.05, n≥4). CONCLUSIONS: Docosahexaenoic acids can activate large conductance calcium-activated potassium channels by the pathway of PLC-IP3-Ca(2+) to increase cytosolic calcium concentration in normal coronary smooth muscle cells, dilate the coronary vessels and bestow protective effects on cardiovascular system.

Therapy-related leukemia and myelodysplasia in small-cell lung cancer. Report of a case and results of morphologic, cytogenetic, and bone marrow culture studies in long-term survivors.

The bone marrow of 11 patients with small-cell lung cancer, who survived more than two years following combined-modality therapy, was subjected to morphologic, cytogenetic, and bone marrow culture studies. One patient, after a prodrome of anemia and thrombocytopenia, developed acute leukemia 60 months after the start of chemotherapy. Four months before frank leukemia developed, bone marrow culture studies showed a marked inability to form colonies. Cytogenetic studies demonstrated an abnormal clone of cells that included the deletion of the long arm of chromosome 5. No morphologic abnormalities were noted in the bone marrow of any other long-term survivor; however, the mean corpuscular volume of peripheral red blood cells was greater than normal in three of four patients who remain alive and disease free. In one of these patients marrow culture studies also failed to grow colonies. The other patients showed a decreased ability to form multilineage colonies and colonies of the granulocyte-macrophage lineage in vitro compared with a control population. All patients showed some degree of aneuploidy on cytogenetic analysis; in two cases approximately 50% of cells were aneuploid. However, no clonal abnormality was detected in any patient. Follow-up for the development of secondary acute leukemia and other long-term complications continues in these patients.