Animals ; Bleomycin ; Epithelial-Mesenchymal Transition ; Lung ; Male ; Pulmonary Fibrosis/drug therapy* ; Rats ; Rats, Sprague-Dawley ; Simvastatin/pharmacology*

Objective To investigate the effects of simvastatin on diabetic neuropathic pain and systematic inflammation in diabetic rats and explore their molecular mechanisms.Methods Totally 24 rats were equally randomized into the normal+vehicle(NV)group,diabetic+vehicle(DV)group,and diabetic+simvastatin(DS)group using the random number table.Streptozotocin(STZ)was used to establish the rat models of diabetes.Blood glucose,body mass,paw withdrawal mechanical threshold(PWMT),and paw withdrawal thermal latency(PWTL)in each group were observed on days 7,14,21,and 28 after STZ injection.On day 28 after STZ injection,rats were sacrificed,and the lumbar spinal dorsal horn and serum were collected.Western blotting was used to detect the expression of receptor for advanced glycation end products(RAGE)and the phosphorylation levels of protein kinase B(AKT),extracellular signal-regulated kinase(ERK),p38,and c-Jun N-terminal kinase(JNK)in the spinal dorsal horn of rats in each group.Enzyme-linked immunosorbent assay was performed to determine the serum concentrations of oxidized low density lipoprotein(ox-LDL)and interleukin-1β(IL-1β).Results On days 14,21 and 28 after STZ injection,the PWMT in DV group were(8.6 ± 0.8),(7.1 ± 1.6),and(7.8 ± 0.8)g respectively,which were significantly lower than (12.0 ± 0.9)(=8.482, =0.000),(11.6 ± 1.5)(=11.309, =0.000),and(11.7 ± 1.5)g(=9.801, =0.000)in NV group.The PWMT in DS group on days 21 and 28 were(9.4 ± 1.4)(=5.780, =0.000)and(9.7 ± 0.9)g(=4.775, =0.003),respectively,which were significantly improved comparing with those of DV group.On days 7,14,21,and 28,there were no significant differences in PWTL among these three groups (all <0.05).The expression of RAGE in the spinal dorsal horn of DV group was significantly higher than those of NV group(=6.299, =0.000)and DS group(=2.891, =0.025).The phosphorylation level of AKT in the spinal dorsal horn of DV group was significantly higher than those of NV group(=8.915,=0.000)and DS group(=4.103,=0.003).The phosphorylation levels of ERK( =8.313,=0.000),p38( =2.965, =0.022),and JNK(=7.459, =0.000)in the spinal dorsal horn of DV group were significantly higher than those of NV group;the phosphorylation level of JNK in the spinal dorsal horn of DS group was significant lower than that of DV group(=3.866, =0.004);however,there were no significant differences in the phosphorylation levels of ERK(=1.987,=0.122)and p38(=1.260,=0.375)in the spinal dorsal horn between DS group and DV group.The serum concentrations of ox-LDL and IL-1β in DV group were(41.86 ± 13.40)ng/ml and(108.16 ± 25.88)pg/ml,respectively,which were significantly higher than those in NV group [(24.66 ± 7.87)ng/ml(=3.606,=0.003)and(49.32 ± 28.35)pg/ml(=5.079,=0.000)] and DS group [(18.81 ± 5.62)ng/ml (=4.833, =0.000)and(32.73 ± 11.73)pg/ml(=6.510, =0.000)].Conclusions Simvastatin can relieve the mechanical allodynia of diabetic rats possibly by inhibiting the activation of RAGE/AKT and the phosphorylation of JNK in the spinal dorsal horn.Simvastatin can also decrease the serum concentrations of ox-LDL and IL-1β in diabetic rats,which may contribute to the relief of systematic inflammation.
Animals ; Diabetes Mellitus, Experimental ; complications ; Hyperalgesia ; Inflammation ; drug therapy ; Interleukin-1beta ; blood ; Lipoproteins, LDL ; blood ; Neuralgia ; drug therapy ; Proto-Oncogene Proteins c-akt ; metabolism ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Receptor for Advanced Glycation End Products ; metabolism ; Simvastatin ; pharmacology

BackgroundOxidized low-density lipoprotein (ox-LDL)-induced oxidative stress and endothelial apoptosis are essential for atherosclerosis. Our previous study has shown that ox-LDL-induced apoptosis is mediated by the protein kinase RNA-like endoplasmic reticulum kinase (PERK)/eukaryotic translation initiation factor 2α-subunit (eIF2α)/CCAAT/enhancer-binding protein homologous protein (CHOP) endoplasmic reticulum (ER) stress pathway in endothelial cells. Statins are cholesterol-lowering drugs that exert pleiotropic effects including suppression of oxidative stress. This study aimed to explore the roles of simvastatin on ox-LDL-induced ER stress and apoptosis in endothelial cells.

MethodsHuman umbilical vein endothelial cells (HUVECs) were treated with simvastatin (0.1, 0.5, or 2.5 μmol/L) or DEVD-CHO (selective inhibitor of caspase-3, 100 μmol/L) for 1 h before the addition of ox-LDL (100 μg/ml) and then incubated for 24 h, and untreated cells were used as a control group. Apoptosis, expression of PERK, phosphorylation of eIF2α, CHOP mRNA level, and caspase-3 activity were measured. Comparisons among multiple groups were performed with one-way analysis of variance (ANOVA) followed by post hoc pairwise comparisons using Tukey's tests. A value of P < 0.05 was considered statistically significant.

ResultsExposure of HUVECs to ox-LDL resulted in a significant increase in apoptosis (31.9% vs. 4.9%, P < 0.05). Simvastatin (0.1, 0.5, and 2.5 μmol/L) led to a suppression of ox-LDL-induced apoptosis (28.0%, 24.7%, and 13.8%, F = 15.039, all P < 0.05, compared with control group). Ox-LDL significantly increased the expression of PERK (499.5%, P < 0.05) and phosphorylation of eIF2α (451.6%, P < 0.05), if both of which in the control groups were considered as 100%. Simvastatin treatment (0.1, 0.5, and 2.5 μmol/L) blunted ox-LDL-induced expression of PERK (407.8%, 339.1%, and 187.5%, F = 10.121, all P < 0.05, compared with control group) and phosphorylation of eIF2α (407.8%, 339.1%, 187.5%, F = 11.430, all P < 0.05, compared with control group). In contrast, DEVD-CHO treatment had no significant effect on ox-LDL-induced expression of PERK (486.4%) and phosphorylation of eIF2α (418.8%). Exposure of HUVECs to ox-LDL also markedly induced caspase-3 activity together with increased CHOP mRNA level; these effects were inhibited by simvastatin treatment.

ConclusionsThis study suggested that simvastatin could inhibit ox-LDL-induced ER stress and apoptosis in vascular endothelial cells.

Apoptosis ; drug effects ; Cells, Cultured ; Endoplasmic Reticulum Stress ; drug effects ; Human Umbilical Vein Endothelial Cells ; drug effects ; metabolism ; Humans ; Lipoproteins, LDL ; pharmacology ; Oligopeptides ; pharmacology ; Simvastatin ; pharmacology

OBJECTIVE: To observe the protective effect of simvastatin on renal injury in diabetic rats and to explore the possible molecular mechanism. METHODS: Twenty-four SD rats were randomly divided into normal control (NC) group (=8) and modeling group (=16).The rats in modeling group were injected with streptozotocin intraperitoneally at a dose of 55 mg/kg to establishing diabetic rat model. After diabetic ratmodel established successfully, the diabetic rats were randomly subdivided into diabetes mellitus (DM) group and diabetes mellitus + simvastatin (DM+Sim) group (=8).Rats in DM+Sim group were given simvastatin at a dose of 40 mg/kg by oral gavages, once a day for 4 weeks. Morphological changes and interstitial fibrosis of kidney were observed by histopathological method. The expressions of relative protein in endoplasmic reticulum stress, inflammatory molecules in renal tissues and cells apoptosis were detected by molecular biology method. RESULTS: ① Compared with NC group, the pathological changes of glomerulus and tubulointerstitium were obvious, and the collagen fibers were obviously erythrophilous and unevenly distributed in DM group. Compared with DM group, the morphological changes and fibrosis were significantly improved in DM+Sim group. ② The expressions of GRP78, p-IRE1α, NF-κB p65 and MCP-1 in DM group were significantly higher than those in NC group (<0.05), while the expressions of GRP78, p-IRE1α, NF-κB p65 and MCP-1in DM + Sim group were decreased (<0.05). ③ There were a small number of apoptotic nuclei in the glomeruli and adjunctive renal tubules in NC group detected by TUNEL assay, while there were a large number of apoptotic nuclei in DM group (<0.01). The number of apoptotic nuclei was decreased significantly in DM+Sim group (<0.01). CONCLUSIONS Morphologicalchanges and fibrosis of renal tissue are improved obviously, and the number of apoptotic cells is decreased significantly after administration of simvastatin in diabetic rats. Simvastatin exertsthe protective effect on diabetic nephropathy by inhibiting endoplasmic reticulum stress and NF-κB inflammatory signaling pathway, and reducing renal cell apoptosis.
Animals ; Diabetes Mellitus, Experimental ; Diabetic Nephropathies ; Kidney ; Rats ; Rats, Sprague-Dawley ; Simvastatin ; pharmacology

BACKGROUND/AIMS: Statins act as antineoplastic agents through the inhibition of cell proliferation. This study sought to demonstrate the effects of statins on extrahepatic bile duct cancer cell apoptosis and to document the changes in protein expression involved in tumor growth and suppression. METHODS: Human extrahepatic bile duct cancer cells were cultured. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays were performed to determine the effect of statins on cell proliferation. Apoptosis was measured by a cell death detection enzyme-linked immunosorbent assay and caspase-3 activity assay, and flow cytometry was used to determine the percentage of cells in each phase of the cell cycle. The protein expression of Bax, Bcl-2, insulin-like growth factor 1 (IGF-1) receptor, extracellular signal-regulated kinase 1/2 (ERK1/2), and Akt was measured by Western blot analysis. RESULTS: Simvastatin suppressed cell proliferation by inducing G1 phase cell cycle arrest in bile duct cancer cells. Furthermore, it induced apoptosis via caspase-3 activation, downregulated the expression of the Bcl-2 protein, and enhanced the expression of the Bax protein. Moreover, simvastatin suppressed the expression of the IGF-1 receptor and IGF-1-induced ERK/Akt activation. CONCLUSIONS: Simvastatin induces apoptosis in bile duct cancer cells, which suggests that it could be an antineoplastic agent for bile duct cancer.
Antineoplastic Agents/pharmacology ; Apoptosis/*drug effects ; Bile Duct Neoplasms/*drug therapy ; Cell Cycle/drug effects ; Cell Line, Tumor ; Cell Proliferation/drug effects ; Enzyme-Linked Immunosorbent Assay ; Flow Cytometry ; Humans ; Hypolipidemic Agents/*pharmacology ; Receptor, IGF Type 1/*drug effects ; Simvastatin/*pharmacology

Capsular fibrosis and contracture occurs in most breast reconstruction patients who undergo radiotherapy, and there is no definitive solution for its prevention. Simvastatin was effective at reducing fibrosis in various models. Peri-implant capsular formation is the result of tissue fibrosis development in irradiated breasts. The purpose of this study was to examine the effect of simvastatin on peri-implant fibrosis in rats. Eighteen male Sprague-Dawley rats were allocated to an experimental group (9 rats, 18 implants) or a control group (9 rats, 18 implants). Two hemispherical silicone implants, 10 mm in diameter, were inserted in subpanniculus pockets in each rat. The next day, 10-Gy of radiation from a clinical accelerator was targeted at the implants. Simvastatin (15 mg/kg/day) was administered by oral gavage in the experimental group, while animals in the control group received water. At 12 weeks post-implantation, peri-implant capsules were harvested and examined histologically and by real-time polymerase chain reaction. The average capsular thickness was 371.2 μm in the simvastatin group and 491.2 μm in the control group. The fibrosis ratio was significantly different, with 32.33% in the simvastatin group and 58.44% in the control group (P < 0.001). Connective tissue growth factor (CTGF) and transforming growth factor (TGF)-β1 gene expression decreased significantly in the simvastatin group compared to the control group (P < 0.001). This study shows that simvastatin reduces radiation-induced capsular fibrosis around silicone implants in rats. This finding offers an alternative therapeutic strategy for reducing capsular fibrosis and contracture after implant-based breast reconstruction.
Administration, Oral ; Animals ; Breast/*drug effects/metabolism/pathology/radiation effects ; *Breast Implants ; Connective Tissue Growth Factor/genetics/metabolism ; Fibrosis ; Gamma Rays ; Male ; Rats ; Rats, Sprague-Dawley ; Real-Time Polymerase Chain Reaction ; Silicone Gels/*chemistry ; Simvastatin/*pharmacology ; Transforming Growth Factor beta1/metabolism

Capsular fibrosis and contracture occurs in most breast reconstruction patients who undergo radiotherapy, and there is no definitive solution for its prevention. Simvastatin was effective at reducing fibrosis in various models. Peri-implant capsular formation is the result of tissue fibrosis development in irradiated breasts. The purpose of this study was to examine the effect of simvastatin on peri-implant fibrosis in rats. Eighteen male Sprague-Dawley rats were allocated to an experimental group (9 rats, 18 implants) or a control group (9 rats, 18 implants). Two hemispherical silicone implants, 10 mm in diameter, were inserted in subpanniculus pockets in each rat. The next day, 10-Gy of radiation from a clinical accelerator was targeted at the implants. Simvastatin (15 mg/kg/day) was administered by oral gavage in the experimental group, while animals in the control group received water. At 12 weeks post-implantation, peri-implant capsules were harvested and examined histologically and by real-time polymerase chain reaction. The average capsular thickness was 371.2 μm in the simvastatin group and 491.2 μm in the control group. The fibrosis ratio was significantly different, with 32.33% in the simvastatin group and 58.44% in the control group (P < 0.001). Connective tissue growth factor (CTGF) and transforming growth factor (TGF)-β1 gene expression decreased significantly in the simvastatin group compared to the control group (P < 0.001). This study shows that simvastatin reduces radiation-induced capsular fibrosis around silicone implants in rats. This finding offers an alternative therapeutic strategy for reducing capsular fibrosis and contracture after implant-based breast reconstruction.
Administration, Oral ; Animals ; Breast/*drug effects/metabolism/pathology/radiation effects ; *Breast Implants ; Connective Tissue Growth Factor/genetics/metabolism ; Fibrosis ; Gamma Rays ; Male ; Rats ; Rats, Sprague-Dawley ; Real-Time Polymerase Chain Reaction ; Silicone Gels/*chemistry ; Simvastatin/*pharmacology ; Transforming Growth Factor beta1/metabolism

OBJECTIVE: To observe the role of aminophylline and simvastatin in preventing and curing chronic obstructive pulmonary disease (COPD), and to explore the underlying mechanisms based on airway inflammation and mucus hypersecretion.
 METHODS: The rat model of COPD was established by combination of cigarette smoking with intratracheal lipopolysaccharide (LPS) injection. Male SD rats were randomly divided into 4 groups (n=10 per group): a control group, a COPD group, an aminophylline group and a simvastatin group. The rats in the control group and the COPD group were treated with normal saline once a day via intragastric administration, while the rats in the aminophylline group and the simvastatin group were treated with aminophylline (5 g/L) and simvastatin (0.5 g/L) 1 mL/100 g once a day via intragastric administration, respectively. Pulmonary function and pathological changes in bronchus and lung were observed. The levels of IL-8, IL-17, and TNF-α in bronchoalveolar lavage fluid (BALF) were measured by enzyme-linked immunosorbent assay (ELISA). The mRNA and protein expressions of TLR4 and mucin 5AC (MUC5AC) in bronchi and lung tissues were detected by real-time PCR and Western blot, respectively.
 RESULTS: Pulmonary function and the pathophysiologic changes in bronchi and lung tissues in the COPD rats were consistent with typical phenotype of COPD. Compared with the control group, lung function indexes were significantly attenuated in the COPD group, while the levels of IL-8, IL-17, and TNF-α in BALF as well as the mRNA and protein levels of MUC5AC and TLR4 were significantly increased. Compared with the COPD group, lung function indexes were significantly increased in the aminophylline group and simvastatin group (P<0.01), while pulmonary pathological damages, the levels of IL-8, IL-17, and TNF-α in BALF as well as the mRNA and protein levels of MUC5AC and TLR4 were significantly decreased (P<0.01). Compared with the aminophylline group, the peak expiratory flow as well as the levels of IL-8, IL-17, and TNF-α in the simvastatin group were elevated (P<0.05). There are no significant difference in the mRNA and protein levels of MUC5AC and TLR4 between the 2 groups (P﹥0.05).
 CONCLUSION Aminophylline and simvastatin can decrease IL-8, IL-17, and TNF-α levels in BALF and inhibit the expression of MUC5AC and TLR4 in airway and lung tissues in COPD rats, suggesting that they may have a preventive and therapeutic effect on COPD through reducing the airway inflammation and mucus hypersecretion.
Aminophylline ; pharmacology ; Animals ; Bronchi ; metabolism ; Bronchoalveolar Lavage Fluid ; chemistry ; Cytokines ; chemistry ; Inflammation ; drug therapy ; Lipopolysaccharides ; Lung ; metabolism ; physiopathology ; Male ; Mucin 5AC ; metabolism ; Mucus ; metabolism ; Pulmonary Disease, Chronic Obstructive ; drug therapy ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Simvastatin ; pharmacology ; Smoke ; adverse effects ; Smoking ; adverse effects ; Toll-Like Receptor 4 ; metabolism

Recently, several studies showed that gastrointestinal tract may be associated with pathophysiology of Parkinson's disease (PD). Intestine tight junction protein zonula occluden-1 (ZO-1) is an important component of intestinal barrier which can be degraded by matrix metallopeptidase 9 (MMP-9). In our previous study, a significant decline in ZO-1 was observed along with enhanced MMP-9 activity in the duodenum and distal colon of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-intoxicated mice. In this study, the protective effect of simvastatin on ZO-1 was investigated using an MPTP mouse model of PD. Seven days after the end of MPTP application, the expression level of ZO-1 was evaluated by immunohistochemistry. The protein expression levels of ZO-1 and MMP9 were detected by Western blotting. Meanwhile, MMP-9 activity was analyzed by gelatin zymography. MPTP treatment led to a decrease in the expression of ZO-1, which was accompanied by elevated MMP-9 activity. Treatment with simvastatin could partly reverse the MPTP-induced changes in ZO-1 expression and reduce MMP-9 protein and activity. Taken together, these findings suggest that simvastatin administration may partially reverse the impairment of ZO-1 induced by MPTP via inhibiting the activity of MMP9, fortify the impaired intestinal barrier and limit gut-derived toxins that pass across the intestinal barrier.
Animals ; Disease Models, Animal ; Hydroxymethylglutaryl-CoA Reductase Inhibitors ; pharmacology ; Mice ; Parkinson Disease ; metabolism ; Simvastatin ; pharmacology ; Zonula Occludens-1 Protein ; drug effects

OBJECTIVETo observe the effect of Buyang Huanwu Decoction (BYHWD), a representative formula of qi benefiting blood activating method on aorta Rho associated coiled-coil forming protein serine/threonine kinase (Rhokinase, ROCK) and nuclear transcription factor kappa B (NF-κB) p65 mRNA expressions and levels of blood lipids in atherosclerosis (AS) model rats.

METHODSThe AS rat model was prepared by vitamin D3 and high fat diet. Totally 60 rats were randomly divided into 6 groups, i.e., the normal control group, the model group, the low dose BYHWD group (10 g/kg), the high dose BYHWD group (20 g/kg), the Simvastatin control group (0.6 mg/kg), and the BYHWD prevention group (10 g/kg), 10 in each group. After successful modeling all medication was intervened for 28 days. Expression levels oxidized low density lipoprotein (ox-LDL) were detected by ELISA. Levels of TG, TC, LDL-C, HDL-C were determined by enzyme method. Pathological changes of aortic tissue were observed under light microscope. mRNA expressions of Rho kinase and NF-κB p65 in aorta were detected by real time (RT) PCR.

RESULTSHigh fat diet and peritoneal injection of vitamin D3 could induce AS rat model. Typical atheromatous plaque formed in aorta of AS model rats. Compared with the normal control group, levels of TC, TG, LDL-C, and ox-LDL significantly increased in the model group, but the HDL-C level decreased (P < 0.01). Compared with the model group, levels of TC, TG, LDL-C, and ox-LDL all decreased, but HDL-C increased in low and high dose BYHWD groups, the Simvastatin control group, and the BYHWD prevention group (P < 0.05, P < 0.01). Compared with the low dose BYHWD group, above-mentioned indices were more obviously lowered in the high dose BYHWD group, the Simvastatin control group, and the BYHWD prevention group (P < 0.05). Compared with the normal control group, mRNA expression levels of Rho kinase and NF-κB p65 significantly increased in the model group (P < 0.01). Compared with the model group, mRNA expressions of Rho kinase and NF-κB p65 obviously decreased in low and high dose BYHWD groups, the Simvastatin control group, and the BYHWD prevention group (P < 0.01). Compared with the low dose BYHWD group, the two indicators were more obviously lowered in the high dose BYHWD group, the Simvastatin control group, and the BYHWD prevention group (P < 0.05). But there was no statistical difference in blood lipids levels, mRNA expression levels of Rho kinase or NF-κB p65 among the high dose BYHWD group, the Simvastatin control group, and the BYHWD prevention group (P >0. 05).

CONCLUSIONSBYHWD could down-regulate mRNA expression levels of Rho kinase and NF-κB p65, lower levels of blood lipids, and fight against AS. Suppressing Rho kinase pathway might be one of its mechanisms.

Animals ; Aorta ; Atherosclerosis ; genetics ; metabolism ; Drugs, Chinese Herbal ; pharmacology ; therapeutic use ; Gene Expression ; drug effects ; Lipids ; Lipoproteins, LDL ; NF-kappa B ; metabolism ; RNA, Messenger ; metabolism ; Rats ; Simvastatin ; Transcription Factor RelA ; metabolism ; rho-Associated Kinases ; metabolism