Concentrations Of Tanshinone IIA Research Articles

Local Application of Tanshinone IIA protects mesenchymal stem cells from apoptosis and promotes fracture healing in ovariectomized mice

BackgroundElderly patients suffering from osteoporotic fractures are more susceptible to delayed union or nonunion, and their bodies then are in a state of low-grade chronic inflammation with decreased antioxidant capacity. Tanshinone IIA is widely used in treating cardiovascular and cerebrovascular diseases in China and has anti-inflammatory and antioxidant effects. We aimed to observe the antioxidant effects of Tanshinone IIA on mesenchymal stem cells (MSCs), which play important roles in bone repair, and the effects of local application of Tanshinone IIA using an injectable biodegradable hydrogel on osteoporotic fracture healing.MethodsMSCs were pretreated with or without different concentrations of Tanshinone IIA followed by H2O2 treatment. Ovariectomized (OVX) C57BL/6 mice received a mid-shaft transverse osteotomy fracture on the left tibia, and Tanshinone IIA was applied to the fracture site using an injectable hydrogel.ResultsTanshinone IIA pretreatment promoted the expression of nuclear factor erythroid 2-related factor 2 and antioxidant enzymes, and inhibited H2O2-induced reactive oxygen species accumulation in MSCs. Furthermore, Tanshinone IIA reversed H2O2-induced apoptosis and decrease in osteogenic differentiation in MSCs. After 4 weeks of treatment with Tanshinone IIA in OVX mice, the bone mineral density of the callus was significantly increased and the biomechanical properties of the healed tibias were improved. Cell apoptosis was decreased and Nrf2 expression was increased in the early stage of callus formation.ConclusionsTaken together, these results indicate that Tanshinone IIA can activate antioxidant enzymes to protect MSCs from H2O2-induced cell apoptosis and osteogenic differentiation inhibition. Local application of Tanshinone IIA accelerates fracture healing in ovariectomized mice.

Neuroprotective effects of tanshinone IIA in experimental model of Parkinson disease in rats

Neurodegenerative diseases including Parkinson's disease (PD) cause many problems for the patient. Treatment options include the administration of chemical drugs, which are associated with many side effects. Therefore, the current study was aimed to investigate tanshinone IIA effects on the experimental model of PD in rats and to study the neuroprotective mechanisms of this natural product. After preparation of rats and induction of PD by 6-hydroxypopamine (6-OHDA), different concentrations of tanshinone IIA were administered to the rats for 10 days. Apomorphine-induced rotation and muscle rigidity behavioral tests were used to evaluate PD induction and to study the effects of tanshinone IIA. After separation of the hippocampus, biochemical parameters such as lipid peroxidation, protein oxidation, succinic dehydrogenase (SDH), super oxide dismutase (SOD), reactive oxygen species (ROS), malate dehydrogenase (MDH), catalase (CAT), reduced glutathione (GSH), lactate dehydrogenase (LDH), IL-1β, TNF-α, INF-γ, IL-10 cytokines and NF-ĸB protein expression were measured. The results showed that PD rats receiving tanshinone IIA improved their performance in behavioral tests. Decreased oxidative stress indices and increased antioxidant capacity were observed in the hippocampal tissue of PD rats receiving tanshinone IIA. Also, administration of tanshinone IIA to PD rats downregulated inflammatory cytokines TNF-α. IL-1β and INF-γ and decreased NF-ĸB protein expression. Tanshinone IIA has therapeutic effects in PD condition as results of antioxidant and anti-inflammatory properties. Therefore, this natural product is recommended in PD treatment.

Tanshinone IIA attenuates high glucose-induced epithelial-to-mesenchymal transition in HK-2 cells through VDR/Wnt/β-catenin signaling pathway.

The progression of diabetic kidney disease (DKD) is closely related to renal tubular epithelial- to-mesenchymal transition (EMT) and tubulointerstitial fibrosis. Tanshinone IIA (TSIIA), extracted from a traditional Chinese medicine named Salvia miltiorrhiza, has been proved to have anti-fibrosis effects. The aim of this study was to investigate the effect of TSIIA on high glucose-induced EMT in human proximal tubular cells (HK-2 cells) and its possible mechanism. The proliferation of cells exposed to different concentrations of glucose was measured by light microscopy and CCK-8 test. The cells were stimulated with 30 mM glucose and different concentrations of TSIIA (5 μM or 10 μM) for 48 h. Vitamin D receptor (VDR)-siRNA was used to transfect cells, and high glucose and TSIIA treatment were further used to treat cells. The expression of alpha smooth muscle actin (a-SMA) mRNA was detected by qPCR to ensure successful induction of EMT, and the expression of VDR mRNA was detected by qPCR to ensure successful transfection of VDR-siRNA. Protein expression of a-SMA, E-cadherin, VDR, b-catenin and glycogen synthase kinase 3b (GSK-3b) was detected by Western blot analysis. The results showed that high glucose concentration inhibited cell proliferation and promoted EMT in HK-2 cells. TSIIA could reverse high glucose-induced EMT by increasing the level of VDR protein and inhibiting the levels of b-catenin and GSK-3b proteins suggestive of a negative correlation between VDR and the Wnt/b-catenin pathway. After VDR-siRNA transfection and incubation of cells at high glucose concentration, the inhibitory effect of VDR on the expression of b-catenin and GSK-3b of Wnt pathway was suppressed and the b-catenin pathway was activated. When VDR level was restored by TSIIA, the inhibitory effect of VDR on the pathway was also restored and the activation of the pathway was suppressed. TSIIA was able to attenuate high glucose-induced EMT in HK-2 cells by up-regulating VDR levels, which might be related to the inhibitory effect of VDR on the Wnt pathway.

Influence of release rate, dose and co-administration on pharmacokinetics, pharmacodynamics and PK-PD relationship of tanshinone IIA and tanshinol

The present study aims to investigate the influence of release rate, dose and co-administration on pharmacokinetics (PK) and pharmacodynamics (PD) of tanshinone IIA (TA) and tanshinol (TS), and reveal the changes in their PK-PD relationships. Sustained and immediate release pellets of TS and TA were prepared respectively, and oral administrated to angina model rabbits according to the experimental design. The administration dose of TS was 50, 35 or 20 mg/kg and that of TA was 30 mg/kg. Then, plasma concentrations of TS and TA were measured to evaluate the pharmacokinetics. Pharmacodynamic biomarkers including cardiac troponin (cTn-I), creatine kinase (CK-MB), superoxide dismutase (SOD) and nitric oxide (NO) were measured to evaluated the effects of cardioprotection, amelioration of oxidative stress and vasorelaxation of TS and TA. Parameters such as maximum plasma concentration (Cmax), maximum effect (Emax), time to Cmax or Emax (TCmax or TEmax), areas under the plasma concentration or effect curves (AUC0-∞ or AUEC) and pharmacodynamic efficiency (EFF) were calculated based on non-compartmental analysis. Beside, PK-PD relationship/hysteresis was evaluated. The TEmax was less sensitive than TCmax to changes in release rate. The Emax, AUEC and EFF showed increasing trend as the decrease of release rate even that the AUC0-∞ showed no significant difference. In addition, slow drug release decreased the magnitude of hysteresis of TS and TA. The sensitivities of Emax and AUEC of four biomarkers to changes in dose were varied and relatively lower than those of Cmax and AUC0-∞. The EFF decreased and the magnitude of hysteresis increased for high dose. The Cmax and AUC0-∞ of TS and TA showed little difference after co-administration. The Emax and AUEC of four biomarkers increased for immediate release pellets (P < 0.05 or P < 0.01) and generally decreased for sustained release pellets (P < 0.05 or P < 0.01) after co-administration. In addition, the magnitudes of hysteresis of four biomarkers decreased for immediate release pellets and generally increased for sustained release pellets after co-administration. In summary, the dissociated and unstable PK-PD relationship should be considered during optimization of dosage forms and regimens to make sure the rationality, safety and efficacy. These findings could also provide some valuable information for the development and clinical therapy of other drugs.

Preparation of water-soluble chitosan/poly-gama-glutamic acid—tanshinone IIA encapsulation composite and its in vitro/in vivo drug release properties

Some diseases could be treated by Tanshinone IIA (TA), which is an isolated component from the Chinese medicinal herb Tanshen (Salvia miltiorrhiza). However, the poor water solubility and low oral bioavailability of TA limited its clinical application. In this paper, TA was encapsulated by water - soluble chitosan/poly - γ - glutamic acid (WCS-γ-PGA) to improve its dissolution and oral bioavailability. The in vitro dissolution and in vivo metabolism of the encapsulated composite in rats were employed to evaluate the efficiency of the improvement. FTIR spectroscopy was applied to confirm the validity of encapsulation for TA by WCS-γ-PGA. The study’s results showed that the optimal ratio of TA to drug carrier (WCS + γ-PGA) was 1:5.5 in weight with a reaction time of 1 h at room temperature for the encapsulation. The proper concentrations for WCS and TA in preparing the encapsulated composite using γ-PGA 0.125 mg ml−1 were 6 mg ml−1 and 1 mg ml−1, respectively; The encapsulation efficiency and drug loading efficiency of WCS-γ-PGA-TA composite were (93.99 ± 2.20)% and (10.73 ± 0.75)%, respectively. The cumulative release of TA from the WCS-γ-PGA-TA encapsulated composite reached to 81% within 60 min, which was 5.56 times of that of the original TA in vitro dissolution. The peak concentration Cmax of TA from the encapsulated composite in rat blood as measured by an ultracentrifugation test of an intra - gastric administration was 4.43 times that of the original TA concentration, and the area under the drug-time curve AUC (0-t) and AUC (0-∞) (p<0.01) of the WCS-γ-PGA-TA encapsulated composite were 4.56 and 4.20 times that of the original TA, respectively. It indicated that the encapsulation of TA with WCS-γ-PGA improved its solubility and bioavailability significantly.

Improved tendon healing by a combination of Tanshinone IIA and miR-29b inhibitor treatment through preventing tendon adhesion and enhancing tendon strength.

Background: Despite significant advances in the materials and methods development used in surgical repair and postoperative rehabilitation, the adhesion formation remains the most common clinical problem in tendon injuries. Therefore, the development of novel therapies is necessary for targeting at preventing tendon adhesion formation and improving tendon strength.Methods: We used rat fibroblasts for in vitro experiments to determine the optimal concentration of TSA in rats, and then set up negative control group, TSA intervention group, mir-29b interference adenovirus intervention group and TSA and mir-29b interference adenovirus co-intervention group. By comparing cell proliferation and protein expression in different group, we verified the effect and mechanism of drugs on fibroblast function. At the same time, the Sprague-Dawley rat Achilles tendon model in vivo was established in this study, which was divided into sham operation group and operation group. Afterwards in the operation group, mir-29b inhibitor and placebo were injected every 3 days respectively. Then the injection inhibitor group was divided into 5 groups which mean TSA was injected into the marked area at 0, 6, 24 and 72 hours after operation for 1 week, finally all of the rats were died at 3 weeks after operation. Through the observation of general properties, histological observation of Achilles tendon injury, biomechanical test and cell and protein expression in rats' tendon cell, the effect of drugs on tendon adhesion formation was analyzed.Results: We demonstrated that the combination of miR-29b inhibitor and tanshinone IIA(TSA) could prevent tendon adhesion and also enhance tendon strength. Mechanically, the miR-29b inhibitor could activate the TGF-β/Smad3 pathway to trigger endogenous pathways and induce a high proliferation of fibroblast. Subsequently, we also found adding TSA after 6 hours of miR-29b treatment gave less cell cytotoxicity in our rat model with better outcome of less tendon adhesion and enhanced strength.Conclusion: We conclude that the use of miR-29b inhibitor at the end of the tendon break could initiate endogenous repair mechanism and subsequently use of TSA should be able to inhibit the exogenous repair mechanism. Therefore, the combination of both treatments could prevent tendon adhesion and ensure tendon strength. Our findings suggested that this approach would be a feasible approach for tendon repair.

Tanshinone IIA Inhibits TGF-β1-Mediated Fibroblasts Proliferation by Down-Regulating lncRNA-HSRL/SNX9/p-Smad3 Signaling

Tanshinone IIA (TSIIA), one of the components of Salvia miltiorrhiza (Danshen), has been reported to inhibit proliferation in hypertrophic scars (HS). However, the molecular mechanisms underlying TSIIA regulating HS still remain largely unknown. In this study, we used different concentrations of TGF-β1 to simulate Human skin fibroblasts (HSFs) to mimic scar formation, followed by treatment with various concentrations of TSIIA. We observed TSIIA could effectively attenuate TGF-β1-mediated fibroblasts proliferation in a dose-dependent manner using CCK-8, Edu staining and colony formation assays. Meanwhile, TSIIA could effectively down-regulated the expression of α-SMA, VEGFA, Collagen 1, HSRL, SNX9 and p-Smad2/3 in TGF-β1-stimulated HSFs in a dose-dependent manner using quantitative real time PCR and western blotting analysis. Through loss-of-function and rescue experiments, we found SNX9 overexpression reversed the effects of HSRL knockdown on TGF-β1- stimulated HSFs. Mechanically, the interaction between HSRL and SNX9 was validated by RNA pull-down assay in HSFs. Using Co-IP assay, we confirmed the p-Smad3 that interaction with SNX9 was weakened after TSIIA treatment in TGF-β1-stimulated HSFs. In conclusions, these findings indicate that specifically targeting HSRL/SNX9/p-Smad3 might enhance the suppressive effects of TSIIA in HS pathogenesis. Funding Statement: Guangdong natural science project [2018A0303130234] and excellent young teachers training project in higher education of Guangdong [YQ2015099] which supported this experiment. Declaration of Interests: The authors declare no conflicts of interest. Ethics Approval Statement: Not required.

Mechanism of the effect of tanshinone IIA epithelial-mesenchymal transition via the TGF-β1/smad pathway in human alveolar epithelial cell line A549

This study aimed to investigate the inhibitory effects of the different concentrations of Tanshinone IIA (Tan IIA) on the proliferation of human alveolar epithelial cell line A549 and its regulatory mechanism in epithelial-mesenchymal transition (EMT). The proliferation activity of cells was examined using the thiazolyl blue tetrazolium bromide (MTT) method. The changes in the expression of epithelial cell marker protein E-cadherin (E-cad) and interstitial marker protein alpha-smooth muscle actin (α-SMA) were detected using the cellular immunochemical method. The changes in cell morphology and ultrastructure were observed under the inverted microscope and transmission electron microscope, respectively. Western blot analysis was used to detect the expression of E-cad, α-SMA, Smad7, and Smad3. The MTT assay showed that the cell viability in the transforming growth factor beta 1 (TGF-β1) induced group was higher than that in the normal group, but the difference was not obvious. However, the cell viability of the Tan IIA-treated groups obviously decreased compared with the TGF-β1-induced and normal groups. Meanwhile, the expression of E-cad and Smad7 decreased, and the expression of α- SMA and Smad3 increased after A549 cells were induced by 5 ng/mL TGF-β1 for 24 h. However, their expression levels were close to the expression level of the control group after the cells were treated with Tan IIA for 24 h. In conclusion, the results demonstrated that Tan IIA could inhibit EMT of alveolar epithelial cells induced by TGF-β1, probably by regulating the expression of TGF-β/Smad pathway protein. Therefore, Tan IIA might serve as a potential anti-fibrosis drug in treating pulmonary fibrosis.

Tanshinone IIA Inhibits VEGF Secretion and HIF-1α Expression in Cultured Human Retinal Pigment Epithelial Cells under Hypoxia

ABSTRACTPurpose: The current work intends to study the activity of tanshinone IIA on secretion of vascular endothelial growth factor (VEGF) and expression of hypoxia inducible factor 1α (HIF-1α) in human retinal pigment epithelial cells (ARPE-19 cells) under hypoxic condition.Methods: The cytotoxicity of tanshinone IIA was tested in ARPE-19 cells by MTT assay. ARPE-19 cells were incubated with different concentrations of cobalt chloride (100, 150, and 200 µM) for 12 h, and levels of expressed HIF-1α and secreted VEGF were quantified through Western blot and ELISA, respectively. Further, ARPE-19 cells were pretreated for 1 h with different concentrations of tanshinone IIA (5, 10, 15, and 18 µM). After 1 h, the cells were subjected to hypoxic condition using 150 µM cobalt chloride for 12 h in the presence and absence of tanshinone IIA. The cells were then harvested, and the secreted VEGF and expressed HIF-1α was studied.Results: Tanshinone IIA at concentrations of 5, 10, 15, and 18 μM did not show cytotoxicity in ARPE-19 cells. Chemical hypoxia induced by cobalt chloride caused a significant increase in VEGF level in a dose-dependent manner, and HIF-1α expression peaked at 150 µM. Based on the data, cobalt chloride concentration was maintained at 150 μM for further studies. Tanshinone IIA decreased the level of HIF-1α and VEGF secretion in a dose-dependent manner under hypoxic condition.Conclusion: Tanshinone IIA could be explored as a new potential candidate for treating wet AMD.

Crosstalk between Beclin-1-dependent autophagy and caspase‑dependent apoptosis induced by tanshinoneIIA in human osteosarcoma MG-63 cells.

The aim of the present study was to ascertain whether or not autophagy is induced by tanshinone IIA (TanIIA), and to explore the crosstalk between autophagy and apoptosis in regards to the antitumor effects of TanIIA on MG-63 cells and the potential mechanism. MG-63 cells were cultured in vitro with various concentrations of TanIIA (0, 2.5, 5, 10 and 20 mg/l) for 0, 24, 48 and 72 h, respectively. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide MTT assay was used to evaluate the inhibition of the proliferation of osteosarcoma MG-63 cells by TanIIA or in the presence/absence of chloroquine (CQ). Autophagic vacuoles and characteristic autophagosomes were observed by transmission electron microscopy (TEM). TanIIA-induced autophagy in MG-63 cells was confirmed by GFP-LC3 punctate fluorescence. The expression levels of apoptosis-related proteins caspase-3, caspase-8, caspase-9 and cleaved-PARP and autophagy-related proteins LC3II/LC3I and Beclin-1 were detected by western blotting. FITC-Annexin V/propidium iodide (PI) staining, flow cytometry and Hoechst 33258 staining were used to analyze the apoptotic rate. Fluorescence intensity of reactive oxygen species (ROS) was examined under a fluorescence microscope using an analysis software system. Cell proliferation was obviously inhibited by TanIIA in a dose- and time-dependent manner. Generation of autophagy was triggered by TanIIA (0–20 mg/l) treatment, and in a Beclin-1-dependent manner. Compared with the control group, the apoptosis ratio following treatment with 2.5 mg/l TanIIA failed to achieve statistical significance. Expression of caspase-3, -8 and -9, and cleaved-PARP in the other groups was gradually enhanced in dose-dependent manner. Our analysis also suggested that the influence of autophagy on TanIIA cytotoxicity had a phase effect; with low-dose drugs and shorter treatment periods, autophagy functioned as a damage repair mechanism. In conrast, when the cells were treated with higher doses of TanIIA for longer treatment periods, autophagic cell death contributed to apoptosis. Furthermore, generation of ROS occurred in a dose-dependent manner and pretreatment with NAC, a selective ROS scavenger, blocked the coexistence of Beclin-1 autophagy and caspase-dependent apoptosis. In conclusion, our findings provide strong evidence that TanIIA may be a potential therapeutic drug against osteosarcoma. Moreover, its cytotoxity can be enhanced with ROS agonists.

Insight into the pharmacokinetic behavior of tanshinone IIA in the treatment of Crohn’s disease: comparative data for tanshinone IIA and its two glucuronidated metabolites in normal and recurrent colitis models after oral administration

1. Previous reports implied that tanshinone IIA (TSA) may offer potential benefits for Crohn’s disease (CD). However, the detailed pharmacokinetic behavior of TSA in the treatment of colitis remain unclear. Herein, a recurrent trinitrobenzene sulfonic acid (TNBS)-colitis mouse model was used to investigate whether TSA possesses favorable pharmacokinetic and colonic distribution profiles to serve as a candidate drug.2. Although the systemic TSA exposures were low (AUC0–t approximately 330 ng*h/ml) in both the normal and colitis models after oral administration TSA 20 mg/kg, high levels of TSA were found in the gastrointestinal tract (GI). Such a GI exposure of TSA in colitis mice is adequate to exert anti-inflammatory effects as observed in various in vitro studies.3. Interestingly, colonic TSA exposure in the colitis mouse model was much lower than that in the normal mice, which may be explained by a significant upregulation of colonic UDP-glucuronosyltransferase (Ugt)1a9 expression and a higher plasma concentration of TSA glucuronides in the model mice at 0.5, 1 and 2 h after TSA administration.4. Together, these results reveal high accumulation at the site of inflammation and minimal systemic concentration of TSA, which are favorable pharmacokinetic behaviors to meet the requirements for CD treatment.

Tanshinone IIA Inhibits Proliferation and Induces Apoptosis Through the Downregulation of Survivin in Keloid Fibroblasts

Keloids are considered benign dermal fibroproliferative tumors. Keloid fibroblasts (KFs) persistently proliferate and fail to undergo apoptosis, and no treatment is completely effective against these lesions. Tanshinone IIA induces apoptosis and inhibits the proliferation of various tumor cell types. In this study, we investigated the effect of tanshinone IIA on the regulation of proliferation, cell cycle, and apoptosis in KFs, and investigated potential mechanisms involved in the effects. First, KFs and normal skin fibroblasts (NSFs) were treated with various concentrations of tanshinone IIA. Cell counting kit-8 (CCK-8) was used to assess the proliferative activity of KFs and NSFs, and flow cytometry was used to investigate the cell cycle and apoptosis in KFs. We found that the proliferation of all tanshinone IIA-treated KFs was significantly decreased after treatment for 72 hours (P < 0.001). Also, NSFs treated with tanshinone IIA did not exhibit noticeable effects compared with KFs. In addition, the percentages of G0/G1 cells in all tanshinone IIA-treated KFs were significantly increased after treatment for 72 hours (P < 0.001). And the percentages of cells undergoing early apoptosis in all tanshinone IIA-treated KFs were significantly increased after treatment for 120 hours (P < 0.001). Furthermore, the apoptosis antibody array kit and Western blot analysis revealed that tanshinone IIA decreased survivin expression in KFs (P < 0.001). In conclusion, tanshinone IIA downregulates survivin and deactivates KFs, thus suggesting that tanshinone IIA could serve as a potential clinical keloid treatment.

Influence of Tanshinone IIA on the Apoptosis of Human Esophageal Ec-109 Cells

The induced-apoptosis effect and mechanism of human esophageal cancer Ec-109 cells via tanshinone IIA was investigated. The Ec-109 cells were cultured in vitro with different concentrations of tanshinone IIA (2 µg/mL, 4 µg/mL, or 8 µg/mL) for 12, 24, 36, and 48 hours. MTT assay was used to evaluate the proliferative inhibition rate of tanshinone IIA on esophageal Ec-109 cells. After 24 hours of culturing in vitro, a control group was assigned. The apoptosis rate was detected by the AO/EB and annexin V-FITC/propidium iodide assay, and the protein levels of Caspase-4 and CHOP were determined by the Western blot technique. MTT data showed that tanshinone IIA could significantly inhibit the proliferation of Ec-109 cells with a dose- and time-dependent mode. Compared with the control group, tanshinone IIA could apparently induce apoptosis of Ec-109 cells, and the level of Caspase-4 and CHOP (p < 0.01) obviously increased. Tanshinone IIA can significantly induce the apoptosis of Ec-109 cells, which may take effect by the stress pathway of the endoplasmic reticulum.

Tanshinone IIA promotes the proliferation of WB-F344 hepatic oval cells via Wnt/β-catenin signaling.

Tanshinone IIA (TSA) is a widely used traditional Chinese medicine, which has been demonstrated to protect damaged liver cells and is currently administered in the treatment of liver fibrosis. Liver precursor cells, also termed oval cells, are key in the repair of liver tissues following injury. However, whether TSA improves the function of liver cells and protects the liver from injury by enhancing the growth and proliferation of hepatic oval cells remains to be elucidated. In the present study, low to moderate concentrations of TSA were observed to stimulate proliferation, did not induce apoptosis in WB-F344 rat hepatic oval cells and the increased expression levels of β-catenin. WB-F344 cells were treated with various concentrations of TSA (0–80 µg/ml) for 24, 48, 72 and 96 h. Cell proliferation was measured using a Cell Counting kit-8 (CCK-8) assay, a 5-ethynyl-2′-deoxyuridine assay and a carboxyfluorescein diacetate succinimidyl ester (CFSE) assay. The CCK-8 assay demonstrated that treatment of WB-F344 cells with 20–40 µg/ml TSA for up to 72 h significantly increased proliferation. Similar results were observed in the subsequent EdU and CFSE assays. Furthermore, a terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling assay demonstrated that 20–40 µg/ml TSA treatment for up to 96 h did not induce apoptosis of the WB-F344 cells. Notably, the results of western blot, immunofluorescence and reverse transcription-quantitative polymerase chain reaction analyses demonstrated that treatment of the WB-F344 cells with 20–40 µg/ml TSA for up to 72 h significantly increased the expression levels of β-catenin. These data indicated that TSA at concentrations between 20 and 40 µg/ml may induce WB-F344 cell proliferation by activating the canonical Wnt signaling pathway. The results of the present study suggest that TSA may be a useful natural agent to enhance repair and regeneration of the injured liver, and improve liver regeneration following orthotopic liver transplantation.

Studies on the active constituents in radix salviae miltiorrhizae and their protective effects on cerebral ischemia reperfusion injury and its mechanism.

Background:To extract, purify and identify the active constituents in ethanol extract of Radix Salviae Miltiorrhizae, and to analyze the protective effects of tanshinone IIA on cerebral ischemia-reperfusion injury in rats.Materials and Methods:Radix Salviae Miltiorrhizae was extracted by ultrasonic extraction, effective parts were extracted by extraction method, compounds were isolated by preparative TLC and preparative HPLC, and structures of compounds were identified by 1H NMR and 13C NMR; the effects of tanshinone IIA on cerebral ischemia-reperfusion injury in rats were determined by establishing rat model of middle cerebral artery occlusion (MCAO).Results:The experimental data show four compounds were isolated, namely tanshinone IIB, hydroxymethylene tanshinone, salvianolic acid B and 9”’-methyl lithospermate B. Tanshinone IIA could alleviate the symptoms of neurological deficit in rats, the neurological deficit alleviating effect became more obvious with the increase of dose; tanshinone IIA experimental groups could reduce the cerebral infarction size and brain water content in rats, different concentrations of tanshinone IIA could decrease the SOD content and increase the MDA content in the frontal and parietal cortices of ischemic hemisphere in the ischemia reperfusion group, the differences were statistically significant compared with the ischemia reperfusion group.Conclusion:Radix Salviae Miltiorrhizae has the protective effects on cerebral ischemia reperfusion injury in rats.

The molecular mechanisms of Tanshinone IIA on the apoptosis and arrest of human esophageal carcinoma cells.

Objective. To explore the possible mechanisms of Tanshinone IIA (TanIIA) on esophageal carcinoma cell lines. Methods. Two human esophageal carcinoma cell lines (EC-1 cells and ECa-109 cells) were treated with different concentrations of TanIIA. Cell proliferation was measured by CCK-8, colony-forming efficiency was calculated, cell cycle and apoptosis were measured, and changes in cell cycle- and apoptosis-related gene expression were measured by Western blotting. Results. The CCK-8 and colony formation assay indicated that TanIIA inhibited the cell proliferation of human esophageal cancer cells (IC50 below 1 μg/mL) at 48 h. Hoechst 33258 and flow cytometry showed that TanIIA induced apoptosis in both esophageal cancer cell lines. Flow cytometry showed that TanIIA arrested cell cycle in S phase and G2/M phase. Western blotting analysis showed that Akt1 and its phosphorylation were inhibited, the Bax/Bcl-2 ratio increased, and both caspase-9 and caspase-3 were activated after treatment with 1.3 μg/mL TanIIA at 48 h. Meanwhile, p53 and p21 protein levels increased, whereas cyclin B1, CDC2, and CDC2 phosphorylation were inhibited. Conclusion. TanIIA inhibits the growth of esophageal cancer cells and induces apoptosis in a time-dependent and concentration-dependent manner, possibly by affecting cell cycle- and apoptosis-related signaling pathways.

Effects of different light quality on growth, active ingredients and enzymes activities of Salvia miltiorrhiza

This study aimed to investigate the effects of different light quality on the growth, accumulation of active ingredients and enzymes activities of Salvia miltiorrhiza. The seedlings of S. miltiorrhiza were treated by different light quality, and relative parameters were measured. The data was statistically processed. Plant height was significantly decreased with supplemental blue light (WB), and the root length, root diameter, root fresh weight and root dry weight were significantly increased with supplemental red light (WR). Salvianolic acid B concentration in S. miltiorrhiza was highly increased by supplemental blue and red light, but tanshinone IIA concentration was not significantly affected by supplemental blue and red light. Enzymes activities of SOD, POD, PAL, TAT and PPO in S. miltiorrhiza were significant increased by supplemental blue light, and enzymes activities of POD, TAT and PPO were significant increased by supplemental red light. The root growth of S. miltiorrhiza was greatly promoted by supplemental red light (WR). Salvianolic acid B concentration in S. miltiorrhiza was highly increased by supplemental blue and red light. Enzymes activities of TAT and PPO in S. miltiorrhiza were significant increased by supplemental blue light and red light.

Effect of tanshinone IIA on the change of calcium current induced by beta-amyloid protein 25-35 in neurons of nucleus basalis of Meynert

To explore the effect of tanshinone IIA (TanIIA) on calcium current induced by beta-amyloid protein 25-35 (Abeta25-35) in neurons of nucleus basalis of Meynert (nbM). Cell acute dissociated technique and the whole-cell recording model of patch-clamp technique of single-cell were used. The voltage-dependent calcium current in neurons of nbM was recorded in SD rats first. Then the effect of TanIIA on the voltage-dependent calcium current in the neurons was assayed. The change of calcium current induced by Abeta25-35 as well as the effect of TanIIA on the change of calcium current induced by Abeta25-35 in neurons of nbM were analyzed. Extracellular fluid containing different concentrations of TanIIA was irrigated, respectively. The peak current did not change obviously. There was no difference in current density between the TanIIA group and the control group at 0 mV (P>0.05). Extracellular fluid containing 200 nmol/L Abeta25-35 was irrigated after the normal calcium current recorded under whole patch clamp, and the peak current changed obviously. There was distinct difference in the current density between the Abeta group and the control group at 0 mV (P<0.05). Extracellular fluid containing Abeta25-35 and different concentrations of TanIIA were irrigated after the normal calcium current was recorded under whole patch clamp, respectively, and the peak current did not change. There was no difference in current density between the TanIIA +Abeta group and the control group at 0 mV (P>0.05). In vitro, TanIIA could inhibit the calcium current amplification induced by Abeta25-35 in neurons of nbM. TanIIA may protect neurons against the toxicity of Abeta and decrease the inward flow of Ca(2+).

Tanshinone IIA induces apoptosis in human lung cancer A549 cells through the induction of reactive oxygen species and decreasing the mitochondrial membrane potential

Tanshinone IIA (Tan-IIA) is extracted from Danshen and known to inhibit proliferation and induce apoptosis in many cancer cells. We aimed to elucidate its anticancer activity and molecular mechanism in human lung cancer A549 cells. The cytotoxicity of Tan-IIA in A549 cells were measured by the MTT assay. The effects of Tan-IIA on the cell cycle, mitochondrial membrane potential (MMP), calcium and reactive oxygen species (ROS) released in A549 cells were detected by flow cytometry. The protein expressions of p53, Bax, Bcl-2 and beta-actin in A549 cells were tested by Western blotting. The proliferative rates of A549 cells were obviously inhibited by Tan-IIA in a dose- and time-dependent manner. The results of FACS showed that the sub-G1 phase was increased when A549 cells were cultured with various concentrations of Tan-IIA (control, 2.5, 5 and 10 microg/ml) for 48 h. Tan-IIA induced the production of ROS, Ca+2 and decreased MMP. The outcome of Western blotting showed that protein expressions of p53 and bax were increased, but proto-oncogene bcl-2 was notably decreased, after culturing with Tan-IIA (5 microg/ml) for 6, 12 and 24 h. Tan-IIA inhibited the proliferation of non-small cell lung cancer A549 cells, possibly by decreasing the MMP and inducing apoptosis due to the induction of a higher ratio of Bax/Bcl-2.

Novel polymeric nanoparticles containing tanshinone IIA for the treatment of hepatoma

Novel polylactic acid nanoparticles containing tanshinone IIA (TS-PLA-NPs) were synthesized by a single oil-in-water emulsion/solvent evaporation method. In this study, the optimized nanoparticles were characterized for morphology, mean particle size, zeta potential, entrapment efficiency, drug-loading content, X-ray diffractometer measurement, and in vitro release. The obtained nanoparticles were spherical and intact. The mean particle size was 192.5 nm with polydispersity index being 0.029 and zeta potential − 26.27 mV. The mean entrapment efficiency and loading of tanshinone IIA (TSIIA) in TS-PLA-NPs were 86.35 and 1.61%, respectively. The in vitro release study was performed at pH 7.4 using a dialysis membrane. Without initial burst effect, the TSIIA sustained release from TS-PLA-NPs for more than 7 days. The mean in vitro cumulative release percentage of TSIIA from TS-PLA-NPs vs. time curve fitted well with the Higuchi Equation (Q = 2.0365 + 13.564 × t1/2, r = 0.9950). In pharmacokinetics and tissue distribution studies, the concentrations of TSIIA are higher in hepatoma and lower in blood, heart, kidney, spleen, and lung at 2 h after TS-PLA-NPs was administered via caudal vein. TS-PLA-NPs were effective in destroying the human liver cancer cells by the Mono-nuclear cell direct cytotoxicity assay (MTT) assay, and the growth-inhibitory effect of TS-PLA-NPs on human liver cancer cells was concentration and time dependent. The effect of TS-PLA-NPs on hepatoma in mice was also performed. The results of TS-PLA-NPs were markedly more effective than both of TSIIA and blank PLA nanoparticles in preventing tumor growth and increasing survival time of mice with hepatoma. This study provided support for the new paradigm, the application of TSIIA for the treatment of hepatoma.