Farnesyl Pyrophosphate Synthase Blocker Ibandronate Reduces Thoracic Aortic Fibrosis in Diabetic Rats.

BACKGROUND: This study assessed the effect of ibandronate (IBN), a farnesyl pyrophosphate synthase (FPPS) inhibitor, on vascular remodeling in diabetic rats. METHODS: A rat model of diabetes was induced by a high-fat and high-sugar diet combined with a small dose of streptozotocin. The diabetic rats received 5 µg/kg of ibandronate solution or normal saline subcutaneously every morning for 16 weeks. The morphology of the thoracic aorta was assessed by hematoxylin and eosin and Masson's trichrome staining techniques. Gene expression levels of connective tissue growth factor (CTGF) and FPPS were assessed by quantitative real-time polymerase chain reaction (qRT-PCR) analysis. CTGF and FPPS protein levels were determined by Western blotting analysis. RESULTS: Rats with diabetes mellitus showed moderate hyperglycemia, insulin resistance, hyperlipidemia and thoracic aortic fibrosis. FPPS was significantly upregulated in the thoracic aorta from diabetic animals. Interestingly, IBN treatment for 16 weeks alleviated the diabetes-induced histopathologic changes in the thoracic aortic wall and reduced CTGF protein and mRNA levels. CONCLUSIONS: These findings provided evidence that FPPS is involved in thoracic aortic fibrosis in diabetic rats. Meanwhile, IBN could alleviate vascular remodeling in diabetic animals.

TNF-α induces Drp1-mediated mitochondrial fragmentation during inflammatory cardiomyocyte injury.

Dynamin-related peptide 1 (Drpl)-mediated mitochondrial fission is an important process associated with cardiac dysfunction under different pathological conditions. The aim of the present study was to investigate the expression of Drpl during inflammatory myocardial injury. Sprague­Dawley rats were treated intraperitoneally with lipopolysaccharides (LPS). Furthermore, cultured H9C2 cardiomyocytes were treated with LPS, interleukin­6 (IL­6) and tumor necrosis factor­α (TNF­α). Total and mitochondrial proteins were isolated from the heart tissue of rats and from the H9C2 cardiomyocytes. Expression levels of Drp1 and RhoA were analyzed by western blotting. Mitochondrial morphology was determined using confocal laser microscopy. The levels of mitochondrial Drp1 and phosphorylated­Drp1 (p­Drp1) Ser616 were revealed to be increased in rats 6 h after injection with LPS (5, 10 or 20 mg/kg). Furthermore, treatment with LPS and IL­6 did not demonstrate a significant effect on the expression of total and mitochondrial Drp1 in H9C2 cardiomyocytes in vitro; however, treatment with TNF­α (20 ng/ml) significantly enhanced the levels of mitochondrial Drp1 and p­Drp1 Ser616. Following TNF­α treatment, the expression of Ras homolog gene family member A (RhoA) was also revealed to increase. Treatment with both Y­27632 and fasudil, [Rho kinase (ROCK) inhibitors], was demonstrated to attenuate the otherwise TNF­α­induced increase in p­Drp1 Ser616 and mitochondrial Drp1. In addition, it was revealed that Y­27632 and fasudil may also attenuate the TNF­α­induced increase in mitochondrial fragmentation and cell viability. Therefore, the findings of the present study suggest that TNF­α is the predominant inducer of Drp1 S616 phosphorylation during sepsis. The results of the present study also suggest that the RhoA/ROCK pathway may be involved in the phosphorylation and mitochondrial translocation of Drp1, which leads to mitochondrial fragmentation.

Isolation of mononuclear cells from CNS tissue using Percoll gradients centrifugation / 中国药理学通报

Aim To establish a rapid method to efficiently iso-late mononuclear cells from central nervous system ( CNS) tis-sues of mice that can be effectively utilized for identification of various immune cell populations in a single sample by flow cy-tometry. Methods For defining the feasibility and practicality of the method, wild-type C57BL/6 mice and two mouse models of CNS disease including EAE mice and APP/PS1 mice were used in this study. After the collection and homogenization of the brain and spinal cord tissues respectively, the mononuclear cells were isolated by spinning the 70% -30% Percoll gradients. Cell activities were detected by trypan blue staining, and the im-mune population that infiltrated CNS was identified by flow cy-tometry. Results The results of trypan blue staining showed that the survival rate of the isolated cells was above 90% in all groups. Flow cytometry analysis showed that the relative num- bers of lymphocytes infiltrating CNS of EAE and AD mice in-creased significantly compared with wild-type C57BL/6 mice. In addition, the relative numbers of Th1 and Th17 cell subsets me-diating the inflammatory response also increased significantly, while the decreased regulatory T cells frequency was observed in the two mouse models of CNS disease. Conclusions The cells isolated by the 70% ~30% Percoll gradients centrifugation can be effectively utilized for the identification of various immune cell populations in a single sample by flow cytometry. The meth-od described in this article is simple and rapid in operation and with high survival rate and activity of the cells, which can be ap-plied to the study of the mononuclear cells in CNS.

Effects of total flavones from Dendranthema morifolium on vasocontraction and proliferation of vascular smooth muscle cells.

Pharmacological studies have shown that the active components in Dendranthema morifolium exhibit protective effects against ischemia/reperfusion injury; however, its pharmacological action on blood vessels has not yet been investigated. The purpose of the present study was to assess the effects of the total flavones extracted from D. morifolium (Ramat.) Tzvel. cv. Hangju (FDM) on the vasocontraction and proliferation of vascular smooth muscle cells (VSMCs). The tension of rat thoracic aortic rings was measured using a mechanical force transducer attached to a recording system. FDM induced a dose­dependent relaxation of rings with endothelium pre­contracted by either phenylephrine (PE; 10(­6) mol/l) or a high concentration of potassium chloride (KCl; 60 mmol/l). FDM did not significantly affect the vasorelaxant effects on mechanically removed endothelium. In endothelium­denuded aortic rings depolarized by 60 mmol/l KCl, FDM inhibited the contraction induced by Ca2+. FDM reduced the transient contraction caused by PE in a Ca2+­free solution, but did not affect the contraction induced by phorbol ester. Furthermore, FDM inhibited the proliferation of VSMCs with or without growth stimulation by insulin. In conclusion, that the vasorelaxation induced by FDM in rat aortic rings is not dependent on the endothelium but is mediated via a reduction of the influx of extracellular Ca2+ through the voltage­dependent and receptor­operated channels and via the inhibition of the release of intracellular Ca2+ in VSMCs. The anti­proliferative activity of FDM suggests that it may be beneficial in inhibiting atherosclerosis.

Inhibition of farnesyl pyrophosphate synthase attenuates angiotensin II-induced fibrotic responses in vascular smooth muscle cells.

Through the regulation of the RhoA/Rho kinase (ROCK) pathway, angiotensin II (Ang II)-induced fibrotic responses contribute to vascular remodeling. Farnesyl pyrophosphate synthase (FPPS) plays an important role in cardiovascular remodeling through the modulation of the above-mentioned pathway. However, the role of FPPS in Ang II-induced fibrotic responses and the related molecular mechanisms have not yet been elucidated. In the present study, vascular smooth muscle cells (VSMCs) from Sprague-Dawley (SD) rats were stimulated with Ang II. Cell proliferation was measusred usin the cell counting kit-8 (CCK-8). The levels of connective tissue growth factor (CTGF), FPPS, and those of phosphorylated and total extracellular signal-regulated kinase (ERK)1/2, p38 and c-Jun N-terminal kinase (JNK) were determined by western blot analysis. RhoA activity was determined using a pull-down assay. The results revealed that stimulation with Ang II enhanced cell proliferation, and increased the protein expression levels of FPPS and CTGF in the VSMCs. The inhibition of FPPS with ibandronate sodium attenuated the Ang II-induced increase in cell proliferation, CTGF expresison and RhoA activity; these effects were partially reversed by treatment with geranylgeraniol and were mimicked by GGTI-286. Furthermore, both SB203580 (a specific inhibitor of p38) and SP600125 (JNK1, JNK2 and JNK3 inhibitor) diminished the Ang II-induced production of CTGF; however, the inhibition of FPPS reduced the Ang II-induced activation of p38 mitogen-activated protein kinase (MAPK) and JNK. In conclusion, our data indicate that FPPS may play an important role in Ang II-induced fibrotic responses in VSMCs, and the underlying mechanisms at least partly involve the modulation of RhoA activity, and the p38 and JNK pathways.

[Effects of metoprolol on electrophysiology of ischemic and anoxic myocardium in diabetic rats].

OBJECTIVE: To investigate the effects of metoprolol on electrophysiology of ischemic and anoxic myocardium in diabetic rats. METHODS: Forty Sprague-Dawley (SD) rats were divided into 4 groups: diabetes group; diabetes and ablation of left sympathetic nerve group; diabetes and metoprolol group and sham group. The diabetes model was induced by intraperitoneal injection of streptozotocin (STZ, 60 mg/kg). The ventricular diastolic effective threshold (DET), effective refractive period (ERP), and Ventricular fibrillation threshold (VFT) were measured. The serum concentration of nerve growth factor (NGF) was measured. RESULTS: Metoprolol increased DET of ischemic and anoxic myocardium in diabetic rats. The ablation of the left sympathetic nerve increased VFT of diabetic rats. VFT in metoprolo group was significantly increased compared to diabetes group after ischemia. The concentrations of NGF in diabetic group and metoprolol group were higher than those in sham group. There were no difference in NGF levels between ablation of left sympathetic nerve group and sham group. CONCLUSION: The remodeling of sympathetic nerve affects the electrophysiology of ischemic myocardium of diabetic rats. Metoprolol can increase the VFT and decrease the excitation threshold of the ischemic myocardium in diabetic rats.

Effects of Radix Ginseng and Radix Ophiopogonis extract (SMF) on protein S-nitrosylation in ischemic myocardial tissue / 中国中药杂志

<p><b>OBJECTIVE</b>To observe the effect of Radix Ginseng and Radix Ophiopogonis extract (SMF) on protein S-nitrosylation in rats myocardial with ischemia/reperfusion injury (MI/RI).</p><p><b>METHOD</b>Myocardial ischemia/reperfusion in rats were produced by occlusion of the left anterior descending coronary artery. To study the cardioprotective effects of SMF on the acute MI/RI rats, the serum levels of creatine kinase (CK), lactate dehydrogenase (LDH), and nitric oxide (NO) were determined. The change of the expression of endothelial nitric oxide synthase (eNOS) was detected by Western blot. The content of related S-nitrosylation proteins in myocardial tissue was measured by Biotin-Switch method.</p><p><b>RESULT</b>SMF significantly decreased the serum levels of CK and LDH as well as increased the serum levels of NO and the expression of eNOS in myocardial tissue. The contents of S-nitrosylation proteins were significantly increased from (4.42 +/- 0.60) micromol x g(-1) to (8.78 +/- 1.37) micromol x g(-1). The molecular weight of the majority S-nitrosylation proteins were in the range of 90 x 10(3)-117 x 10(3).</p><p><b>CONCLUSION</b>Increased expression of eNOS and NO induced by SMF may activate S-nitrosylation of many proteins in rat hearts. The change of the activities or functions of those proteins by S-nitrosylation may be an important mechanism for myocardial protective effects of SMF.</p>

[Involvement of potassium channel in hemin-induced cardioprotection in rat hearts].

OBJECTIVE: To investigate the effects of heme oxygenase 1 inducer hemin on protection of ischemia-reperfusion injury in rats and its mechanisms. METHODS: The Langendorff model of isolated rat heart was used; the left anterior descending coronary artery was occluded for 30 min and subsequently reperfused for 2 h. Then the ventricular function and infarct size were measured. RESULT: Hemin preconditioning prevented the increase in LVEDP, decrease in LVDP and +/- dp/dt(max) in the isolated ischemia-reperfusion rat hearts. The leakage of LDH and CK in the coronary effluent was significantly declined in hemin-treated rat hearts. And the infarct size was also reduced. Administration of a blocker of mitochondrial ATP-sensitive potassium channel (mitoK(ATP)) 5-HD (5 mg/kg) before hemin preconditioning increased the LVEDP, and reduced the LVDP and +/- dp/dt(max). The leakage of LDH and CK in the coronary effluent and the infarct size were also increased compared with only hemin-treated rat hearts. Pretreatment of the rats with a blocker of sarcolemmal ATP-sensitive potassium channel (sarcK(ATP)) HMR-1098 (6 mg/kg) before hemin preconditioning also abolished the protective effect. Infusion of paxilline (1 micromol/L), a blocker of calcium activated potassium channel (K(Ca)) for 10 min before ischemia/reperfusion led to larger infarct size and poorer myocardial performance as compared with the hemin group. The leakage of LDH and CK in the coronary effluent was also increased. CONCLUSION: Both mitoK(ATP)and sarcK(ATP)channels activation are required for the delayed cardioprotection induced by hemin. The opening of K(Ca) channels-dependent mechanism may be involved in the protection.

[Role of reactive oxygen species, nitric oxide and mitochondrial K(ATP) channels in TNF-alpha induced cardioprotection].

AIM: To explore the cardiac effect of TNF-alpha in postischemic heart and the possible mechanism. METHODS: Langendorff perfused rat heart was used to evaluate the contractile properties of myocardium by intraventricular pressure measurement. The isolated rat heart underwent 20 min of global ischemia followed by 20 min of reperfusion. The level of lactate dehydrogenase (LDH) in the coronary effluent was measured to evaluate the cardiac injury. And the activity of manganese superoxide dismutase (Mn-SOD) in myocardium was measured. RESULTS: Perfusion with TNF-alpha (104 U/L) attenuated the inhibitory effects induced by ischemia/reperfusion on left ventricular developed pressure (LVDP), left ventricular end-diastolic pressure(LVEDP), maximal rise/fall rate of left ventricular pressure (+/- dP/dtmax) and rate pressure product (LVDP multiplied by heart rate, LVDP x HR). TNF-alpha significantly decreased the release of LDH (P < 0.05) and increased the activity of Mn-SOD in the myocardium (P < 0.05). Antioxidant 2-MPG (0.3 mmol/L), NOS inhibitor L-NAME (0.5 mmol/L) or mitochondrial selective KATP channel inhibitor 5-HD (100 micromol/L) attenuated the increase in LVDP, +/- dP/dtmax and LVDP x HR, and decrease in LVEDP induced by TNF-alpha in ischemia/reperfusion heart, respectively. And the effects of TNF-alpha in reducing the levels of LDH and increasing the Mn-SOD activity were also attenuated by 2-MPG, L-NAME or 5-HD, respectively. CONCLUSION: TNF-alpha pretreatment attenuates the myocardial injury induced by ischemia/reperfusion, which coincides with the increasing of myocardial Mn-SOD activity. Reactive oxygen species, nitric oxide and mitochondrial KATP channels are involved in the cardioprotection induced by TNF-alpha.

[Modulation of iron on the vasodilating effect of interleukin-2 in the isolated aortic ring].

OBJECTIVE: To explore the effect and mechanism of iron on the vasodilating effect of interleukin-2 (IL-2) in the isolated aortic ring. METHODS: Isometric tension was recorded in response to drugs in organ bath. Ferric ammonium citrate (FAC) was added to the bath 30 min before phenylephrine (1 micromol/L), which was followed by IL-2 in a cumulative fashion. Spectrophotometry was used to determine the activity of nitric oxide synthase (NOS) of the thoracic aorta. RESULTS: FAC (0.1 - 10 micromol/L) alone did not affect the tension of rings,but inhibited the vasodilating effect of IL-2 (1 - 1,000 U/ml) in a dose dependent manner. IL-2(1, 10, 100, 1000 U/ml) decreased the aortic tension to (78.47+/-4.31)%, (66.86+/-5.55)%, (52.62+/-4.51)% and (42.39+/-4.27)% of pre-drug control, respectively. However, after incubation with 10 micromol/L FAC in the presence of IL-2, the aortic tension was reduced to (89.81+/-1.94)%, (86.13+/-3.11)%, (77.16+/-5.66)% and (68.76+/-5.69)% of pre-drug control, respectively. Pretreatment with L-arginine (1 mmol/L) abolished the inhibitory effect of FAC. Pretreatment with FAC attenuated the increased activity of NOS induced by IL-2 from (22.10+/-1.87)U/mg prot to (15.71+/-0.89)U/mg prot. High Ca(2+) (2.5 mmol/L) incubation did not change the inhibitory effect of FAC. Pretreatment with FAC attenuated the increased caffeine-releasable pool of Ca(2+) by IL-2. High K(+) (10 mmol/L) incubation abolished the inhibitory effect of FAC. CONCLUSION: FAC inhibits the vasodilating effect of IL-2 in the isolated aortic ring,which may be mediated by decreasing the activity of NOS. Intracellular calcium release and inward rectifier potassium channel are involved in the inhibitory effect of FAC.

[Treatment of interferon-alpha in reducing the endothelium-dependent relaxation of rat thoracic aorta].

OBJECTIVE: To investigate the vascular effect of acute and chronic treatment of interferon-alpha (IFN-alpha) in rat aortic rings. METHODS: Isolated thoracic aortic rings were mounted on the organ bath and the tension of the vessel was recorded. RESULTS: IFN-alpha(10, 100, 1,000 and 10,000 U/ml) caused concentration -dependent relaxation of endothelium-intact aorta rings preconstricted with phenylephrine (PE,10(-6)mol/L), to(90.1+/-0.91)%, (65.1+/-5.21)%, (39.5+/-8.22)% and (35.3+/-8.27)% of pre-drug control, respectively. Removal of the endothelium inhibited the relaxation by IFN-alpha. The vasorelaxant effect of IFN-alpha (100 U/ml ) was attenuated by pretreatment with L-NAME (10(-4)mol/L), methylene blue (10(-5)mol/L) or AMG (10(-4)mol/L), to (97.2+/-5.34)%, (95.1+/-6.25)% and (93.7+/-8.82)% of the control, respectively. Pretreatment with IFN-alpha (1,000,000 U/d, i.p.) for five days markedly inhibited the endothelium-dependent relaxation of the aortic rings to acetylcholine. But the endothelium-dependent relaxation to acetylcholine was not changed by pretreatment of IFN-alpha (10,000 U/ml) with the isolated aorta rings for 2 h. CONCLUSION: The vasorelaxation induced by IFN-alpha in rat aorta rings is endothelium-dependent and is possibly mediated by inducible nitric oxide synthase. Chronic treatment of IFN-alpha may impair the endothelium or NO-sGC pathway.

[NO/cGMP signal pathway involved in the disturbance of calcium homeostasis in vascular smooth muscle during the late phase of sepsis].

OBJECTIVE: To evaluate the alterations in calcium metabolism of the vascular smooth muscle of rat thoracic aorta in the late phase of sepsis and to investigate the involvement of nitric oxide (NO)/cyclic-GMP(cGMP) signal transduction pathway in the sepsis-induced vascular hyporeactivity. METHODS: Male Sprague-Dawley rats were subjected to sepsis by cecal ligation and puncture (CLP). Eighteen hours post CLP, rat aortic rings were removed for measurement of contractile responses to vasoconstrictors by using organ bath technique. RESULT: In endothelium intact aortic rings from CLP rats, concentration-contraction curves to phenylephrine (PE) and high KCl were significantly decreased when compared with those from control rats. The transient contraction induced by PE in calcium-free Krebs solution and the concentration-dependent contraction to CaCl(2)in KCl-depolarized medium were also markedly reduced. The hyporeactivity to vasoconstrictors was completely reversed by pretreatment either with aminoguanidine (AMG), a selective inducible nitric oxide synthase inhibitor, or with 1H [1,2,4] oxadiazolo[4,3-a] quininoxalin-1-one(ODQ), an inhibitor of NO-sensitive guanylyl cyclase. CONCLUSION: A generalized impairment in calcium handling in vascular smooth muscle,including the calcium influx through the voltage-operated and receptor-operated channels and calcium release from intracellular calcium stores, is involved in vascular hyporeactivity during the late phase of sepsis. The NO/cGMP signal transduction pathway might be involved in this defect in vascular smooth muscle.

The association between interleukin-1 receptor antagonist genotype and chronic periodontitis of Uighur patients / 中华口腔医学杂志

<p><b>OBJECTIVE</b>To investigate interleukin-1 receptor antagonist (IL-1ra) genotype and its association with the susceptibility of chronic periodontitis in Uighur patients of Xinjiang.</p><p><b>METHODS</b>Genomic DNA was obtained from buccal swabs of 41 subjects with severe chronic periodontitis (CP), 43 subjects with moderate CP, 49 subjects with mild CP and 92 ethnically matched healthy control individuals. Genotypes of IL-1RN intron 2 VNTR was analyzed by SSP-PCR method. Then compared the differences in distribution of each genotype.</p><p><b>RESULTS</b>A significant over-representation of IL-1RN intron 2 VNTR allele 2 was found in severe chronic periodontitis group.</p><p><b>CONCLUSION</b>IL-1RN intron 2 VNTR allele 2 may be a risk indicator for the susceptibility of severe chronic periodontitis in Uighur patients of Xinjiang.</p>