Salvianolic acid A ameliorates AGEs-induced glomerular endothelial dysfunction and protects against diabetic nephropathy

OBJECTIVE Diabetic nephropathy(DN)has been one of the most common complications of diabetes and the leading cause of end-stage renal disease.Glomerular hyperfiltrationis central in earlystage of DN and leads to the progression of renal architectonic and functional abnormalities.Salvianolic acid A(SalA)has been proved to protect diabetic complications such as hepatic fibrosis and neuropathy.The present study was designed to investigate the effects of SalA on glomerular endothelial dysfunctionand diabetic nephropathy.METHODS Primary glomerular endothelial cells were subjected to assess permeability under injury of advanced glycation end-products(AGEs).AGEs-induced changes of Rho A/ROCK pathway and cytoskeleton rearrangement were assessed bywestern blotandimmunofluorescence.The beneficial effects of SalA on diabetic nephropathy were investigated in a rat model induced by high-fat and high-glucose diet combined with low dose of streptozocin(35 mg·kg^(-1),ip).Renal function and architectonic changes were evaluated by biochemical assay and PAS staining.RESULTS SalA 3μMameliorated AGEs-induced glomerular endothelial permeability(P<0.05)and suppressed rearrangement of cytoskeleton through inhibiting AGE-RAGE-Rho A/ROCK pathway.SalA1 mg·kg^(-1)markedly reduced endothelium loss(P<0.01)and glomerular hyperfiltration(P<0.05)in diabetic kidney.Subsequently,SalA 1 mg·kg^(-1) suppressed glomerular hypertrophy and mesangial matrix expansion,eventually reduced 24 h-urinary albumin and ameliorated renal function by decreasing blood urine nitrogen(BUN),serum creatinine(Scr)and serum n-acetyl-β-d-glucosaminidase(NAG).AGEs-RAGE-Nox4-induced oxidative stress was suppressed by the treatment of SalA 1 mg·kg^(-1).CONCLUSION SalA ameliorated AGEs-induced glomerular endothelial hyperpermeability,and effectively protected against early-stage diabetic nephropathy by reducing hyperfiltration and alleviating renal structural deterioration through inhibiting AGEs and its downstream pathway.Thus,SalA might be a promising therapeutic agent for the treatment of diabetic nephropathy.

Preparation and Certification of a New Salvianolic Acid A Reference Material for Food and Drug Research

Salvianolic acid A(Sal A),a water-soluble ingredient in Danshen,has various biological activities.Sal A and its impurities have similar physical and chemical properties,as well as strong reducibility;therefore,they are difficult to prepare and purify.In this study,high-purity Sal A was obtained by purification of sephadex chromatography and preparative chromatography.Furthermore,HPLC-DAD tandem ECD and HPLC-DAD tandem MS methods were used for non-volatile organic impurity analysis,ICP-MS method was used for non-volatile inorganic impurities and mass balance method and quantitative nuclear magnetic resonance were employed to certify the product.The structures of Sal A and its relative impurities were validated by nuclear magnetic resonance spectroscopy and mass spectrometry,and their contents were quantified as well.Following the principles of ISO Guides 34:2009 and 35:2005,a Sal A reference material was certified,covering homogeneity studies,stability studies,characterization,and uncertainty estimations.

The Synthesis of Hexamethyl Ether of Salvianolic Acid A Benzyl Ester

A salvianolic acid A derivative, hexamethyl ether of salvianolic acid A benzyl ester, was synthesized in fourteen steps. The overall yield was 3.4 parts per thousand. The compound was identified by (1)HNMR and MS. This is the first report of building this structural skeleton.

Salvianolic acid A attenuates isoproterenol-induced myocardial infarction in mice through PI3K/Akt signal pathway

OBJECTIVE To investigate the protective effect of salvianolic acid A(Sal A)on isoproterenol-induced myocardial infarction in mice and its possible mechanisms.METHODS The mice were subcutaneously injected with isopropranol(ISO 8 mg·kg-1)to induce myocardial infarction and evaluated the myocardial protective effect of Sal A from mortality rate,electrocardiogram(ECG),heart function,myocardial infarction index,serum myocardial enzymes and explored its possible mechanisms from inflammatory,antioxidant and cells apoptosis.RESULTS Sal A can dose-dependently enhanced the heart function of myocardial infarction mice,reduced the heart index,inhibited the myocardial enzyme leakage,showed obvious myocardial protection effects.ELISA results showed that Sal A can reduce the expression of myocardial inflammatory cytokines such as IL-6,TNF-α.Western blotting confirmed that Sal A can increase the expression of anti-apoptotic proteins Bcl-2,reduce the expression of apoptosis protein Bax,and raise the phosphorylation level of PI3K and Akt.CONCLUSION Sal A have displayed significant protective effect against isoproterenol-induced myocardial infarction and its mechanism may be related to increasing of PI3K/Akt signal pathway and inhibition of cell apoptosis and inflammatory reaction.

Salvianolic acid A attenuates vascular remodeling in a pulmonary arterial hypertension rat model

OBJECTIVE The current therapeutic approaches have a limited effect on the dysregulated pulmonary vascular remodeling,which is characteristic of pulmonary arterial hypertension(PAH).In this study we exam-ined whether salvianolic acid A(SAA)extracted from the traditional Chinese medicine′Dan Shen′attenuated vascular remodeling in a PAH rat model,and elucidated the underlying mechanisms.METHODS PAH was induced in rats by injecting a single dose of monocrotaline(MCT 60 mg·kg-1,sc).The rats were orally treated with either SAA(0.3,1,3 mg·kg-1·d-1)or a positive control bosentan(30 mg·kg-1·d-1)for 4 weeks.Echocardiography and hemodynamic measurements were performed on d 28.Then the hearts and lungs were harvested,the organ indices and pulmonary artery wall thickness were calculated,and biochemical and histochemical analysis were conducted.The levels of apoptotic and signaling proteins in the lungs were measured using immunoblotting.RESULTS Treatment with SAA or bosentan effectively ameliorated MCTinduced pulmonary artery remodeling,pulmonary hemodynamic abnormalities and the subsequent increases of right ventricular systolic pressure(RVSP).Furthermore,the treatments significantly attenuated MCT-induced hypertrophic damage of myocardium,parenchymal injury and collagen deposition in the lungs.Moreover,the treatments attenuated MCT-induced apoptosis and fibrosis in the lungs.The treatments partially restored MCT-induced reductions of bone morphogenetic protein typeⅡreceptor(BMPRⅡ)and phosphorylated Smad1/5 in the lungs.CONCLUSION SAA ameliorates the pulmonary arterial remodeling in MCT-induced PAH rats most likely via activating the BMPRⅡ-Smad pathway and inhibiting apoptosis.Thus,SAA may have therapeutic potential for the patients at high risk of PAH.

Salvianolic acid A alleviates renal injury in systemic lupus erythematosus induced by pristane in BALB/c mice

OBJECTIVE To investigate the effects of salvianolic acid A(SAA)in systemic lupus erythematosus(SLE)induced by pristane in BALB/c mice,this study was performed.METHODS Lupus mice were established by confirming elevated levels of autoantibodies and IL-6 after intraperitoneal injection of pristane.Micewere then treated with daily oral doses of SAA for 5months in parallel with mice treated with prednisone and aspirin as positive controls.The levels of autoantibodies were monitored at monthly intervals and nephritic symptoms observed by hematoxylin and eosin(H&E)and periodic acid-Schiff(PAS)staining.Western blot analysis of renal tissue was also employed.RESULTS SAA treatment caused a significant reduction in the levels of anti-Sm autoantibodies and reduced renal histopathological changes and pathological effects.SAA treatment also significantly inhibited the phosphorylation of IKK,IκB and NFκB in renal tissues of lupus mice.CONCLUSION The results suggest that SAA alleviates renal injury in pristane-induced SLE in BALB/c mice through inhibition of phosphorylation of IKK,IκB and NFκB.

Salvianolic acid A reduces endothelial-mesenchymal transition of HPAECs

OBJECTIVE Salvianolic acid A(SAA),a polyphenols acid,is a bioactive ingredient from a traditional Chinese medicine named Danshen(Salvia Miltiorrhiza Bunge).According to previous studies,it was shown to possess various effects such as anti-oxidative stress,anti-diabetic complications and anti-pulmonary hypertension.This study is aimed to investigate the effect of SAA on pulmonary arterial endothelial-mesenchymal transition(endoMT)induced by hypoxia and the underlying mechanisms.METHODS Primary cultured human pulmonary arterial endothelial cells(HPAECs)were exposed to 1%O2 for 48 h with or without SAA treatment.RESULTS SAA treatment improved the morphology of HPAECs and inhibited the cytoskeleton remodeling and reduced migration distances.It was observed that the produc⁃tion of ROS in cells was significantly reduced by the treatment of SAA.Meanwhile,SAA alleviated the loss of CD31 and slightly inhibited the expression ofα-SMA.The mechanisms study shows that SAA treatment increased the phosphoryla⁃tion levels of Smad1/5,but inhibited that of Smad2/3.Furthermore,SAA attenuated the phosphorylation levels of ERK and Cofilin,which were enhanced by hypoxia.CONCLUSION SAA treatment can protect HPAECs from endoMT induced by hypoxia,which may perform via the downstream effectors of BMPRs or TGFβR including Smads,ERK and ROCK/cofilin pathways.

Research Progress on Neuroprotective Effects of Salvianolic Acid B in an Animal Model of Parkinson's Disease

As an active ingredient extracted from Salvia miltiorrhiza,the neuroprotective effects of salvianolic acid B in Parkinson's disease include antioxidation,improvement of mitochondrial function,modulation of neuroinflammation,inhibition of apoptosis,promotion of neuronal differentiation and proliferation,and influence on intestinal flora.As an adjuvant drug,salbutamol B can be used in combination with conventional therapeutic drugs to enhance the efficacy and minimize the side effects,which provides a method and basis for the early diagnosis and treatment of Parkinson's disease in clinical practice.

Determination of Salvianolic Acid B in Yiqi Huayu Prescription by HPLC

[Objectives]To determine the content of salvianolic acid B in Yiqi Huayu Prescription by HPLC.[Methods]The chromatographic column was ZORBAX Eclipse Plus C 18(4.6 nm×250 nm,5μm);the mobile phase was acetonitrile-0.1%phosphoric acid(21:79),the detection wavelength was 286 nm,the column temperature was 30℃,and the flow rate was 1.0 mL/min.A method for determination of salvianolic acid B in Yiqi Huayu Prescription was established.[Results]The linear relationship of salvianolic acid B was good in the range of 0.0214-0.4064 mg/mL.The regression equation was Y=5995.98984 X-0.07332,r=0.9999.The average recovery rate was 98.88%(RSD=1.6%).[Conclusions]The method is reliable,accurate and specific,and can be used for the determination of salvianolic acid B in Yiqi Huayu Prescription.

Effect of salvianolic acid B-loaded mesoporous silica nanoparticles on myocardial ischemia-reperfusion injury

Background:Currently,no drugs can specifically improve clinical cardiac ischemia-reperfusion injury or the prognosis of hemodialysis.Salvianolic acid B(SalB)is a widely used cardiac protectant;however,its clinical application is limited by its low oral bioavailability and poor intestinal absorption.The exploration of its preparation and clinical applications has become a research hotspot in recent years.Methods:To determine whether mesoporous silica nanoparticles(MSNs)efficiently delivered SalB to the heart and SalB@MSNs-RhB reduced myocardial ischemia-reperfusion injury,we constructed a myocardial ischemia-reperfusion male rat model,hypoxia/reoxygenation cardiomyocytes,and treated them with SalB@MSNs-RhB.Results:SalB@MSNs-RhB showed improved bioavailability,therapeutic effect,heightened JAK2/STAT3-dependent pro-survival signaling,and antioxidant responses,thereby protecting cardiomyocytes from ischemia-reperfusion injury-induced oxidative stress and apoptosis.Conclusion:This use of SalB-loaded nanoparticles and investigation of their mechanism of action may provide a new strategy for treating cardiomyocytes.Thus,hypoxia/reoxygenation promotes the clinical application of SalB.

Salvianolic acid A attenuates ischemia reperfusion induced rat brain damage by protecting the blood brain barrier through MMP-9 inhibition and anti-inflammation

Salvianolic acid A （SAA） is a water-soluble component from the root of Salvia Miltiorrhiza Bge, a traditional Chinese medicine, which has been used for the treatment of cerebrovascular diseases for centuries. The present study aimed to determine the brain protective effects of SAA against cerebral ischemia reperfusion injury in rats, and to figure out whether SAA could protect the blood brain barrier （BBB） through matrix metallopeptidase 9 （MMP-9） inhibition. A focal cerebral ischemia reperfusion model was induced by middle cerebral artery occlusion （MCAO） for 1.5-h followed by 24-h reperfusion. SAA was administered intravenously at doses of 5, 10, and 20 mg.kg-l. SAA significantly reduced the infarct volumes and neurological deficit scores. Immunohistochemical analyses showed that SAA treatments could also improve the morphology of neurons in hippocampus CA1 and CA3 regions and increase the number of neurons. Western blotting analyses showed that SAA downregulated the levels of MMP-9 and upregulated the levels of tissue inhibitor of metalloproteinase 1 （TIMP-1） to attenuate BBB injury. SAA treatment significantly prevented MMP-9-induced degradation of ZO-1, claudin-5 and occludin proteins. SAA also prevented cerebral NF-κB p65 activation and reduced inflammation response. Our results suggested that SAA could be a promising agent to attenuate cerebral ischemia reperfusion injury through MMP-9 inhibition and anti-inflammation activities.

Salvianolic acid A alleviates renal injury in systemic lupus erythematosus induced by pristane in BALB/c mice

The purpose of this study was to investigate the effects of salvianolic acid A (SAA) in systemic lupus erythematosus (SLE) induced by pristane in BALB/c mice. Lupus mice were established by confirming elevated levels of autoantibodies and IL-6 after intraperitoneal injection of pristane. Mice were then treated with daily oral doses of SAA for 5 months in parallel with mice treated with prednisone and aspirin as positive controls. The levels of autoantibodies were monitored at monthly intervals and nephritic symptoms observed by hematoxylin and eosin(H&E) and periodic acid-Schiff (PAS) staining. Western blot analysis of renal tissue was also employed. SAA treatment caused a significant reduction in the levels of anti-Sm autoantibodies and reduced renal histopathological changes and pathological effects. SAA treatment also significantly inhibited the phosphorylation of IKK, I kappa B and NF kappa B in renal tissues of lupus mice. In conclusion, the results suggest that SAA alleviates renal injury in pristane-induced SLE in BALB/c mice through inhibition of phosphorylation of IKK, I kappa B and NF kappa B. (C) 2017 Chinese Pharmaceutical Association and Institute of Materia Medica, Chinese Academy of Medical Sciences. Production and hosting by Elsevier B.V.

Blocking Effect of Salvianolic Acid A on Calcium Channels in Isolated Rat Ventricular Myocytes

Objective： To study the effect of salvianolic acid A （SAA） on L-type calcium current （I-CaL） in isolated ventdcular myocytes of Sprague-Dawley rats. Methods： SPA powder was dissolved in normal Tyrode＇s solution to reach the concentrations of 1, 10, 100, and 1000 μmol/L. The traditional whole-cell patch-clamp recording technique was employed to evaluate the effects of SAA on I-CaL in single ventricular myocytes which were prepared by Langendorff perfusion apparatus from Sprague-Dawley rats. Results： SPA （1, 10, 100, and 1000 μmol/L） inhibited I-CaL peak value by 16.23%± 1.3% （n=6, P〈0.05）, 22.9% ± 3.6% （n=6, P〈0.05）, 53.4% ± 3.0% （n=8, P〈0.01）, and 62.26% ± 2.9% （n=8, P〈0.01）, respectively. SAA reversibly inhibited I-CaL in a dose-dependent manner and with a half-blocking concentration （IC50） of 38.3 μmol/L. SAA at 100 μmol/L elevated the I-V curve obviously, and shifted the half-active voltage （V0.5） from （-15.78± 0.86） mV to （-11.24 ± 0.77） mV （n=6, P〈0.05） and the slope （K） from 5.33 ±0.74 to 4.35±0.74 （n=6, P〉0.05）. However, it did not alter the shapes of I-V curve, steady-state inactivation curve, or recovery from inactivation curve. Conclusions： SAA inhibited I-CaL in a dose-dependent manner. It shifted the steady-state activation curve to a more positive voltage, which indicated that the drug affected the activated state of calcium channels, and suggested that the Ca2. antagonistic effect of SPA be beneficial in the treatment of myocardial ischemia reperfusion injury.

Protective Effect of Salvianolic Acid A on Brain Endothelial Cells after Treatment with Deprivation and Reperfusion of Oxygen-glucose

Objective Salvianolic acid A（SAA） has a significant protective effect on ischemia/reperfusion injury of brain. However, it is not clear for SAA to exert its cerebral protection by targeting at the microvascular endothelial cells of blood brain barrier（BBB）. Our previous study demonstrated that SAA could hardly pass through the BBB. This present study was therefore designed to investigate the protective effect of SAA on brain microvascular endothelial cells（BMECs） induced by deprivation and reperfusion with oxygen-glucose. Methods Rat BMECs were treated with oxygen glucose deprivation（OGD）, followed by reperfusion（OGD/R）. Cell viability was assessed by MTT and the content of reactive oxygen species（ROS） in cells after OGD/R in the absence or presence of SAA. GC-MS based metabolomic platform was applied to evaluate the regulation of SAA on the cellular metabolic perturbation induced by OGD/R. Results OGD/R significantly increased the production of intracellular reactive oxygen species（ROS）, and decreased the activity of cells. SAA significantly reduced ROS and improve the cell viability. Metabolomic study revealed distinct perturbation of metabolic pathways of energy metabolism in the BMEC induced by OGD/R, while SAA significantly regulated the perturbed metabolism involved in energy metabolism pathways, especially for intermediates in TCA cycle. Conclusion SAA shows protective effects on BMECs involved in central nervous system.

Study on Optimization of Ethanol Reflux Extraction of Phenolic Acids from Salvia miltiorrhiza

The extraction technology of phenolic acid compounds from Salvia miltiorrhiza by ethanol reflux was studied. In this experiment, salvianolic acid B standard was used to make the standard curve. Single factor experiment and orthogonal experiment were used to study the extraction of different ethanol concentrations, reflux times and material-to-liquid ratios. The OD value of salvianolic acid compounds was measured with a spectrophotometer. The extraction rate of phenolic acid compounds under different extraction conditions was calculated through a regression equation, so as to obtain the optimal conditions for the ethanol reflux extraction process of Salvia miltiorrhiza. The experimental data can provide a reference for the ethanol reflux extraction process of salvianolic acids in the industry. According to the experiment, the extraction rate of phenolic acids in Salvia miltiorrhiza was the highest when the ethanol concentration was 60%, the reflux time was 1.5 hours, and the ratio of material-to-liquid was 1:10.

Effect of salvianolic acid B on Smad3 expression in hepatic stellate cells

BACKGROUND: Salvianolic acid B (SA-B), one of water soluble compounds derived from Radix salviae miltiorrhizae, had good action against liver fibrosis of patients with chro- nic hepatitis. Hepatic stellate cells (HSCs) is the cellular re- source for liver fibrogenesis, while transforming growth factor-β1 (TGF-β1) is most potent fibrogenic factor. In this study we investigated the mechanism of SA-B action against liver fibrosis relating to the interference with TGF- β1 signaling at HSC. METHODS: Hepatic stellate cells (HSCs) were isolated, cultured, and incubated with SA-B. The TGF-β1 content in the supernatant of subcultured HSCs was assayed with ELISA. Type I collagen and Smad3 protein in TGF-β1-sti- mulated primarily cultured HSCs for 4 days were detected by Western blot. RESULTS: TGF-β1 secreted in activated HSCs was more than in primary HSCs, and SA-B significantly decreased TGF-β1 secretion in activated HSCs. TGF-β1 increased the expression of type I collagen and Smad3 protein in d4 pri- mary HSCs, while SA-B inhibited their expression. CONCLUSIONS: SA-B inhibits TGF-β1 secretion in activa- ted HSCs and counteracts the expression of TGF-β1 stimu- lated type I collagen and Smad3. These actions are associat- ed with the effect of SA-B on liver fibrosis.

Salvianolic acid B modulates the expression of drug-metabolizing enzymes in HepG2 cells

BACKGROUND: Enzymes involved in drug and xenobiotic metabolism have been considered to exist in two groups: phase I and phase II enzymes. Cytochrome P450 isoenzymes (CYPs) are the most important phase I enzymes in the metabolism of xenobiotics. The products of phase I metabolism are then acted upon by phase II enzymes, including glutathione S-transferases (GSTs). Herbs that inhibit CYPs such as CYP3A4 or that induce GSTs may have the potential to protect against chemical carcinogenesis since the mutagenic effects of carcinogens are often mediated through an excess of CYP-generated reactive intermediates. This study was designed to investigate the effects of salvianolic acid B (Sal B), a pure compound extracted from Radix Salviae Miltiorrhizae, a Chinese herb, on cell proliferation and CYP1A2 and CYP3A4 mRNA expression in the presence or absence of rifampicin, a potent inducer of CYPs and GST protein expression in HepG2 cells. METHODS: HepG2 cells were incubated with different concentrations of Sal B. Cell proliferation was determined by SYTOX-Green nucleic acid staining. CYP3A4 and CYP1A2 mRNA expression was assayed by real-time PCR. GST protein expression was analyzed by Western blotting. RESULTS: Low concentrations of Sal B (0-20 μmol/L) had no significant effects on cell proliferation, while higher concentrations (100-250 μmol/L) significantly inhibited proliferation in a concentration-dependent manner. Ten μmol/L Sal B, but not 1 μmol/L, down-regulated CYP3A4 and CYP1A2 mRNA expression after 24 hours of incubation, whereas both 1 and 10 μmol/L Sal B down-regulated CYP3A4mRNA expression after 96 hours of incubation; moreover, 1 and 10 μmol/L Sal B inhibited CYP3A4 mRNA expression induced by rifampicin. Both 1 μmol/L and 10 μmol/L Sal B increased GST expression. CONCLUSION: Sal B inhibits CYP3A4 and CYP1A2 mRNA expression and induces GST expression in HepG2 cells.

Role of CD36 in central nervous system diseases

CD36 is a highly glycosylated integral membrane protein that belongs to the scavenger receptor class B family and regulates the pathological progress of metabolic diseases.CD36 was recently found to be widely expressed in various cell types in the nervous system,including endothelial cells,pericytes,astrocytes,and microglia.CD36 mediates a number of regulatory processes,such as endothelial dysfunction,oxidative stress,mitochondrial dysfunction,and inflammatory responses,which are involved in many central nervous system diseases,such as stroke,Alzheimer’s disease,Parkinson’s disease,and spinal cord injury.CD36 antagonists can suppress CD36 expression or prevent CD36 binding to its ligand,thereby achieving inhibition of CD36-mediated pathways or functions.Here,we reviewed the mechanisms of action of CD36 antagonists,such as Salvianolic acid B,tanshinone IIA,curcumin,sulfosuccinimidyl oleate,antioxidants,and small-molecule compounds.Moreover,we predicted the structures of binding sites between CD36 and antagonists.These sites can provide targets for more efficient and safer CD36 antagonists for the treatment of central nervous system diseases.

SALVIANOLIC ACID H,A NEW DEPSIDE FROM SALVIA CAVALERIEI VAR.SIMPLICIFOLIA

A new depside named salvianolic acid H was isolated from the aqueous extract of Salvia cavaleriei vat.simplicifolia,along with salvianolic acid A,B,C,isosalvianolic acid C,lithospermic acid, rosmarinic acid,R-(+)-β-(3,4-dihydroxyphenyl)lacticacid and protocatechualdehyde.Rosmarinic acid was obtained as the major component.

SALVIANOLIC ACID G,A CAFFEIC ACID DIMER WITH A NOVEL TETRACYCLIC SKELETON

Salvianolic acid G,a caffeic acid dimer with a novel tetracyclic skeleton was isolated from the roots of Salvia miltiorrhiza.Its structure was elucidated by chemical and spectral analysis,especially by 2D NMR analysis.