Clinical Non-inferiority Trial on Treatment of Coronary Heart Disease Angina Pectoris of Xin-blood Stasis Syndrome Type with Lyophilized Salvia Salt of Lithospermic Acid Powder for Injection

Objective： To evaluate the effectiveness and safety of lyophilized Salvia salt of lithospermic acid powder for injection （SSLA） in treating coronary heart diseases angina pectoris （CHD-AP） of Xin-blood stasis syndrome type, and to conduct the non-inferiority trial with Danshen injection （丹参注射液, DSI） as positive control. Methods： An non-inferiority clinical layered, segmented, randomized, and blinded trial on three parallel and multiple centered groups was conducted in 480 patients with stable effort angina grade Ⅰ , Ⅱand Ⅲ, who had two or more times of attack every week. The 240 patients in test group A were treated with SSLA 200 mg added in 250 ml of 5% glucose solution for intravenous dripping every day; the 120 patients in test group B were treated with SSLA but the dosage doubled; and the 120 patients in the control group were treated with DSI 20 ml daily in the same method as SSLA was given. The clinical effectiveness and safety were evaluated after the patients were treated for 14 days. Results： The results showed that the markedly effective rate in test groups A, B and control group was 37.45 %, 36.75 % and 30.09 % respectively, while the total effective rate in them was 88.09%, 89.74% and 67.26% respectively. Statistical significance was shown in comparisons of the therapeutic effect between control group with test group A and test group B, with that in the two test groups superior to that in the control group, and non-inferiority trial showed eligibility （P〈0.01）. Adverse reaction appeared in 8 patients in the test groups and 2 in the control group. Conclusion： SSLA has definite therapeutic effect in treating patients with CHD-AP, with its effect not inferior to that of DSI, and no evident toxic-adverse reaction.

Pretreatment with Lithospermic Acid Attenuates Oxidative Stress-induced Apoptosis in Bone Marrow-derived Mesenchymal Stem Cells via Anti-oxidation and Activation of PI3K/Akt Pathway

Objective Despite the potential therapeutic approaches of bone marrow-derived mesenchymal stem cells(BMSCs)in orthopaedic,their applications are hampered by harsh oxidative stress conditions after transplantation.In this study,the antiapoptotic and anti-oxidative properties of lithospermic acid(LSA)on BMSCs exposed to hydrogen peroxide(H2O2)were investigated.Methods In the present study,we used H2O2 to induce oxidative injury on BMSCs.Reactive oxygen species(ROS)staining and superoxide dismutase(SOD)assay were performed.The expression levels of phosphorylated(p)-Akt,Bcl-2-associated X protein(Bax)and B-cell lymphoma 2(Bcl-2)were measured by Western blotting.Results LSA can significantly reduce H2O2-induced chromatin condensation and intracellular ROS levels,enhance the activity of SOD.Moreover,it can alleviate H2O2-induced apoptosis by upregulating Bcl-2 and p-Akt,down-regulating Bax,which was blocked by the PI3K inhibitor,LY294002.Conclusions Our results demonstrated that pretreatment with LSA could attenuate oxidative stress-induced apoptosis in BMSCs,which may be related with anti-oxidant properties and partly via modulating PI3K/Akt pathway,suggesting that pharmacologically manipulating BMSCs with LSA could be a promising drug to increase cell survival for BMSCs transplantation in musculoskeletal disorders of orthopaedic.

Pharmacokinetic investigation on nteraction between hydrophilic lithospermic acid B and lipophilic tanshinone IIA in rats:an experimental study

OBJECTIVE:To elucidate the interaction between hydrophilic lithospermic acid B and lipophilic tanshinone Ⅱ A in rats.METHODS:A reliable high-performance liquid chromatography method was adopted for simultaneous determination of lithospermic acid B and tanshinone Ⅱ A in rat plasma,through which the pharmacokinetic interaction between lithospermic acid B and tanshinone Ⅱ A by intravenous injection was investigated.RESULTS:The simultaneous intravenous injection of tanshinone Ⅱ A and lithospermic acid B significantly altered the pharmacokinetic parameters of both compounds when compared with the individual intravenous administration of each compound.The area under the concentration-time curve of tanshinone Ⅱ A and lithospermic acid B increased by 18.35 and 59.31%,respectively.The mean retention time of tanshinone Ⅱ A and lithospermic acid B increased,respectively,from 9.3 to 32.8 h and20.2 to 49.1 h.The concomitant use of tanshinoneⅡ A magnified the volume of distribution at steady state(V_(ss)) and time for the drug in the plasma to reduce the highest concentration by half(t_(1/2)) of lithospermic acid B,while at the same time the V_(ss) and t_(1/2)of tanshinone Ⅱ A changed significantly in the presence of lithospermic acid B.CONCLUSION:Lithospermic acid B and tanshinone D A interact with each other following simultaneous intravenous injection in rats and this observation may expand the clinical use of Danshen(Radix Salviae Miltiorrhizae).

丹参成分Magnesium lithospermate B对大鼠急性肾衰竭治疗作用的实验研究

目的 :探讨丹参成分MagnesiumlithospermateB对急性肾衰竭大鼠的治疗作用及其可能机理。 方法 :采用双侧大腿肌肉注射 5 0 %甘油造成的急性肾衰竭大鼠模型。观察MagnesiumlithospermateB对此模型肾功能的改善作用 ,并分析肾组织中活性氧清除酶类的改变。结果 :二组不同剂量治疗组的血清尿素氮、肌酐、内生肌酐清除率、血钾、尿渗透压、尿糖、钠排泄分数以及肾组织丙二醛、超氧化物岐化酶、过氧化氢酶的改变均较模型组为轻。结论 :MagnesiumlithospermateB对甘油所致急性肾衰竭大鼠有治疗作用 ,作用机制可能与其改善肾组织活性氧清除酶活性有关。

Magnesium lithospermate B inhibits lipopolysaccharide-induced endothelial activation through NF-KB pathway

Aim Magnesium lithospermate B （MLB） is the most abundant hydrophilic active component of Salvia rniltiorrhiza Radix, a traditional Chinese herbal medicine mainly used to treat cardiovascular diseases. Studies have shown that endothelial activation contributes to the pathophysiology of cardiovascular diseases such as atherosclero- sis, diabetic vasculopathy, heart failure and hypertension. In the present study, the effects of MLB on endothelial activation were investigated. Lipopolysaccharide （LPS） 1 mg L^-1 was employed to induce endothelial activation, which was determined by relative gene expression and endothelial adhesion assay. Results showed that pretreatment with MLB attenuated LPS-induced ICAM1, VCAM1 and TNF-α upregulation in human dermal microvascular endo- thelial cells （HMEC-1） in dose-dependent manner, which contributed to the reduction of THP-1 adhesion to HMEC-1. Furthermore, it was revealed that 100 μmol · L^-1 MLB significantly decreased the nuclear translocation of NF-KB p65, a critical transcription factor in LPS-indueed inflammatory response, through the inhibition of IKBμ degradation. Besides, the transcriptional activity of NF-KB p65 was also inhibited by the pretreatment of MLB. Mo- reover, MLB pretreatment considerably inhibited LPS-induced p38 phosphorylation, which at least partly contribu- ted to the reduction of ICAM1 expression. In conclusion, these findings suggest that MLB inhibits LPS-induced nu- clear translocation and transcripitional activity of NF-KB, thus attenuates the increased expression of adhesion mole- cules and inflammatory factors, protects endothelial cells from LPS-induced activation.

Ethyl Lithospermate Reduces Lipopolysaccharide-Induced Inflammation through Inhibiting NF-κB and STAT3 Pathways in RAW 264.7 Cells and Zebrafish

Objective:To explore the anti-inflammatory effects of ethyl lithospermate in lipopolysaccharide(LPS)-stimulated RAW 264.7 murine-derived macrophages and zebrafish,and its underlying mechanisms.Methods:3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazoliumbromide(MTT)assays were performed to investigate the toxicity of ethyl lithospermate at different concentrations(12.5–100μmol/L)in RAW 264.7 cells.The cells were stimulated with LPS(100 ng/mL)for 12 h to establish an inflammation model in vitro,the production of proinflammatory cytokines interleukin(IL)-6 and tumor necrosis factorα(TNF-α)were assessed by enzyme linked immunosorbent assay(ELISA).Western blot was used to ascertain the protein expressions of signal transducer and activator of transcription 3(STAT3),nuclear factor kappa B(NF-κB)p65,phospho-STAT3(p-STAT3,Tyr705),inhibitor of NF-κB(IκB)α,and phospho-IκBα(p-IκBα,Ser32),and confocal imaging was used to identify the nuclear translocation of NF-κB p65 and p-STAT3(Tyr705).Additionally,the yolk sacs of zebrafish(3 days post fertilization)were injected with 2 nL LPS(0.5 mg/mL)to induce an inflammation model in vivo.Survival analysis,hematoxylin-eosin(HE)staining,observation of neutrophil migration,and quantitative real-time polymerase chain reaction(qR T-PCR)were used to further study the anti-inflammatory effects of ethyl lithospermate and its probable mechanisms in vivo.Results:The non-toxic concentrations of ethyl lithospermate have been found to range from 12.5 to 100μmol/L.Ethyl lithospermate inhibited the release of IL-6 and TNF-α(P<0.05 or P<0.01),decreased IκBαdegradation and phosphorylation(P<0.05)as well as the nuclear translocation of NF-κB p65 and p-STAT3(Tyr705)in LPS-induced RAW 264.7 cells(P<0.01).Ethyl lithospermate also decreased inflammatory cells infiltration and neutrophil migration while increasing the survival rate of LPS-stimulated zebrafish(P<0.05 or P<0.01).In addition,ethyl lithospermate also inhibited the mR NA expression levels of of IL-6,TNF-α,IκBα,STAT3,and NF-κB in LPS-stimulated zebrafish(P<0.01).Conclusion:Ethyl lithospermate exerts anti-Inflammatory effected by inhibiting the NF-κB and STAT3 signal pathways in RAW 264.7 macrophages and zebrafish.

Antioxidant properties of magnesium lithospermate B contribute to the cardioprotection against myocardial ischemia/reperfusion injury in vivo and in vitro

OBJECTIVE： To determine the cardioprotective ef- fect of magnesium lithospermate B （MLB） on myo- cardial ischemia/reperfusion （MI/R） injury and to in- vestigate the antioxidant potential in vivo and in vitro. METHODS： MI/R injury was induced by the occlu- sion of left anterior descending coronary artery for 30 min followed by reperfusion for 3 h in rats. After reperfusion, hearts were harvested to assess infarct size, histopathological damages, the levels of superoxide dismutase （SOD）, catalase （CAT）, glutathione peroxidase （GPx）, reduced glutathione （GSH） and malondialdehyde （MDA）. Blood samples were collected to determine serum levels of creatine kinase-MB （CK-MB）, cardiac troponin （cTnl） and lactate dehydrogenase （LDH）. Furthermore, simulatedischemia/reperfusion （SI/R） injury in vitro was established by oxygen and glucose deprivation （OGD） for 2 h followed by 24-hour recovery period in cardiomyocytes. The activity of LDH in the cultured su- pernatant and the levels of intracellular reactive oxygen species （ROS）, SOD and MDA in cardiomyo- cytes were also measured. Finally, cardiomyocytes apoptosis was determined with flow cytometry. RESULTS： MLB significantly limited infarct size, ameliorated histopathological damages and prevented leakage of CK-MB, cTnl and LDH. Additional- ly, SOD, CAT, GPx and GSH activities were notably increased by MLB, along with the MDA content decreased as compared with the model group in rats. In vitro study, MLB also decreased LDH activity in the cultured supernatant, increased SOD activity in cardiomyocytes, reduced intracellular ROS and MDA levels, and significantly suppressed cardiomyocytes apoptosis. CONCLUSION： MLB possessed remarkably cardioprotective effects on MI/R injury in vivo and in vitro. The protection of MLB may contribute to its antioxidant properties.