| 1IntroductionAtherosclerosis (AS) damages not only the large and medium-sized arteries, but also the small artery and arteriole, which leads to morphological and functional abnormalities of multiple organs. Heart, brain and kidney are the main target organs of AS, and AS could cause myocardial infarction, ischemic cardiomyopathy, stroke and chronic kidney diseases, finally resulting in multiple organ failure and death. Therefore, it is of important significance in theory and practice to study mechanisms and intervention targets of macrovascular and micro vascular lesions of AS.Atherosclerotic plaque rupture is the major cause of acute coronary syndrome (ACS) that leads to unstable angina, acute myocardial infarction and sudden death. Pathologically, plaques vulnerable to rupture are characteristic of a large lipid core and a thin fibrous cap. As collagen is the major component of fibrous caps, the content of collagen in plaques determines plaque vulnerability. Prolyl-4-Hydroxylase (P4H) is one of the key enzymes essential for the synthesis of all known types of collagen. P4H catalyzes proline located in repeating X-Pro-Gly triplets to hydroxyproline during posttranslational processing of collagen production. As an isoenzyme of P4H, P4Hal, a rate-limiting enzyme that folds the procollagen polypeptide chains into stable triple helical molecules, which is essential for collagen maturation and secretion. The low expression of P4Hal leads to reduce the content of collagen and then leads to the unstability of AS plaque. Previous studies found that upregulated the expression of P4Hα1whereas smoking induced P4Hα1degradation. Our studies also found that TNF-a and IL-6suppressed P4Hal expression via ASK1-JNK-NonO pathway and ERK1/2-Spl pathway, respectively.It is known that oxidized low density lipoprotein (ox-LDL) played the key role in the development of AS. Previous studies found that ox-LDL may. induce plaque instability by increasing lipid accumulation, initiating oxidative stress, and activating matrix metalloproteinases (MMPs). However, the direct effects of ox-LDL on P4Hα1expression and collagen synthesis is unclear. Statins have become the drug of first choice for the primary and secondary prevention of atherosclerotic disease. A wealth of evidence indicates that statins have pleiotropic effects such as lowered serum lipid level, improved endothelial function, reduced local inflammation and inhibited atherothrombosis. Previous studies reported that statins enhanced the collagen content in plaques by decreasing MMPs expression and increasing tissue inhibitors of metalloproteinase-1(TIMP-1) expression. However, the direct effects of statins on P4Hal expression and collagen production are unknown.2Objectives(1) To investigate whether ox-LDL suppresses the expression of P4Hα1and elucidate the underlying mechanisms and to detect the effect of simvastatin on it;(2) To investigate the effect of simvastatin on the expression of P4Hal and the underlying mechanisms and to explore a novel target of statins on stabilizing plaques.3Methods3.1Cell culture and treatment(1) To study the time-response of P4Hα1expression after ox-LDL stimulation, human aortic smooth muscle cells (HASMCs) were treated with50ug/ml ox-LDL at0h and fresh medium containing50ug/ml ox-LDL was used every8h till24h. The mRNA and protein expression of P4Hα1expression was assayed at0,4,8,12and24h after ox-LDL stimulation. To study the dose-response of P4Hal expression after ox-LDL stimulation, HASMCs were stimulated with0,25and50ug/ml ox-LDL for8h, respectively.(2) To examine the role of MAPK pathways in ox-LDL-mediated effects on P4Hα1expression, HASMCs pretreated with or without simvastatin for lh were stimulated with50ug/ml ox-LDL for0,5,15,30min and2,4,8h, and then the phosphorylation levels of p38MAPK, JNK and ERK1/2were assayed by western blot. Then, HASMCs were treated with the inhibitors or siRNA of p38MAPK, JNK and ERK1/2and then were incubated with ox-LDL for8h before cells were harvested for measurement.(3) To further study the effects of simvastatin on the impact of ox-LDL on P4Hal expression, HASMCs were cultured with or without50ug/ml ox-LDL for8h after pretreatment with or without10μmol/1simvastatin for1h. And then cells were harvested for measurement.3.2Animal modelOne hundred and twenty male ApoE-/-mice on a C57BL/6background (8weeks old) were randomly divided into two groups:a normal diet group (n=40) fed with a normal chaw and a high-fat diet group (n=80) fed with a diet containing0.25%cholesterol and15%cocoa butter. Two weeks later, a constrictive silastic tube was placed on the right common carotid artery in all mice after anesthesia. Six weeks after the surgery, mice in the normal diet group were randomly divided into two subgroups (n=20mice in each group):Mockl group that received oral methylcellulose alone, and Siml group that received an intragastric administration of50mg/kg·d simvastatin in0.5%methylcellulose. Mice in the high-fat diet group were randomly divided into four subgroups (n=20in each group):Mock2group that received oral methylcellulose alone, SB group that received an intraperitoneal injection of2mg/kg·d SB203580, PD group that received an intraperitoneal injection of2mg/kg-d PD98059, and Sim2group that received an intragastric administration of50mg/kg-d simvastatin. At the end of14weeks, all the mice were weighted and euthanized.3.3Quantitative real-time PCRTotal RNA was extracted from HASMCs to detect the expression level of P4Hα1mRNA.3.4Western blot analysisTotal proteins were extracted from HASMCs or the right common carotid arteries of ApoE-/-mice. The protein expression of P4Hal, type I and type III collagen, P-/T-p38MAPK, P-/T-JNK, P-/T-ERK1/2were analyzed by western blot.3.5ELISAThe content of soluble type I and III collagen in culture supernatants was assayed by ELISA.3.6Dil-ox-LDL uptakeHASMCs pretreated with or without simvastatin for1h were stimulated with50ug/ml Dil-ox-LDL for8h. After fixed and DAPI staining, the cells were placed for confocal microscopy.3.7Serum lipidsAt the end of the experiment, blood samples were collected by cardiac puncture in the mice fasted overnight to measure the serum levels of total cholesterol (TC), triglycerides (TG), low-density lipoprotein cholesterol (LDL-C) and high-density lipoprotein cholesterol (HDL-C) by an enzymatic assay.3.8Histopathological analysisSuccessive transverse cryosections were cut at5μm thickness and selectively stained with hematoxylin and eosin (H&E) at50μm intervals to select the point of maximal stenosis of arteries used for morphological analysis. Sirius red and Oil-red O staining were also performed. Frozen sections were stained for immunohistochemical analysis including macrophages (MOMA-2), SMCs (a-smooth muscle actin) and ox-LDL. The vulnerability index was calculated as (macrophage%+lipid%)/(collagen%+a-SM actin%).4Results4.1The in vitro experiment4.1.1ox-LDL-mediated suppression of P4Hal expressionIn the in vitro time-response study, ox-LDL suppressed the mRNA and protein expression of P4Hal significantly and the peak effect occurred after ox-LDL stimulation for8hours. In the in vitro dose-response study, ox-LDL also suppressed the expression of P4Hα1significantly and the peak effect was observed when HASMCs were treated with50ug/ml ox-LDL. Ox-LDL treatment also significantly reduced the expression levels of type I and III collagen in HASMCs and ELISA showed that ox-LDL also significantly reduced the protein levels of type I and III collagen in cell culture medium.4.1.2p38MAPK and ERKl/2pathways were involved in ox-LDL-induced downregulation of P4Hal expressionox-LDL induced the phosphorylation of p38MAPK and ERKl/2with the effect peaking at5min and lasting for8h. However, ox-LDL had no effect on the phosphorylation levels of JNK. To validate these results, HASMCs were pretreated with the inhibitors or siRNAs of p38MAPK, JNK and ERKl/2respectively before ox-LDL treatment. As a result, blockade or gene silencing of p38MAPK and ERKl/2attenuated ox-LDL-induced downregulation of P4Hal expression, whereas blockade or gene silencing of JNK had no effect on P4Hα1expression.4.1.3Simvastatin attenuated the suppressive effect of ox-LDL on P4Hα1expressionSimvastatin significantly inhibited the suppressive effect of ox-LDL on P4Hal expression, furthermore, simvastatin also largely reversed ox-LDL-induced reduction of type I and III collagen expression. However, simvastatin treatment alone without ox-LDL stimulation had no effect on P4Hal and collagen expression. Furthermore, simvastatin substantially reduced the phosphorylation levels of p38MAPK and ERKl/2in ox-LDL-stimulated HASMCs but had no effect on the phosphorylation of JNK. Furthermore, we found that simvastatin significantly decreased ox-LDL accumulation in the cells, suggesting that simvastatin inhibits the activation of p38and ERK probably via inhibiting ox-LDL uptake by HASMCs.4.2Animal model4.2.1Body weight and serum lipids in miceAt the end of the animal experiment, there were no significant difference in body weight among the six subgroups of mice. The serum levels of TC, TG and LDL-C increased while HDL-C decreased dramatically in the four subgroups of mice fed with a high diet relative to the Mockl subgroup but these serum lipid parameters did not differ among subgroups of mice fed with a high fat diet or with a normal diet. 4.2.2Simvastatin and p38MAPK and ERKl/2inhibitors enhanced plaque stabilityIn the normal diet group, there was no significant difference in vulnerability index between mice treated with and without simvastatin. The vulnerability index in mice fed with a high-fat diet only (Mock2group) was significantly higher than that in Mockl subgroup, indicating that high-fat diet feeding alone may increase plaque vulnerability in ApoE-/-mice. We also found that the vulnerability index was significantly decreased in mice receiving both high-fat diet and treatment with simvastatin or inhibitors of p38MAPK and ERKl/2, compared with the Mock2subgroup. These results demonstrated that simvastatin and p38and ERK1/2inhibitors had a plaque stabilizing effect in apoE-/-mice.4.2.3Simvastatin upregulated P4Hal expression in carotid plaquesThe levels of p38and ERK1/2phosphorylation and P4Hα1protein expression showed no significant difference between the two subgroups in mice fed with a normal diet. In contrast, the protein expression level of P4Hal was substantially decreased whereas the phosphorylation levels of p38and ERKl/2were significantly increased in the Mock2subgroup relative to the Mockl subgroup. The protein expression levels of P4Hα1and the content of collagen in the carotid plaques were significantly increased whereas the phosphorylation levels of p38and ERK1/2were reduced in mice receiving both a high-fat diet and treatment with simvastatin or inhibitors of p38MAPK and ERKl/2, in comparison with the Mock2subgroup. Furthermore, simvastatin treatment significantly reduced the relative content of ox-LDL in plaques relative to the Mock2subgroup. These results showed that simvastatin upregulated P4Hal expression by inhibiting ox-LDL uptake and inactivating p38and ERK1/2pathways in the carotid plaques.5Conclusion(1) Ox-LDL suppressed the expression of P4Hal and collagen via activating p38MAPK and ERK1/2signaling pathway.(2) Statins attenuated the suppressive effect of ox-LDL on P4Hal.The underlying mechanisms were that statins decreased the uptake of ox-LDL in HASMCs and then suppressed the activation of p38MAPK and ERKl/2.(3) In ApoE-/- mice, statins increased the expression of P4Hal and the content of collagen in AS plaques and increased the plaque stability. 1IntroductionAtherosclerotic plaque vulnerable to rupture is the major cause of acute coronary syndrome (ACS), which is identified by a thin, weakened fibrous cap, a large lipid core, accumulation of inflammatory cells and the imbalance between extracellular matrix (ECM) synthesis and degradation. As collagens are the major component of the fibrous cap of atherosclerotic plaques, the strength of plaque depends on a dynamic balance of collagen synthesis and degradation.Prolyl-4-Hydroxylase (P4H) is one of the key enzymes essential for the synthesis of all known types of collagen. P4H could catalyze proline located in repeating X-Pro-Gly triplets to hydroxyprolin which folds the procollagen polypeptide chains into stable triple helical molecules. As an isoenzyme of P4H, P4Hα1, is rate-limiting and essential for collagen maturation and secretion. The suppressed expression of P4Hal led to the decreased content of collagen in AS plaques. On the contrary, overexpression of P4Hal led to collagen synthesis.NonO, originally identified as a non-POU-domain-containing, octamer-binding protein, is55kDa ubiquitously expressed protein which contains two kinds of nucleic acid binding domains which could bind to both DNA and RNA. NonO could regulate the expression of many genes not only through directly binding to DNA as a transcriptional factor but also through binding to other protein as a cofactor. So, NonO could regulate many genes through different ways, among which some genes are in close relationship with atherosclerosis. NonO acts as a component of the cAMP-signaling pathway and is necessary for the activation of cAMP response element binding protein (CREB) target genes which contains TNF-a, IL-2and IL-6, and so on, suggesting that NonO may participate in promoting inflammation. NonO can bind to the auxiliary upstream sequence elements of cyclooxygenase-2(COX-2) and regulate its expression which also participate in the progression of AS. So, these studies suggests that NonO may take part in inflammation and atherosclerosis. Especially in recent years, Zhang C et al found that in HASMCs, NonO silencing dramatically attenuated TNF-a suppression of P4Hal gene expression and collagen synthesis. However, the exact role of NonO in vulnerable plaque is unclear.2Objectives(1) In the in vivo experiments, we delivered NonO-lentivirus (LV) or si-NonO-LV into the carotid plaques of apolipoprotein E-deficient (ApoE-/-) mice to detect its role in plaque disruption and plaque composition.(2) To elucidate the underlying molecular mechanisms of NonO-mediated detrimental effects on vulnerable plaque.3Methods3.1Preparation of Lentiviral vectors and Target screening of siRNANonO-overexpression lentivirus (NonO-LV) and three si-NonO lentivirus (si-A, si-B, si-C) were transduced into RAW264.7cells at a multiplicity of infection (MOI) of40.3days after transduction, cells were harvested for western blot to detect the overexpression effect and screen the most effective siRNA of NonO.3.2Animal model(1)20male ApoE-/-mice (8weeks of age) were fed with a high-fat diet (0.25%cholesterol and15%cocoa butter) for2weeks, and then randomly divided into two groups:Control group (n=10) which got no surgery; Mock group (n=10) which were placed a constrictive silastic tube around the right common carotid artery.8weeks later, mice in Mock group were restrained in a50-ml plastic tube with multiple holes on the wall to ensure sufficient ventilation and sound transmission, and then the tubes were put into a noise generator that emits noise every5min at110dB for3sec. The noise and restraint stress lasted for6h per day for4weeks. At the end of14weeks, all the mice were euthanized.(2)15male ApoE-/-mice were placed a constrictive silastic tube around the right common carotid artery.8weeks after surgery, the mice were transfected with lentivirus and euthanized before transfection (n=5),2weeks after transfection (n=5) and4weeks after transfection (n=5) to detect the tansfection efficiency of lentivirus.(3)125male ApoE-/-mice (8weeks of age) were fed with a high-fat diet for2weeks. Then all the mice were placed a constrictive silastic tube on the right common carotid artery to induce atherosclerotic lesion as described previously.8weeks after surgery, all the mice were divided into5groups (n=25per group) named:Control group, si-N.C group, si-NonO group, N.C group and NonO group, respectively, which were delivered to plaques with physiological saline, siRNA-N.C-LV, si-NonO-LV, pGC-GFP-LV and NonO-LV respectively as previously described.3days after lentiviral transfection, all the mice underwent stress stimulation as mentioned above. At the end of14weeks, all the apoE-/-mice were euthanized to collect the right common carotid arteries and blood from left ventricular for further analysis.3.3Body weight and Serum lipid profileBefore and at the end of the experiment, body weight of all the mice was measured. The serum concentrations of total cholesterol (TC), triglycerides (TG), low-density lipoprotein cholesterol (LDL-C) and high-density lipoprotein cholesterol (HDL-C) were measured by an enzymatic assay.3.4Histopathological analysisSerial cryosections of the carotid arteries were stained by H&E, oil-red O, sirius red. In addition, frozen sections were stained for immunohistochemical analysis included macrophages, SMCs, MMP-2, MMP-9, IL-1β,IL-6. The vulnerable index was calculated by the following formula:the relative positive staining area of (macrophages%+lipid%)/the relative positive staining area of (α-SMCs%+collagen%).3.5Cell treatment(1) For the time course study, we treated RAW264.7cells with100ng/ml TNF-a for0,6,12,24and48h. For the dose-dependent effects, we treated RAW264.7cells with0,25,50and100ng/ml for24h. All the cells were harvested for measurement of protein expression.(2) RAW264.7cells were cultured in6-well plates (2x106) in DMEM supplemented with10%FBS without antibiotics. Overnight after plating, cells were divided into five groups:①Control group:cells without transfection;②si-N.C group:cells transfected with siRNA-N.C-LV;③si-NonO group:cells transfected with si-NonO-LV;④N.C group:cells transfected with pGC-GFP-LV;⑤NonO-LV group:cells transfected with NonO-LV.3days after transduction, all the cells were stimulated with100ng/ml TNF-a for24h and then harvested for further analysis.3.6Quantitative real-time PCRTotal mRNA was extracted from the right common carotid arteries of ApoE-/-mice to detect the expression levels of NonO, MMP-2and MMP-9.3.7Western blotTotal proteins were extracted from the right common carotid arteries and RAW264.7cells. The protein expression levels of NonO, MMP-2, MMP-9, P4Hα1, LOX-1and COX-2in carotid plaques and NonO, MMP-2, MMP-9, IL-1β, MCP-1, ICAM-1, VCAM-1, p/t-NF-KB and IκBα in RAW264.7cells were detected by western blot.3.8Co-immunoprecipitationCo-immunoprecipitation assays were performed in RAW264.7cells treated with or without TNF-a to detect the combination of NonO and NF-κB.3.9ImmunofluorescenceImmunofluorescence were performed in RAW264.7cells to detect the effect of NonO on NF-κB p65nuclear translocation.4Results4.1Upregulation of NonO expression in atherosclerotic plaque and TNF-a-induced RAW264.7cellsIn ApoE-/-mice, compared with the control group, both mRNA and protein expression of NonO in Mock group were significantly increased, which suggested that a potential role of NonO in the pathogenesis of atherosclerosis.We also examined NonO expression in TNF-α-stimulated RAW264.7cells. In the time-response study, TNF-α upregulated the protein expression of NonO and the peak effect occurred at24h. In the dose-response study, TNF-α also increased the protein expression of NonO and the peak effect was observed when RAW264.7cells were treated with100ng/ml TNF-α.4.2Efficiency and effect of lentiviral transfection in vitroMice macrophage RAW264.7cells were transfected with three si-NonO-LVs (si-A, B and C) and the NonO-LV to detect their effects. Si-A, B and C exhibited57%,43%and44%reduction, respectively, in NonO protein expression. So, si-A were selected for further studies. NonO-LV increased the protein expression level of NonO by70%. So, si-NonO-LV and NonO-LV effectively knockdown and overexpressed the expression of NonO, respectively.4.3Efficiency of lentiviral transfection in vivoSince GFP expression provides a convenient monitor for detecting the transfection efficiency of lentivirus, the GFP fluorescence in the plaques was examined in0,2and4weeks after transfection.2weeks after transfection, carotid artery plaques had the obvious GFP fluorescence, which suggested a successful transfection of lentivirus.4weeks after transfection, GFP fluorescence was still visible in plaques, although their densities became weak. These results showed that lentivirus was efficiently transfected into atherosclerotic plaques.4.4The effect of NonO silencing and overexpression in carotid plaquesAt the end of our study, the expression levels of mRNA and protein of NonO in the carotid arterial plaques were detected by RT-PCR and western blot. Compared with them in control group, the levels of mRNA and protein of NonO were significantly decreased in si-NonO group by about47%and43%, respectively and increased in NonO-LV group by61%and59%, respectively.4.5Body weight and Serum lipid profileThere was no significant difference in body weight among the five groups of ApoE-/-mice, which suggested lentiviral transfection was safe in our experimental animals. Similarly, serum levels of TC, TG, LDL-C and HDL-C did not change significantly in all the experimental groups.4.6The effect of NonO on carotid plaque disruptionH&E staining revealed that in carotid arteries of ApoE-/-mice, the plaque rupture rate was46.67%(7/15) in the control group, the si-N.C group and the N.C group respectively,13.33%(2/15) in the si-NonO group and66.67%(10/15) in the NonO group. Perl’s staining also verified the results by indicating intraplaque thrombi and hemorrhage.4.7NonO changed carotid plaque compositionWe detected whether NonO changed the compositions of carotid plaques including macrophages, lipid, SMCs and collagen. NonO knockdown significantly decreased the content of macrophages and lipid, but increased the content of SMCs and collagen in carotid plaques, whereas NonO overexpression exerted opposite effects. Accordingly, the plaque vulnerability index was significantly reduced by NonO knockdown but elevated by NonO overexpression. These results showed that NonO played a critical role in plaque destabilization.4.8Effect of NonO on the expression of P4Hal in vivoWestern blot showed that the protein level of P4Hal were significantly increased in the NonO-silencing mice, whereas they were remarkably decreased in the NonO-overexpression mice. The result confirmed that NonO participated in inhibiting the expression of P4Hα1.4.9Effect of NonO on the expression of MMP-2and MMP-9both in vivo and in vitroWe also detected the expression levels of MMP-2and MMP-9both in vivo and in vitro.In ApoE-/-mice and RAW264.7cells, the silencing of NonO downregulated the expression levels of MMP-2and MMP-9, while the overexpression of NonO upregulated the expression levels of them. In RAW264.7cells, we also detected the activity of MMP-2and MMP-9and found that NonO knockdown significantly suppressed the activity of MMP-2and MMP-9, whereas NonO overexpresion obviously increased them. 4.10Effect of NonO on proinflammatory cytokine expression both in vivo and in vitroIn ApoE-/-mice, the relative contents of IL-1β and IL-6in the carotid plaques analysed by immunohistochemistry were significantly lower in si-NonO group, whereas significantly higher in NonO-LV group than those in the control group. Western blot showed that NonO significantly increased the protein expression levels of LOX-1and COX-2, whereas NonO downregulation decreased their expression.In cultured RAW264.7cells, the protein expression levels of IL-1β, MCP-1, ICAM-1and VCAM-1were remarkably decreased in si-NonO group, whereas increased in NonO group compared with those in the control group. So, NonO promoted the expression of inflammatory cytokines both in vivo and in vitro.4.11NonO combined with NF-κB and influenced its nuclear translocation and phosphorylationCo-immunoprecipitation assays were performed in RAW264.7cells treated with or without TNF-a. Whole cell lysates were analyzed in SDS/PAGE along with anti-NF-κB p65or anti-NonO immunoprecipitations. Western blot showed that in the nonstimulated state, NonO coimmunoprecipitated with NF-κB p65, although the interaction was weak; after TNF-α stimulation, the interaction was significantly enhanced between NonO and NF-κB p65.We detected the effect of NonO on the nuclear translocation of NF-κB p65. Compared with the control group, si-NonO group showed significantly suppressed p65nuclear translocation, whereas NonO group remarkably increased this translocation. In addition, compared with the control group, NonO silencing could suppress the level of p-p65, while NonO overexpression could enhance it. We also detected the expression of IkBα and found that NonO overexpression significantly decreased the expression level of IκBα, whereas NonO silencing enhaced its protein level.5Conclusion(1) In ApoE-/-mice, the expression of NonO was upregulated in its atherosclerotic plaque. (2) NonO played an important role in plaque destabilization, and gene silencing of NonO could increase the stability of plaque.(3) NonO destabilizes atherosclerotic plaques via increasing the expression of MMP-2and MMP-9, suppressing the expression of P4Hα1and exaggerating inflammatory responses mediated by NF-κB. 1IntroductionDiabetic nephropathy (DN) has become the most common cause of end-stage renal disease and represents an increasing global public health problem. A wealth of evidence indicates that renin-angiotensin system (RAS) plays a key role in the pathogenesis of DN, and a galaxy of clinical trials have proven that angiotensin-converting enzyme (ACE) inhibitors and type1angiotensin receptor antagonists are effective in attenuating the development of DN. These protective effects were originally thought to result from blocking angiotensin Ⅱ-dependent pathways. However, the recent discovery of a homologue of ACE, ACE2, revealed a new pathway for angiotensin peptide metabolism. ACE2generates angiotensin(1-7)[Ang(1-7)] from angiotensin Ⅱ and plays a protective role against DN. However, the effect of Ang(1-7), the primary product of ACE2, on DN remains poorly understood.Ang(1-7) as a heptapeptide is mainly derived from the degradation of Angiotensin Ⅱ by ACE2in the kidney. Consistent with its site of synthesis, Ang(1-7) exerts important effects on renal homeostasis. In proximal tubular cells, Ang(1-7) activates tyrosine phosphatase and inhibits high-glucose-stimulated p38MAPK, thereby suppressing high-glucose-induced protein synthesis. Ang(1-7) attenuates renal vascular dysfunction by alleviating NADPH oxidase (NOX)-mediated oxidative stress. However, an recent study showed that an injection of moderate dose of Ang(1-7) paradoxically accelerated STZ-induced diabetic renal injury.So far, several important issues remain unsolved. First, whether Ang(1-7) treatment is beneficial or harmful to DN is controversial. Second, the effect of different doses of Ang(1-7) on DN is to be defined. Third, it is unclear whether combined treatment with Ang(1-7) and an angiotensin receptor blocker (ARB) is superior to either treatment alone for DN. Finally, t are only poorly understood.2Objectives(1) To assess the effect of Ang(1-7) and the combined treatment of Ang(1-7) and ARB on STZ-induced diabetic renal injury.(2) To elucidate the possible mechanisms underlying the effect of Ang(1-7) on DN.3Methods3.1Animal Model120male Wistar rats (10weeks old,200to250g) were randomly divided into two groups:control group (n=15) that received an intraperitoneal injection of normal saline and DM group (n=105) that received intraperitoneal injection of65mg/kg STZ. The status of DM in rats was confirmed by a tail-blood glucose level higher than16.7mmol/148h after STZ injection. All diabetic rats received an intraperitoneal injection of insulin (2-3U) every3days to maintain blood glucose levels between16.7and25mmol/1to prevent mouse death induced by excessively high blood glucose levels. Twelve weeks after STZ injection, diabetic rats were further randomly divided into seven subgroups (n=15per subgroup) for treatment:no treatment subgroup (NT group); large-dose, moderate-dose and small-dose Ang(1-7) subgroups that received a subcutaneous injection of800ng/kg-min,400ng/kg-min and200ng/kg-min of Ang(1-7), respectively, by an embedded mini-osmotic pump; valsartan subgroup given by intragastric administration at a dose of30mg/kg per day; large-dose Ang(1-7)+valsartan (30mg/kg per day) subgroup and large-dose Ang(1-7)+A779(both800ng/kg-min) subgroup. After treatment for4weeks, all rats underwent euthanasia.3.2General condition of rats At the end of our experiment, the blood glucose, systolic pressure (SBP), body weight (BW), kidney weight (KW),24h urine volume, creatinine levels of blood and urine were measured and KW/BW and creatinine clearance were calculated.3.3ELISAELISA was performed to test Ang(1-7) levels of serum and urine, the level of urinary albumin, and24-h urinary albumin were calculated.3.4Measurement of MDA content and SOD activityThe content of MDA and the activity of SOD in glomeruli were measured according to the specifications of kits.3.5Histopathological analysisTissue section were stained with PAS staining and glomeruli sclerosis index (GSI) were calculated. Immunohistochemistry were performed to detect the content of type IV collagen, TGF-(31, VEGF, PCNA and macrophages in glomeruli.3.6Cell culture and treatmentRat glomerular mesangial HBZY-1cells were pretreated with three doses of Ang(1-7)(50,100and200nmol/1) or10-6mol/1valsartan or200nmol/1Ang(1-7)+10-6mol/1valsartan or200nmol/1Ang(1-7)+200nmol/1A779for1h, and then treated with high glucose (25mmol/1) for24h. In addition, in the group treated by Ang(1-7)+A779, cells was pretreated with A779for30min before Ang(1-7) treatment. After stimulation, cells were harvested for further study.3.7Western blot analysisWestern blot was performed to detect the expression levels of NOX4, p47phox, PKCα, PKCpβ2, TGF-β1, p-Smad3in glomeruli and NOX4. p47phox、TGF-β1、 p-Smad3、VEGF'type IV collagen in HBZY-1cells. In addition, PKC proteins in the membranous and cytosolic fractions of HBZY-1cells were purified using membrane and cytosol protein extraction kit and western blot were performed to detect their expression levels in membrane and cytoplasm.3.8DHE staining, DCF staining and EdU proliferation assayDHE and DCF staining were performed to detect the level of ROS in HBZY-1cells. Proliferating HBZY-1cells were detected by use of an EdU labeling kit. 4Results4.1Serum and urine levels of Ang(1-7)The serum and urinary levels of Ang(1-7) in seven groups of DM rats were significantly lower than in the control group of rats before Ang(1-7) treatment. However, these levels were dose-dependently increased in the six treatment groups.4.2Blood pressure and blood glucose levelsAt the end of experiment, systolic blood pressure (SBP) was significantly lower in all seven groups of DM rats than in the control group of rats, but there was no significant difference in SBP among seven groups of DM rats. The blood glucose levels in all seven groups of DM rats were markedly higher than in the control group of rats, but did not differ among seven groups of DM rats.4.3Ang(1-7) dose-dependently ameliorated renal functionBody weight and creatinine clearance were significantly lower in seven groups of DM rats than those in the control group of rats. However, these parameters were dose-dependently increased in all treatment groups of DM rats except for the large-dose Ang(1-7)+A779group in which the body weight and creatinine clearance were similar with those in the no treatment group. The ratio of kidney weight to body weight,24-h urinary volume, plasma creatinine level and24-h urinary albumin excretion were all significantly higher in seven groups of DM rats than in the control group of rats. These values were decreased in all treatment groups of DM rats except for the large-dose Ang(1-7)+A779group in which these values returned to the level of the no treatment grou... |
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