Wu-tou Decoction Research Articles

The Chinese Medicine Wu-Tou Decoction Relieves Neuropathic Pain by Inhibiting Hippocampal Microglia Activation

The comorbidity between the nociceptive and mental syndromes adds to the refractoriness of neuropathic pain (NP). Wu-Tou decoction (WTD) has been prescribed for chronic pain for thousands of years in China. Recently, we reported that WTD was helpful for hippocampus and co-curative for the nociceptive, depressive and anxiety behaviors in the spinal cord ligation (SNL) mice. However, the mechanism underlying the rescue of hippocampus, as well as the roles hippocampus assumed in co-curation remain unexplored. In this study, we validated that in SNL mice, the long-lasting damages to limbic system were mainly limited to hippocampus. In addition, hippocampal neurons were proven sensitive to harms induced by microglia and rescued by WTD, which in sum indicated hippocampal microglia as the critical modulator of co-curation. To validate this hypothesis the hippocampal microglia were mal-activated in shamed mice, in which the atrophy of hippocampus and the development of NP syndromes were consolidated and proven rescued by WTD. On the contrary, in the SNL mice, the failure to control hippocampal microglia was sufficient to void all the rescues mediated by WTD. In sum, our study points out that the effective modulation of microglia in hippocampus is of pivotal importance for the co-curation by WTD.

Optimizing Personalized Therapy for Rheumatoid Arthritis Using a Novel Drug Combination of Representative Bioactive Compounds of a Classic Chinese Herbal Formula

Background: Personalized therapy of rheumatoid arthritis (RA) based on traditional Chinese medicine cold and hot syndromes is extremely essential for optimizing therapeutic efficacy. Wutou Decoction (WTD) achieves favorable therapeutic response in treating RA-cold syndrome. However, its material basis and potential mechanisms remain unclear. Herein, we aimed to identify the representative bioactive compounds (BACs) of WTD against RA-cold syndrome. Methods: Microarray analysis was performed to screen RA-cold and RA-hot-syndrome-related genes, and WTD effect genes. Representative BACs and candidate targets of WTD were identified by integrating network analysis, chemical profiling, absorption-distribution-metabolism-excretion evaluation and molecular docking simulation. Both in vivo and in vitro experiments were performed to evaluate therapeutic effects of the BAC-combination against RA-cold syndrome. Findings: ALOX15B-PPAR-γ-PTGS2-FGF2-IL-1β-c-JUN-MMP13-TGF-β1 signal axis, mainly involved into regulating thermogenesis and energy metabolism, as well as maintaining the balance of inflammation-immune system, was demonstrated to be a candidate network target of WTD against RA-cold syndrome. The four-BAC-combination, including Beiwutine/10-OHmesaconitine, Talatizidine, Paeoniflorin and Glabrene, regulating the signal axis, were formed to be a potential drug for treating RA-cold syndrome. Further experiments demonstrated that this combination efficiently reversed the pathological changes of RA-cold syndrome by regulating the ALOX15B-PPAR-γ-PTGS2-FGF2-IL-1β-c-JUN-MMP13-TGF-β1 signal axis. Interpretation: This study identified the novel four-BAC-combination, which may represent the therapeutic efficacy and syndrome differentiation-based treatment characteristics of WTD against RA. The findings shed light into the modernization of TCM formulae and promoted new drug discovery. Funding Statement: National Natural Science Foundation of China (81630107 & 81673834), and Fundamental Research Funds for the Central public welfare research institutes (L2017018). Declaration of Interests: The authors declare that there is no conflict of interests regarding the publication of this paper. Ethics Approval Statement: All procedures in the current study were performed in accordance with the ethical standards of the Center for Laboratory Animal Care, China Academy of Chinese Medical Sciences.

Effect of Wutou decoction on depression and anxiety induced by neuropathic pain mice

Objective To observe the effects of Wutou decoction (WTD) on Anxiety and Depression induced by the L5 spinal nerve ligation (SNL) mice. Methods The ICR male mice were randomly divided into sham, SNL, high-dose, medium-dose and low-dose group of Wutou decoction, and Pregabalin group, with 24 in each group. Except the sham group, SNL models were prepared in the remaining groups of mice. After successful modeling, the high, medium, and low dosages were administered with 12.60, 6.30, 3.15 g/kg of Wutou decoction, and pregabalin group was administered with 0.25 g/kg of pregabalin, and the sham group and the model group was given an equal volume of normal saline. All the administration was once per day, continuously last for 21 days. On the 7/14/21th days after the operation, the depressive and eanxiety behavior were evaluated by the tests of the sugar water consumption, the open field and forced swimming detection. In addition, the number of the positive cells expressing the extracellular signal-regulated kinase (p-ERK) in hippocampus were analyzed on Day 7/14/21 in each group by Immunohistochemistry and the expression of both p-ERK and postsynaptic density protein 95 (PSD95) in the hippocampus were analyzed on Day 21 by Western blot. Results At 7, 14, and 21th day after administration, compared with the model group, the paw withdrawal mechanical threshold of mice (0.76 ± 0.21 vs. 0.05 ± 0.03), (0.93 ± 0.33 vs. 0.05 ± 0.01), (1.12 ± 0.20 vs. 0.04 ± 0.01) in the high dose group of Wutou decoction respectively increased (P<0.01), and sugar consumption (1.86 ± 0.44 g vs. 0.84± 0.23 g), (1.84 ± 0.10 g vs. 1.02 ± 0.18 g), (1.63 ± 0.31 g vs. 0.75 ± 0.23 g) significantly increased (P<0.01), and immobility time 4 min after swimming (128.90 ± 35.27 s vs. 180.30 ± 21.81 s), (125.50 ± 23.07 s vs. 195.30 ± 27.70 s), (169.50 ± 23.07 s vs. 207.50 ± 7.46 s) significantly decreased (P<0.01), and the retention time in the central area of the open field (35.35 ± 7.61 s vs. 13.90 ± 2.27 s), (26.26 ± 3.61 s vs.13.08 ± 1.98 s), (24.04 ± 6.57 s vs. 10.62 ± 3.38 s) significantly increased (P<0.01). After 21 days, the number of p-ERK positive cells in the high-dose group of Wutou decoction (11.00 ± 4.89 vs. 33.67 ± 7.35 ) was siginificantly lower than that of the model group, and the hippocampus p-ERK protein (0.15 ± 0.09 vs. 0.54 ± 0.04) siginificantly decreased, and the expression of PSD95 protein (0.32 ± 0.04 vs. 0.57 ± 0.09) siginificantly decreased (P<0.01). Conclusions The Wutou decoction achieved significant anti-depressant and anti-anxiety effects, and regulated p-ERK expression levels of hippocampus in the late stage of NP. Key words: Wu Tou Tang; Pain; Depression; Anxiety; Extracellular signal-regulated kinase; Postsynaptic density protein 95; Mice

Wu-Tou Decoction Inhibits Angiogenesis in Experimental Arthritis by Targeting VEGFR2 Signaling Pathway.

Wu-tou decoction (WTD) is a classic traditional Chinese medicine formula and has been extensively used for the treatment of rheumatoid arthritis (RA). Previous reports indicate that WTD possesses anti-inflammatory and antinociceptive activities, and inhibits the development of arthritic joints and disease severity of collagen-induced arthritis (CIA) or adjuvant-induced rats; however, its action on angiogenesis of RA has not been clarified. This study aims to determine the anti-angiogenic activity of WTD in CIA rats and in various angiogenesis models. Our data showed that WTD (0.95, 1.9, and 3.8 g/kg) markedly reduced the immature blood vessels in synovial membrane tissues of inflamed joints from CIA rats. It also inhibited in vivo angiogenesis in chick embryo and VEGF165-induced microvessel sprout formation ex vivo. Meanwhile, WTD suppressed VEGF165-/MH7A-induced migration, invasion, adhesion, and tube formation of human umbilical vein endothelial cells (HUVECs). Moreover, WTD significantly reduced the expression of angiogenic activators, including vascular endothelial growth factor (VEGF), VEGFR2, interleukin (IL)-1β, IL-17, transforming growth factor-β, platelet-derived growth factor, placenta growth factor, angiopoietin (Ang) I and Ang II in synovium of CIA rats, and/or in HUVECs. More interestingly, WTD blocked the autophosphorylation of VEGF165-induced VEGFR2 and consequently downregulated the signaling pathways of activated AKT, ERK1/2, JNK, and p38 in VEGF165-induced HUVECs. These findings suggest for the first time that WTD possesses the anti-angiogenic effect in RA in vivo, ex vivo, and in vitro by interrupting the targeting of VEGFR2 activation.

Wu-tou decoction attenuates neuropathic pain via suppressing spinal astrocytic IL-1R1/TRAF6/JNK signaling.

Neuropathic pain (NP) caused by nerve injuries continues to be an intractable challenge due to inadequate therapeutic strategies. Recent study demonstrated glia-induced neuro-inflammation in the spinal cord, especially the activation of astrocytes, plays an essential role in the development of NP, which opens new avenues for NP treatment. In this study, we explored the anti-hyperalgesia properties of Wu-tou decoction (WTD) and showed that WTD potently attenuates mechanical allodynia and heat hyperalgesia in lumbar 5 (L5) spinal nerve ligation (SNL)-induced NP without noticeable side effect or affecting basal pain perception of mice. Mechanistically, initial targets screening tests indicated WTD's analgesic action may be centrally mediated within the spinal cord, which further verified by its inhibitory actions on glia-releasing factors of IL-1β, CCL2 and CXCL1. Meanwhile, WTD significantly reduced spinal IL-1R1, TRAF6 expressions, p-JNK levels, and number of GFAP/IL-1R1, GFAP/TRAF6, GFAP/p-JNK positive astrocytes in the superficial lamina of spinal cord. Additionally, co-administration of IL-1Ra increased the anti-hyperalgesia effects of WTD and further decreased CCL2 and CXCL1 expressions, while no synergistic effects were detected when TRAF6 or JNK inhibitors were co-administrated with WTD. Thus, our data suggested that the effective inhibition of spinal astrocytic IL-1R1/TRAF6/JNK signaling (especially IL-1R1) contributes, at least in part, to WTD's anti-hyperalgesia action. It also indicates that WTD might be a promising candidate for the treatments of chronic pain, especially under NP-related neurological disorders.

Evidence that CA3 is Underling the Comorbidity Between Pain and Depression and the Co-curation by Wu-Tou decoction in Neuropathic Pain

In neuropathic pain (NP), the atrophy of hippocampus contributes to the comorbidity between pain, depression and the cognitive deficits. However, the exact mechanism underling the comorbidity, the effective control of the degenerations in hippocampus and the remission of the accompanied depressive symptoms are still lacking. Wu-Tou decoction (WTD) has been prescribed for inflammatory pain for thousands of years. In this study, we manifested the effects of WTD on the pain, depression and anxiety co-curative symptoms of NP. Moreover, we reported that WTD rescued the mal-regulated BDNF and TNF-α in hippocampal CA3 alone, which is proven contributing to the pain and induced psychiatric symptoms. Finally, analysis of biochemistry, morphology and electrophysiology exhibited the potential mechanism of WTD in CA3. We found that, in the late stage of SNL condition, WTD mediated the rescue of the down-regulated glutamate as well as its pre-synaptic vesicular glutamate transporters (VGLuT1) and the post-synaptic α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors in CA3. In sum, the targeted mediation of glutamatergic system in CA3 suggest that WTD may be responsible for the remission of the hypo-functioned CA3 glutamatergic neurons and further contribute to the co-curative effects of WTD.

Molecular mechanisms of the analgesic action of Wu-tou Decoction on neuropathic pain in mice revealed using microarray and network analysis.

Wu-tou Decoction (WTD) is a classic herbal formula in traditional Chinese medicine for the treatment of joint diseases, neuropathic pain (NP) and inflammatory pain. In this study we investigated whether WTD produced analgesic action in a mouse spinal nerve ligation (SNL) model and elucidated the underlying molecular mechanisms. Mice were subjected to SNL and orally treated with WTD (3.15, 6.30 or 12.60 g·kg-1·d-1) for 21 d. SNL induced mechanical hyperalgesia and heat hyperalgesia characterized by rapid and persistent pain hypersensitivity. In addition, the expression levels of IL-1β, TNF-α, CCL2 and CXCL1 in the spinal cord dorsal horn were dramatically increased on the 10th d post-surgery. Oral administration of WTD dose-dependently suppressed both mechanical and heat hyperalgesia as well as the expression levels of inflammatory cytokines in the spinal cord dorsal horn on the 21st d post-surgery. Then whole-genome microarray analyses were conducted to detect the gene expression profiles of spinal cord dorsal horn in SNL mice with or without WTD treatment. After construction of the WTD-SNL-network and topological analysis, a list of candidate target genes of WTD acting on SNL-induced NP was identified and found to be functionally enriched in several glial cell activation-related pathways and neuroinflammatory pathways. Our data have clarified the gene expression patterns in the mouse spinal cord under the NP condition. We also demonstrate the analgesic action of WTD through suppression of glial cell activation and neuroinflammation, which suggest the potential of WTD as a promising candidate for the treatment of NP.

Wu-Tou Decoction in Rheumatoid Arthritis: Integrating Network Pharmacology and In Vivo Pharmacological Evaluation.

Purpose: This study aimed to explore underlying action mechanism of Wu-Tou decoction (WTD) in rheumatoid arthritis (RA) through network pharmacology prediction and experimental verification.Methods: Chemical compounds and human target proteins of WTD as well as RA-related human genes were obtained from TCM Database @ Taiwan, PubChem and GenBank, respectively. Subsequently, molecular networks and canonical pathways presumably involved in the treatment of WTD on RA were generated by ingenuity pathway analysis (IPA) software. Furthermore, experimental validation was carried out with MIP-1β-induced U937 cell model and collagen induced arthritis (CIA) rat model.Results: CCR5 signaling pathway in macrophages was shown to be the top one shared signaling pathway associated with both cell immune response and cytokine signaling. In addition, protein kinase C (PKC) δ and p38 in this pathway were treated as target proteins of WTD in RA. In vitro experiments indicated that WTD inhibited MIP-1β-induced production of TNF-α, MIP-1α, and RANTES as well as phosphorylation of CCR5, PKC δ, and p38 in U937 cells. WTD treatment maintained the inhibitory effects on production of TNF-α and RANTES in MIP-1β-induced U937 cells after CCR5 knockdown. In vivo experiments demonstrated that WTD ameliorated symptoms in CIA rats, decreased the levels of IL-1β, IL-2, IL-6, TNF-α, MIP-1α, MIP-2, RANTES, and IP-10 in serum of CIA rats, as well as mRNA levels of MIP-1α, MIP-2, RANTES, and IP-10 in ankle joints of CIA rats. Furthermore, WTD also lowered the phosphorylation levels of CCR5, PKC δ and p38 in both ankle joints and macrophages in ankle joints from CIA rats.Conclusion: It was demonstrated in this research that WTD played a role in inhibiting inflammatory response in RA which was closely connected with the modulation effect of WTD on CCR5 signaling pathway in macrophages.

Systematically characterize the absorbed effective substances of Wutou Decoction and their metabolic pathways in rat plasma using UHPLC-Q-TOF-MS combined with a target network pharmacological analysis

Wu-tou Decoction (WTD) is a famous traditional Chinese medicine (TCM) formula which is applied to treat arthritis and pain of joints. In this study, a sensitive and rapid method was developed for the separation and identification of the absorbed constituents and metabolites of WTD in the rats plasma by ultra high-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UHPLC-Q-TOF-MS). A total of 22 absorbed prototype constituents and 49 metabolites were identified or tentatively characterized in dosed plasma. The possible metabolic pathways of these constituents involved sulfation, glucuronidation, demethylation, hydroxylation and so on. What’s more, we optimized the conventional process ways of network pharmacology and proposed a new concept called target-network pharmacology (T-NP). T-NP method used the absorbed constituents and the corresponding target proteins to generate compound-target network, and compare to the conventional method indifferent using the compounds collected from herbs, it could reduce the false positive results. We found that the following proteins were related to the WTD therapeutic effects, such as PTGS2, PTGS1, MAPK3, PPARG, TNF, IL4 and IL6. On the whole, the proposed method clearly presented the metabolic processes of WTD and the results gave a comprehensive metabolic profile of WTD in vivo for the first time. The combining use of the T-NP method could discover potential drug targets and disclose the biological processes of WTD, which will open up a new approach in the study of TCM in future.

Pathway of PPAR-gamma coactivators in thermogenesis: a pivotal traditional Chinese medicine-associated target for individualized treatment of rheumatoid arthritis.

Traditional Chinese medicine (TCM) syndromes have been regarded as the crucial clinical manifestations for individualized diagnosis and treatment of complex diseases, including rheumatoid arthritis (RA) and cancer. Especially, RA patients are classified into cold and hot syndromes with different clinical manifestations, interventions and molecular mechanisms. Better effectiveness of a classic cold syndrome-specific herbal formula Wu-tou decoction (WTD) has been achieved. To explore molecular mechanisms of syndrome-specific formulae is of great clinical significance to improve the effectiveness and pertinence of treatment for the complex diseases with personalized conditions. However, the scientific basis of WTD treatment on RA with the cold syndrome remains unclear. Here, we predicted the putative targets for composite compounds contained in WTD using drugCIPHER-CS and constructed a WTD herbs-putative targets-RA related genes network. Next, a list of major WTD targets was identified based on their topological features, including the degree, node betweenness, closeness and k-coreness in the above pharmacological network. Importantly, pathway enrichment analysis revealed that these major WTD targets were significantly associated with the pathway of peroxisome proliferator-activated receptor (PPAR)-gamma (PPAR-γ) coactivators in thermogenesis. These computational findings were subsequently verified by experiments on a rat model of collagen-induced arthritis (CIA) with cold or hot syndromes, and on human fibroblast-like synoviocytes-rheumatoid arthritis (HFLS-RA) cell line. In conclusion, the pathway of PPAR-γ coactivators in thermogenesis might be one of the potential pharmacological targets of WTD to alleviate RA with the TCM cold syndrome. These findings may open new avenues for designing individualized treatment regimens for RA patients.

Effects of Wutou Decoction on DNA Methylation and Histone Modifications in Rats with Collagen‐Induced Arthritis

Background. Wutou decoction (WTD) has been wildly applied in the treatment of rheumatoid arthritis and experimental arthritis in rats for many years. Epigenetic deregulation is associated with the aetiology of rheumatoid arthritis; however, the effects of WTD on epigenetic changes are unclear. This study is set to explore the effects of WTD on DNA methylation and histone modifications in rats with collagen-induced arthritis (CIA). Methods. The CIA model was established by the stimulation of collagen and adjuvant. The knee synovium was stained with hematoxylin and eosin. The DNA methyltransferase 1 (DNMT1) and methylated CpG binding domain 2 (MBD2) expression of peripheral blood mononuclear cells (PBMCs) were determined by Real-Time PCR. The global DNA histone H3-K4/H3-K27 methylation and total histones H3 and H4 acetylation of PBMCs were detected. Results. Our data demonstrated that the DNMT1 mRNA expression was significantly lowered in group WTD compared to that in group CIA (P < 0.05). The DNA methylation level was significantly reduced in group WTD compared to that in group CIA (P < 0.05). Moreover, H3 acetylation of PBMCs was overexpressed in WTD compared with CIA (P < 0.05). Conclusions. WTD may modulate DNA methylation and histone modifications, functioning as anti-inflammatory potential.

Pain and Inflammation: Update on Emerging Phytotherapy, Zootherapy, and Nutritional Therapies.

Pain is the most common health challenge that drives patients to consult physicians. Pain and inflammation are usually symptoms of many diseases. Inability to perceive sensory pain may lead to shortened life expectancy and yet excessive pain may result in low quality of life. Despite the arrays of therapies available for the treatment of pain, it is still not tamed and the currently available drugs have their drawbacks. The challenges to amelioration of pain and inflammation have become an impetus for research into alternative and complementary medicines for the treatment of these ailments. The roles of phytotherapy and nutritional therapies in the treatment of varied types of ailments cannot be overemphasized. Actually, most of the commonly available therapeutic drugs for pain, such as opioids and nonsteroidal anti-inflammatory drugs (NSAIDs), were derived from phytotherapy. Hence, giving attention to this issue is highly recommended as it provides a medium for knowledge acquisition for improvement of the treatment of the twin ailment-pain and inflammation. The researches published in this issue are broad and they accomplished the aim for which the issue was set out. Many manuscripts were received but only those contributing significantly to the subject matter were accepted after thorough peer review. The new insights include topics on systemic models of inflammation contributed by Y. Liu et al., “Effects of Wutou Decoction on DNA Methylation and Histone Modifications in Rats with Collagen-Induced Arthritis,” and X. Wu et al., “Protective Effect of Tetrandrine on Sodium Taurocholate-Induced Severe Acute Pancreatitis.” Z. Shu et al. reported the anti-inflammatory effects of plant extract “Antibacterial and Anti-Inflammatory Activities of Physalis alkekengi var. franchetii and Its Main Constituents” as well as R. Boukhary et al., “Anti-Inflammatory and Antioxidant Activities of Salvia fruticosa: An HPLC Determination of Phenolic Contents.” In another study, I. J. M. Santos et al. reported “Topical Anti-Inflammatory Activity of Oil from Tropidurus hispidus (Spix, 1825).” A review was accepted for inclusion in the issue on effectiveness of acupuncture for treating sciatica by Z. Qin et al. A. Rauf et al. reported on a potential cyclooxygenase inhibitor named daturaolone while W. Kim et al. evaluated the anti-inflammatory potential of a new Ganghwaljetongyeum (N-GHJTY) on adjuvant-induced inflammatory arthritis in rats. This issue therefore is a rich resource for all interested in pain and inflammation research and treatment. Bamidele V. Owoyele Musa T. Yakubu Roi Treister

The significant inhibition on CYP3A caused by radix Aconiti single herb is not observed in the Wutou decoction: The necessity of combination therapy of radix Aconiti

Ethnopharmacological relevanceWutou (WT, Radix Aconiti), the mother root of Aconitum carmichaelii Debx., is a famous Chinese herb against rheumatoid arthritis. In Chinese clinics, PWT is often prepared as a decoction in combination with other herbs, such as Wutou decoction (WTD). The present study aimed to compare the effects of PWT single herb and WTD on CYP3A activity ex vivo and in vivo. Materials and methodsIn the ex vivo study, CYP3A activity was determined by using testosterone (Tes) as a specific probe. Levels of Tes and its metabolite 6β-hydroxytestosterone (6β-OH-Tes) were measured using a validated ultra-performance liquid chromatography (UPLC) method. CYP3A protein and mRNA levels were measured by using Western blot and real-time PCR, respectively. In the in vivo study, CYP3A activity was determined by using buspirone (BP) as a specific probe. The plasma concentrations of BP and its primary metabolites, namely, 1-(2-pyrimidinyl) piperazine (1-PP) and 6′-hydroxybuspirone (6′-OH-BP), were determined using a validated UPLC-tandem mass spectrometry (UPLC/MS/MS) method. ResultsCompared with the control group, the formation rates of 6β-OH-Tes from Tes ex vivo significantly decreased in groups treated with PWT at the tested doses, and this decrease was accompanied by a striking decrease in CYP3A protein and mRNA levels. However, a significant increase was observed in the ratios in the WTD groups compared with PWT single herb groups. In vivo, both formation ratios of 6′-OH-BP and 1-PP from BP showed no significant change in the WTD group. ConclusionsPWT can significantly inhibit CYP3A activity ex vivo at the tested doses because of the down-regulation of CYP3A protein and mRNA expression levels. WTD can significantly reverse the inhibition caused by PWT. WTD also had no significant effect on CYP3A activity in vivo. Results implied that the use of PWT as a part of the WTD prescription rather than PWT single herb is more appropriate in clinics.

Uncovering pharmacological mechanisms of Wu-tou decoction acting on rheumatoid arthritis through systems approaches: drug-target prediction, network analysis and experimental validation.

Wu-tou decoction (WTD) has been extensively used for the treatment of rheumatoid arthritis (RA). Due to lack of appropriate methods, pharmacological mechanisms of WTD acting on RA have not been fully elucidated. In this study, a list of putative targets for compositive compounds containing in WTD were predicted by drugCIPHER-CS. Then, the interaction network of the putative targets of WTD and known RA-related targets was constructed and hub nodes were identified. After constructing the interaction network of hubs, four topological features of each hub, including degree, node betweenness, closeness and k-coreness, were calculated and 79 major hubs were identified as candidate targets of WTD, which were implicated into the imbalance of the nervous, endocrine and immune (NEI) systems, leading to the main pathological changes during the RA progression. Further experimental validation also demonstrated the preventive effects of WTD on inflammation and joint destruction in collagen-induced arthritis (CIA) rats and its regulatory effects on candidate targets both in vitro and in vivo systems. In conclusion, we performed an integrative analysis to offer the convincing evidence that WTD may attenuate RA partially by restoring the balance of NEI system and subsequently reversing the pathological events during RA progression.

Wu-tou decoction inhibits chronic inflammatory pain in mice: participation of TRPV1 and TRPA1 ion channels.

Wu-tou decoction (WTD) is a classic traditional Chinese medicine formula and has been used effectively to treat joint diseases clinically. Previous reports indicated that WTD possesses anti-inflammatory activity; however, its actions on pain have not been clarified. Here, we investigated the antinociceptive activity of WTD in CFA-induced mice, and its possible mechanism of the action associated with transient receptor potential (TRP) ion channels was also explored. Our results showed that 1.58, 3.15, and 6.30 g/kg WTD significantly attenuated mechanical, cold, and heat hypersensitivities. Moreover, WTD effectively inhibited spontaneous nociceptive responses to intraplantar injections of capsaicin and cinnamaldehyde, respectively. WTD also effectively suppressed jumping and wet-dog-shake behaviors to intraperitoneal injection of icilin. Additionally, WTD significantly reduced protein expression of TRPV1 and TRPA1 in dorsal root ganglia and skins of injured paw. Collectively, our data demonstrate firstly that WTD exerts antinociceptive activity in inflammatory conditions by attenuating mechanical, cold, and heat hypersensitivities. This antinociceptive effect may result in part from inhibiting the activities of TRPV1, TRPA1, and TRPM8, and the suppression of TRPV1 and TRPA1 protein by WTD was also highly effective. These findings suggest that WTD might be an attractive and suitable therapeutic agent for the management of chronic inflammatory pain.

Studies of the intestinal absorption of the alkaloids in the Wu-tou decoction combined with different incompatible medicinal herbs in a Caco-2 cell culture system using UPLC–MS/MS

Abstract In this study, seven alkaloids were detected in Wu-tou decoction using ultra performance liquid chromatography coupled with tandem mass spectrometry (UPLC–MS n ). The aim of this study was to investigate the effect of Fritillariae Cirrhosae Bulbus , Fritillariae Thunbergii Bulbus , Pinelliae Rhizoma in different ratios with Wu-tou decoction (2:1, 1:1, 1:2) by measuring the therapeutic effects in Wu-tou decoction of main seven alkaloids including benzoylaconitine (BA), benzoylmesaconitine (BM), benzoylhypaconitine (BH), hypaconitine (HA), fuziline (FU), niaolin (NE) and deoxyaconitine (DA). The permeability of aconitum alkaloids extract through a Caco-2 cell monolayer was analyzed in the absence and presence of Fritillariae Cirrhosae Bulbus , Fritillariae Thunbergii Bulbus , and Pinelliae Rhizoma , respectively. The results showed that Pinelliae Rhizoma could reduce the absorption of the alkaloids and increase the excretion of the alkaloids, which would attenuate the therapeutic effects of Wu-tou decoction. Therefore, Pinelliae Rhizoma is an incompatible herb of Wu-tou decoction because of the inhibition of the absorption of alkaloids in the intestine. And that Fritillariae Cirrhosae Bulbus and Fritillariae Thunbergii Bulbus showed the effects to improve the permeability of the alkaloids in Wu-tou decoction. These effects of these two herbs were similar, but the former was stronger than the latter, which most likely is due to the fact that the compositions of these two traditional Chinese medicines are similar. The in vitro data suggests that the compounds such as fritillary presented in alkaloids in the formula maybe improve the therapeutic function caused by the increased bioavailability of alkaloids in intestine.

Metabonomic study of Wu-tou decoction in adjuvant-induced arthritis rat using ultra-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry

A urinary metabonomics method based on the ultra-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF-MS) had been established to investigate the holistic efficacy of Wu-tou decoction (WTD), a traditional Chinese medicine (TCM) formula used to treat rheumatoid arthritis (RA), in adjuvant-induced arthritis (AIA) rat model. Multivariate statistical approaches, such as principal component analysis (PCA) and orthogonal projection to latent structures squares-discriminant analysis (OPLS-DA) were used to distinguish healthy control group, AIA model group and WTD treated group and find potential biomarkers. There was a clear separation among the three groups in PCA model. Sixteen potential biomarkers had been identified using OPLS-DA, and 11 of them was considered to be in response to therapeutic effects of WTD involved in tryptophan metabolism, phenylalanine metabolism, tricarboxylic acid (TCA) cycle, bile acid biosynthesis, steroid hormone biosynthesis and valine metabolism. In this study, WTD also showed good anti-inflammatory and antioxidant activities in vivo, and it could suppress histopathological changes of AIA rats. There might be a correlation between these results and the regulation of the disturbed metabolites in urine. This study demonstrates that metabonomics is a powerful methodology to gain insight in the mechanism of TCM formula in therapy.

Chemical profiling of Wu-tou decoction by UPLC–Q-TOF-MS

Wu-tou decoction (WTD), a traditional Chinese medicine (TCM) formula, is composed of Aconiti Radix Cocta, Ephedrae Herba, Paeoniae Radix Alba, Astragali Radix and Glycyrrhiza Radix Preparata, and it has been used for more than a thousand years to treat rheumatic arthritis, rheumatoid arthritis and pain of joints, while the active constitutions of WTD are unclear. In this research, an ultra performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF-MS) method in both positive and negative ion mode was established to investigate the major constitutions in WTD. A Waters ACQUITY UPLC BEH C18 column was used to separate the aqueous extract of WTD. Acetonitrile and 0.1% aqueous formic acid (v/v) were used as the mobile phase. 74 components including alkaloids, monoterpene glycosides, triterpene saponins, flavones and flavone glycosides were identified or tentatively characterized in WTD based on the accurate mass within 15 ppm error and tandem MS behavior. All the constitutions were also detected in the corresponding individual herbs. These results will provide a basis for further study in vivo of WTD and the information of potential new drug structure for treating rheumatic arthritis and rheumatoid arthritis.

Comparative study of regulation of TLR / TRAF signaling pathway of classicpriscriptions that deals with arthralgia syndrome based on releventtheory of priscription and syndrome

Based on relevent theory of prescription and syndrome, to compare the gene expression differences of TLR2, TRAF6, and Faslg with adjuvant arthritis in rat spleen among Wutou decoction, Guizhi Shaoyao Zhimu decoction and Baihu Guizhi decoction. The experiment animal model of adjuvant arthritis in rats was established. Relative expression amount of TLR2, TRAF6, and Faslg in rats spleen was detected by SYBR Green I dye methods and implementation of fluorescence quantitative PCR technology with 18sRNA as an internal gene. 2(delta delta CT) method was used for computing and data analysis. TLR2, TRAF6, and Faslg gene in adjuvant arthritis rat spleen was significantly higher than those in the blank group. The various doses of Wutou decoction, Guizhi Shaoyao Zhimu decoction and Baihu Guizhi decoction can significantly inhibit or reduce the abnormally high expression of TLR2, TRAF6, and Faslg genes. The gene expression level caused by three decoctions mentioned above was followed by strong to weak as Wutou decoction Guizhi Shaoyao Zhimu decoction and Baihu Guizhi decoction with the clinical equivalent dose, but the strength of the trend to reduce the role of TRAF6 is just the opposite with the TLR2 and Faslg genes. Wutou decoction, Guizhi Shaoyao Zhimu decoction and Baihu Guizhi decoction can reduce the abnormally high expression of TLR2, TRAF6 and Faslg in rat spleen with adjuvant arthritis, but the differences of intensity exist and remain relatively consistent with that of pharmacodynamics and regulation trends of T cell subsets. Results suggest that the suppression of TLR2/TRAF6 signal pathway and apoptosis Faslg receptor gene may be the reasons that the pharmacodynamics of three decoctions on peripheral T cell subsets in regulating intensity was different.