Traditional Chinese Medicine Danshen Research Articles

Salvianolic acid C promotes renal gluconeogenesis in fibrotic kidneys through PGC1α.

Impaired renal gluconeogenesis is recently identified as a hallmark of chronic kidney disease. However, the therapeutic approach to promote renal gluconeogenesis in CKD is still lacking. We aimed to study whether Salvianolic acid C (SAC), a nature compound extracted from the traditional Chinese medicine Danshen, inhibits renal fibrosis through promotion of gluconeogenesis. TGF-β stimulated HK2 human renal epithelial cells and mice with unilateral ureteral obstruction (UUO) were used as in vitro and in vivo models to study renal fibrosis. Fibrotic and gluconeogenic changes were determined by Western blotting analysis, quantitative PCR and Masson staining. Glucose and lactate concentrations were measured in cell culture and renal tissues. We found that SAC treatment inhibits the deposition of extracellular matrix proteins and the expression of fibrotic markers such as fibronectin, N-cadherin, Vimentin, aSMA, pSmad3, and Snail in UUO kidneys or renal cells. Inhibition of these fibrotic markers by SAC treatment was associated with enhanced expression of three gluconeogenic enzymes such as PCK1, G6PC and FBP1 in renal tissues or cells. SAC increase the concentration of glucose in the supernatant of renal cells. Lactate concentration was reduced by SAC in renal tissues or cells. Pyruvate and glucose tolerance tests showed that SAC improve the impaired glucose metabolism systemically in UUO mice. Peroxisome proliferator activated receptor gamma coactivator 1 alpha (PGC1ɑ) was downregulated in mouse kidneys after UUO operation, which was increased by SAC treatment. Moreover, PGC1α inhibitor SR-18292 reversed the anti-fibrotic effect and pro-gluconeogenic effect caused by SAC in renal cells. In conclusion, SAC inhibits renal fibrosis through promotion of PGC1α-mediated renal gluconeogenesis.

Tanshinone I improves renal fibrosis by promoting gluconeogenesis through upregulation of peroxisome proliferator-activated receptor-γ coactivator 1α

Background Renal fibrosis, a hallmark of chronic kidney disease, is closely associated with dysregulated gluconeogenesis. Tanshinone I (Tan I), a bioactive compound derived from the traditional Chinese medicine Danshen, exhibits antifibrotic and anti-inflammatory properties. However, its effects on gluconeogenesis and the mechanisms through which it alleviates renal fibrosis remain unclear. This study aimed to investigate whether Tan I promotes gluconeogenesis and mitigates renal fibrosis. Methods Both in vivo and in vitro experiments were conducted. A unilateral ureteral obstruction (UUO) mouse model was used. Masson’s trichrome, HE, and immunofluorescence staining, along with Western blotting, were employed. Lactate concentrations and a pyruvate tolerance test were conducted to assess glucose metabolism. In vitro, HK2 cells and primary renal tubular cells were treated with transforming growth factor-β (TGFβ) to induce fibrosis, and the effects of Tan I on glucose and lactate levels were examined. Results In the UUO model, Tan I reduced fibrosis, decreased lactate accumulation, and modulated fibrosis markers while upregulating gluconeogenesis markers. Tanshinone I restored impaired renal gluconeogenesis, as evidenced by increased pyruvate levels. In vitro, Tan I inhibited fibrosis, reduced lactate levels, and increased glucose levels in cell supernatants. It also restored gluconeogenesis protein expression and decreased fibrotic protein levels. Peroxisome proliferator-activated receptor-γ coactivator (PGC1α) expression was downregulated in UUO and TGFβ-stimulated models, and Tan I reversed this downregulation. Inhibition of PGC1α in TGFβ-stimulated cells counteracted the antifibrotic and gluconeogenesis-promoting effects of Tan I. Conclusions Tanshinone I ameliorated renal fibrosis by enhancing gluconeogenesis through upregulation of PGC1α.

Research Progress on the Pharmacological Mechanism of Traditional Chinese Medicine Danshen in the Treatment of Cerebral Ischemic Stroke

Cerebral ischemic stroke (CIS) is an acute cerebrovascular disease characterized by focal neurological deficits. Many modern scholars believe that the pathogenesis of CIS is related to factors such as endothelial injury, oxidative stress imbalance, neuroinflammation, cell apoptosis and autophagy, calcium overload, and excessive accumulation of oxygen free radicals. Traditional Chinese medicine believes that the pathogenesis of CIS is obstructed meridians, phlegm and blood stasis, and obstruction of the brain and orifices. Danshen, as a commonly used traditional Chinese medicine in clinical practice, has the effects of promoting blood circulation and removing blood stasis, unblocking meridians and relieving pain, clearing the heart and eliminating restlessness, cooling blood and eliminating carbuncle. In addition, modern pharmacological research has found that the specific pharmacological components of Salvia miltiorrhiza include tanshinone, salvianolic acid, shikimic acid, danshensu, protocatechualdehyde, etc. It has antioxidant stress, regulation of cell autophagy, anti-inflammatory factors, antiplatelet aggregation, and protection of vascular endothelium. This article aims to analyze and summarize the pharmacological mechanism of salvia miltiorrhiza in treating CIS, providing scientific basis for clinical application of salvia miltiorrhiza in treating CIS.

Elucidating the molecular mechanisms of sepsis: Identifying key aging-related biomarkers and potential therapeutic targets in the treatment of sepsis.

Sepsis remains a crucial global health issue characterized by high mortality rates and a lack of specific treatments. This study aimed to elucidate the molecular mechanisms underlying sepsis and to identify potential therapeutic targets and compounds. High-throughput sequencing data from the GEO database (GSE26440 as the training set and GSE13904 and GSE32707 as the validation sets), weighted gene co-expression network analysis (WGCNA), Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis, alongside a combination of PPI and machine learning methods (LASSO and SVM) were utilized. WGCNA identified the black module as positively correlated, and the green module as negatively correlated with sepsis. Further intersections of these module genes with age-related genes yielded 57 sepsis-related genes. GO and KEGG pathway enrichment analysis, PPI, LASSO, and SVM selected six hub aging-related genes: BCL6, FOS, ETS1, ETS2, MAPK14, and MYC. A diagnostic model was constructed based on these six core genes, presenting commendable performance in both the training and validation sets. Notably, ETS1 demonstrated significant differential expression between mild and severe sepsis, indicating its potential as a biomarker of severity. Furthermore, immune infiltration analysis of these six core genes revealed their correlation with most immune cells and immune-related pathways. Additionally, compounds were identified in the traditional Chinese medicine Danshen, which upon further analysis, revealed 354 potential target proteins. GO and KEGG enrichment analysis of these targets indicated a primary enrichment in inflammation and immune-related pathways. A Venn diagram intersects these target proteins, and our aforementioned six core genes yielded three common genes, suggesting the potential efficacy of Danshen in sepsis treatment through these genes. This study highlights the pivotal roles of age-related genes in the molecular mechanisms of sepsis, offers potential biomarkers, and identifies promising therapeutic compounds, laying a robust foundation for future studies on the treatment of sepsis.

Protective effect of salvianolic acid B against myocardial ischemia/reperfusion injury: preclinical systematic evaluation and meta-analysis.

Salvianolic acid B is the most abundant water-soluble component in the traditional Chinese medicine Danshen and can reduce myocardial ischemia-reperfusion (MI/R) injury through multiple targets and pathways. However, the role of SalB in protecting the myocardium from ischemia/reperfusion injury remains unclear. To perform a preclinical systematic review and meta-analysis to assess the efficacy of Sal B in an animal model of myocardial infarction/reperfusion (MI/R) and to summarize the potential mechanisms of Sal B against MI/R. Studies published from inception to March 2024 were systematically searched in PubMed, Web of Science, Embase, China National Knowledge Infrastructure Wanfang, and VIP databases. The methodological quality was determined using the SYRCLE RoB tool. The R software was used to analyze the data. The potential mechanisms are categorized and summarized. 32 studies containing 732 animals were included. The results of the meta-analysis showed that Sal B reduced myocardial infarct size (p < 0.01), and the cardiological indices of CK-MB (p < 0.01), CK (p < 0.01), LDH (p < 0.01), and cTnI (p < 0.01) compared to the control group. In addition, Sal B increased cardiac function indices, such as LVFS (p < 0.01), -dp/dt max (p < 0.01), +dp/dt max (p < 0.01), and cardiac output (p < 0.01). The protective effects of Sal B on the myocardium after I/R may be mediated by attenuating oxidative stress and inflammation, promoting neovascularization, regulating vascular function, and attenuating cardiac myocyte apoptosis. Publication bias was observed in all the included studies. Further studies are required to elucidate the extent of the cardioprotective effects of SalB and the safety of its use. To the best of our knowledge, this is the first meta-analysis of Sal B in the treatment of MI/R injury, and Sal B demonstrated a positive effect on MI/R injury through the modulation of key pathological indicators and multiple signaling pathways. Further studies are needed to elucidate the extent to which SalB exerts its cardioprotective effects and the safety of its use. https://www.crd.york.ac.uk/PROSPERO/.

Cytotoxic diterpenoids from Salvia glutinosa and comparison with the tanshinone profile of danshen (Salvia miltiorrhiza).

The roots of Salvia miltiorrhiza are the source of the traditional Chinese medicine danshen and the class of tanshinones, particular quinoid nor-diterpenoids of the abietane type. Of these compounds, cryptotanshinone, dihydrotanshinone I, tanshinone I, and tanshinone IIA, have been extensively studied for their anticancer potential, not only but as well because of their high abundance in S. miltiorrhiza and their thus easy availability. However, also additional Salvia species are known to contain tanshinones, mainly such of the subgenus Glutinaria, of which S. glutinosa is the only species widely occurring in Europe. Using UHPLC-DAD-MS, the tanshinone profile of S. glutinosa roots collected from two different locations was compared to the profile in S. miltiorrhiza roots. In addition, tanshinone IIA and another six diterpenoids from S. glutinosa were investigated for their antiproliferative and cytotoxic potential against MDA-MB-231 and HL-60 cells. Apart from dihydrotanshinone I, which has been previously characterized due to its anticancer properties, we determined danshenol A as a highly antiproliferative and cytotoxic agent, significantly surpassing the effects of dihydrotanshinone I. With regard to the diterpenoid profile, S. miltiorrhiza showed a higher concentration for most of the tanshinones, except for (+)-danshexinkun A, which was present in comparable amounts in both species. Danshenol A, in contrast, was only present in S. glutinosa as were dehydroabietic acid and (+)-pisiferic acid. The results of our study underlines the long traditional use of danshen due to its high amount on tanshinones, but also demonstrates the potential value of investigating closely related species for the discovery of new biologically active lead compounds.

Protective effects of tanshinone ⅡA on sepsis-induced multiple organ dysfunction: a literature review.

TanshinoneⅡA (TanⅡA) is a noteworthy lipophilic diterpene compound derived from the dried roots of the Traditional Chinese Medicine Danshen () that has various pharmacological properties, including anti-inflammatory, antibacterial, and antioxidative effects. Sepsis is a life-threatening organ dysfunction induced by a dysregulated host response to infection. Recently, increasing attention has been paid to sepsis-induced dysfunction of the intestine, car-diovascular system, lungs, kidneys, liver, and other organs. Experimental studies have shown that TanⅡA has therapeutic potential for sepsis-induced organ dysfunction owing to its anti-inflammatory, anti-apoptotic and regulatory effects on multiple signalling pathways. The purpose of this article is to evaluate the potential multiorgan protective effects of TanⅡA in sepsis.

Cryptotanshinone inhibits ovarian tumor growth and metastasis by degrading c-Myc and attenuating the FAK signaling pathway.

Cryptotanshinone (CT), a natural compound derived from Salvia miltiorrhiza Bunge that is also known as the traditional Chinese medicine Danshen, exhibits antitumor activity in various cancers. However, it remains unclear whether CT has a potential therapeutic benefit against ovarian cancers. The aim of this study was to test the efficacy of CT in ovarian cancer cells in vitro and using a xenograft model in NSG mice orthotopically implanted with HEY A8 human ovarian cancer cells and to explore the molecular mechanism(s) underlying CT’s antitumor effects. We found that CT inhibited the proliferation, migration, and invasion of OVCAR3 and HEY A8 cells, while sensitizing the cell responses to the chemotherapy drugs paclitaxel and cisplatin. CT also suppressed ovarian tumor growth and metastasis in immunocompromised mice orthotopically inoculated with HEY A8 cells. Mechanistically, CT degraded the protein encoded by the oncogene c-Myc by promoting its ubiquitination and disrupting the interaction with its partner protein Max. CT also attenuated signaling via the nuclear focal adhesion kinase (FAK) pathway and degraded FAK protein in both cell lines. Knockdown of c-Myc using lentiviral CRISPR/Cas9 nickase resulted in reduction of FAK expression, which phenocopies the effects of CT and the c-Myc/Max inhibitor 10058-F4. Taken together, our studies demonstrate that CT inhibits primary ovarian tumor growth and metastasis by degrading c-Myc and FAK and attenuating the FAK signaling pathway.

Tanshinone IIA Has a Potential Therapeutic Effect on Kawasaki Disease and Suppresses Megakaryocytes in Rabbits With Immune Vasculitis.

Background and ObjectiveIt is urgent to find out an alternative therapy for Kawasaki disease (KD) since around 20% patients are resistant to intravenous immunoglobulin (IVIG) or aspirin. Tanshinone IIA is the active component of the traditional Chinese medicine Danshen (Salvia miltiorrhiza), which has anti-inflammatory and anti-platelet properties; however, whether or not tanshinone IIA has a therapeutic effect on KD remains unclear. Therefore, the present study aimed to examine the effect of tanshinone IIA on KD patients and rabbits with immune vasculitis, and to identify the potential mechanisms with special emphasis on megakaryopoiesis and megakaryocytic apoptosis.MethodsKawasaki disease patients were recruited and prescribed with tanshinone IIA in the absence or presence of aspirin and IVIG, and the inflammatory responses and platelet functions were determined. Megakaryocytes (MKs) isolated from rabbits with immune vasculitis and human megakaryocytic CHRF-288-11 cells were treated with tanshinone IIA to examine the colony forming unit (CFU) and apoptosis, respectively. Microarray assay was conducted to identify potential targets of tanshinone IIA-induced apoptosis.ResultsTanshinone IIA reduced the serum levels of C-reactive protein (CRP), interleukin (IL)-1β, IL-6, and P-selectin in KD patients; such inhibitory effect was more significant compared to aspirin and IVIG. It also dose-dependently lowered the levels of tumor necrosis factor (TNF)-α and IL-8 in peripheral blood mononuclear cells isolated from KD patients. In rabbits with immune vasculitis, tanshinone IIA significantly reduced the serum levels of proinflammatory cytokines and platelet functions. In addition, tanshinone IIA significantly decreased the number of bone marrow MKs and inhibited the Colony Forming Unit-Megakaryocyte (CFU-MK) formation. In human megakaryocytic CHRF-288-11 cells, tanshinone IIA induced caspase-dependent apoptosis, probably through up-regulating TNF receptor superfamily member 9 (TNFRSF9) and the receptor (TNFRSF)-interacting serine/threonine-protein kinase 1 (RIPK1), which may contribute to its anti-platelet and anti-inflammatory properties.ConclusionTanshinone IIA exerts better anti-inflammatory and anti-platelet effects in treating KD patients than aspirin and IVIG. It attenuates immune vasculitis likely by inhibiting IL-mediated megakaryopoiesis and inducing TNFRSF9/RIPK1/caspase-dependent megakaryocytic apoptosis. The findings therefore suggest that tanshinone IIA may be a promising alternative therapy for the treatment of KD.

Inhibition of PI3K/AKT molecular pathway mediated by membrane estrogen receptor GPER accounts for cryptotanshinone induced antiproliferative effect on breast cancer SKBR-3 cells

BackgroundBreast cancer is the most frequently diagnosed malignancy among women and the second leading cause of cancer death worldwide. Among which nuclear estrogen receptor (nER) negative breast cancer is always with much poor prognosis. Recently, membrane G protein coupled estrogen receptor (GPER), a newly recognized estrogen receptor has been documented to take essential part in the development and treatment of breast cancer. The present study was designed to investigate the anti nER negative breast cancer effect of cryptotanshinone (CPT), an important active compound of traditional Chinese medicine Danshen and its possible molecular pathway.MethodsThe following in vitro tests were performed in nER negative but GPER positive breast cancer SKBR-3 cells. The effect of CPT on cell proliferation rate and cell cycle distribution was evaluated by MTT cell viability test and flow cytometry assay respectively. The role of PI3K/AKT pathway and the mediated function of GPER were tested by western blot and immunofluorescence. Technique of gene silence and the specific GPER agonist G-1 and antagonist G-15 were employed in the experiments to further verify the function of GPER in mediating the anticancer role of CPT.ResultsThe results showed that proliferation of SKBR-3 cells could be blocked by CPT in a time and dose dependent manner. CPT could also exert antiproliferative activities by arresting cell cycle progression in G1 phase and down regulating the expression level of cyclin A, cyclin B, cyclin D and cyclin-dependent kinase 2 (CDK2). The antiproliferative effect of CPT was further enhanced by G-1 and attenuated by G-15. Results of western blot and immunofluorescence showed that expression of PI3K and p-AKT could be downregulated by CPT and such effects were mediated by GPER which were further demonstrated by gene silence test.ConclusionThe current study showed that the antiproliferative action of CPT on SKBR-3 cells was realized by inhibition of GPER mediated PI3K/AKT pathway. These findings provide further validation of GPER serving as useful therapeutic target.

Tanshinone IIA reduces palmitate-induced apoptosis via inhibition of endoplasmic reticulum stress in HepG2 liver cells.

Research has indicated that stress on the endoplasmic reticulum (ER) of a cell affects the pathogenesis of metabolic disorders such as obesity, type 2 diabetes mellitus, and non-alcoholic fatty liver disease (NAFLD). Palmitate, a saturated fatty acid, is known to induce toxicity and cell death in various types of cells. Tanshinone IIA (Tan IIA), one of the effective components of the traditional Chinese medicine Danshen, was reported to exhibit a variety of biochemical activities, including amelioration of ER stress-mediated apoptosis in renal preservation. To address the hypothesis that tan IIA attenuates apoptosis and triglycerides (TG) accumulation via reducing ER stress, we studied the effect of tan IIA on experimentally induced ER stress using palmitate in HepG2 cells. Palmitate led to cytotoxicity, TG accumulation, and apoptosis in HepG2 cells and also strongly induced ER stress indicated by increased GRP78, phosphorylation of eIF2α, ATF6, and CHOP. Pretreatment with tan IIA (10μm) significantly increased cell viability, decreased apoptotic cell death, and reduced the activity of caspase-3. Meanwhile, tan IIA significantly decreased palmitate-induced TG accumulation. Moreover, tan IIA significantly suppressed the phosphorylation of eIF2α, and inhibited GRP78, ATF6, and CHOP expression. In conclusion, tan IIA protects the HepG2 cells exposed to palmitate partially by inhibiting excessive ER stress, ER stress-induced apoptosis, and hepatic steatosis. Therefore, tan IIA has therapeutic potential in the treatment of NAFLD.

Synthesis of (+)-salvianolic acid A from sodium Danshensu

(+)-Salvianolic acid A, one of the most active components in the traditional Chinese medicine Danshen, has been synthesized over 10 steps in 6.5% overall yield. Starting from inexpensive ortho-vanillin and sodium Danshensu (synthesized via asymmetric catalysis in our group), the process consists of the following: A Wittig reaction that gives the desire product with absolute E-configuration, a demethylation reaction with AlCl3 in a satisfactory yield, and a practical deprotection of allylic groups to afford the terminal product (+)-salvianolic acid A. The current synthetic technology possesses the advantages of using inexpensive starting materials, mild reaction conditions and has the potential for use in large scale synthesis.

Salvianolic acids from antithrombotic Traditional Chinese Medicine Danshen are antagonists of human P2Y1 and P2Y12 receptors

Many hemorheologic Traditional Chinese Medicines (TCMs) that are widely-used clinically lack molecular mechanisms of action. We hypothesized that some of the active components of hemorheologic TCMs may function through targeting prothrombotic P2Y1 and/or P2Y12 receptors. The interactions between 253 antithrombotic compounds from TCM and these two G protein-coupled P2Y receptors were evaluated using virtual screening. Eleven highly ranked hits were further tested in radioligand binding and functional assays. Among these compounds, salvianolic acid A and C antagonized the activity of both P2Y1 and P2Y12 receptors in the low µM range, while salvianolic acid B antagonized the P2Y12 receptor. These three salvianolic acids are the major active components of the broadly-used hemorheologic TCM Danshen (Salvia militorrhiza), the antithrombotic molecular mechanisms of which were largely unknown. Thus, the combination of virtual screening and experimental validation identified potential mechanisms of action of multicomponent drugs that are already employed clinically.

Potentiation of the anticancer effect of doxorubicinin drug-resistant gastric cancer cells by tanshinone IIA

BackgroundGastric cancer is the fifth commonest cancer and the third cause of cancer-related deaths all over the world. The effectiveness of chemotherapy is still limited by drug resistance in gastric cancer. Tanshinones, abietane diterpenes isolated from the traditional Chinese medicine Danshen (Salvia miltiorrhiza), have exhibited versatile anticancer activities in particular the ability to overcome drug resistance in different cancers. PurposeThe current study aimed to explore the capacity of tanshinone IIA, the most abundant tanshinone found in the plant Danshen, to overcome drug resistance of gastric cancer cells to a commonly used anticancer drug doxorubicin. Study designSensitivity of cell lines to doxorubicin was determined by MTT assay. Doxorubicin resistant gastric cancer cell lines was established by step selection with increasing concentrations of doxorubicin. Cell cycle arrest, apoptosis and doxorubicin efflux were analyzed by flow cytometry. The expression of MRP1 was determined by realtime PCR and western-blot. ResultsBased on the IC50 values of doxorubicin, we identified the doxorubicin-sensitive gastric cancer cell lines SNU-719 and SNU-610 as well as the cell lines relatively resistant to doxorubicin including SNU-638, SNU-668, SNU-216 and SNU-620. We also established two drug-resistant cell lines SNU-719R and SNU-610R. Despite the fact that tanshinone IIA alone showed no cytotoxicity on these gastric cells, we found the potentiation of the anticancer effect of doxorubicin in drug-resistant gastric cancer cells by tanshinone IIA. Furthermore, using doxorubicin-sensitive cell line SNU-719 and doxorubicin-resistant cell lines SNU-719R and SNU-620, we revealed the pivotal roles of MRP1. Its overexpression impaired cell cycle arrest and suppressed apoptosis in the development of both intrinsic and acquired drug resistance in gastric cancer cells to doxorubicin. Importantly, inhibition of MRP1 function enhanced cell cycle arrest, increased apoptosis and induced autophagic cell death which contributed to the capability of tanshinone IIA to potentiate the anticancer effect of doxorubicin in drug-resistant gastric cancer cells. ConclusionTanshinone IIA is an interesting agent with potential to treat drug-resistant gastric cancer in combination therapy.

Tanshinone IIA Elicits Neuroprotective Effect Through Activating the Nuclear Factor Erythroid 2-Related Factor-Dependent Antioxidant Response.

Tanshinone IIA (TSA), a principal component derived from the Traditional Chinese Medicine Danshen has been suggested to exert neuroprotective effect against experimental cerebral ischemic/reperfusion injury. But the associated underlying mechanisms still have not been understood. The current study characterized the role of nuclear factor erythroid two-related factor-induced antioxidant response in the neuroprotective efficacy of TSA treatment. The focal cerebral ischemia/reperfusion model was established by 60-minute middle cerebral artery occlusion. At the onset during reperfusion, mice were treated with 10 mg/kg TSA intraperitoneally. The mRNA and nuclear factor erythroid 2 (Nrf2) protein expression, the antioxidant enzymes, and oxidative production levels were measured. To further verify the role of Nrf2 in the neuroprotective effect induced by TSA, the Nrf2 small silenced RNA and Nrf2 knockout mice were used, the neurological function, brain infarct volume, and cellular apoptosis examination were assessed. TSA treatment improved neurological scores, reduced infarct volume, and attenuated the cellular apoptosis. TSA treatment upregulated the expression of Nrf2 mRNA and the contents of Nrf2 protein in nuclear extract. Nrf2 activation by TSA treatment increased the contents of antioxidant enzymes, and reduced the generation of oxidative productions. Either Nrf2 knockdown or Nrf2 knockout abolished the antioxidative and neuroprotective effect of TSA treatment. These results demonstrate that the Nrf2 activation contributes to TSA-induced neuroprotection from experimental ischemic stroke through maintaining antioxidant effect.

Tanshinone IIA Exerts an Antinociceptive Effect in Rats with Cancer-induced Bone Pain

Background: Cancer-induced bone pain (CIBP) is a common chronic pain characterized by 2 components, ongoing pain and breakthrough pain. Tanshinone IIA (TSN IIA) is a bioactive constituent of the traditional Chinese medicine Danshen, which has been reported to have an antinociceptive effect on neuropathic and inflammatory pain through downregulation of the late proinflammatory cytokine high-mobility group protein B1 (HMGB1). Objective: To assess the antinociceptive effect of TSN IIA on CIBP. Study Design: A randomized, double-blind, controlled animal trial was performed. Setting: University lab in China. Methods: A rat CIBP model was established by injecting Walker 256 mammary gland carcinoma cells into the intramedullary cavity of the tibia. Both ongoing pain, e.g., flinching and guarding, and breakthrough pain, e.g., limb use and von Frey threshold, were evaluated. The effects of intraperitoneally administered TSN IIA on pain behavior and the expression levels of spinal HMGB1, interleukin (IL)-1β, tumor necrosis factor (TNF)-α, and IL-6 were determined. The effect of TSN IIA on the electrically evoked response of spinal wide-dynamic range (WDR) neurons was performed in vivo. Results: TSN IIA dose-dependently inhibited cancer-induced ongoing pain and breakthrough pain. The expression levels of spinal HMGB1 and other inflammatory factors (IL-1β, TNF-α, and IL-6) were increased in the rat model, but they were suppressed by TSN IIA in a dose-dependent manner. Moreover, TSN IIA significantly inhibited the neuronal responses of WDR neurons in spinal deep layers. Limitations: Further studies are warranted to ascertain how TSN IIA attenuates cancer-induced ongoing pain. Conclusions: Our results indicate that TSN IIA attenuates cancer-induced ongoing pain and breakthrough pain, possibly via suppression of central sensitization in CIBP rats. Therefore, we have provided strong evidence supporting TSN IIA as a potential and effective therapy for relieving CIBP. Key words: Cancer-induced bone pain, high-mobility group protein B1, Tanshinone IIA, ongoing pain, breakthrough pain

Tanshinone IIA Attenuates Chronic PancreatitisInduced Pain in Rats via Downregulation of HMGB1 and TRL4 Expression in the Spinal Cord

Background: Chronic pancreatitis (CP) is a long-standing inflammation of the exocrine pancreas, which typically results in severe and constant abdominal pain. Previous studies on the mechanisms underlying CP-induced pain have primarily focused on the peripheral nociceptive system. A role for a central mechanism in the mediation or modulation of abdominal pain is largely unknown. Tanshinone IIA (TSN IIA), an active component of the traditional Chinese medicine Danshen, exhibits anti-inflammatory properties via downregulation of the expression of high-mobility group protein B1 (HMGB1), a late proinflammatory cytokine. HMGB1 binds and activates toll-like receptor 4 (TLR4) to induce spinal astrocyte activation and proinflammatory cytokine release in neuropathic pain. Objective: In this study, we investigated the effect of TSN IIA on pain responses in rats with trinitrobenzene sulfonic acid (TNBS)-induced CP. The roles of central mechanisms in the mediation or modulation of CP were also investigated. Study Design: A randomized, double-blind, placebo-controlled animal trial. Methods: CP was induced in rats by intrapancreatic infusion of trinitrobenzene sulfonic acid (TNBS). Pancreatic histopathological changes were characterized with semi-quantitative scores. The abdomen nociceptive behaviors were assessed with von Frey filaments. The effects of intraperitoneally administered TSN IIA on CP-induced mechanical allodynia were tested. The spinal protein expression of HMGB1 was determined by western blot. The spinal mRNA and protein expression of proinflammatory cytokines IL-1β, TNF-α, and IL-6 were determined by RT-PCR and western blot, respectively. The spinal expression of the HMGB1 receptor TRL4 and the astrocyte activation marker glial fibrillary acidic protein (GFAP) were determined by western blot or immunohistological staining after intraperitoneal injection of TSN IIA or intrathecal administration of a neutralizing anti-HMGB1 antibody. Results: TNBS infusion resulted in pancreatic histopathological changes of chronic pancreatitis and mechanical allodynia in rats. TSN IIA significantly attenuated TNBS-induced mechanical allodynia in a dose-dependent manner. TNBS significantly increased the spinal expression of HMGB1 and proinflammatory cytokines IL-1β, TNF-α, and IL-6. These TNBS-induced changes were significantly inhibited by TSN IIA in a dose-dependent manner. Furthermore, TSN IIA, but not the neutralizing anti-HMGB1 antibody, significantly inhibited TNBS-induced spinal TLR4 and GFAP expression. Limitations: In addition to TLR4, HMGB1 can also bind to toll-like receptor-2 (TLR2) and the receptor for advanced glycation end products (RAGE). Additional studies are warranted to ascertain whether HMGB1 contributes to CP-induced pain through activation of these receptors. Conclusions: Our results suggest that spinal HMGB1 contributes to the development of CPinduced pain and can potentially be a therapeutic target. TSN IIA attenuates CP-induced pain via downregulation of spinal HMGB1 and TRL4 expression. Therefore, TSN IIA may be a potential anti-nociceptive drug for the treatment of CP-induced pain. Key words: Chronic pancreatitis, HMGB1, proinflammatory cytokine, Tanshinone IIA, spinal cord, astrocyte, TLR4

Tanshinone IIA inhibits viral oncogene expression leading to apoptosis and inhibition of cervical cancer

Human papilloma virus (HPV) is the well-established etiological factor of cervical cancer. E6 and E7 oncoproteins expressed by HPV are known to inactivate tumor suppressor proteins p53 and pRb, respectively. Tanshinone IIA (Tan IIA) is a diterpenoid naphthoquinone found in the traditional Chinese medicine Danshen (Salvia sp.). Tan IIA has been shown to possess anti-tumor activity against several cancer types. In this study we show that Tan IIA potently inhibited proliferation of the human cervical cancer CaSki, SiHa, HeLa and C33a cells. Mechanistically in HPV positive CaSki cells, Tan IIA was found to (i) downregulate expression of HPV E6 and E7 genes and modulate associated proteins E6AP and E2F1, (ii) cause S phase cell cycle arrest, (iii) induce accumulation of p53 and alter expression of p53-dependent targets, (iv) modulate pRb and related proteins, and (v) cause p53-mediated apoptosis by moderating Bcl2, Bax, caspase-3, and PARP cleavage expressions. In vivo, Tan IIA resulted in over 66% reduction in tumor volume of cervical cancer xenograft in athymic nude mice. Tan IIA treated tumor tissues had lower expression of proliferation marker PCNA and changes in apoptosis targets were in agreement with in vitro studies, further confirming reduced proliferation and involvement of multiple targets behind anti-cancer effects. This is the first demonstration of Tan IIA to possess significant anti-viral activity by repressing HPV oncogenes leading to inhibition of cervical cancer. Together, our data suggest that Tan IIA can be exploited as a potent therapeutic agent for the prevention and treatment of cervical and other HPV-related cancers.

Detecting metabolites of different transition metal-lithospermate B complexes after intravenous injection in rats.

Lithospermate B (LSB) isolated from the traditional Chinese medicine danshen (Salvia miltiorrhiza) is an effective Na(+)/K(+)-ATPase inhibitor and used to treat congestive heart failure. The inhibition of LSB on Na(+)/K(+)-ATPase is potentiated by forming complexes with transition metal ions. Here we investigated the safety and metabolites of different transition metal-LSB complexes in rats. LSB complexed with six different transition metal ions (Mg(2+), Zn(2+), Cr(3+), Co(2+), Ni(2+) and Mn(2+)) were prepared. Adult male SD rats were injected with the different metal-LSB complexes (50 mg/kg, iv), and their bile and blood samples were collected. The metabolites of the metal-LSB complexes in the samples were analyzed using mass spectroscopy. In rats injected with LSB complexed with Mg(2+), Zn(2+), Cr(3+), Ni(2+) or Mn(2+), LSB and its four putative metabolites were equivalently detected in their bile samples. Mn(2+)-LSB exhibited distinct metabolite profiles compared with the other four metal-LSB complexes. The four putative metabolites were identified as 3-monomethyl-LSB, 3,3''-dimethyl-LSB, 3,3'''-dimethyl-LSB and 3,3'',3'''-trimethyl-LSB. The tracking of successive bile samples of rats injected with Mg(2+)-LSB, Zn(2+)-LSB and Mn(2+)-LSB concurrently demonstrated that LSB was firstly methylated at position 3, then at position 3'', and, finally, the 3''' hydroxyl group. All rats injected with Co(2+)-LSB died. Zn(2+)-LSB, Cr(3+)-LSB, Ni(2+)-LSB or Mn(2+)-LSB produces identical four methylated metabolites of LSB in rats, and seemed to be as safe as LSB or Mg(2+)-LSB.

Proteomic Study on the Protective Effect of Tanshinone IIA Against Oxidative Injury to Human Umbilical Vein Endothelial Cells by Hydrogen Peroxide

Atherosclerosis is considered a chronic inflammatory disease and injury to vascular endothelial cells is an important event at the onset of its pathogenesis. Oxidative stress is regarded as a pivotal pathogenic factor in the development of endothelial cell injury and apoptosis. Tanshinone IIA is considered the main active lipophilic component of the traditional Chinese medicine Danshen. It has been demonstrated that Tanshinone IIA protects endothelial cells from oxidative stress-triggered injury and apoptosis, but many of its anti-oxidant and anti-apoptotic mechanisms still remain to be elucidated. In this paper, our results show that HUVECs incubated with 1.1mM H2O2 for 12h had significantly decreased the viability of endothelial cells, which was accompanied with obviously cell apoptosis. However, treatment with Tanshinone IIA (8 mM, 24h) resulted in a significant resistance to H2O2-induced apoptosis. We used 2D-DIGE (Difference in Gel Electrophoresis) analysis followed by MALDI-TOF-TOF-MS to identify the proteins differentially expressed after human umbilical vein endothelial cells (HUVECs) were protected with Tanshinone IIA against oxidative damage by hydrogen peroxide. There were various differently expressed proteins, we picked out 32 proteins to analysis, and discovered that Tanshinone IIA could protect endothelial cells by its anti-inflammatory or antioxidant effects, regulating proliferation and differentiation, maintaining the cytoskeleton, and maintaining intracellular calcium homeostasis. Keywords: HUVEC, H2O2, atherosclerosis, proteome, Tanshinone IIA.