Groundwater chromate removal by autotrophic sulfur disproportionation.

Chromate [Cr(VI)] contamination in groundwater is a global environmental challenge. Traditional elemental sulfur-based biotechnologies for Cr(VI) removal depend heavily on the synthesis of dissolved organic carbon to fuel heterotrophic Cr(VI) reduction, a bottleneck in the remediation process. Here we show an alternative approach by leveraging sulfur-disproportionating bacteria (SDB) inherent to groundwater ecosystems, offering a novel and efficient Cr(VI) removal strategy. We implemented SDB within a sulfur-packed bed reactor for treating Cr(VI)-contaminated groundwater, achieving a notable removal rate of 6.19 mg L-1 h-1 under oligotrophic conditions. We identified the chemical reduction of Cr(VI) via sulfide, produced through sulfur disproportionation, as a key mechanism, alongside microbial Cr(VI) reduction within the sulfur-based biosystem. Genome-centric metagenomic analysis revealed a symbiotic relationship among SDB, sulfur-oxidizing, and chromate-reducing bacteria within the reactor, suggesting that Cr(VI) detoxification by these microbial communities enhances the sulfur-disproportionation process. This research highlights the significance of sulfur disproportionation in the cryptic sulfur cycle in Cr(VI)-contaminated groundwater and proposes its practical application in groundwater remediation efforts.

A novel sulfidogenic process via sulfur reduction to remove arsenate in acid mine drainage: Insights into the performance and microbial mechanisms.

Biological sulfidogenic processes based on sulfate-reducing bacteria (SRB) are not suitable for arsenic (As)-containing acid mine drainage (AMD) treatment because of the formation of the mobile thioarsenite during sulfate reduction. In contrast, biological sulfidogenic processes based on sulfur-reducing bacteria (S0RB) produce sulfide without pH increase, which could achieve more effective As removal than the SRB-based process. However, the reduction ability and toxicity tolerance of S0RB to As remains mysterious, which may substantially affect the practical applicability of this process when treating arsenate (As(V))-containing AMD. Thus, this study aims to develop a biological sulfur reduction process driven by S0RB, and explore its long-term performance on As(V) removal and microbial community evolution. Operating under moderately acidic conditions (pH=4.0), the presence of 10 mg/L As(V) significantly suppressed the activity of S0RB, leading to the failure of As(V) removal. Surprisingly, a drop in pH to 3.0 enhanced the tolerance of S0RB to As toxicity, allowing for efficient sulfide production (396±102 mg S/L) through sulfur reduction. Consequently, effective and stable removal of As(V) (99.9 %) was achieved, even though the sulfidogenic bacteria were exposed to high levels of As(V) (42 mg/L) in long-term trials. Spectral and spectroscopic analysis showed that As-bearing sulfide minerals were present in the bioreactor. Remarkably, the presence of As(V) induced notable changes in the microbial community composition, with Desulfurella and Clostridium identified as predominate sulfur reducers. The qPCR result further revealed an increase in the concentration of functional genes related to As transport (asrA and arsB) in the bioreactor sludge as the pH decreased from 4.0 to 3.0. This suggests the involvement of microorganisms carrying asrA and arsB in an As transport process. Furthermore, metagenomic binning demonstrated that Desulfurella contained essential genes associated with sulfur reduction and As transportation, indicating its genetic potential for sulfide production and As tolerance. In summary, this study underscores the effectiveness of the biological sulfur reduction process driven by S0RB in treating As(V)-contaminated AMD. It offers insights into the role of S0RB in remediating As contamination and provides valuable knowledge for practical applications.

Adaptability of sulfur-disproportionating bacteria for mine water remediation under the pressures of heavy metal ions and high sulfate content.

Biological sulfide production processes mediated by sulfate/sulfur reduction have gained attention for metal removal from industrial wastewater (e.g., mine water (MW) and metallurgical wastewater) via forming insoluble metal sulfides. However, these processes often necessitate the addition of external organic compounds as electron donors, which poses a constraint on the broad application of this technology. A recent proof of concept study reported that microbial sulfur disproportionation (SD) produced sulfide with no demand for organics, which could achieve more cost-benefit MW treatment against the above-mentioned processes. However, the resistance of SD bioprocess to different metals and high sulfate content in MW remains mysterious, which may substantially affect the practical applicability of such process. In this study, the sulfur-disproportionating bacteria (SDB)-dominated consortium was enriched from a previously established SD-driven bioreactor, in which Dissulfurimicrobium sp. with a relative abundance of 39.9 % was the predominated SDB. When exposed to the real pretreated acidic MW after the pretreatment process of pH amelioration, the sulfur-disproportionating activity remained active, and metals were effectively removed from the MW. Metal tolerance assays further demonstrated that the consortium had a good tolerance to different metal ions (i.e., Pb2+, Cu2+, Ni2+, Mn2+, Zn2+), especially for Mn2+ with a concentration of approximately 20 mg/L. It suggested the robustness of Dissulfurimicrobium sp. likely due to the presence of genes encoding for the enzymes associated with metal(loid) resistance/uptake. Additionally, although high sulfate content resulted in a slight inhibition on the sulfur-disproportionating activity, the consortium still achieved sulfide production rates of 27.3 mg S/g VSS-d on average under an environmentally relevant sulfate level (i.e., 1100 mg S/L), which is comparable to those reported in sulfate reduction. Taken together, these findings imply that SDB could ensure sustainable MW treatment in a more cost-effective and organic-free way.

Hydrogen sulfide control in sewer systems: A critical review of recent progress.

In sewer systems where anaerobic conditions are present, sulfate-reducing bacteria reduce sulfate to hydrogen sulfide (H2S), leading to sewer corrosion and odor emission. Various sulfide/corrosion control strategies have been proposed, demonstrated, and optimized in the past decades. These included (1) chemical addition to sewage to reduce sulfide formation, to remove dissolved sulfide after its formation, or to reduce H2S emission from sewage to sewer air, (2) ventilation to reduce the H2S and humidity levels in sewer air, and (3) amendments of pipe materials/surfaces to retard corrosion. This work aims to comprehensively review both the commonly used sulfide control measures and the emerging technologies, and to shed light on their underlying mechanisms. The optimal use of the above-stated strategies is also analyzed and discussed in depth. The key knowledge gaps and major challenges associated with these control strategies are identified and strategies dealing with these gaps and challenges are recommended. Finally, we emphasize a holistic approach to sulfide control by managing sewer networks as an integral part of an urban water system.

Sulfur disproportionation realizes an organic-free sulfidogenic process for sustainable treatment of acid mine drainage.

Biological sulfidogenic processes (BSPs) have been considered effective biotechnologies for the treatment of organic-deficit acid mine drainage (AMD) and heavy metal recovery. However, high-rate sulfide production relies on the continuous addition of exogenous organic substrates as electron donors to facilitate dissimilatory sulfate reduction, which substantially increases the operational cost and CO2 emission and also limits the wide application of BSPs in AMD treatment. In this study, we proposed a novel chemoautotrophic elemental sulfur disproportionation (SD) process as an alternative to conventional BSPs for treating AMD, in which sulfur-disproportionating bacteria (SDB) disproportionates sulfur to sulfide and sulfate without organic substrate supplementation. During the 393-day lab-scale test, we observed that the sulfur-disproportionating reactor (SDR) achieved a stable high-rate sulfide production, with a maximal rate of 21.10 mg S/L-h at an organic-substrate-free condition. This high rate of sulfide production suggested that the SD process could provide sufficient sulfide to precipitate metal ions from AMD. Thermodynamics analysis and batch tests further revealed that alkalinity rather than sulfate was the critical factor influencing the SD process, suggesting that the abundant sulfate present in AMD would not inhibit the SD process. The critical condition of SD in the SDR was therefore determined. Microbial community analysis showed that Dissulfurimicrobium sp. was the dominant SDB during the long-term operation regardless of dynamic sulfate and/or alkalinity concentrations, which provides evidence that SDB can be employed for sustainable and high-rate sulfide production for engineering purposes. A multi-stage AMD treatment system equipped with a SDR removed over 99% of the influent metals (i.e., Fe, Al, Zn, Cu, Pb) from AMD except for Mn. This study demonstrated that the novel SD process is a green and promising biotechnology for the sustainable treatment of organic-deficient metal-laden wastewater, such as AMD.

Treatment and remediation of metal-contaminated water and groundwater in mining areas by biological sulfidogenic processes: A review.

Heavy metal pollution in the mining areas leads to serious environmental problems. The biological sulfidogenic process (BSP) mediated by sulfidogenic bacteria has been considered an attractive technology for the treatment and remediation of metal-contaminated water and groundwater. Notwithstanding, BSP driven by different sulfidogenic bacteria could affect the efficiency and cost-effectiveness of the treatment performance in practical applications, such as the microbial intolerance of pH and metal ions, the formation of toxic byproducts, and the consumption of organic electron donors. Sulfur-reducing bacteria (S0RB)-driven BSP has been demonstrated to be a promising alternative to the commonly used sulfate-reducing bacteria (SRB)-driven BSP for treating metal-contaminated wastewater and groundwater, due to the cost-saving in chemical addition, the high efficiency in sulfide production and metal removal efficiency. Although the S0RB-driven BSP has been developed and applied for decades, the present review works mainly focus on the developments in SRB-driven BSP for the treatment and remediation of metal-contaminated wastewater and groundwater. Accordingly, a comprehensive review for metal-contaminated wastewater treatment and groundwater remediation should be provided with the incorporation of the SRB- and S0RB-driven BSP. To identify the bottlenecks and to improve BSP performance, this paper reviews sulfidogenic bacteria presenting in metal-contaminated water and groundwater; highlight the critical factors for the metabolism of sulfidogenic bacteria during BSP; the ecological roles of sulfidogenic bacteria and the mechanisms of metal removal by sulfidogenic bacteria; and the application of the present sulfidogenic systems and their drawbacks. Accordingly, the research knowledge gaps, current process limitations, and future prospects were provided for improving the performance of BSP in the treatment and remediation of metal-contaminated wastewater and groundwater in mining areas.

Research progress of mechanisms for tight junction damage on blood-brain barrier inflammation.

Inflammation in the central nervous system (CNS) contributes to disease pathologies by disrupting the integrity of the blood-brain barrier (BBB). Tight junctions (TJ) are a key component of the BBB. Following hypoxic-ischaemic or mechanical injury to the brain, inflammatory mediators are released such as cytokines, chemokines, and growth factors. Simultaneously, matrix metalloproteinases (MMPs) are released which can degrade TJ proteins. Subsequently, the function and morphology of the BBB are disrupted, which allows immune cells an opportunity to enter into the brain parenchyma. This review summarises the information on the role of TJ protein families in the BBB and provides a comprehensive summary of the mechanisms whereby inflammation breaks down the BBB by increasing degradation of TJ proteins.

Elemental sulfur as electron donor and/or acceptor: Mechanisms, applications and perspectives for biological water and wastewater treatment.

Biochemical oxidation and reduction are the principle of biological water and wastewater treatment, in which electron donor and/or acceptor shall be provided. Elemental sulfur (S0) as a non-toxic and easily available material with low price, possesses both reductive and oxidative characteristics, suggesting that it is a suitable material for water and wastewater treatment. Recent advanced understanding of S0-respiring microorganisms and their metabolism further stimulated the development of S0-based technologies. As such, S0-based biotechnologies have emerged as cost-effective and attractive alternatives to conventional biological methods for water and wastewater treatment. For instance, S0-driven autotrophic denitrification substantially lower the operational cost for nitrogen removal from water and wastewater, compared to the conventional process with exogenous carbon source supplementation. The introduction of S0 can also avoid secondary pollution commonly caused by overdose of organic carbon. S0 reduction processes cost-effectively mineralize organic matter with low sludge production. Biological sulfide production using S0 as electron acceptor is also an attractive technology for metal-laden wastewater treatment, e.g. acid mine drainage. This paper outlines an overview of the fundamentals, characteristics and advances of the S0-based biotechnologies and highlights the functional S0-related microorganisms. In particular, the mechanisms of microorganisms accessing insoluble S0 and feasibility to improve S0 bio-utilization efficiency are critically discussed. Additionally, the research knowledge gaps, current process limitations, and required further developments are identified and discussed.

A pilot-scale sulfur-based sulfidogenic system for the treatment of Cu-laden electroplating wastewater using real domestic sewage as electron donor.

Elemental sulfur (S0) reduction process has been demonstrated as an attractive and cost-efficient approach for metal-laden wastewater treatment in lab-scale studies. However, the system performance and stability have not been evaluated in pilot- or large-scale wastewater treatment. Especially, the sulfide production rate and microbial community structure may significantly vary from lab-scale system to pilot- or large-scale systems using real domestic sewage as carbon source, which brings questions to this novel technology. In this study, therefore, a pilot-scale sulfur-based sulfidogenic treatment system was newly developed and applied for the treatment of Cu-laden electroplating wastewaters using domestic sewage as carbon source. During the 175-d operation, >99.9% of Cu2+ (i.e., 5580 and 1187 mg Cu/L for two types of electroplating wastewaters) was efficiently removed by the biogenic hydrogen sulfide that produced through S0 reduction. Relatively high level of sulfide production (200 mg S/L) can be achieved by utilizing organics in raw domestic sewage, which was easily affected by the organic content and pH value of the domestic sewage. The long-term feeding of domestic sewage significantly re-shaped the microbial community in sulfur-reducing bioreactors. Compared to the reported lab-scale bioreactors, higher microbial community diversity was found in our pilot-scale bioreactors. The presence of hydrolytic, fermentative and sulfur-reducing bacteria was the critical factor for system stability. Accordingly, a two-step ecological interaction among fermentative and sulfur-reducing bacteria was newly proposed for sulfide production: biodegradable particulate organic carbon (BPOC) was firstly degraded to dissolved organic carbon (DOC) by the hydrolytic and fermentative bacteria. Then, sulfur-reducing bacteria utilized the total DOC (both DOC degraded from BPOC and the original DOC present in domestic sewage) as electron donor and reduced the S0 to sulfide. Afterwards, the sulfide precipitated Cu2+ in the post sedimentation tank. Compared with other reported technologies, the sulfur-based treatment system remarkable reduced the total chemical cost by 87.5‒99.6% for the same level of Cu2+ removal. Therefore, this pilot-scale study demonstrated that S0 reduction process can be a sustainable technology to generate sulfide for the co-treatment of Cu-laden electroplating wastewater and domestic sewage, achieving higher Cu2+removal and higher cost-effectiveness than the conventional technologies.

Elemental sulfur-driven sulfidogenic process under highly acidic conditions for sulfate-rich acid mine drainage treatment: Performance and microbial community analysis.

Elemental sulfur-driven sulfidogenic process has been demonstrated to be more economical and energy-efficient than sulfate-driven sulfidogenic process when treating metal-laden wastewater. In previous studies, we observed that the polysulfide-involved indirect sulfur reduction ensured the superiority of sulfur over sulfate as the electron acceptor in the sulfidogenic process under neutral or weak-alkaline conditions. However, realizing high-rate sulfur reduction process for acid mine drainage (AMD) treatment without pH amelioration is still a great challenge because polysulfide cannot exist under acidic conditions. In this study, a laboratory-scale sulfur-packed bed reactor was therefore continuously operated with a constant sulfate concentration (~1300 mg S/L) and decreasing pH from 7.3 to 2.1. After 400 days of operation, a stable sulfide production rate (38.2 ± 7.6 mg S/L) was achieved under highly acidic conditions (pH 2.6-3.5), which is significantly higher than those reported in sulfate reduction under similar conditions. In the presence of high sulfate content, elemental sulfur reduction could dominate over sulfate reduction under neutral and acidic conditions, especially when the pH ≥ 6.5 or ≤ 3.5. The decreasing pH significantly reduced the diversity of microbial community, but did not substantially influence the abundance of functional genes associated with organic and sulfur metabolisms. The predominant sulfur-reducing genera shifted from Desulfomicrobium under neutral conditions to Desulfurella under highly acidic conditions. The high-rate sulfur reduction under acidic conditions could be attributed to the combined results of high abundance of Desulfurella and low abundance of sulfate-reducing bacteria (SRB). Accordingly, sulfur reduction process can be developed to achieve efficient and economical treatment of AMD under highly acidic conditions (pH ≤ 3.5).

Overlooked pathways of denitrification in a sulfur-based denitrification system with organic supplementation.

Elemental sulfur-driven autotrophic denitrification (SADN) is a cost-effective approach for treating secondary effluent from wastewater treatment plants (WWTPs). Additional organics are generally supplemented to promote total nitrogen (TN) removal, reduce nitrite accumulation and sulfate production, and balance the pH decrease induced by SADN. However, understanding of the impacts of organic supplementation on microbial communities, nitrogen metabolism, denitrifier activity, and SADN rates in sulfur-based denitrification reactors is still limited. Here, a sulfur-based denitrification reactor was continuously operated for 272 days during which six different C/N ratios were tested successively (2.7, 1.5, 0.7, 0.5, 0.25, and 0). Organic supplementation improved TN removal and decreased NO2- accumulation, but reduced the relative abundance of denitrifiers and the contribution of autotrophic nitrate-reducing bacteria (aNRB) to TN removal during the long-term operation of reactor. Predictive functional profiling showed that nitrogen metabolism potential increased with decreasing C/N ratios. SADN was the predominant removal process when the C/N ratio was ≤0.7 (achieving 60% contribution when C/N = 0.7). Although organic supplementation weakened the dominant role of aNRB in denitrification, batch tests for the first time demonstrated that it could accelerate the SADN rate, attributed to the improvement of sulfur bioavailability, likely via the formation of polysulfide. A possible nitrogen removal pathway with multiple electron donors (i.e., sulfur, organics, sulfide, and polysulfide) in a sulfur-based denitrification reactor with organic supplementation was therefore proposed. However, supplementation with a high level of organics could increase the operational cost and effluent concentrations of sulfide and organics as well as enrich heterotrophic denitrifiers. Moreover, microbial community had substantial changes at C/N ratios of >0.5. Accordingly, an optimal C/N ratio of 0.25-0.5 was suggested, which could simultaneously minimize the additional operating cost associated with organic supplementation and maximize TN removal and SADN rates.

Ma Xing Shi Gan Decoction Attenuates PM2.5 Induced Lung Injury via Inhibiting HMGB1/TLR4/NFκB Signal Pathway in Rat.

Ma Xing Shi Gan Decoction (MXD), a classical traditional Chinese medicine prescription, is widely used for the treatment of upper respiratory tract infection. However, the effect of MXD against particulate matters with diameter of less than 2.5 µm (PM2.5) induced lung injury remains to be elucidated. In this study, rats were stimulated with PM2.5 to induce lung injury. MXD was given orally once daily for five days. Lung tissues were harvested to assess pathological changes and edema. Myeloperoxidase (MPO) activity and malonaldehyde (MDA) content in lung were determined to evaluate the degree of injury. To assess the barrier disruption, the bronchoalveolar lavage fluid (BALF) was collected to determine the total protein content and count the number of neutrophils and macrophages. For evaluating the activation of macrophage in lung tissue, CD68 was detected using immunohistochemistry (IHC). The levels of inflammatory factors including tumor necrosis factor-alpha (TNF-α), interleukin-1beta (IL-1ß), and interleukin-6 (IL-6) in BALF and serum were measured. In vitro, a PM2.5-activated RAW 264.7 macrophages inflammatory model was introduced. To evaluate the protective effect of MXD-medicated serum, the cell viability and the release of inflammatory factors were measured. The effects of MXD on the High mobility group box-1/Toll-like receptor 4/Nuclear factor-kappa B (HMGB1/TLR4/NFκB) pathway in lung tissue and RAW 264.7 cells were assessed by Western blot. For further confirming the protective effect of MXD was mediated by inhibiting the HMGB1/TLR4/NFκB pathway, RAW 264.7 cells were incubated with MXD-medicated serum alone or MXD-medicated serum plus recombinant HMGB1 (rHMGB1). MXD significantly ameliorated the lung injury in rats, as evidenced by decreases in the pathological score, lung edema, MPO activity, MDA content, CD68 positive macrophages number, disruption of alveolar capillary barrier and the levels of inflammatory factors. In vitro, MXD-medicated serum increased cell viability and inhibited the release of inflammatory cytokines. Furthermore, MXD treatment was found to inhibit HMGB1/TLR4/NFκB signal pathway both in vivo and in vitro. Moreover, the protection of MXD could be reversed by rHMGB1 in RAW 264.7. Taken together, these results suggest MXD protects rats from PM2.5 induced acute lung injury, possibly through the modulation of HMGB1/TLR4/NFκB pathway and inflammatory responses.

Research Progress of Mechanisms and Drug Therapy For Atherosclerosis on Toll-Like Receptor Pathway.

Recent reports have established atherosclerosis (AS) as a major factor in the pathogenetic process of cardiovascular diseases such as ischemic stroke and coronary heart disease. Although the possible pathogenesis of AS remains to be elucidated, a large number of investigations strongly suggest that the inhibition of toll-like receptors (TLRs) alleviates the severity of AS to some extent by suppressing vascular inflammation and the formation of atherosclerotic plaques. As pattern recognition receptors, TLRs occupy a vital position in innate immunity, mediating various signaling pathways in infective and sterile inflammation. This review summarizes the available data on the research progress of AS and the latest antiatherosclerotic drugs associated with TLR pathway.

Realizing a high-rate sulfidogenic reactor driven by sulfur-reducing bacteria with organic substrate dosage minimization and cost-effectiveness maximization.

Biological sulfur reduction is an attractive sulfidogenic technology for the treatment of organics-deficient metal-laden wastewater, because it theoretically reduces the electron donor consumption by 75%, compared to sulfate reduction. However, reducing the external organic substrate dosage may lower the sulfur reduction rate. Supplying with a more biodegradable organic substrate could possibly enhance sulfidogenic activity but also increase the chemical cost. Therefore, the sulfide production performance of a sulfur-reducing bioreactor feeding with varied levels of organic supply, and different types of organic substrates were investigated. The results showed that high-rate sulfide production (12.30 mg S/L/h) in a sulfur-reducing bioreactor can be achieved at the minimal dosage of organic substrate as low as 39 mg C/L of organic carbon in the influent. Changing the type of organic substrate posed a significant effect on the sulfidogenic activity in the sulfur-reducing bioreactor. Sodium acetate was found to be the optimal substrate to achieve the highest sulfide production rate (28.20 mg S/L/h) by sulfur-reducing bacteria (S0RB), followed by ethanol, methanol, glycerol, pyruvic acid, acetic acid, glucose, sucrose, malic acid, sodium formate, formic acid, N-propanol, N-butanol, lactic acid, sodium lactate, propionic acid and sodium propionate (2.87 mg S/L/h as the lowest rate). However, the cost-effectiveness analysis showed that glucose was the most cost-effective organic substrate to realize the sulfur reduction process in high sulfide production rate (20.13 mg S/L/h) and low chemical cost (5.94 kg S/$). The utilization pathway of the different organic substrates in the sulfur-reducing bioreactor was also discussed.

JLX001 Modulated the Inflammatory Reaction and Oxidative Stress in pMCAO Rats via Inhibiting the TLR2/4-NF-κB Signaling Pathway.

Inflammatory reactions and oxidative stress play critical roles in cerebral ischemic injuries. Microglia are activated after ischemic injury. Activated microglia produce neurotoxic proinflammatory factors and reactive oxygen species (ROS), which have been demonstrated closely related TLR2/4-NF-κB signal pathways. This study was to evaluate the effect of JLX001 against ischemic injury and investigate the mechanisms. The permanent middle cerebral artery occlusion (pMCAO) model was employed in rats. The neurobehavioral score, brain infarction rate, brain water content, pathological changes, immunohistochemical staining, biochemical index (T-AOC, SOD, and MDA), proinflammatory factors (IL-1ß, TNF-α, and NO), expression of TLR2/4 and nuclear translocation of NF-κB p65 were determined. To explore probable underlying mechanism of the neuroprotective effect of JLX001, BV-2 cells were exposed to in oxygen-glucose deprivation (OGD) for 4 h to mimic ischemic injury in vitro. The result showed that JLX001 significantly decreased neurological deficit score, infarct size, and brain edema, attenuated pathological changes, inhibited the activation of microglia, improved the process of oxidative stress, reduced the release of proinflammatory cytokines and downregulated TLR2/4-NF-κB signal pathway. Moreover, OGD reduced BV2 cell viability, induced oxidative damage, increased the release of proinflammatory factors and activated TLR2/4-NF-κB signal pathway, which was significantly reversed by the intervention of JLX001. This study demonstrates that JLX001 is effective in protecting the brain from ischemic injury, which may be mediated by regulating oxidative stress, inflammation and inhibiting TLR2/4-NFκB signal pathway.

Salvia miltiorrhiza Bunge (Danshen) extract attenuates permanent cerebral ischemia through inhibiting platelet activation in rats.

ETHNOPHARMACOLOGICAL RELEVANCE: Danshen is a crude herbal drug isolated from dried roots of Salvia miltiorrhiza Bunge. This plant is widely used in oriental medicine for the treatment of cardiovascular and cerebrovascular diseases. The supercritical CO2 extract from Danshen (SCED) (57.85%, 5.67% and 4.55% for tanshinone IIA, tanshinone I and cryptotanshinone respectively) was studied in this article, whose potential molecular mechanism remains unclear, especially in anti-thrombosis. AIM OF THE STUDY: The present study was designed to observe the protective effect of SCED on ischemic stroke in rats and to explore the underlying anti-thrombosis mechanism. MATERIALS AND METHODS: Following induction of cerebral ischemia in rats by permanent middle cerebral artery occlusion (pMCAO). Neurological defect score, cerebral blood flow, infarct size, and brain edema were measured to evaluate the injury. Arteriovenous shunt thrombosis model and adenosine 5'-diphosphate (ADP) induced acute pulmonary embolism model were conducted to estimate the antithrombotic effect of SCED. In order to investigate the effects of SCED on platelet aggregation, rat platelet-rich-plasma (PRP) were incubated with SCED prior to the addition of the stimuli (ADP or 9, 11-dideoxy-11α, 9α-epoxymethanoprostaglandin F2α (U46619)). Aggregation was monitored in a light transmission aggregometer. Inhibitory effect of SCED on thromboxane A2 (TXA2) release was detected by ELISA kit. Phospholipase C (PLC)/ Protein kinase C (PKC) signaling pathway was analyzed by a Western blot technique. The effect of the SCED was also studied in vivo on bleeding time in mice. RESULTS: SCED improved the neurological defect score, increased cerebral blood flow, reduced infarct size and alleviated brain edema in rats exposed to pMCAO. After administration of SCED, thrombosis formation in arteriovenous shunt was inhibited and recovery time in pulmonary embolism was shortened. The inhibitory effect of SCED on platelet activation was further confirmed by TXB2 ELISA kit and Western blot analysis of PLC/PKC signaling pathway. CONCLUSIONS: SCED attenuates cerebral ischemic injury. The possible mechanism is that SCED inhibits thrombosis formation, platelet aggregation and activation of PLC/PKC pathway. On this basis, this new extract could be a promising agent to inhibit thrombosis formation and protect against cerebral ischemia injury.