The Toll-like Receptor-2/4 Antagonist, Sparstolonin B, and Inflammatory Diseases: A Literature Mining and Network Analysis.

BACKGROUND: Sparstolonin B (SsnB) is characterized as a new toll-like receptor (TLR)-2/4 antagonist. However, the effects of SsnB on different inflammatory diseases have not been systemically reviewed. METHODS: We investigated the effects of SsnB on inflammatory diseases with data mining and network analysis of literature, including frequency description, cluster analysis, association rule mining, functional enrichment, and protein-protein interaction (PPI) mining. RESULTS: A total of 27 experimental reports were included. The ARRIVE 2.0 guidelines were used to evaluate the quality of animal studies. Frequency analysis revealed 13 different diseases (cardio-cerebrovascular system diseases account for 23.53%), 12 pharmacological effects (anti-inflammatory effect accounts for 53.85%), and 67 therapeutic targets. The overview of investigation sequence of SsnB studies was depicted by Sankey diagram. Cluster analysis classified the therapeutic targets for SsnB into four main categories: (1) NF-κB; (2) IL-1ß, IL-6, and TNF-α; (3) TLR2, TLR4, and MyD88; (4) the other targets. Moreover, the Apriori association discovered two main association pairs: (1) TNF-α, IL-1ß, and IL-6 and (2) TLR2, TLR4, and MyD88 (support range 33.33-50%, confidence range 83.33-88.89%). Functional enrichment of the therapeutic targets for SsnB showed that the top enriched items in the biological process were mainly the response to lipopolysaccharide (LPS)/bacterial origin and regulation of cytokine production. Finally, the PPI network and hub gene selection by maximal clique centrality (MCC) algorithm indicated the top ranked proteins were TNF-α, IL-1ß, IL-6, AKT1, PPAR-γ, TLR4, CCL2, and TLR2. CONCLUSION: These results emphasized the importance of TLR2/TLR4-MyD88-NF-κB-IL-1ß/IL-6/TNF-α pathways as therapeutic targets of SsnB in inflammatory diseases.

Sparstolonin B inhibits pancreatic adenocarcinoma through the NF-κB signaling pathway.

Pancreatic adenocarcinoma is a highly lethal malignant gastrointestinal tumor. Sparstolonin B is an isocoumarin whose anticancer activity has recently received increasing attention. This study aimed to investigate Sparstolonin B's potential antitumor effect on pancreatic adenocarcinoma. The effect of Sparstolonin B on pancreatic cancer target genes and molecular mechanism was predicted via network pharmacology; Sparstolonin B significantly decreased Panc-1 and SW1990 cell viability and effectively suppressed the proliferation, migration, and invasion of pancreatic cancer cells as shown by CCK-8, colony formation, and Transwell assays. Flow cytometry showed that it induced cell cycle arrest and apoptosis. Sparstolonin B also upregulated Bax levels but decreased those of MMP2 and Bcl-2, downregulated IκBα expression, and upregulated p65 and IκBα phosphorylation; however, it had no effect on total NF-κB p65 levels. The NF-κB pathway inhibitor QNZ reversed these effects. The treatment group (26 µmol/L) had reduced graft volume and weight and fewer Ki-67-positive cells than the control group. Therefore, Sparstolonin B can inhibit the growth and induce the apoptosis of pancreatic cancer cells via the NF-κB signaling pathway and may be a potential novel drug for pancreatic cancer treatment.

Sparstolonin B suppresses free fatty acid palmitate-induced chondrocyte inflammation and mitigates post-traumatic arthritis in obese mice.

Abnormal lipid metabolism, such as systemic increased free fatty acid, results in overproduction of pro-inflammatory enzymes and cytokines, which is crucial in the development of obesity-related osteoarthritis (OA). However, there are only a few drugs that target the lipotoxicity of OA. Recent researches have documented that the traditional Chinese medicine, Sparstolonin B (Ssn B), exerted anti-inflammatory effects in various diseases, but not yet in OA. On the basis of this evidence, our works purposed to evaluate the effect of Ssn B on free fatty acid (FFA) palmitate (PA)-stimulated human osteoarthritic chondrocytes and obesity-associated mouse OA model. We found that Ssn B suppressed PA-triggered inflammatory response and extracellular matrix catabolism in a concentration-dependent approach. In vivo, Ssn B treatment inhibited cartilage degeneration and subchondral bone calcification caused by joint mechanical imbalance and alleviated metabolic inflammation in obesity. Mechanistically, co-immunoprecipitine and molecular docking analysis showed that the formation of toll-like receptor 4 (TLR4)/myeloid differentiation protein-2 (MD-2) complex caused by PA was blocked by Ssn B. Subsequently, it leads to inactivation of PA-caused myeloid differentiation factor 88 (MyD88)-dependent nuclear factor-kappaB (NF-κB) cascade. Together, these findings demonstrated that Ssn B is a potential treatment agent for joint degenerative diseases in obese individuals.

Anti-Inflammatory Effect of Sparstolonin B through Inhibiting Expression of NF-κB and STAT-1.

Sparstolonin B (SsnB), which is found in Sparganium stoloniferum, prevents the synthesis of inflammatory mediators and is related to functional pathways of survival. In this study, we assessed the possible protective functions of SsnB on lipopolysaccharide (LPS)-induced inflammatory responses. We determined the functions of SsnB on controlling heme oxygenase (HO)-1, cyclooxygenase (COX-)2, and inducible nitric oxide synthase (iNOS) in LPS-activated human umbilical vein endothelial cells (HUVECs). Furthermore, the distinct function of SsnB on the expression of iNOS and well-known pro-inflammatory mediators, such as tumor necrosis factor (TNF)-α and interleukin (IL)-1ß, were assessed in the pulmonary histological status of LPS-injected mice. SsnB upregulated the HO-1 production, inhibited luciferase-NF-κB interaction, and lowered COX-2/PGE2 and iNOS/NO, which lead to the reduction of STAT-1 phosphorylation. Moreover, SsnB enhanced the nuclear translocation of Nrf2, elevated the binding activity between Nrf2 and antioxidant response elements (AREs), and weakened IL-1ß expression on LPS-treated HUVECs. SsnB-suppressed iNOS/NO synthesis was restored by the process of the RNAi inhibition of HO-1. In experiment with an LPS-injected animal model, SsnB remarkably decreased the iNOS expression in the pulmonary biostructure and TNF-α level in the bronchoalveolar lavage fluid (BALF). Therefore, these results demonstrate that SsnB is responsible for inflammation ameliorative activity by controlling iNOS through inhibition of both NF-κB expression and p-STAT-1. Therefore, SsnB could be a candidate for promoting novel clinical substances to remedy pathologic inflammation.

Sparstolonin B exerts beneficial effects on prostate cancer by acting on the reactive oxygen species-mediated PI3K/AKT pathway.

Prostate cancer is a major health concern in males worldwide, owing to its high incidence. Sparstolonin B (SsnB), a component of the Chinese herbal medicine Sparganium stoloniferum, is used to treat many diseases. However, the effects and mechanisms of action of SsnB in prostate cancer have not yet been reported. In this study, we evaluated the effects of SsnB on cellular processes and tumour growth. In particular, we verified that SsnB could inhibit the proliferation, migration and invasion of prostate cancer cells and induce apoptosis by activating G2/M phase arrest in vitro based on a series of cytological experiments. In vivo, we found that SsnB could inhibit tumour growth in nude mouse xenograft models. We further confirmed that SsnB could repress the PI3K/AKT pathway by increasing reactive oxygen species (ROS) accumulation and oxidative stress. Collectively, SsnB inhibits tumour growth and induces apoptosis in prostate cancer via the suppression of the ROS-mediated PI3K/AKT pathway and may be a new alternative to adjuvant therapy for prostate cancer.

Protective effects of Sparstolonin B, a selective TLR2 and TLR4 antagonist, on mouse endotoxin shock.

Sepsis is characterized by an overwhelming systemic inflammation and multiple organ injury. Toll-like receptors (TLRs) 2 and 4 mediate these inflammatory responses. Sparstolonin B (SsnB), isolated from Chinese herb Scirpus yagara, is a new selective TLR2/4 antagonist. Herein, we report that SsnB inhibited the expression of various inflammatory mediators such as tumor necrosis factor (TNF-α), interleukin (IL)-1ß, IL-6, and chemokine (C-C motif) ligand 2 (CCL-2) in lipopolysaccharide (LPS)- or Pam3csk4-stimulated macrophages. Moreover, in LPS-stimulated macrophages, the downregulation of peroxisome proliferator-activated receptor γ (PPAR-γ) was reversed by SsnB dose-dependently; and SsnB had synergistic inhibitory effects with rosiglitazone, a PPAR-γ agonist, on TNF-α and IL-6 expression in LPS-stimulated macrophages. The effects of SsnB were further evaluated in a mouse endotoxin shock model. When intraperitoneal injected in mice 2 days before or 1-2h after LPS challenge, SsnB attenuated the body temperature reduction and decreased the mortality. SsnB pre-treatment significantly suppressed LPS-induced increase of TNF-α and IL-6 in serum, lungs and livers, and substantially attenuated lung dysfunction in mice. In vivo toxicity test showed that at doses as high as 500 mg/kg, SsnB did not cause death of mice. These results suggest that SsnB protects mice against endotoxin shock by inhibiting production of multiple cytokines and lung dysfunction. In conclusion, our findings indicate that SsnB may be used in the prevention and treatment of endotoxin shock.

Determination of sparstolonin B by ultra-high performance liquid chromatography coupled with triple quadrupole mass spectrometry: application to pharmacokinetic study of sparstolonin B in rat plasma.

Sparstolonin B (SsnB), a spontaneous isocoumarin compound isolated from the tuber of Scirpus yagara Ohwi. (Cyperaceae), possesses potent anti-inflammatory and antitumor activity. In the present study, a rapid and simple UHPLC/MS/MS method for determination of SsnB in rat plasma was developed and validated. Plasma samples were pretreated by liquid-liquid extraction with ethyl acetate containing rhein as an internal standard and separated on a C18 column at 35 °C, with a gradient mobile phase consisting of acetonitrile and water containing 0.2% (v/v) formic acid within 2.1 min. MS/MS detection was accomplished in multiple reaction monitoring mode with negative electrospray ionization. The precursor-product ion transitions were m/z 266.9 [M-H](-) → m/z 211.0 for SsnB and m/z 283.2 [M-H](-) → m/z 239.0 for IS. The intra- and inter-day precision (RSD) was <8.98% and the accuracy (RE) ranged from -7.40 to 4.50%. The extraction recoveries ranged from 96.28 to 97.30%. The pharmacokinetic parameters were calculated using Win Nonlin53 software. The absolute bioavailability of SsnB was estimated to be 6.98%. The proposed method was successfully applied to a pharmacokinetic study of SsnB in rats after intravenous administration with a dose of 0.5 mg/kg and oral administration at a dose of 5 mg/kg.

Inhibition of factor Xa activity, platelet aggregation, and experimentally induced thrombosis by Sparstolonin B.

BACKGROUND: Sparstolonin B (SsnB) is an isocumarin compound extracted from medicinal plants such as Sparganium stoloniferum and Scirpus yagara with well documented anti-inflammatory activity. Here we examined if SsnB also possesses antithrombotic activity and the underlying mechanisms. METHODS: Anti-thrombotic effects of SsnB were determined by measuring in vitro/ex vivo/in vivo clotting times, platelet aggregation assay, production and activity of factor Xa, nitric oxide, and expressions of relative proteins. RESULTS: Treatment with SsnB prolonged the clotting time of human platelet-poor serum at concentrations comparable to the clinical anticoagulant rivaroxaban (as a positive control) and inhibited human platelet aggregation induced by adenosine diphosphate (ADP) or the thromboxane A2 analog U46619. SsnB also inhibited U46619-induced and ADP-induced phosphorylation of phospholipase C (PLC)γ2/protein kinase C (PKC) and intracellular calcium mobilization, both of which are required for platelet aggregation. In addition, SsnB inhibited expression of the cell adhesion factors P-selectin and PAC-1. SsnB increased production of the vasodilator nitric oxide and suppressed secretion of the vasoconstrictor endothelin-1 from ADP- or U46619-treated human umbilical vein endothelial cells. Further, SsnB reduced coagulation factor Xa (FXa) catalytic activity and production by endothelial cells as well as FXa-induced platelet aggregation. CONCLUSION: Finally, SsnB injection reduced thrombus formation time, number, size, and related mortality in mouse models of thromboembolism. SsnB is a promising antithrombotic agent targeting both FXa and platelet aggregation pathways, which can overcome the side effects of existing antithrombotic agents.

Abrogation of LRRK2 dependent Rab10 phosphorylation with TLR4 activation and alterations in evoked cytokine release in immune cells.

LRRK2 protein is expressed prominently in immune cells, cell types whose contribution to LRRK2-associated genetic Parkinson's disease (PD) is increasingly being recognised. We investigated the effect of inflammatory stimuli using RAW264.7 murine macrophage cells as model systems. A detailed time course of TLR2 and TLR4 stimulation was investigated through measuring LRRK2 phosphorylation at its specific phospho-sites, and Rab8 and Rab10 phosphorylation together with cytokine release following treatment with LPS and zymosan. LRRK2 phosphorylation at Ser935, Ser955 and Ser973 was increased significantly over untreated conditions at 4-24h in both WT-LRRK2 and T1348N-LRRK2 cell lines to similar extents although levels of Ser910 phosphorylation were maintained at higher levels throughout. Importantly we demonstrate that LPS stimulation significantly decreased phospho-Rab10 but not phospho-Rab8 levels over 4-24h in both WT-LRRK2 and T1348N-LRRK2 cell lines. The dephosphorylation of Rab10 was not attributed to its specific phosphatase, PPM1H as the levels remained unaltered with LPS treatment. MAPK phosphorylation occurred prior to LRRK2 phosphorylation which was validated by blocking TLR4 and TLR2 receptors with TAK242 or Sparstolonin B respectively. A significant decrease in basal level of TNFα release was noted in both T1348N-LRRK2 and KO-LRRK2 cell lines at 48h compared to WT-LRRK2 cell line, however LPS and zymosan treatment did not cause any significant alteration in the TNFα and IL-6 release between the three cell lines. In contrast, LPS and zymosan caused significantly lower IL-10 release in T1348N-LRRK2 and KO-LRRK2 cell lines. A significant decrease in phospho-Rab10 levels was also confirmed in human IPS-derived macrophages with TLR4 activation. Our data demonstrates for the first time that LRRK2-dependent Rab10 phosphorylation is modulated by LPS stimulation, and that cytokine release may be influenced by the status of LRRK2. These data provide further insights into the function of LRRK2 in immune response, and has relevance for understanding cellular dysfunctions when developing LRRK2-based inhibitors for clinical treatment.

Sparstolonin B improves neurological outcomes following intracerebral hemorrhage in mice.

Inflammation serves an important role in inducing secondary injury following intracerebral hemorrhage (ICH). It has been demonstrated that sparstolonin B (SsnB) is able to attenuate the lipopolysaccharide-induced inflammatory response in sepsis. Mouse ICH models were used to explore the efficacy of SsnB on the ICH-induced inflammatory response. Mice underwent a working memory version of Morris water maze (MWM) test. They underwent 5 successive days of training consisting of 4 trials each day. The ICH model was established on the last training day. Mice were injected intraperitoneally either with vehicle or SsnB once a day for 3 consecutive days following the establishment of the ICH model. The MWM was used to determine the effect of SsnB on short-term memory following ICH. Neurological deficit scores and brain water content were measured following the MWM. Furthermore, the expression of inflammatory factors and signaling molecules downstream of TLR4 were measured. The results demonstrated that 5 mg/kg SsnB significantly improved the MWM path and time latency (P<0.05). Furthermore, neurological deficit scores were decreased in SsnB-treated mice compared with vehicle-treated mice (P<0.01). Brain water content, levels of inflammatory cytokines and the expression of inflammation-associated proteins were also significantly reduced in the SsnB-treated group (P<0.05). These results indicate that SsnB treatment stimulates short-term neurobehavioral recovery and reduces neurological deficits and this may inhibit the inflammatory response. Therefore, SsnB may attenuate the inflammatory response following ICH.

Sparstolonin B attenuates spinal cord injury­induced inflammation in rats by modulating TLR4­trafficking.

The present study used a spinal cord injury (SCI) model to evaluate whether sparstolonin B was able to prevent SCI, and to investigate the underlying signaling mechanism. Sparstolonin B attenuated the SCI­induced Batto, Beattie and Bresnahan score and water content in rats. Sparstolonin B attenuated the mRNA expression of proinflammatory cytokines interleukin (IL)­18, IL­6, IL­1ß, and IL­23, decreased the levels of tumor necrosis factor­α and interferon­Î³, and decreased caspase­3 activity and apoptosis regulator Bax protein expression in SCI rats. Similarly, sparstolonin B inhibited monocyte chemoattractant protein­1 mRNA levels, and Toll­like receptor (TLR) 4, myeloid differentiation primary response protein MyD88 (MyD88) and nuclear factor (NF)­κB protein levels in SCI rats. The present results suggested that sparstolonin B may attenuate SCI­induced inflammation and apoptosis in rats by modulating the TLR4/MyD88/NF­κB signaling pathway.

Sparstolonin B suppresses rat vascular smooth muscle cell proliferation, migration, inflammatory response and lipid accumulation.

Vascular smooth muscle cells (VSMCs) play a crucial role in atherosclerotic lesion formation. Sparstolonin B (SsnB) is a TLR2/TLR4 antagonist that inhibits inflammatory responses in multiple cell types. Herein, we investigated if SsnB inhibited VSMC proliferation, migration, inflammatory response and lipid accumulation. We found that SsnB suppressed VSMC proliferation and migration induced by PDGF. SsnB significantly suppressed the expression of MCP-1, TNFα and IL-6 in VSMCs stimulated by either lipopolysaccharide (LPS) or PDGF. Erk1/2 and Akt signaling pathways, which are responsible for the VSMC inflammatory response, were activated by LPS or PDGF stimulation, and SsnB significantly inhibited their activation. SsnB also substantially suppressed the intracellular cholesterol accumulation in VSMCs loaded with acetylated LDL. Mechanistically, SsnB remarkably repressed LPS-induced up-regulation of CD36, which is responsible for lipid uptake, and dramatically reversed LPS-induced inhibition of ABCA1, which promotes the efflux of intracellular free cholesterol. In conclusion, our results indicate that SsnB significantly inhibits VSMC proliferation, migration, inflammatory responses and lipid accumulation. Along with the previously reported anti-inflammatory activities of SsnB on macrophages and vascular endothelial cells, our data strongly suggest that SsnB may be developed as a new anti-atherogenic therapy.

Sparstolonin B inhibits lipopolysaccharide-induced inflammation in 3T3-L1 adipocytes.

Sparstolonin B (SsnB), an isocoumarin compound isolated from the tubers of both Sparganium stoloniferum and Scirpus yagara, has been reported to have anti-inflammatory effects. However, whether SsnB has anti-inflammatory effects on LPS-stimulated 3T3-L1 adipocytes remains unclear. In this study, we investigated the effects of SsnB on adipocyte inflammation in 3T3-L1 adipocytes and anti-obesity properties in high fat diet (HFD)-induced obese rats. 3T3-L1 adipocytes were pretreated with SsnB 1h before LPS treatment. The expression of MCP-1, IL-6, TNF-α, and IL-10 were measured by qRT-PCR and ELISA. The expression of PPAR-γ, TLR4 and NF-κB were detected by western blotting. SsnB was administered to HFD-induced obese rats to confirm its effects in vivo. Our results showed that SsnB dose-dependently inhibited LPS-induced MCP-1, IL-6, and TNF-α production. SsnB was found to inhibit LPS-induced TLR4 expression and NF-κB activition. Furthermore, SsnB was found to activate PPAR-γ and the inhibitory effects of SsnB on MCP-1, IL-6, and TNF-α production can be reversed by PPAR-γ antagonist GW9662. In vivo, SsnB was found to reduce the body weight of rats fed with HFD. SsnB also inhibited the levels of serum triglyceride (TG) and cholesterol (TC) induced by HFD. In conclusion, the results suggested that SsnB could reduce HFD-induced obesity in rats and inhibited LPS-induced cytokines production in 3T3-L1 adipocytes by activating PPAR-γ.

Sparstolonin B attenuates hypoxia-reoxygenation-induced cardiomyocyte inflammation.

Myocardial ischemia-reperfusion (MIR) injury is characterized by a rapid increase in cytokines and chemokines and an infiltration of inflammatory cells. Toll-like receptors (TLRs) 2 and 4 mediate these inflammatory responses. Herein we investigated the ability of Sparstolonin B (SsnB), a new selective TLR2/4 antagonist, to inhibit the TLR2/4-mediated inflammatory responses during cardiomyocyte hypoxia-reoxygenation injury as well as the responsible mechanisms. Lactate dehydrogenase (LDH) assay was performed to measure the cytotoxicity of SsnB on H9c2 cardiomyocytes. Quantitative real-time PCR (qRT-PCR) confirmed that TLR2 and TLR4 expression was elevated during hypoxia-reoxygenation, and that their up-regulation in cardiomyocytes was significantly inhibited by SsnB (P < 0.05). Both the mRNA and protein levels of monocyte chemotactic protein-1 and high mobility group box 1 were up-regulated during hypoxia-reoxygenation and were significantly attenuated by SsnB (P < 0.05). Next we found that extracellular signal-regulated kinase 1 or 2 (ERK1/2) and c-Jun NH2-terminal kinase (JNK) signaling pathways were activated during hypoxia-reoxygenation and SsnB significantly inhibited their activation (P < 0.05). Moreover, transwell migration assays revealed that the migration of mouse macrophages to hypoxia-reoxygenation injured cardiomyocytes was significantly reduced by SsnB (P < 0.05). In conclusion, our data indicate that the new selective TLR2 and TLR4 antagonist, SsnB, can substantially attenuate hypoxia-reoxygenation-induced inflammation of cardiomyocytes via inhibiting ERK1/2 and JNK signaling pathways. Accordingly, SsnB has the potential to serve as a therapeutic agent for the prevention of MIR injury.

Protective effects of Sparstolonin B on mouse with intracerebral haemorrhage / 中国药学杂志

OBJECTIVE: To study the protective effects of sparstolonin B(8,5'-dihydroxy-4-phenyl-5,2'-oxidoisocoumarin, SsnB) on mouse with intracerebral haemorrhage (ICH). METHODS: The effect of SsnB on neuronal cell death induced by Hb using a microglia-neuron transwell system was investigated. Autologous nonanticoagulated blood was collected from the tail artery of the mouse and then injected into the striatum using a syringe pump. Neurological deficit of the mouse with ICH was assessed 1,3,5 and 7 d after SsnB administration using modified neurological severity score system, and brain water content of the mouse cerebral tissues after ICH was measured at the same day. The concentrations of pro-inflammatory cytokines in perihematoma tissues, including interleukin-6, interleukin-1 p and tumor necrosis factor-α, were measured by ELISA assay. RESULTS: The results show that SsnB significantly reduced the neurological deficit scores and the brain water content, and inhibited the levels of IL-6, IL-1β and TNF-α at first day and third day after ICH. CONCLUSION: SsnB can improve the brain injury in mouse induced by ICH. The mechanism may involve increased the neuronal survival rate and decreased expression of pro-inflammatory cytokines of mouse after ICH.