Objective To investigate the effect of simvastatin on inducing endothel ial progenitor cells (EPCs) homing and promoting bone defect repair, and to explore the mechanism of local implanting simvastatin in promoting bone formation. Methods Simvastatin (50 mg) compounded with polylactic acid (PLA, 200 mg) or only PLA (200 mg) was dissolved in acetone (1 mL) to prepare implanted materials (Simvastatin-PLA material, PLA material). EPCs were harvested from bone marrow of 2 male rabbits and cultured with M199; after identified by immunohistochemistry, the cell suspension of EPCs at the 3rd generation (2 × 106 cells/mL) was prepared and transplanted into 12 female rabbits through auricular veins(2 mL). After 3 days, the models of cranial defect with 15 cm diameter were made in the 12 female rabbits. And the defects were repaired with Simvastatin-PLA materials (experimental group, n=6) and PLA materials (control group, n=6), respectively. The bone repair was observed after 8 weeks of operation by gross appearance, X-ray film, and histology; gelatin-ink perfusion and HE staining were used to show the new vessels formation in the defect. Fluorescence in situ hybridization (FISH) was performed to show the EPCs homing at the defect site. Results All experimental animals of 2 groups survived to the end of the experiment. After 8 weeks in experimental group, new bone formation was observed in the bone defect by gross and histology, and an irregular, hyperdense shadow by X-ray film; no similar changes were observed in control group. FISH showed that the male EPC containing Y chromosome was found in the wall of new vessels in the defect of experimental group, while no male EPC containing Y chromosome was found in control group. The percentage of new bone formation in defect area was 91.63% ± 4.07% in experimental group and 59.45% ± 5.43% in control group, showing significant difference (P lt; 0.05). Conclusion Simvastatin can promote bone defect repair, and its mechanism is probably associated with inducing EPCs homing and enhancing vasculogenesis.
Objective To confirm the stimulating effect of simvastatin on BMSCs of SD rats osteogenic differentiation, and to further study the role of Wnt signal ing pathway in this process. Methods BMSCs derived from the tibia and femur of 6-week-old female SD rats were cultured in vitro．Two groups were establ ished: control group and experimental group. After the 2nd passage, the cells of experimental group were treated with simvastatin (1 × 10-7mol/L) and the cells of control group with absolute ethyl alcohol and PBS. ALP staining was used at 7 days and von Kossa staining was appl ied at 28 days to assess osteoblastic differentiation and mineral ization. Real-time quantitative PCR was performed to evaluate theexpressions of Axin2, β-catenin, osteocalcin (OC), frizzled-2, Lef-1, and Wnt5a mRNA at 7 days and 14 days after simvastatin treatment. Results The observation of inverted phase contrast microscope showed that the majority of cells were polygonal and triangular in the experimental group, and were spindle-shaped in the control group at 7 days. The ALP staining showed blue cytoplasm, the positive cells for ALP staining in the experimental group were more than those in the control group at 7 days. The von Kossa staining showed that mineral ization of extracelluar matrix at 28 days in two groups, but the mineral ization in the experimental group was more obvious than that in the control group. The expression of Axin2 mRNA was significantly lower, and frizzled-2, Lef-1 mRNA were significantly higher in the experimental group than in the control group (P lt; 0.05) at 7 days, while the mRNA expressions of Axin2, OC, frizzled-2, Lef-1, and Wnt5a were significantly higher in the experimental group than in the control group at 14 days (P lt; 0.05). Conclusion Simvastatin can promote the osteogenic differentiation of BMSCs and change the expression of mRNA of some components of Wnt signal ing pathway.
ObjectiveTo investigate the effect of simvastatin and mechanical pretreatment on intimal hyperplasia of venous graft and its mechanism.MethodsTwelve New Zealand rabbits were selected and randomly divided into 4 groups: a blank control group, a simvastatin topical treatment group, a mechanical precondition group and a combined group (n=3 in each group). Ultrasound was used to evaluate the changes of graft wall and blood flow velocity in the graft, and pathological section was used to evaluate the intimal hyperplasia. Human umbilical cord endodermal cells were cultured in vitro. A simvastatin group and a solvent control group were set to detect YAP phosphorylation, downstream target gene expression and cell proliferation.ResultsVascular ultrasound showed that except the simvastatin topical treatment group, the flow velocity in vein grafts in the other three groups significantly increased 21 days after surgery compared with 7 days after surgery (P<0.01). Pathological sections showed that the thickness of new intima in the simvastatin topical treatment group, mechanical precondition group, combined group and blank control group were 45.56±4.11 μm, 201.28±16.71 μm, 143.57±7.82 μm, 249.45±13.33 μm, respectively, and there were statistical differences compared with the blank control group (P<0.05). In vitro results showed that compared with the solvent control group, cell death was observed in high concentration simvastatin (5 mmol/L) group, cell proliferation was inhibited in low concentration simvastatin (2.5 mmol/L) group (P<0.05), the expression of YAP protein in the simvastatin group was unchanged, but the expression of phosphorylated YAP protein significantly increased (P<0.05), and the expression of downstream target gene ccn1 was down-regulated (P<0.001).ConclusionIntravascular local application of simvastatin and mechanical preconditioning alone or in combination can inhibit intimal hyperplasia of venous graft. High concentration of simvastatin has cytotoxicity, while low concentration of simvastatin has inhibitory effect on cell proliferation. Simvastatin can inhibit the formation of new intima by inhibiting the entry of YAP into the nucleus and reducing the transcription of cell proliferation-related target gene ccn1.
OBJECTIVE: To study the effect of simvastatin on the expression of bone morphogenetic protein-2 (BMP-2) and alkaline phosphates (ALP) activity in the primary cultured bone marrow stromal cells, and to elucidate the mechanism of the anabolic osteogenetic effect of simvastatin. METHODS: Bone marrow stromal cells in femur and tibia of adult mouse were cultured in vitro. after treated with different concentrations of simvastatin (0, 0.1, 0.2, 0.5 and 1.0 mumol/L) or recombinant human BMP-2 for 72 hours, ALP activity of bone marrow stromal cells was determined. BMP-2 expression of bone marrow stromal cells was analyzed by using immunocytochemistry and Western blotting. RESULTS: After treated with simvastatin for 72 hours, BMP-2 expression increased, while little BMP-2 expression could be observed in the control group. ALP activity also increased in a dose-dependent manner; t-test showed that ALP activity in the group which concentrations of simvastatin were 0.5 mumol/L (t = 2.35, P = 0.041), 1.0 mumol/L (t = 2.348, P = 0.041) had significant difference when compared with control group. CONCLUSION: Simvastatin lead to high expression of BMP-2 in bone marrow stromal cells, via the increased auto- or para-crine of BMP-2, and ALP activity increased. These may be parts of the mechanism on the anabolic osteogenetic effect of simvastatin.
ObjectiveTo investigate the regulatory effect of simvastatin on osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) at middle/late stages by p38MAPK pathway under condition of osteoinductive environment.
MethodsThe bone marrow of bilateral femur and tibia were harvested from 20 4-week-old female Sprague Dawley rats. BMSCs were isolated and cultured with whole bone marrow culture method; the second generation of cells were randomly divided into 5 groups: control group (complete medium, CM), simvastatin group (simvastatin medium, SIM), osteogenic induction group (osteogenic induction medium, OM), simvastatin and osteogenic induction group (simvastatin+osteogenic induction medium, OM+SIM), and blocker group (SB203580+simvastatin+osteogenic induction medium, OM+SIM+SB). MTT assay was used to detect the cell activity in CM group and SIM group at 2, 3, 4, 5, and 6 days, ELISA method to measure the content of alkaline phosphatase (ALP) in OM group and OM+SIM group at 7 and 14 days. The mRNA and protein expressions of osteocalcin (OCN) were detected by real-time quatitative PCR and Western blot after 1, 12, and 24 hours of osteogenic induction at 21 and 28 days. The protein expressions of phospho-p38 (p-p38) and p38 in OM group, OM+SIM group, and OM+SIM+SB group were detected by Western blot at the best induction time of simvastatin.
ResultsMTT assay showed that no significant difference was found in absorbance (A) value between CM group and SIM group at each time point (P > 0.05), indicating no effect of 1×10-7 mol/L simvastatin on cell viability. ELISA results showed that ALP content significantly increased in OM+SIM group when compared with OM group at 7 and 14 days; the ALP content was significantly higher at 7 days than 14 days in OM group and OM+SIM group (P < 0.05). OCN mRNA and protein expressions at 12 hours were significantly higher than those at other time points in each group (P < 0.05), and the expressions of OM+SIM group was significantly higher than those of OM group (P < 0.05). The best induction time of simvastatin was 12 hours. At 12 hours after blocking intervention, the p-p38/p38 in OM+SIM+SB group was significantly lower than that in OM group and OM+SIM group (P < 0.05), and the p-p38/p38 in OM+SIM group was significantly higher than that in OM group (P < 0.05).
ConclusionSimvastatin can increase the mRNA and protein expression levels of OCN and the protein of p-p38 in osteogenic differentiation of BMSCs at middle/ late stages, and its best induction time is 12 hours.
Objective To investigate the effects of simvastatin on pulmonary function and vascular endothelial growth factor ( VEGF) levels in induced sputumof patients with COPD exacerbation( AECOPD) .Methods Thirty-eight patients with AECOPD were divided into two groups randomly, ie. a routine medical treatment( RT) group( n =30) and a routine + statin medical treatment( RST) group( n =28) . The VEGF levels in serumand induced sputum were detected by ELISA on the first day and after a week treatment in hospital, respectively. Meanwhile, the pulmonary function measurements were performed. Results There were no significant differences in the pulmonary function ( FEV1% pred and FEV1 /FVC) and VEGF levels in induced sputumbetween the two groups before treatment( P gt;0. 05) . The RT group showed no significantchanges in any parameters before and after a week treatment( P gt; 0. 05) . FEV1% pread, FEV1 /FVC and VEGF levels in induced sputum in the RST group after a week treatment significantly increased compared with those before treatment and the RT group( P lt;0. 01, P lt;0. 01, P lt;0. 05) . But There were no significant differences in serumVEGF levels between the two groups before and after a week treatment. The VEGF levels in induced sputum were positively correlated to FEV1% pread and FEV1 /FVC after a week treatment( r =0. 430, P lt;0. 05; r = 0. 388, P lt; 0. 05) . Conclusions Simvastatin may reduce the decline in pulmonary function and decrease the levels of VEGF in induced sputum of patients with AECOPD. Improvement in pulmonary function may be related to down-expression of lung VEGF
Objective To investigate the effects of simvastatin on monocrotaline-induced pulmonary hypertension in rats, and explore the potential mechanism of simvastatin by blocking heme oxygenase-1( HO-1) expression. Methods 52 male Sprague-Dawley rats were randomly divided into five groups, ie. a control group, a simvastatin control group, a pulmonary hypertension model group, a simvastatin treatment group, a ZnPP ( chemical inhibitor of HO) group. Mean pulmonary arterial pressure ( mPAP) and right ventricular systolic pressure ( RVSP) were detected by right heart catheter at 5th week. Right ventricular hypertrophy index ( RVHI) was calculated as the right ventricle to the left ventricle plus septum weight. Histopathology changes of small intrapulmonary arteries were evaluated via image analysis system.Immunohistochemical analysis was used to investigate the expression and location of HO-1. HO-1 protein level in lung tissue were determined by western blot. Results Compared with the model group, simvastatin treatment decreased mPAP and RVHI significantly [ ( 35. 63 ±5. 10) mm Hg vs. ( 65. 78 ±15. 51) mm Hg,0. 33 ±0. 05 vs. 0. 53 ±0. 06, both P lt; 0. 05 ] . Moreover, simvastatin treatment partially reversed the increase of arterial wall area and arterial wall diameter [ ( 50. 78 ±9. 03 ) % vs. ( 65. 92 ±7. 19) % ,( 43. 75 ±4. 23) % vs. ( 52. 00 ±5. 35) % , both P lt; 0. 01) . In the model group, HO-1 staining was primarily detected in alveolar macrophages. Simvastatin treatment increased HO-1 protein expression significantly, especially in the thickened smooth muscle layer and alveolar macrophages. Inhibiting HO-1 expression using ZnPP resulted in a loss of the effects of simvastatin. mPAP in the ZnPP group was ( 52. 88±17. 45) mm Hg, while arterial wall area and arterial wall diameter were ( 50. 78 ±9. 03) % and ( 52. 00 ±5. 35) % , respectively. Conclusions Simvastatin attenuates established pulmonary arterial hypertension andpulmonary artery remodeling in monocrotaline-induced pulmonary hypertension rats. The effect of simvastatin is associated with HO-1.
Objective To evaluate the effectiveness and safety of simvastatin 40 mg daily use in treatment of coronary heart disease. Methods The study was designed as before-after study in the same patients. One hundred and sixty seven patients with coronary heart disease were prescribed simvastatin 40 mg daily for 3 and 6 months. Total cholestero (TC), low-density lipoproteins cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), triglycerldes (TG), ALT and creatine kinase (CK) in serum before therapy and at the end of 3 months and 6 months treatment were dectected. Continuous data were analyzed by standard difference of blocked randomization and described by mean±SD. Dunnet-t test was used for multiple comparison of trial and control groups. Statistical difference was set up at P＜0.05. Success rate was assessed by chi square test at the end of 3 and 6 months treatment. Results Simvastatin 40 mg/d significantly decreased the level of TC (P＜0.000 5), LDL-C (P＜0.000 5), TG (P＜0.05), and could elevate HDL-C (P＜0.05). There were 39.5% of patients whose LDL-C reduced below 70 mg/dl. One patient whose CK raised 5.6 times of upper line of normal range and 4 patients whose ALT raised more than 2 times of upper line of normal range withdrew. The reliability of simvastatin 40 mg/d was relatively good. Conclusions Simvastatin 40 mg/d could significantly improve the lipid profile, and is relatively reliable in treatment of coronary heart disease.
Objective To observe the protective effects of simvastatin at different stages on monocrotaline (MCT) induced pulmonary arteral hypertension (PAH) in rats and evaluate the early preventive effect of simvastatin. Methods Twenty-four male SD rats were randomized into a control group, a PAH group, an early intervention group, and a late intervention group, with 6 rats in each group. The rats in the control group received intraperitoneal injection of normal saline (NS) on d0. The rats in the PAH group received one-off intraperitoneal injection of MCT (50 mg/kg) on d0. The rats in the early intervention group were pretreated with oral gavage of simvastatin (20 mg·kg–1·d–1)(d–7––1) before the intraperitoneal one-off injection of MCT (50 mg/kg, d0) and continued with oral gavage of simvastatin for 14 days (d1~14). The rats in the late intervention group received one-off intraperitoneal injection of MCT (50 mg/kg)(d0) and oral gavage of simvastatin (20 mg·kg–1·d–1) for the next 21 days (d15~35). Thirty-five days after the MCT injection (d36), mean pulmonary arterial pressure (mPAP) and right ventricular systolic pressure (RVSP) were measured by right heart catheter. Then the rats were sacrificed for separating the heart and lung, the right ventricular hypertrophy index (RVHI) and percentage of small pulmonary arteries media thickness (WT%), the inflammation score around the small pulmonary arterial were recorded. Results Compared with those in the PAH group, RVSP, mPAP, RVHI and WT% in two simvastatin interventiongroups got much better (P＜0.01), and the inflammation score around the small pulmonary arterial declined (P＜0.05). Compared with those in the late intervention group, RVSP, mPAP in the early intervention group improved (P＜0.05) and WT% decreased more significantly (P＜0.01). However RVHI and the inflammation score around the small pulmonary arterial were not different between two simvastatin intervention groups. Conclusions Both early intervention and late intervention with simvastatin can reduce RVSP, mPAP and WT% in MCT induced PAH rats. Compared with later intervention, early intervention can prevent PAH more remarkably.
