Effect of Salvia officinalis aqueous infusion on copper sulfate-induced inflammatory response and oxidative stress imbalance in mice liver and kidney.

Extracts of Salvia officinalis (S. officinalis) have been described to have many therapeutic properties. However, the effect of S. officinalis on copper sulfate toxicity has not been previously reported. The aim of this study was to investigate the toxicity of copper sulfate and the potential beneficial effects of S. officinalis aqueous infusion on proinflammatory response and antioxidant status. 56 male mice were used and equally divided into 6 groups: control group, copper sulfate treated group (40 mg/kg), S. officinalis aqueous infusion treated groups (200 mg/kg and 400 mg/kg) separately or in combination with copper. IL-6 (interleukine-6) and TNF-α (Tumor necrosis factor alpha) were assessed by Elisa. Catalase (CAT), superoxide dismutase (SOD) and acetylcholinesterase (AChE) activities, malondialdehyde (MDA) and oxygen peroxide levels were determined. Serum biochemical parameters were analyzed. Copper enhanced aspartate aminotransferase (AST), alanine aminotransferase (ALT) and Lactate dehydrogenase (LDH) (p < 0.05). Copper enhances significantly IL-6, TNF-α and MDA levels in liver and kidney and reduced CAT, SOD and AChE activities (p < 0.05). Aqueous infusion of S. officinalis at 400 mg/kg abolished copper-induced changes in AST and ALT activity. S. officinalis aqueous infusion at 200 mg/kg reversed copper-induced IL-6 in kidney and TNF-α in liver at both doses. S. officinalis aqueous infusion at 400 mg/kg restored SOD in kidney and CAT and AChE activities in both liver and kidney. S. officinalis aqueous infusion may be useful in partially ameliorating tissue disorders induced by copper exposure such as inflammatory response, oxidative stress imbalance and organ dysfunction through its phenolic compounds and higher antioxidant capacity.

Effects of Lupinus luteus on hepatic and renal extracellular compounds turnover under diabetes in rat.

Hepatic and renal extracellular matrix (ECM) turnover associated with diabetes and potential beneficial effects of yellow lupin extract (YLE) need further investigations. The aim of this study was to explore the effect of yellow Lupinus luteus extract (YLE) on renal and hepatic ECM under diabetes. Composition of YLE performed by LC-ESI-MS. Diabetes (DM) was induced in rats by alloxan (250 mg/kg, ip). Normal and diabetic rats received 100 mg/kg of YLE for 1 month. ECM was assessed by ELISA. Gelatinases and collagenases were analyzed by a colorimetric assay. Histology was performed on sections of liver and kidney. In the liver, diabetes increases collagen, laminin, and fibronectin contents, respectively, by 49% (p < .01), 56% (p < .01), and 67% (p < .05) compared to control rats. In the kidney, total collagen and laminin contents were increased by 91% (p < .01) and 35% (p < .01) in the DM group, while fibronectin content in diabetic animals and those treated with YLE remains similar to the control group. Collagenases and gelatinases activities were significantly increased by diabetes in liver and kidney. While YLE treatment abrogates diabetes-enhanced MMPs activities in liver. In diabetic rats, the liver shows signs of diffuse dilatation of the sinusoid veins and steatosis. However, the liver of diabetic rats treated with yellow lupine extract showed a normal histological aspect similar to controls. Diabetes causes hepatic and renal ECM turnover. YLE can be useful to partially improve tissue disorders induced by diabetes.

Cardiac extracellular matrix modulation in a rat-diabetic model: biochemical and anti-oxidant beneficial effect of pomegranate (Punica granatum) peel extract.

CONTEXT: Disorders associated with diabetes and the beneficial effects of pomegranate peel extract (PPE) were widely reported. However effect of diabetes and PPE on extracellular matrix (ECM) remodelling needs further investigation. OBJECTIVES: The focus of this study was to investigate the involvement of diabetes in cardiac ECM and the beneficial effects of PPE. METHODS: Diabetes was induced by alloxan. PPE group was injected with 100 mg/kg of PPE. The phenolic profile of PPE was analyzed by HPLC. ECM was detected by ELISA. MMP-1, -8, -13 were determined by a colorimetric assay. RESULTS: Compared to control fibronectin and laminin plasma content was higher respectively by 69% and 42% (p < 0.05) in diabetes. LV content of hydroxyproline and total collagen was higher by 195% (p < 0.01) and 56% (p < 0.05) in the diabetic group compared to control and restored at a similar level to controls in the PPE group. Compared to control, collagenase activity was significantly reduced by 32% (p < 0.05) and 35% (p < 0.05) respectively in ALX and PPF groups. There is no significant difference in collagenase activities in diabetic rats after and before PPE injection. CONCLUSION: Diabetes is involved in cardiac ECM remodelling which can be improved by PPE. These findings will be useful for more understanding diabetes-induced cardiac disorders.

The detoxifying effect of Polygonum equisetiforme extracts against dichlorvos (DDVP)-induced oxidative stress and neurotoxicity in the commercial clam Ruditapes decussatus.

Effects of Polygonum equisetiforme extracts against dichlorvos were investigated in the commercial clam Ruditapes decussatus. The toxicity of this pesticide was firstly tested in R. decussatus gill and digestive gland tissues using five doses varying from 0.05 to 1 mg/l during 2, 4, and 7 days. Results showed that 0.05 mg/l of DDVP induced oxidative stress and neurotoxicity in R. decussatus after 2 days of exposure. Investigations of the effects of P. equisetiforme extracts in R. decussatus exposed to 0.25 mg/l of DDVP were made in clams receiving three concentrations (0.009, 0.045, and 0.09 g/l) during 4 and 7 days. Antioxidant enzymes SOD and CAT as well as H2O2 content and AChE were quantified by colorimetric method. Four days of exposure to DDVP increased SOD and CAT activities and enhances H2O2 content. AChE levels decreased considerably following DDVP exposure, although a restoration in the enzyme activity was observed with P. equisetiforme extract (E3 = 0.09 g/l). Overall, P. equisetiforme extract at concentration (E1 = 0.009 g/l) prevents oxidative stress caused by DDVP, while 0.09 g/l of P. equisetiforme extract induced an effect similar to that obtained with DDVP alone. Nevertheless, this concentration was found effective for the restoration of the AChE activity.

Computational identification of potential transcriptional regulators of TGF-ß1 in human atherosclerotic arteries.

TGF-ß is protective in atherosclerosis but deleterious in metastatic cancers. Our aim was to determine whether TGF-ß transcriptional regulation is tissue-specific in early atherosclerosis. The computational methods included 5 steps: (i) from microarray data of human atherosclerotic carotid tissue, to identify the 10 best co-expressed genes with TGFB1 (TGFB1 gene cluster), (ii) to choose the 11 proximal promoters, (iii) to predict the TFBS shared by the promoters, (iv) to identify the common TFs co-expressed with the TGFB1 gene cluster, and (v) to compare the common TFs in the early lesions to those identified in advanced atherosclerotic lesions and in various cancers. Our results show that EGR1, SP1 and KLF6 could be responsible for TGFB1 basal expression, KLF6 appearing specific to atherosclerotic lesions. Among the TFs co-expressed with the gene cluster, transcriptional activators (SLC2A4RG, MAZ) and repressors (ZBTB7A, PATZ1, ZNF263) could be involved in the fine-tuning of TGFB1 expression in atherosclerosis.

Hypercholesterolemic diet induces vascular smooth muscle cell apoptosis in sympathectomized rats via intrinsic pathway.

In this study, we intend to investigate the role of hypercholesterolemic diet, a high risk factor for atherosclerosis, on vascular cell apoptosis in rats that have been previously sympathectomized. Thus, newborn male Wistar rats received injections of guanethidine for sympathectomy. Sham received injections of vehicle. The two groups were fed 1% cholesterol diet for 3months. Sympathectomy alone group was also exploited. Apoptosis in abdominal aortic tissue was identified by TUNEL method and conventional agarose gel electrophoresis to detect specific DNA fragmentation. Caspases 3 and 9, Bcl-2, Bax and cytochrome c were examined by immunoblotting. Oil Red O staining was used to reveal lipid in the arterial wall. Vascular smooth muscle cells (VSMCs) and macrophages were identified by immunostaining for α-smooth muscle actin and rat macrophage marker (ED1), respectively. The efficacy of sympathectomy was evaluated by analysis of perivascular sympathetic fibers. Our study showed that hypercholesterolemic diet, when performed in rats with neonatal sympathectomy, 1) increased aortic TUNEL-positive cells compared to sham and sympathectomy alone groups, 2) illustrated a typical apoptotic DNA ladder on agarose gel electrophoresis, 3) induced Bax translocation from cytosol to mitochondria, 4) enhanced cytochrome c release from mitochondria to cytosol, 5) increased expression of active caspases 3 and 9, and 6) decreased Bcl-2 expression. VSMCs are identified as the major cell type exhibiting apoptosis in this model. Taken together, it can be concluded that hypercholesterolemic diet, when performed in rats with neonatal sympathectomy, induces vascular cell apoptosis in an intrinsic pathway.

Physiological regulation of pro-inflammatory cytokines expression in rat cardiovascular tissues by sympathetic nervous system and angiotensin II.

Pro-inflammatory cytokines regulation by sympathetic nervous system (SNS) and angiotensin II (ANG II) was widely described in cardiovascular system, but the role of such neuro-humoral interaction needs further investigation in this context. We tested SNS-ANG II interaction on IL-6 and TNF-α mRNA expression in left ventricle and aorta from normotensive rats by sympathectomy with guanethidine and blockade of the ANG II AT1 receptors (AT1R) antagonist with losartan. mRNA synthesis of IL-6 and TNF-α were performed by Q-RT-PCR. In the left ventricle, IL-6 mRNA increased by 63% (p < 0.01) after sympathectomy, still unchanged after losartan treatment and decreased by 38% (p < 0.05) after combined treatment. TNF-α mRNA decreased by 44% (p < 0.01), only after combined treatment. In the aorta, IL-6 mRNA increased equally by 65% (p < 0.05) after sympathectomy or losartan treatment. TNF-α mRNA decreased by 28, 41, and 42% (p < 0.05) after sympathectomy, losartan and combined treatments, respectively. Our data suggest that ANG II stimulates directly (via AT1R) and indirectly (via SNS) IL-6 mRNA synthesis in left ventricle and aorta and TNF-α mRNA in left ventricle. ANG II seems unable to influence directly TNF-α mRNA synthesis in the aorta but can stimulate this cytokine via SNS. The results are relevant to prevent or reduce proinflammatory cytokines overexpression seen in cardiovascular diseases.

Regulation of aortic extracellular matrix synthesis via noradrenergic system and angiotensin II in juvenile rats.

CONTEXT: Extracellular matrix (ECM) synthesis regulation by sympathetic nervous system (SNS) or angiotensin II (ANG II) was widely reported, but interaction between the two systems on ECM synthesis needs further investigation. OBJECTIVE: We tested implication of SNS and ANG II on ECM synthesis in juvenile rat aorta. MATERIALS AND METHODS: Sympathectomy with guanethidine (50 mg/kg, subcutaneous) and blockade of the ANG II AT1 receptors (AT1R) blocker with losartan (20 mg/kg/day in drinking water) were performed alone or in combination in rats. mRNA and protein synthesis of collagen and elastin were examined by Q-RT-PCR and immunoblotting. RESULTS: Collagen type I and III mRNA were increased respectively by 62 and 43% after sympathectomy and decreased respectively by 31 and 60% after AT1R blockade. Combined treatment increased collagen type III by 36% but not collagen type I. The same tendency of collagen expression was observed at mRNA and protein levels after the three treatments. mRNA and protein level of elastin was decreased respectively by 63 and 39% and increased by 158 and 15% after losartan treatment. Combined treatment abrogates changes induced by single treatments. DISCUSSION AND CONCLUSION: The two systems act as antagonists on ECM expression in the aorta and combined inhibition of the two systems prevents imbalance of mRNA and protein level of collagen I and elastin induced by single treatment. Combined inhibition of the two systems prevents deposit or excessive reduction of ECM and can more prevent cardiovascular disorders.

Chemical sympathectomy induces arterial accumulation of native and oxidized LDL in hypercholesterolemic rats.

The aim of the present study was to examine the effect of sympathectomy on plasmatic and arterial native and oxLDL levels, as well as arterial LDL receptors (LDLR) and scavenger receptors in hypercholesterolemic rats, which are normally protected against atherosclerosis. Neonatal Wistar rats received subcutaneous injections of either guanethidine for sympathectomy (Gua+HC) or vehicle (HC), then were fed 1% cholesterol for three months. Intact normocholesterolemic rats were used as control of the HC group. Total cholesterol (TC) and LDL-cholesterol were evaluated in the plasma and the abdominal aorta by an auto-analyzer. Plasmatic and aortic oxLDL and native LDL-apo B100 were assessed by a sandwich ELISA. Aortic and hepatic native LDLR and aortic scavenger receptors (CD36 and SR-A) were quantified at mRNA and protein levels by real time PCR and western immunoblot. The effect of hypercholesterolemia was limited to an increase in plasmatic TC and LDL-cholesterol and a decrease in aortic apoB100 and aortic and hepatic LDLR. Hypercholesterolemia and sympathectomy in combination increased markedly plasmatic and aortic TC, LDL-cholesterol, apo B100 and oxLDL together with aortic scavenger receptors, but reduced markedly aortic and hepatic LDLR. Sympathectomy broke down the rat's protection against hypercholesterolemia by promoting accumulation of native and oxLDL in the aorta via scavenger receptors.

Physiological regulation of extracellular matrix collagen and elastin in the arterial wall of rats by noradrenergic tone and angiotensin II.

The interactions between the effects of the sympathetic nervous system (SNS) and angiotensin II (ANG II) on vascular extracellular matrix (ECM) synthesis were determined in rats. The mRNA and protein content of collagen I, collagen III and elastin in the abdominal aorta (AA) and femoral artery (FA) was investigated in Wistar-Kyoto rats treated for 5 weeks with guanethidine, a sympathoplegic, losartan, an ANG II AT1 receptor (AT1R) blocker, or both. The effects of noradrenaline (NE) and ANG II on collagen III and elastin mRNA, and the receptor involved, were tested in cultured vascular smooth muscle cells (VSMCs) in vitro. Guanethidine increased collagen types I and III and decreased elastin, while losartan had an opposite effect, although without effect on collagen III. The combination of treatments abrogated changes induced by simple treatment with collagen I and elastin, but increased collagen III mRNA in AA and not in FA. NE stimulated collagen III mRNA via ß receptors and elastin via α1 and α2 receptors. ANG II stimulated collagen III but inhibited elastin mRNA via AT1R. Overall, SNS and ANG II exert opposite and antagonistic effects on major components of ECM in the vascular wall. This may be of relevance for the choice of a therapeutic strategy in vascular diseases.

Physiological regulation of MMPs and tPA/PAI in the arterial wall of rats by noradrenergic tone and angiotensin II.

The interactions between the sympathetic nervous system (SNS) and angiotensin II (ANG II), and their direct effects in vitro on the enzymes involved in vascular extracellular matrix (ECM) degradation, were examined. Rats were treated with guanethidine, losartan or the combined treatments. mRNA, protein and activity of matrix metalloproteinase (MMP)-2 and MMP-9 and mRNA of tissue plasminogen activator (t-PA) and plasminogen activator inhibitor 1 (PAI-1) were quantified in abdominal aorta (AA) and femoral artery (FA). Norepinephrine (NE) or ANG II with adrenergic (ß, α1 and α2) or losartan antagonists was tested for MMP mRNA response in cultured vascular smooth muscle cells (VSMCs). Combined treatment enhances the inhibition of MMP-2 mRNA and protein level induced by simple treatment in AA. However MMP-9 in AA and MMP mRNA in FA were reduced in the same order by treatments. MMP activities were not affected by treatments. The t-PA/PAI-1 ratio, which reflects the fibrinolytic balance, remained higher after treatments. In cultured VSMCs, NE induced stimulation of MMP mRNA via α2 and ß adrenergic receptors and MMP-2 activity via ß adrenergic receptors, while ANG II-induced stimulation was abrogated by losartan. Overall, there is a synergic inhibition of both systems on the level of MMP-2 in AA.

Interaction between sympathetic nervous system and renin angiotensin system on MMPs expression in juvenile rat aorta.

The aim of our present study is to investigate the interaction between angiotensin II (ANG II) and sympathetic nervous system (SNS) on matrix metalloproteinase MMP-2 and MMP-9 expression and activity in juvenile rat aorta under normal conditions. Sympathectomy with guanethidine and blockade of the ANG II receptors (AT1R) by losartan were performed alone or in combination on new-born rats. mRNA, protein expression and activity of MMP-2 and MMP-9 were examined by Q-RT-PCR, immunoblotting and zymography, respectively. MMP-2 mRNA and protein amount were decreased after sympathectomy or AT1R blockade and an additive effect was observed after combined treatment. However, MMP-9 expression was reduced to the same level in the three treated groups. There were some detectable gelatinolytic activity of the MMPs in both control and treated rats. We concluded that ANG II stimulates directly and indirectly (via sympathostimulator pathway) the MMP-2 expression but seems unable to affect MMP-9 expression through direct pathway. Combined inhibition of SNS and ANG II were more efficient than a single inhibition in reducing MMP amounts in rat vessels.

The profile of the extracellular matrix changes in the aorta after sympathectomy in the hypercholesterolemic rats.

We previously showed that sympathectomy induces thickened intima and decreases the expression of cytoskeletal proteins associated with a differentiated smooth muscle cell (SMC) phenotype in hypercholesterolemic rats. In the present study, we sought to determine the effect of sympathectomy on various components of the extracellular matrix (ECM) in the aorta from these animals, since the state of SMC differentiation depends on the nature of ECM components. Collagen types I and III, previously reported to be associated with SMC dedifferentiation, and collagen VI, elastin, laminin and elastin-laminin receptor (E/L-R), previously reported to be associated with SMC differentiation, were analyzed by western immunoblot and confocal microscopy in abdominal aortae from sham rats and hypercholesterolemic rats sympathectomized with guanethidine. Both western immunoblot and immunohistological analysis showed an increase in collagens I and III (more for collagen I), with abundant labeling in the media, adventitia and thickened intima in sympathectomized aortae. Collagen IV labeling was decreased in the media and adventitia and was weak in the thickened intima in sympathectomised aortae. The E/L-R increased and was abundantly labeled in the media and weakly in the thickened intima in sympathectomized aortae. Elastin and laminin decreased and appeared less labeled in the media in the sympathectomised aortae. In the thickened intima, laminin was slightly labeled while elastin was not obviously labeled. These data show that sympathectomy favors the ECM features reported in association with a dedifferentiated/immature SMC phenotype and intimal thickening, probably by actions on both SMCs and fibroblasts.

Influence of antagonist sensory and sympathetic nerves on smooth muscle cell differentiation in hypercholesterolemic rat.

The effect of sympathectomy and sensory denervation on vascular smooth muscle cell (SMC) differentiation was investigated in hypercholesterolemic rats. Newborn rats received injections of guanethidine, capsaicin or both for denervations. Shams received injections of vehicles. The four groups were fed 1% cholesterol diet for 3 months. Intact normocholesterolemic rats were also exploited. Serum total cholesterol and systolic blood pressure (SBP) were measured. Lipid presence in the arterial wall was shown by Red-Oil-O staining. Catecholamine- and CGRP-containing fibres, vimentin and the adult SMC markers alpha-SMC-actin, desmin and h-caldesmon were analysed in abdominal aorta by western blot and confocal microscope. The sympathetic (catecholamine) fibres and SBP increased after sensory denervation while the sensory (CGRP) fibres increased and SBP decreased after sympathectomy. SBP was not changed after double denervation. Total cholesterol increased in sham and rose further after sympathectomy. Vimentin and the three adult SMC markers were not influenced by hypercholesterolemia. However, in the sympathectomized aorta, vimentin increased, desmin did not change, whereas alpha-SMC-actin and h-caldesmon decreased. In the sensory-denervated aorta, vimentin decreased, desmin increased, alpha-SMC-actin did not change and h-caldesmon decreased but less than in sympathectomized aorta. In the doubly denervated aorta, vimentin did not change and the three adult SMC markers decreased, although less than in sympathectomized aorta for alpha-SMC-actin and h-caldesmon. Thickened intima was identified by Red-Oil-O staining in the sympathectomized and (less remarkably) doubly denervated aortas containing SMCs not fully dedifferentiated. Our findings suggest that sympathectomy induces intimal thickening and favours SMC dedifferentiation, whereas sensory denervation favours SMC differentiation.

Differential control of collagen synthesis by the sympathetic and renin-angiotensin systems in the rat left ventricle.

In the present study, we tested the hypothesis of the indirect (via the sympathetic nervous system (SNS)) and direct (via AT1 receptors) contributions of Angiotensin II (Ang II) on the synthesis of collagen types I and III in the left ventricle (LV) in vivo. Sympathectomy and blockade of the Ang II receptor AT1 were performed alone or in combination in normotensive rats. The mRNA and protein synthesis of collagen types I and III were examined by Q-RT-PCR and immunoblotting in the LV. Collagen types I and III mRNA were decreased respectively by 53% and 22% after sympathectomy and only collagen type I mRNA was increased by 52% after AT1 receptor blockade. mRNA was not changed for collagen type I but was decreased by 25% for collagen type III after double treatment. Only collagen protein type III was decreased after sympathectomy by 12%, but collagen proteins were increased respectively for types I and III by 145% and 52% after AT1 receptor blockade and by 45% and 60% after double treatment. Deducted interpretations from our experimental approach suggest that Ang II stimulates indirectly (via SNS) and inhibits directly (via AT1 receptors) the collagen type I at transcriptional and protein levels. For collagen type III, it stimulates indirectly the transcription and inhibited directly the protein level. Therefore, the Ang II regulates collagen synthesis differently through indirect and direct pathways.

Differential control of MMP and t-PA/PAI-1 expressions by sympathetic and renin-angiotensin systems in rat left ventricle.

In the present study, we tested the hypothesis that angiotensin II (Ang II) has both direct (via AT1 receptors) and indirect (via sympathostimulator pathway) actions on the synthesis and activity of the enzymes involved in the extracellular matrix degradation in vivo. For this purpose, sympathectomy and blockade of the Ang II receptor AT1 were performed alone or in combination in normotensive rats. The mRNA of the plasminogen activator (t-PA) and its inhibitor (PAI-1), the mRNA, protein and activity of the matrix metalloproteinases MMP-2 and MMP-9 were examined by Q-RT-PCR, immunoblotting and zymographic methods in the left ventricle. t-PA and PAI-1 mRNA were decreased after sympathectomy and remained unchanged after AT1 receptors blockade. mRNA was increased for t-PA and decreased by similar degree for PAI-1 after double treatment. MMPs mRNA and protein levels were decreased either after sympathectomy or AT1 receptors blockade and an additive effect was acquired after double treatment. MMPs activity was decreased by similar degree in the three treated groups. Deducted interpretations from our experimental approach suggest that Ang II inhibits directly (via AT1 receptors) and indirectly (via sympathostimulator pathway) t-PA mRNA synthesis. It seems unable to influence directly PAI-1 mRNA, but stimulates indirectly PAI-1 mRNA synthesis. Ang II stimulates directly (via AT1 receptors) and indirectly (via sympathostimulator pathway) MMPs synthesis at both transcriptional and protein levels. The enzymatic activity of MMPs does not seem to be influenced directly by Ang II but it could be stimulated indirectly (via sympathostimulator pathway).