Effect of aldosterone on rat peritoneal fibrosis induced by peritoneal dia-lysis

AIM: To investigate the pathologic role of aldosterone and protective effect of aldosterone receptor antagonist on peritoneal fibrosis in peritoneal dialysis rats. METHODS: A peritoneal fibrosis rat model was established by intraperitoneal injection of lipopolysaccharide(at 1 d, 3 d, 5 d and 7 d, 0.6 mg/kg) and dialysate(daily intraperitoneal injection of 4.25% dialysate, 100 mL/kg). At the same time, spironolactone(an aldosterone receptor antagonist, 100 mg·kg^-1·d^-1) was given to the model rats. After 4 weeks, the expression of aldosterone synthase CYP11B2, 11β-hydroxysteroid dehydrogenase type 2(11β-HSD2), mineralocorticoid receptor(MCR), and inflammatory factors were detected by immunohistochemistry, real-time PCR and Western blotting. RESULTS: The rat model of peritoneal fibrosis was successfully established. At the same time, the injury of mesothelial cells, deposition of collagen fibers and thickness of peritoneal were increased. Moreover, the infiltration of macrophages in the peritoneum/dialysate was increased. The level of aldosterone and the expression of MCR, 11β-HSD2 and CYP11B2 in fibrotic peritoneum were obviously up-regulated as compared with normal rats. The expression of NF-κB/MCP-1 was also increased. However, treatment with spironolactone alleviated peritoneal fibrosis and reduced the expression of NF-κB/MCP-1. CONCLUSION: Local aldosterone is involved in the process of peritoneal fibrosis via NF-κB/MCP-1 pathway. Spironolactone alleviates peritoneal fibrosis of peritoneal dialysis.     

Effect of leonurine on expression of miR-1 in rats with myocardial fibrosis induced by isoproterenol

AIM: To investigate effect of leonurine on the expression of microRNA-1 (miR-1) in rats with myocardial fibrosis induced by isoproterenol (ISO). METHODS: SD rats (n=10) were used as normal control group, and 80 rats were given ISO by intraperitoneal injection daily for 2 weeks to establish the model of myocardial fibrosis. The model rats were randomly divided into 5 groups:model group, low-dose (7.5 mg·kg^-1·d^-1) leonurine group, middle-dose (15 mg·kg^-1·d^-1) leonurine group, high-dose (30 mg·kg^-1·d^-1) leonurine group and p38 mitogen-activated protein kinase (p38 MAPK) inhibitor (0.3 mg·kg^-1·d^-1) group. After the treatment for 2 weeks, the ultrastructure of left ventricular myocardial tissues was observed under electron microscope. Masson staining was used to detect collagen fibrosis, and the expression of collagen I and collagen Ⅲ was determined by the method of immunohistochemistry. The contents of endothelin-1 (ET-1) and angiotensin Ⅱ (Ang Ⅱ) were measured by ELISA. The expression of miR-1 and ET-1 mRNA was detected by real-time PCR, and the protein expression of p38 MAPK, β-myosin heavy chain (MHC) and α-MHC was determined by Western blot. RESULTS: Compared with model group, the ultrastructure of left ventricular myocardial tissues in high-dose leonurine group was attenuated, and the expression of miR-1 and the protein expression of α-MHC in left ventricular myocardial tissues of high-dose leonurine group were increased (P<0.05). Collagen volume fraction, collagen I, collagen Ⅲ, the ratio of collagen Ⅰ/collagen Ⅲ, the contents of ET-1 and Ang Ⅱ, the mRNA expression of ET-1, and the protein expression of p38 MAPK and β-MHC in high-dose leonurine group were lower than those in model group (P<0.05). CONCLUSION: Leonurine attenuates myocardial fibrosis in the rats induced by ISO, and it is potentially associated with affecting the expression of miR-1, and inhibiting ET-1/p38 MAPK signaling pathway.     

Effect of imatinib on myocardial fibrosis in DOCA-salt hypertensive rats

AIM: To investigate the effects of imatinib (IMA), one of platelet-derived growth factor receptor (PDGFR) inhibitors, on myocardial fibrosis in deoxycorticosterone acetate (DOCA)-salt hypertensive rats. METHODS: Sixty male uninephrectomized SD rats were treated with 1% NaCl and 0.2% KCl in the drinking water for 4 weeks and assigned to 3 groups: vehicle control group (control group), DOCA treatment group (DOCA group), DOCA and IMA treatment group (DOCA+IMA group). Systolic blood pressure (SBP) was measured using the tail-cuff method. Myocardial tissue inflammation was analyzed by hematoxylin-eosin staining. Collagen volume fraction (CVF) and perivascular collagen area (PVCA) were analyzed by Sirius red staining. Ectodermal dysplasia-1 (ED-1) was analyzed by immunohistochemistry. Expression levels of platelet-derived growth factors (PDGF-A and PDGF-C), PDGF receptor α (PDGFRα) and phosphorylated PDGFRα (p-PDGFRα) were detected by Western blotting. RESULTS: SBP in DOCA group and DOCA+IMA group were signficantly higher than that in control group. No significant difference of SBP between DOCA group and DOCA+IM group was observed. In DOCA group, severe myocardial fibrosis was found, and CVF and PVCA were higher than those in control group. The differences of the CVF and PVCA between DOCA+IMA group and control group were detected, but the CVF and PVCA in DOCA+IMA group were significantly lower than those in DOCA group. Compared with control group, different degrees of myocardial tissue inflammation and monocyte/macrophage infiltration were observed in DOCA group and DOCA+IMA group. The expression levels of PDGF-A, PDGF-C and PDGFRα in DOCA group and DOCA+IMA group were much higher than those in control group, but the expression of p-PDGFRα in DOCA+IMA group were signficantly lower than that in DOCA group. CONCLUSION: Mineralocorticoid-induced myocardial fibrosis is related to cardiac tissue inflammatory response, excessive monocyte/macrophage infiltration and expressions of PDGF-A,PDGF-C and PDGFRα. Imatinib has an inhibitory effect on the myocardial fibrosis. The mechanisms may be associated with the inhibition of PDGFRα activity on the surface of fibroblast, thus interrupting PDGFs signaling-induced fibroblast division and proliferation.     

Effects of atorvastatin on cardiac myocyte hypertrophy of neonatal rat induced by angiotensinⅡ in vitro and TLR4 expression

AIM: To assess effects of atorvastatin (Ator) on cardiac myocytes (CM) hypertrophy of neonatal rat induced by angiotensinⅡ (AngⅡ) in vitro and Toll like receptor 4 (TLR4) expression for theoretical bases of preventing and treating myocardial hypertrophy.METHODS: CM of neonatal Sprague-Dawley (SD) rats were isolated with trypsin digestion method and those growth-arrested cells were stimulated with 10^-7 mol/L AngⅡ in the presence of various concentrations of Ator.The method of coomassie brilliant blue was adopted to evaluate the protein contents of CM.The changes in β-MHC,AT₁ receptor and TLR4 mRNA expression were observed by RT-PCR.RESULTS: ① AngⅡ increased the protein content of CM and β-MHC mRNA expression significantly,upregulated AT₁ receptor and TLR4 gene expression.② In a dose-dependent manner,Ator inhibited the increases in the protein contents of CM and β-MHC mRNA expression induced by AngⅡ.③ In a dose-dependent manner,Ator downregulated the AT₁ receptor and TLR4 mRNA expression of CM hypertrophy of neonatal rat induced by AngⅡ.CONCLUSION: Ator inhibits CM hypertrophy,downregulates the AT₁ receptor and TLR4 gene expression.     

Effect of bFGF on promoting angiogenesis in infarct area and improving myocardial fibrosis in mouse myocardial infarction model

AIM: To investigate the protective effect of basic fibroblast growth factor (bFGF) on the heart of mice with myocardial infarction and its mechanism. METHODS: The model of myocardial infarction was established by the ligation of left anterior descending artery of C57/B6 mice (8~12 weeks old) after lateral thoracotomy. The mice were divided into sham operation group, myocardial infarction group and bFGF administration group. bFGF at 0.5 μg was intraperitoneally injected on alternate days after myocardial infarction for 7 d. Cardiac Doppler ultrasonography was used to detect cardiac function after myocardial infarction for 28 d, and left ventricular end-diastolic diameter, left ventricular end-systolic diameter, left ventricular ejection fraction and left ventricular shortening fraction (LVFS) were used to evaluate cardiac function. After myocardial infarction for 28 d, the mice were sacrificed and the hearts were collected for preparing pathological sections. The degrees of myocardial fibrosis and angiogenesis in the myocardial infarction area were observed. Western blot was used to detect the indicators of angiogenesis. RESULTS: The results of Masson staining showed that bFGF administration significantly reduced myocardial fibrosis at 28 d after myocardial infarction. Cardiac ultrasound data showed that cardiac functions in myocardial infarction group were poorer than those in sham group, and bFGF administration significantly improved cardiac functions. Immunofluorescence staining showed that neovascularization in myocardial infarction area of bFGF administration group was more than that in myocardial infarction group. The results of Western blot showed that bFGF activated AKT/HIF-1α/VEGF signaling pathway. CONCLUSION: Intraperitoneal injection of bFGF reduces myocardial fibrosis and improves cardiac function in myocardial infarction mice. bFGF may promote angiogenesis by activating AKT/HIF-1α/VEGF signaling pathway.     

Fasudil ameliorates myocardial fibrosis by regulating polarization of macrophages in diabetic mice

AIM: To investigate the effect of hydroxyl fasudil (HF) on myocardial fibrosis and macrophage polarization in the diabetic (D) mice. METHODS: C57BL/6 mice (n=60) were randomly divided into normal saline group (NS group), normal+hydroxyl fasudil group (N+HF group), diabetes group (D+NS group), diabetes+low dose of HF group (D+LHF group), diabetes+middle dose of HF group (D+MHF group) and diabetes+high dose of HF group (D+HHF group). A mouse model of type 1 diabetes mellitus was established by intraperitoneal injection of streptozotocin (STZ). The mice in treatment groups received different doses of fasudil through intraperitoneal injection for 8 weeks. At the end of the study, the effects of fasudil at different doses on the body weight and blood glucose were observed. The histopathological changes of the cardiac tissues were observed by HE staining. The myocardial collagen volume fraction (CVF) was calculated by Masson staining. Immumohistochemical staining was used to test the macrophage polarization and protein expression of interleukin-6 (IL-6), tumor necrosis factor-α(TNF-α) and IL-10 and Western blot was applied to determine the protein levels of p-MYPT1 Thr853, inducible nitric oxide synthase (iNOS) and Arginase-1 (Arg-1).RESULTS: Compared with NS group, the body weight of the mice in D+NS group was decreased and the blood glucose was increased significantly(P<0.05). However, no statistically difference of blood glucose and body weight between the treatment groups and D+NS group was observed. Compared with NS group, CVF, the number of M1-type macrophages and the protein levels of IL-6, TNF-α, p-MYPT1 Thr853 and iNOS were increased markedly, while M2-type macrophages and the expression of IL-10 and Arg-1 were decreased in D+NS group (P<0.05). Compared with D+NS group, CVF, the number of M1-type macrophages, and the protein levels of IL-6 and TNF-α were relatively decreased, conversely the number of M2-type macrophages and the protein level of IL-10 was increased in treatment groups (P<0.05). Moreover, the protein levels of p-MYPT1 Thr853 and iNOS were reduced and the protein level of Arg-1 was increased in D+MHF and D+HHF group compared with D+NS group (P<0.05). No statistical difference in above mentioned indexes between NS group and N+HF group was observed. CONCLUSION: Fasudil significantly attenuates the myocardial fibrosis of diabetic cardiomyopathy in mice, which is possibly related to increased polarization of M2-type macrophages, decreased polarization of M1-type macrophages and inflammation.     

Effect of bitter melon on liver fibrosis induced by carbon tetrachloride

AIM: To investigate the effects of bitter melon (BM) on liver fibrosis induced by CCl₄ in Wistar rats. METHODS: Healthy male Wistar rats were randomly divided into 4 groups (with 8 each): olive oil control group (group C), olive oil CCl₄ model group (group M), CCl₄+BM at low concentration (BM 100 g/kg, group BM-L), CCl₄+ BM at high concentration (BM 200 g/kg, group BM-H). All rats except those in group C were subcutaneously injected with CCl₄ twice a week for 8 weeks to induce liver fibrosis. After injection of CCl₄ for 8 weeks, all rats were sacrificed and the samples of blood and livers were collected. The weight ratio of liver to body was measured. The serum level of MDA and the activity of SOD were tested. The contents of total protein and albumin, the activity of GSH-Px, the content of hydroxyproline and the activity of monoamine oxidase in the liver homogenate were determined. Hepatic inflammation and collagen deposition were observed under microscope with Masson staining. RESULTS: In the rats treated with BM, the weight ratio of liver to body, the serum level of MDA, the content of hydroxyproline and the activity of monoamine oxidase in the liver homogenate were lower than those in group M (P<0.01). The serum activity of SOD, the contents of total protein and albumin, and the activity of GSH-Px in the liver homogenate were enhanced (P<0.01). The livers of the model rats had remarkable inflammatory necrosis, collagen accumulation and fibrosis. The rats in BM-treated group showed slighter hepatic injury and collagen deposition, and the liver functions were much better than those in the model group. High dose of BM showed more obvious liver-protective effects. CONCLUSION: BM attenuates liver fibrosis by its antioxidant effect and the mechanisms of reducing hydroxyproline content and monoamine oxidase activity.     

Progress in monocytes/macrophages regulating cardiac fibrosis after myocardial infarction

Myocardial fibrosis is the main pathophysiology following myocardial infarction, which leads to cardiac remodeling and heart failure ultimately. The inflammatory response of monocyte/macrophage recruitment is essential to heart repair after myocardial infarction. However, macrophages in different periods after myocardial infarction are multifaceted, and participate in the process of myocardial fibrosis. This article mainly elucidates the relationship between monocytes/macrophages and cardiac fibrosis after myocardial infarction.     

Effects of atorvastatin on release of endothelial microparticles and myocardial apoptosis in rats with acute myocardial infarction

AIM: To investigate the effect of atorvastatin(AT) on the release of endothelial microparticles(EMP) and myocardial apoptosis in the rats with myocardial infarction. METHODS: SD male rats(n=24) were randomly divided into 3 groups:sham operation(sham) group, myocardial infarction(MI) group and MI+AT group. The rat model of acute myocardial infarction was prepared by coronary artery ligation. At 2 h and 24 h after modeling, the peripheral blood was collected to detect creatine kinase-MB(CK-MB) and cardiac troponin T(cTnT). The circulating levels of EMP were measured by flow cytometry. The myocardial apoptosis was detected by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling(TUNEL) assay. RESULTS: At 2 h after modeling, the level of CK-MB was significantly increased in MI group compared with sham group, and the level of EMP and the myocardial apoptotic rate were significantly increased in MI group and MI+AT group compared with sham group. At 24 h after modeling, the level of EMP was significantly increased in MI group compared with sham group. The levels of CK-MB, cTnT, EMP and the myocardial apoptotic rate were significantly decreased in MI+AT group compared with MI group. Moreover, the level of CK-MB in MI group was significantly increased at 24 h compared with that at 2 h after modeling. The levels of CK-MB, cTnT and EMP were significantly decreased in MI+AT group at 24 h compared with those at 2 h after modeling. CONCLUSION: Ator-vastatin may reduce the level of EMP and the myocardial apoptotic rate in the rats with acute myocardial infarction, indicating that atorvastatin plays a role in protecting endothelium.     

Imatinib attenuates DOCA/salt-induced rat myocardial fibrosis via PDGFs/PDGFRs signaling pathway

AIM：To investigate the role of imatinib (IMA) in reducing myocardial fibrosis and its relationship with platelet-derived growth factors (PDGFs)/PDGF receptors (PDGFRs) signaling pathway in deoxycorticosterone acetate (DOCA)/salt-induced hypertension in rats. METHODS:Sixty male uninephrectomized SD rats were treated with 1% NaCl and 0.2% KCl in the drinking water for 4 weeks and randomly divided into vehicle control (CON) group, DOCA treatment (DOCA) group and DOCA plus IMA treatment (DOCA+IMA) group. Systolic blood pressure (SBP) was mea-sured weekly using the tail-cuff method. The extent of myocardial interstitial fibrosis and the ratio of perivascular collagen area/vascular area (PVCA/VA) were analyzed by picro-Sirius red staining. The mRNA expression of fibroblast-specific protein 1 (FSP-1), α-smooth muscle actin (α-SMA), procollagenⅠ, procollagenⅢ, PDGFRα and PDGFRβ in the myocardium was measured by real-time PCR. The protein levels of PDGFRα, PDGFRβ, p-PDGFRβ, vimentin and α-SMA were analyzed by immunohistochemical staining. The cell types of PDGFRα and PDGFRβ localizations were examined by immunofluorescence method. RESULTS：(1) SBP in DOCA and DOCA+IMA groups was higher than that in CON group on day 14 and day 28, and no difference of SBP between DOCA group and DOCA+IMA group was found. The extent of myocardial interstitial fibrosis and the ratio of PVCA/VA in DCOA group were higher than those in CON group, and those in DOCA+IMA group were significantly lower than those in DOCA group on day 28. (2) Compared with CON group, the expression of PDGFRα and PDGFRβ in DOCA group was increased on day 14. Compared with DOCA group, the expression of PDGFRα and PDGFRβ in DOCA+IMA group was attenuated. Compared with CON group, the expression of PDGFRβ, p-PDGFRβ, FSP-1, α-SMA, procollagenⅠand procollagen Ⅲ in DOCA group was increased significantly on day 28, but the expression of PDGFRα was not increased significantly, and the expression of PDGFRβ was higher than PDGFRα. Compared with DOCA group, the expression of PDGFRβ, p-PDGFRβ, FSP-1, α-SMA, procollagenⅠand procollagen Ⅲ in DOCA+IMA group was attenuated on day 28. (3) The fibroblasts in the myocardial interstitium of CON group were mainly vimentin positive, but not α-SMA positive on day 28,while the number of α-SMA-positive spindle-shaped cells (myofibroblasts) increased significantly in DOCA group. Compared with DOCA group, the number of myofibroblasts in DOCA+IMA group was attenuated on day 28. (4) PDGFRα was located in fibroblasts and myofibroblasts but not in vascular smooth muscle cells (VSMCs). PDGFRβ was not only located in myofibroblasts but also in VSMCs. CONCLUSION:PDGFRα takes effect in the early phase of myocardial fibrosis in the DOCA/salt hypertensive rats, while PDGFRβ takes effect in the entire process. PDGFRα and PDGFRβ are expressed in cardiac fibroblasts and myofibroblasts. Imatinib reduces the proliferation and transformation of fibroblasts, thereby attenuating myocardial fibrosis by inhibiting PDGFs/PDGFRs signaling pathway.     

Effect of atorvastatin on HMG-CoA reductase expression in spontaneously hypertensive rats

AIM: To evaluate the effect of atorvastatin on HMG-CoA reductase (HMGR) expression level in spontaneously hypertensive rats (SHR). METHODS: Twelve eight-week-old SHR was randomized into distilled water group (SHR_DW group, n=6) and atorvastatin-treated group (SHR_ATV group, n=6). The age-matched Wistar-Kyoto rats (WKY) were used as control (WKY group, n=6). RT-PCR and Western blotting were used to detect HMGR mRNA and protein expression levels, respectively. Meanwhile, systolic blood pressure (SBP) and serum lipid levels were examined. RESULTS: Ten weeks a treatment, SBP in SHR_ATV group was markedly decreased compared with before treatment and SHR_DW group (P<0.05). The levels of TC, TG, LDL-C and HDL-C in SHR_ATV group were also markedly lower than those in SHR_DW group and WKYgroup (P<0.05). 10 weeks later, HMGR mRNA levels decreased markedly in SHR_ATV group compared to WKY group and SHR_DW group (P<0.05). The similar results were found in HMGR protein expression levels. CONCLUSIONS: Atorvastatin downregulates mRNA and protein expression levels of HMGR in SHR, which not only decreases the serum lipids levels, but also impacts blood pressure to a certain extent.     

Protective effects of total saponins of panax notoginseng on myocardial hypertrophy and fibrosis induced by isoproterenol in rats

AIM: To investigate the protective effects of total saponins of panax notoginseng (PNS) on myocardial hypertrophy and fibrosis induced by isoproterenol (ISO) in rats.METHODS: Myocardial hypertrophy and fibrosis model of rats were induced by injection of ISO (5 mg·kg^-1·d^-1,sc) for 7 days.From day 2,the rats were administered with PNS at dose of 25 and 50 mg·kg^-1·d^-1,ip for 14 days,the control and ISO model group were received saline injection.Then,the heart-weight (HW),left ventricular weight (LVW),the ratio of HW/BW and LVW/BW (LVI) were measured;the hydroxyproline and malondialdehyde (MDA) and angiotensin (AngII) content of left ventricle.The level of nitric oxide (NO),nitric oxide synthase (NOS),superoxide disrnutase (SOD) and glutathione peroxidase (GSH-Px) activities in left ventricle were determined by spectrophotemetry and radioimmunoassay,respectively.RESULTS: Compared with NS control group,the ratio of HW/BW,LVW/BW and the content of hydroxyproline,AngII,MDA and iNOS activity in the left ventricle were significantly increased.The cNOS,SOD,GSH-Px activities and NO content were obriously decreased in the ISO model group.After treatment with PNS,the left ventricular NO content,cNOS,SOD and GSH-Px activities were markedly higher than those in ISO model group.The content of MDA,AngII and iNOS activities and the ratio of HW/BW,LVI were significantly lower than those in ISO model group.CONCLUSION: PNS reverses the myocardial hypertrophy and fibrosis induced by isoproterenol in rats.This effect may be related to eliminating the oxygen free radicals and raising NO level.     

Effect of atorvastatin on cardiac function and HGF/c-Met signaling pathway after acute myocardial infarction in diabetic rats

AIM: To investigate the effect of atorvastatin on myocardial apoptosis, ventricular remodeling and cardiac function after acute myocardial infarction (AMI) in diabetic rats, and to explore whether the effect is mediated by hepatocyte growth factor (HGF)/c-Met signaling pathway. METHODS: Diabetes in 70 male SD rats was induced by intraperitoneal injection of streptozotocin (STZ, 65 mg/kg). After 8 weeks, AMI was induced by the ligation of the left anterior descending coronary artery in the diabetic rats, and 32 surviving rats were divided into AMI group (n=16) and AMI＋atorvastatin group (n=16, 20 mg·kg-1·d-1) at random. The similar surgical procedure was completed in sham group (n=11) without coronary ligation. Atorvastatin was given daily by gavage from the first day after AMI. Two weeks later, the cardiac function, pathological changes of myocardial tissues, myocardial apoptosis, and the expression of HGF and c-Met were compared among groups. RESULTS: AMI significantly reduced cardiac function, increased collagen volume fraction (CVF) and myocardial apoptotic index, and up-regulated the expression of HGF and c-Met at mRNA and protein levels in AMI control group (P<0.05). The cardiac function was improved, and CVF and myocardial apoptotic index were reduced by the treatment with atorvastatin, which also up-regulated the expression of HGF and c-Met (P<0.05). CONCLUSION: Atorvastatin significantly attenuates myocardial apoptosis and cardiac remodeling, and improves cardiac function after AMI in diabetic rats by further enhancing the activation of HGF/c-Met pathway.     

Cyclosporin A induces myocardial fibrosis and affects expression of matrix metalloproteinases and their inhibitors in rats

AIM: To investigate the changes of matrix metalloproteinases (MMP2 and MMP9) and tissue inhibitor of metalloproteinases (TIMP2 and TIMP1) in the myocardial fibrosis induced by cyclosporin A (CsA), and to explore the underlying mechanism. METHODS: Female Wistar rats (n=64, 6~8 weeks old) weighing (200±25) g were randomly divided into 4 groups[control, low dose of CsA (5 mg·kg^-1·d^-1), medium dose of CsA (12.5 mg·kg^-1·d^-1) and high dose of CsA (25 mg·kg^-1·d^-1)]. The rats were intraperitoneally injected with saline and different doses of CsA, respectively. CsA was continuously administered for 1, 2 and 3 weeks, and then the animals were killed to collect samples. HE staining was used to observe the morphological structure of myocardium. Masson staining was used to observe the deposition of myocardial collagen and the degree of myocardial fibrosis. The protein expression levels of MMP2, MMP9, TIMP2 and TIMP1 were determined by immunohistochemistry and Western blot. RESULTS: HE staining showed that CsA induced cardiomyocyte edema, eosinophilic changes in the cytoplasm, fine granular and vacuolar changes in cardiomyocytes, disappearance of myocardial striae, nuclear condensation, and myocardial focal necrosis and fibrosis. Masson staining showed that the degree of myocardial interstitial fibrosis was getting worse with the increase in the duration and doses of CsA exposure. The results of immunohistochemistry and Western blot showed that the expression of MMP2 in CsA groups was significantly increased in the process of CsA-induced myocardial fibrosis. In each CsA dose group, MMP2 was highly expressed at the first week, and the expression was gradually decreased over time. In contrast, the expression of TIMP2 was increased gradually in a time-and dose-dependent manner (P<0.05). Compared with control group, MMP9 expression level was low at the first week, increased at the second week, and decreased at the third week (P<0.05). Compared with control group, the expression of TIMP1 was dose-dependently increased (P<0.05). CONCLUSION: CsA induces myocardial injury and interstitial fibrosis in rats in a dose-and time-dependent manner. CsA-induced myocardial fibrosis is associated with changes of MMPs and TIMPs, and is affected by the imbalance of MMPs/TIMPs.     

Effect of atorvastatin on progression of heart failure and association with sodium-calcium exchanger expression

AIM: To investigate the effect of atorvastatin on the development of heart failure after myocardial infarction and the association with sodium-calcium exchanger (NCX) expression.METHODS: The model of heart failure was established by ligation of the anterior descending coronary artery for 8 weeks. The rats were randomly divided into control group, heart failure group, and atorvastatin group. Either atorvastatin (10 mg·kg^-1·d^-1) or vehicle was orally administered to the rats on the next day after the surgery. The left ventricular function and NCX expression were analyzed 8 weeks after ligation. Neonatal rat cardiomyocytes were cultured to investigate the effect of atorvastatin on the changes of calcium concentration induced by hypoxemia.RESULTS: A decrease in left ventricular diastolic dimension, an increase in left ventricular fractional shortening, and reductions of BNP level and NCX expression were observed in atorvastatin group. The hypoxemia-induced calcium overload in cultured cardiomyocytes was inhibited by atorvastatin, and it was inhibited by the inhibitor of NCX.CONCLUSION: Atorvastatin treatment improves cardiac function, which may be related to the expression and function of sodium calcium exchanger in heart failure.     

Effect of Chaihushugansan on pancreatic fibrosis in mice with chronic pancreatitis induced by DBTC plus ethanol and its anti-oxidation mechanism

AIM:To explore the role of superoxide dismutase (SOD) and malondialdehyde (MDA) in chronic pancreatitis (CP) induced by dibutyltin dichloride (DBTC) combined with ethanol, and the mechanisms for prevention and treatment of pancreatic fibrosis by Chaihushugansan. METHODS:The KM mice were randomly divided into control group, CP group (DBTC combined with ethanol) and Chaihushugansan group (CP+Chaihushugansan). Except for control group, the mice in other groups were intravenously injected in tail with DBTC (8 mg/kg) and drank 10% ethanol. The mice in Chaihushugansan group were administered intragastrically with Chaihushugansan (6 g·kg-1·d-1) at the following experimenal period. Before modeling and 1 week, 2 weeks, 4 weeks and 8 weeks after modeling, the mice were anesthetized and sacrificed. The activity of amylase and the content of hyaluronic acid in the serum were measured. The morphology and the degree of fibrosis in the pancreas were observed by HE staining. The activity of SOD and the level of MDA in the pancreas homogenate were analyzed. The protein of pancreas was extracted to detect the expression of type I collagen by Western blotting. RESULTS:DBTC combined with ethanol induced CP with increased serum amylase and hyaluronic acid levels， while the serum amylase and hyaluronic acid levels in Chaihushugansan group were significantly lowered (P<0.05). In 1 week, 2 weeks, 4 weeks and 8 weeks, the pancreas were obviously injured and appeared different degrees of fibrosis. The content of MDA and the expression of type I collagen in the increased significantly, but the SOD was decreased. In Chaihushugansan group, the pathological damage and the degree of fibrosis of the pancreas were improved. The level of MDA and type I collagen expression in the pancreas were significantly reduced, but the SOD was increased. CONCLUSION: The oxidative stress may take part in the development of CP. Inhibition of oxidative stress in the pancreas is one of the mechanisms that Chaihushugansan attenuates the development of CP.     

Effects of atorvastatin on expression of PAPP-A induced by TNF-α and IL-1β in rat aortic endothelial cells

AIM: To investigate the effects of atorvastatin on the expression of pregnancy-associated plasma protein A(PAPP-A)induced by TNF-α and IL-1β in endothelial cells. METHODS: The rat aortic endothelial cells were isolated from thoracic aortas and cultured by the tissue explant method. The cells in passage 3-4 were used in the experiment and were randomly divided into 4 groups: blank control group: the cells were treated without any intervention; atorvastatin concentration groups: the cells were incubated with atorvastatin at the concentrations of 0.1, 1 and 10 μmol/L for 24 h; atorvastatin time groups: the cells were incubated with atorvastatin at the concentration of 10 μmol/L for 6 h,12 h and 24 h; atorvastatin+inflammatory factors groups: the cells were pre-incubated with 60 μg/L TNF-α or 20 μg/L IL-1β for 1 h, then different concentrations of atorvastatin (0.1, 1.0, 10 μmol/L) were added for 6 h,12 h and 24 h. MTT reduction assay was used to observe the cell proliferation. The mRNA expression of PAPP-A was detected by RT-PCR. The protein level of PAPP-A in the supernatants of cultured cells was measured by ELISA. RESULTS: Compared with blank control group, no significant change of cell proliferation was observed after the intervention of atorvastatin and TNF-α/IL-1β for 3 h, 6 h, 12 h, 24 h and 48 h, indicating that the drugs had no toxic effects on the cells. No significant difference of PAPP-A expression between atorvastatin groups and blank control groups was found. Compared with TNF-α groups and IL-1β groups, PAPP-A expressions in atorvastatin intervention groups significantly decreased. The protein level of PAPP-A was gradually decreased with the raised concentration of atorvastatin and the prolonged time in a concentration- and time-dependent manner. CONCLUSION: Atorvastatin doesn't influence the PAPP-A expression, but inhibits the expression of PAPP-A activated by inflammatory factors in a concentration- and time-dependent manner in primary cultured rat aortic endothelial cells.     

Up-regulation of miR-133a expression attenuates myocardial fibrosis in spontaneously hypertensive rats

AIM: To investigate the effect of up-regulated expression of microRNA-133a (miR-133a) on myocardial fibrosis in spontaneously hypertensive rats (SHR). METHODS: Wistar-Kyoto (WKY) rats with homologous normal blood pressure served as the normal control group. SHR were divided into SHR group, SHR+ adeno-associated virus (AAV) group and SHR+miR-133a-AAV group randomly. miR-133a carried by miR-133a-AAV was transfected into SHR heart by coronary perfusion. The rat tail artery pressure was monitored. The myocardial collagen deposition was observed by Masson staining. The expression of miR-133a in myocardial tissue was detected by real-time PCR. The protein levels of transforming growth factor-β1 (TGF-β1) and connective tissue growth factor (CTGF) were determined by immunohistochemistry and Western blot. RESULTS: Compared with the WKY rats, the tail artery pressure of the SHR increased significantly. The expression of miR-133a in heart decreased, and the expression levels of TGF-β1 and CTGF increased (P<0.05), and myocardial fibrosis occurred. After up-regulating the expression level of miR-133a in the heart of SHR, the myocardial fibrosis was significantly reduced, and the expression levels of TGF-β1 and CTGF decreased (P<0.05). CONCLUSION: Up-regulation of the miR-133a expression improves myocardial fibrosis induced by hypertension, which may be related to inhibiting the protein expression of TGF-β1 and CTGF in myocardium.     

Effects of Ginkgo biloba extract on myocardial TGF-β1 and collagen expression and interstitial fibrosis in type I diabetic cardiomyopathy rats

AIM:To study the effects of Ginkgo biloba extract (EGB) on myocardial TGF-β1 and collagen expression and interstitial fibrosis in type I diabetic cardiomyopathy rats. METHODS:Thirty male SD rats were randomly divided into normal control group (CON), diabetes mellitus group (DM) and EGB treatment group (EGB). Streptozocin was intraperitoneally injected into the animals in the latter 2 groups to induce type I diabetic rat model. The rats in EGB group were intraperitoneally injected with EGB. At the end of the 12th week, the body weight of each rat and its left ventri-cular weight, blood glucose, glycosylated hemoglobin and serum insulin concentration were measured. The left ventricular end-diastolic volume (LVEDV), the left ventricular end-systolic volume (LVESV), the left ventricular ejection fraction (LVEF) and the stroke volume (SV) were determined by echocardiography. The content of collagen in left ventricular myocardium, and the expression of transforming growth factor β1 (TGF-β1), procollagen type I and collagen type III were assayed by Sirius red staining, immunohistochemical staining and RT-PCR, respectively. Left ventricular myocardial cells of the neonatal SD rats were isolated and cultured in vitro with low-glucose culture medium (LG group), high-glucose culture medium (HG group) or high-glucose culture medium plus EGB (HG+EGB group). The mRNA levels of TGF-β1, procollagen type I and collagen type III were detected by RT-PCR. RESULTS:Compared with CON group, blood glucose, glycosylated hemoglobin, left ventricular weight index, the content of collagen, and the expression of TGF-β1, procollagen type I and collagen type III in left ventricular myocardial tissues of DM group were significantly increased, while the levels of blood insulin, LVEDV and SV were significantly decreased. However, compared with DM group, blood glucose, glycosylated hemoglobin, left ventricule weight index, the content of collagen, and the expression levels of TGF-β1, procollagen type I and collagen type III in the left ventricular myocardial tissues of EGB-treated rats were significantly decreased, while the levels of blood insulin, LVEDV and SV were significantly increased. Compared with LG group, the mRNA expression levels of TGF-β1, procollagen type I and collagen type III were significantly increased. However, compared with HG group, the mRNA expression levels of TGF-β1, procollagen type I and collagen type III were significantly decreased after treated with EGB. CONCLUSION: EGB retards the process of myocardial fibrosis and improves the cardiac functions in type I diabetic cardiomyopathy rats by down-regulating the expression of TGF-β1, reducing the synthesis and deposition of collagen type I and collagen type III.